ASMS Poster collection Clinical, Forensic and Pharmaceutical Applications • Page 4 • Page 54 Rapid development of analytical method for anti- Application of a sensitive liquid chromatography- epileptic drugs in plasma using UHPLC method tandem mass spectrometric method to pharma- scouting system coupled to LC/MS/MS cokinetic study of telbivudine in humans

• Page 11 • Page 60 Determination of ∆9-tetrahydrocannabinol and Accelerated and robust monitoring for immu- two of its metabolites in whole blood, plasma nosuppressants using triple quadrupole mass and urine by UHPLC-MS/MS using QuEChERS spectrometry sample preparation • Page 66 • Page 17 Highly sensitive quantitative analysis of felodip- Determination of opiates, amphetamines and ine and hydrochlorothiazide from plasma using cocaine in whole blood, plasma and urine by LC/MS/MS UHPLC-MS/MS using a QuEChERS sample prepa- ration • Page 73 Highly sensitive quantitative estimation of geno- • Page 23 toxic impurities from API and drug formulation Simultaneous analysis for forensic drugs in using LC/MS/MS human blood and urine using ultra-high speed LC-MS/MS • Page 80 Development of 2D-LC/MS/MS method for quan- • Page 29 titative analysis of 1␣,25-Dihydroxylvitamin D3 in Simultaneous screening and quantitation of human serum amphetamines in urine by on-line SPE-LC/MS method • Page 86 Analysis of polysorbates in biotherapeutic prod- • Page 36 ucts using two-dimensional HPLC coupled with Single step separation of plasma from whole mass spectrometer blood without the need for centrifugation ap- plied to the quantitative analysis of warfarin • Page 93 A rapid and reproducible Immuno-MS platform • Page 42 from sample collection to quantitation of IgG Development and validation of direct analysis method for screening and quantitation of • Page 99 amphetamines in urine by LC/MS/MS Simultaneous determinations of 20 kinds of common drugs and pesticides in human blood • Page 48 by GPC-GC-MS/MS Next generation plasma collection technology for the clinical analysis of temozolomide by • Page 103 HILIC/MS/MS Low level quantitation of loratadine from plasma using LC/MS/MS PO-CON1452E

Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS

ASMS 2014 ThP 672

Miho Kawashima1, Satohiro Masuda2, Ikuko Yano2, Kazuo Matsubara2, Kiyomi Arakawa3, Qiang Li3, Yoshihiro Hayakawa3 1 Shimadzu Corporation, Tokyo, JAPAN, 2 Kyoto University Hospital, Kyoto, JAPAN, 3 Shimadzu Corporation, Kyoto, JAPAN Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS

Introduction Method development for therapeutic drug monitoring quadrupole mass spectrometer used in this study can (TDM) is indispensable for managing drug dosage based on dramatically shorten the total time for optimization of the drug concentration in blood in order to conduct a analytical conditions because this system can make rational and ef cient drug therapy. Liquid chromatography enormous combinatorial analysis methods and run batch coupled with tandem quadrupole mass spectrometry is program automatically. In this study, we developed a increasingly used in TDM because it can perform selective high-speed and sensitive method for measurement of and sensitive analysis by simple sample pretreatment. The seventeen antiepileptics in plasma by UHPLC coupled with UHPLC method scouting system coupled to tandem tandem quadrupole mass spectrometer.

O O + N H N O O- O N H3C NH O N N H N O O NH N 2 2 Cl N CH3 O O NH2 O O O NH2 Cl CH3 Carbamazepine Carbamazepine- 10,11-epoxide Clonazepam Diazepam Ethomuximide Felbamate

O O NH + 2 N H N N O O NH NH O- H N N N OH Cl N O H2N O O NH2 NH2 N 3CH O N Cl O H CH3 O Gabapentin Lamotrigine Levetiracetam Nitrazepam Phenobarbial Phenytoin

CH3 CH3 S O O H 3CH CH O N 3 O 3CH N S N OH O O O O NH O O O CH S 3 O S H2C OH 3CH H2 ON O O CH3 NH2 Primidone Tiagabine Topiramate Vigabatrin Zonisamide

Figure 1 Antiepileptic drugs used in this assay

Experimental Instruments UHPLC based method scouting system (Nexera X2 Method dedicated software was newly developed to control the Scouting System, Shimadzu Corporation, Japan) is system (Method Scouting Solution, Shimadzu Corporation, configured by Nexera X2 UHPLC modules. For the detection, Japan), which provides a graphical aid to configure the tandem quadrupole mass spectrometer (LCMS-8050, different type of columns and mobile phases. The software Shimadzu Corporation, Japan) was used. The system can be is integrated into the LC/MS/MS workstation (LabSolutions, operated at a maximum pressure of 130 MPa, and it enables Shimadzu Corporation, Japan) so that selected conditions to automatically select up to 96 unique combinations of are seamlessly translated into method files and registered to eight different mobile phases and six different columns. A a batch queue, ready for analysis instantly.

2 Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS

Figure 2 Nexera Method Scoutuing System and LCMS-8050 triple quadrupole mass spectrometer

Calibration standards and QC samples

The main standard mixture was prepared in methanol added to 990μL methanol) before injection. from individual stock solutions. The calibration standards Next step of preparation procedure was divided into three were prepared by diluting the standard mixture with groups by the intensity of each compound. For methanol. ethomuximide, phenobarbial and phenytoin, the QC sample was prepared by adding 4 volume of supernatant was used for the LC/MS/MS analysis without acetonitrile to 1 volume of control plasma, thereby further dilution. For zonisamide, 10 μL supernatant was precipitating proteins, and subsequently adding the further diluted with 990 μL methanol. For others, 100 μL standard mixture to the supernatant to contain plasma supernatant was further diluted with 900 μL methanol. concentration equivalents stated in Table 4. The QC The diluted solutions were used for the LC/MS/MS samples were further diluted 100 times (10 μL sample analysis.

Result MRM condition optimization The MS condition optimization was performed by flow MRM optimization function. The transition that gave highest injection analysis (FIA) of ESI positive and negative ionization intensity was used for quantification. The MRM transitions mode, and the compound dependent parameters such as used in this assay are listed in Table 1. CID and pre-bias voltage were adjusted using automatic

3 Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS

Table 1 Compounds, Ionization polarity and MRM transition

Compound Retaintion (min) Polarity Precursor m/z Product m/z Carbamazepine 3.84 + 237.1 194.2 Carbamazepine-10,11-epoxide 3.24 + 253.1 180.15 Clonazepam 3.93 + 316.1 269.55 Diazepam 4.79 + 284.9 154.15 Ethomuximide 2.50 + 239.3 117.20 Felbamate 2.86 + 172.2 154.25 Gabapentin 2.27 + 256.2 211.05 Lamotrigine 2.96 + 171.2 126.15 Levetiracetam 2.32 + 281.9 236.20 Nitrazepam 3.90 + 219.2 162.15 Phenobarbial 3.06 + 376.2 111.15 Phenytoin 3.64 + 130.2 71.15 Primidone 2.83 + 213.1 132.10 Tiagabine 4.28 - 140.0 42.00 Topiramate 3.14 - 231.0 42.05 Vigabatrin 0.82 - 337.9 78.00 Zonisamide 2.58 - 143.1 143.10

UHPLC condition optimization 36 analytical conditions, comprising combinations of 9 ammonium acetate water and methanol for mobile phase mobile phase and 4 columns, were automatically and Inertsil-ODS4 for separation column were selected. investigated using Method Scouting System. Schematic Using this combination of mobile phase and column, the representation of scouting system was shown in Figure 3. gradient condition was further optimized. The final analytical From the result of scouting, the combination of 10 mM condition was shown in Table 2.

Kinetex XB-C18 (Phenomenex) 2.1 x 50 mm Kinetex PFP (Phenomenex) 2.1 x 50 mm Pump A InertsilODS-4 (GL Science) 2.1 x 50 mm Discovery HS F5-5 (SPELCO) 2.1 x 50 mm 1 2 3 4 Auto Sampler LPGE Unit LCMS-8050

Column Oven

Pump B (A) 1 – 10mM Ammonium Acetate 2 – 10mM Ammonium Formate 3 – 0.1%FA - 10mM Ammonium Acetate (B) 1 – Methanol 2 – Acetonitrile 1 2 3 4 3 – Methanol/Acetonitrile=1/1

Figure. 3 Schematic representation and features of the Nexera Method Scouting System.

4 Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS

Table.2 UHPLC analytical conditions

Column : Inertsil ODS-4 (50 mmL. x 2.1mmi.d., 2um) Mobile phase : A) 10mM Ammonium Acetate B) Methanol Binary gradient : B conc. 3% (0.65 min) → 40% (1.00 min) → 85% (5.00 min) → 100% (5.01-8.00 min) → 3% (8.01-10.00 min) Flow Rate : 0.4 mL/min Injection vol. : 1 μL Column Temp. : 40 deg. C

Precision, accuracy and linearity of AEDs Figure 4 shows MRM chromatograms of the 17 AEDs. It took only 10 minutes per one UHPLC/MS/MS analysis, including column rinsing.

Vigabatrin Felbamate Carbamazepine 130.20>71.15(+) 239.30>117.20(+) 237.10>194.20(+)

0.0 1.0 2.0 3.0 4.0 5.0 min 0.0 1.0 2.0 3.0 4.0 5.0 min 0.0 1.0 2.0 3.0 4.0 5.0 min

Gabapentin Lamotrigine Nitrazepam 172.20>154.25(+) 256.20>211.05(+) 281.90>236.20(+)

0.0 1.0 2.0 3.0 4.0 5.0 min 0.0 1.0 2.0 3.0 4.0 5.0 min 0.0 1.0 2.0 3.0 4.0 5.0 min

Levetiracetam Phenobarbial Clonazepam 171.20>126.15(+) 231.00>42.05(-) 316.10>269.55(+)

0.0 1.0 2.0 3.0 4.0 5.0 min 0.0 1.0 2.0 3.0 4.0 5.0 min 0.0 1.0 2.0 3.0 4.0 5.0 min

Ethomuximide Topiramate Tiagabine 140.00>42.00(-) 337.85>78.00(-) 376.20>111.15(+)

0.0 1.0 2.0 3.0 4.0 5.0 min 0.0 1.0 2.0 3.0 4.0 5.0 min 0.0 1.0 2.0 3.0 4.0 5.0 min

Zonisamide Carbamazepine-10,11-epoxide Diazepam 213.10>132.10(+) 253.10>180.15(+) 284.90>154.15

0.0 1.0 2.0 3.0 4.0 5.0 min 0.0 1.0 2.0 3.0 4.0 5.0 min 0.0 1.0 2.0 3.0 4.0 5.0 min

Primidone Phenytoin 219.20>162.15(+) 251.00>208.20(-)

0.0 1.0 2.0 3.0 4.0 5.0 min 0.0 1.0 2.0 3.0 4.0 5.0 min

Figure. 4 Chromatogram of 17 AEDs calibration standards

5 Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS

Table 3 illustrates linearity of 17 AEDs and Table 4 illustrates and accuracy were within +/- 15%. Excellent linearity, accuracy and precision of the QC samples at three accuracy and precision for all 17 AEDs were obtained at concentration levels. Determination coefficient (r2) of all only 1 μL injection volume. calibration curves was larger than 0.995, and the precision

Table.3 Linearity of 17 AEDs QC sample

Compound Linarity (ng/mL) r2 Carbamazepine 0.25 - 50 0.999 Carbamazepine-10,11-epoxide 0.25 - 50 0.998 Clonazepam 0.005 - 2.5 0.998 Diazepam 0.01 - 5 0.999 Ethomuximide 25 - 2500 0.998 Felbamate 0.5 - 100 0.998 Gabapentin 2 - 50 0.999 Lamotrigine 0.25 - 50 0.999 Levetiracetam 0.5 - 100 0.999 Nitrazepam 0.005 - 1 0.999 Phenobarbial 5 - 500 0.996 Phenytoin 5 - 500 0.998 Primidone 0.25 - 10 0.996 Tiagabine 0.25 - 50 0.998 Topiramate 0.5 - 100 0.998 Vigabatrin 0.5 - 50 0.998 Zonisamide 0.5 - 20 0.996

6 Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS

Table.4 Accuracy and precision of 17 AEDs QC sample

Plasma concentration Precision (%) Accuracy (%) Compound equivalents (µg/mL) Low Middle High Low Middle High Low Middle High Carbamazepine 1.8 18 71 2.2 0.9 0.9 106.1 103.9 95.8 Carbamazepine-10,11-epoxide 1.8 18 71 2.4 1.9 1.3 104.2 105.0 98.2 Clonazepam 0.04 0.9 1.8 3.3 0.7 0.5 106.7 102.1 90.1 Diazepam 0.1 0.7 2.9 3.2 1.7 1.4 105.8 106.6 100.6 Ethomuximide 18 446 714 7.8 1.5 1.4 104.3 99.9 97.0 Felbamate 3.6 89 179 1.7 0.4 0.8 97.1 106.3 91.7 Gabapentin 18 36 143 1.3 0.7 0.7 85.8 98.8 89.5 Lamotrigine 1.8 45 71 10.5 1.2 1.7 107.7 98.4 99.2 Levetiracetam 3.6 89 179 2.1 0.5 1.1 99.5 104.9 90.4 Nitrazepam 0.04 0.4 1.4 3.3 1.4 1.5 105.0 105.2 97.9 Phenobarbial 3.6 71 143 3.5 6.2 1.6 100.9 108.4 95.8 Phenytoin 3.6 89 143 7.8 1.9 1.2 103.2 100.1 96.2 Primidone 1.8 18 45 3.2 0.7 0.7 99.5 112.6 97.1 Tiagabine 1.8 18 71 1.8 1.8 1.0 107.6 105.7 97.5 Topiramate 3.6 36 143 12.5 1.5 1.2 105.4 101.6 96.1 Vigabatrin 8.9 18 89 1.4 1.1 2.1 105.9 101.6 88.8 Zonisamide 36 89 179 3.3 1.3 1.6 111.7 100.4 95.2

Conclusions • We could select the most suitable combination of mobile phase and column from 36 analytical condition without time-consuming investigation. • We have measured plasma sample as it is after 100-10,000 times dilution by methanol without making tedious sample pretreatment. Excellent linearity, precision and accuracy for all 17 AEDs were obtained at only 1 uL injection volume.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1446E

Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation

ASMS 2014 ThP600

Sylvain DULAURENT1, Mikaël LEVI2, Jean-michel GAULIER1, Pierre MARQUET1,3 and Stéphane MOREAU2 1 CHU Limoges, Department of Pharmacology and Toxicology, Unit of clinical and forensic toxicology, Limoges, France ; 2 Shimadzu France SAS, Le Luzard 2, Boulevard Salvador Allende, 77448 Marne la Vallée Cedex 2 3 Univ Limoges, Limoges, France Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation

Introduction In France, as in other countries, cannabis is the most and GC-MS. The use of LC-MS/MS for this application is widely used illicit drug. In forensic as well as in clinical relatively recent, due to the low response of these contexts, ∆9-tetrahydrocannabinol (THC), the main active compounds in LC-MS/MS while low limits of quantication compound of cannabis, and two of its metabolites need to be reached. Recently, on-line [11-hydroxy-∆9-tetrahydrocannabinol (11-OH-THC) and Solid-Phase-Extraction coupled with UHPLC-MS/MS was 11-nor-∆9-tetrahydrocannabinol-9-carboxylic acid described, but in our hands it gave rise to signicant (THC-COOH)] are regularly investigated in biological uids carry-over after highly concentrated samples. We propose for example in Driving Under the In uence of Drug here a highly sensitive UHPLC-MS/MS method with context (DUID) (gure 1). straightforward QuEChERS sample preparation (acronym Historically, the concentrations of these compounds were for Quick, Easy, Cheap, Effective, Rugged and Safe). determined using a time-consuming extraction procedure

CH3

OH H

H

3CH O 3CH THC

OH O OH

2CH

OH OH H H

H H CH 3CH O 3 O 3CH 3CH

11-OH-THC THC-COOH

Figure 1: Structures of THC and two of its metabolites

Methods and Materials Isotopically labeled internal standards (one for each target citrate dehydrate/Sodium citrate sesquihydrate) and 200 compound in order to improve method precision and µL of acetonitrile. Then the mixture was shaken and accuracy) at 10 ng/mL in acetonitrile, were added to 100 centrifuged for 10 min at 12,300 g. Finally, 15 µL of the µL of sample (urine, whole blood or plasma) together upper layer were injected in the UHPLC-MS-MS system.

with 50 mg of QuEChERS salts (MgSO4/NaCl/Sodium The whole acquisition method lasted 3.4 min.

2 Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation

UHPLC conditions (Nexera MP system)

Column : Kinetex C18 50x2.1 mm 2.6 µm (Phenomenex) Mobile phase A : 5mM ammonium acetate in water

B : CH3CN Flow rate : 0.6 mL/min Time program : B conc. 20% (0-0.25 min) - 90% (1.75-2.40 min) - 20% (2.40-3.40 min) Column temperature : 50 °C

MS conditions (LCMS-8040)

Ionization : ESI, negative MRM mode Ion source temperatures : Desolvation line: 300°C Heater Block: 500°C Gases : Nebulization: 2.5 L/min Drying: 10 L/min MRM Transitions: Compound MRM Dwell time (msec) THC 313.10>245.25 (Quan) 60 313.10>191.20 (Qual) 60 313.10>203.20 (Qual) 60

THC-D3 316.10>248.30 (Quan) 5 316.10>194.20 (Qual) 5 11-OH-THC 329.20>311.30 (Quan) 45 329.20>268.25 (Qual) 45 329.20>173.20 (Qual) 45

11-OH-THC-D3 332.30>314.40 (Quan) 5 332.30>271.25 (Qual) 5 THC-COOH 343.20>245.30 (Quan) 50 343.20>325.15 (Qual) 50 343.20>191.15 (Qual) 50 343.20>299.20 (Qual) 50

THC-COOH-D3 346.20>302.25 (Quan) 5 346.20>248.30 (Qual) 5 Pause time : 3 msec Loop time : 0.4 sec (minimum 20 points per peak for each MRM transition)

3 Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation

Results Chromatographic conditions A typical chromatogram of the 6 compounds is presented in figure 1.

Figure 1: Chromatogram obtained after an injection of a 15 µL whole blood extract spiked at 50 µg/L

Extraction conditions As described by Anastassiades et al. J. AOAC Int 86 (2003) obtained with whole blood and plasma-serum where 412-31, the combination of acetonitrile and QuEChERS salts deproteinization occurred and allowed phase separation, allowed the extraction/partitioning of compounds of interest but also with urine as presented in figure 2. from matrix. This extraction/partitioning process is not only

A B

Figure 2: in uence of QuEChERS salts on urine extraction/partitioning: A: acetonitrile with urine sample lead to one phase / B: acetonitrile, QuEChERS salts and urine lead to 2 phases.

4 Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation

Validation data One challenge for the determination of cannabinoids in lower limit of quantification was fixed at 0.5 µg/L for the blood using LC-MS/MS is the low quantification limits that three compounds (3.75 pg on column). The corresponding need to be reached. The French Society of Analytical extract ion chromatograms at this concentration are Toxicology proposed 0.5 µg/L for THC et 11-OH-THC and presented in figure 3. 2.0 µg/L for THC-COOH. With the current application, the

THC-COOH 11-OH-THC THC

Figure 3: Chromatogram obtained after an injection of a 15 µL whole blood extract spiked at 0.5 µg/L (lower limit of quantication).

The upper limit of quantification was set at 100 µg/L. expected cannabinoids concentration were constructed Calibration graphs of the cannabinoids-to-internal standard using a quadratic with 1/x weighting regression analysis peak-area ratios of the quantification transition versus (figure 4).

THC-COOH 11-OH-THC THC

Figure 4: Calibration curves of the three cannabinoids

Contrary to what was already observed with on-line injected after patient urine samples with concentrations Solid-Phase-Extraction no carry-over effect was noted using exceeding 2000 µg/L for THC-COOH. the present method, even when blank samples were

5 Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation

Conclusions • Quick sample preparation based on QuEChERS salts extraction/partitioning, almost as short as on-line Solid Phase Extraction. • Low limit of quantication compatible with determination of DUID. • No carry over effect noticed.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1445E

Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation

ASMS 2014 ThP599

Sylvain DULAURENT1, Mikaël LEVI2, Jean-michel GAULIER1, Pierre MARQUET1,3 and Stéphane MOREAU2 1 CHU Limoges, Department of Pharmacology and Toxicology, Unit of clinical and forensic toxicology, Limoges, France ; 2 Shimadzu France SAS, Le Luzard 2, Boulevard Salvador Allende, 77448 Marne la Vallée Cedex 2 3 Univ Limoges, Limoges, France Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation

Introduction The determination of drugs of abuse (opiates, coupled with off-line sample preparation. Recently, on-line amphetamines, cocaine) in biological uids is still an Solid-Phase-Extraction coupled with UHPLC-MS/MS was important issue in toxicology, in cases of driving under the described, but in our hands it gave rise to signicant in uence of drugs (DUID) as well as in forensic toxicology. carry-over after highly concentrated samples. We propose At the end of the 20th century, the analytical methods able here another approach based on the QuEChERS (acronym to determine these three groups of narcotics were mainly for Quick, Easy, Cheap, Effective, Rugged and Safe) sample based on a liquid-liquid-extraction with derivatization preparation principle, followed by UHPLC-MS/MS. followed by GC-MS. Then LC-MS/MS was proposed,

Methods and Materials This method involves 40 compounds of interest (13 metabolites) and 18 isotopically labeled internal standards opiates, 22 amphetamines, as well as cocaine and 4 of its (designed with *) (Table1).

Table 1: list of analyzed compounds with their associate internal standard (*)

Amphetamines or related Cocaine and metabolites Opiates compounds

• Anhydroecgonine methylester • 2-CB • 6-monoacetylmorphine* • Benzoylecgonine* • 2-CI • Dextromethorphan • Cocaethylene* • 4-MTA • Dihydrocodeine* • Cocaine* • Ritalinic acid • Ethylmorphine • Ecgonine methylester* • Amphetamine* • Hydrocodone • BDB • Hydromorphone • Ephedrine* • Methylmorphine* • MBDB • Morphine* • m-CPP • Naloxone* • MDA* • Naltrexone* • MDEA* • Noroxycodone* • MDMA* • Oxycodone* • MDPV • Pholcodine • Mephedrone • Metamphetamine* • Methcathinone • Methiopropamine • Methylphenidate • Norephedrine • Norfen uramine • Norpseudoephedrine • Pseudoephedrine

2 Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation

To 100 µL of sample (urine, whole blood or plasma) were sesquihydrate) were added and the mixture was shaken added isotopically labeled internal standards (in order to again for 15 s and centrifuged for 10 min at 12300 g. The improve method precision and accuracy) at 20 µg/L in upper layer was diluted (1/3; v/v) with a 5 mM ammonium acetonitrile (20 µL), and 200 µL of acetonitrile. After a 15 s formate buffer (pH 3). Finally, 5 µL were injected in the shaking, the mixture was placed at -20°C for 10 min. Then UHPLC-MS/MS system. The whole acquisition method approximately 50 mg of QuEChERS salts lasted 5.5 min.

(MgSO4/NaCl/Sodium citrate dehydrate/Sodium citrate

UHPLC conditions (Nexera MP system, gure 1)

Column : Restek Pinnacle DB PFPP 50x2.1 mm 1.9 µm Mobile phase A : 5mM Formate ammonium with 0.1% formic acid in water

B : 90% CH3OH/ 10% CH3CN (v/v) with 0.1 % formic acid Flow rate : 0.474 mL/min Time program : B conc. 15% (0-0.16 min) - 20% (1.77 min) - 90% (2.20 min) – 100% (4.00 min) – 15% (4.10-5.30 min) Column temperature : 50 °C

MS conditions (LCMS-8040, gure 1)

Ionization : ESI, Positive MRM mode Ion source temperatures : Desolvation line: 300°C Heater Block: 500°C Gases : Nebulization: 2.5 L/min Drying: 10 L/min MRM Transitions : 2 Transitions per compounds were dynamically scanned for 1 min except pholcodine (2 min) Pause time : 3 msec Loop time : 0.694 sec (minimum 17 points per peak for each MRM transition)

Figure 1: Shimadzu UHPLC-MS/MS Nexera-8040 system

3 Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation

Results Chromatographic conditions The analytical conditions allowed the chromatographic (figure 2). A typical chromatogram of the 58 compounds is separation of two couples of isomers: norephedrine and presented in figure 3. norpseudoephedrine; ephedrine and pseudoephedrine

A B

Figure 2: Chromatograms obtained after an injection of a 5 µL whole blood extract spiked at 200 µg/L. Order of retention - A: norephedrine and norpseudoephedrine / B: ephedrine and pseudoephedrine

Figure 3: Chromatogram obtained after an injection of a 5 µL whole blood extract spiked at 200 µg/L

4 Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation

Extraction conditions As described by Anastassiades et al. J. AOAC Int 86 (2003) obtained with whole blood and plasma-serum where 412-31, the combination of acetonitrile and QuEChERS salts deproteinization occurred and allowed phase separation, allowed the extraction/partitioning of compounds of interest but also with urine as presented in figure 4. from matrix. This extraction/partitioning process is not only

A B

Figure 4: in uence of QuEChERS salts on urine extraction/partitioning: A: acetonitrile with urine sample lead to one phase / B: acetonitrile, QuEChERS salts and urine lead to 2 phases.

Validation data Among the 40 analyzed compounds, 38 filled the validation concentrations obtained with a reference (GC-MS) method conditions in term of intra- and inter-assay precision and in positive patient samples were compared with those accuracy were less than 20% at the lower limit of obtained with this new UHPLC-MS/MS method and showed quantification and less than 15% at the other satisfactory results. concentrations. Contrary to what was already observed with on-line Despite the quick and simple sample preparation, no Solid-Phase-Extraction, no carry-over effect was noted using significant matrix effect was observed and the lower limit of the present method, even when blank samples were quantification was 5 µg/L for all compounds, while the injected after patient urine samples with analytes upper limit of quantification was set at 500 µg/L. The concentrations over 2000 µg/L.

5 Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation

Conclusions • Separation of two couples of isomers with a run duration less than 6 minutes and using a 5 cm column. • Quick sample preparation based on QuEChERS salts extraction/partitioning, almost as short as on-line Solid Phase Extraction. • Lower limit of quantication compatible with determination of DUID. • No carry over effect noticed.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1442E

Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS

ASMS 2014 ThP-592

Toshikazu Minohata1, Keiko Kudo2, Kiyotaka Usui3, Noriaki Shima4, Munehiro Katagi4, Hitoshi Tsuchihashi5, Koichi Suzuki5, Noriaki Ikeda2 1Shimadzu Corporation, Kyoto, Japan 2Kyushu University, Fukuoka, Japan 3Tohoku University Graduate School of Medicine, Sendai, Japan 4Osaka Prefectural Police, Osaka, Japan 5Osaka Medical Collage, Takatsuki, Japan Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS

Introduction In Forensic Toxicology, LC/MS/MS has become a preferred speed triple quadrupole mass spectrometry with a new method for the routine quantitative and qualitative analysis extraction method for pretreatment in forensic analysis. of drugs of abuse. LC/MS/MS allows for the simultaneous The system has a sample preparation utilizing modi ed analysis of multiple compounds in a single run, thus QuEChERS extraction combined with a short enabling a fast and high throughput analysis. In this study, chromatography column that results in a rapid run time we report a developed analytical system using ultra-high making it suitable for routine use.

Methods and Materials Sample Preparation Whole blood sample preparation was carried out by the 4) Vigorously shake the tube by hand several times, agitate modified QuEChERS extraction method (1) using Q-sep™ well using the vortex mixer for approximately 20 QuEChERS Sample Prep Packets purchased from RESTEK seconds. Then centrifuge the tube for 10 minutes at (Bellefonte, PA). 3000 rpm. 5) Move the supernatant to a different 15 mL centrifugal 1) Add 0.5 mL of blood and 1 mL of distilled water into tube and add 100 µL of 0.1 % TFA acetonitrile solution. the 15 mL centrifugal tube and agitate the mixture Then, dry using a nitrogen-gas-spray concentration and using a vortex mixer. drying unit or a similar unit. 2) Add two 4 mm stainless steel beads, 1.5 mL of 6) Reconstitute with 200 µL of methanol using the vortex acetonitrile and 100 µL of acetonitrile solution mixer. Then move it to a microtube, and centrifuge for containing 1 ng/µL of Diazepam-d5. Then agitate using 5 minutes at 10,000 rpm. the vortex mixer. 7) Transfer 150 µL of the supernatant to a 1.5 mL vial for 3) Add 0.5 g of the filler of the Q-sep™ QuEChERS HPLC provided with a small-volume insert. Extraction Salts Packet.

[ ref.] (1) Usui K et al, Legal Medicine 14 (2012), 286-296

Water 1 mL ACN 1.5 mL Diazepam-d5 (IS) 100ng Transfer supernatant Stainless-Steel Beads (4mm x 2) Add 100uL of 0.1% TFA

Dry

Reconstitution with 200 uL MeOH Q-sep QuEChERS Extraction Salts LC/MS/MS analysis (MgSO4,NaOAc) Sample [Shake] 0.5 mL [Centrifuge]

Figure 1 Scheme of the modi ed QuEChERS procedure

2 Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS

LC-MS/MS Analysis Treated samples were analyzed using a Nexera UHPLC provides Synchronized Survey Scan® parameters (product system coupled to a LCMS-8050 triple quadrupole mass ion spectral data acquisition parameters based on the spectrometer (Shimadzu Corporation, Japan) with MRM intensity as threshold) optimized for screening LC/MS/MS Rapid Tox. Screening Database. The Database analysis. contains product ion scan spectra for 106 forensic and Samples were separated on a YMC Triart C18 column. A toxicology-related compounds of Abused drugs, ow rate of 0.3 mL/min was used together with a gradient Psychotropic drugs and Hypnotic drugs etc (Table 1) and elution.

Analytical Conditions

HPLC (Nexera UHPLC system)

Column : YMC Triart C18 (100x2mm, 1.9μm) Mobile Phase A : 10 mM Ammonium formate - water Mobile Phase B : Methanol Gradient Program : 5%B (0 min) - 95%B (10 min - 13min) - 5%B (13.1 min - 20 min) Flow Rate : 0.3 mL / min Column Temperature : 40 ºC Injection Volume : 5 uL

Mass (LCMS-8050 triple quadrupole mass spectrometry)

Ionization : heated ESI Polarity : Positive & Negative Probe Voltage : +4.5 kV (ESI-Positive mode); -3.5 kV (ESI-Negative mode) Nebulizing Gas Flow : 3 L / min Drying Gas Pressure : 10 L / min Heating gas ow : 10 L / min DL Temperature : 250 ºC BH Temperature : 400 ºC MRM parameter :

Collision Collision Analytes Ret. Time Q1 m/z Q3 m/z Energy Analytes Ret. Time Q1 m/z Q3 m/z Energy 290.15 154.05 -27 411.20 191.05 -28 Diazepam-d5 9.338 Risperidone 7.993 290.15 198.20 -34 411.20 69.05 -55 309.10 281.10 -24 343.05 315.00 -27 Alprazolam 8.646 Triazolam 8.573 309.10 205.10 -41 343.05 308.20 -25 290.15 124.15 -23 Amobarbital 225.15 42.00 25 Atropine 5.378 8.093 290.15 93.20 -30 (neg) 225.15 182.00 14 295.05 267.15 -24 Barbital 183.10 42.10 21 Estazolam 8.408 5.243 295.05 205.25 -37 (neg) 183.10 140.10 15 361.15 259.10 -30 Phenobarbital 231.10 42.20 19 Ethyl lo azepate 9.350 6.762 361.15 287.15 -19 (neg) 231.10 85.10 14 343.05 314.10 -24 Thiamylal 253.00 58.10 23 Etizolam 8.786 8.883 343.05 138.15 -36 (neg) 253.00 101.00 16 376.15 165.15 -24 Haloperidol 8.253 376.15 123.10 -39

3 Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS

positive

negative

Figure 2 LCMS-8050 triple quadrupole mass spectrometer

Results and Discussion

Alprazolam Etizolam Risperidone Triazolam

(x103) 309.10>281.10(+) (x103) 343.05>314.10(+) (x103) 411.20>191.05(+) (x102) 343.05>315.00(+) 2.0

0.01 S/N 39.5 1.0 S/N 145.5 2.5 S/N 107.6 S/N 18.8 2.5 ng/mL 1.0

0.0 0.0 0.0 0.0 (x104) 309.10>281.10(+) (x104) 343.05>314.10(+) (x104) 411.20>191.05(+) (x103) 343.05>315.00(+) 1.0 0.1 0.5 2.5 2.5 ng/mL 0.5

0.0 0.0 0.0 0.0 8.0 8.5 9.0 9.5 8.0 8.5 9.0 9.5 7.0 7.5 8.0 8.5 8.0 8.5 9.0 9.5 Area Ratio Area Ratio (x0.1) Area Ratio Area Ratio (x0.1) 2 2 5.0 2 4.0 2 1.0 r =0.998 7.5 r =0.998 r =0.998 r =0.998 3.0 5.0 0.5 2.5 2.0 2.5 1.0 0.0 0.0 0.0 0.0 0.00 0.25 0.50 0.75 Conc. Ratio 0.00 0.25 0.50 0.75 Conc. Ratio 0.00 0.25 0.50 0.75 Conc. Ratio 0.00 0.25 0.50 0.75 Conc. Ratio

Conc. Area Accuracy %RSD Conc. Area Accuracy %RSD Conc. Area Accuracy %RSD Conc. Area Accuracy %RSD 9,004 112.1 4,865 114.4 29,832 108.4 3,047 107.0 0.01 8,288 105.1 6.57 0.01 5,109 119.9 8.71 0.01 32,436 116.7 5.14 0.01 3,064 109.2 5.63 9,519 119.3 4,321 105.7 30,461 110.8 3,356 118.5 75,236 89.6 48,038 84.0 335,202 91.3 27,991 94.8 0.1 75,983 89.6 6.04 0.1 49,152 85.1 1.82 0.1 309,273 83.7 4.74 0.1 25,542 85.7 7.83 74,023 80.6 54,497 87.0 343,172 85.6 26,317 81.5 829,519 99.9 604,640 103.7 3,826,373 102.8 288,776 99.0 1 831,098 99.6 2.53 1 581,207 99.2 2.22 1 3,718,854 99.4 1.66 1 297,332 101.5 1.96 849,597 104.2 579,390 101.2 3,705,165 101.4 294,788 102.9

4 Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS

Amobarbital (neg) Barbital (neg) Phenobarbital (neg) Thiamylal (neg)

(x102) 225.15>42.00(-) (x10) 183.10>42.10(-) (x102) 231.10>42.20(-) (x102) 253.00>58.10(-) 5.0 S/N 40.2 S/N 15.3 1.0 S/N 38.2 S/N 167.9 2.5 5.0 1 2.5 ng/mL 0.5

0.0 0.0 0.0 0.0 (x103) 225.15>42.00(-) (x102) 183.10>42.10(-) (x103) 231.10>42.20(-) (x103) 253.00>58.10(-) 5.0 5.0 1.0 10 2.5 2.5 ng/mL 2.5 0.5

0.0 0.0 0.0 0.0 7.5 8.0 8.5 9.0 4.5 5.0 5.5 6.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

Area Ratio (x0.1) Area Ratio (x0.01) Area Ratio (x0.1) Area Ratio (x0.1) r2=0.999 r2=0.999 1.00 r2=0.999 4.0 r2=0.999 2.0 5.0 0.75 3.0 0.50 2.0 1.0 2.5 0.25 1.0 0.0 0.0 0.00 0.0 0.0 25.0 50.0 Conc. Ratio 0.0 25.0 50.0 Conc. Ratio 0.0 25.0 50.0 Conc. Ratio 0.0 25.0 50.0 Conc. Ratio

Conc. Area Accuracy %RSD Conc. Area Accuracy %RSD Conc. Area Accuracy %RSD Conc. Area Accuracy %RSD 1,837 100.2 521 108.7 725 106 2,520 107 1 1,862 99.1 4.53 1 464 96.6 7.10 1 693 100.2 9.82 1 2,192 95.3 8.99 2,041 105.8 509 103.4 617 91 2,288 97.5 21,685 99.6 5,078 95.6 7,909 98.8 30,808 101.4 10 22,169 102.4 5.30 10 5,033 95.4 2.38 10 8,564 107.5 5.82 10 29,623 98.3 1.68 20,654 92.5 5,424 99.4 7,939 96.7 31,379 100.6 227,698 101.3 55,420 101.4 81,987 99.2 318,233 100.7 100 223,480 98.3 1.62 100 55,658 100.8 1.42 100 83,274 99.7 0.85 100 317,214 99.3 0.71 225,079 100.9 53,484 98.7 82,656 100.8 313,399 100

Figure 3 Results of 8 drugs spiked in human whole blood using LCMS-8050

In this experiment, two different matrices consisting of Haloperidol, Nimetazepam, Risperidone and Triazolam) and human whole blood and urine were prepared and 18 from 1 to 100 ng/mL for 6 drugs (Bromovalerylurea, drugs were spiked into extract solution. Calibration curves Amobarbital, Barbital, Loxoprofen, Phenobarbital and constructed in the range from 0.01 to 1 ng/mL for 12 Thiamylal). All calibration curves displayed linearity with an drugs (Alprazolam, Aripiprazole, Atropine, Brotizolam, R2 > 0.997 and excellent reproducibility was observed for Estazolam, Ethyl loazepate, Etizolam, Flunitrazepam, all compounds (CV < 12%) at low concentration level.

5 Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS

Amobarbital (neg) Barbital (neg) Phenobarbital (neg) Thiamylal (neg)

(x102) 225.15>42.00(-) (x102) 183.10>42.10(-) (x102) 231.10>42.20(-) (x102) 253.00>58.10(-)

5.0 2.5 S/N 14.7 S/N 9.4 S/N 18.3 S/N 97.4 1 1.0 1.0 2.5 ng/mL

0.0 0.0 0.0 (x103) 225.15>42.00(-) (x102) 183.10>42.10(-) (x103) 231.10>42.20(-) (x103) 253.00>58.10(-)

1.0 5.0 2.5 10 5.0 0.5 2.5 ng/mL 2.5

0.0 0.0 0.0 0.0 7.5 8.0 8.5 9.0 4.5 5.0 5.5 6.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 Area Ratio (x0.1) Area Ratio (x0.1) Area Ratio (x0.1) Area Ratio (x0.1) 2 2 2 2 3.0 r =0.999 r =0.999 r =0.999 r =0.999 0.75 1.0 5.0 2.0 0.50 0.5 2.5 1.0 0.25

0.0 0.00 0.0 0.0 0.0 25.0 50.0 Conc. Ratio 0.0 25.0 50.0 Conc. Ratio 0.0 25.0 50.0 Conc. Ratio 0.0 25.0 50.0 Conc. Ratio

Conc. Area Accuracy %RSD Conc. Area Accuracy %RSD Conc. Area Accuracy %RSD Conc. Area Accuracy %RSD 1,468 102.2 651 93.6 612 103.6 3,142 95.1 1 1,233 86.6 12.73 1 695 96.1 2.77 1 545 89.4 8.16 1 3,470 100.5 4.54 1,245 87.6 654 89 609 99.3 3,153 91.4 17,241 104.4 4,989 105.2 5,656 97.9 27,257 94.9 10 20,546 114.7 5.10 10 5,613 109.6 2.07 10 6,632 106.1 4.24 10 34,377 110.8 8.15 18,689 106.9 5,443 108.6 6,384 104.4 32,933 108.5 211,917 96.8 55,392 92.6 71,965 95.2 365,563 98.5 100 251,963 103 3.34 100 69,481 104 5.98 100 88,685 105 4.95 100 431,826 104.1 4.15 234,789 97.9 66,327 101.3 82,091 99.1 390,719 96.1

Figure 4 Results of 4 drugs spiked in human urine using LCMS-8050

Conclusions • The validated sample preparation protocol can get adequate recoveries in quantitative works for all compounds ranging from acidic to basic. • The combination of the modi ed QuEChERS extraction method and high-speed triple quadrupole LC/MS/MS with a simple quantitative method enable to acquire reliable data easily.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1460E

Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method

ASMS 2014 ThP587

Helmy Rabaha1, Lim Swee Chin1, Sun Zhe2, Jie Xing2 & Zhaoqi Zhan2 1Department of Scienti c Services, Ministry of Health, Brunei Darussalam; 2Shimadzu (Asia Paci c) Pte Ltd, Singapore, SINGAPORE Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method

Introduction Amphetamines belong to stimulant drugs and are also analysis of ve amphetamines in urine without sample controlled as illicit drugs worldwide. The conventional pre-treatment except dilution with water. The compounds analytical procedure of amphetamines in human urine studied include amphetamine (AMPH), methamphetamine includes initial immunological screening followed by GCMS (MAMP) and three newly added MDMA, MDA and MDEA con rmation and quantitation [1]. With new SAMHSA by the new SAMHSA guideline (group A in Table 1). Four guidelines effective in Oct 2010 [2], screening, potential interferences (group B in) and PMPA (R) as a con rmation and quantitation of illicit drugs including control reference were also included to enhance the amphetamines were allowed to employ LC/MS and method reliability in identi cation of the ve targeted LC/MS/MS, which usually does not require a derivatization amphetamines from those structurally similar analogues step as used in the GCMS method [1]. The objective of this which potentially present in forensic samples. study was to develop an on-line SPE-LC/MS method for

Experimental The test stock solutions of the ten compounds (Table 1) a normal SPE cartridge. The injected sample rst passed were prepared in the toxicology laboratory in the through the trapping column where the amphetamines Department of Scienti c Services (MOH, Brunei). Five urine were trapped, concentrated and washed by pure water for specimens were collected from healthy adult volunteers. 3 minutes followed by switching to the analytical ow line. The urine samples used as blank and matrix to prepare The trapped compounds were then eluted out with a spiked amphetamine samples were not pre-treated off-line gradient program: 0.01min, valve at position 0 & B=5%; 3 by any means except dilution of 10 times with pure water. min, valve at position 1; 3.01-10 min, B=5% → 15%; An on-line SPE-LC/MS was set up on the LCMS-2020, a 10.5-12 min, B=65%; 12.1 min, B=5%; 14 min stop, valve single quadrupole system, with a switching valve and a to position 0. The mobile phases A and B were water and trapping column kit (Shimadzu Co-Sense con guration) MeOH both with 0.1% formic acid and mobile C was pure installed in the column oven and controlled by the water. The total ow rates of the trapping line and LabSolutions workstation. The analytical column used was analytical line are 0.6 and 0.3 mL/min, respectively. The Shim-pack VP-ODS 150 x 2mm (5um) and the trapping injection volume was 20uL in all experiments. column was Synergi Polar-RP 50 x 2mm (2.5um), instead of

2 Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method

Table 1: Amphetamines & relevant compounds

No Name Abbr. Name Formula Structure

A1 Amphetamine AMPH C9H13N

A2 Methampheta-mine MAMP C10H15N

A3 3,4-methylene-dioxyamphetamine MDA C10H13NO2

A4 3,4-methylene-dioxymetham phetamine MDMA C11H15NO2

A5 3,4-methylene dioxy-N-ethyl amphetamine MDEA C12H17NO2

B1 Nor pseudo-ephedrine Nor pseudo-E C9H13NO

B2 Ephedrine Ephe C10H15NO

B3 Pseudo-Ephedrine Pseudo-E C10H15NO

B4 Phentermine Phent C10H15N

R Propyl-amphetamine PAMP C12H19N

SPE Trapping Manual Pump A Mixer Column injector Analytical LCMS-2020 column

5 3 1

Waste

Pump B Switching Auto Pump C Valve sampler

Figure 1: Schematic diagram of on-line SPE-LC/MS system

3 Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method

Results and Discussion Development of on-line SPE-LC/MS method With ESI positive SIM and scan mode, all of the 10 (10-30 mmL) for on-line SPE could not trap all of the ten compounds formed protonated ions [M+H]+ which were compounds. With using a 50mmL C18-column to replace used as quantifier ions. The scan spectra were used for the SPE cartridge, the ten compounds studied were confirmation to reduce false positive results. Mixed trapped efficiently. Furthermore, the trapped compounds standards of the ten compounds in Table 1 spiked in urine were well-separated and eluted out in 8~13 minutes as was used for method development. An initial difficulty sharp peaks (Figure 2) by the fully automated on-line encountered was that the normal reusable SPE cartridges SPE-LC/MS method established.

(x1,000,000) (x1,000,000) 2.0 2:136.10(+) 2.0 2:136.10(+) 2:150.10(+) 2:150.10(+) 2:178.10(+) 2:178.10(+) 2:180.10(+) (a) Urine blank (b) spiked samples 2:180.10(+) MDEA

2:194.10(+) 2:194.10(+) PAMP 2:208.20(+) 1.5 1.5 2:208.20(+) MAMP 2:166.10(+) MDMA 2:152.10(+) 2:166.10(+) AMPH 2:152.10(+) Phent Ephedrine 1.0 1.0 MDA Pseudo

0.5 0.5 Norpseudo

0.0 0.0 0.0 2.5 5.0 7.5 10.0 12.5 min 0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 2: SIM chromatograms of urine blank (a) and ve amphetamines and related compounds (125 ppb each) spiked in urine (b) by on-line SPE-LC/MS.

Calibration curves of the on-line SPE-LC/MS method were curves with R2> 0.999 were obtained for every compound established using mixed standard samples with (Figure 3 & Table 2). concentrations from 2.5 ppb to 500 ppb. Linear calibration

Area (x1,000,000) Area (x10,000,000) Area (x10,000,000) Area (x10,000,000) Area (x10,000,000) 3.0 7.5 AMPH MAMP 1.0 MDA MDMA MDEA 1.5 2.0 2.0 5.0 1.0 0.5 1.0 2.5 0.5 1.0

0.0 0.0 0.0 0.0 0.0 0 250 Conc. 0 250 Conc. 0 250 Conc. 0 250 Conc. 0 250 Conc. Area (x1,000,000) Area (x10,000,000) Area (x10,000,000) Area (x10,000,000) Area (x10,000,000) Nor pseudo-E 1.0 Ephedrine 1.5 Pseudo-E Phent PAMP 5.0 1.5 2.0

1.0 1.0 0.5 2.5 1.0 0.5 0.5

0.0 0.0 0.0 0.0 0.0 0 250 Conc. 0 250 Conc. 0 250 Conc. 0 250 Conc. 0 250 Conc.

Figure 3: Calibration curves of ve amphetamines and ve related compounds with concentrations from 2.5 ppb to 500 ppb by on-line SPE-LC/MS method

4 Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method

Table 2: Peak detection, retention, calibration curves and method performance evaluation

SIM ion RT Conc. range Linearity Rec. % M.E % RSD%(n=6) S/N LOD/LOQ Name (+) (min) (ppb) (r2) (62.5ppb) (62.5ppb) (62.5ppb) (2.5ppb) (ppb)

Norpseudo-E 152.1 8.0 2.5 - 500 0.9982 97.3 69.3 1.67 11.3 0.71/2.17 Ephe 166.1 8.4 2.5 - 500 0.9960 84.4 111.0 0.54 33.7 0.25/0.76 Pseudo-E 166.1 9.0 2.5 - 500 0.9976 78.9 109.2 0.41 28.5 0.29/0.88 AMPH 136.1 9.6 2.5 - 500 0.9983 85.6 71.1 0.98 17.5 0.48/1.46 MAMP 150.1 10.2 2.5 - 500 0.9968 76.5 96.8 0.94 30.3 0.26/0.80 MDA 180.1 10.4 2.5 - 500 0.9989 71.8 70.3 1.94 18.2 0.45/1.36 MDMA 194.1 10.8 2.5 - 500 0.9973 72.2 116.3 1.08 36.6 0.23/0.70 MDEA 208.1 12.2 2.5 - 500 0.9908 74.8 107.1 2.18 41.9 0.19/0.57 Phent 150.1 12.4 2.5 - 500 0.9960 74.5 69.9 1.82 12.7 0.66/2.01 PAMP (Ref) 178.1 12.7 2.5 - 500 0.9912 69.5 96.8 5.30 37.7 0.22/0.66

Performance evaluation of on-line SPE-LCMS method The trapping efficiency of the on-line SPE is critical and urine specimens did not show significant differences. must be evaluated first, because it determines the recovery Repeatability was evaluated with spiked mixed samples of of the method. In this study, the recovery of the on-line 62.5 ppb and 250 ppb. The results of 62.5 ppb is shown in SPE was determined by injecting a same mixed standard Table 2, RSD between 0.41% and 5.3%. The sensitivity of sample from a manual injector installed before the the on-line SPE-LC/MS method was evaluated with spiked analytical column (by-pass on-line SPE) and also from the sample of 2.5 ppb level. The SIM chromatograms are Autosampler (See Figure 1). The peaks areas obtained by shown in Figure 4. The S/N ratios obtained ranged the two injections were used to calculate recovery value of 11.3~42, which were suitable to determine LOQ (S/N = 10) the on-line SPE method. As shown in Table 2, the recovery and LOD (S/N = 3). Since the urine samples were diluted obtained with 62.5 ppb mixed standards are at 69.5% ~ for 10 times with water before injection, the LOD and LOQ 97.3%. The recovery with 250 ppb and 500 ppb mixed of the method for source urine samples were at 1.9~7.1 samples were also determined and similar results were and 5.7~21.7 ng/mL, respectively. The confirmation cutoff obtained. values of the five targeted amphetamines (Group A) in Matrix effect was determined with 62.5 ppb and 250 ppb urine enforced by the new SMAHSA guidelines are 250 levels of mixed samples in clear solution and in urine. The ng/mL [2]. The on-line SPE-LC/MS method established has results (Table 2) show a variation between 69.3% and sufficient allowance in terms of sensitivity and confirmation 116% with compounds. The matrix effect with different reliability for analysis of actual urine samples.

(x10,000) 6.0 2:136.10(+) 2:150.10(+)

2:178.10(+) PAMP 2:180.10(+) 5.0 2:194.10(+) 2:208.20(+)

2:166.10(+) MDEA 4.0 2:152.10(+)

3.0 MDMA Phent Ephedrine MDA Pseudo 2.0 MAMP AMPH 1.0 Norpseudo

7.5 10.0 12.5 min

Figure 4: SIM chromatograms of 10 compounds with 2.5 ppb each by on-line SPE-LC/MS method.

5 Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method

Durability of on-line SPE trapping column The durability of the trapping column was tested purposely spiked sample. The results show that the variations of peak by continuous injections of spiked urine samples (125 ppb) area and retention time of the 200th injection compared to for 200 times in a few days. Figure 5 shows the the 1st injection were at 89.5%~117.8% and chromatograms of the first and 200th injections of a same 89.5%~99.8% respectively.

(x1,000,000) (x1,000,000) 2:136.10(+) 2.0 2:136.10(+) 2.0 2:150.10(+) 2:150.10(+) th 2:178.10(+) PAMP 2:178.10(+) st PAMP 200 injection 2:180.10(+) 1 injection 2:180.10(+) 2:194.10(+) 2:194.10(+) spiked mixed std 125ppb in urine 2:208.20(+) spiked mixed std 125ppb in urine 1.5 2:208.20(+)

1.5 MDEA 2:166.10(+) 2:166.10(+) inj vol: 20 µL 2:152.10(+) inj vol: 20 µL 2:152.10(+) MDEA 1.0 1.0 MDMA MDMA Ephedrine Ephedrine Pseudo MAMP Phent MAMP Pseudo 0.5 0.5 MDA Phent MDA AMPH AMPH Norpseudo Norpseudo

0.0 0.0 0.0 2.5 5.0 7.5 10.0 12.5 min 0.0 2.5 5.0 7.5 10.0 12.5 min

Figure 5: Durability test of on-line SPE-LC/MS method, comparison of 1st and 200th injections.

Con rmation Reliability Confirmation reliability of LC/MS and LC/MS/MS methods simultaneously is used for excluding false-positive. In this must be proven to be equivalent to the GCMS method work, the confirmation reliability was evaluated using five according to the SMAHSA guidelines [2]. Validation of different urine specimens as matrix to prepare spiked confirmation reliability of the on-line SPE-LC/MS method samples of 2.5 ppb (correspond 25 ng/mL in source urine) has not be carried out systematically. The high sensitivity of and above. The results show that false-positive and false MS detection in SIM mode is a key factor to ensure no negative results were not found. false-negative and the scan spectra acquired

Conclusions A novel high sensitivity on-line SPE-LC/MS method was SPE cartridges. The method performance was evaluated developed for screening, conformation and quanti cation thoroughly with urine spiked samples. The results of ve amphetamines: AMPH, MAMP, MDMA, MDA and demonstrate that the on-line SPE-LC/MS method is suitable MDEA in urines. The recovery of the on-line SPE by for direct analysis of the amphetamines and relevant employing a 50mmL Synergi Polar-RP column was at compounds in urine samples without off-line sample 72%~86% for the ve amphetamines, which are pre-treatment. considerably high if comparing with conventional on-line

6 Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method

References 1. Kudo K, Ishida T, Hara K, Kashimura S, Tsuji A, Ikeda N, J Chromatogr B, 2007, 855, 115-120. 2. SAMHSA “Manual for urine laboratories, National laboratory certi cation program”, Oct 2010, US Department of Health and Human Services.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1481E

Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin

ASMS 2014 MP762

Alan J. Barnes1, Carrie-Anne Mellor2, Adam McMahon2, Neil J. Loftus1 1Shimadzu, Manchester, UK 2WMIC, University of Manchester, UK Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin

Introduction Dried plasma sample collection and storage from whole result of vitamin-K recycling being inhibited, hepatic blood without the need for centrifugation separation and synthesis is in-turn inhibited for blood clotting factors as refrigeration opens new opportunities in blood sampling well as anticoagulant proteins. Whilst the measurement of strategies for quantitative LC/MS/MS bioanalysis. Plasma warfarin activity in patients is normally measured by samples were generated by gravity ltration of a whole prothrombin time by international normalized ratio (INR) in blood sample through a laminated membrane stack some cases the quantitation of plasma warfarin allowing plasma to be collected, dried, transported and concentration is needed to con rm patient compliance, analysed by LC/MS/MS. This novel plasma separation card resistance to the anticoagulant drug, or diet related issues. (PSC) technology was applied to the quantitative In this preliminary evaluation, warfarin concentration was LC/MS/MS analysis of warfarin, in blood samples. Warfarin measured by LC/MS/MS to evaluate if PSC technology is a coumarin anticoagulant vitamin-K antagonist used for could complement INR when sampling patient blood. the treatment of thrombosis and thromboembolism. As a

Materials and Methods Sample preparation Warfarin standard was dissolved in water containing 50% 40uL methanol, followed by centrifugation 16,000g 5 min. ethanol + 0.1% formic acid, spiked (60uL) to whole human 20uL supernatant was added directly to the LCMS/MS blood (1mL) and mixed gently. 50uL of spiked blood was sample vial already containing 80uL water (2uL analysed). deposited onto the PSC. After 3 minutes, the primary Control plasma comparison was prepared by centrifuging filtration overlay was removed followed by 15 minutes air remaining blood at 1000g for 10min. 2.5uL supernatant drying at room temperature. The plasma sample disc was plasma was taken, 40uL methanol added, and prepared as prepared directly for analysis after drying. LC/MS/MS PSC samples. LCMS/MS sample injection volume, 2uL. sample preparation involved vortexing the sample disk in

LC-MS/MS analysis Warfarin was measured by MRM, positive negative switching mode (15msec).

LC/MS/MS System : Nexera UHPLC system + LCMS-8040 Shimadzu Corporation Flow rate : 0.4mL/min (0-7.75min), 0.5mL/min (7.5-14min), 0.4mL/min (15min) Mobile phase : A= Water + 0.1% formic acid B= Methanol + 0.1% formic acid Gradient : 20% B (0-0.5 min), 100% B (8-12 min), 20% B (12.01-15 min) Analytical column : Phenomenex Kinetex XB C18 100 x 2.1mm 1.7um 100A Column temperature : 50ºC Ionisation : Electrospray, positive, negative switching mode Desolvation line : 250ºC Drying/Nebulising gas : 10L/min, 2L/min Heating block : 400ºC

2 Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin

Design of plasma separator technology

Control Spot: Spreading Layer [Determines whether enough [Lateral spreading layer rapidly spreads blood so it will blood was placed on the card]. enter the ltration layer as a front while adding buffers and anticoagulants. The lateral spreading rate is 150um/sec].

Filtration Layer Isolation Screen [Filtration layer captures blood [Precludes lateral wicking along the cells by a combination of ltration card surface]. and adsorption. The average Collection Layer linear vertical migration rate is [Loads with a speci c aliquot of plasma onto a 6.35mm disc]. Although ow through approximately 1um/sec]. the ltration membrane is unlikely to be constant throughout the plasma extraction process, the average loading rate of the Collection Disc was 13 nL/sec. This corresponds to a volumetric ow rate into the Collection Disc of 400 pL/mm2/sec.

Plasma separation work ow

1 2 3 4

The collection disc is removed from the card and is ready for extraction for LC-MS/MS analysis.

A NoviPlex card is Approximately 50uL After 3 minutes, the The collection disc removed from foil of whole blood is top layer is completely contains 2.5uL of packaging. added to the test removed (peeled plasma. Card is air area. back). dried for 15 minutes.

Figure 1. Noviplex workow.

3 Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin

Figure 2. Applying a blood sample, either as a nger prick or by accurately measuring the blood volume, to the laminated membrane stack retains red cells and allows a plasma sample to be collected. The red cells are retained by a combination of adsorption and ltration whilst plasma advances through the membrane stack by capillary action. After approximately three minutes the plasma Collection Disc was saturated with an aliquot of plasma and was ready for LC/MS/MS analysis.

Results Comparison between plasma separation cards (PSC) and plasma

(x100,000) Plasma separation card (x100,000) Plasma 3.00 2.00 Positive ion Positive ion Warfarin m/z 309.20 > 163.05 2.75 Warfarin m/z 309.20 > 163.05 2.50 1.75 Q1 (V) -22 Q1 (V) -22 2.25 1.50 Collision energy -15 Collision energy -15 Q3 (V) -15 2.00 Q3 (V) -15 1.25 1.75 1.00 1.50 2.5ug/mL 1.25 2.5ug/mL 0.75 Calibration standard 1.00 Calibration standard 0.50 0.4ug/mL 0.75 0.4ug/mL Calibration standard 0.50 Calibration standard 0.25 0.25 0.00 0.00 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min (x100,000) Plasma separation card (x100,000) Plasma 1.2 Negative ion 1.1 1.50 Negative ion 1.0 Warfarin m/z 307.20 > 161.25 Warfarin m/z 307.20 > 161.05 Q1 (V) 14 0.9 1.25 Q1 (V) 14 Collision energy 19 0.8 Collision energy 19 Q3 (V) 30 1.00 0.7 Q3 (V) 30 0.6 2.5ug/mL 0.75 2.5ug/mL 0.5 Calibration standard Calibration standard 0.4 0.50 0.3 0.4ug/mL 0.4ug/mL 0.2 Calibration standard 0.25 Calibration standard 0.1 0.0 0.00 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

Figure 3. Comparison between the warfarin response in both positive and negative ion modes for warfarin calibration standards at 2.5ug/mL and 0.4ug/mL extracted from the plasma separation cards and a conventional plasma sample. There is a broad agreement in ion signal intensity between the 2 sample preparation techniques.

4 Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin

800,000 Plasma separation card 450000 Plasma separation card Positive ion Negative ion 700,000 Warfarin m/z 309.20 > 163.05 400000 Warfarin m/z 309.20 > 163.05 Replicate calibration points at 350000 Replicate calibration points at 600,000 2.5ug/mL and 0.4ug/mL (n=3) 2.5ug/mL and 0.4ug/mL (n=3) 300000 500,000 250000 400,000 200000 300,000 Linear regresson analysis 150000 200,000 Linear regression analysis y = 246527x + 14796 100000 y = 133197x + 15795 R² = 0.9986 R² = 0.9954 100,000 50000

0 0 0 0.5 1 1.5 2 2.5 3 3.5 0 0.5 1 1.5 2 2.5 3 3.5 Blood concentration ( ug/mL) Blood concentration ( ug/mL)

Figure 4. In both ion modes, the calibration curve was linear over the therapeutic range studied for warfarin extracted from PSC’s (calibration range 0-3ug/mL, single point calibration standards at each level with the exception of replicate calibration points at 2.5ug/mL and 0.4ug/mL (n=3); r2>0.99 for PSC analysis [r2>0.99 for a conventional plasma extraction]).

(x10,000) (x10,000) Matrix blank comparison 1.75 Matrix blank comparison 1.75 Positive ion Negative ion 1.50 1.50 Plasma separation card matrix blank Plasma separation card matrix blank Plasma matrix blank 1.25 Plasma matrix blank 1.25 1.00 1.00 0.75 0.75 0.50 0.50 0.25 0.25 0.00 0.00 0.0 2.5 5.0 min 2.5 5.0 min

Figure 5. Matrix blank comparison. In both ion modes, the MRM chromatograms for PSC and plasma are comparable. Warfarin ion signals were not detected in the any PSC or plasma matrix blank.

Plasma separation card comparison The drive to work with smaller sample volumes offers instability (some enzyme labile compounds, particularly significant ethical and economical advantages in prodrugs, analyte stability can be problematic), hematocrit pharmaceutical and clinical workflows and dried blood effect and background interferences of DBS. DBS also spot sampling techniques have enabled a step change shows noticeable effects on many lipids dependent on the approach for many toxicokinetic and pharmacokinetic sample collection process. To compare PSC to plasma lipid studies. However, the impressive growth of this technique profiles the same blood sample extraction procedure in the quantitative analysis of small molecules has also applied for warfarin analysis was measured by a high mass discovered several limitations in the case of sample accuracy system optimized for lipid profiling.

5 Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin

Monoacylglycerophosphoethanolamines Ceramide Diacylglycero- Monoacylglycerophosphocholines phosphocholines phosphocholines

Plasma separation card Conventional plasma sample sample Positive ion Positive ion LCMS-IT-TOF LCMS-IT-TOF Lipid pro ling Lipid pro ling

7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min

Figure 6. Lipid pro les from the same human blood sample extracted using a plasma separation card (left hand pro le) compared to a conventional plasma samples (centrifugation). Both lipid pro les are comparable in terms of distribution and the number of lipids detected (the scaling has been normalized to the most intense lipid signal). Conclusions • In this limited study, plasma separation card (PSC) sampling delivered a quantitative analysis of warfarin spiked into human blood. • PSC generated a linear calibration curve in both positive and negative ion modes (r2>0.99; n=5); • The warfarin plasma results achieved by using the PSC technique were in broad agreement with conventional plasma sampling data. • The plasma generated by the ltration process appears broadly similar to plasma derived from conventional centrifugation. • Further work is required to consider the robustness and validation in a routine analysis.

References • Jensen, B.P., Chin, P.K.L., Begg, E.J. (2011) Quanti cation of total and free concentrations of R- and S-warfarin in human plasma by ultra ltration and LC-MS/MS. Anal Bioanal Chem., 401, 2187-2193 • Radwan, M.A., Bawazeer, G.A., Aloudah, N.M., Aluadeib, B.T., Aboul-Enein, H.Y. (2012) Determination of free and total warfarin concentrations in plasma using UPLC MS/MS and its application to patient samples. Biochemical Chromatography, 26, 6-11

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1462E

Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS

ASMS 2014 MP535

Zhaoqi Zhan1, Zhe Sun1, Jie Xing1, Helmy Rabaha2 and Lim Swee Chin2 1Shimadzu (Asia Paci c) Pte Ltd, Singapore, SINGAPORE; 2Department of Scienti c Services, Ministry of Health, Brunei Darussalam Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS

Introduction Amphetamines are among the most commonly abused demand and many such efforts have been reported drugs type worldwide. The conventional analytical recently [3]. The objective of this study is to develop a fast procedure of amphetamines in human urine in forensic LC/MS/MS method for direct analysis of amphetamines in laboratory involves initial immunological screening urine without sample pre-treatment (except dilution with followed by GCMS con rmation and quantitation [1]. The water) on LCMS-8040, a triple quadrupole system featured new guidelines of SAMHSA under U.S. Department of as ultra fast mass spectrometry (UFMS). The compounds Health and Human Services effective in Oct 2010 [2] studied include amphetamines (AMPH), methamphetamine allowed use of LC/MS/MS for screening, con rmation and (MAMP) and three newly added MDMA, MDA and MDEA quantitation of illicit drugs including amphetamines. One by the new SAMHSA guidelines, four potential of the advantages by using LC/MS/MS is that derivatization interferences as well as PMPA as a control reference (Table of amphetamines before analysis is not needed, which was 1). Very small injection volumes of 0.1uL to 1uL was a standard procedure of GCMS method. Since analysis adopted in this study, which enabled the method suitable speed and throughput could be enhanced signi cantly, for direct injection of untreated urine samples without development and use of LC/MS/MS methods are in causing signi cant contamination to the ESI interface.

Experimental The stock standard solutions of amphetamines and related were water (A) and MeOH (B), both with 0.1% formic acid. compounds as listed in Table 1 were prepared in the A fast gradient elution program was developed for analysis Toxicology Laboratory in the Department of Scienti c of the ten compounds: 0-1.6min, B=2%->14%; Services (MOH, Brunei). Five urine specimens were 1.8-2.3min, B=70%; 2.4min, B=2%; end at 4min. The collected from healthy adult volunteers. The urine samples total ow rate was 0.6 mL/min. Positive ESI ionization used as blank and spiked samples were not pre-treated by mode was applied with drying gas ow of 15 L/min, any means except dilution of 10 times with Milli-Q water. nebulizing gas ow of 3 L/min, heating block temperature An LCMS-8040 triple quadrupole coupled with a Nexera of 400 ºC and DL temperature of 250 ºC. Various injection UHPLC system (Shimadzu Corporation) was used. The volumes from 0.1 uL to 5 uL were tested to develop a analytical column used was a Shim-pack XR-ODS III UHPLC method with a lower injection volume to reduce column (1.6 µm) 50mm x 2mm. The mobile phases used contamination of untreated urine samples to the interface.

Results and Discussion Method development of direct injection of amphetamines in urine MRM optimization of the ten compounds (Table 1) was pre-treatment while it should not cause significant performed using an automated MRM optimization contamination to the interface. The Nexera SIL-30A program with LabSolutions workstation. Two MRM auto-sampler enables to inject as low as 0.10 uL of sample transitions were selected for each compound, one for with excellent precision. quantitation and second one for confirmation (Table 1). Figure 1 shows a few selected results of direct injection of The ten compounds were separated and eluted in urine blank (a) and mixed standards spiked in urine with 1 0.75~2.2 minutes as sharp peaks as shown in Figure 1. In uL (c and d) and 0.1 uL (b) injection. It can be seen that all addition to analysis speed and detection sensitivity, this compounds (12.5 ppb each in urine) could be detected method development was also focused on evaluation of with 0.1uL injection except MDA and Norpseudo-E. With small to ultra-small injection volumes to develop a method 1uL injection, all of them were detected. suitable for direct injection of urine samples without any

2 Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS

Table 1: MRMs of amphetamines and related compounds

Cat. Compound Abbr. RT (min) MRM CE (V) 152>134 -13 B1 Nor pseudo ephedrine Nor pseudo-E 0.75 152>115 -23 166>148 -14 B2 Ephedrine Ephe 0.94 166>91 -31 166>148 -14 B3 Pseudo ephedrine Pseudo-E 1.01 166>91 -30 136>91 -20 A1 Amphetamine AMPH 1.20 136>119 -14 150>91 -20 A2 Methampheta-mine MAMP 1.42 150>119 -14 180>163 -12 A3 3,4-methylenedi oxyamphetamine MDA 1.49 180>163 -38 194>163 -13 A4 3,4-methylene dioxymeth amphetamine MDMA 1.59 194>105 -22 208>163 -12 A5 3,4-methylene dioxy-N-ethyl amphetamine MDEA 1.94 208>105 -24 150>91 -20 B4 Phentermine Phent 1.93 150>119 -40 178>91 -22 R Propyl amphetamine PAMP 2.20 178>65 -47

(x10,000) (x10,000) (a) Urine blank, 1 uL inj (b) 12.5ppb in urine, 0.1uL inj PAMP 3.0 3.0

2.0 MDA

2.0 MAMP Phent MDEA AMPH 1.0 1.0 Ephedrine MDMA Pseudo Norpseudo

0.0 0.0 0.0 0.5 1.0 1.5 2.0 2.5 min 0.0 0.5 1.0 1.5 2.0 2.5 min (x100,000) (x1,000,000) (c) 12.5ppb, 1uL inj 1.5 3.0 PAMP (d) 62.5ppb in urine, 1uL inj PAMP

2.0 1.0 MAMP MAMP MDEA MDEA Phent Phent

1.0 AMPH 0.5 MDMA AMPH MDMA Pseudo MDA Ephedrine Ephedrine Pseudo MDA Norpseudo Norpseudo 0.0 0.0 0.0 0.5 1.0 1.5 2.0 2.5 min 0.0 0.5 1.0 1.5 2.0 2.5 min Figure 1: MRM chromatograms of urine blank (a) and spiked samples of amphetamines and related compounds in urine by LC/MS/MS method with 1uL and 0.1uL injection volumes.

3 Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS

Calibration curves with small and ultra-small injection volumes Linear calibration curves were established for the ten volume. The calibration curves with 0.1 uL injection volume compounds spiked in urine with different injection are shown in Figure 2. The linearity (r2) of all compounds volumes: 0.1, 0.2, 0.5, 1, 2 and 5 uL. Good linearity of with 0.1 uL and 1 uL injection volumes are equivalently calibration curves (R2>0.999) were obtained for all good as shown in Table 2. injection volumes including 0.1uL, an ultra-small injection

Area (x100,000) Area (x1,000,000) Area (x100,000) Area (x100,000) 1.25 Area (x100,000) 7.5 5.0 AMPH MAMP MDA MDMA 7.5 MDEA 1.00 5.0 5.0 0.75 5.0 2.5 0.50 2.5 2.5 2.5 0.25

0.0 0.00 0.0 0.0 0 250 Conc. 0.0 0 250 Conc. 0 250 Conc. 0 250 Conc. 0 250 Conc.

Area (x100,000) Area (x100,000) Area (x100,000) Area (x100,000) Area (x1,000,000) 3.0 Nor pseudo-E Ephedrine Pseudo-E 7.5 PAMP 5.0 Phent 5.0 1.5

2.0 5.0 1.0 2.5 2.5 1.0 2.5 0.5

0.0 0.0 0.0 0.0 0.0 0 250 Conc. 0 250 Conc. 0 250 Conc. 0 250 Conc. 0 250 Conc.

Figure 2: Calibration Curves of amphetamines spiked in urine with 0.1uL injection

Performance validation Repeatability of peak area was evaluated with a same LOD and LOQ of the ten compounds in urine are shown in loading amount (6.25 pg) but with different injection Table 3. Since the working samples (blank and spiked) volumes. The RSD shown in Table 2 were 1.6% ~ 7.9% were diluted for 10 times with water before injection, the and 1.6 ~ 7.8% for 0.1uL and 1uL injection, respectively. It concentrations and LOD/LOQ of the method described is worth to note that the repeatability of every compounds above for source urine samples have to multiply a factor of with of 0.1uL injection is closed to that of 1uL injection as 10. Therefore, the LOQs of the method for urine specimens well as 5uL injection (data not shown). are at 2.1-17.1 ng/mL for AMPH, PAMP, MDMA and Matrix effect of the method was determined by MDEA and 53 ng/mL for MDA. The LOQs for the potential comparison of peak areas of mixed standards in pure water interferences (Phentermine, Ephedrine, Pseudo-Ephedrine and in urine matrix. The results of 62.5ppb with 1uL and Norpseudo-Ephedrine) are at 17-91 ng/mL, 2.4 ng/mL injection were at 102-115% except norpseudoephedrine for the internal reference MAMP. The sensitivity of the (79%) as shown in Table 2. direct injection LC/MS/MS method are significantly higher Accuracy and sensitivity of the method were evaluated than the confirmation cutoff (250 ng/mL) required by the with spiked samples of low concentrations. The results of SAMHSA guidelines.

4 Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS

Table 2: Method Performance with different inj. volumes

Calibration curve, R2 RSD% area (n=6) M.E. %1 Name (ppb)2 (0.1uL) (1uL) (0.1uL) (1uL) (1uL) Norpseudo-E 1-500 0.9992 0.9996 4.5 5.7 79 Ephe 2.5-500 0.9995 0.9998 3.2 2.9 115 Pseudo-E 1-500 0.9994 0.9986 3.7 3.3 113 AMPH 1-500 0.9997 0.9998 3.5 2.4 102 MAMP 1-500 0.9998 0.9999 1.6 2.3 110 MDA 2.5-500 0.9978 0.9995 7.9 7.8 103 MDMA 1-500 0.9993 0.9998 1.8 4.5 115 MDEA 1-500 0.9996 0.9998 3.5 2.9 115 Phent 2.5-500 0.9998 0.9998 4.1 1.6 106 PAMP 1-500 0.9998 0.9932 2.9 2.0 102 1: Measured with mixed stds of 62.5 ppb in clear solution and spiked in urine 2: For 0.1uL injection, the lowest conc. is 2.5 or 12.5 ppb

Table 3: Method performance: sensitivity & accuracy (1uL)

Conc. (ppb) Accuracy Sensitivity (ppb) Name Prep. Meas. (%) S/N LOD LOQ Norpseudo-E 1.0 1.2 118.7 2.3 1.53 5.09 Ephe 2.5 2.2 88.2 2.7 2.41 8.04 Pseudo-E 1.0 1.0 99.5 5.9 0.50 1.67 AMPH 1.0 1.1 114.1 6.7 0.51 1.71 MAMP 1.0 1.0 103.6 21.8 0.14 0.47 MDA 2.5 2.4 96.3 4.5 1.60 5.34 MDMA 1.0 1.1 106.4 51.9 0.06 0.21 MDEA 1.0 1.1 111.8 28.5 0.12 0.39 Phent 2.5 2.6 105.3 2.9 2.73 9.10 PAMP 1.0 1.0 101.7 42.2 0.07 0.24

Method operational stability The method operational stability with 1uL injection was 120th injection of the same spiked sample (S1) as well as tested with spiked samples of 25 ppb in five urine other spiked samples (S2, S3, S4 and S5) in between. specimens, corresponding to 250 ng/mL in the source urine Decrease in peak areas of the compounds occurred, but the samples. Continuous injections of accumulated 120 times degree of the decrease in average was about 17% from the was carried out in about 10 hours. The purpose of the first injection to the last injection. This result indicates that it experiment was to evaluate the operational stability against is possible to carry out direct analysis of urine samples (10 the ESI source contamination by urine samples without times dilution with water) by the high sensitivity LC/MS/MS pre-treatment. Figure 3 shows the first injection and the method with a very small injection volume.

5 Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS

(x100,000) (x100,000) (x100,000) st th 7.5 st 7.5 S1 (1 inj) 7.5 S2 (11 inj) S3 (21 inj) PAMP PAMP PAMP MAMP MAMP 5.0 5.0 5.0 MAMP Phent MDEA MDEA MDEA Phent Phent AMPH MDMA PseudoEphedrine AMPH PseudoEphedrine MDMA Pseudo Ephedrine MDMA 2.5 2.5 2.5 AMPH MDA MDA MDA Norpseudo Norpseudo Norpseudo 0.0 0.0 0.0 0.0 1.0 2.0 min 0.0 1.0 2.0 min 0.0 1.0 2.0 min (x100,000) (x100,000) (x100,000) S4 (31st inj) S5 (41st inj) S1 (110th inj) PAMP PAMP PAMP

5.0 5.0 5.0 MAMP MAMP MDEA Phent MAMP Phent MDEA MDEA Phent

2.5 MDMA 2.5 2.5 AMPH Pseudo Pseudo MDA MDMA MDMA Pseudo MDA Ephedrine MDA Norpseudo AMPH AMPH Norpseudo Norpseudo Ephedrine 0.0 Ephedrine 0.0 0.0 0.0 1.0 2.0 min 0.0 1.0 2.0 min 0.0 1.0 2.0 min

Figure 3: Selected chromatograms of continuous injections of spiked samples (25 ppb) with 1 µL injection. Five urine specimens S1, S2, S3, S4 and S5 were used to prepare these spiked samples.

Conclusions In this study, we developed a fast LC/MS/MS method for operational stability. The good performance results direct analysis of ve amphetamines and related observed reveals that screening and con rmation of compounds in human urine for screening and quantitative amphetamines in human urine by direct injection to con rmation. Very small injection volumes of 0.1~1.0 uL LC/MS/MS is possible and the method could be an were adopted to minimize ESI contamination and enhance alternative choice in forensic and toxicology analysis.

References 1. Kudo K, Ishida T, Hara K, Kashimura S, Tsuji A, Ikeda N, J Chromatogr B, 2007, 855, 115-120. 2. Mandatory guidelines for Federal Workplace Drug Testing Program, 73 FR 71858-71907, Nov. 25, 2008. 3. Huei-Ru Lina, Ka-Ian Choia, Tzu-Chieh Linc, Anren Hu,, Journal of Chromatogr B, 2013, 929, 133–141.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1482E

Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS

ASMS 2014 WP641

Alan J. Barnes1, Carrie-Anne Mellor2, Adam McMahon2, Neil Loftus1 1Shimadzu, Manchester, UK 22WMIC, University of Manchester, UK Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS

Introduction Plasma extraction technology is a novel technique achieved Under physiological conditions TMZ is rapidly converted to by applying a blood sample to a laminated membrane 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) stack which allows plasma to ow through the asymmetric which in-turn degrades by hydrolysis to lter whilst retaining the cellular components of the blood 5-aminoimidazole-4-carboxamide (AIC). Storage of plasma sample. has previously shown that both at -70C and 4C Plasma separation card technology was applied to the degradation still occurs. In these experiments, whole blood quantitative analysis of temozolomide (TMZ); an oral containing TMZ standard was applied to NoviPlex plasma imidazotetrazine alkylating agent used for the treatment of separation cards (PSC). The aim was to develop a robust Grade IV astrocytoma, an aggressive form of brain tumour. LC/MS/MS quantitative method for TMZ.

Materials and Methods Plasma separation TMZ spiked human blood calibration standards (50uL) were applied to the PSC as described below in figure 1.

1 2 3 4

The collection disc is removed from the card and is ready for extraction for LC-MS/MS analysis.

A NoviPlex card is Approximately 50uL After 3 minutes, the The collection disc removed from foil of whole blood is top layer is completely contains 2.5uL of packaging. added to the test removed (peeled plasma. Card is air area. back). dried for 15 minutes.

Figure 1. Noviplex plasma separation card work ow

2 Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS

Control Spot: Spreading Layer [Determines whether enough [Lateral spreading layer rapidly spreads blood so it will blood was placed on the card]. enter the ltration layer as a front while adding buffers and anticoagulants. The lateral spreading rate is 150um/sec].

Filtration Layer Isolation Screen [Filtration layer captures blood [Precludes lateral wicking along the cells by a combination of ltration card surface]. and adsorption. The average Collection Layer linear vertical migration rate is [Loads with a specic aliquot of plasma onto a 6.35mm disc]. Although ow through approximately 1um/sec]. the ltration membrane is unlikely to be constant throughout the plasma extraction process, the average loading rate of the Collection Disc was 13 nL/sec. This corresponds to a volumetric ow rate into the Collection Disc of 400 pL/mm2/sec.

Figure 1. Noviplex plasma separation card work ow (Cont'd)

Figure 2. Applying a blood sample, either as a nger prick or by accurately measuring the blood volume, to the laminated membrane stack retains red cells and allows a plasma sample to be collected. The red cells are retained by a combination of adsorption and ltration whilst plasma advances through the membrane stack by capillary action. After approximately three minutes the plasma Collection Disc was saturated with an aliquot of plasma and was ready for LC/MS/MS analysis.

Sample preparation TMZ was extracted from the dried plasma collection discs As a control, conventional plasma samples were prepared by adding 40uL acetonitrile + 0.1% formic acid, followed by centrifuging the human blood calibration standards at by centrifugation 16,000g for 5 min. 30uL supernatant 1000g for 10min. TMZ was extracted from 2.5uL of plasma was added directly to the LC/MS/MS sample vial for using the same extraction protocol as applied for PSC. analysis.

3 Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS

LC/MS/MS analysis

Ionisation : Electrospray, positive mode MRM 195.05 >138.05 CE -10

Desolvation line : 300ºC Drying/Nebulising gas : 10L/min, 2L/min Heating block : 400ºC

HPLC : HILIC Reverse Phase Nexera UHPLC system Nexera UHPLC system Flow rate : 0.5mL/min (0-7min), 1.8mL/min (7.5min-17.5min) 0.4mL/min Mobile phase : A= Water + 0.1% formic acid A= Water + 0.1% formic acid B= Acetonitrile + 0.1% formic acid B= methanol + 0.1% formic acid Gradient : 95% B – 30%% B (6.5 min), 5% B – 100%% B (3 min), 30% B (7.5 min), 95% B (18 min) 100% B (7 min), 5% B (10 min) Analytical column : ZIC HILIC 150 x 4.6mm 5um 200ª Phenomenex Kinetex XB C18 100 x 2.1mm 1.7um 100A Column temperature : 40ºC 50ºC Injection volume : 10uL 2µL

Results HILIC LC/MS/MS Temozolomide is known to be unstable under physiological a nonenzymatic, chemical degradation process. Previous conditions and is converted to studies have used HILIC to analyze the polar compound 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) by and to avoid degradation in aqueous solutions.

(x10,000) 5.0 Peak Area Plasma separation card 700000 Plasma separation card HILIC analysis HILIC analysis TMZ m/z 195.05> 138.05 600000 TMZ 4.0 Q1 (V) -20 Single point calibration standards Collision energy -10 Calibration curve 0.2-10ug/mL Q3 (V) -12 500000 3.0 400000

2.0 300000

200000 1.0 8.0ug/mL calibn std Linear regression analysis 100000 y = 64578x + 18473 0.5ug/mL calibn std R² = 0.9988

0.0 0 0 2 4 6 8 10 12 0.0 2.5 5.0 min Blood Concentration (ug/mL)

Figure 3. HILIC LC/MS/MS chromatograms for PSC TMZ analysis at 0.5 and 8ug/mL. The PSC calibration curve was linear between 0.2-10ug/mL (r2>0.99). HILIC was considered in response to previous published data and to minimize potential stability issues. However, to reduce sample cycle times a reverse phase method was also developed.

4 Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS

Reversed Phase LC/MS/MS

(x10,000)

9.0 Plasma separation card Peak Area Plasma separation card RP analysis RP analysis 8.0 800,000 TMZ m/z 195.05 > 138.05 TMZ calibration curve 7.0 Q1 (V) -20 700,000 Replicate calibration points at Collision energy -10 0.5ug/mL and 8ug/mL (n=3) 6.0 Q3 (V) -12 600,000

5.0 500,000

4.0 8.0ug/mL 400,000 Calibration standard 3.0 300,000 0.5ug/mL 2.0 Calibration standard 200,000 Linear regression analysis y = 72219x - 355.54 1.0 100,000 R² = 0.9997

0.0 0 0 2 4 6 8 10 12 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min Blood Concentration (ug/mL)

Figure 4. Reverse phase LC/MS/MS chromatograms for PSC TMZ analysis at 0.5 and 8ug/mL. The PSC calibration curve was linear between 0.2-10ug/mL (r2>0.99; replicate samples were included in the liner regression analysis at 0.5 and 8ug/mL; n=3).

Due to the relatively long cycle time (18 min), a faster added directly to the LC/MS sample vial plus 80uL water + reversed phase method was developed (10 min). Sample 0.1% formic acid. In addition to reversed phase being preparation was modified with PSC sample disk placed in faster, the sample injection volume was reduced to just 2uL 40uL methanol + 0.1% formic acid, followed by as a result of higher sensitivity from narrower peak width centrifugation 16,000g 5 min. 20uL supernatant was (reversed phase,13 sec; HILIC, 42 sec).

Comparison between PSC and plasma

(x100) Matrix blank comparison (x1,000) 500ng/mL comparison

4.0 MRM 195.05>67.05 1.50 MRM 195.05>67.05 Plasma separation card Plasma separation card 3.5 matrix blank 500ng/mL calibration standard 1.25 Plasma Plasma 3.0 matrix blank 500ng/mL calibration standard 1.00 2.5

2.0 0.75

1.5 0.50

1.0 0.25 0.5

0.0 0.00

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

Figure 5. Human blood TMZ calibration standards were prepared using PSC and conventional plasma. Using the conrmatory ion transition 195.05>67.05 both the PSC and plasma sample are in broad agreement with regard to matrix ion signal distribution.

5 Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS

(x10,000) (x10,000) Matrix peak Matrix blank comparison Matrix peak 500ng/mL comparison MRM 195.05>138.05 MRM 195.05>138.05 1.50 1.50 Plasma separation card Plasma separation card matrix blank 500ng/mL calibration standard 1.25 1.25 Plasma Plasma matrix blank 500ng/mL calibration standard 1.00 1.00

0.75 TMZ 0.75

0.50 0.50 TMZ 0.25 Rt 0.25 1.7mins

0.00 0.00

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

Figure 6. Human blood TMZ calibration standards were prepared using PSC and conventional plasma. Using the quantitation ion transition 195.05>138.05 both the PSC and plasma sample are in broad agreement in signal distribution and intensity including the presence of a matrix peak at 2.4mins.

Conclusions This technology has the potential for a simplified clinical sample collection by the finger prick approach, with future work aimed to evaluate long term sample stability of PSC samples, stored at room temperature. Quantitation of drug metabolites MTIC and AIC also could help provide a measure of sample stability.

References • Andrasia, M., Bustosb, R., Gaspara,A., Gomezb, F.A. & Kleknerc, A. (2010) Analysis and stability study of temozolomide using capillary electrophoresis. Journal of Chromatography B. Vol. 878, p1801-1808 • Denny, B.J., Wheelhouse, R.T., Stevens, M.F.G., Tsang, L.L.H., Slack, J.A., (1994) NMR and molecular modeliing investigation of the mechanism of activation of the antitumour drug temozolomide and its Interaction with DNA. Biochemistry, Vol. 33, p9045-9051

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1475E

Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans

ASMS 2014 WP 629

Bicui Chen1, Bin Wang1, Xiaojin Shi1, Yuling Song2, Jinting Yao2, Taohong Huang2, Shin-ichi Kawano2, Yuki Hashi2 1 Pharmacy Department, Huashan Hospital, Fudan University, 2 Shimadzu Global COE, Shimadzu (China) Co., Ltd. Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans

Introduction Telbivudine is a synthetic L-nucleoside analogue, which is of 5’-triphosphorylated-telbivudine into viral DNA obligates phosphorylated to its active metabolite, 5’-triphosphate, by DNA chain termination, resulting in inhibition of HBV cellular kinases. The telbivudine 5’-triphosphate inhibits replication. The objectives of the current studies were to HBV DNA polymerase (a reverse transcriptase) by develop a selective and sensitive LC-MS/MS method to competing with the natural substrate, dTTP. Incorporation determine of telbivudine in human plasma.

Method Sample Preparation (1) Add 100 μL of plasma into the polypropylene tube, add 40 μL of internal standard working solution (33 µg/mL, with thymidine phosphorylase) to all other tubes. (2) Incubate the tubes for 1 h at 37 ºC in dark. (3) Add 200 μL of acetonitrile to all tubes, seal and vortex for 1 minutes. (4) Centrifuge the tubes for 5 minutes at 13000 rpm. (5) Transfer 200 μL supernatant to a clean glass bottle and inject into the HPLC-MS/MS system.

LC-MS/MS Analysis The analysis was performed on a Shimadzu Nexera UHPLC mmL.) with the column temperature at 40 ºC. A triple instrument (Kyoto, Japan) equipped with LC-30AD pumps, quadruple mass spectrometer (Shimadzu LCMS-8050,

CTO-30A column oven, DGU-30A5 on-line egasser, and Kyoto, Japan) was connected to the UHPLC instrument via SIL-30AC autosampler. The separation was carried out on an ESI interface. GL Sciences InertSustain C18 column (3.0 mmI.D. x 100

Analytical Conditions

HPLC (Nexera UHPLC system)

Column : InertSustain (3.0 mmI.D. x 100 mmL., 2 μm, GL Sciences) Mobile Phase A : water with 0.1% formic acid Mobile Phase B : acetonitrile Gradient Program : as shown in Table 1 Flow Rate : 0.4 mL/min Column Temperature : 40 ºC Injection Volume : 2 µL

Table 1 Time Program

Time (min) Module Command Value 0.00 Pumps Pump B Conc. 5 4.00 Pumps Pump B Conc. 80 4.10 Pumps Pump B Conc. 5 6.00 Controller Stop

2 Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans

MS (LCMS-8050 triple quadrupole mass spectrometer)

Ionization : ESI Polarity : Positive Ionization Voltage : +0.5 kV (ESI-Positive mode) Nebulizing Gas Flow : 3.0 L/min Heating Gas Flow : 8.0 L/min Drying Gas Flow : 12.0 L/min Interface Temperature : 250 ºC Heat Block Temperature : 300 ºC DL Temperature : 350 ºC Mode : MRM

Table 2 MRM Parameters

Precursor Product Dwell Time Q1 Pre Bias Q3 Pre Bias Compound CE (V) m/z m/z (ms) (V) (V) Telbivudine 243.10 127.10 100 -26 -10 -13 Telbivudine-D3 246.10 130.10 100 -16 -9 -25

Results and Discussion Human plasma samples containing telbivudine ranging intra-day and inter-day precision and accuracy of the from 1.0 to 10000 ng/mL were prepared and extracted assay were investigated by analyzing QC samples. by protein precipitation and the nal extracts were Intra-day precision (%RSD) at three concentration levels analyzed by LC-MS/MS. MRM chromatograms of (3, 30, and 8000 ng/mL) were below 2.5% and inter-day telbivudine (1 ng/mL) and deuterated internal standard precision (%RSD) was below 2.5%. The recoveries of are presented in Fig. 1 (blank) and Fig. 2 (spiked). The telbivudine were 100.6±2.5 %, 104.5±1.5% and linear regression for telbivudine was found to be 104.3±1.6% at three concentration levels, respectively. >0.9999. The calibration curve with human plasma as The stability data showed that the processed samples the matrix were shown in Fig. 3. Excellent precision and were stable at the room temperature for 8 h, and there accuracy were maintained for four orders of magnitude, was no signicant degradation during the three demonstrating a linear dynamic range suitable for freeze/thaw cycles at -20 ºC. The reinjection real-world applications. LLOQ for telbivudine was 1.0 reproducibility results indicated that the extracted ng/mL, which met the criteria for bias (%) and precision samples could be stable for 72 h at 10 ºC. within ±15% both within run and between run. The

3 Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans

(x100) (x1,000) 4.0 1:Telbivudine 243.10>127.10(+) CE: -10.0 2:Telbivudine-D3 246.10>130.10(+) CE: -9.0 4.0

3.0 3.0

2.0 2.0

1.0 1.0

0.0 0.0

0.0 1.0 2.0 3.0 4.0 5.0 min 1.0 2.0 3.0 4.0 5.0 min

Figure 1 Representative MRM chromatograms of blank human plasma (left: transition for telbivudine, right: transition for internal standard)

(x100) (x1,000,000) 1:Telbivudine 243.10>127.10(+) CE: -10.0 1.50 2:Telbivudine-D3 246.10>130.10(+) CE: -9.0

1.25 7.5

Telbivudine 1.00 Telbivudine-D3 5.0 0.75

0.50 2.5 0.25

0.0 0.00

0.0 1.0 2.0 3.0 4.0 5.0 min 1.0 2.0 3.0 4.0 5.0 min

Figure 2 Representative MRM chromatograms of telbivudine (left, 1 ng/mL) and internal standard (right) in human plasma

Area Ratio

2.5

2.0

1.5

1.0

0.5

0.0 0 2500 5000 7500 Conc. Ratio

Figure 3 Calibration curve of telbivudine in human plasma

4 Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans

Calibration Linear Range Accuracy Compound r Curve (ng/mL) (%) Telbivudine Y = (2.77×10-4)X + (3.39×10-5) 1~10000 93.1~116.6% 0.9998

Table 3 Accuracy and precision for the analysis of amlodipine in human plasma (in pre-study validation, n=3 days, six replicates per day)

Added Conc. Intra-day Precision Inter-day Precision Accuracy (ng/mL) (%RSD) (%RSD) (%) 3 2.18 2.11 107.7~114.4 400 1.52 1.58 91.6~95.9 8000 1.76 1.68 95.4~101.3

Table 4 Recovery for QC samples (n=6)

Concentartion Recovery QC Level (ng/mL) (%) LQC 3 100.6 MQC 400 104.5 HQC 8000 104.3

Table 5 Matrix effect for QC samples (n=6)

Added Conc. IS-Normalized QC Level Matrix Factor (ng/mL) Matrix Factor LQC 3 82.3% 99.0% MQC 400 81.7% 101.0% HQC 8000 90.8% 101.5%

(x10,000) (x1,000,000) 1:Telbivudine 243.10>127.10(+) CE: -10.0 2:Telbivudine-D3 246.10>130.10(+) CE: -9.0 3.0 1.00

0.75 2.0

0.50

1.0 0.25

0.0 0.00

0.0 1.0 2.0 3.0 4.0 5.0 min 1.0 2.0 3.0 4.0 5.0 min

Figure 4 Representative MRM chromatograms of real-world sample

5 Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans

Conclusion Results of parameters for method validation such as dynamic range, linearity, LLOQ, intra-day precision, inter-day precision, recoveries, and matrix effect factors were excellent. The sensitive LC-MS/MS technique provides a powerful tool for the high-throughput and highly selective analysis of telbivudine in clinical trial study.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1449E

Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry

ASMS 2014 WP628

Natsuyo Asano1, Tairo Ogura1, Kiyomi Arakawa1 1 Shimadzu Corporation. 1, Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto 604–8511, Japan Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry

Introduction Immunosuppressants are drugs which lower or suppress analysis. Therefore, it is important to analyze these drugs in activity of the immune system. They are used to prevent blood by using ultra-fast mass spectrometer to accelerate the rejection after transplantation or treat autoimmune monitoring with high quantitativity. We have developed disease. To avoid immunode ciency as adverse effect, it is analytical method for four immunosuppressants recommended to monitor blood level of therapeutic drug (Tacrolimus, Rapamycin, Everolimus and Cyclosporin A) with high throughput and high reliability. There are several with two internal standards (Ascomycin and Cyclosporin D) analytical technique to monitor drugs, LC/MS is superior in using ultra-fast mass spectrometer. terms of cross-reactivity at low level and throughput of

O HO HO HO

O O O O

O OH O O OH O O OH N N N O O O O O O O O O O O O O HO HO HO O O O O O

H O O Tacrolimus Rapamycin Everolimus MW: 804.02 MW: 914.17 MW: 958.22

OH O N OH HO N O O O O H H H O N N N N N N N O O HO O HN H O NO O O O O O O N O O N O O O N N O N O H H H HO H N N N N N O N O N O H H O O O O

Cyclosporin A Ascomycin (IS) Cyclosporin D (IS) MW: 1202.61 MW: 792.01 MW: 1216.64

Figure 1 Structure of immunosuppressants and internal standards (IS)

2 Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry

Methods and Materials Standard samples of each compound were analyzed to optimize conditions of liquid chromatograph and mass spectrometer. Whole blood extract was prepared based on liquid-liquid extraction described bellow.

2.7 mL of Whole blood and 20.8 mL of Water ↓ Vortex for 15 seconds ↓ Add 36 mL of MTBE/Cyclohexane (1:3) ↓ Vortex for 15 seconds and Centrifuge with 3000 rpm at 20 ºC for 10 minutes ↓ Extract an Organic phase ↓ Evaporate and Dry under a Nitrogen gas stream ↓ Redissolve in 1.8 mL of 80 % Methanol solution with 1 mmol/L Ammonium acetate ↓ Vortex for 1 minute and Centrifuge with 3000 rpm at 4 ºC for 5 minutes ↓ Filtrate and Transfer into 1 mL glass vial

Table 1 Analytical conditions UHPLC

Liquid Chromatograph : Nexera (Shimadzu, Japan) Analysis Column : YMC-Triart C18 (30 mmL. × 2 mmI.D.,1.9 μm) Mobile Phase A : 1 mmol/L Ammonium acetate - Water Mobile Phase B : 1 mmol/L Ammonium acetate - Methanol Gradient Program : 60 % B. (0 min) – 75 % B. (0.10 min) – 95 % B. (0.70 – 0.90 min) – 60 % B. (0.91 – 1.80 min) Flow Rate : 0.45 mL/min Column Temperature : 65 ºC Injection Volume : 1.5 µL

MS

MS Spectrometer : LCMS-8050 (Shimadzu, Japan) Ionization : ESI (negative) Probe Voltage : -4.5 ~ -3 kV Nebulizing Gas Flow : 3.0 L/min Drying Gas Flow : 5.0 L/min Heating Gas Flow : 15.0 L/min Interface Temperature : 400 ºC DL Temperature : 150 ºC HB Temperature : 390 ºC

3 Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry

Result Immunosuppressants, which we have developed a method each compound was detected as deprotonated molecule in for monitoring of, has been often observed as ammonium negative mode by using heated ESI source of LCMS-8050 or sodium adduct ion by using positive ionization. In (Table 2). general, protonated molecule (for positive) or The separation of all compounds was achieved within 1.8 deprotonated molecule (for negative) is more preferable min, with a YMC-Triart C18 column (30 mmL. × 2 for reliable quantitation than adduct ions such as mmI.D.,1.9 μm) and at 65 ºC of column oven temperature. ammonium, sodium, and potassium adduct. In this study,

(x100,000)

5 1.4

1.2 6

1.0

0.8

0.6

0.4 4 3 0.2 2 1 0.0

0.75 1.00 1.25 min

Figure 2 MRM chromatograms of immnosuppresants in human whole blood (50 ng/mL)

Table 2 MRM transitions

Peak No. Compound Porality Precursor ion (m/z) Product ion (m/z) 1 Ascomysin (IS) neg 790.40 548.20 2 Tacrolimus neg 802.70 560.50 3 Rapamycin neg 912.70 321.20 4 Everolimus neg 956.80 365.35 5 Cyclosporin A neg 1200.90 1088.70 6 Cyclosporin D (IS) neg 1215.10 1102.60

4 Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry

a) Tacrolimus

0.5 ng/mL

Ascomycin 40 ng/mL 0.5 – 1000 ng/mL

b) Rapamycin

0.5 ng/mL

Ascomycin 40 ng/mL 0.5 – 500 ng/mL

c) Everolimus

0.5 ng/mL

Ascomycin 40 ng/mL 0.5 – 100 ng/mL

d) Cyclosporin A

0.5 ng/mL

Cyclosporin D 0.5 – 1000 ng/mL 100 ng/mL

Figure 3 MRM chromatograms at LLOQ and ISTD (left), and calibration curves (right) for four immnosuppresants in human whole blood

5 Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry

Figure 3 illustrates both a calibration curve and chromatogram at the lowest calibration level for all immunosuppressants analyzed. Table 3 lists both the reproducibility and accuracy for each immunosuppressant that has been simultaneously measured in 1.8 minutes. Table 3 Reproducibility and Accuracy

Compound Concentration CV % (n = 6) Accuracy % Low (0.5 ng/mL) 18.0 99.4 Tacrolimus Low-Mid (2 ng/mL) 13.0 99.5 High (1000 ng/mL) 2.87 88.7 Low (0.5 ng/mL) 6.87 95.6 Rapamycin Low-Mid (5 ng/mL) 2.88 109.3 High (500 ng/mL) 3.41 90.0 Low (0.5 ng/mL) 10.4 95.3 Everolimus Low-Mid (5 ng/mL) 5.11 104.4 High (100 ng/mL) 2.26 93.3 Low (0.5 ng/mL) 7.31 95.1 Cyclosporin A Low-Mid (10 ng/mL) 2.36 99.9 High (1000 ng/mL) 2.67 94.9

In high speed measurement condition, we have achieved high sensitivity and wide dynamic range for all analytes. Additionally, the accuracy of each analyte ranged from 88 to 110 % and area reproducibility at the lowest calibration level of each analyte was less than 20%.

Conclusions • Monitoring with negative mode ionization permitted more sensitive, robust and reliable quantitation for four immunosuppressants. • A total of six compounds were measured in 1.8 minutes. The combination of Nexera and LCMS-8050 provided a faster run time without sacri cing the quality of results. • Even with a low injection volume of 1.5 μL, the lower limit of quantitation (LLOQ) for all compounds was 0.5 ng/mL. • In this study, it is demonstrated that LCMS-8050 is useful for the rugged and rapid quantitation for immunosuppressants in whole blood.

Acknowledgement We appreciate suggestions from Prof. Kazuo Matsubara and Assoc. Prof. Ikuko Yano from the department of pharmacy, Kyoto University Hospital, and Prof. Satohiro Masuda from the department of pharmacy, Kyusyu University Hospital.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1468E

Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS

ASMS 2014 TP497

Shailendra Rane, Rashi Kochhar, Deepti Bhandarkar, Shruti Raju, Shailesh Damale, Ajit Datar, Pratap Rasam, Jitendra Kelkar Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh Chambers, Makwana Road, Marol, Andheri (E), Mumbai-400059, Maharashtra, India. Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS

Introduction Felodipine is a calcium antagonist (calcium channel methods of quantitation for these two drugs using blocker), used as a drug to control hypertension[1]. LCMS-8050 system from Shimadzu Corporation, Japan. Hydrochlorothiazide is a diuretic drug of the thiazide class Presence of heated Electro Spray Ionization (ESI) probe in that acts by inhibiting the kidney’s ability to retain water. It LCMS-8050 ensured good quantitation and repeatability is, therefore, frequently used for the treatment of even in the presence of a complex matrix like plasma. Ultra hypertension, congestive heart failure, symptomatic high sensitivity of LCMS-8050 enabled development edema, diabetes insipidus, renal tubular acidosis and the quantitation method at low ppt level for both Felodipine prevention of kidney stones[2]. and Hydrochlorthiazide. Efforts have been made here to develop high sensitive

Felodipine Felodipine is a calcium antagonist (calcium channel blocker). Felodipine is a dihydropyridine derivative that is chemically described as ± ethyl methyl 4-(2,3-dichlorophenyl)1,4-dihydro-2,6-dimethyl-3,5-pyridin

edicarboxylate. Its empirical formula is C18H19Cl2NO4 and its structure is shown in Figure 1. Figure 1. Structure of Felodipine

Hydrochlorothiazide Hydrochlorothiazide, abbreviated HCTZ (or HCT, HZT), is a diuretic drug of the thiazide class that acts by inhibiting the kidney‘s ability to retain water. Hydrochlorothiazide is 6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine-

7-sulfonamide.Its empirical formula is C7H8ClN3O4S2 and its Figure 2. Structure of Hydrochlorothiazide structure is shown in Figure 2.

Method of Analysis Preparation of matrix matched plasma by protein precipitation method using cold acetonitrile To 100 µL of plasma, 500 µL of cold acetonitrile was added at 12000 rpm for 15 minutes. Supernatant was collected for protein precipitation then put in rotary shaker at 20 and evaporated to dryness at 70 ºC and finally rpm for 15 minutes for uniform mixing. It was centrifuged reconstituted in 200 µL Methanol.

2 Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS

Preparation of matrix matched plasma by liquid-liquid extraction method using diethyl ether and hexane mixture (1:1 v/v) To 500 µL plasma, 100 µL sodium carbonate (1.00 mol/L) minutes. Supernatant was collected and evaporated to and 5 mL of diethyl ether : hexane (1:1 v/v) was added. It dryness at 60 ºC. It was finally reconstitute in 1000 µL was placed in rotary shaker at 20 rpm for 15 minutes for Methanol. uniform mixing and centrifuged at 12000 rpm for 15

Preparation of calibration standards in matrix matched plasma Response of Felodipine and Hydrochlorothiazide were Felodipine and Hydrochlorothiazide molecules respectively. checked in both above mentioned matrices. It was found Calibration standards were thus prepared in respective that cold acetonitrile treated plasma and diethyl ether: matrix matched plasma. hexane (1:1 v/v) treated plasma were suitable for

• Felodipine Calibration Std : 5 ppt, 10 ppt, 50 ppt, 100 ppt, 500 ppt, 1 ppb and 10 ppb • HCTZ Calibration Std : 2 ppt, 5 ppt, 10 ppt, 50 ppt, 100 ppt, and 500 ppt

Figure 3. LCMS-8050 triple quadrupole mass spectrometer by Shimadzu Figure 4. Heated ESI probe

LCMS-8050 triple quadrupole mass spectrometer by In order to improve ionization efficiency, the newly Shimadzu (shown in Figure 3), sets a new benchmark in developed heated ESI probe (shown in Figure 4) combines triple quadrupole technology with an unsurpassed high-temperature gas with the nebulizer spray, assisting in sensitivity (UFsensitivity), Ultra fast scanning speed of the desolvation of large droplets and enhancing ionization. 30,000 u/sec (UFscanning) and polarity switching speed of This development allows high-sensitivity analysis of a wide 5 msecs (UFswitching). This system ensures highest quality range of target compounds with considerable reduction in of data, with very high degree of reliability. background.

LC/MS/MS analysis Compounds were analyzed using Ultra High Performance Corporation, Japan), The details of analytical conditions are Liquid Chromatography (UHPLC) Nexera coupled with given in Table 1 and Table 2. LCMS-8050 triple quadrupole system (Shimadzu

3 Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS

Table 1. LC/MS/MS conditions for Felodipine

• Column : Shim-pack XR-ODS (75 mm L x 3 mm I.D.; 2.2 µm) • Flow rate : 0.3 mL/min • Oven temperature : 40 ºC • Mobile phase : A: 10 mM ammonium acetate in water B: methanol • Gradient program (%B) : 0.0 – 3.0 min → 90 (%); 3.0 – 3.1 min → 90 – 100 (%); 3.1 – 4.0 min → 100 (%); 4.0– 4.1 min → 100 – 90 (%) 4.1 – 6.5 min → 90 (%) • Injection volume : 10 µL • MS interface : ESI • Nitrogen gas ow : Nebulizing gas 1.5 L/min; Drying gas 10 L/min; • Zero air ow : Heating gas 10 L/min • MS temperature : Desolvation line 200 ºC; Heating block 400 ºC Interface 200 ºC

Table 2. LC/MS/MS conditions for Hydrochlorothiazide

• Column : Shim-pack XR-ODS (100 mm L x 3 mm I.D.; 2.2 µm) • Flow rate : 0.2 mL/min • Oven temperature : 40 ºC • Mobile phase : A: 0.1% formic acid in water B: acetonitrile • Gradient program (%B) : 0.0 – 1.0 min → 80 (%); 1.0 – 3.5 min → 40 – 100 (%); 3.5 – 4.5 min → 100 (%); 4.5– 4.51min → 100 – 80 (%) 4.51 – 8.0 min → 90 (%) • Injection volume : 25 µL • MS interface : ESI • Nitrogen gas ow : Nebulizing gas 2.0 L/min; Drying gas 10 L/min; • Zero air ow : Heating gas 15 L/min • MS temperature : Desolvation line 250 ºC; Heating block 500 ºC Interface 300 ºC

Results LC/MS/MS analysis results of Felodipine LC/MS/MS method for Felodipine was developed on ESI respectively. Calibration curves as mentioned with R2 = positive ionization mode and 383.90>338.25 MRM 0.998 were plotted (shown in Figure 7). Also as seen in transition was optimized for it. Checked matrix matched Table 3, % Accuracy was studied to confirm the reliability plasma standards for highest (10 ppb) as well as lowest of method. Also, LOD as 2 ppt and LOQ as 5 ppt was concentrations (5 ppt) as seen in Figure 5 and Figure 6 obtained.

4 Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS

(x100,000) (x1,000) 5.0 383.90>338.25(+) 2.0 383.90>338.25(+)

1.5

2.5 FELODIPINE 1.0

0.5 FELODIPINE 0.0 0.0

0.0 2.5 5.0 0.0 2.5 5.0

Figure 5. Felodipine at 10 ppb in matrix matched plasma Figure 6. Felodipine at 5 ppt in matrix matched plasma

Table 3: Results of Felodipine calibration curve

Nominal Concentration Measured Concentration % Accuracy % RSD for area counts Sr. No. Standard (ppb) (ppb) (n=3) (n=3)

1 STD-FEL-01 0.005 0.005 97.43 9.87 2 STD-FEL-02 0.01 0.010 103.80 8.76 3 STD-FEL-03 0.05 0.053 104.47 2.24 4 STD-FEL-04 0.1 0.103 103.13 1.23 5 STD-FEL-05 0.5 0.469 94.88 1.33 6 STD-FEL-06 1 0.977 97.33 0.95 7 STD-FEL-07 10 10.023 100.90 0.60

Area (x1,000,000) 2.0 7

Area (x10,000) 3.0 1.5 2.5

2.0 4

1.0 1.5

3 1.0

0.5 0.5 2 1 6 0.0 5 0.05 0.10 Conc. 34 0.012 0.0 2.5 5.0 7.5 Conc.

Figure 7. Calibration curve of Felodipine

LC/MS/MS analysis results of Hydrochlorothiazide LC/MS/MS method for Hydrochlorothiazide was developed Calibration curves as mentioned with R2=0.998 were on ESI negative ionization mode and 296.10>204.90 MRM plotted (shown in Figure 10). Also as seen in Table 4, % transition was optimized for it. Checked matrix matched Accuracy was studied to confirm the reliability of method. plasma standards for highest (500 ppt) as well as lowest (2 Also, LOD as 1 ppt and LOQ as 2 ppt were obtained. ppt) concentrations as seen in Figures 8 and 9 respectively.

5 Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS

(x10,000) (x100) 296.10>204.90(-) 2.5 296.10>204.90(-) 1.5 2.0 HCTZ

1.0 1.5 HCTZ 1.0 0.5 0.5

0.0 0.0

0.0 2.5 5.0 7.5 0.0 2.5 5.0 7.5

Figure 8. Hydrochlorothiazide at 500 ppt in matrix matched plasma Figure 9. Hydrochlorothiazide at 2 ppt in matrix matched plasma

Table 4. Results of Hydrochlorothiazide calibration curve

Nominal Concentration Measured Concentration % Accuracy % RSD for area counts Sr. No. Standard (ppb) (ppb) (n=3) (n=3)

1 STD-HCTZ-01 0.002 0.0020 102.03 6.65 2 STD-HCTZ-02 0.005 0.0048 95.50 3.53 3 STD-HCTZ-03 0.01 0.0099 100.07 3.80 4 STD-HCTZ-04 0.05 0.0512 102.67 1.60 5 STD-HCTZ-05 0.1 0.1019 102.11 3.58 6 STD-HCTZ-06 0.5 0.4944 102.13 1.68

Area (x100,000) 1.00 6

Area (x10,000) 0.75 1.5

4 0.50 1.0

0.5 3 2 0.25 5 1 0.0 4 0.000 0.025 0.050 Conc. 23 0.00 1 0.0 0.1 0.2 0.3 0.4 Conc.

Figure 10. Calibration curve of Hydrochlorothiazide

Conclusion • Highly sensitive LC/MS/MS method for Felodipine and Hydrochlorothiazide was developed on LCMS-8050 system. • LOD of 2 ppt and LOQ of 5 ppt was achieved for Felodipine and LOD of 1 ppt and LOQ of 2 ppt was achieved for Hydrochlorothiazide by matrix matched methods. • Heated ESI probe of LCMS-8050 system enables drastic augment in sensitivity with considerable reduction in background. Hence, LCMS-8050 system from Shimadzu is an all rounder solution for bioanalysis.

6 Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS

References [1] YU Peng; CHENG Hang, Chinese Journal of Pharmaceutical Analysis, Volume 32, Number 1, (2012), 35-39(5). [2] Hiten Janardan Shah, Naresh B. Kataria, Chromatographia, Volume 69, Issue 9-10, (2009), 1055-1060.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1467E

Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS

ASMS 2014 TP496

Shruti Raju, Deepti Bhandarkar, Rashi Kochhar, Shailesh Damale, Shailendra Rane, Ajit Datar, Pratap Rasam, Jitendra Kelkar Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh Chambers, Makwana Road, Marol, Andheri (E), Mumbai-400059, Maharashtra, India. Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS

Introduction The toxicological assessment of Genotoxic Impurities (GTI) benzofuran}, DRN-IB and the determination of acceptable limits for such {5-amino-3-[4-(3-di-n-butylamino-propoxy)benzoyl}-2-n-but impurities in Active Pharmaceutical Ingredients (API) is a yl benzofuran} and BHBNB {2-n-butyl-3-(4-hydroxy dif cult issue. As per European Medicines Agency (EMEA) benzoyl)-5-nitro benzofuran}. Structures of Dronedarone guidance, a Threshold of Toxicological Concern (TTC) value and its GTI are shown in Figure 1. of 1.5 µg/day intake of a genotoxic impurity is considered As literature references available on GTI analysis are to be acceptable for most pharmaceuticals[1]. minimal, the feature of automatic MRM optimisation in Dronedarone is a drug mainly used for indications of LCMS-8040 makes method development process less cardiac arrhythmias. GTI of this drug has been tedious. In addition, the lowest dwell time and pause time quantitated here. Method has been optimized for and ultrafast polarity switching of LCMS-8040 ensures simultaneous analysis of DRN-IA uncompromised and high sensitive quantitation. {2-n-butyl-3-[4-(3-di-n-butylamino-propoxy)benzoyl]-5-nitro

C4H9 C4H9 N N O O O O C H 4 9 C4H9

NHSO2Me NO2

C4H9 C4H9 O O

Dronedarone DRN-IA C4H9

N O O O OH C4H9

NO 2

NH2 C 4H 9

C4H9 O

O DRN-IB BHBNB

Figure 1. Structures of Dronedarone and its GTI

2 Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS

Method of Analysis Sample Preparation • Preparation of DRN-IA and DRN-IB and BHBNB stock solutions 20 mg of each impurity standard was weighed separately and dissolved in 20 mL of methanol to prepare stock solutions of each standard.

• Preparation of calibration levels GTI mix standards (DRN-IA, DRN-IB and BHBNB) at concentration levels of 0.5 ppb, 1 ppb, 5 ppb, 10 ppb, 40 ppb, 50 ppb and 100 ppb were prepared in methanol using stock solutions of all the three standards.

• Preparation of blank sample 400 mg of Dronedarone powder sample was weighed and mixed with 20 mL of methanol. Mixture was sonicated to dissolve sample completely.

• Preparation of spiked (at 12 ppb level) sample 400 mg of sample was weighed and spiked with 60 µL of 1 ppm stock solution. Solution was then mixed with 20 mL of methanol. Mixture was sonicated to dissolve sample completely.

LC/MS/MS Analytical Conditions Analysis was performed using Ultra High Performance effective concentration of 40 ppb. For analytical method Liquid Chromatography (UHPLC) Nexera coupled with development it is desirable to have LOQ at least 30 % of LCMS-8040 triple quadrupole system (Shimadzu limit value, which in this case corresponds to 12 ppb. The Corporation, Japan), shown in Figure 2. Limit of GTI for developed method gives provision for measuring GTI at Dronedarone is 2 mg/kg. However, general dosage of much lower level. However, recovery studies have been Dronedarone is 400 mg, hence, limit for GTI is 0.8 µg/400 done at 12 ppb level. mg. This when reconstituted in 20 mL system, gives an

Figure 2. Nexera with LCMS-8040 triple quadrupole system by Shimadzu

3 Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS

Below mentioned table shows the analytical conditions used for analysis of GTI.

Table 1. LC/MS/MS analytical conditions

• Column : Shim-pack XR-ODS II (75 mm L x 3 mm I.D.; 2.2 µm) • Mobile phase : A: 0.1% formic acid in water B: acetonitrile • Flow rate : 0.3 mL/min • Oven temperature : 40 ºC • Gradient program (B%) : 0.0–2.0 min → 35 (%); 2.0–2.1 min → 35-40 (%); 2.1–7.0 min → 40-60 (%); 7.0–8.0 min → 60-100 (%); 8.0–10.0 min → 100 (%); 10.0–10.01 min → 100-35 (%); 10.01–13.0 min → 35 (%) • Injection volume : 1 µL • MS interface : Electro Spray Ionization (ESI) • MS analysis mode : MRM • Polarity : Positive and negative • MS gas ow : Nebulizing gas 2 L/min; Drying gas 15 L/min • MS temperature : Desolvation line 250 ºC; Heat block 400 ºC

Note: Flow Control Valve (FCV) was used for the analysis to divert HPLC ow towards waste during elution of Dronedarone so as to prevent contamination of Mass Spectrometer.

Results LC/MS/MS analysis LC/MS/MS method was developed for simultaneous calibration method. Calibration graphs of each GTI are quantitation of GTI mix standards. MRM transitions used shown in Figure 5. LOQ was determined for each GTI for all GTI are given in Table 2. No peak was seen in diluent based on the following criteria – (1) % RSD for area < 15 (methanol) at the retention times of GTI for selected MRM %, (2) % Accuracy between 80-120 % and (3) Signal to transitions which confirms the absence of any interference noise ratio (S/N) > 10. LOQ of 0.5 ppb was achieved for from diluent (shown in Figure 3). MRM chromatogram of DRN-IB and BHBNB whereas 1 ppb was achieved for GTI mix standard at 5 ppb level is shown in Figure 4. DRN-IA. Results of accuracy and repeatability for all GTI are Linearity studies were carried out using external standard given in Table 3.

Table 2: MRM transitions selected for all GTI

Name of GTI MRM transition Retention time (min) Mode of ionization DRN-IB 479.15>170.15 1.83 Positive ESI DRN-IA 509.10>114.10 5.85 Positive ESI BHBNB 338.20>244.05 8.77 Negative ESI

4 Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS

1000 1:DRA-IB 479.15>170.15(+) CE: -29.0 2:DRA-IA 509.10>114.10(+) CE: -41.0 3:BHBNB 338.20>244.05(-) CE: 20.0 750

500

250

0 0.0 2.5 5.0 7.5 10.0 min

Figure 3. MRM chromatogram of diluent (methanol)

1:DRA-IB 479.15>170.15(+) CE: -29.0 2:DRA-IA 509.10>114.10(+) CE: -41.0 40000 3:BHBNB 338.20>244.05(-) CE: 20.0 35000

30000

25000

20000

15000 DRN-IA 509.10>114.10 DRN-IB 479.15>170.15 10000

5000 BHBNB 338.20>244.05 0

0.0 2.5 5.0 7.5 10.0 min

Figure 4. MRM chromatogram of GTI mix standard at 5 ppb level

Area Area Area 750000 DRN-IB 1250000 DRN-IA BHBNB 2 2 R -0.9989 R2-0.9943 150000 R -0.9922 1000000 500000

750000 100000

250000 500000 50000 250000

0 0 0 0.0 25.0 50.0 75.0 Conc. 0.0 25.0 50.0 75.0 Conc. 0.0 25.0 50.0 75.0 Conc.

Figure 5. Calibration graphs for GTI

5 Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS

Table 3: Results of accuracy and repeatability for all GTI

Calculated concentration Standard concentration % Accuracy % RSD for area counts Sr. No. Name of GTI from calibration graph (ppb) (ppb) (n=6) (n=6) (n=6)

0.5 0.492 98.40 9.50 1 1.044 104.40 6.62 5 4.961 99.22 3.10 1 DRN-IB 12 12.014 100.12 2.97 40 38.360 95.90 1.17 50 49.913 99.83 1.08 100 103.071 103.07 0.86 1 0.994 99.40 5.02 5 4.916 98.32 2.82 12 11.596 96.63 2.43 2 DRN-IA 40 37.631 94.08 1.27 50 48.605 97.21 1.40 100 100.138 100.14 0.99 0.5 0.486 97.20 4.88 1 1.062 106.20 6.97 5 4.912 98.24 2.16 3 BHBNB 12 11.907 99.23 1.31 40 37.378 93.45 0.37 50 48.518 97.04 0.43 100 96.747 96.75 0.91

Recovery studies For recovery studies, samples were prepared as described of recovery studies have been shown in Table 4. Recovery previously. MRM chromatogram of blank and spiked could not be calculated for DRN-IB as blank sample samples are shown in Figures 6 and 7 respectively. Results showed higher concentration than spiked concentration.

1:DRA-IB 479.15>170.15(+) CE: -29.0 400000 2:DRA-IA 509.10>114.10(+) CE: -41.0 3:BHBNB 338.20>244.05(-) CE: 20.0 350000

300000

250000

200000

150000 DRN-IB 479.15>170.15

100000

50000 DRN-IA 509.10>114.10

0 BHBNB 338.20>244.05

0.0 2.5 5.0 7.5 10.0 min Figure 6. MRM chromatogram of blank sample

6 Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS

1:DRA-IB 479.15>170.15(+) CE: -29.0 2:DRA-IA 509.10>114.10(+) CE: -41.0 3:BHBNB 338.20>244.05(-) CE: 20.0 125000

100000

75000

50000 DRN-IB 479.15>170.15 25000 DRN-IA 509.10>114.10 BHBNB 338.20>244.05 0

0.0 2.5 5.0 7.5 10.0 min Figure 7. MRM chromatogram of spiked sample

Table 4. Results of the recovery studies

Concentration of Average concentration Average concentration obtained Name of % Recovery = GTI mix standard spiked obtained from calibration graph from calibration graph Impurity in blank sample (ppb) for blank sample (ppb) (A) (n=3) for spiked sample (ppb) (B) (n=3) (B-A)/ 12 * 100 DRN-IB 12 94.210 NA NA DRN-IA 12 3.279 12.840 79.678 BHBNB 12 1.241 12.723 95.689

Conclusion • A highly sensitive method was developed for analysis of GTI of Dronedarone. • Ultra high sensitivity, ultra fast polarity switching (UFswitching) enabled sensitive, selective, accurate and reproducible analysis of GTI from Dronedarone powder sample.

References [1] Guideline on The Limits of Genotoxic Impurities, (2006), European Medicines Agency (EMEA).

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1470E

Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum

ASMS 2014 WP449

Daryl Kim Hor Hee1, Lawrence Soon-U Lee1, Zhi Wei Edwin Ting2, Jie Xing2, Sandhya Nargund2, Miho Kawashima3 & Zhaoqi Zhan2 1 Department of Medicine Research Laboratories, National University of Singapore, 6 Science Drive 2, Singapore 117546 2 Customer Support Centre, Shimadzu (Asia Paci c) Pte Ltd, 79 Science Park Drive, #02-01/08, Singapore 118264 3 Global Application Development Centre, Shimadzu Corporation, 1-3 Kanda Nishihiki-cho, Chiyoda-ku, Tokyo 101-8448, Japan Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum

Introduction Developments of LC/MS/MS methods for accurate amount of serum required was normally rather high from quantitation of low pg/mL levels of 1α,25-dihydroxy 0.5mL to 2mL, which is not favourite in the clinical vitamin D2/D3 in serum were reported in recent years, applications. Direct analysis methods with using smaller because their levels in serum were found to be important amount of serum are in demand. Research efforts have indications of several diseases associated with vitamin D been reported in literatures to enhance ionization metabolic disorder in clinical research and diagnosis [1]. ef ciency by using different interfaces such as ESI, APCI or However, it has been a challenge to achieve the required APPI and ionization reagents to form purposely NH3 sensitivity directly, due to the intrinsic dif culty of adduct or lithium adduct [4,5]. Here, we present a novel ionization of the compounds and interference from matrix 2D-LC/MS/MS method with APCI interface for direct [2,3]. Sample extraction and clean-up with SPE and analysis of 1α,25-diOH-VD3 in serum. The method immunoaf nity methods were applied to remove the achieved a detection limit of 3.1 pg/mL in spiked serum interferences [4] prior to LC/MS/MS analysis. However, the samples with 100 uL injection.

Experimental High purity 1α,25-dihydroxyl Vitamin D3 and deuterated separations and MS conditions are compiled into Table 1. 1α,25-dihydroxyl-d6 Vitamin D3 (as internal standard) were The procedure of sample preparation of spiked serum obtained from Toronto Research Chemicals. samples is shown in Figure 1. It includes protein Charcoal-stripped pooled human serum obtained from precipitation by adding ACN-MeOH solvent into the serum Bioworld was used as blank and matrix to prepare spiked in 3 to 1 ratio followed by vortex and centrifuge at high samples in this study. A 2D-LC/MS/MS system was set up speed. The supernatant collected was ltered before on LCMS-8050 (Shimadzu Corporation) with a column standards with IS were added (post-addition). The clear switching valve installed in the column oven and controlled samples obtained were then injected into the 2-D by LabSolutions workstation. The details of columns, LC/MS/MS system. mobile phases and gradient programs of 1st-D and 2nd-D LC

Table 1: 2D-LC/MS/MS analytical conditions

LC condition MS Interface condition

Column 1st D: FC-ODS (2.0mml.D. x 75mm L, 3μm) Interface APCI, 400ºC 2nd D: VP-ODS (2.0mmI.D. x 150mm L, 4.6μm) MS mode Positive, MRM Mobile Phase A: Water with 0.1% formic acid Heat Block & DL Temp. 300ºC & 200ºC of 1st D B: Acetontrile CID Gas Ar (270kPa) Mobile Phase C: Water with 0.1% formic acid Nebulizing Gas Flow N2, 2.5 L/min of 2nd D D: MeOH with 0.1% formic acid Drying Gas Flow N2, 7.0 L/min

B: 40% (0 to 0.1min) → 90% (5 to 7.5min) 1st D gradient pro- → 15% (11 to 12min) → 40% (14 to 25min); gram & ow rate Total ow rate: 0.5mL/min

D: 15% (0min) → 80% (20 to 22.5min) → 2nd D gradient pro- 15% (23 to 25min); Peak cutting: 3.15 to 3.40; gram & ow rate Total ow rate: 0.5 mL/min

Oven Temp. 45ºC Injection Vol. 100 uL

2 Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum

150µL of serum 450µL of ACN/MeOH (1:1)

Shake and Vortex 10mins

Centrifuge for 10 minutes at 13000rpm

480µL of Supernatant

0.2µm nylon lter

400µL of ltered protein precipitated Serum

50µL of of Std stock 500µL of calibrate 50µL of IS stock

Figure 1: Flow chart of serum sample pre-treatment method

Results and Discussion Development of 2D-LC/MS/MS method An APCI interference was employed for effective ionization (Table 2), the first one for quantitation and the second one

of 1α,25-diOH-VitD3 (C27H44O3, MW 416.7). A MRM for confirmation. The parent ion of 1α,25-diOH-VitD3 was quantitation method for 1α,25-diOH-VitD3 with its the dehydrated ion, as it underwent neutral lost easily in deuterated form as internal standard (IS) was developed. ionization with ESI and APCI [2,3]. The MRM used for MRM optimization was performed using an automated quantitation (399.3>381.3) was dehydration of the second MRM optimization program with LabSolutions workstation. OH group in the molecule. Two MRM transitions for each compound were selected

Table 2: MRM transitions and CID parameters of 1α,25-diOH-VitD3 and deuterated IS

CID Voltage (V) Name RT1 (min) Transition (m/z) Q1 Pre Bias CE Q3 Pre Bias 399.3 > 381.3 -20 -13 -14 1α,25-dihydroxyl Vitamin D3 22.74 399.3 > 157.0 -20 -29 -17 402.3 > 366.3 -20 -12 -18 1α,25-dihydroxyl-d6 Vitamin D3 (IS) 22.71 402.3 > 383.3 -20 -15 -27 1, Retention time by 2D-LC/MS/MS method

3 Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum

The reason to develop a 2-D LC separation for a LC/MS/MS 1:OH2D3 399.30>381.30(+) CE: -13.0 1:OH2D3 399.30>157.00(+) CE: -29.0 method was the high background and interferences 5000 1:OH2D3 399.30>105.00(+) CE: -44.0 occurred with 1D LC/MS/MS observed in this study and 4000 also reported in literatures. Figure 2 shows the MRM 3000 OH2-VD3 chromatograms of 1D-LC/MS/MS of spiked serum sample. 2000

It can be seen that the baseline of the quantitation MRM 1000 (399.3>381.3) rose to a rather high level and interference 0 peaks also appeared at the same retention time. 0.0 2.5 5.0 7.5 10.0 min The 2-D LC/MS/MS method developed in this study 2:OH2D3-D6 402.30>383.30(+) CE: -15.0 700 2:OH2D3-D6 402.30>366.30(+) CE: -12.0 involves “cutting the targeted peak” in the 1st-D separation 600

precisely (3.1~3.4 min) and the portion retained in a 500 OH2-VD3-D3 stainless steel sample loop (200 uL) was transferred into 400 the 2nd-D column for further separation. The operation was 300 200 accomplished by switching the 6-way valve in and out by a 100 st nd time program. Both 1 -D and 2 -D separations were 0 carried out in gradient elution mode. The organic mobile 2.5 5.0 7.5 10.0 min st phase of 2nd-D (MeOH with 0.1% formic acid) was Peak cutting (125 uL) in 1 D separation and transferred to 2nd D LC different from that of 1st-D (pure ACN). The interference peaks co-eluted with the analyte in 1st-D were separated Figure 2: 1D-LC/MS/MS chromatograms of 22.7 pg/mL from the analyte peak (22.6 min) as shown in Figure 3. 1α,25-diOH-VitD3 (top) and 182 pg/mL internal standard (bottom) in serum (injection volume: 50uL)

Calibration curve (IS), linearity and accuracy Two sets of standard samples were prepared in serum and note that the calibration curve has a non-zero Y-intercept, in clear solution (diluent). Each set included seven levels of indicating that the blank (serum) contains either residual 1 1α,25-diOH-VitD3 from 3.13 pg/mL to 200 pg/mL, each α,25-diOH-VitD3 or other interference which must be added with 200 pg/mL of IS (See Table 3). The deducted in the quantitation method. The peak area ratios chromatograms of the seven spiked standard samples in shown in Table 3 are the results after deduction of the serum are shown in Figure 3. A linear IS calibration curve background peaks. The accuracy of the method after this (R2 > 0.996) was established from these 2D-LC/MS/MS correction is between 92% and 117%. analysis results, which is shown in Figure 4. It is worth to

Area Ratio R2 = 0.9967 4000 5.0 4000 1α,25-diOH-VitD3

3000 4.0 3000

2000 3.0 2000

1000 2.0

1000 1.0 Non-zero intercept 22.0 23.0 min 0 0.0 0 10 20 min 0.00 0.25 0.50 0.75 Conc. Ratio

Figure 3: Overlay of 2nd-D chromatograms of 7 levels Figure 4: Calibration curves of from 3.13 pg/mL to 200 pg/mL spiked in serum. 1α,25-diOH VD3 in serum by IS method.

4 Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum

Table 3: Seven levels of standard samples for calibration curve and performance evaluation

Conc. Level 1α,25-diOH VD3 Conc. Ratio1 Area Ratio2 Area Ratio2 Accuracy3 Matrix Effect of Std. (pg/mL) (Target/IS) (in serum) (in clear solu) (%) (%) L1 3.13 0.0156 0.243 0.414 103.8 58.7 L2 6.25 0.0313 0.321 0.481 100.0 66.8 L3 12.5 0.0625 0.456 0.603 117.3 75.6 L4 25.0 0.1250 0.757 0.914 115.9 82.9 L5 50.0 0.2500 1.188 1.354 95.5 87.7 L6 100.0 0.5000 2.168 2.580 92.15 84.0 L7 200.0 1.0000 4.531 4.740 102.0 95.6

1, Target = 1α,25-diOH VD3; 2, Area ratio = area of target / area of IS; 3, Based on the data of spiked serum samples

Matrix effect, repeatability, LOD/LOQ and speci city Matrix effect of the 2D-LC/MS/MS method was determined by from either the residue of 1α,25-diOH VD3 or other comparison of peak area ratios of standard samples in diluent interference present in the serum. Due to this background and in serum at the seven levels. The results are compiled into peak, the actual S/N ratio could not be calculated. Therefore, Table 3. The matrix effect of the method are between 58% it is difficult to determine the LOD and LOQ based on the and 95%. It seems that the matrix effect is stronger at lower S/N method. Tentatively, we propose a reference LOD and concentrations than at higher concentrations. Repeatability of LOQ of the method for 1α,25-diOH VD3 to be 3.1 pg/mL peak area of the method was evaluated with L2 and L3 spiked and 10 pg/mL, respectively. serum samples for both target and IS. The Results of RSD (n=6) The specificity of the method relies on several criteria: two are displayed in Table 4. MRMs (399>381 and 399>157), their ratio and RT in 2nd-D The MRM peaks of 1α,25-diOH VD3 in clear solution and in chromatogram. The MRM chromatograms shown in Figure serum are displayed in pairs (top and bottom) in Figure 5. It 5 demonstrate the specificity of the method from L1 (3.1 can be seen from the first pair (diluent and serum blank) pg/mL) to L7 (200 pg/mL). It can be seen that the results of that a peak appeared at the same retention of 1α,25-diOH spiked serum samples (bottom) meet the criteria if VD3 in the blank serum. As pointed out above, this peak is compared with the results of samples in the diluent (top).

750 750 750 1:399.30>381.30(+) 1:399.30>381.30(+) 1:399.30>381.30(+) 1:399.30>381.30(+) 1:399.30>381.30(+) 1:399.30>157.00(+) 1:399.30>157.00(+) 1:399.30>157.00(+) 1:399.30>157.00(+) 4000 1:399.30>157.00(+) 1000 Diluent L1 L3 L5 L7 500 500 500 3000 750 OH2VD3/22.622 OH2VD3/22.602 OH2VD3/22.619 500 2000 OH2VD3/22.630 250 250 250 250 1000

0 0 0 0 0

22.5 24.7 22.5 24.7 22.5 24.7 22.5 24.7 22.5 24.7

750 750 750 1:399.30>381.30(+) 1:399.30>381.30(+) 1:399.30>381.30(+) 1:399.30>381.30(+) 1:399.30>381.30(+) 1:399.30>157.00(+) 1:399.30>157.00(+) 1:399.30>157.00(+) 1:399.30>157.00(+) 4000 1:399.30>157.00(+) Serum L1 L3 L5 L7 1000 500 blank 500 500 3000 OH2VD3/22.565 OH2VD3/22.565 2000 OH2VD3/22.595 OH2VD3/22.598 OH2VD3/22.573 250 250 250 500 1000

0 0 0 0 0

22.5 24.7 22.5 24.7 22.5 24.7 22.5 24.7 22.5 24.7

Figure 5: MRM peaks of 1α,25-diOH-VitD3 spiked in pure diluent (top) and in serum (bottom) of L1, L3, L5 and L7 (spiked conc. refer to Table 3)

5 Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum

Table 4: Repeatability Test Results (n=6)

Sample Compound Spiked Conc. (pg/mL) %RSD 1α,25-diOH VD3 6.25 10.10 L2 IS 200 7.66 1α,25-diOH VD3 12.5 9.33 L3 IS 200 6.28

Conclusions A 2D-LC/MS/MS method with APCI interface has been repeatability, matrix effect, LOD/LOQ and speci city. The developed for quantitative analysis of results indicate that the 2D-LC/MS/MS method is sensitive 1α,25-dihydroxylvitamin D3 in human serum without and reliable in detection and quantitation of trace ofine extraction and cleanup. The detection and 1α,25-dihydroxylvitamin D3 in serum. Further studies to quantitation range of the method is from 3.1 pg/mL to 200 enable the method for simultaneous analysis of both pg/mL, which meets the diagnosis requirements in clinical 1α,25-dihydroxylvitamin D3 and 1α,25-dihydroxylvitamin applications. The performance of the method was D2 are needed. evaluated thoroughly, including linearity, accuracy,

References 1. S. Wang. Nutr. Res. Rev. 22, 188 (2009). 2. T. Higashi, K. Shimada, T. Toyo’oka. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. (2010) 878, 1654. 3. J. M. El‐Khoury, E. Z. Reineks, S. Wang. Clin. Biochem. 2010. DOI: 10.1002/jssc.20200911. 4. Chao Yuan, Justin Kosewick, Xiang He, Marta Kozak and Sihe Wang, Rapid Commun. Mass Spectrom. 2011, 25, 1241–1249 5. Casetta, I. Jans, J. Billen, D. Vanderschueren, R. Bouillon. Eur. J. Mass Spectrom. 2010, 16, 81.

For Research Use Only. Not for use in diagnostic procedures.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1450E

Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer

ASMS 2014 WP 182

William Hedgepeth, Kenichiro Tanaka Shimadzu Scienti c Instruments, Inc., Columbia MD Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer

Introduction Polysorbate 80 is commonly used for biotherapeutic them require time-consuming sample pretreatment such as products to prevent aggregation and surface adsorption, as derivatization and alkaline hydrolysis because polysorbates well as to increase the solubility of biotherapeutic do not have suf cient chromophores. Those methods also compounds. A reliable method to quantitate and require an additional step to remove biotherapeutic characterize polysorbates is required to evaluate the quality compounds. Here we report a simple and reliable method and stability of biotherapeutic products. Several methods for quantitation and characterization of polysorbate 80 in for polysorbate analysis have been reported, but most of biotherapeutic products using two-dimensional HPLC.

Materials Reagents and standards Reagents: Tween® 80 (Polysorbate 80), IgG from human phosphate monobasic and 871 mg of potassium serum, potassium phosphate monobasic, potassium phosphate dibasic in 1 L of water. phosphate dibasic, and ammnonium formate were Polysorbate 80 was diluted with 10 mmol/L phosphate purchased from Sigma-Aldrich. Water was made in house buffer (pH 6.8) to 200, 100, 50, 20, 10 mg/L and using a Millipore Milli-Q Advantage A10 Ultrapure Water transferred to 1.5 mL vials for analysis. Purification System. Isopropanol was purchased from Sample solutions: A model sample was prepared by Honeywell. dissolving 2 mg of IgG in 0.1 mL of a 100 mg/L polysorbate Standard solutions: 10 mmol/L phosphate buffer (pH 6.8) 80 standard solution. The sample was centrifuged and was prepared by dissolving 680 mg of potassium transferred to a 1.5 mL vial for analysis.

O O wO O OH O x OH OH O O z y w+x+y+z=approx. 20

CH3

Fig.1 Typical structure of polysorbate 80

2 Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer

System The standard and sample solutions were injected into a detector was used to check protein removal. Shimadzu Co-Sense for BA system consisting of two Fig. 2 shows the flow diagram of the Co-Sense for BA LC-20AD pumps and a LC-20AD pump equipped with a system. In step 1, a sample pretreatment column solvent switching valve, DGU-20A5R degassing unit, “Shim-pack MAYI-ODS” traps polysorbate 80 in the SIL-20AC autosampler, CTO-20AC column oven equipped sample. Proteins (antibody) cannot enter the pore interior with a 6-port 2-position valve, and a CBM-20A system that is blocked by a hydrophilic polymer bound on the controller. Polysorbate 80 was detected by a LCMS-2020 outer surface. Other additives and excipients such as single quadrupole mass spectrometer or a LCMS-8050 sugars, salts, and amino acids cannot be retained by the triple quadrupole mass spectrometer because polysorbates ODS phase of the inner surface due to their polarity. In do not have any chromophores and are present at low step 2, the trapped polysorbate 80 is introduced to the concentrations in antibody drugs. A SPD-20AV UV-VIS analytical column by valve switching.

Step 1 : Protein removal

Mass spectrometer

Pump 2 Mobile phase C Analytical column Valve (Position 0) Mobile phase A (solution for sample injection)

Autosampler Mobile phase D

Protein, Salts, Polysorbate Amino acids, UV-VIS detector 80 Sugars Pump 1

Sample pretreatment column Mobile phase B (solution for rinse) Step 2 : Analyzing the trapped polysorbate

Polysorbate 80 Mass spectrometer Pump 2 Mobile phase C Analytical column Valve (Position 1) Mobile phase A (solution for sample injection)

Autosampler Mobile phase D

UV-VIS detector Pump 1

Sample pretreatment column Mobile phase B (solution for rinse)

Fig.2 Flow diagram of Co-Sense for BA

3 Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer

Results Quantitation method A fast analysis for quantitation will be shown here. Table 1 mmol/L phosphate buffer pH6.8). Polysorbate 80 in the shows the analytical conditions and Fig. 3 shows the TIC model sample was successfully analyzed. The peak at 4.4 chromatogram of a 100 mg/L polysorbate 80 standard min was used for quantitation. solution and the mass spectrum of the peak at 4.4 min. Six replicate injections for the model sample were made to Polysorbates contain many by-products, so several peaks evaluate the reproducibility. The relative standard appeared on the TIC chromatogram. The peak at 4.4 min deviations of retention time and peak area were 0.034 % was identified as polyoxyethylene sorbitan monooleate and 1.11 %, respectively. The recovery ratio was obtained (typical structure of polysorbate 80) based on E. Hvattum by comparing the peak area of the model sample and a et al 2011. The ion at 783 was used as a marker for 100 mg/L polysorbate 80 standard solution and was 99 %. detection in selected ion mode (SIM). This ion is Five different levels of polysorbate 80 standard solutions + attributable to the 2NH4 adduct of polyoxyethylene ranging from 10 to 200 mg/L were used for the linearity sorbitan monooleate containing 25 polyoxyethylene evaluation. The correlation coefficient (R2) of determination groups. Fig. 4 shows the SIM chromatogram of the model was higher than 0.999. sample (20 g/L of IgG, 100 mg/L of polysorbate 80 in 10

Table 1 Analytical Conditions

System : Co-Sense for BA equipped with LCMS-2020 [Sample Injection] [UV Detection] Column : Shim-pack MAYI-ODS (5 mm L. x 2.0 mm I.D., 50 μm) Detection : 280 nm Mobile Phase : A: 10 mmol/L ammonium formate in water Flow Cell : Semi-micro cell B: Isopropanol [MS Detection] Solvent Switching : A (0-1.5 min), B (1.5-3.5 min), A (3.5-9 min) Ionization Mode : ESI Positive Flow Rate : 0.6 mL/min Applied Voltage : 4.5 kV Valve Position : 0 (0-1 min, 7-9 min), 1 (1-7 min) Nebulizer Gas Flow : 1.5 mL/min Injection Volume : 1 µL DL Temperature : 250 ºC [Separation] Block Heater Temp. : 400 ºC Column : Kinetex 5u C18 100A (50 mm L. x 2.1 mm I.D., 5 μm) Scan : m/z 300-2000 Mobile Phase : A: 10 mmol/L ammonium formate in water SIM : m/z 783 B: Isopropanol Time Program : B. Conc 5 % (0-1 min) - 100 % (6-7 min) -5 % (7.01-9 min) Flow Rate : 0.3 mL/min Column Temperature : 40 ºC

4 Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer

4000000

3000000

2000000

1000000

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min

Inten.(x100,000) Triply charged ions 1.5

587601 616 631 572 Doubly charged ions 1.0 645 557 660 675 783 0.5 543 805 827 689 739 761 849 528 871 893 704717 915 0.0 500 550 600 650 700 750 800 850 900 950 m/z

Fig.3 TIC Chromatogram of 100 mg/L polysorbate 80 standard solution and mass spectrum of the peak at 4.4 min

100000

75000

50000

25000

0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min

Fig.4 SIM chromatogram of the model sample

Characterization method An analysis for characterization will be shown here. Table 2 and others. The peaks on the TIC chromatogram are shows the analytical conditions and Fig. 5 shows the TIC assumed to correspond to those by-products. For example, chromatogram of the model sample and mass spectra of the peaks from 10 to 22 min correspond to the peaks from 10 to 30 min. A longer column and polyoxyethylene and polyoxyethylene isosorbide and the gradient were applied to obtain better resolution. peaks from 22 to 30 min correspond to polyoxyethylene Polysorbate 80 consists of not only monooleate (typical sorbitan. This method is helpful for characterization as well structure of polysorbate 80), but also many by-products as checking degradation such as auto-oxidation and such as polyoxyethylene, polyoxyethylene sorbitan, hydrolysis. polyoxyethylene isosorbide, dioleate, trioleate, tetraoleate

5 Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer

Table 2 Analytical Conditions

System : Co-Sense for BA equipped with LCMS-8050 [Sample Injection] [UV Detection] Column : Shim-pack MAYI-ODS (5 mm L. x 2.0 mm I.D., 50 μm) Detection : 280 nm Mobile Phase : A: 10 mmol/L ammonium formate in water Flow Cell : Semi-micro cell B: Isopropanol [MS Detection] Solvent Switching : A (0-1.5 min), B (1.5-3.5 min), A (3.5-9 min) Ionization Mode : ESI Positive Flow Rate : 0.6 mL/min (0-10 min, 95.01-110 min), 0.1 mL/min (10.01-95 min) Applied Voltage : 4.5 kV Valve Position : 0 (0-3 min, 100-110 min), 1 (3-100 min) Nebulizer Gas Flow : 2 mL/min Injection Volume : 5 µL Drying Gas Flow : 10 mL/min [Separation] Heating Gas Flow : 10 mL/min Column : Kinetex 5u C18 100A (100 mm L. x 2.1 mm I.D., 5 μm) Interface Temperature : 300 ºC Mobile Phase : A: 10 mmol/L ammonium formate in water DL Temperature : 250 ºC B: Isopropanol Block Heater Temp. : 400 ºC Time Program : B. Conc 5 % % (0-3min) – 35% (15min) – 100% (100min) – 5% (100.01-110min) Q1 Scan : m/z 300-2000 Flow Rate : 0.2 mL/min Column Temperature : 40 ºC

(x100,000,000) 1:TIC(+) (x10,000,000) 4.0 1:TIC(+) 7.5

3.0 5.0

2.5

2.0 0.0

10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min

1.0

0.0

0 10 20 30 40 50 60 70 80 90 100 min

Inten.(x100,000) Inten.(x100,000) 6.0

3.0 513.6528.3 692.8 5.0 498.9 648.8 543.0 736.8 604.7 484.2 4.0 557.6 2.0 469.5 560.7 780.9 421.7443.8 3.0 628.9651.0673.0695.0 399.7 465.8 564.7 608.8 572.3 652.8 454.8 717.1 377.6 516.6520.7 824.9 606.9 696.9 587.0 739.0 445.4 2.0 761.1 1.0 355.6 423.5 740.9 440.2 379.5401.6 869.0 783.1 784.9 805.1 913.0 1.0 425.4 827.1

0.0 0.0 300 400 500 600 700 800 900 m/z 400 500 600 700 800 m/z

O OH O O w OH Polyoxyethylene O OH Polyoxyethylene y OH H O O OH x Polyoxyethylene x O O OH OH z isosorbide O O sorbitan z y

Fig.5 TIC chromatogram of the model sample

6 Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer

Con rmation of protein removal Fig. 6 shows the chromatogram of elution from the sample pretreatment column. Protein (IgG) was successfully removed from the sample by using the MAYI-ODS column.

uV

1250000 5uL injection of model sample 1uL injection of model sample 1000000

750000

500000

250000

0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min

Fig.6 Chromatogram of elution from the sample pretreatment column

Conclusions 1. Co-Sense for BA system automatically removed protein from the sample and enabled quantitation and characterization of polysorbate 80 in a protein formulation. 2. The quantitation method was successfully applied to the model sample with excellent reproducibility and recovery. 3. The high-resolution chromatogram was obtained by the characterization method. This method is helpful for characterization as well as checking degradation such as auto-oxidation and hydrolysis.

Reference E. Hvattum, W.L. Yip, D. Grace, K. Dyrstad, Characterization of polysorbate 80 with liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy: Specific determination of oxidation products of thermally oxidized polysorbate 80, J Pharm Biomed Anal 62, (2012) 7-16

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1457E

A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG

ASMS 2014 WP161

Rachel Lieberman1, David Colquhoun1, Jeremy Post1, Brian Feild1, Scott Kuzdzal1, Fred Regnier2, 1Shimadzu Scienti c Instruments, Columbia, MD, USA 2Novilytic L.L.C, North Webster, IN, USA A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG

Novel Aspect Using rapid, automated processing, coupled to the speed and sensitivity of the LCMS-8050 allows for improved analysis of Immunoglobulin G.

Introduction Dried blood spot analysis (DBS) has provided clinical avoid variability caused by the hematocrit. This laboratories a simple method to collect, store and transport presentation focuses on an ultra-fast-immuno-MS platform samples for a wide variety of analyses. However, sample that combines next generation plasma separator cards stability, hematocrit effects and inconsistent spotting (Novilytic L.L.C., North Webster, IN) with fully automated techniques have limited the ability for wide spread immuno-afnity enrichment and rapid digestion as an adoption in clinical applications. Dried plasma spots (DPS) upfront sample preparation strategy for mass spectrometric offer new opportunities by providing stable samples that analysis of immunoglobulins.

Sample Work ow

Plasma Affinity Buffer Enzyme Desalting LC/MS/MS Generation Selection Exchange Digestion

NoviplexTM Card Perfinity Workstation LCMS-8050 Triple Quadrupole MS

Rapid plasma extraction technology Automates and integrates key - Ultrafast MRM methods from whole blood (~ 18 minutes) proteomic workflow steps: - Up to 555 MRM transitions per run - 2.5 uL of plasma collected (3 min) - Affinity Selection (15 min) - Heated electrospray source - Air dry for 15 minutes - Trypsin digestion (1-8 min) - Scan speeds up to 30,000 u/sec - Extract plamsa disc for analysis - Online Desalting - Polarity switching 5 msec - Reversed phase LC Exceptional reproducibility (CV less than 10%)

2 A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG

Methods IgG was weighed out and then diluted in 500 μL of 0.5% reduced and alkylated to yield a total sample volume of BSA solution. Approximately15 uL of IgG standard was 100 uL. IgG standards and QC samples were directly spiked into mouse whole blood and processed using the injected onto the Pernity-LCMS-8050 platform for afnity Noviplex card. The resulting plasma collection disc was pulldown with a Protein G column followed by trypsin extracted with 30 uL of buffer and each sample was digestion and LC/MS/MS analysis.

Conc. Amount on Amount on Time (min) %B Level 100 (μg/mL) column (μg) column (pmol) 0 2 80 1 465 34.88 581.25 0.2 2 60

2 315 23.63 393.75 8 50 %B 40 3 142.5 10.69 178.13 9.5 50 20 4 127.5 9.56 159.37 0 10 90 0 2 4 6 8 10 12 14 16 5 102 7.65 127.50 12.5 90 Time (minutes) 6 60 4.50 75.00 12.51 2 7 22.5 1.69 28.12 16 2

IgG concentrations for calibration levels. LCMS gradient conditions.

Q1 Rod Bias Q3 Rod Bias Compound Name Transitions +/- CE (V) (V) (V) 937.70>836.25 + -27 -28 -26 TTPPVLDSDGSFFLYSK 937.70>723.95 + -27 -30 -22 VVSVLTVLHQDWLNGK 603.70>805.7 + -22 -16 -13

MRM transitions on LCMS-8050 for two IgG peptides monitored.

Noviplex Cards (2) (3) (4)

(1)

Approximately 50 uL of the spiked whole blood was disc. The plasma collection disc was allowed to dry for an pipetted onto the Noviplex card test area (1). The spot was additional 15 minutes. Once the disc was dry (4), it was allowed to dry for 3 minutes (2). The top layer of the card placed into an eppendorf tube for solvent extraction. was then peeled back (3) to reveal the plamsa collection

3 A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG

Results - Chromatograms

300000000

275000000

250000000

225000000

200000000

175000000

150000000

125000000

100000000

75000000 Optimization of Collision Energies for peptides of interest

50000000

25000000

0 1250000 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min Range CE: -50 to -10 V

1000000 TTPPVLDSDGFFLYSK Total Ion Chromatogram for IgG 750000

500000

250000

0

6.200 6.225 6.250 6.275 6.300 6.325 6.350 6.375 6.400 6.425 6.450 6.475 6.500 6.525 6.550 6.575 6.600 6.625 6.650 6.675 min

Inten. 2.00 938 +2 1.75 [M+2H]

1.50 1.25 [P1+2H]+2 5000 1.00 937 4500 TTPPVLDSDGSFFLYSK 938 VVSVLTVLHQDWLNGK 0.75 4000 +2 837 [P2+2H] 836 0.50 3500 397 0.25 352 724 836 3000 283295 337 397369 407 524 561 836 915 1163 510 724 836 1046 283 407 466 591 640658 723 756 379397 443449 809 851 891 2500 0.00 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z

2000

1500

1000

500

0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 min

MRM Chromatogram for Level 4 standard of spiked IgG in whole blood.

Carryover Assessment

1100 90 1000 Control - Mouse blood 80 Blank Injection 900

70 800

700 60

600 50

500 40

400 30 300 20 200

10 100

0 0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 min

4 A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG

Results - Calibration Curves

Calibration Curve and MS Chromatograms

TTPPVLDSDGSFFLYSK VVSVLTVLHQDWLNGK

603.70>805.70(+) 25000 937.70>836.25(+) 603.70>805.70(+) 937.70>836.25(+) 10000 Level 1 937.70>723.95(+) Level 1 2000 937.70>723.95(+) Level 7 600 Level 7

20000 500 7500 1500 400 15000 5000 1000 300 10000 200 500 2500 5000 100

0 0 0 0 6.00 6.25 6.50 6.75 5.50 5.75 6.00 6.25 6.50 5.50 5.75 6.00 6.25 6.50 6.00 6.25 6.50 6.75

Area Area 30000 r2 = 0.979 r2 = 0.989 25000 50000 20000

15000

25000 10000

5000

0 0 0 100 200 300 400 Conc. 0 100 200 300 400 Conc.

Results - Tables and Replicates

QC data and Calculations for IgG Peptides

VVSVLTVLHQDWLNGK Sample Ret. Time Area Calc. Conc. Std. Conc. % Accuracy QC 1 6.49 32,492 502.804 465 108.1 QC 2 6.516 11,726 167.189 142.5 117.3 QC 3 6.514 8,507 115.155 102 112.9 QC 4 6.492 2,727 21.745 22.5 96.6

TTPPVLDSDGSFFLYSK Sample Ret. Time Area Calc. Conc. Std. Conc. % Accuracy QC 1 6.029 61,525 416.447 465 89.6 QC 2 6.052 25,355 155.568 142.5 109.2 QC 3 6.047 16,900 94.58 102 92.7 QC 4 6.029 6,502 19.587 22.5 87.1

5 A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG

Skyline Data - Retention Time Replicates

VVSVLTVLHQDWLNGK TTPPVLDSDGSFFLYSK

y14 - 805.4385++ y15 - 836.4169++ 6.65 6.20

6.60 6.15

6.55 6.10

6.50 6.05

6.45 6.00 Retention Time Retention Time 6.40 5.95

6.35 5.90 4...L6...006 M_2252014...L7...004 839 AM_2262014...L2...004 839 AM_2262014...L3...003 839 AM_2262014...L4...002 839 AM_2262014...L3...003 839 AM_2262014...L4...002 839 AM_2262014...L1...005 839 AM_2262014...L1...005 839 AM_2262014...L2...004 1433 PM_2252014...L6...006 1433 P 1433 PM_2252014...L5...008 1433 PM_2252014...L5...008 1433 PM_2252014...L7...004 1433 PM_225201

Replicate Replicate

Integration of Skyline Software into LabSolutions allows for further interrogation of data. Here are representative gures showing the retention time reproducibility for each peptide monitored during the analysis.

Conclusions Combining the sample collection technique of next generation plasma separator Noviplex cards for quick plamsa collection from whole blood, with the automated afnity selection and trypsin digestion of the Pernity workstation coupled to LCMS-8050, provides a very rapid and reproducible Immuno-MS platform for quantitation of IgG peptides. Furthermore, this rapid immuno-MS platform can be applied to many other peptide/protein applications.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1473E

Simultaneous Determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS

ASMS 2014 TP 757

Qian Sun, Jun Fan, Taohong Huang, Shin-ichi Kawano, Yuki Hashi, Shimadzu Global COE, Shanghai, China Simultaneous Determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS

Introduction On-line gel permeation chromatography-gas techniques provide much better selectivity thus signi cantly chromatography/mass spectrometry (GPC-GC-MS) is a lower detection limits. In this work, a new method was unique technique to cleanup sample that reduce the time developed for rapid determination of 20 common drugs of sample preparation. GPC can ef ciently separates fats, and pesticides in human blood by GPC-GC-MS/MS. The protein and pigments from samples, due to this advantage, modi ed QuEChERS method was used for sample on-line GPC is widely used for pesticide analysis. preparation. Meanwhile, compared to widely used GC-MS, GC-MS/MS

Experimental The human blood samples were extracted with acetonitrile, macromolecular interference material, such as protein and

then was puri ed by PSA, C18 and MgSO4 to remove most cholesterol, the background interference brought about by of the fats, protein and pigments in samples, then after the complex matrix in samples was effectively reduced. on-line GPC-GC-MS/MS analysis which further removed

Sample pretreament

human blood 2 mL

CH3CN vortex

PSA/C18/MgSO4

vortex centrifuge

supernatant

evaporate

set volume using moblie phase

GPC-GC-MS/MS

Figure 1 Schematic ow diagram of the sample preparation

2 Simultaneous Determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS

Instrument

GPC

Mobile phase : acetone/cyclohexane (3/7, v/v) Flow rate : 0.1mL/min Column : Shodex CLNpak EV-200 (2 mmI.D. x 150 mmL.) Oven temperature : 40 ºC Injection volume : 10 μL

GCMS-TQ8030

Column : deactivated silica tubing [0.53 mm(ID) x 5 m(L)] +pre-column Rtx-5ms [0.25 mm(ID) x 5 m(L)] Rtx-5ms [0.25mm(ID) x 30 m(L), Thickness: 0.25 μm] Injector : PTV Injector time program : 120 ºC(4.5min)-(80 ºC/min)-280 ºC(33.7 min) Oven temperature program : 82 ºC(5min)-(8 ºC/min)-300 ºC(7.75 min) Linear velocity : 48.8 cm/sec Ion Source temperature : 210 ºC Interface temperature : 300 ºC

Results For all of analytes, recoveries in the acceptable range of The method is simple, rapid and characterized with 70~120% and repeatability (relative standard deviations, acceptable sensitivity and accuracy to meet the RSD)≤5% (n=3) were achieved for matrices at spiking levels requirements for the analysis of common drugs and of 0.01 µg/mL. The limitis of detection were 0.03~4.4 µg/L. pesticides in the human blood.

(x10,000,000)

1.00

0.75

0.50

0.25

0.00

15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5

Figure 2 MRM chromatograms of standard mixture

3 Simultaneous Determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS

Table 1 Results of method validation for drugs and pesticides (Concentration range: 5-100 μg/L, LODs: S/N≥3, LOQs: S/N≥10, RSDs: n=3)

t Correlation LOD LOQ 0.01 µg/mL No. Compound Name R (min) Coef cient* (µg/L) (µg/L) Recovery (%) RSD (%) 1 Dichlorvos 10.795 0.9993 0.103 0.345 72.9 2.99 2 Methamidophos 11.800 0.9994 0.023 0.076 85.3 3.58 3 Barbital 15.210 0.9994 0.018 0.058 72.4 1.72 4 Sulfotep 17.580 0.9995 0.011 0.037 110.7 2.27 5 Dimethoate 18.310 0.9993 0.400 1.333 103.7 3.10 6 Malathion 21.555 0.9997 0.005 0.016 82.7 2.52 7 Chlorpyrifos 21.715 0.9996 0.010 0.033 85.7 3.57 8 Phenobarbital 22.000 0.9995 0.353 1.177 79.6 3.25 9 Parathion 22.180 0.9993 0.003 0.009 92.3 3.17 10 Triazophos 25.675 0.9994 0.046 0.155 87.7 1.32 11 Zopiclone deg. 26.025 0.9993 0.189 0.631 83.5 1.28 12 Diazepam 27.635 0.9992 0.007 0.022 98.3 1.55 13 Midazolam 29.250 0.9994 0.048 0.160 87.1 2.01 14 Zolpidem 31.225 0.9993 1.298 4.325 99.3 1.01 15 Clonazepam 31.795 0.9995 0.432 1.440 110.0 1.57 16 Estazolam 32.335 0.9994 0.092 0.305 103.7 1.37 17 Clozapine 32.400 0.9991 0.050 0.167 100.6 3.12 18 Alprazolam 32.730 0.9993 0.028 0.095 103.3 1.48 19 Zolpidem 33.095 0.9995 1.027 3.425 87.3 1.75 20 Triazolam 33.700 0.9992 0.027 0.091 81.3 2.56

Conclusion A very quick, easy, effective, reliable method in human validation criteria. The method has been successfully blood based on modi ed QuEChERS method was applied for determination of human blood samples and developed using GPC-GCMS-TQ8030. The performance of ostensibly has further application opportunities, e.g. the method was very satisfactory with results meeting biological samples.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1466E

Low level quantitation of Loratadine from plasma using LC/MS/MS

ASMS 2014 TP498

Shailesh Damale, Deepti Bhandarkar, Shruti Raju, Rashi Kochhar, Shailendra Rane, Ajit Datar, Pratap Rasam, Jitendra Kelkar Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh Chambers, Makwana Road, Marol, Andheri (E), Mumbai-400059, Maharashtra, India. Low level quantitation of Loratadine from plasma using LC/MS/MS

Introduction Loratadine is a histamine antagonist drug used for the of heated Electro Spray Ionization (ESI) interface in treatment of itching, runny nose, hay fever and such other LCMS-8050 ensured good quantitation and repeatability allergies. Here, an LC/MS/MS method has been developed even in the presence of a complex matrix like plasma. Ultra for high sensitive quantitation of this molecule from high sensitivity of LCMS-8050 enabled development of a plasma using LCMS-8050, a triple quadrupole mass low ppt level quantitation method for Loratadine. spectrometer from Shimadzu Corporation, Japan. Presence

Loratadine Loratadine, a piperidine derivative, is a potent long-acting, non-sedating tricyclic antihistamine with selective peripheral H1-receptor antagonist activity. It is used for relief of nasal and non-nasal symptoms of seasonal allergies and skin rashes[1,2,3]. Due to partial distribution in central nervous system, it has less sedating power compared to traditional H1 blockers. Loratadine is given orally, is well absorbed from the gastrointestinal tract, and has rapid rst-pass hepatic metabolism; it is metabolized by Ethyl 4- (8-chloro-5, 6-dihydro-11H-benzo [5, 6] isoenzymes of the cytochrome P450 system, including cyclohepta [1, 2-b] pyridin-11-ylidene) CYP3A4, CYP2D6, and, to a lesser extent, several others. -1-piperidinecarboxylate Loratadine is almost totally (97–99 %) bound to plasma Figure 1. Structure of Loratadine proteins and reaches peak plasma concentration (Tmax) in ~ 1–2 h[4,5].

Method of Analysis This bioanalytical method was developed for measuring simple and accurate method for estimation of Loratadine in Loratadine in therapeutic concentration range for the human plasma. analysis of routine samples. It was important to develop a

Preparation of matrix matched plasma by protein precipitation method using cold acetonitrile To 100 µL of plasma 500 µL cold acetonitrile was added was centrifuged at 12000 rpm for 15 minutes. Supernatant for protein precipitation. It was placed in rotary shaker at was taken and evaporated to dryness at 70 ºC . The 20 rpm for 15 minutes for uniform mixing. This solution residue was reconstituted in 200 µL Methanol.

Preparation of calibration standards in matrix matched plasma 1 ppt, 5 ppt, 50 ppt, 100ppt, 500 ppt, 1 ppb, 5 ppb and in cold acetonitrile treated matrix matched plasma. 10 ppb of Loratadine calibration standards were prepared

2 Low level quantitation of Loratadine from plasma using LC/MS/MS

LC/MS/MS analysis LCMS-8050 triple quadrupole mass spectrometer by range of target compounds with considerable reduction Shimadzu Corporation, Japan (shown in Figure 2A), sets a in background. new benchmark in triple quadrupole technology with an Presence of heated Electro spray interface in LCMS-8050 unsurpassed sensitivity (UFsensitivity) with Scanning speed (shown in Figure 2B) ensured good quantitative sensitivity of 30,000 u/sec (UFscanning) and polarity switching even in presence of a complex matrix like plasma. speed of 5 msecs (UFswitching). This system ensures The parent m/z of 382.90 giving the daughter m/z of highest quality of data, with very high degree of 337.10 in the positive mode was the MRM transition used reliability. for quantitation of Loratadine. MS voltages and collision In order to improve ionization ef ciency, the newly energy were optimized to achieve maximum transmission developed heated ESI probe combines high-temperature of mentioned precursor and product ion. Gas ow rates, gas with the nebulizer spray, assisting in the desolvation source temperature conditions and collision gas were of large droplets and enhancing ionization. This optimized, and linearity graph was plotted for 4 orders of development allows high-sensitivity analysis of a wide magnitude.

Figure 2A. LCMS-8050 triple quadrupole mass spectrometer by Shimadzu Figure 2B. Heated ESI probe

Table 1. LC conditions Table 2. LCMS conditions

Column Shim-pack XR-ODS (100 mm L x 2.0 mm ID ; 2.2 µm) MS Interface ESI Mobile Phase A : 0.1% formic acid in water Polarity Positive B : acetonitrile Nebulizing Gas Flow 2.0 L / min (nitrogen) Gradient Program Drying Gas Flow 10.0 L / min (nitrogen) Time (min) A conc. (%) B conc. (%) Heating Gas Flow 15.0 L / min (zero air) 0.01 40 60 Interface Temp. 300 ºC 1.50 0 100 Desolvation Line Temp. 250 ºC 4.00 0 100 Heater Block Temp. 400 ºC 4.10 40 60 MRM Transition 382.90 > 337.10 13.00 Stop

Flow Rate 0.15 mL/min Oven Temperature 40 ºC Injection Volume 20 µL

3 Low level quantitation of Loratadine from plasma using LC/MS/MS

Results LC/MS/MS Analysis LC/MS/MS method for Loratadine was developed on ESI Calibration curve was plotted for Loratadine concentration +ve ionization mode and 382.90>337.10 MRM transition range. Also as seen in Table 3, % Accuracy was studied to was optimized for Loratadine. Checked matrix matched confirm the reliability of method. plasma standards for highest (10 ppb) as well as lowest Linear calibration curves were obtained with regression (0.001 ppb) concentrations as seen in Figures 4A and 4B coefficients R2 > 0.998. % RSD of area was within 15 % respectively. Optimized MS method to ensure no plasma and accuracy was within 80-120 % for all calibration levels. interference at the retention time of Loratadine (Figure 5).

(x1,000,000) (x10,000) 3.5 382.90>337.10(+) 382.90>337.10(+) 5.0 3.0

2.5 4.0 LORATADINE/3.391

2.0 3.0

1.5 2.0 1.0 1.0 0.5 LORATADINE/3.377 0.0 0.0

-0.5 -1.0 0.0 2.5 5.0 7.5 0.0 2.5 5.0 7.5 Figure 4A. Mass chromatogram 10 ppb Figure 4B. Mass chromatogram 0.001 ppb

Speci city and interference

(x10,000) 1.2 1:LORATIDINE 382.90>337.10(+) CE: -23.0 LORA_PLASMA_003.lcd 1:LORATIDINE 382.90>337.10(+) CE: -23.0 LORA_PLASMA_002.lcd 1.1 ------LOQ Level 1.0 0.9 ------Blank 0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

-0.1

-0.2

-0.3

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 min

Figure 5. Overlay chromatogram

4 Low level quantitation of Loratadine from plasma using LC/MS/MS

Area (x10,000,000) 2.0 8 Area (x100,000)

2.0 4 7 1.0 3 1.0

2 6 1 5 0.0 1234 0.0 0.05 0.10 Conc. 0.0 2.5 5.0 7.5 Conc.

Figure 6. Loratadine calibration curve

Result Table Table 3. Results of Loratadine calibration curve

Nominal Concentration Measured Concentration % RSD for area counts % Accuracy Sr. No. Standard (ppb) (ppb) (n=3) (n=3)

1 STD-01 0.001 0.00096 0.62 95.83 2 STD-02 0.005 0.0050 5.24 100.73 3 STD-03 0.05 0.057 0.98 114.83 4 STD-04 0.1 0.095 1.81 95.40 5 STD-05 0.5 0.048 1.40 95.70 6 STD-06 1.0 0.986 0.11 98.53 7 STD-07 5.0 5.077 1.07 101.53 8 STD-08 10.0 9.983 1.96 99.37

Conclusion • Highly sensitive LC/MS/MS method for Loaratadine was developed on LCMS-8050 system. • Calibration was plotted from 10 ppb to 0.001 ppb, and LOQ was computed as 0.001 ppb.

5 Low level quantitation of Loratadine from plasma using LC/MS/MS

References [1] Bhavin N. Patel, Naveen Sharma, Mallika Sanyal, and Pranav S. Shrivastav, Journal of chromatographic Sciences, Volume 48, (2010), 35-44. [2] J. Chen, YZ. Zha, KP. Gao, ZQ. Shi, XG. Jiang, WM. Jiang, XL. Gao, Pharmazie, Volume 59, (2004), 600-603. [3] M. Haria, A. Fitton, and D.H. Peters, Drugs, Volume 48, (1994), 617-637. [4] J. Hibert, E. Radwanski, R. Weglein, V. Luc, G. Perentesis, S. Symchowicz, and N. Zampaglione, J.clin. Pharmacol, Volume 27, (1987), 694-698. [5] S.P.Clissold, E.M. Sorkin, and K.L. Goa, Drugs, Volume 37,(1989), 42-57.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 Food • Page 111 • Page 141 An LCMS method for the detection of cocoa High sensitivity quantitation method of dicyandi- butter substitutes, replacers, and equivalents amide and melamine in milk powders by liquid in commercial chocolate-like products chromatography tandem mass spectrometry

• Page 116 • Page 147 Highly sensitive and robust LC/MS/MS method Multiresidue pesticide analysis from dried chili for quantitative analysis of articial sweeteners powder using LC/MS/MS in beverages • Page 154 • Page 122 Multi pesticide residue analysis in tobacco by Highly sensitive and rapid simultaneous method GCMS/MS using QuEChERS as an extraction for 45 mycotoxins in baby food samples by method HPLC-MS/MS using fast polarity switching • Page 161 • Page 129 Simultaneous quantitative analysis of 20 amino High sensitivity analysis of acrylamide in potato acids in food samples without derivatization chips by LC/MS/MS with modified QuEChERS using LC-MS/MS sample pre-treatment procedure

• Page 135 Determination of benzimidazole residues in animal tissue by ultra high performance liquid chromatography tandem mass spectrometry PO-CON1458E

An LCMS Method for the Detection of Cocoa Butter Substitutes, Replacers, and Equivalents in Commercial Chocolate-like Products

ASMS 2014 ThP632

Jared Russell, Liling Fang and Willard Bankert Shimadzu Scienti c Instruments., Columbia, MD An LCMS Method for the Detection of Cocoa Butter Substitutes, Replacers, and Equivalents in Commercial Chocolate-like Products

Introduction There is increasing demand for genuine cocoa butter (CB) for the TAGs and typical GC analyses of this type can take in chocolate products in developed nations, however, this 40 minutes. LCMS is able to not only provide faster demand has created a shortage of CB and raised its costs. throughput, but also has the additional advantage of To overcome this, chocolate manufactures sometimes add allowing characterization of the TAG, including qualitative vegetable-derived fats to some chocolate products to regiospeci c analysis. We have developed a single, UHPLC reduce costs while still maintaining desirable physical column-based LCMS method to analyze the TAG characteristics. It is of current interest to have a reliable components in commercial chocolate and chocolate-like method to detect, identify, and quantify the triacylglycerol products. This analysis has a runtime of 17minutes, making (TAG) components of cocoa butter substitutes, replacers, it suitable for relatively high throughput. Additionally, the and equivalents (CBEs) in chocolate products. Traditionally method was very repeatable, with an interday variability of GC was used for this task, but due to the low volatility of <7% for the absolute area counts of the three major TAGs triacylglycerides and their susceptibility to thermal in CB (POP,POS,SOS). decomposition, retention time is the only identifying factor

Materials and Method A Shimadzu Nexera UHPLC coupled to a Shimadzu reference. Chocolate and chocolatey products were LCMS-8040 triple quadrupole mass spectrometer was purchased in retail stores over a range of cocoa content. utilized for this analysis. A pure CB standard was used as a

Sample Preparation For analysis, we slightly modified a sample preparation mixture for 5 minutes. The solution was filtered through a method originally used for algal oils. For analysis, 5mg of Thomson filter vial (P/N 35538-100) to remove sugars and sample was weighed and then dissolved in a 3:1 other insoluble materials and diluted 5-fold using 3:1 Toluene-Isopropyl Alcohol solution. We then sonicated the Toluene-IPA and injected into the UHPLC-MS system.

Chromatography

Instrument : Shimadzu Nexera UHPLC system Column : Shimadzu Shim-Pack XR-ODSIII (200x2.1mm,) Mobile Phase A : LC/MS Acetonitrile Mobile Phase B : 1:1 Dichloromethane-Isopropyl Alcohol Gradient Program : 48% B (initially) – gradient to 51% B (0-8.0 min) – gradient to 54% B (8.0 – 11.0 min) – gradient to 74% B (11.0-14.0 min) – hold at 74% B (14.0-15.0 min) – reequilibrate at 48% B (15.1-17 min) Flow Rate : 0.33 mL/min Column Temperature : 30°C Injection Volume : 1 μL

Mass Spectrometry

Instrument : Shimadzu LCMS-8040 Triple Quadrupole Mass Spectrometer Ionization : APCI Polarity : Positive Scan Mode : Q3 Scan

2 An LCMS Method for the Detection of Cocoa Butter Substitutes, Replacers, and Equivalents in Commercial Chocolate-like Products

Results Retail Chocolates from Hershey’s, Lindt and Tcho, as well match. In order to identify usage of CBEs, we applied the as a chocolatey candy - Charleston Chew - were compared equation: %POP<44.025-0.733*%SOS, which was against pure cocoa butter. The chocolates used were determined by the European Commission Joint Research selected to cover a range of Cocoa content and purity. We Centre, which can detect around 2% CBE usage in CB speci cally chose to use Hershey’s Mr. Goodbar and content, or approximately 0.4% CBE content in chocolate. Charleston Chews because they listed the use of vegetable The chocolate products we tested all agreed with the oils in their ingredients list. As you can see in the expected results: All of the dark chocolate products we chromatograms, the products that market themselves as tested passed this speci cation, as well as Hershey’s Milk pure chocolate have similar chromatograms in comparison Chocolate. The two products which had a higher %POP to the pure CB. than is allowable, Mr. Goodbar and Charleston Chew, We used an MS library that was provided to us by Dr. John were selected speci cally for the inclusion of vegetable Carney and Mona Koutchekinia to identify the types of oils. It may be informative to further test the accuracy of TAGs contained in the chocolates using the spectral this testing method by adulterating cocoa butter with information captured in the Q3 scans. A minimum known quantities of CBEs. The data has been summarized similarity of 70 was required for a result to be considered a in Table 1.

Table 1: Percentage of the major TAGs in CB in various chocolate products

%POP needs to Product %POP %POS %SOS be less than Cocoa Butter 23.7% 46.9% 29.5% 43.8 Lindt 85% Cocoa 16.9% 46.4% 36.6% 43.8 TCHO 70% from Ghana 17.8% 46.1% 36.1% 43.8 TCHO 65% from Ecuador 20.9% 46.2% 32.9% 43.8 Hershey's Special Dark 20.0% 47.1% 32.9% 43.8 Hershey's Milk Chocolate 18.6% 46.6% 34.8% 43.8 Hershey's Mr Goodbar 44.8% 21.1% 34.1% 43.8 Charleston Chew 100.0% 0.0% 0.0% 44.0

3 An LCMS Method for the Detection of Cocoa Butter Substitutes, Replacers, and Equivalents in Commercial Chocolate-like Products

(x100,000,000) 1:TIC(+) Cocoa Butter.lcd 1:TIC(+) Lindt 85% Cocoa.lcd 1.5 1:TIC(+) TCHO 70% from Ghana.lcd POS 1:TIC(+) TCHO 65% from Ecuador.lcd 1.4 1:TIC(+) Hershey's Special Dark 45% Cacao.lcd 1:TIC(+) Hershey's Milk Chocolate.lcd SOS* 1.3

1.2 POP

1.1

1.0 OO S PLP 0.9 OO P

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 min

(x10,000,000) 9.0 1:TIC(+) Cocoa Butter.lcd 1:TIC(+) Hershey's Mr. Goodbar.lcd

8.5 1:TIC(+) Charleston Chew.lcd POS

8.0

7.5

7.0 SOS*

6.5

6.0

5.5 POP

5.0

4.5

4.0

3.5

3.0 OO S PLP 2.5 OO P

2.0

1.5

1.0

0.5

0.0

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 min

Figure 1. Chromatograms of the various chocolate products analyzed versus pure cocoa butter

4 An LCMS Method for the Detection of Cocoa Butter Substitutes, Replacers, and Equivalents in Commercial Chocolate-like Products

Conclusions We have developed a 17 minute method for the rapid determination of CBE usage in chocolate products by using a UHPLC column and Q3 ion scans to analyze samples and then matching spectral information with an MS library of ion ratios for identifying TAGs. Further studies could add a calibration curve to enable quanti cation of TAGs. This method should also provide a base method which can be modi ed to support TAG analysis in other product types.

References Co ED, Koutchekinia M, Carney J et al. Matching the Functionality of Single-Cell Algal Oils with Different Molecular Compositions. 2014. Buchgraber M and Anklam E. Validation of a Method for the Detection of Cocoa Butter Equivalents in Cocoa Butter and Plain Chocolate. 2003.

Acknowledgements Dr. John Carney and Mona Koutchekinia for the invaluable information they provided.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1471E

Highly Sensitive and Robust LC/MS/MS Method for Quantitative Analysis of Arti cial Sweeteners in Beverages

ASMS 2014 MP351

Jie Xing1, Wantung Liw1, Zhi Wei Edwin Ting1, Yin Ling Chew*2 & Zhaoqi Zhan1 1 Customer Support Centre, Shimadzu (Asia Paci c) Pte Ltd, 79 Science Park Drive, #02-01/08, SINTECH IV, Singapore Science Park 1, Singapore 118264 2 Department of Chemistry, Faculty of Science, National University of Singapore, 21 Lower Kent Ridge Road, Singapore 119077, *Student Highly Sensitive and Robust LC/MS/MS Method for Quantitative Analysis of Arti cial Sweeteners in Beverages

Introduction Arti cial sweeteners described as intense, low-calorie and sweeteners are found as emerging environmental non-nutritive are widely used as sugar substitutes in contaminants in surface water and waste water [3]. beverages and foods to satisfy consumers’ desire to sweet Initially, HPLC analysis method with ELSD detection was taste while concerning about obesity and diabetes. As adopted, because many arti cial sweeteners are non-UV synthetic additives in food, the use of arti cial sweeteners absorption compounds [2]. Recently, LC/MS/MS methods must be approved by authority for health and safety have been developed and used for identi cation and concerns. For example, Aspartame, Acesulfame-K, quantitation of arti cial sweeteners in food and beverages Saccharin, Sucralose and Neotame are the FDA approved as well as water for its high sensitivity and selectivity [3, 4]. arti cial sweeteners on the US market. However, there are Here we report a high sensitivity LC/MS/MS method for also many other arti cial sweeteners allowed to use in EU identi cation and quantitation of ten arti cial sweeteners and many other countries (Table 2), but not in the US. In (Table 2) in beverage samples. An ultra-small injection this regard, analysis of arti cial sweeteners in beverages volume was adopted in this study to develop a very robust and foods has become essential due to the relevant LC/MS/MS method suitable for direct injection of beverage regulations in protection of consumers’ bene ts and safety samples without any sample pre-treatment except dilution concerns in many countries [1, 2]. Recently, arti cial with solvent.

Experimental Ten arti cial sweeteners of high purity as listed in Table 2 except dilution with the diluent prior to injection into were obtained from chemicals suppliers. Stock standard LCMS-8040 (Shimadzu Corporation, Japan), a triple solutions and a set of calibrants were prepared from the quadrupole LC/MS/MS system. The front-end LC system chemicals with methanol/water (50/50) solvent as the connected to the LCMS-8040 is a high pressure binary diluent. Three brand soft-drinks and a mouthwash bought gradient Nexera UHPLC. The details of analytical conditions from local supermarket were used as testing samples in of LC/MS/MS method are shown in Table 1. this study. The samples were not pretreated by any means

Table 1: LC/MC/MS analytical conditions of arti cial sweeteners on LCMS-8040

Column Synergi, Polar-RP C18 (100 x 2 mm, 2.5µm ) Flow Rate 0.25 mL/min A: water with 0.1% Formic acid - 0.03% TA Mobile Phase B: MeOH with 0.1% FA - 0.03% Trimethylamine Gradient program B: 10% (0.01 to 0.5 min) → 95% (8 to 9 min) → 10% (9.01 to 11min) MS mode ESI, MRM, positive-negative switching ESI condition Nebulizing gas: 3L/min, Drying gas: 15L/min, Heating block: 400ºC, DL: 250ºC Inj. Vol. 0.1uL, 0.5uL, 1uL, 5uL and 10uL

2 Highly Sensitive and Robust LC/MS/MS Method for Quantitative Analysis of Arti cial Sweeteners in Beverages

Results and Discussion Method development First, precursor selection and MRM optimization of the ten in Table 2. We also investigated the performance of the sweeteners studied was carried out using an automated LC/MS/MS method established by employing very small MRM optimization program of the LabSolutions. Six injection volumes (0.1, 0.5, 1 and 5 uL). This is because compounds were ionized in negative mode and four in actual beverages usually contain very high contents of positive mode as shown in Table2. For each compound, sweeteners (>>1ppm) to MS detection. Analysts normally two optimized MRM transitions were selected and used, dilute the samples before injection into LC/MS/MS. An with the first one for quantitation and the second one for alternative way is to inject a very small volume of samples confirmation. even without dilution. Figs 2 & 3 show a chromatogram The ten compounds were well-separated as sharp peaks and calibration curves established with 0.1uL injection, between 2 min and 8.2 min as shown in Figure 1. Linear which demonstrates the feasibility of an ultra-small calibration curves of wide concentration ranges were injection volume combined with high sensitivity LC/MS/MS. established with mixed standards in diluent as summarized

Table 2: Arti cial Sweeteners, MRM transitions and calibration curves on LCMS-8040

MRM parameter RT & Calibration Curve4 Cat1 Compd. & Abbr. Name Trans. (m/z) Pola. (+/-) Q1 (V) CE (V) Q3 (V) RT (min) Conc. R. (ug/L) R2 161.9 >82.1 - 11 14 29 A2 Acesulfame K (Ace-K) 1.99 1 - 20000 0.9999 161.9 >78.0 - 11 32 28 178.3 >80.1 - 19 24 30 A5 Cyclamate (CYC)3 2.87 5 - 20000 0.9996 178.3 >79.0 - 12 27 10 181.9 >106.1 - 13 20 15 A3 Saccharin (SAC) 3.28 1 - 20000 0.9984 181.9 >42.1 - 13 36 13 441.0 >395.1 - 20 11 25 A4 Sucralose2 (SUC) 4.61 5 - 20000 0.9983 441.0 >359.1 - 20 15 23 295.1 >120.1 + -19 -25 -25 A1 Aspartame (ASP) 5.15 0.1 - 2000 0.9999 295.1 >180.1 + -19 -14 -20 379.3 >172.2 + -18 -23 -20 A6 Neotame (NEO) 7.51 0.05 - 1000 0.9998 379.3 >319.3 + -18 -18 -24 332.2 >129.1 + -23 -19 -26 B1 Alitame (ALI) 5.44 0.1 - 2000 0.9995 332.2 >187.1 + -23 -16 -21 181.1 >108.1 + -22 -25 -21 B3 Dulcin (DUL) 5.58 5 - 10000 0.999 181.1 >136.1 + -21 -18 -26 Neohespiridin 611.3 >303.1 - 30 38 30 B2 6.71 0.5 - 2000 0.9988 Dihydrochalcone (NHDC) 611.3 >125.3 - 30 47 20 821.5 >351.2 - 22 46 20 C1 Glycyrrhi-Zinate (GLY) 8.19 5 - 1000 0.9996 821.5 >193.2 - 22 52 19

1. A1~A6: US FDA, EU and others approval; B1~B3: only EU and other countries approval. C1: natural sweetener, info not available. 2. Sucralose precursor ion m/z 441.0 is formic acid adduct ion. 3. Sodium cyclamate known as “magic sugar” was initially banned in the US in 2000. FDA lifted the ban in 2013. 4. Injection volume: 10 uL

3 Highly Sensitive and Robust LC/MS/MS Method for Quantitative Analysis of Arti cial Sweeteners in Beverages

(x10,000)

5.0 Dulcin Neotame

4.0

3.0 Acesulfame K Sucralose Aspartame

2.0 Alitame

1.0 Saccharin Glycyrrhizic Cyclamate NHDC 0.0 0.0 2.5 5.0 7.5 10.0 min Figure 1: MRM Chromatogram of ten sweeteners by LC/MS/MS with 10uL injection: Asp & Ali 1ppb, Neo 0.5ppb, Dul, Gly, Ace-K, Sac, Suc and Cyc 10ppb, NHDC 1ppb.

Area (x100,000) Area (x10,000) Area (x10,000) Area (x100,000) Area(x100,000) 4.0 1.5 Ace-K CYC SAC 1.5 SUC ASP r2=0.9977 r2=0.9948 r2=0.9977 r2=0.9991 r2=0.9983 2.0 3.0 5.0 1.0 1.0 Area(x1,000) Area(x10,000) Area(x1,000) 2.0 5.0 Area(x1,000) Area(x1,000) 1.0 2.5 1.0 1.0 2.5 0.5 5.0 0.5 1.0 0.5 2.5

0.0 0.0 0.0 0.0 0 500 Conc. 0 500 Conc. 0.0 0.0 0.0 0.0 24 Conc. 0.0 0 Conc. 0.0 0 Conc. 0 10000 Conc. 0 10000 Conc. 0 10000 Conc. 0 10000 Conc. 0 1000 Conc.

Area (x100,000) Area (x100,000) Area (x100,000) Area (x10,000) Area (x10,000) 5.0 7.5 NEO 1.5 ALI DUL 4.0 NHDC GLY r2=0.9982 r2=0.9990 3.0 r2=0.9987 r2=0.9991 r2=0.9997 3.0 5.0 1.0 Area(x10,000) Area(x10,000) 2.0 Area(x10,000) Area(x1,000) 2.5 1.0 2.0 1.0 Area(x1,000) 2.5 1.0 1.0 0.5 0.5 0.5 2.5 1.0 1.0

0.0 0.0 0.0 0.0 0.0 0.0 25.0 Conc. 0 Conc. 0 500 Conc. 0.0 25.0 Conc. 0.0 0.0 0.0 0.0 0.0 0 500 Conc. 0 500 Conc. 0 1000 Conc. 0 10000 Conc. 0 1000 Conc. 0 10000 Conc. Figure 3: Calibration curves of arti cial sweeteners on LCMS-8040 with an ultra-small injection volume (0.1 uL) of same set of calibrants as shown in Table 2.

(x1,000) 4.5

4.0 Neotame 3.5 Acesulfame K 3.0 Dulcin 2.5

2.0 Alitame 1.5 Sucralose

1.0 Saccharin Glycyrrhizic Aspartame 0.5 Cyclamate NHDC 0.0 0.0 2.5 5.0 7.5 10.0 min Figure 2: MRM Chromatogram of ten sweeteners by LC/MS/MS with 0.1uL injection: Asp & Ali 0.1ppm, Neo 0.05ppm, Dul, Gly, Ace-K, Sac, Suc and Cyc 1ppm, NHDC 0.1ppm.

4 Highly Sensitive and Robust LC/MS/MS Method for Quantitative Analysis of Arti cial Sweeteners in Beverages

Method performance Table 3 summarizes the results of repeatability and matrix effect and interferences can be ignored for high sensitivity of the method with mixed standards. The method sensitivity LC/MS/MS analysis. The results indicate that the was not evaluated with beverage spiked samples. However, method with ultra-small injection volume exhibits good because beverage samples are normally diluted many times, linearity, repeatability and sensitivity.

Table 3: Repeatability and Sensitivity of LC/MS/MS method of arti cial sweeteners

Repeatability (peak area), 10uL Sensitivity (ug/L) Name Conc. (ug/L) RSD% Conc. (ug/L) RSD% LOQ/LOD (0.1 µL inj) LOQ/LOD (0.5 µL inj) LOQ/LOD 10 (µL inj) Ace-K 20 5.1 100 5.2 200 50 40 10 4.0 1.33 CYC 20 11.7 100 8.1 800 500 200 90 14 4.5 SAC 20 8.0 100 5.8 250 100 50 20 4.5 1.5 SUC 20 7.5 100 2.7 200 100 50 15 2.4 0.8 ASP 2 7.8 10 3.0 80 20 20 4 0.5 0.17 NEO 1 5.3 5 1.0 5 3 2 1 0.03 N.A. ALI 2 8.6 10 1.7 40 25 10 5 0.2 N.A. DUL 20 7.5 100 3.1 160 50 30 10 1.4 0.5 NHDC 2 9.2 10 4.6 100 25 40 6 0.5 0.18 GLY 20 8.2 100 5.4 400 150 15 5 5.0 1.8

Analysis of beverage samples The LC/MS/MS method established was applied for S4. The results are shown in Figure 4 and Table 4. It is screening and quantitation of the targeted sweeteners in interested to note that glycyrrizinate was found in the three brand beverages: S1, S2 and S3, and a mouthwash mouthwash.

Table 4: Screening and quantitation results for ten arti cial sweeteners in beverages and mouthwash (mg/L)

Arti cial Sweetener S1 S2 S3 S4 ASP 116.9 127.9 ND ND Ace-K 143.9 165.9 97.2 ND Saccharin ND ND ND 208.7 SUC 55.1 ND 183.4 ND GLY ND ND ND 449.3 Others ND ND ND ND 1. S2 was diluted 100 times, the rests were diluted 10 times. 1 uL injection. 2. ND = not detected.

5 Highly Sensitive and Robust LC/MS/MS Method for Quantitative Analysis of Arti cial Sweeteners in Beverages

(x1,000,000) (x100,000) 5.0 5.0 4.0 S1 S2 4.0 Aspartame 3.0 Aspartame 3.0 2.0 Acesulfame K (x10) Acesulfame K (x10) 2.0 Sucralose (x10)

1.0 1.0

0.0 0.0 0.0 2.5 5.0 7.5 10.0 min 0.0 2.5 5.0 7.5 10.0 min

(x100,000) (x100,000) 3.0 S3 1.5 S4 Saccharin Sucralose 2.0 Glycyrrhizic Acesulfame K 1.0

1.0 0.5

0.0 0.0 0.0 2.5 5.0 7.5 10.0 min 0.0 2.5 5.0 7.5 10.0 min

Figure 4: Screening and quantitation for 10 targeted arti cial sweeteners in beverage and mouthwash samples by LC/MS/MS with 1uL injection.

Conclusions A MRM-based LC/MS/MS method was developed and pre-treatment (except dilution). The method is expected to evaluated for screening and quantitation of ten arti cial be applicable to surface water and drinking water samples. sweeteners in beverages. This high sensitivity LC/MS/MS For wastewater and various foods, sample pre-treatment is method combined with small or ultra-small injection usually required. However, the advantages of the method volume (0.1~1.0 uL) was proven to be feasible and reliable in high sensitivity and ultra-small injection volume are in actual samples analysis of the targeted sweeteners in expected to enable it tolerates relatively simple sample beverages, achieving high throughput and free of sample pre-treatment procedures.

References 1. http://en.wikipedia.org/wiki/Sugar_substitute and EU directive 93/35/EC, 96/83/EC, 2003/115/EC, 2006/52/EC and 2009/163/EU. 2. Buchgraber and A. Wasik, Report EUR 22726 EN (2007). 3. F.T. Large, M. Scheurer and H.-J Brauch, Anal Bioanal Chem, 403: 2503-2518 (2012) 4. Ho-Soo Lim, Sung-Kwan Park, In-Shim Kwak, Hyung-Ll Kim, Jun-Hyun Sung, Mi-Youn Byun and So-Hee Kim, Food Sci, Biotechnol, 22(S):233-240 (2013)

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1480E

Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching

ASMS 2014 MP345

Stéphane MOREAU1 and Mikaël LEVI2 1 Shimadzu Europe, Albert-Hahn Strasse 6-10, Duisburg, Germany 2 Shimadzu France SAS, Le Luzard 2, Boulevard Salvador Allende, 77448 Marne la Vallée Cedex 2, France Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching

Introduction Mycotoxins are toxic metabolites produced by fungal Therefore, a sensitive method to assay mycotoxins in molds on food crops. For consumer food safety, quality complex matrices is mandatory. In order to ensure control of food and beverages has to assay such productivity of laboratory performing such assays, a unique contaminants. Depending on the potency of the mycotoxin rapid method able to measure as much mycotoxins as and the use of the food, the maximum allowed level is possible independently of the sample origin is also needed. de ned by legislation. Baby food is particularly critical. For In this study, we tested three kind of samples: baby milk example, European Commission has xed the maximum powder, milk thickening cereals (our, rice and tapioca) level of Aatoxin B1 and M1 to 0.1 and 0.025 µg/kg, and a vegetable puree mixed with cereals. respectively, in baby food or milk.

Materials and Methods Sample preparation Sample preparation was performed by homogenization Then column was washed with 3 mL of water followed by followed by solid phase extraction using specific cartridges 3 mL of water/acetonitrile (9/1 v/v). After drying, (Isolute® Myco, Biotage, Sweden) covering a large compounds were successively eluted with 2 mL of spectrum of mycotoxins. acetonitrile with 0.1% of formic acid and 2 mL of Sample (5g) was mixed with 20 mL of water/acetonitrile methanol. (1/1 v/v), sonicated for 5 min and agitated for 30 min at The eluate was evaporated under nitrogen flow at 35 ºC room temperature. After centrifugation at 3000 g for 10 until complete drying (Turbovap, Biotage, Sweden). min, the supernatant was diluted with water (1/4 v/v). The sample was reconstituted in 150 µL of a mixture of Columns (60mg/3 mL) were conditioned with 2 mL of water/methanol/acetonitrile 80/10/10 v/v with 0.1% of acetonitrile then 2 mL of water. 3 mL of the diluted formic acid. supernatant were loaded at the lowest possible flow rate.

LC-MS/MS analysis Extracts were analysed on a Nexera X2 (Shimadzu, Japan) carried out using selected reaction monitoring acquiring 2 UHPLC system and coupled to a triple quadrupole mass transitions for each compound. spectrometer (LCMS-8050, Shimadzu, Japan). Analysis was

2 Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching

Table 1 – LC conditions

Analytical column : Shimadzu GLC Mastro™ C18 150x2.1 mm 3µm Mobile phase : A = Water 2mM ammonium acetate and 0.5% acetic acid B = Methanol/Isopropanol 1/1 + 2mM ammonium acetate and 0.5% acetic acid Gradient : 2%B (0.0min), 10%B (0.01min), 55%B (3.0min), 80%B (7.0 -8.0min), 2%B (8.01min), Stop (11.0min) Column temperature : 50ºC Injection volume : 10 µL Flow rate : 0.4 mL/min

Table 2 – MS/MS conditions

Ionization mode : Heated ESI (+/-) Temperatures : HESI: 400ºC Desolvation line: 250ºC Heat block: 300ºC Gas ows : Nebulizing gas (N2): 2 L/min Heating gas (Air): 15 L/min Drying gas (N2): 5 L/min CID gas pressure : 270 kPa (Ar) Polarity switching time : 5 ms Pause time : 1 ms Dwell time : 6 to 62 ms depending on the number of concomitant transitions to ensure a minimum of 30 points per peak in a maximum loop time of 200 ms (including pause time and polarity switching)

3 Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching

Table 3 – MRM transitions

Name Ret. Time (min) MRM Quan MRM Qual 15-acetyldeoxynivalenol (15ADON) [M+H]+ 3.37 339 > 297.1 339 > 261 3-acetyldeoxynivalenol (3ADON) [M+H]+ 3.37 339 > 231.1 339 > 231.1 Aatoxine B1 (AFB1) [M+H]+ 3.78 312.6 > 284.9 312.6 > 240.9 Aatoxine B2 (AFB2) [M+H]+ 3.57 315.1 > 259 315.1 > 286.9 Aatoxine G1 (AFG1) [M+H]+ 3.46 329.1 > 242.9 329.1 > 199.9 Aatoxine G2 (AFG2) [M+H]+ 3.26 330.9 > 244.9 330.9 > 313.1 Aatoxine M1 (AFM1) [M+H]+ 3.30 329.1 > 273 329.1 > 229 Alternariol [M-H]- 4.78 257 > 214.9 257 > 213.1 Alternariol monomethyl ether [M-H]- 5.81 271.1 > 255.9 271.1 > 228 Beauvericin (BEA) [M+H]+ 8.03 784 > 244.1 784 > 262 Citrinin (CIT) [M+H]+ 4.16 251.3 > 233.1 251.3 > 205.1 D5-OTA (ISTD) 5.22 409.2 > 239.1 N/A Deepoxy-Deoxynivalenol (DOM-1) [M-H]- 3.02 279.2 > 249.3 279.2 > 178.4 Deoxynivalenol (DON) [M-CH3COO]- 2.61 355.3 > 295.2 355.3 > 265.1 Deoxynivalenol 3-Glucoside (D3G) [M+CH3COO]- 2.45 517.5 > 457.1 517.5 > 427.1 Deoxynivalenol 3-Glucoside (D3G) [M+CH3COO]- 2.45 517.5 > 457.1 517.5 > 427.1 Diacetoxyscirpenol (DAS) [M+NH4]+ 1.20 384 > 283.3 384 > 343 Enniatin A (ENN A) [M+H]+ 8.51 699.2 > 682.2 699.3 > 210 Enniatin A1 (ENN A1) [M+H]+ 8.22 685.3 > 668.3 685.3 > 210.1 Enniatin B (ENN B) [M+H]+ 7.57 657 > 640.4 657 > 195.9 Enniatin B1 (ENN B1) [M+H]+ 7.92 671.2 > 654.2 671.2 > 196 Fumagillin (FUM) [M+H]+ 6.16 459.2 > 131.1 459.2 > 338.7 Fumonisine B1 (FB1) [M+H]+ 4.10 722.1 > 334.2 722.1 > 352.2 Fumonisine B2 (FB2) [M+H]+ 4.71 706.2 > 336.3 706.2 > 318.1 Fumonisine B3 4.38 706.2 > 336.2 706.2 > 688.1 Fusarenone-X (FUS-X) [M+H]+ 2.84 355.1 > 247 355.1 > 175 HT2 Toxin [M+Na]+ 4.58 446.9 > 344.9 446.9 > 285 Moniliformin (MON) [M-H]- 1.16 97.2 > 40.9 N/A Neosolaniol (NEO) [M+NH4]+ 2.90 400.2 > 215 400.2 > 185 Nivalenol (NIV) [M+CH3COO]- 2.41 371.2 > 280.9 371.2 > 311.1 Ochratoxin A (OTA) [M+H]+ 5.53 404.2 > 239 404.2 > 358.1 Ochratoxin B (OTB) [M+H]+ 4.83 370.2 > 205.1 370.2 > 187 Patulin (PAT) [M-H]- 2.35 153 > 81.2 153 > 53 Sterigmatocystin (M+H]+ 5.60 325.3 > 310 325.3 > 281.1 T2 Tetraol [M+CH3COO]- 1.64 356.8 > 297.1 356.8 > 59.1 T2 Toxin [M+NH4]+ 4.94 484.2 > 215 484.2 > 305 Tentoxin [M-H]- 4.77 413.1 > 140.9 413.1 > 271.1 Tenuazonic acid (TEN) [M-H]- 4.51 196.1 > 138.8 196.1 > 112 Wortmannin (M-H) 3.95 426.9 > 384 426.9 > 282.1 Zearalanol (alpha) (ZANOL) [M-H]- 5.17 321.3 > 277.2 321.3 > 303.2 Zearalanol (beta) (ZANOL) [M-H]- 4.85 321.3 > 277.2 321.3 > 303.1 Zearalanone (ZOAN) [M-H]- 5.43 319 > 275.1 319 > 301.1 Zearalenol (alpha) (ZENOL) [M-H]- 5.25 319.2 > 275.2 319.2 > 160.1 Zearalenol (beta) (ZENOL) [M-H]- 4.94 319.2 > 275.2 319.2 > 160.1 Zearalenone (ZON) [M-H]- 5.52 316.8 > 174.9 316.8 > 131.1

4 Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching

Results and discussion Method development LC conditions were transferred from a previously described Small adjustments in the mobile phase and in the gradient method (Tamura et al., Poster TP-739, 61st ASMS). In program were made to handle more mycotoxins, especially particularly, the column was chosen to provide very good the isobaric ones. These modifications are reported in the peak shape for chelating compounds like fumonisins Table 1. thanks to its inner PEEK lining.

Stainless steel Body Polymer lining

Polymer frit Stationary phase

Figure 1 – Structure of the Mastro™ column

Also, autosampler rinsing conditions were kept to ensure time consuming. Therefore, new assistant software carry-over minimisation of some difficult compounds. (Interface Setting Support) was used to generate all Electrospray parameters (gas flows and temperatures) were possible combinations and generate a rational batch cautiously optimized to find the optimal combination for analysis. Optimal combination was found in the most critical mycotoxins (aflatoxins). Since these chromatographic conditions. The difference observed parameters act in a synergistic way, a factorial design between optimum and default or worst parameters was of experiment is needed to find it. Manually testing all 200 and 350%, respectively. combinations in the chromatographic conditions is very

Figure 2 – Parameters selection view in the Interface Setting Support Software

5 Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching

Results Extraction and ionisation recovery for aflatoxins was 4 showed that the total recovery was quite acceptable to measured in the three matrices by comparing peak areas of ensure accurate quantification. Results from other matrices the raw sample extract to extract spiked at 50 ppb after or were not significatively different. before extraction and to standard solution. Results in table

Table 4 – Extraction and ionisation recoveries in puree

AFB1 AFB2 AFG1 AFG2 AFM1 Extraction recovery 101% 109% 104% 114% 118% Ionisation recovery 49% 90% 96% 106% 91% Total recovery 49% 98% 100% 121% 108%

Repeatability was evaluated at low level for aflatoxins. Figure 3 shows an overlaid chromatogram (n=4) for aflatoxins.

(x10,000) 2.50 2.25 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0.00 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 min Figure 3 – Chromatogram of aatoxins at 0.1 ppb in milk thickening cereals

12000000 11500000 11000000 10500000 10000000 9500000 9000000 8500000 8000000 7500000 7000000 6500000 6000000 5500000 5000000 4500000 4000000 3500000 3000000 2500000 2000000 1500000 1000000 500000 0 -500000 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 min

Figure 4 – Chromatogram of the 45 mycotoxins in standard at 50 ppb (2 ppb for aatoxins and ochratoxines)

6 Highly sensitive and rapid simultaneous method for 45 mycotoxins in baby food samples by HPLC-MS/MS using fast polarity switching

Conclusion • A very sensitive method for multiple mycotoxines was set up to ensure low LOQ in baby food sample, • Thanks to high speed polarity switching, a high number of mycotoxines can be assayed using the same method in a short time, • The extraction method demonstrate good recoveries to ensure accurate quanti cation.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1461E

High Sensitivity Analysis of Acrylamide in Potato Chips by LC/MS/MS with Modi ed QuEChERS Sample Pre-treatment Procedure

ASMS 2014 MP342

Zhi Wei Edwin Ting1; Yin Ling Chew*2; Jing Cheng Ng*2; Jie Xing1; Zhaoqi Zhan1 1 Shimadzu (Asia Paci c) Pte Ltd, Singapore, SINGAPORE; 2 Department of Chemistry, Faculty of Science, National University of Singapore, 21 Lower Kent Ridge Road, Singapore119077, *Student High Sensitivity Analysis of Acrylamide in Potato Chips by LC/MS/MS with Modi ed QuEChERS Sample Pre-treatment Procedure

Introduction Acrylamide was found to form in fried foods like present a novel LC/MS/MS method for quantitative potato-chips via the so-called Maillard reaction of determination of acrylamide in potato chips with using a asparagine and glucose (reducing sugar) at higher modi ed QuEChERS procedure for sample extraction and temperature (120ºC) in 2002 [1,2]. The health risk of clean-up, achieving high sensitivity and high recovery. A acrylamide present in many processing foods became a small sample injection volume (1uL) was adopted purposely concern immediately, because it is known that the to reduce the potential contamination of samples to the compound is a neurotoxin and a potential carcinogen to interface of MS system, so as to enhance the operation humans [3]. Various analytical methods, mainly LC/MS/MS stability in a laboratory handling food samples with high and GC/MS based methods, were established and used in matrix contents. analysis of acrylamide in foods in recent years [4]. We

Experimental Acrylamide and isotope labelled acrylamide-d3 (as internal Method development and performance evaluation were standard) were obtained from Sigma-Aldrich. The carried out using spiked acrylamide samples in the QuEChERS kits were obtained from RESTEK. A modi ed extracted potato chip matrix. A LCMS-8040 triple procedure of the QuEChERS was optimized and used in the quadrupole LC/MS/MS (Shimadzu Corporation, Japan) was sample extraction of acrylamide (Q-sep Q100 packet, used in this work. A polar-C18 column of 2.5µm particle original unbuffered) in potato chips and clean-up of matrix size was used for fast UHPLC separation with a gradient with d-SPE tube (Q-sep Q250, AOAC 2007.01). Acrylamide elution method. Table 1 shows the details of analytical and acrylamide-d3 (IS) stock solutions and diluted conditions on LCMS-8040 system,. calibrants were prepared using water as the solvent.

Table 1: LC/MS/MS analytical conditions of LCMS-8040 for acrylamide

LC condition MS Interface condition

Phenomenex Synergi 2.5u Polar-Rp 100A Interface ESI Column (100 x 2.00mm) MS mode Positive, MRM, 2 transitions each compound Flow Rate 0.2 mL/min Block Temp. 400ºC A: water DL Temp. 200ºC Mobile Phase B: 0.1% formic acid in Methanol CID Gas Ar (230kPa)

Gradient elution, B%: 1% (0 to 1 min) → Nebulizing Gas Flow N2, 1.5L/min Elution Mode 80% (3 to 4.5 min) → 1% (5.5 to 10min) Drying Gas Flow N2, 10.0L/min Oven Temp. 40ºC Injection Vol. 1.0 µL

2 High Sensitivity Analysis of Acrylamide in Potato Chips by LC/MS/MS with Modi ed QuEChERS Sample Pre-treatment Procedure

Results and Discussion

QuEChERS Sample Pre-treatment [1] Weigh 2.0g of sample in a 50mL centrifuge tube The details of a modified QuEChERS procedure for potato Add 5mL hexane, 10mL water chips are shown in Figure 1. Hexane was used to defat and 10mL acetonitrile [2] Vortex and shake vigorously for 1min potato chips, removing oils and non-polar components. In Add Q-sep Q100Packet salt

the extraction step with Q-sep Q100Packet extraction salt Additional 4g MgSO4 (anhydrous) [3] Vortex and shake vigorously for 5min (contain 4g MgSO4 & 0.5g NaCl), additional 4g of MgSO4 was added to absorb the water completely (aqueous phase [4] Discard the hexane (top layer) disappeared). Acrylamide is soluble in both aqueous and [5] Transfer the solution into a 20mL volumetric ask organic phases. With this modification, high recovery of wash extraction salt with ACN acrylamide was obtained. It is believed that this is because in the centrifuge tube complete removal of water in the mixed extract solution [6] Combine the washing solution into the volumetric ask (above) could promote acrylamide transferring into the organic phase. Dispersive SPE tube was used as PSA to remove [7] Transfer 1mL of solution into the 2mL Q-sep Q250 organic acids which may decompose acrylamide in the QuEChERS dSPE tube process. [8] Vortex and centrifuge for 10min at 13000rpm

[9] Transfer 500uL extract to a 1.5mL vial Evaporate to dryness by N2 blow [10] Reconstitute with 250uL of Milli Q water

Method Development [11] Analyze by Shimadzu LCMS-8040 As acrylamide is a more polar compound, a Polar-RP type column was selected. Isotope labeled internal Figure 1: Flow chart of sample pre-treatment with modi ed QuEChERS.

standard (acrylamide-d3) was used to compensate the variation of acrylamide peak area caused by system Table 2: MRM transitions and CID voltages fluctuation and inconsistency in sample preparation of CID Voltage (V) different batches. Name MRM (m/z) Q1 CE Q3 The precursor ions of acrylamide and acrylamide-d3 (IS) were their protonated ions (m/z72.1 and m/z75.1). The 75.1 > 58.0* -29 -15 -22 Acrylamide-d3 MRM optimization was carried out using an automated 75.1 > 30.1 -29 -24 -30 program of the LabSolutions workstation, which could 72.1 > 55.0* -17 -16 -24 Acrylamide generate a list of all MRM transitions with optimized CID 72.1 > 27.1 -17 -22 -30 voltages accurate to (+/-) 1 volt in minutes. Two MRM *MRM transition as quanti er

transitions of acrylamide and acryl-amide-d3 were selected as quantifier and confirmation ion as shown in Table 3: Acrylamide spiked samples and peak Table 2. area ratios of measured by IS method

The obtained extract solution of potato chips was used Acrylamide IS post- Conc. Ratio Area Ratio as “blank” and also matrix for preparation of post-spiked spiked Calculated measured* post-spiked calibrants for establishment of calibration L0, Blank 0 0.6033 curve with IS (acrylamide-d ). To obtain reliable results, 3 L1, 1ppb 0.02 0.6120 the blank and each post-spiked calibrant as shown in Table 3 were injected three times and the average peak L2, 5ppb 0.10 0.6786 area ratios were calculated and used. L3, 10ppb 50ppb 0.20 0.8239 L4, 50ppb 1.00 1.7686 L5 100ppb 2.00 2.8196 L6, 500ppb 10.00 11.8330 *= Area (acrylamide) / Area (IS)

3 High Sensitivity Analysis of Acrylamide in Potato Chips by LC/MS/MS with Modi ed QuEChERS Sample Pre-treatment Procedure

It was found that the potato chips used as “blank” in this 0.594 at zero spiked concentration (L0) as shown in Figure study was not free of acrylamide. Instead, it contained 27.1 2. Good linearity with correlation coefficient (R2) greater ng/mL of acrylamide in the extract solution. A linear than 0.9999 across the range of 1.0 ng/mL– 500.0 ng/mL calibration curve was established with an intercept of was obtained.

Area Ratio (x10) 1.25 2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 1ppb 01a.lcd 2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb 01a.lcd Y= 1.1239X + 0.594168 2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 10ppb 01a.lcd R2 = 0.9999 2:Acrylamide150000 72.10>55.00(+) CE: -15.0 Acrylamide 50ppb 01a.lcd 1.00 300000 2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 100ppb 01a.lcd 2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 500ppb 01a.lcd

0.75 Area Ratio 100000 200000 1.0 0.50 50000 0.5 100000 0.25 0.0 0 0.00 Conc. Ratio 0 0.00 1.5 2.0 2.5 min 0.0 2.5 5.0 7.5 Conc. Ratio 2.5 5.0 7.5 min

Figure 2: Calibration curve (left) and MRM peaks (right) of acrylamide spiked into potato chips matrix, 1-500 ppb with 50 ppb IS added.

Method Performance Evaluation It was hard to estimate the LOD and LOQ of the analytical Figure 3. The peak area %RSD of acrylamide and IS were method due to the presence of acrylamide (27.1 ng/mL) in below 4%. the “blank” (extract of potato chips). However, as reported The matrix effect (M.E.), recovery efficiency (R.E.) and also by other researchers, it is difficult to obtain potato process efficiency (P.E.) of the method were determined chips free of acrylamide actually. To obtain actual with a duplicate set of spiked samples of 50 ng/mL level concentration, it is normally subtracting the background except for the non-spiked sample. The chromatograms of content of acrylamide of a “blank” sample used as “set 2”, i.e., non-spiked extract, pre-spiked, post-spiked reference from a measurement of testing sample. The and the standard in neat solution are shown in Figure 4. same way was used to estimate actual S/N value in this Noted that, the existing acrylamide in the extract of the work. As a result, the LOD and LOD of acrylamide of this potato chips used as reference was accounted for 27.1 method with 1ul injection volume were estimated to be ng/mL, corresponding to 135.5 ng per gram of potato lower than 1ng/mL and 3ng/mL, respectively. This is chips. The average R.E, M.E and P.E of the method for consistence with the results estimated with the IS. extraction and analysis of acrylamide obtained are shown The repeatability of the method was evaluated with L2 and in Table 6. L4 spiked samples. The results are shown in Table 4 and

Table 4: Repeatability Test Results (n=6)

spiked Sample Compound Conc. (ng/mL) %RSD Acrylamide 5 3.5 L2 Acrylamide-d3 50 3.8 Acrylamide 50 3.9 L4 Acrylamide-d3 50 3.6

4 High Sensitivity Analysis of Acrylamide in Potato Chips by LC/MS/MS with Modi ed QuEChERS Sample Pre-treatment Procedure

2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R01.lcd 2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R02.lcd 2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R03.lcd 30000 2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R04.lcd 2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R05.lcd 2:Acrylamide 72.10>55.00(+) CE: -15.0 Acrylamide 5ppb R06.lcd

20000

10000

1.00 1.5 2.0 2.5

2.5 5.0 7.5 min

Figure 3: Overlay MRM chromatograms of 5 ng/mL acrylamide spiked in potato chips extract (total: 27.1+5 = 32.1 ng/mL)

50000 (a) Extract 50000 (b) standard 50000 (c) post-spiked 50000 (d) pre-spiked (non-spiked) 40000 40000 40000 40000

30000 30000 30000 30000

20000 20000 20000 20000

10000 10000 10000 10000

0 0 0 0 1.5 2.0 2.5 1.5 2.0 2.5 1.5 2.0 2.5 1.5 2.0 2.5

Figure 4: The MRM peaks of acrylamide detected in “blank” extract of potato chips (a), neat standard of 50ppb (b) post-spiked sample of 50ppb (c) and pre-spiked sample of 50ppb.

Table 6: Method evaluation of at 50.0ng/mL concentration in potato chips matrix

Parameter Set 1 Set 2 Average R.E. 104.7% 112.0% 108.4% M.E. 96.5% 84.6% 90.5% P.E. 100.8% 94.5% 97.6%

Conclusions Acrylamide is formed unavoidably in starch-rich food in related to the outstanding performance of the LC/MS/MS cooking and processing at high temperature like potato used which features ultra fast mass spectrometry (UFMS) chips, French fries, cereals and roasted coffee etc. The technology. The high sensitivity of the method allows the analysis method established in this work can be used to analysis to be performed with a very small injection volume monitor the levels of acrylamide in processing food (1µL or below), which would be a great advantage in accurately and reliably. The QuEChERS method is proven to running heavily food samples with high matrix contents be fast and effective in extraction of acrylamide from and strong matrix effects. Maintenance of the interface of potato chips. The excellent performance of the method in a mass spectrometer could also be reduced signi cantly. terms of sensitivity, linearity, repeatability and recovery are

5 High Sensitivity Analysis of Acrylamide in Potato Chips by LC/MS/MS with Modi ed QuEChERS Sample Pre-treatment Procedure

References [1] Swedish National Food Administration. “Information about acrylamide in food, 24 April 2002”, http://www.slv.se [2] Mottram, D.S., & Wedzicha, B.L., Nature, 419 (2002), 448-449. [3] Ahn, J.S., Castle, J., Clarke, D.B., Lloyd, A.S., Philo, M.R., & Speck, D.R., Food Additives and Contaminants, 19 (2002), 1116-1124. [4] Mastovska, K., & Lehotary, S.J., J. Food Chem., 54 (2006), 7001-7998.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1472E

Determination of Benzimidazole Residues in Animal Tissue by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry

ASMS 2014 TP 281

Yin Huo, Jinting Yao, Changkun Li, Taohong Huang, Shin-ichi Kawano, Yuki Hashi Shimadzu Global COE, Shimadzu (China) Co., Ltd., China Determination of Benzimidazole Residues in Animal Tissue by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry

Introduction Benzimidazoles are broad-spectrum, high efciency, low This poster employed a liquid chromatography-electrospray toxicity anthelmintic. Because some benzimidazoles and ionization tandem mass spectrometry (LC-ESI-MS/MS) their metabolites showed teratogenic and mutagenic method to determinate 16 benzimidazole residues in effects in animal and target animal safety evaluation animal tissue. The method is simple, rapid and high experiment, many countries have already put sensitivity, which meets the requirements for the analysis benzimidazoles and metabolites as the monitoring object. of veterinary drug residue in animal tissue.

Method Sample Preparation (1) Animal tissue samples were extracted with ethyl acetate-50% potassium hydroxide-1% BHT (2) The samples were treated with n-hexane for defatting and further cleaned-up on MCX solid phase (SPE) cartridge. (3) The separation of benzimidazoles and their metabolites was performed on LC-MS/MS instrument.

LC/MS/MS Analysis The analysis was performed on a Shimadzu Nexera UHPLC instrument (Kyoto, Japan) equipped with LC-30AD pumps, a CTO-30A column oven, a DGU-30A5 degasser, and an SIL-30AC autosampler. The separation was carried out on a Shim-pack XR-ODS III (2.0 mmI.D. x 50 mmL., 1.6 μm, Shimadzu) with the column temperature at 30 ºC. A triple quadrupole mass spectrometer (Shimadzu LCMS-8040, Kyoto, Japan) was connected to the UHPLC instrument via an ESI interface.

Analytical Conditions

UHPLC (Nexera system)

Column : Shim-pack XR-ODS III (2.0 mmI.D. x 50 mmL., 1.6 μm) Mobile phase A : water with 0.1% formic acid Mobile phase B : acetonitrile Gradient program : as in Table 1 Flow rate : 0.4 mL/min Column temperature : 30 ºC Injection volume : 20 µL

Table 1 Time program

Time (min) Module Command Value 0.01 Pumps Pump B Conc. 5 3.50 Pumps Pump B Conc. 80 4.00 Pumps Pump B Conc. 80 4.01 Pumps Pump B Conc. 5 6.00 Controller Stop

2 Determination of Benzimidazole Residues in Animal Tissue by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry

MS/MS (LCMS-8040 triple quadrupole mass spectrometer)

Ionization : ESI Polarity : Positive Ionization voltage : +4.5 kV Nebulizing gas ow : 3.0 L/min Heating gas pressure : 15.0 L/min DL temperature : 200 ºC Heat block temperature : 350 ºC Mode : MRM

Table 2 MRM parameters of 16 benzimidazoles (*: for quantitation)

Precursor Product Dwell Time Q1 Pre Bias Q3 Pre Bias Compound CE (V) m/z m/z (ms) (V) (V) 268.05* 50 -15.0 -21.0 -18.0 Fenbendazole 300.10 159.05 50 -15.0 -36.0 -30.0 240.10* 10 -14.0 -12.0 -17.0 Albendazole sulfoxide 282.00 208.05 10 -14.0 -23.0 -22.0 175.10* 10 -30.0 -24.0 -18.0 Thiabendazole 202.00 131.15 10 -30.0 -31.0 -25.0 191.05* 50 -30.0 -23.0 -13.0 Thiabendazole-5-hydroxy 218.00 147.10 50 -30.0 -32.0 -27.0 159.15* 20 -11.0 -34.0 -30.0 Oxfendazole 316.20 191.15 20 -11.0 -22.0 -20.0 234.10* 8 -30.0 -19.0 -25.0 Albendazole 266.30 191.10 8 -30.0 -33.0 -20.0 133.20* 50 -15.0 -27.0 -24.0 Albendazole -2-aminosulfone 240.30 198.10 50 -15.0 -18.0 -21.0 159.10* 20 -13.0 -37.0 -30.0 Albendazole sulfone 298.30 224.05 20 -13.0 -27.0 -23.0 264.15* 10 -13.0 -21.0 -27.0 Mebendazole 296.30 105.25 10 -13.0 -35.0 -19.0 105.20* 10 -15.0 -26.0 -20.0 Mebendazole-amine 238.30 133.20 10 -15.0 -36.0 -25.0 266.10* 10 -30.0 -22.0 -18.0 5-Hydroxymebendazole 298.30 160.15 10 -30.0 -35.0 -30.0 282.15* 10 -14.0 -22.0 -19.0 Flubendazole 314.30 123.15 10 -14.0 -35.0 -24.0 123.20* 10 -16.0 -26.0 -22.0 2-Aminoubendazole 256.30 95.20 10 -16.0 -41.0 -18.0 217.15* 5 -30.0 -28.0 -23.0 Cambendazole 303.20 261.10 5 -30.0 -17.0 -28.0 218.15* 5 -30.0 -17.0 -23.0 Oxibendazole 250.30 176.15 5 -30.0 -27.0 -18.0 300.10* 10 -15.0 -22.0 -21.0 Oxfendazole 332.20 159.05 10 -15.0 -39.0 -30.0

3 Determination of Benzimidazole Residues in Animal Tissue by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry

Results and Discussion A liquid chromatography-electrospray ionization tandem 16 drugs mixture are presented in Fig.1. The correlation mass spectrometry (LC-ESI-MS/MS) method has been coefcients for 16 drugs (0.5 – 50 ng/mL) were found to developed to identify and quantify trace levels of 16 0.9993~0.9999. MRM chromatograms of pork samples benzimidazoles residue (fenbendazole, albendazole and pork samples spiked with standards are shown in sulfoxide, thiabendazole, thiabendazole- 5-hydroxy, Fig.2. By analyzing 16 drugs at three levels including 0.5 oxfendazole, albendazole, albendazole-2-aminosulfone, ng/mL, 5 ng/mL, 50 ng/mL, excellent repeatability was albendazole sulfone, mebendazole, mebendazole-amine, demonstrated with the %RSD being better than 5% for all 5-hydroxymebendazole, ubendazole, the compound within six injections as shown in Table 3. 2-aminoubendazole, cambendazole, oxibendazole, Results of recovery test were good as shown in Table 4. oxfendazole) in animal tissue. The MRM chromatograms of

1:218.00>191.05(+)(10.00)

70000 2:240.30>133.20(+)(2.00) 9 3:202.00>175.10(+) 4:238.30>105.20(+)(3.00) 5:256.30>123.20(+)(2.00)

60000 6:298.30>266.10(+) 6 7:282.00>240.10(+) 8 8:303.20>217.15(+) 9:250.30>218.15(+) 1

10:316.20>159.15(+)(2.00) 2

50000 11:298.30>159.10(+)(2.00) 3

12:266.30>234.10(+) 4 13:296.30>264.15(+) 14:332.20>300.10(+)(2.00) 15:314.30>282.15(+) 40000 16:300.10>268.05(+) 5 7 12 30000 10 13 20000 11 16 14 10000 15

0

0.0 1.0 2.0 3.0 4.0 min

Figure 1 MRM chromatograms of standard 16 drugs (1 ng/mL) (1: Thiabendazole-5-hydroxy; 2: Albendazole -2-Aminosulfone; 3: Thiabendazole; 4: Mebendazole-amine; 5: 2-Aminoubendazole;6: 5-Hydroxymebendazole; 7: Albendazole Sulfoxide; 8: Cambendazole; 9: Oxibendazole; 10: Oxfendazole; 11: Albendazole sulfone; 12: Albendazole; 13: Mebendazole; 14: Oxfendazole; 15: Flubendazole; 16: Fenbendazole)

4 Determination of Benzimidazole Residues in Animal Tissue by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry

Table 3 Repeatability of 16 drugs in pork sample (n=6)

%RSD (0.5 ng/mL) %RSD (5.0 ng/mL) %RSD (50 ng/mL) Compound R.T. Area R.T. Area R.T. Area Fenbendazole 0.059 3.01 0.064 1.48 0.082 0.34 Albendazole Sulfoxide 0.202 4.26 0.084 2.86 0.153 0.92 Thiabendazole 0.272 4.52 0.180 2.85 0.132 2.58 Thiabendazole-5-hydroxy 0.526 4.44 0.249 3.91 0.158 1.41 Oxfendazole 0.121 2.71 0.089 2.91 0.105 0.97 Albendazole 0.073 2.07 0.090 1.29 0.099 0.92 Albendazole -2-Aminosulfone 0.392 4.36 0.162 2.08 0.177 1.72 Albendazole sulfone 0.103 3.95 0.126 0.63 0.113 0.64 Mebendazole 0.093 4.95 0.095 1.69 0.094 0.74 Mebendazole-amine 0.363 3.95 0.149 2.72 0.243 0.94 5-Hydroxymebendazole 0.091 2.31 0.099 0.79 0.140 1.17 Flubendazole 0.107 4.22 0.058 1.52 0.091 1.00 2-Aminoubendazole 0.339 4.30 0.177 2.53 0.166 1.43 Cambendazole 0.150 4.90 0.123 3.38 0.121 1.87 Oxibendazole 0.091 3.46 0.108 1.31 0.125 1.20 Oxfendazole 0.170 3.23 0.044 3.09 0.084 0.80

1:218.00>191.05(+) 1:218.00>191.05(+)(10.00) 9 2:240.30>133.20(+) 2:240.30>133.20(+) 3:202.00>175.10(+) 3:202.00>175.10(+)

50000 4:238.30>105.20(+) 50000 4:238.30>105.20(+) 6 5:256.30>123.20(+) 5:256.30>123.20(+) 6:298.30>266.10(+) 6:298.30>266.10(+) 7:282.00>240.10(+) 7:282.00>240.10(+) 8:303.20>217.15(+) 8:303.20>217.15(+) 9:250.30>218.15(+) 9:250.30>218.15(+) 10:316.20>159.15(+) 10:316.20>159.15(+) 40000 11:298.30>159.10(+) 40000 11:298.30>159.10(+) 8

12:266.30>234.10(+) 12:266.30>234.10(+) 1 13:296.30>264.15(+) 13:296.30>264.15(+) 3 14:332.20>300.10(+) 14:332.20>300.10(+) 2 15:314.30>282.15(+) 15:314.30>282.15(+) 16:300.10>268.05(+) 16:300.10>268.05(+) 12 30000 30000 4 5 7

20000 20000 13 16 10 11 15 10000 10000 14

0 0 0.0 1.0 2.0 3.0 4.0 min 0.0 1.0 2.0 3.0 4.0 min

Figure 2 MRM chromatograms of pork sample (left) and spiked pork sample (right) (1: Thiabendazole-5-hydroxy; 2: Albendazole -2-Aminosulfone; 3: Thiabendazole; 4: Mebendazole-amine; 5: 2-Aminoubendazole;6: 5-Hydroxymebendazole; 7: Albendazole Sulfoxide; 8: Cambendazole; 9: Oxibendazole; 10: Oxfendazole; 11: Albendazole sulfone; 12: Albendazole; 13: Mebendazole; 14: Oxfendazole; 15: Flubendazole; 16: Fenbendazole)

5 Determination of Benzimidazole Residues in Animal Tissue by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry

Table 4 Recovery of 16 drugs in pork sample

Sample Conc. Spike Conc. Measured Conc. Recovery Compound (µg/kg) (µg/kg) (µg/kg) (%) Fenbendazole N.D. 10.0 9.5 94.5 Albendazole Sulfoxide N.D. 10.0 8.1 80.9 Thiabendazole N.D. 10.0 9.8 98.2 Thiabendazole-5-hydroxy N.D. 10.0 10.0 99.8 Oxfendazole N.D. 10.0 11.4 113.8 Albendazole N.D. 10.0 9.6 96.3 Albendazole -2-Aminosulfone N.D. 10.0 9.6 96.1 Albendazole sulfone N.D. 10.0 11.8 118.5 Mebendazole N.D. 10.0 11.3 112.8 Mebendazole-amine N.D. 10.0 11.8 118.3 5-Hydroxymebendazole N.D. 10.0 9.8 97.8 Flubendazole N.D. 10.0 10.4 103.6 2-Aminoubendazole N.D. 10.0 9.3 92.6 Cambendazole N.D. 10.0 10.8 107.8 Oxibendazole N.D. 10.0 9.6 96.1 Oxfendazole N.D. 10.0 9.1 90.7

Conclusion The sensitive and reliable LC/MS/MS technique was 0.9993~0.9999. The LODs of the 16 benzimidazoles successfully applied for determination of 16 were 1 -2.2 µg/kg. The recoveries were in the range of benzimidazoles residue. The calibration curves of 16 80.9%~118.5% for pork samples, with relative standard benzimidazoles ranging from 0.5 to 50 ng/mL were deviations less than 5%. established and the correlation coefcients were

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1459E

High Sensitivity Quantitation Method of Dicyandiamide and Melamine in Milk Powders by Liquid Chromatography Tandem Mass Spectrometry

ASMS 2014 TP275

Zhi Wei Edwin Ting1, Jing Cheng Ng2*, Jie Xing1 & Zhaoqi Zhan1 1 Customer Support Centre, Shimadzu (Asia Paci c) Pte Ltd, 79 Science Park Drive, #02-01/08, SINTECH IV, Singapore Science Park 1, Singapore 118264 2 Department of Chemistry, Faculty of Science, National University of Singapore, 21 Lower Kent Ridge Road, Singapore 119077, *Student High Sensitivity Quantitation Method of Dicyandiamide and Melamine in Milk Powders by Liquid Chromatography Tandem Mass Spectrometry

Introduction Melamine was found to be used as a protein-rich also to be potentially used as protein-rich adulterants, adulterant rst in pet-food in 2007, and then in infant including amidinourea, biuret, cyromazine, dicyandiamide, formula in 2008 in China [1]. The outbreak of the triuret and urea [2]. Recently, low levels of dicyandiamide melamine scandal that killed many dogs and cats as well as (DCD) residues were found in milk products from New led to death of six infants and illness of many had caused Zealand [3]. Instead of addition directly as an adulterant, panic in publics and great concerns in food safety the trace DCD found in milk products was explained to be worldwide. Melamine was added into raw milk because of relating to the grass “contaminated by DCD”. its high nitrogen content (66%) and the limitation of the Dicyandiamide has been used to promote the growth of Kjeldahl method for determination of protein level pastures for cows grazing. We report here an LC/MS/MS indirectly by measuring the nitrogen content. In fact, in method for sensitive detection and quanti cation of both addition to melamine and its analogues (cyanuric acid etc), dicyandiamide (DCD) and melamine in infant milk powder a number of other nitrogen-rich compounds was reported samples.

Experimental High purity dicyandiamide (DCD) and melamine were modi cation as illustrated in Figure 1. The nal clear obtained from Sigma Aldrich. Amicon Ultra-4 (MWCO 5K) sample solution was injected into LC/MS/MS for analysis. centrifuge ltration tube (15 mL) obtained from Millipore Stock solutions of DCD and melamine were prepared in was used in sample pre-tretment. The milk powder sample pure water. was pre-treated according to a FDA method [1] with some

Table 1: Analytical conditions of DCD and melamine Weigh 2.0g of milk powder sample in milk powders on LCMS-8040 LC conditions Add 14mL of 2.5% formic acid Column Alltima HP HILIC 3µ, 150 x 2.10mm (1) Sonicate for 1hr Flow Rate 0.2 mL/min (2) Centrifuge at 6000rpm for 10min A: 0.1 % formic acid in H O/ACN (5:95 v/v) Mobile Phase 2 B: 20mM Ammonium Formate in H2O/ACN (50:50 v/v) Transfer 4mL of supernatant to Amicon Ultra-4 Gradient elution: 5% (0.01 to 3.0 min) → (MWCO 5K) centrifuge ltration tube (15mL) Elution Mode 95% (3.5 to 5.0 min) → 5% (5.5 to 9.0 min) Centrifuge at 7500rpm for 10min Oven Temperature 40ºC Collect clear ltrate Injection Volume 5 µL

To 50uL of ltrate added 950uL of ACN MS conditions Interface ESI Filter the ltrate by a 0.2um PTFE syringe lter MS mode Positive Block Temperature 400ºC Further 10x dilution with ACN DL Temperature 300ºC CID Gas Ar (230kPa) LC/MS/MS analysis Nebulizing Gas Flow N2, 2.0L/min

Fig 1: Sample pre-treatment workow Drying Gas Flow N2, 15.0L/min

2 High Sensitivity Quantitation Method of Dicyandiamide and Melamine in Milk Powders by Liquid Chromatography Tandem Mass Spectrometry

An LCMS-8040 triple quadrupole LC/MS/MS (Shimadzu melamine with a gradient program developed (Table 1). Corporation, Japan) was used in this work. The system is The details of the LC and MS conditions are shown in consisted of a high pressure binary gradient Nexera UHPLC Table 1. A set of calibrants (0.5, 1.0, 2.5, 5 and 10 ppb) coupled with a LCMS-8040 MS system. An Alltima HP was prepared from the stock solutions using of ACN/water HILIC column was used for separation of DCD and (90/10) as diluent.

Results and Discussion MRM optimization Table 2: MRM transitions and optimized parameters MRM optimization of DCD and melamine were performed Voltage (V) Name RT (min) Transition (m/z) using an automated MRM optimization program of the Q1 Pre Bias CE Q3 Pre Bias LabSolutions. The precursors were the protonated ions of 85.1 > 68.1 -15 -21 -26 DCD 2.55 DCD and melamine. Two optimized MRM transitions of 85.1 > 43.0 -15 -17 -17 each compound were selected and used for quantitation 127.1 > 85.1 -26 -20 -17 and confirmation. The MRM transitions and parameters are MEL 6.29 127.1 > 68.1 -26 -27 -26 shown in Table 2.

Method Development A LC/MS/MS method was developed for quantitation of calibration curves of DCD and melamine standard in neat DCD and melamine based on the MRM transitions in Table solutions and in milk matrix solutions (spiked). The linearity 2. Under the HILIC separation conditions (Table 1), DCD with correlation coefficient (R2) greater than 0.997 across and melamine eluted at 2.55 min and 6.29 min as sharp the calibration range of 0.5~10.0 ng/mL was obtained for peaks (see Figures 4 & 5). Figures 2 and 3 show the both compounds in both neat solution and matrix (spiked).

Area(x10,000) Area(x100,000)

3.5 7.5 DCD (85.1>68.1) Melamine (127.1>85.1) R2 = 0.997 3.0 R2 = 0.999

2.5 5.0 2.0

1.5

2.5 1.0

0.5

0.0 0.0 0.0 2.5 5.0 7.5 Conc. 0.0 2.5 5.0 7.5 Conc.

Figure 2: Calibration curves of DCD and melamine in neat solution

3 High Sensitivity Quantitation Method of Dicyandiamide and Melamine in Milk Powders by Liquid Chromatography Tandem Mass Spectrometry

Area(x10,000) Area(x100,000) 2.5 DCD (85.1>68.1) 5.0 Melamine (127.1>85.1) R2 = 0.998 2.0 R2 = 0.997 4.0

1.5 3.0

1.0 2.0

0.5 1.0

0.0 0.0 0.0 2.5 5.0 7.5 Conc. 0.0 2.5 5.0 7.5 Conc.

Figure 3: Calibration curves of DCD and melamine spiked in milk powder matrix

Performance Evaluation The repeatability of the method was evaluated at the levels injections of 0.5 ng/mL level with and without matrix. The of 0.5 ng/mL and 1.0 ng/mL. Figures 4 & 5 show the MRM peak area %RSD for the two analytes were lower than chromatograms of DCD and melamine of six consecutive 9.2% (see Table 3).

(x100) (x1,000) (x1,000) (x1,000) 4.5 1.00 6.0 DCD Melamine DCD 5.0 Melamine 4.0 (127.1>85.1) (85.1>68.1) (127.1>85.1) (85.1>68.1) 5.0 3.5 0.75 4.0 4.0 3.0 2.5 3.0 3.0 0.50 2.0

2.0 2.0 1.5 0.25 1.0 1.0 1.0 0.5 0.00 0.0 0.0 0.0 2.00 2.25 2.50 2.75 min 5.5 6.0 6.5 min 2.00 2.25 2.50 2.75 min 5.5 6.0 6.5 min

Figure 4: Overlapping of six MRM peaks of 0.5 ng/mL Figure 5: Overlapping of six MRM peaks of 0.5 ng/mL DCD and melamine in neat solution DCD and melamine in milk powder matrix

Table 3: Results of repeatability and sensitivity evaluation of DCD and melamine (n=6)

Sample Compd. Conc. (ng/mL) %RSD LOD (ng/mL) LOQ (ng/mL) 0.5 5.9 DCD 0.03 0.10 1.0 5.3 In solvent 0.5 5.5 MEL 0.03 0.09 1.0 2.6 0.5 5.9 DCD 0.05 0.16 1.0 8.2 In matrix 0.5 9.2 MEL 0.05 0.15 1.0 2.4

4 High Sensitivity Quantitation Method of Dicyandiamide and Melamine in Milk Powders by Liquid Chromatography Tandem Mass Spectrometry

The LOD and LOQ were estimated from the results of and 62%~73%, respectively. 0.5 ng/mL in both neat and matrix solution. The LOD The recovery was determined by comparing the results and LOQ results were summarized in Table 3. The of pre-spiked and post-spiked mixed samples of DCD method achieved LOQs (in matrix) of 0.16 and 0.15 and melamine in the milk powder matrix (2.5 ng/mL ng/mL (ppb) for DCD and melamine, respectively. Tables each compound). The chromatograms of these samples 4 & 5 show the results of matrix effect and recovery of are shown in Figure 6. The recovery of DCD and the method. The matrix effects for DCD and melamine melamine were determined to be 103% and 105% in the whole concentration ranges were at 64%~70% respectively.

Table 4: Matrix effect (%) of DCD and melamine Table 5: Recovery of DCD and melamine determined in milk powder matrix with spiked sample of 2.5 ng/mL

Conc. (ng/mL) 0.5 1 2.5 5 10 Compound Pre-spiked Area Post-spiked Area Recovery (%) DCD 70.4 65.4 66.9 64.8 66.6 DCD 14,393 13,987 102.9 MEL 62.2 62.5 73.1 68.9 68.0 MEL 65,555 62,659 104.6

1:85.10>68.05(+) 1:85.10>68.05(+) 1:85.10>68.05(+) 1:85.10>43.00(+) 1:85.10>43.00(+) 7000 7000 7000 1:85.10>43.00(+)

6000 6000 6000

5000 5000 5000 4000 4000 DCD 4000 DCD

Blank matrix of Dicyandiamide Dicyandiamide 3000 milk powder 3000 Pre-spiked 3000 Post-spiked 2000 2000 2000 1000 1000 1000 0 0 0 2.00 2.25 2.50 2.75 3.00 2.00 2.25 2.50 2.75 3.00 2.00 2.25 2.50 2.75 3.00

2:127.10>85.10(+) 2:127.10>85.10(+) 2:127.10>85.10(+) 2:127.10>68.05(+) 2:127.10>68.05(+) 2:127.10>68.05(+) 17500 17500 17500 Melamine 15000 15000 15000 Melamine

12500 12500 12500 Blank matrix of Melamine Melamine 10000 10000 milk powder Pre-spiked 10000 Post-spiked 7500 7500 7500

5000 5000 5000

2500 2500 2500

0 0 0 6.00 6.25 6.50 6.75 6.00 6.25 6.50 6.75 6.00 6.25 6.50 6.75

Figure 6: MRM peaks of DCD and melamine in pre- and post-spiked samples of 2.5 ng/mL (each). DCD and melamine were not detected in blank matrix of milk powder.

5 High Sensitivity Quantitation Method of Dicyandiamide and Melamine in Milk Powders by Liquid Chromatography Tandem Mass Spectrometry

Conclusions A high sensitivity LC/MS/MS method was developed on ng/mL for both compounds in the matrix, allowing its LCMS-8040 for detection and quantitation of application in simultaneous analysis of melamine, a protein dicyandiamide (DCD) and melamine in milk powders. The adulterant in relatively high concentration, and method performance was evaluated using infant milk dicyandiamide residue in trace level in milk powders powders as the matrix. The method achieved LOQ of 0.16 samples.

References 1. S. Turnipseed, C. Casey, C. Nochetto, D. N. Heller, FDA Food, LIB No. 4421, Volume 24, October 2008. 2. S. MachMahon, T. H. Begley, G. W. Diachenko, S. A. Stromgren, Journal of Chromatography A, 1220, 101-107 (2012). 3. http://www.naturalnews.com/041834_Fonterra_milk_powder_dicyandiamide.html

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1465E

Multiresidue pesticide analysis from dried chili powder using LC/MS/MS

ASMS 2014 WP350

Deepti Bhandarkar, Shruti Raju, Rashi Kochhar, Shailesh Damale, Shailendra Rane, Ajit Datar, Jitendra Kelkar, Pratap Rasam Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh Chambers, Makwana Road, Marol, Andheri (E), Mumbai-400059, Maharashtra, India. Multiresidue pesticide analysis from dried chili powder using LC/MS/MS

Introduction Pesticide residues in foodstuffs can cause serious health matrix. Therefore, this approach was used to obtain more problems when consumed. LC/MS/MS methods have been reliable and accurate data as compared to quantitation increasingly employed in sensitive quanti cation of against neat (solvent) standards[1]. pesticide residues in foods and agriculture products. Multiresidue, trace level analysis in complex matrices is However, matrix effect is a phenomenon seen in Electro challenging and tedious. Feature of automatic MRM Spray Ionization (ESI) LC/MS/MS analysis that impacts the optimization in LCMS-8040 makes method development data quality of the pesticide analysis, especially for complex process less tedious. In addition, the lowest dwell time and matrix like spice/herb. pause time along with ultra fast polarity switching Chili powder is one such complex matrix that can exhibit (UFswitching) enables accurate, reliable and high sensitive matrix effect (either ion suppression or enhancement). A quantitation. UFsweeperTM II technology in the system calibration curve based on matrix matched standards can ensures least crosstalk, which is very crucial for demonstrate true sensitivity of analyte in presence of multiresidue pesticide analysis.

Method of Analysis Sample Preparation Commercially available red chili was powdered using mixer pesticide matrix matched standards at concentration levels grinder. To 1 g of this chili powder, 20 mL water:methanol of 0.01 ppb, 0.02 ppb, 0.05 ppb, 0.1 ppb, 0.2 ppb, 0.5 (1:1 v/v) was added and the mixture was sonicated for 10 ppb, 1 ppb, 2 ppb, 5 ppb, 10 ppb and 20 ppb. Each mins. The mixture was centrifuged and supernatant was concentration level was then filtered through 0.2 µ nylon collected. This supernatant was used as diluent to prepare filter and used for the analysis.

LC/MS/MS Analytical Conditions Pesticides were analyzed using Ultra High Performance Corporation, Japan), shown in Figure 1. The details of Liquid Chromatography (UHPLC) Nexera coupled with analytical conditions are given in Table 1. LCMS-8040 triple quadrupole system (Shimadzu

Table 1. LC/MS/MS analytical conditions

• Column : Shim-pack XR-ODS (75 mm L x 3 mm I.D.; 2.2 µm) • Guard column : Phenomenex SecurityGuard ULTRA Cartridge • Mobile phase : A: 5 mM ammonium formate in water:methanol (80:20 v/v) B: 5 mM ammonium formate in water:methanol (10:90 v/v) • Flow rate : 0.2 mL/min • Oven temperature : 40 ºC • Gradient program (B%) : 0.0–1.0 min → 45 (%); 1.0–13.0 min → 45-100 (%); 13.0–18.0 min → 100 (%); 18.0–19.0 min → 100-45 (%); 19.0–23.0 min → 45 (%) • Injection volume : 15 µL • MS interface : ESI • Polarity : Positive and negative • Nitrogen gas ow : Nebulizing gas 2 L/min; Drying gas 15 L/min • MS temperature : Desolvation line 250 ºC; Heat block 400 ºC • MS analysis mode : Staggered MRM

2 Multiresidue pesticide analysis from dried chili powder using LC/MS/MS

Figure 1. Nexera with LCMS-8040 triple quadrupole system by Shimadzu

Results LC/MS/MS method was developed for analysis of 80 LOQ achieved for 80 pesticides have been summarized in pesticides belonging to different classes like carbamate, Table 2 and results for LOQ and linearity for each pesticide organophosphate, urea, triazines etc. in a single run[2]. LOQ have been given in Table 3. Representative MRM was determined for each pesticide based on the following chromatogram of pesticide mixture at 1 ppb level is shown criteria – (1) % RSD for area < 16 % (n=3), (2) % Accuracy in Figure 2. Representative MRM chromatograms at LOQ between 80-120 % and (3) Signal to noise ratio (S/N) > 10. level for different classes of pesticides are shown in Figure 3.

Table 2: Summary of LOQ achieved

LOQ (ppb) 0.01 0.02 0.05 0.1 0.2 0.5 1 Number of pesticides 1 1 3 8 17 24 26

Table 3. Results of LOQ and linearity for pesticide analysis

Sr. No. Name of compound MRM Transition Polarity LOQ (ppb) Linearity (R2) 1 Spinosyn D 746.20>142.10 Positive 0.01 0.9987 2 Fenpyroximate 421.90>366.10 Positive 0.02 0.9915 3 Bifenazate 301.00>198.00 Positive 0.05 0.9947 4 Spinosyn A 732.20>142.10 Positive 0.05 0.9974 5 Spiromesifen 371.00>273.10 Positive 0.05 0.9957 6 Acetamiprid 222.90>126.00 Positive 0.1 0.9910 7 Carbofuran 221.70>123.00 Positive 0.1 0.9971 8 Dimethoate 229.80>198.90 Positive 0.1 0.9970 9 Dimethomorph I 387.90>301.00 Positive 0.1 0.9991 10 Dimethomorph II 387.90>301.00 Positive 0.1 0.9992 11 Isoproturon 207.00>72.10 Positive 0.1 0.9984 12 Pirimiphos methyl 305.70>108.00 Positive 0.1 0.9997 13 Trioxystrobin 408.90>186.00 Positive 0.1 0.9989

3 Multiresidue pesticide analysis from dried chili powder using LC/MS/MS

Sr. No. Name of compound MRM Transition Polarity LOQ (ppb) Linearity (R2) 14 Anilophos 367.70>198.85 Positive 0.2 0.9974 15 Atrazine 215.90>174.00 Positive 0.2 0.9985 16 Carboxin 235.90>143.00 Positive 0.2 0.9952 17 Cyazofamid 324.85>108.10 Positive 0.2 0.9971 18 Edifenphos 310.60>111.00 Positive 0.2 0.9997 19 Ethion 384.70>198.80 Positive 0.2 0.9957 20 Fipronil 434.70>330.00 Negative 0.2 0.9973 21 Linuron 248.80>159.90 Positive 0.2 0.9945 22 Metolachlor 283.90>252.00 Positive 0.2 0.9966 23 Oxycarboxin 267.90>174.90 Positive 0.2 0.9995 24 Phosalone 367.80>181.90 Positive 0.2 0.9987 25 Phosphamidon 299.90>173.90 Positive 0.2 0.9997 26 Thiacloprid 252.90>126.00 Positive 0.2 0.9976 27 Thiobencarb 257.90>125.10 Positive 0.2 0.9977 28 Thiodicarb 354.90>88.00 Positive 0.2 0.9906 29 Triadimefon 293.90>196.90 Positive 0.2 0.9994 30 Tricyclazole 189.90>162.90 Positive 0.2 0.9977 31 Aldicarb 208.10>116.05 Positive 0.5 0.9962 32 Benfuracarb 411.10>190.10 Positive 0.5 0.9981 33 Bitertanol 338.00>99.10 Positive 0.5 0.9935 34 Buprofezin 305.70>201.00 Positive 0.5 0.9933 35 Clodinafop propargyl 349.90>266.00 Positive 0.5 0.9978 36 Chlorantraniliprole 483.75>452.90 Positive 0.5 0.9994 37 Diclofop methyl 357.90>280.80 Positive 0.5 0.9976 38 Flufenacet 363.70>193.90 Positive 0.5 0.9997 39 Flusilazole 315.90>247.00 Positive 0.5 0.9983 40 Hexaconazole 313.90>70.10 Positive 0.5 0.9996 41 Hexythiazox 352.90>227.90 Positive 0.5 0.9909 42 Iodosulfuron methyl 507.70>167.00 Positive 0.5 0.9971 43 Iprobenfos 288.70>205.00 Positive 0.5 0.9981 44 Malaoxon 314.90>99.00 Positive 0.5 0.9996 45 Malathion 330.90>284.90 Positive 0.5 0.9997 46 Mandipropamid 411.90>356.20 Positive 0.5 0.9952 47 Metalaxyl 280.00>220.10 Positive 0.5 0.9996 48 Methabenzthiazuron 221.70>150.00 Positive 0.5 0.9957 49 Methomyl 162.90>88.00 Positive 0.5 0.9988 50 Oxadiazon 362.15>303.00 Positive 0.5 0.9963 51 Penconazole 283.90>70.10 Positive 0.5 0.9992 52 Phorate 260.80>75.00 Positive 0.5 0.9987 53 Phorate sulfoxide 276.80>96.90 Positive 0.5 0.9991 54 Thiophanate methyl 342.90>151.00 Positive 0.5 0.9996 55 Avermectin B1a 890.30>305.10 Positive 1 0.9990 56 Carpropamid 333.70>139.00 Positive 1 0.9985

4 Multiresidue pesticide analysis from dried chili powder using LC/MS/MS

Sr. No. Name of compound MRM Transition Polarity LOQ (ppb) Linearity (R2) 57 Clomazone 241.90>127.00 Positive 1 0.9967 58 Clorimuron ethyl 415.30>186.00 Positive 1 0.9965 59 Cymoxanil 198.90>128.10 Positive 1 0.9949 60 Diafenthiuron 385.00>329.10 Positive 1 0.9961 61 Diubenzuron 310.80>158.00 Positive 1 0.9982 62 Dodine 228.10>60.00 Positive 1 0.9980 63 Emamectin benzoate 886.30>158.10 Positive 1 0.9983 64 Fenamidone 311.90>236.10 Positive 1 0.9997 65 Fenarimol 330.70>268.00 Positive 1 0.9900 66 Fenazaquin 306.95>57.10 Positive 1 0.9992 67 Flonicamid 229.90>202.70 Positive 1 0.9971 68 Flubendiamide 680.90>254.05 Negative 1 0.9993 69 Forchlorfenuron 247.90>129.00 Positive 1 0.9956 70 Kresoxim methyl 331.00>116.00 Positive 1 0.9996 71 Paclobutrazol 293.90>70.10 Positive 1 0.9974 72 Pencycuron 328.90>125.00 Positive 1 0.9943 73 Pendimethalin 281.90>212.10 Positive 1 0.9932 74 Profenofos 372.70>302.70 Positive 1 0.9966 75 Propargite 368.00>231.10 Positive 1 0.9950 76 Propoxur 209.90>110.90 Positive 1 0.9987 77 Pyrazosulfuron ethyl 414.90>182.00 Positive 1 0.9992 78 Pyriproxyfen 321.90>96.10 Positive 1 0.9975 79 Simazine 201.90>103.90 Positive 1 0.9992 80 Thiomethon 246.80>89.10 Positive 1 0.9989

50000

40000

30000

20000

10000

0

5.0 10.0 15.0 min

Figure 2. MRM chromatogram of pesticide mixture at 1 ppb level

5 Multiresidue pesticide analysis from dried chili powder using LC/MS/MS

15:229.80>198.90(+) 33:221.70>123.00(+) 44:207.00>72.10(+) 4000 Organophosphorus N-Methyl Urea 1000 4000 Carbamate 3000 750 3000 Dimethoate 2000 Isoproturon 500 Carbofuran 2000

250 1000 1000

0 0 2.0 3.0 4.0 5.0 5.0 6.0 7.0 8.0 7.0 8.0 9.0 10.0

4000 42:215.90>174.00(+) 115:746.20>142.10(+) 121:421.90>366.10(+) Triazine 300 Macrocyclic 1250 Pyrazole Lactone 3000 1000 200 750 Fenpyraoximate Spinosyn D

2000 Atrazine 500 100 1000 250

0 0

6.0 7.0 8.0 9.0 16.0 17.0 18.0 19.0 15.0 16.0 17.0 18.0

126:680.90>254.05(-) 80:283.90>70.10(+) 70:283.90>252.00(+) 5000 7500 6000 Anthranilic Azole Chloroacetanilide 4000 Diamide 5000 5000 3000 4000 Flubendiamide Penconazole 3000 Metolachlor 2000 2000 2500 1000 1000 0 0 0 10.0 11.0 12.0 13.0 11.0 12.0 13.0 14.0 10.0 11.0 12.0 13.0

Figure 3. Representative MRM chromatograms at LOQ level from different classes of pesticides

Conclusion • A highly sensitive method was developed for analysis of 80 pesticides belonging to different classes, from dried chili powder in a single run. • Ultra high sensitivity, ultra fast polarity switching (UFswitching), low pause time and dwell time along with UFsweeperTM II technology enabled sensitive, selective, accurate and reproducible multiresidue pesticide analysis from complex matrix like dried chili powder.

6 Multiresidue pesticide analysis from dried chili powder using LC/MS/MS

References [1] Kwon H, Lehotay SJ, Geis-Asteggiante L., Journal of Chromatography A, Volume 1270, (2012), 235–245. [2] Banerjee K, Oulkar DP et al., Journal of Chromatography A, Volume 1173, (2007), 98-109.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1463E

Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method

ASMS 2014 TP762

Durvesh Sawant(1), Dheeraj Handique(1), Ankush Bhone(1), Prashant Hase(1), Sanket Chiplunkar(1), Ajit Datar(1), Jitendra Kelkar(1), Pratap Rasam(1), Kaushik Banerjee(2), Zareen Khan(2) (1) Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh Chambers, Makwana Road, Marol, Andheri (E), Mumbai-400059, Maharashtra, India. (2) National Referral Laboratory, National Research Centre for Grapes, P.O. Manjri Farm, Pune-412307, Maharashtra, India. Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method

Introduction India is the world’s second largest producer (after China) levels in tobacco, the Guidance Residue Levels (GRL) of 118 and consumer (after Brazil) of tobacco with nearly $ pesticides have been issued by the Agro-Chemical Advisory 1001.54 million revenue generated annually from its Committee (ACAC) of the Cooperation Center for export.[1] In countries like India, with tropical-humid Scienti c Research Relative to Tobacco (CORESTA). climate, the incidences of insect attacks and disease Tobacco is a complex matrix and hence requires selective infestations are frequent and application of pesticides for extraction and extensive cleanup such as QuEChERS (Quick their management is almost obligatory. Like any other Easy Cheap Effective Rugged Safe) to ensure trace level crop, tobacco (Nicotiana tabacum Linn.), one of the detection with adequate precision and accuracy. The world’s leading high-value crops, is also prone to pest objective of the present study was to develop an effective, attacks, and the farmers do apply various pesticides as a sensitive and economical multi-pesticide residue analysis control measure. method for 203 pesticides in tobacco as listed in Table 1. The residues of pesticides applied on tobacco during its cultivation may remain in the leaves at harvest that may even sustain post harvest processing treatments and could appear in the nal product. Thus, monitoring of pesticide residues in tobacco is an important issue of critical concern from public health and safety point of view demanding implementation of stringent regulatory policies.[2] To protect the consumers by controlling pesticide residue Figure 1. Dried tobacco

Method of Analysis Extraction of pesticides from tobacco Extraction of pesticides was done using QuEChERS method, as described below.[3]

Take 2 g of dry powdered tobacco leaves (Figure 1). Add 18 mL of water containing 0.5 % acetic acid. Homogenize the sample and Keep it for 30 min.

Add 10 mL ethyl acetate. Immediately, put 10 g sodium sulfate.

Homogenize it thoroughly at 15000 rpm for 2 min.

Centrifuge at 5000 rpm for 5 min for phase separation.

Draw 3 mL of ethyl acetate upper layer from the extract for further cleanup.

2 Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method

Add 1 mL toluene to it and vortex for 0.5 min.

Add cleanup mixture [PSA (150 mg), C18 (150 mg), GCB (75 mg) and

anhydrous MgSO4 (300 mg)] and vortex for 2 min.

Centrifuge the mixture at 7000 rpm for 7 min.

Collect the supernatant and lter through a 0.2 µm PTFE membrane lter.

Inject 2.0 µL of the clean extract into GCMS-TQ8030 (Figure 2).

Figure 2. GCMS-TQ8030 Triple quadrupole system by Shimadzu

Key Features of GCMS-TQ8030 • ASSP™ (Advanced Scanning Speed Protocol) enables high-speed scan and data acquisition for accurate quantitation at 20,000 u/sec • Capable of performing simultaneous Scan/MRM • UFsweeper® technology efficiently sweeps residual ions from the collision cell for fast, efficient ion transport ensuring no cross-talk • Two overdrive lenses reduce random noise from helium, high-speed electrons and other factors to improve S/N ratio • Flexible platform with EI (Electron Ionization), CI (Chemical Ionization), and NCI (Negative Chemical Ionization) techniques • Full complement of acquisition modes including MRM, Scan/MRM, Precursor Ion, Product Ion and Neutral Loss Scan

3 Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method

Table 1. List of pesticides Sr. No. Pesticide Sr. No. Pesticide Sr. No. Pesticide Sr. No. Pesticide 1 2,6-Dichlorobenzamide 52 Cyuthrin-3 103 Fipronil sulphone 154 Permethrin-1 2 2-Phenylphenol 53 Cyuthrin-4 104 Flucythrinate-1 155 Permethrin-2 3 3,4-Dichloraniline 54 Cyhalofop-butyl 105 Flucythrinate-2 156 Pethoxamid 4 3-Chloroaniline 55 Cypermethrin-2 106 Flufenacet 157 Phosalone 5 4-Bromo 2-Chloro phenol 56 Cypermethrin-3 107 Flumoixazine 158 Phosmet 6 4,4-Dichlorobenzophenone 57 Cypermethrin-4 108 Fluquinconazole 159 Pirimicarb 7 Acetochlor 58 Cyprodinil 109 Flurochloridone-1 160 Pretilachlor 8 Acrinathrin 59 Delta-HCH 110 Flurochloridone-2 161 Procymidone 9 Alachlor 60 Demeton-s-methyl 111 Flutolanil 162 Profenofos 10 Aldrin 61 Demeton-S-methyl sulphone 112 Flutriafol 163 Propanil 11 Azinphos-ethyl 62 Dialifos 113 Fluxapyoxad 164 Propaquizafop 12 Azinphos-methyl 63 Diazinon 114 Folpet 165 Propazine 13 Azoxystrobin 64 Dichlobenil 115 Fuberidazole 166 Propham 14 Barban 65 Dichlouanid 116 Heptachlor 167 Propiconazole-1 15 Beubutamid 66 Diclofop 117 Hexaconazole 168 Propisoclor 16 Benuralin 67 Dicloran 118 Iprobenfos 169 Propyzamide 17 Benoxacor 68 Dieldrin 119 Isoprocarb 170 Proquinazid 18 Beta-endosulfan 69 Diethofencarb 120 Isoprothiolane 171 Pyraufen-ethyl 19 Bifenox 70 Difenoconazole-1 121 Isopyrazam 172 Pyrazophos 20 Bifenthrin 71 Difenoconazole-2 122 Isoxaben 173 Pyrimethanil 21 Bitertanol 72 Diubenzuron 123 Lactofen 174 Pyriprooxyfen 22 Boscalid 73 Diufenican 124 Lambda-cyhalothrin 175 Pyroquilon 23 Bromacil 74 Dimethipin 125 Malaoxon 176 Quinoxyfen 24 Bromophos-ethyl 75 Dimethomorph-1 126 Malathion 177 Simazine 25 Bromopropylate 76 Dimethomorph-2 127 Mepanipyrim 178 Spirodiclofen 26 Bromuconazole-1 77 Dimoxystrobin 128 Mepronil 179 Sulfotep 27 Bromuconazole-2 78 Diniconazole 129 Metalaxyl 180 Swep 28 Butralin 79 Dinoseb 130 Metalaxyl M 181 Tebufenpyrad 29 Butylate 80 Dinoterb 131 Metazachlor 182 Tebupirimfos 30 Carbaryl 81 Dioxathion 132 Metconazole 183 Tebuthiuron 31 Carbofuran 82 Edifenfos 133 Methabenzthiazuron 184 Teuthrin 32 Carfentrazone 83 Endosulfan sulphate 134 Methacrifos 185 Terbacil 33 Chlordane-trans 84 Endrin 135 Methidathion 186 Tetraconazole 34 Chlordecone 85 Epoxiconazole 136 Methiocarb 187 Tetradifon 35 Chlorfenvinphos 86 Ethaluralin 137 Metholachlor-s 188 Thiobencarb 36 Chlormephos 87 Ethoprophos 138 Methoxychlor 189 Tolyluanid 37 Chlorobenzilate 88 Etoxazole 139 Metribuzin 190 Tralkoxydim 38 Chloroneb 89 Etridiazole 140 Mevinphos 191 Triadimefon 39 Chlorothalonil 90 Etrimfos 141 Monolinuron 192 Tri-allate 40 Chlorpyriphos-ethyl 91 Famoxadone 142 Myclobutanyl 193 Triazophos 41 Chlorpyriphos-methyl 92 Fenamidone 143 Napropamide 194 Tricyclazole 42 Chlorpyriphos-oxon 93 Fenarimol 144 Nitrapyrin 195 Trioxystrobin 43 Chlorthal-dimethyl 94 Fenbuconazole 145 Oxadiargyl 196 Triumizole 44 Cinidon-ethyl 95 Fenchlorphos 146 Oxadiazon 197 Triumuron 45 Cis-1,2,3,6 tetrahydrophthalimide 96 Fenchlorphos oxon 147 Oxycarboxin 198 Triuralin 46 Clodinafop propargyl 97 Fenhexamid 148 p,p-DDE 199 Triusulfuron 47 Clomazone 98 Fenobucarb 149 Parathion-ethyl 200 Triticonazole 48 Crimidine 99 Fenoxycarb 150 Parathion-methyl 201 Valifenalate 49 Cyanophos 100 Fenthion sulphoxide 151 Penconazole 202 Vinclozolin 50 Cyuthrin-1 101 Fenvalerate 152 Pencycuron (Deg.) 203 Zoxamide (Deg.) 51 Cyuthrin-2 102 Fipronil 153 Pendimethalin

4 Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method

GCMS/MS Analytical Conditions The analysis was carried out on Shimadzu GCMS-TQ8030 as per the conditions given below.

Chromatographic parameters

• Column : Rxi-5Sil MS (30 m L x 0.25 mm I.D.; 0.25 µm) • Injection Mode : Splitless • Sampling Time : 2.0 min • Split Ratio : 5.0 • Carrier Gas : Helium • Flow Control Mode : Linear Velocity • Linear Velocity : 40.2 cm/sec • Column Flow : 1.2 mL/min • Injection Volume : 2.0 µL • Injection Type : High Pressure Injection • Total Program Time : 41.87 min • Column Temp. Program : Rate (ºC /min) Temperature (ºC) Hold time (min) 70.0 2.00 25.00 150.0 0.00 3.00 200.0 0.00 8.00 280.0 10.00

Mass Spectrometry parameters

• Ion Source Temp. : 230.0 ºC • Interface Temp. : 280.0 ºC • Ionization Mode : EI • Acquisition Mode : MRM

Results For MRM optimisation, well resolved pesticides were corresponding CE value was selected, thereby assigning a grouped together. Standard solution mixture of characteristic MRM transition to every pesticide. Based on approximately 1 ppm concentration was prepared and MRM transitions, the mixture of 203 pesticides was analyzed in Q3 scan mode to determine the precursor ion analyzed in a single run (Figure 3). for individual pesticides. Selected precursor ions were Method was partly validated for each pesticide with allowed to pass through Q1 & enter Q2, also called as respect to linearity (0.5 to 25 ppb), reproducibility, LOQ Collision cell. In Collision cell, each precursor ion was and recovery. The validation summary for two pesticides bombarded with collision gas (Argon) at different energies namely Mevinphos and Parathion-ethyl (Sr. Nos.140 and (called as Collision Energy-CE) to produce fragments 149 in Table 1) is shown in Figures 4 and 5. The summary (product ions). These product ions were further scanned in data of linearity and LOQ for 203 pesticides is given in Q3 to obtain their mass to charge ratio. For each precursor Table 2 and 3 respectively. ion, product ion with highest intensity and its

5 Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method

(x100,000) 6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

-0.5

-1.0 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 min Figure 3. MRM Chromatogram for 203 pesticides mixture

Calibration overlay Linearity curve Recovery overlay

Area(x100,000) (x10,000) 2.5 (x10,000) MRM : 192.00>127.00 MRM : 192.00>127.00 Post extraction spike 5.0 Pre extraction spike 2.0 1.00 4.0 1.5 0.75 3.0 0.50 1.0 2.0

0.25 1.0 0.5

0.0 0.00 0.0 7.0 7.5 8.0 8.5 9.0 min 0.0 5.0 10.0 15.0 20.0 Conc. 7.25 7.50 7.75 8.00 8.25 8.50 min

% RSD at LOQ % Recovery Linearity (R2) LOD (ppb) LOQ (ppb) S/N at LOQ (n=6) at LOQ 0.9999 0.3 1 173 6.93 89.28

Figure 4. Summary data for mevinphos

Calibration overlay Linearity curve Recovery overlay

Area(x100,000) (x10,000) (x1,000) MRM : 291.10>137.00 1.50 8.0 3.5 MRM : 291.10>137.00 Post extraction spike 7.0 Pre extraction spike 3.0 1.25 6.0 2.5 1.00 5.0 2.0 4.0 0.75 1.5 3.0

1.0 0.50 2.0

0.5 1.0 0.25 0.0 0.0 0.00 15.0 15.5 16.0 16.5 min 0.0 5.0 10.0 15.0 20.0 Conc. 15.0 15.5 16.0 16.5 min

% RSD at LOQ % Recovery Linearity (R2) LOD (ppb) LOQ (ppb) S/N at LOQ (n=6) at LOQ 0.9993 1.5 5 93 4.05 109.10

Figure 5. Summary data for parathion-ethyl

6 Multi pesticide residue analysis in tobacco by GCMS/MS using QuEChERS as an extraction method

Table 2. Linearity Summary Table 3. LOQ Summary

Number of Number of % RSD range S/N Ratio % Recovery Sr. No. Linearity (R2) Sr. No. LOQ (ppb) pesticides pesticides (n=6) range range 1 0.9950 - 1.0000 193 1 1 15 6 – 15 16 – 181 2 0.9880 - 0.9950 10 2 5 18 3 – 15 19 – 502 70 – 130 3 10 158 0.95 – 15 10 – 14255 4 25 12 1 – 10 19 – 660

Conclusion • A highly sensitive method was developed for quantitation of 203 pesticides in complex tobacco matrix by using Shimadzu GCMS-TQ8030. • The MRM method developed for 203 pesticides can be used for screening of pesticides in various food commodities. For 90 % of the pesticides, the LOQ of 10 ppb or below was achieved. • Ultra Fast scanning, UFsweeper® and ASSP™ features enabled sensitive, selective, fast, reproducible, linear and accurate method of analysis.

Reference [1] Tobacco Board (Ministry of Commerce and Industry, Government of India), Exports performance during 2013-14, (2014), 1. http://tobaccoboard.com/admin/statistics les/Exp_Perf_Currentyear.pdf [2] CORESTA GUIDE Nº 1, The concept and implementation of cpa guidance residue levels, (2013), 4. http://www.Coresta.org/Guides/Guide-No01-GRLs%283rd-Issue-July13%29.pdf [3] Zareen S Khan, Kaushik Banerjee, Rushali Girame, Sagar C Utture et al., Journal of Chromatography A, Volume 1343, (2014), 3.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1453E

Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS

ASMS 2014 TP 510

Keiko Matsumoto1; Jun Watanabe1; Itaru Yazawa2 1 Shimadzu Corporation, Kyoto, Japan; 2 Imtakt Corporation, Kyoto, Japan Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS

Introduction In order to detect many kinds of amino acids with high acids, it is expected to develop the method without using selectivity in food samples, the LC/MS analysis have been reagents mentioned above. used widely. Amino acids are high polar compound, so This time, we tried to develop a simultaneous high sensitive they are hard to be retained to reverse-phased column analysis method of 20 amino acids by LC/MS/MS with such as ODS (typical method in LC/MS analysis). It needs mix-mode column (ion exchange, normal-phase) and the their derivartization or addition of ion pair reagent in typical volatile mobile phase suitable for LC/MS analysis. mobile phase to retain them. For easier analysis of amino

Methods and Materials Amino acid standard regents and food samples were quadrupole mass spectrometer (Nexera with LCMS-8050, purchased from the market. Standards of 20 kinds of Shimadzu Corporation, Kyoto, Japan). Sample was eluted amino acids were optimized on each with a binary gradient system and LC-MS/MS with compound-dependent parameter and MRM transition. electrospray ionization was operated in As an LC-MS/MS system, HPLC was coupled to triple multiple-reaction-monitoring (MRM) mode.

High Speed Mass Spectrometer

UF-MRM High-Speed MRM at 555ch/sec

UFswitching High-Speed Polarity Switching 5msec

Figure 1 LCMS-8050 triple quadrupole mass spectrometer

Result Method development First, MRM method of 20 amino acids was optimized. As a optimized. Even though amino acids were not derivartized result, all compounds were able to be detected high and ion-pairing reagent wasn’t used, 20 amino acids were sensitively and were detected in positive MRM transitions. As retained by using a mixed-mode stationary phase structure the setting temperature of ESI heating gas was found to and separated excellently on the below-mentioned affected on the sensitivity of amino acids, it was also condition.

2 Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS

HPLC conditions (Nexera system)

Column : Intrada Amino Acid (3.0mmI.D. x 50mm, 3um, Imtakt Corporation, Kyoto, Japan) Mobile phase Case1 A : Acetonitrile / Formic acid = 100 / 0.1 B : 100mM Ammonium formate Time program : B conc.14%(0-3 min) -100%(10min) - 14%(10.01-15min) Case2 (High Resolution condition) A : Acetonitrile / Tetrahydrofuran / 25mM Ammonium formate / formic acid = 9 / 75 / 16 / 0.3 B : 100mM Ammonium formate / Acetonitrile = 80 / 20 Time program : B conc.0%(0-2 min) - 5%(3min) - 30%(6.5min) - 100%(12min) - 0%(12.01-17min) Flow rate : 0.6 mL/min Injection volume : 2 uL Column temperature : 40 °C

MS conditions (LCMS-8050)

Ionization : ESI, Positive MRM mode MRM transition are shown in Table 1.

Case1

Mobile Phase A: Acetonitrile / Formic acid = 100 / 0.1 B: 100mM Ammonium formate

Thr Phe Pro Asp Ser His Trp Ara Gly

4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 min Arg Lys Ile Gln Leu Val Met Glu Tyr Asn (Cys)2

2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

Figure 2 Mass Chromatograms of 20 Amino acids (concentration of each compound : 10nmol/mL)

3 Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS

Case2 (High Resolution condition)

Mobile Phase A: Acetonitrile / Tetrahydrofuran / 25mM Ammonium formate / formic acid = 9 / 75 / 16 / 0.3 B: 100mM Ammonium formate / Acetonitrile = 80 / 20

Thr

Pro

Asp Ara Ser Gly

Ile 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 min

Trp Phe Arg Leu Met Val Lys Tyr His Glu Gln (Cys)2 Thr Asn

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 min

Figure 3 Mass Chromatograms of 20 Amino acids (concentration of each compound : 10nmol/mL)

In this study, two conditions of mobile phase were the result of case1 was sufficiently well, case1 analytical investigated. It was found that 20 amino acids were condition was used for quantitative analysis. The dilution separated with higher resolution in case2. series of these compounds were analyzed. All amino acids As the mobile phase condition of case1 is more simple and were detected with good linearity and repeatability (Table1).

4 Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS

Table1 Linearity and Repeatability of 20 amino acids

Linearity Repeatability* MRM Transition Range (nmol/mL) Coef cient (r2) %RSD Trp 205.10>188.10 0.01-100 0.9950 1.4 Phe 166.10>120.10 0.01-100 0.9971 1.2 Tyr 182.10>136.00 0.05-100 0.9900 1.7 Met 150.10>56.10 0.05-200 0.9963 0.1 Lue, Lle 132.10>86.15 0.01-100 0.9955 0.7 Val 118.10>72.05 0.05-100 0.9991 1.9 Glu 148.10>84.10 0.05-10 0.9965 4.5 Pro 116.10>70.10 0.01-50 0.9933 1.5 Asp 134.20>74.10 0.5-500 0.9953 1.4 Thr 120.10>74.00 0.1-50 0.9923 4.5 Ala 90.10>44.10 0.5-500 0.9989 16.2 Ser 106.10>60.20 0.5-500 0.9988 6.5 Gln 147.10>84.10 0.05-1 0.9959 3.9 Gly 76.20>29.90 5-200 0.9974 11.0 Asn 133.10>74.05 0.05-20 0.9939 6.1 (Cys)2 241.00>151.95 0.05-20 0.9909 2.3 His 156.10>110.10 0.05-200 0.9983 1.7 Lys 147.10>84.10 0.05-5 0.9908 0.9 Arg 175.10>70.10 0.01-100 0.9956 0.5

*@ 0.5nmol/mL : except for Gly, 5nmol/mL : for Gly

The analysis of 20amino acids in food samples The analysis of the amino acids contained in sports beverage on the market was carried out. In the case of sports beverage, all amino acids written in the package were detected.

Pro Sports Beverage Thr Lys Gly Ser Asp Phe Ara

4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 min His Trp Tyr Arg Leu Ile Val Glu Met Thr

2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

Figure 4 Mass Chromatograms of Sports Beverage (100 fold dilution with 0.1N HCl)

5 Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS

Furthermore, Japanese Sake, Beer and sweet cooking rice preparation. These were filtered through a 0.2um filter and wine (Mirin) were analyzed using this method. Japanese then analyzed. MRM chromatograms of each food samples Sake and Beer were diluted with 0.1N HCl. Sweet cooking are shown in Figure 5,6,7. Amino acids of each sample were rice wine was diluted in the same way after a deproteinizing detected with high sensitivity.

Pro Ala Japanese Sake Phe Arg Thr Gly Ser

4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2min Leu Tyr His Gln Lys Trp Ile Val Glu Met Ala Asn (Cys)2

2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

Figure 5 Mass Chromatograms of Japanese Sake (100 fold dilution with 0.1N HCl)

Pro Beer

Asn

Asp Trp Ala Thr Gly Ser

Phe 4.5 4.6 4.7 4.8 4.9 5.0 5.1 min Arg Leu Tyr His Ile Lys Glu Gln Met Val (Cys)2

2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

Figure 6 Mass Chromatograms of Beer (10 fold dilution with 0.1N HCl)

Trp Sweet Cooking Rice Wine Asn Thr Asp Ser Ala Phe Gly

Pro 4.5 4.6 4.7 4.8 4.9 5.0 5.1 min Tyr Gln Leu Ile Glu Arg Val His Lys Met (Cys)2 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

Figure7 Mass Chromatograms of Sweet Cooking Rice Wine (100 fold dilution with 0.1N HCl)

6 Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS

Conclusions • 20 amino acids could be separated without derivatization using a typical volatile mobile phase suitable for LC/MS analysis and detected with high sensitivity. • This methods was able to be applied to the analysis of amino acids in various food samples.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 Environment

• Page 170 Rapid screening and confirmation analysis of polycyclic aromatic hydrocarbons (PAHs) with DART mass spectrometry

• Page 176 Fast and highly sensitive analysis of multiple drugs in ground-, surface- and wastewater

• Page 182 Multi-residue analysis of pyrethroids in soil and sediment using QuEChERS by LC/MS/MS PO-CON1455E

Rapid Screening and con rmation analysis of polycyclic aromatic hydrocarbons (PAHs) with DART mass spectrometry

ASMS 2014 MP 551

Yu Takabayashi1, Jun Watanabe2, Motoshi Sakakura3, Teruhisa Shiota3 1 SHIMADZU TECHNO-RESEARCH, INC., Tokyo, Japan; 2 Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan; 3 AMR Inc., Meguro-ku, Tokyo, Japan Rapid Screening and con rmation analysis of polycyclic aromatic hydrocarbons (PAHs) with DART mass spectrometry

Introduction Recently, the regulation of the content of the polycyclic the compounds which are not ionized by ESI. Since PAHs is aromatic hydrocarbons (PAHs) in goods which may put into ionizable by DART, PAHs can be quickly screened by a mouth or may contact is advanced and the technologies holding up a sample directly to DART. In this research, the of measuring PAHs quickly are being developed. technique detected by DART-MS was developed coupling The ionizing principle of DART (Direct Analysis in Real Time) with LC and DART analysis after carrying out LC separation using the excitation helium gas is able to widely ionize the was performed. wide-range compounds and it may also be able to ionize

Methods and Materials Commercial PAHs were used for the sample. The samples LC-DART MS analysis was conducted by loading an eluate were applied to DART MS with the solution formed in to a DART ionization area continuously. suitable concentration or the powder formed. Small DART OS ion source and single/triple quadrupole type mass amount of the samples were picked up and held in the spectrometer were used for this experiment. PAHs DART ionization gas stream using glass capillaries. In measured in the detection mode which performs a full LC-DART MS analysis, the mixed-solution of PAHs standard scan mode with positive and negative simultaneous was prepared and applied to HPLC. After carrying out ionization. chromatogram separation using a reverse phased column,

MS condition (LCMS-2020; Shimadzu Corporation)

Ionization : DART (Direct Analysis in Real Time) Heater Temperature (DART) : 300°C to 500°C Measuring mode (MS) : Positive/Negative scanning simultaneously

High Speed Mass Spectrometer

Ufswitching High-Speed Polarity Switching 15msec Ufscanning High-Speed Scanning 15,000u/sec

Figure 1 DART-OS ion source & LCMS-2020

2 Rapid Screening and con rmation analysis of polycyclic aromatic hydrocarbons (PAHs) with DART mass spectrometry

Result First, in order to verify whether PAHs ionizes in DART, PAH standard reagents were analyzed in DART-MS. Benzo[a]anthracene, acenaphthene, anthracene, etc. were used as typical PAHs. When benzo[a]anthracene was analyzed, in the positive spectrum, the signal at m/z 229 which is equivalent to [M+H]+ was detected. Moreover, in the negative spectrum, the signal at m/z 243 which is equivalent to [M+O-H]- was detected. Similarly, acenaphthene and anthracene could also be ionized by DART-MS and were able to be assigned as molecular related ion. Additionally pyrene and uoranthene were also examined. As each of these is structural isomers mutually in structural-formula C16H10, in the negative spectrum, the signal of [M+O-H]- is detected by m/z 217 in each other, and either was not able to identify whether the detected signal is pyrene or uoranthene in analysis by DART-MS without chromatogram separation.

12500000 1:BPC(+) Positive TIC m/z 100-300 10000000

7500000 A 5000000

2500000

0 2:BPC(-) 750000 Negative TIC m/z 100-300

500000 B

250000

0 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 min

Inten.(x1,000,000) 7.0 Positive 229.3 [M+H]+ 6.0 Benzo[a]anthracene

5.0 4.0 M+ 3.0 C

2.0 245.2 1.0 C18H12 0.0 261.3 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z Fw 228 Inten.(x100,000) 4.5 243.2 4.0 Negative [M+O-H]- 3.5

3.0

2.5 2.0 D 1.5

1.0 259.3 0.5 275.1 291.2 125.0 179.3 277.1 0.0 220.6 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z

Figure 2 DART mass chromatogram and mass spectrum of Benzo[a]anthracene A: positive mass chromatogram, B: negative mass chromatogram (The area with the orange dashed line is the time when sample was held in DART.) C: positive mass spectrum, D: negative mass spectrum

3 Rapid Screening and con rmation analysis of polycyclic aromatic hydrocarbons (PAHs) with DART mass spectrometry

Inten.(x100,000) 9.0 Acenaphthene 8.0 M+154.2 [M+H]+ Positive 7.0 155.2 C12H10 6.0 Fw 154 5.0 4.0 3.0 2.0 171.2 102.3 1.0 202.3 130.2 187.3 220.2 253.3 0.0 142.3 268.9 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z

Inten.(x1,000,000) 3.0 179.2 [M+H]+ Positive Anthracene 2.5 M+ C14H10 2.0 Fw 178 1.5

1.0

195.2 0.5

158.3 211.2 225.1 0.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z

Inten.(x10,000,000)

1.00 204.2 Positive Pyrene 0.75 C16H10 Fw 202 0.50

0.25

193.1 218.2 0.00 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z

Inten.(x100,000) 1.75 217.2 Negative 1.50 [M+O-H]- 1.25

1.00

0.75 190.3 0.50

0.25 101.1 179.2 233.3 298.1 226.3 253.3 269.3 115.5 137.1 165.2 205.2 255.6 287.3 0.00 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z

Inten.(x1,000,000) 2.5 208.3 Positive

2.0 194.2 uoranthene

1.5 122.3 C16H10

220.3 Fw 202 1.0 169.2

222.3 0.5 183.2 108.2 136.3 236.3 0.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z

Inten.(x100,000) 4.0 217.1 Negative 3.5 [M+O-H]- 3.0

2.5

2.0

1.5 167.1 1.0 233.3 208.3 247.0 0.5 194.2 181.1 222.7 165.8 256.2 270.9 283.0 0.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z

Figure 3 DART mass spectra of acenaphthene (positive), anthracene (positive), pyrene (positive/negative) and uoranthene (positive/negative)

4 Rapid Screening and con rmation analysis of polycyclic aromatic hydrocarbons (PAHs) with DART mass spectrometry

Then, it examined the sample applied to DART located between column and DART ionization stage. separating with LC in order to perform chromatogram Furthermore, the closed interface was adopted for separation. As the suitable flow rate for DART ionization sensitivity improvement. was thought to be approximately 10uL/min, the splitter

Analytical Condition

Column : Unison UK-C8 (2.0mmI.D. x 100mm, 3um, Imtakt Corporation, Kyoto, Japan) Mobile phase : 1mM Ammonium formate / Acetonitrile=75/25 Flow rate : 0.2mL/min (to DART: 0.01mL/min) DART heater temperature : 500°C Ionization : Positive/Negative SIM mode

Injector

Pump Column

splitter

Mobile phase

Figure 4 DART devices integrated with HPLC (AMR Inc.)

5 Rapid Screening and con rmation analysis of polycyclic aromatic hydrocarbons (PAHs) with DART mass spectrometry

2:325.00(+) 60000 50000 (a) 40000 30000 SIM 325(+) Quinine 20000

10000

0 0.0 2.5 5.0 7.5 10.0 12.5 min

3:202.00(+) 50000 (b) SIM 202(+) pyrene 25000

0 4:217.00(-) 7000 6000 SIM 217(-) pyrene 5000 4000

12500 3:228.00(+) 10000 SIM 228(+) 7500 benzo[a]anthracene 5000

5000 3:154.00(+) 4000 SIM 154(+) acenaphthene 3000

25000 3:178.00(+) 20000 15000 SIM 178(+) anthracene 10000 5000

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

Figure 4 LC-DART mass chromatogram (a) Typical compound for DART; Quinine (b) PAH mixture (4 compounds)

As a result, by measurement of each PAHs standard sample. The conclusion of this examination was reagent, each retention time was able to be confirmed understood that DART MS is effective in quick screening, and also each PAH was able to be detected in each and also LC-DART MS is effective in the confirmation retention time in the measurement using a PAH mixed analysis of detected PAHs in analysis of PAHs.

Conclusions DART mass spectrometer coupled with HPLC was valuable for confirmation analysis of polycyclic aromatic hydrocarbons (PAHs)

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1448E

Fast and highly sensitive analysis of multiple drugs in ground-, surface- and wastewater

ASMS 2014 TP 583

Klaus Bollig1; Sven Vedder2, Anja Grüning2 1 Shimadzu Deutschland GmbH, Duisburg, Germany; 2 Shimadzu Europe GmbH, Duisburg, Germany Fast and highly sensitive analysis of multiple drugs in ground-, surface- and wastewater

Introduction Many pharmaceuticals from medical treatments are not understood in detail so far. The requirement for metabolized or partially degraded in the body. An even universal, reliable and fast methods for drug determination larger amount of these compounds is excreted intact and in water is steadily increasing. Highly sensitive pollutes the aquatic environment. Relevant classes of drugs triple-quad-MS systems are suitable tools for the analysis of are human or veterinary antibiotics, antiepileptics, residues in ground-, surface- and wastewater, but analgetics and lipid lowering drugs or radio-opaque development of a simple, rapid and robust method for substances. The extent of damage caused to the simultaneous measurement of trace levels of various environment and the resulting health risk for humans or different classes of analytes in complex matrices is a animals should not be underestimated, even though it is challenge.

Figure 1. LCMS-8050 triple quadrupole mass spectrometer

Method This study describes a fast LC-MS/MS method for the solvent blending function was further used for method determination of trace levels of different classes of drugs in development. High speed values for MRM recording and water. With online SPE no further sample pretreatment is the fastest polarity switching time of 5 ms are essential necessary. The quaternary system with low pressure physical parameters for a maximum of data points on gradient eluent (LPGE) and solvent blending functionality various classes of analytes in different polarities during one renders addition of a third LC-Pump unnecessary. The single analysis.

LC-MS/MS Method Optimization One of the first steps during this automated process is the adjusted. Six optimization steps were performed via flow precursor ion selection, followed by m/z adjustment of the injection analysis, each taking 30 seconds (Figure 2). The precursor. The collision energy is optimized for the most result of these automated steps was the automatic abundant fragments and finally the fragment m/z is generation of a final MRM method (Table 1).

2 Fast and highly sensitive analysis of multiple drugs in ground-, surface- and wastewater

(x1,000,000) (x1,000,000) 6.25 1:Sulfamethazin 278.70(+) 7.25 1:Sulfamethazin 279.10(+) 1:Sulfamethazin 278.80(+) 1:Sulfamethazin 279.10(+) 1:Sulfamethazin 278.90(+) Inten. (x100,000) 6.00 7.00 1:Sulfamethazin 279.10(+) 1:Sulfamethazin 279.00(+) 1:Sulfamethazin 279.10(+) 1:Sulfamethazin 279.10(+) 6.75 1:Sulfamethazin 279.10(+) 5.75 1:Sulfamethazin 279.20(+) 1:Sulfamethazin 279.10(+) 4.0 186.1 1:Sulfamethazin 279.30(+) 1:Sulfamethazin 279.10(+) 1:Sulfamethazin 279.40(+) 6.50 1:Sulfamethazin 279.10(+) 5.50 1:Sulfamethazin 279.50(+) 1:Sulfamethazin 279.10(+) 1:Sulfamethazin 279.60(+) 1:Sulfamethazin 279.10(+) 1:Sulfamethazin 279.70(+) 6.25 5.25 1:Sulfamethazin 279.10(+) 1:Sulfamethazin 279.10(+) 6.00 1:Sulfamethazin 279.10(+) 186.2 5.00 1:Sulfamethazin 279.10(+) 3.5 5.75 1:Sulfamethazin 279.10(+) 1:Sulfamethazin 279.10(+) 4.75 1:Sulfamethazin 279.10(+) 5.50 1:Sulfamethazin 279.10(+) 4.50 1:Sulfamethazin 279.10(+) 5.25 1:Sulfamethazin 279.10(+) 1:Sulfamethazin 279.10(+) 3.0 4.25 5.00

4.00 4.75

3.75 4.50 2.5 3.50 4.25

3.25 4.00 124.2

3.75 3.00 92.2 3.50 2.0 2.75 3.25 92.2 124.2 2.50 3.00 108.2 2.25 108.2 2.75 1.5 92.2 2.00 92.2 156.1 2.50 156.1 186.1 1.75 2.25 124.2 1.50 108.2 186.2 2.00 1.0 1.25 92.2 1.75 65.2 108.2 124.2 148.9 65.2 124.2 1.00 1.50 65.1 108.2 156.1 186.1 0.75 1.25 92.2 0.5 107.2 124.3 213.3 0.50 1.00 80.2 65.2 149.4 213.2 0.25 0.75 80.1 92.3 108.2 80.080.1 108.1 124.2 156.0 201.2204.1205.5 0.50 53.2 92.3 124.2 143.3 168.2 190.8197.4 0.00 0.0 0.25 -0.25 50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 m/z 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 min 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 min 1st Step: m/z Precursor adjustment 2nd Step: Setting Q1 Prerod Bias 3rd Step: Product Ion / CE selection

(x1,000,000) (x1,000,000) (x1,000,000) 1:Sulfamethazin 279.10>91.50(+) CE: -35.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 1.7 1:Sulfamethazin 279.10>92.20(+) CE: -35.0 1.45 1:Sulfamethazin 279.10>91.60(+) CE: -35.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 1:Sulfamethazin 279.10>92.20(+) CE: -34.0 1:Sulfamethazin 279.10>91.70(+) CE: -35.0 1.5 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 1:Sulfamethazin 279.10>92.20(+) CE: -33.0 1.40 1:Sulfamethazin 279.10>91.80(+) CE: -35.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 1:Sulfamethazin 279.10>92.20(+) CE: -32.0 1:Sulfamethazin 279.10>91.90(+) CE: -35.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 1.6 1:Sulfamethazin 279.10>92.20(+) CE: -31.0 1.35 1:Sulfamethazin 279.10>92.00(+) CE: -35.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 1:Sulfamethazin 279.10>92.10(+) CE: -35.0 1.4 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 1:Sulfamethazin 279.10>92.20(+) CE: -29.0 1:Sulfamethazin 279.10>92.20(+) CE: -35.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 1:Sulfamethazin 279.10>92.20(+) CE: -28.0 1.30 1:Sulfamethazin 279.10>92.30(+) CE: -35.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 1.5 1:Sulfamethazin 279.10>92.20(+) CE: -27.0 1:Sulfamethazin 279.10>92.40(+) CE: -35.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 1:Sulfamethazin 279.10>92.20(+) CE: -26.0 1.25 1:Sulfamethazin 279.10>92.50(+) CE: -35.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 1:Sulfamethazin 279.10>92.20(+) CE: -25.0 2:Sulfamethazin 279.10>185.50(+) CE: -18.0 1.3 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 2:Sulfamethazin 279.10>186.10(+) CE: -20.0 1.20 2:Sulfamethazin 279.10>185.60(+) CE: -18.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 1.4 2:Sulfamethazin 279.10>186.10(+) CE: -19.0 2:Sulfamethazin 279.10>185.70(+) CE: -18.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 2:Sulfamethazin 279.10>186.10(+) CE: -18.0 1.15 2:Sulfamethazin 279.10>185.80(+) CE: -18.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 2:Sulfamethazin 279.10>186.10(+) CE: -17.0 2:Sulfamethazin 279.10>185.90(+) CE: -18.0 1.2 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 2:Sulfamethazin 279.10>186.10(+) CE: -16.0 2:Sulfamethazin 279.10>186.00(+) CE: -18.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 1.3 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 1.10 2:Sulfamethazin 279.10>186.10(+) CE: -18.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 2:Sulfamethazin 279.10>186.10(+) CE: -14.0 2:Sulfamethazin 279.10>186.20(+) CE: -18.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 2:Sulfamethazin 279.10>186.10(+) CE: -13.0 1.05 2:Sulfamethazin 279.10>186.30(+) CE: -18.0 1.1 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 2:Sulfamethazin 279.10>186.10(+) CE: -12.0 2:Sulfamethazin 279.10>186.40(+) CE: -18.0 1:Sulfamethazin 279.10>92.20(+) CE: -30.0 1.2 2:Sulfamethazin 279.10>186.10(+) CE: -11.0 1.00 2:Sulfamethazin 279.10>186.50(+) CE: -18.0 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 2:Sulfamethazin 279.10>186.10(+) CE: -10.0 3:Sulfamethazin 279.10>64.50(+) CE: -50.0 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 3:Sulfamethazin 279.10>65.20(+) CE: -55.0 0.95 3:Sulfamethazin 279.10>64.60(+) CE: -50.0 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 1.1 3:Sulfamethazin 279.10>65.20(+) CE: -54.0 3:Sulfamethazin 279.10>64.70(+) CE: -50.0 1.0 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 3:Sulfamethazin 279.10>65.20(+) CE: -53.0 3:Sulfamethazin 279.10>64.80(+) CE: -50.0 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 3:Sulfamethazin 279.10>65.20(+) CE: -52.0 0.90 3:Sulfamethazin 279.10>64.90(+) CE: -50.0 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 3:Sulfamethazin 279.10>65.20(+) CE: -51.0 3:Sulfamethazin 279.10>65.00(+) CE: -50.0 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 1.0 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 0.85 3:Sulfamethazin 279.10>65.10(+) CE: -50.0 0.9 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 3:Sulfamethazin 279.10>65.20(+) CE: -49.0 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 3:Sulfamethazin 279.10>65.20(+) CE: -48.0 0.80 3:Sulfamethazin 279.10>65.30(+) CE: -50.0 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 3:Sulfamethazin 279.10>65.20(+) CE: -47.0 3:Sulfamethazin 279.10>65.40(+) CE: -50.0 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 0.9 3:Sulfamethazin 279.10>65.20(+) CE: -46.0 0.75 3:Sulfamethazin 279.10>65.50(+) CE: -50.0 0.8 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 3:Sulfamethazin 279.10>65.20(+) CE: -45.0 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 0.70 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 0.8 0.7 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 0.65 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 0.60 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 0.7 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 0.6 0.55 2:Sulfamethazin 279.10>186.10(+) CE: -15.0 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 0.6 0.50 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 0.5 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 0.45 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 0.5 0.40 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 0.4 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 0.35 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 0.4 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 0.30 0.3 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 0.25 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 0.3 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 0.20 0.2 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 0.2 0.15 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 0.10 0.1 3:Sulfamethazin 279.10>65.20(+) CE: -50.0 0.1 0.05

0.00 0.0 0.0

-0.05

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 min 0.000 0.025 0.050 0.075 0.100 0.125 0.150 0.175 0.200 0.225 0.250 0.275 0.300 0.325 min 0.000 0.025 0.050 0.075 0.100 0.125 0.150 0.175 0.200 0.225 0.250 0.275 0.300 0.325 min 4th Step: m/z Product Ion adjustment 5th Step: Setting Q3 Prerod Bias 6th Step: CE ne tuning

Figure 2. Automated MRM Optimization of the drug Sulfamethazin

3 Fast and highly sensitive analysis of multiple drugs in ground-, surface- and wastewater

Table 1. Optimized MRM transitions of 9 drugs

Compound Mode MRM transitions Collision energy (kV) Sulfamethazin ESI positive 279,10>186,10 / 279,10>92,20 -17 / -31 Sulfamethoxazol ESI positive 253,90>92,20 / 253,90>156,15 -26 / -15 Beza brat ESI positive 362,10>139,15 / 362,10>316,25 -25 / -15 Carbamazepine ESI positive 237,10>194,20 / 237,10>179,20 -19 / -34 Diclofenac ESI positive 296,00>214,15 / 296,00>215,15 -34 / -19 Clo bric acid ESI negative 213,00>127,00 / 213,00>85,00 15 / 15 Ibuprofen ESI negative 205,10>161,30 7 Iopamidol ESI negative 775,80>126,95 22 Iopromid ESI negative 790,00>127,00 26

Solvent Blending The solvent blending functionality entails automated method without physically changing the solvents. mobile phase preparation on a LPGE (low pressure Therefore solvent blending is a powerful tool for easy and gradient) unit which is integrated in the binary LC pumps. efficient elucidation of the SPE, the gradient and the The blending function eliminates the need of mobile phase starting conditions. During this study the solvent blending pre-mixing, as necessary with ordinary binary pumps. function was used for optimization of the SPE conditions. Mobile phase composition can simply be changed in the A second LPGE unit was used for the analytical gradient.

Traditional method Step 1 Step 2 Step 3

1: prepare 5 mmol/L Ammonium formate (pH 8.5)

200 mL 800 mL 200 mL 800 mL

2: prepare H2O

3: prepare MeOH Set these to system 5: prepare mobile phase A 6: prepare mobile phase B1 and B2 (SPE loading condition); (analytical condition and gradient) different conditions tested ! 4: prepare 0,0025%NH4OH

Mobile phase blending function LPGE Unit: 1: prepare 5 mmol/L Ammonium formate (pH 8.5) Mobile phase composition for SPE loading, solvent blending allows to change conditions automatically

2: prepare H2O Only step 1!

3: prepare MeOH nd 2 LPGE Unit: Set these to system Gradient for SPE release and separation

4: prepare 0,0025%NH4OH

Figure 3. Solvent blending functionality

4 Fast and highly sensitive analysis of multiple drugs in ground-, surface- and wastewater

HPLC/MS Work ow

Pump 1 A SPE-Column Pump 1 B SPE-Column

Analytical-Column Analytical-Column + LCMS 8050 + LCMS 8050 Autosampler Autosampler

Pump 2 Pump 2 Waste Waste

Figure 4. Scheme of online-SPE extraction (A) and analytical separation (B)

Final method

SPE Conditions

Injection volume : 250 µl SPE-column : Strata-X , 25 µm , 20*2 mm SPE-ow rate : 1 ml/min SPE-loading buffer : 1 mmol/L ammonium formate (LPGE Pump B)

Analytical Conditions (LPGE Pump A)

Column : Kinetex C8, 2.6 µm, 100*2.1 mm Flow rate : 0.5 ml/min

Solvent A : 0.0025% NH4OH Solvent B : MeOH 1 min – 2.5 min analytical separation Gradient : 0 min : 30% B : 1 min : 30% B : 1.5 min : 95% B : 4.5 min : 95% B : 4.51 min : 30% B : 6 min : 30% B (Stop)

LCMS Conditions

Interface : ESI Nebulizing Gas Flow : 2.2 L/min Heating Gas Flow : 12 L/min Interface Temperature : 400 ºC Desolvation Line Temperature : 150 ºC Heat Block Temperature : 400 ºC Drying Gas Flow : 6 L/min Polaritiy Switching Time : 5 ms

5 Fast and highly sensitive analysis of multiple drugs in ground-, surface- and wastewater

Results In this study we developed a fast method for direct online separation. Each compound was quanti ed in a SPE LC-MS/MS analysis of 9 different drugs in water with a concentration range from 0.05 ng/ml up to 2 ng/ml. minimal LC con guration of two binary pumps equipped Measurements were performed on Shimadzu’s LCMS-8050 with LPGE units. The solvent blending function was used Triple Quad MS System. The calibration curves and lowest for method development of the SPE extraction. The second calibration point is shown in gure 5. LPGE unit was used for SPE release and analytical gradient

Figure 5. Calibration curve and lowest calibration point at 0.05 ng/ml of each compound

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1444E

Multi-residue analysis of pyrethroids in soil and sediment using QuEChERS by LC/MS/MS

ASMS 2014 TP 560

Yuka Fujito1, Kiyomi Arakawa1, Yoshihiro Hayakawa1 1 Shimadzu Corporation. 1, Nishinokyo-Kuwabaracho Nakagyo-ku, Kyoto 604–8511, Japan Multi-residue analysis of pyrethroids in soil and sediment using QuEChERS by LC/MS/MS

Introduction Pytrethroids are one of the most widely used commercial to the in uence on the insects and aquatic invertebrates. household insecticides in agricultural or non-agricultural Therefore, quick, high-sensitive and universal analysis application sites. Synthetic pyrethroids are poorly methods are required. The analysis of pyrethroids is water-soluble, but are strongly adsorbed to soil, therefore typically performed by GC or GC/MS because of their these compounds are increasingly being found in soil or hydrophobicity. In this study, we report the development sediments. Recently, soil and sediment contamination by of a simultaneous analysis technique for trace amounts of pyrethroids has been detected in both urban and pyrethroids by LC/MS/MS. agricultural area, and it’s becoming a global concern due

Materials and Methods Materials

Sample Sampling point Soil Residential garden (Kyoto, Japan) Sediment Lake Biwa (Shiga, Japan) Pyrethrin

I : R=CH3

II : R=CO2CH3 Cyhalothrin

Permethrin

Te uthrin

Esfenvalerate

Figure 1 Chemical structure of pyrethroids

Sample preparation Sample preparation was carried out by the use of the amount of the soil and water added, we nally adopted a QuEChERS method. In case of the soil samples, hydration combination of 5 g soil (or 10 g sediment) and 5 mL water, of sample with water before acetonitrile extraction is and the following procedures were based on the original required to improve the recovery and operability. Result of QuEChERS method. several different extraction methods that changed the

2 Multi-residue analysis of pyrethroids in soil and sediment using QuEChERS by LC/MS/MS

Step 1 : Acetonitrile extraction Step 2 : Clean-up

Weigh 5 g soil / 10 g sediment Transfer 6mL Extract 1 into dSPE tube*2 (Add STDs solution) • 900 mg MgSO4 • 150 mg PSA • 45 mg GCB Add 5mL water

Shake vigorously by hand 1min. Add 10mL acetonitrile

Centrifuge for 5min. Add salt mixture*1

• 4g MgSO4 • 1g NaCl • 1g Trisodium citrate dehydrate • 0.5g Disodium hydrogencitrate sesquihydrate Transfer the supernatant into a vial

Shake vigorously by hand 1min. Filtration using disposable lter

Centrifuge for 10min. (Extract 1) LC/MS/MS analysis

*1 : Q-sep QuEChERS Extraction Salts (RESTEK) *2 : Q-sep QuEChERS dSPE Tubes (RESTEK)

LC/MS/MS analsis

HPLC conditions (Nexera UHPLC system, Shimadzu)

Column : Phenomenex Kinetex 2.6 µm PFP 100Å (100 mm x 2.1 mm I.D.) Mobile phase : A 5mM ammonium acetate - water : B Methanol Gradient program : 40 % B (0 min.) → 100 % B (10 -12 min.) → 40 % B (12.01-15 min.) Flow rate : 0.2 mL / min. Column temperature : 40 ºC Injection volume : 1 μL

MS conditions (LCMS-8050, Shimadzu)

Ionization : ESI (positive / negative) Interface temperature : 100 ºC DL temperature : 100 ºC Heat block temperature : 400 ºC Nebulizing gas : 3.0 L / min. Drying gas : 15.0 L / min. Heating gas : 3.0 L / min.

3 Multi-residue analysis of pyrethroids in soil and sediment using QuEChERS by LC/MS/MS

Table 1 MRM transitions of pyrethroids

Compounds Polarity Quantitative ion (m/z) Con rmation ion (m/z) pyrethrin-I + 329.20>161.20 329.20>105.20 pyrethrin-II + 373.20>161.20 373.20>105.20 fenpropathrin + 367.20>125.20 367.20>181.20 cycloprothrin + 498.90>181.10 498.90>229.20 deltamethrin + 522.80>280.90 522.80>181.10 esfenvalrate + 437.10>167.30 437.10>125.30 cypermethrin + 433.10>191.10 433.10>181.20 cy uthrin + 450.90>191.00 450.90>206.10 ethofenprox + 394.20>177.30 394.20>107.20 permethrin + 408.10>183.30 408.10>355.20 cyhalothrin + 467.10>225.10 467.10>141.10 bifenthrin + 440.00>181.20 440.00>166.10 acrinathrin + 559.00>208.20 559.00>181.10 acrinathrin - 540.10>372.20 540.10>345.30 sila uofen + 426.20>287.10 426.20>168.20

High Speed Mass Spectrometer Ultra Fast Scanning - 30,000 u / sec. Ultra Fast Polarity Switching - 5 msec. Ultra Fast MRM - Max. 555 transitions / sec

Figure 2 LCMS-8050 triple quadrupole mass spectrometer

Result MRM of pyrethroid standards In this study, we selected and evaluated 15 pyrethroids agrocultural insecticides worldwide. (pyrethrin, fenpropathrin, cycloprothrin, deltamethrin, Except for tefluthrin, which was not ionized by LC/MS under esfenvarelate, cypermethrin, cyfluthrin, ethofenprox, conditions tested, all other 14 compounds were successfully permethrin, cyhalothrin, bifenthrin, acrinathrin, tefluthrin, detected in ESI positive mode or in both positive and silafruofen) which are the most widely used for household or negative mode.

4 Multi-residue analysis of pyrethroids in soil and sediment using QuEChERS by LC/MS/MS

Table 2 Calibration curves 1500000 compounds min. conc. max. conc. r2 1400000 pyrethrin I 0.5 500 0.9996 1300000 pyrethrin II 0.5 500 0.9997 1200000 fenpropathrin 0.02 100 0.9993 1100000 pyrethrin-II cycloprothrin 0.5 100 0.9991 1000000 pyrethrin-I deltamethrin 0.05 100 0.9992 900000 fentropathrin esfenvalerate 0.5 100 0.9990 800000 cycloprothrin cypermethrin 0.05 100 0.9986

700000 deltamethrin cy uthrin 0.5 100 0.9976 esfenvalrate 600000 ethofenprox 0.01 100 0.9993 cypermethrin trans-permethrin 0.02 100 0.9996 500000 cy uthrin cis-permethrin 0.02 100 0.9994 400000 ethofenprox cyhalothrin 0.1 100 0.9993 300000 permethrin bifenthrin 0.02 100 0.9995 200000 cyhalothrin bifenthrin acrinathrin (+) 0.1 100 0.9987 100000 acrinathrin acrinathrin (-) 0.5 500 0.9993 0 sila uofen sila uofen 0.01 100 0.9999

7.0 8.0 9.0 10.0 min (ppb)

Figure 3 MRM chromatograms

fenpropathrin permethrin bifenthrin silauofen 0.02 ppb 0.02 ppb 0.02 ppb 0.01 ppb (x1,000) (x1,000) (x1,000) (x1,000) 1.25 2.50 1.75 2.25 2.25 1.00 1.50 cis- 2.00 2.00 1.75 1.25 trans- 1.75 0.75 1.50 1.50 1.00 1.25 1.25 0.50 1.00 0.75 1.00 0.75 0.50 0.75 0.50 0.25 0.50 0.25 0.25 0.25 0.00 0.00 0.00 0.00

9.0 9.5 9.5 10.0 9.5 10.0 10.5 10.0 10.5 11.0

Figure 4 MRM chromatograms of the LOQs of typical pyrethroids

5 Multi-residue analysis of pyrethroids in soil and sediment using QuEChERS by LC/MS/MS

Recovery from soil and sediment matrices All target compounds showed good recoveries from soil and method. Neither matrix effect (Ion suppression or sediment matrices in the range 70-120% by the QuEChERS enhancement) nor sample preparation losses were observed.

soil (residential garden) sediment (lake)

140 140 STDs spiked after prep STDs spiked before prep 120 120

100 100

80 80

60 60

Recovery (%) 40 40

20 20

0 0

Cyuthrin Bifenthrin Cyuthrin Bifenthrin Pyrethrin-2Pyrethrin-1 CyhalothrinAcrinathrin Silauofen Pyrethrin-2Pyrethrin-1 CyhalothrinAcrinathrin Silauofen CycloprothrinDeltamethrinEsfenvalrate Ethofenprox CycloprothrinDeltamethrinEsfenvalrate Ethofenprox Fenpropathrin Cypermethrin cis-Permethrin Fenpropathrin Cypermethrin cis-Permethrin trans-Permethrin trans-Permethrin

Figure 5 Recovery of 14 pyrethroids from soil and sediment matrices (10 ppb spiked)

Quantitative analysis of soil and sediment The quantitative analysis of the soil and sediment sample from the soil sample at approximately 0.02 and 0.06 μg / was performed. Ethofenprox and permethrin was detected kg, respectively.

ethofenprox permethrin Table 3 Result of quantitative analysis in the soil and sediment soil sediment pyrethrin-I n.d. n.d. soil blank pyrethrin-II n.d. n.d. fenpropathrin n.d. n.d. cycloprothrin n.d. n.d. solvent blank deltamethrin n.d. n.d. esfenvalrate n.d. n.d. cypermethrin n.d. n.d. cy uthrin n.d. n.d. ethofenprox 0.01 ppb* n.d. Figure 6 Chromatograms of prethroids in the soil permethrin 0.03 ppb n.d. cyhalothrin n.d. n.d. bifenthrin n.d. n.d. acrinathrin n.d. n.d. sila uofen n.d. n.d. n.d. : not detected * :

6 Multi-residue analysis of pyrethroids in soil and sediment using QuEChERS by LC/MS/MS

Conclusions • A method for quantication of 14 pyrethroids in soil and sediment at ppt-level concentrations was developed by LC/MS/MS. • In this study, neither matrix effect nor sample preparation losses were observed in the recovery test, demonstrating the applicability of QuEChERS method to sample preparation of soil and sediment.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 Metabolism • Page 197 High sensitivity analysis of metabolites in serum using simultaneous SIM and MRM modes in a triple quadrupole GC/MS/MS

• Page 202 Analysis of D- and L-amino acids using auto- mated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry

• Page 208 Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/ time-of-flight mass spectrometry

• Page 213 Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta- fluorophenylpropyl column PO-CON1443E

High Sensitivity Analysis of Metabolites in Serum Using Simultaneous SIM and MRM Modes in a Triple Quadrupole GC/MS/MS

ASMS 2014 ThP 641

Shuichi Kawana1, Yukihiko Kudo2, Kenichi Obayashi2, Laura Chambers3, Haruhiko Miyagawa2 1 Shimadzu, Osaka, Japan, 2 Shimadzu, Kyoto, Japan, 3 Shimadzu Scienti c Instruments, Columbia, MD High Sensitivity Analysis of Metabolites in Serum Using Simultaneous SIM and MRM Modes in a Triple Quadrupole GC/MS/MS

Introduction Gas chromatography / mass spectrometry (GC–MS) and a gas chromatography-tandem mass spectrometry (GC-MS/MS) are highly suitable techniques for metabolomics because of the chromatographic separation, reproducible retention times and sensitive mass detection. MRM measurement mode Some compounds with low CID ef ciency produce insuf cient product ions for MRM transitions, and the MRM mode is consequently less sensitive than SIM for these compounds. Our suggestion SIM, MRM, and simultaneous SIM/MRM modes are evaluated for analysis of metabolites in human serum.

Materials and Method Sample and Sample preparation

Sample • Human serum

Sample Preparation1)

50uL serum Supernatant 250 µL

Add 250 µL water / methanol / chloroform (1 / 2.5 / 1) Freeze-dry Add internal standard (2-Isopropylmalic acid) Stir, then centrifuge Residue

Extraction solution 225 µL Add 40 µL methoxyamine solution (20 mg/mL, pyridine) Heat at 30 ºC for 90 min Add 200 µL Milli-Q water Add 20 µL MSTFA Stir, then centrifuge Heat at 37 ºC for 30 min

Sample

1) Nishiumi S et. al. Metabolomics. 2010 Nov;6(4):518-528 Instrumentation

GC-MS : GCMS-TQ8040 (SHIMADZU) Data analysis : GCMSsolution Ver.4.2 Database : GC/MS Metabolite Database Ver.2 (SHIMADZU) Column : 30m x 0.25mm I.D., df=1.00µm (5%-Phenyl)-methylpolysiloxane

2 High Sensitivity Analysis of Metabolites in Serum Using Simultaneous SIM and MRM Modes in a Triple Quadrupole GC/MS/MS

Simultaneous SIM and MRM modes in GC/MS/MS Figure 1 shows the theory of Simultaneous SIM and MRM modes. This analysis mode can measure SIM and MRM data in a single analysis.

Q1 Collision Cell Q3

SIM

SIM MRM

MRM SIM CID SIM

Figure 1 The concept of simultaneous SIM and MRM analysis mode.

Precursor ion (or SIM) Product ion % % 100 100 361 CID 169 75 73 75 50 50 217 103 25 147 73 103 271 437 25 243 191 243 319 361 0 0 100 200 300 400 100 200 300

Figure 2 Mass Spectrum of Precursor (or SIM) and Product ion

Poor sensitivity of MRM in some compounds because of low CID ef ciency

Method Creation using Database and SmartMRM Figure 3 shows the GC/MS Metabolites Database Ver.2. This database involves conditions of SIM and MRM in 186 metabolites and a method creation function we call SmartMRM. SmartMRM creates MRM, SIM, SIM/MRM methods from Database automatically.

Figure 3 GC/MS Metabolites Database Ver.2

• Select the MRM, SIM and SIM/MRM conditions of 186 TMS derivatization metabolites from GC/MS Metabolites Database Ver.2. • Select the two transitions (or ions) each metabolite.

3 High Sensitivity Analysis of Metabolites in Serum Using Simultaneous SIM and MRM Modes in a Triple Quadrupole GC/MS/MS

Results Comparison of the chromatogram between SIM and MRM in human serum

a) Glucuronic acid-meto-5TMS(2)

(x100,000) (x10,000) 333.10 333.10>143.10 SIM 3.5 160.10 MRM 1.75 333.10>171.10 3.0 1.50 2.5 1.25 2.0 1.00 1.5 0.75 1.0 0.50 0.5 0.25

21.00 21.25 21.00 21.25

Detected the peak in MRM because of high selectivity

b) S-Benzyl-Cysteine-4TMS (x100) (x100,000) (x10,000) 1.75 238.10>91.00 2.00 238.10 218.10>73.00 SIM 218.10 MRM 7.5 238.10>91.00 1.50 1.75 1.25 1.50 1.00 0.75 5.0 1.25 0.50 1.00 0.25 21.00 21.25 21.50 0.75 2.5 0.50 0.25

21.25 21.50 21.00 21.25 21.50

Peak was not detected in MRM because of low CID ef ciency.

A number of Identi cation metabolites in serum Table 1 shows the identi cation results of metabolites in human serum using SIM, MRM and simultaneous SIM/MRM analysis modes in GC/MS/MS. In SIM/MRM, the metabolites, which were insuf cient sensitivity in MRM, were measured by SIM and the other metabolites were measured by MRM.

Table 1 The number of identi ed metabolites each analysis mode

Modes A B C Total SIM 57 51 8 116 MRM 131 14 1 146 SIM/MRM 133 22 1 156

note) A:Target and Con rmation ions were detected.; B: Either Target or Con rmation ion was detected. Another one was overlapped by contaminants.; C: Either Target or Con rmation ion was detected.

4 High Sensitivity Analysis of Metabolites in Serum Using Simultaneous SIM and MRM Modes in a Triple Quadrupole GC/MS/MS

Fig.4 shows a number of metabolites in each mode can be measured. In metabolites with low CID ef ciency, SIM are superior to MRM if there are no interfering substances to the target metabolites. MRM SIM

40 106 10

Metabolites with Metabolites with low CID interference in SIM ef ciency in MRM

Figure 4 Detected metabolites in human serum each analysis mode.

The reproducibility(n=6) in MRM and SIM/MRM

Table 2 Comparison of the reproducibility results from MRM and SIM/MRM analysis. A number of detected metabolites involves A, B and C in Table 1.

%RSD MRM SIM/MRM Improvement - 4.99% 73 76 +3 5 - 9.99% 26 30 +4 10 - 14.99% 8 10 +2 15 - 19.99% 9 10 +1 > 20% 30 30 0 146 156 +10

Conclusions • Analytical results from the SIM and MRM modes identi ed 116 and 146 metabolites, respectively. • In metabolites with poor CID ef ciency, the sensitivity of SIM is more than 10 times higher than MRM. • Simultaneous SIM and MRM modes in a single analysis (SIM/MRM) improves the sensitivity and reproducibility for analysis of metabolites in human serum compared to MRM alone. • A novel SIM/MRM expands the utility of a triple quadrupole GC/MS/MS

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1451E

Analysis of D- and L-amino acids using automated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry

ASMS 2014 MP739

Kenichiro Tanaka1; Hidetoshi Terada2; Yoshiko Hirao2; Kiyomi Arakawa2; Yoshihiro Hayakawa2 1. Shimadzu Scienti c Instruments, Inc., Columbia, MD; 2. Shimadzu Corporation, Kyoto, Japan Analysis of D- and L-amino acids using automated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry

Introduction Recently, several species of D- amino acids have been good reliability. One of the drawbacks of pre-column found in mammals including humans and their derivatization is less reproducibility due to the tedious physiological functions have been elucidated. Quantitating manual procedure and human errors. We have launched each enantiomer of amino acids is indispensable for such an autosampler for a UHPLC system equipped with an studies. In order to diagnose diseases, it is desirable that D- automated pretreatment function that allows overlapping and L-amino acid can be separately quantitated and injections in which the next derivatization proceeds during applied to metabolic analysis. the current analysis for saving total analytical time. We Pre-column derivatization with o-phthalaldehyde (OPA) and have applied this autosampler and its function to fully N-acetyl-L-cysteine(NAC) is widely utilized for the analysis automate pre-column derivatization for the determination of D- and L- amino acids since the method can be of amino acids. In this study, we developed a methodology performed with a rapid reversed phase separation on a which enabled the automated procedure of pre-column relatively simple hardware (U)HPLC con guration with chiral derivatization of D- and L- amino acids.

Experimental Instruments The system used was a SHIMADZU UHPLC Nexera workstation (LabSolutions, Shimadzu Corporation, Japan) pre-column Amino Acids (AAs) system consisting of so that selected conditions can be seamlessly translated LC-30AD solvent delivery pump, DGU-20A5R degassing into method les and registered to a batch queue, ready unit, SIL-30AC autosampler, CTO-30A column oven, and for instant analysis. A 1.9um YMC-Triart C8 column (2.0 SHIMADZU triple quadrupole mass spectrometer mm x 150 mm L.) was used for the analysis. LCMS-8040. The software is integrated in the LC/MS/MS

Derivatization Method Derivatizing solutions: 0.1 mol/L boric acid buffer was prepared by dissolving 6.18 mg of o-phthalaldehyde in 0.3 mL of ethanol, adding 0.7 g of boric acid and 2.00 g of sodium hydroxide in 1 L of mL of the 0.1 mol/L boric acid buffer and 4 mL of water. water. Fig.1 shows the schematic procedure for amino acids 10 mmol/L NAC solution was prepared by dissolving 16.3 derivatization with the SIL-30AC. mg of N-acetyl-L-cysteine in 10 mL of the 0.1 mol/L boric Samples, including the derivatized amino acids, were acid buffer. injected onto the UHPLC and separated under the 10 mmol/L OPA solution was prepared by dissolving 6.7 conditions shown in Table 1.

2 Analysis of D- and L-amino acids using automated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry

(1) (2) (3) (4) (5)

Take 20 μL of 10 mmol/L Supply 20 μL of Take 20μL of Supply 20 μL of Take 1 μL of NAC solution NAC solution to the 10 mmol/L OPA solution 10 mmol/L OPA solution sample solution vial for mixing to the vial for mixing

(6) (7) (8) (9) (10)

Supply 1 μL of Mix the sample solution Wait for 3min until Take 1μL of the mixed Inject 1μL of the mixed sample solution and derivatizing solutions the derivatization ends solution solution to the column to the vial for mixing

Fig.1 Schematic procedure of automated pre-column derivatization

Table 1 UHPLC and MS analytical conditions

Mobile Phase : A : 10 mmol/L Ammonium Bicarbonate solution B : Acetonitrile/Methanol = 1/1(v/v) Initial B Conc. : 0% Flow Rate : 0.4 mL/min Column Temperature : 40 ºC Injection Volume : 1 μL LC Time Program : 0 -> 5%(0.01min), 5%(0.01-1.00min), 5 ->20%(1.00 - 15.00min), 20 - 25%(15.00 - 24.00min), 25 – 90%(24.00 - 24.50min), 90%(24.50 - 27.50min), 90 - 0% (27.50 – 28.50min) Ionization Mode : ESI Nebulizing Gas Flow Rate : 3 L/min Drying Gas Flow Rate : 15 L/min DL Temperature : 300 ºC Heating Block Temperature : 450 ºC

Result Analysis of Standard Solution A standard solution containing 27 amino acids was using the function for automatic MRM optimization. The prepared at 1 mmol/L concentration each in 0.1 mol/L HCl transition that provided the highest intensity was used for solution. The MS conditions such as ESI positive and quanti cation. negative ionization modes were optimized in parallel with Table 2 shows the MRM transition of each derivatized the column separation, and compound dependent amino acid. parameters such as CID and pre-bias voltage were adjusted The MRM chromatogram is illustrated in Fig.2.

3 Analysis of D- and L-amino acids using automated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry

Table 2 Compounds, Ionization polarity and MRM transition

Compound Polarity Precursor m/z Product m/z Aspartic acid + 395.00 130.00 Glutamic acid + 409.10 130.05 Serine + 367.00 130.00 Glutamine + 408.20 130.05 Glycine + 337.00 130.00 Histidine + 417.10 244.05 Threonine + 381.20 130.05 Arginine + 436.10 263.10 Tyrosine + 443.00 130.05 Valine + 379.10 250.05 Tryptophan + 466.20 337.10 Isoleucine + 393.00 264.05 Phenylalanine + 427.20 298.05

250000 9 225000 1 2 200000 6

175000 5 3 4 11 15 7 150000 16 8 14 17 125000 10 13 20 100000 12 18 19 24 75000 21 27 22 23 50000 26 25000 25

0

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

■Peaks 1. D-Aspartic acid, 2. L-Aspartic acid, 3. L-Glutamic acid, 4. D-Glutamic acid, 5. D-Serine, 6. L-Serine, 7. L-Glutamine 8. D-Glutamine, 9. Glycine, 10. L-Histidine, 11. D-Histidine ,12. D-Threonine, 13. L-Threonine, 14. L-Arginine 15. D-Arginine, 16. D-Alanine, 17. L-Alanine, 18. D-tyrosine, 19. L-Tyrosine, 20. L-Valine, 21. D-Valine 22. L-Tryptophan, 23. D-Tryptophan, 24. L-Isoleucine, 25. D-Phenylalanine, 26. L-Phenylalanine, 27.D-Isoleucine

Fig. 2 Chromatogram of a 27 amino acid standard solution

4 Analysis of D- and L-amino acids using automated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry

Method Validation Reproducibility and linearity in this analysis were evaluated spiked sample concentration from 1 to 100 μmol/L with a plasma spiked standard solution. As a result, less standard solution were used for the linearity evaluation. than 5% relative standard deviation of peak areas were The coef cients of determination (r2) were approximately obtained. Table 3 shows the reproducibility of repeated 0.999. Table 4 shows the summary for the linearity results. analysis of spiked sample (n=6). Five different levels of

Table 3 Reproducibility

Repeatability (%RSD) Compound 5 μmol/L 25 μmol/L D-Aspartic acid 3.5 2.5 D-Glutamic acid 3.7 3.1 D-Serine 4.8 3.0 D-Glutamine 4.1 3.4 D-Histidine 4.3 1.8 D-Threonine 3.8 2.6 D-Arginine 3.4 1.7 D-Alanine 4.0 2.3 D-Tyrosine 3.2 2.9 D-Valine 3.3 2.2 D-Tryptophan 3.9 3.2 D-Isoleucine 3.1 2.9 D-Phenylalanine 3.5 1.8

Table 4 Linearity

Compound Cali.F r2 D-Asparic acid Y = (44661.8)X + (1829.61) 0.998 D-Glutamic acid Y = (12191.8)X + (10390.7) 0.999 D-Serine Y = (22319.5)X + (-2869.30) 0.999 D-Glutamine Y = (3458.60)X + (1521.83) 0.999 D-Histidine Y = (5778.33)X + (-341.182) 0.998 D-Threonine Y = (10800.6)X + (-1874.07) 0.999 D-Arginine Y = (10535.7)X + (-1298.12) 0.998 D-Alanine Y = (15349.1)X + (-4719.98) 0.999 D-Tyrosine Y = (17098.7)X + (-1812.69) 0.999 D-Valine Y = (23707.7)X + (772.548) 0.999 D-Tryptophan Y = (18089.1)X + (-3620.41) 0.998 D-Isoleucine Y = (44017.1)X + (67903.1) 0.999 D-Phenylalanine Y = (22426.0)X + (-736.090) 0.999

5 Analysis of D- and L-amino acids using automated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry

Table 5 Recovery

Recovery (100%) Compound 5 μmol/L 25 μmol/L D-Asparic acid 100.3 107.1 D-Glutamic acid 92.8 97.8 D-Serine 97.9 100.6 D-Glutamine 103.2 104.3 D-Histidine 104.8 100.4 D-Threonine 101.1 98.8 D-Arginine 102.4 99.6 D-Alanine 93.5 99.5 D-Tyrosine 98.1 101.0 D-Valine 101.0 99.2 D-Tryptophan 97.8 100.4 D-Isoleucine 98.8 102.4 D-Phenylalanine 104.5 100.9

Considering the frequency of amino acids analysis in physiological samples, the recovery of spiked samples were con rmed. In addition, the results indicated that the recovery ratio of most amino acids are around 100%. Table 5 shows the summarized results for the recovery of each amino acid.

Conclusions • The combination of Shimadzu triple quadrupole mass spectrometer and Nexera UHPLC provides reliable pre-column derivatized AAs analysis with enhanced productivity. • An established method was successfully applied to the separation of D- and L- amino acids with excellent reliability.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1476E

Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/time-of- ight mass spectrometry

ASMS 2014 WP 739

Cuiping Yang1, Changkun Li2, Tianhong Zhang1, Qian Sun2, Yueqi Li2, Guixiang Yang2, Taohong Huang2, Shin-ichi Kawano2, Yuki Hashi2, Zhenqing Zhang1,* 1Beijing Institute of Pharmacology & Toxicology, 2Shimadzu Global COE, Shimadzu (China) Co., Ltd., China Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/time-of- ight mass spectrometry

Introduction Aconitine (AC) is a bioactive alkaloid from plants of the metabolites of AC in liver microsomes are limited. The genus Aconitum, some of which have been widely used as study of metabolic pathways is very important for ef cacy medicinal herbs for thousands of years. AC is also well of therapy and evaluation of toxicity for those with narrow known for its high toxicity that induces severe arrhythmias therapy window. leading to death. Although numerous studies have raised The aim of our work was to obtain the metabolic pathways on its pharmacology and toxicity, data on the identi cation of AC by the human liver microsomes.

Methods and Materials Sample Preparation The typical reaction mixture incubation contained 10 μ 60 min. The reactions were terminated by adding 3-volume mol/L aconitine and was preincubated at 37 ºC for 3 min. of ice-cold acetonitrile, then vortexed and centrifuged to Reactions were initiated by adding 50 μL of NADPH (20 remove precipitated protein. mmol/L), then incubated at 37 ºC in a waterbath shaker for

Instrument : LCMS-IT-TOF (Shimadzu Corporation, Japan); UFLCXR system (Shimadzu Corporation, Japan); Column : Shim-pack XR-ODS II (2.0 mmI.D. x 75 mmL.,2.2 μm) Mobile phase : A: water (0.1% formic acid+5 mmol ammonium formate), B: acetonitrile Gradient program : 30%B (0-4 min)-80%B (8 min)-80%B (8-11 min)-30%B (11.01-17 min) Flow rate : 0.3 mL/min

Results

(x1,000,000) 7.5 1:TIC (1.00)

A 5.0

2.5

0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0

(x1,000,000) 7.5 B 5.0 M10

M3 M5 M2 2.5 M6 M12 M15 M16 M4 M7 M11 M13 M14 M0 M1 M9 M8 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5

Fig.1 TIC chromatogram (A) and mass chromatograms of the metabolites of AC in the microsomal incubation mixture of human (B)

2 Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/time-of- ight mass spectrometry

OH O O OH OH+ O OH O O O O O HN OH O N OH OH O + H N OH O OH O O H C H NO + O OH 29 36 8 O + H O O Exact Mass: 526.2441 O

OH + C H NO O 34 48 11 C H NO + O Exact Mass: 646.3227 32 44 9 Exact Mass: 586.3016 O N OH

OH + H O O + OH C31H40NO8 O OH OH O O Exact Mass: 554.2754 OHH+ O O

O O OHH+ N OH O O N HN OH OH + H OH O O O HN + H H O O O C H NO + 25 36 9 H C H NO + Exact Mass: 494.2390 C H NO + HO 25 34 8 22 26 4 C H NO + Exact Mass 368.1862 21 25 4 Exact Mass: 476.2284 Exact Mass 354.1705

Fig. 2 Proposed fragmentation pathway of AC

OH O O O OH OH OH OH O OH O O OH O N O O O OH O N HO O OH O H HOH2C O N OH O OH HO HO N O O OH O H O O OH O HO O H O M6 O O O H O O O M2 M9 O O O M8 N OH HO O H OH O OH OH O O O O O OO M4

HOH C OH 2 O O O N OH O OH OH N OH OH O OH O OH O H O O H O N O OH O O O O M13 O N M11 OH OH O H O O HO O M7 O H O OH HO HOH C O 2 O OH O O OH O O M10 O O O O

O N OH O O N N OH OH HO O H HO O OH O O H O H O O O M14 O HO O O M15 M0 OH OH O OO O OO OH O O O O N OH O HN OH O OH H O O N OH O OH O H O M12 O OH O OH H O O M3 M1

OH OH O O O O O O

O O N N OH OH

OH O H H O O O M5 O M16

Fig. 3 Proposed metabolic pro le of AC in the human liver microsomes

3 Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/time-of- ight mass spectrometry

Table1 Mass data for characterization of metabolites in of AC in the microsomal incubation mixture of human

RT Meas.MW Pred.MW mDa ppm No. MS2 data Formula Biotransformation (min) (m/z) (m/z) error error 586.3000, 554.2752, 526.2785, 494.2536, M0 22.3 646.3230 646.3222 0.8 1.26 C34H47NO11 Parent 476.2431, 404.2432, 368.1847, 354.1687 558.2710, 498.2469, 480.2378, 436.2093, M1 10.5 618.2922 618.2909 1.3 2.10 C32H43NO11 deethylation 354.1725 556.2510, 554.2335, 494.2106, 478.2321, bidemethylation+ M2 11.2 616.2754 616.2752 0.2 0.26 C32H41NO11 434.1908, 402.1682 dehydrogenation

M3 11.3 604.3140 604.3116 2.4 3.94 554.2744, 522.2398, 434.1898 C32H45NO10 deacetylation

demethylation+ M4 11.8 630.2930 630.2909 2.1 3.35 570.2686, 552.2576, 510.2457, 492.2381 C33H43NO11 dehydrogenation 568.2938, 554.2705, 522.2537, 466.2168, deacetylation+ M5 12.2 586.3005 586.3011 0.6 0.96 C32H43NO9 434.1922 dehydration bidemethylation+ M6 13.3 616.2769 616.2752 2.3 3.68 584.2477, 524.2316, 434.1941 C32H41NO11 dehydrogenation 572.2866, 512.2638, 494.2468, 480.2283, M7 13.5 632.3035 632.3065 3.0 4.81 C33H45NO11 demethylation 462.2214, 290.2236, 354.1652, 340.1871 588.2702, 570.2654, 528.2566, 510.2434, oxidation+ M8 13.7 648.3016 648.3015 0.1 0.23 C33H45NO12 406.2161 demethylation

M9 13.8 618.2935 618.2909 3.0 4.88 558.2714, 494.2109, 476.2400, 340.1548 C32H43NO11 bidemethylation

M10 14.1 618.2890 618.2909 1.5 2.43 558.2722, 494.2127, 476.2009, 354.1635 C32H43NO11 bidemethylation

602.2964, 570.2654, 542.2750, 510.2434, M11 15.0 662.3179 662.3171 0.8 1.21 C34H47NO12 oxidation 420.2416 deacetylation+ M12 15.1 602.2948 602.2960 1.6 2.66 584.2533, 524.2249, 510.2179, 406.1582 C32H43NO10 dehydrogenation 572.2853, 512.2661, 480.2368, 476.2445, M13 16.0 632.3054 632.3065 1.1 1.80 C33H45NO11 demethylation 436.2082, 368.1812 602.2947, 570.2654, 542.2766, 510.2434, M14 17.3 662.3209 662.3171 3.8 5.74 C34H47NO12 oxidation 478.2187

M15 17.6 632.3068 632.3065 0.3 0.42 586.2973, 526.2738, 508.2273, 494.2490 C33H45NO11 demethylation

deacetylation+dehydration+ M16 17.9 584.2826 584.2854 2.8 4.82 552.2669, 492.2111, 460.2063 C32H41NO9 dehydrogenation

4 Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/time-of- ight mass spectrometry

Conclusions In this study, totaling 16 metabolites were found and characterized in the humam liver microsomes incubation mixture, including O-demethylation, oxidation, bidemethylation, dehydrogenation, N-deethylation, deacetylation, dehydration and besides M1, M3, M4, M9, M13 and M15, all the left ten of them were rst identi ed and reported. Collectively, these data provide a foundation for the clinical use of AC and contributes to a wider understanding of xenobiotic metabolism and toxicity evaluation.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1447E

Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta uorophenylpropyl column

ASMS 2014 WP 613

Tsuyoshi Nakanishi1, Takako Hishiki2, Makoto Suematsu2,3 1 Shimadzu Corporation, Kyoto, Japan, 2 Department of Biochemistry, School of Medicine, Keio University, Tokyo, Japan, 3 Japan Science and Technology Agency, Exploratory Research for Advanced Technology, Suematsu Gas Biology Project, Tokyo, Japan Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta uorophenylpropyl column

Introduction Various metabolic pathways are controlled to keep a simultaneous measurement of 97 metabolites by triple biological function in the cell and to monitor the rapid quadrupole LC/MS/MS using pentauorophenylpropyl and slight changes of these metabolism, a simple column. In this experiment, MRM transitions of these simultaneous analysis is required for quanti cation of metabolites were optimized and this method was primary metabolites. A typical LC/MS system with an applied to biological samples. Furthermore, to evaluate ODS column is not effective to measure primary the accuracy of developed method for quanti cation, metabolites because of low af nity of ODS column to simultaneous analysis by PFPP column was compared to hydrophilic metabolites. Here we report the measurement of ion-paring chromatography.

Methods and materials Commercially available compounds were used as acid (MES) as internal standards. After a general standards to optimize MRM transition and LC condition chloroform/methanol extraction, upper aqueous layer for separation. Mixed standard solutions were diluted to ltered through 5-kDa cutoff lter. The ltrate was dried a range of 10 nM~10000 nM for a calibration curve and up and dissolved in 0.1 mL puri ed water. Further, the an aliquot of 3 µL was subjected to LC/MS/MS solution was diluted to 20-100 folds in puri ed water. measurement. An aliquot of 3 µL was analyzed to measure primary Mice were sacri ced under anesthesia and the isolated metabolites by LC/MS instrument, Nexera UHPLC system heart/liver tissues were rapidly frozen in liquid nitrogen. and LCMS-8030/LCMS-8040 triple quadrupole mass Frozen liver or heart tissues (>50 mg) from mice were spectrometer. The following is detailed conditions of homogenized in 0.5 mL methanol including LC/MS mesurement. L-methionine sulfone and 2-morpholinoethanesulfonic

2 Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta uorophenylpropyl column

UHPLC conditions (Nexera system using a PFPP column)

Column : Discovery HS F5 150 mm×2.1 mm, 3.0 µm Mobile phase A : 0.1% Formate/water B : 0.1% Formate/acetonitrile Flow rate : 0.25 mL/min Time program : B conc.0%(0-2.0 min) - 25%(5.0 min) - 35%(11.0 min) - 95%(15.0.-20.0 min) - 0%(20.1-25.0 min) Injection vol. : 3 µL Column temperature : 40°C

MS conditions (LCMS-8030/LCMS-8040)

Ionization : Positive/Negative, MRM mode DL Temp. : 250°C HB Temp : 400°C Drying Gas : 10 L/min Nebulizing Gas : 2.0 L/min

Result Optimization of MRM transition The MRM transitions for 97 standard compounds were condition described in Figure 1. The linearity of this optimized on both positive and negative mode by flow method was also confirmed by the simultaneous analysis of injection analysis (FIA). The MRM transitions of the 97 a serial of diluted calibration curve. metabolites were determined as described in Table 1. Subsequently, LC condition was investigated to separate Figure 1 shows the MRM chromatogram of 97 metabolites the 97 metabolites with a good resolution. As a at a concentration of 5 µM. In this figure, we can see the consequence, the 97 metabolites were eluted from a PFPP peak from all metabolites with a good separation. column with a gradient of acetonitrile for <15 min in the

3 Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta uorophenylpropyl column

Table 1 MRM transition of 97 metabolites

No. Name Product ion Precursor ion Polarity Linearity (R2) No. Name Product ion Precursor ion Polarity Linearity (R2) 1 2-Aminobutyrate 104.10 58.05 + 0.99 51 Inosine 269.10 137.05 + 0.99 2 Acetylcarnitine 204.10 85.05 + 0.99 52 Kynurenine 209.10 192.05 + 0.99 3 Acetylcholine 147.10 87.05 + 0.99 53 Leu 132.10 86.05 + 0.99 4 Adenine 136.00 119.05 + 0.98 54 L-Norepinephrine 170.10 152.15 + 0.99 5 Adenosine 268.10 136.05 + 0.99 55 Lys 147.10 84.10 + 0.99 6 Adenylsuccinate 464.10 252.10 + 0.99 56 Met 149.90 56.10 + 0.99 7 ADMA 203.10 70.10 + 0.99 57 Methionine-sulfoxide 166.00 74.10 + 0.99 8 Ala 89.90 44.10 + 0.99 58 Nicotinamide 123.10 80.05 + 0.99 9 AMP 348.00 136.05 + 0.99 59 Nicotinic acid 124.05 80.05 + 0.99 10 Arg 175.10 70.10 + 0.99 60 Ophthalmic acid 290.10 58.10 + 0.99 11 Argininosuccinate 291.00 70.10 + 0.99 61 Ornitine 133.10 70.10 + 0.99 12 Asn 133.10 87.15 + 0.99 62 Pantothenate 220.10 90.15 + 0.99 13 Asp 134.00 74.05 + 0.99* 63 Phe 166.10 120.10 + 0.99 14 cAMP 330.00 136.05 + 0.99 64 Pro 115.90 70.10 + 0.99 15 Carnitine 162.10 103.05 + 0.99 65 SAH 385.10 134.00 + 0.98 16 Carnosine 227.10 110.05 + 0.99* 66 SAM 399.10 250.05 + 0.99* 17 cCMP 306.00 112.10 + 0.99 67 SDMA 203.10 70.15 + 0.99 18 cGMP 346.00 152.05 + 0.99 68 Ser 105.90 60.10 + 0.99* 19 Choline 104.10 60.05 + 0.99 69 Serotonin 177.10 160.10 + 0.99 20 Citicoline 489.10 184.10 + 0.99* 70 Thr/Homoserine 120.10 74.15 + 0.99 21 Citrulline 176.10 70.05 + 0.99 71 Thymidine 243.10 127.10 + 0.99 22 CMP 324.00 112.05 + 0.99 72 Thymine 127.10 54.05 + 0.99* 23 Creatine 132.10 44.05 + 0.99 73 TMP 322.90 81.10 + 0.99* 24 Creatinine 114.10 44.05 + 0.99 74 Trp 205.10 188.15 + 0.99 25 Cys 122.00 76.05 + 0.99* 75 Tyr 182.10 136.10 + 0.99 26 Cystathionine 223.00 88.05 + 0.99 76 Uracil 113.00 70.00 + 0.99* 27 Cysteamine 78.10 61.05 + 0.98* 77 Uridine 245.00 113.05 + 0.99 28 Cystine 241.00 151.95 + 0.99 78 Val 118.10 72.15 + 0.99 29 Cytidine 244.10 112.05 + 0.99 79 2-Oxoglutarate 144.90 101.10 - 0.98* 30 Cytosine 112.00 95.10 + 0.99 80 Allantoin 157.00 97.10 - 0.98* 31 Dimethylglycine 104.10 58.05 + 0.99 81 Cholate 407.20 343.15 - 0.99** 32 DOPA 198.10 152.10 + 0.99* 82 cis-Aconitate 172.90 85.05 - 0.99 33 Dopamine 154.10 91.05 + 0.99* 83 Citrate 191.20 111.10 - 0.99* 34 Epinephrine 184.10 166.10 + 0.99 84 FMN 455.00 97.00 - 0.99 35 FAD 786.15 136.10 + 0.99* 85 Fumarate 115.10 71.00 - 0.99** 36 GABA 104.10 87.05 + 0.99 86 GSSG 611.10 306.00 - 0.99* 37 gamma-Glu-Cys 251.10 84.10 + 0.99* 87 Guanine 150.00 133.00 - 0.99* 38 Gln 147.10 84.15 + 0.99 88 Isocitrate 191.20 111.10 - 0.99* 39 Glu 147.90 84.10 + 0.99* 89 Lactate 89.30 89.05 - 0.97* 40 Gly 75.90 30.15 + 0.99* 90 Malate 133.10 114.95 - 0.99* 41 GMP 364.00 152.05 + 0.99 91 NAD 663.10 541.05 - 0.99* 42 GSH 308.00 179.10 + 0.99* 92 Orotic acid 155.00 111.10 - 0.99 43 Guanosine 284.00 152.00 + 0.99 93 Pyruvate 86.90 87.05 - 0.99* 44 His 155.90 110.10 + 0.99 94 Succinate 117.30 73.00 - 0.99* 45 Histamine 112.10 95.05 + 0.99* 95 Taurocholate 514.20 107.10 - 0.99* 46 Homocysteine 136.00 90.10 + 0.99* 96 Uric acid 167.10 123.95 - 0.99* 47 Homocystine 269.00 136.05 + 0.99 97 Xanthine 151.00 108.00 - 0.99* 48 Hydroxyproline 132.10 86.05 + 0.99 49 Hypoxanthine 137.00 55.05 + 0.98* 50 Ile 132.10 86.20 + 0.99

Calibration curve was obtained at a range of concentration from 10 nM to 10000 nM. * Calibration curve was obtained at a range of concentration from 100 nM to 10000 nM. ** Calibration curve was obtained at a range of concentration from 1000 nM to 10000 nM.

4 Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta uorophenylpropyl column

4500000

4000000

3500000

3000000

2500000

2000000

1500000

1000000

500000

0

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

Figure 1 MRM chromatogram of 97 compounds

Application to tissue extracts as biological samples Simultaneous analysis of 99 compounds was performed for simultaneous analysis of biological samples. As shown in heart / liver tissue extracts as biological samples. Figure 2 the resulting MRM chromatogram, some major peaks were shows MRM chromatograms of 99 compounds from tissue derived from the metabolites which were known to be extracts (liver/heart). In this measurement, 83/97 characteristic to each tissue. Furthermore, this metabolites were detected from liver tissue extracts and characteristic difference in each tissue was also confirmed 88/97 metabolites were confirmed from heart tissue in some faint peaks (e.g., cholate, cystine and extracts. These results show this method is also effective to homocysteine).

5 Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta uorophenylpropyl column

10000000 9000000 GSH 8000000 Liver Tissue 7000000 6000000 Guanosine 5000000 Ophtalmic acid 4000000 AMP 3000000 GSSG 2000000 1000000 0

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

30000000 Creatine 25000000 Heart Tissue S-Adenosylhomocysteine 20000000

Acetylcarnitine 15000000

10000000

5000000

0

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

Figure 2 MRM chromatogram of liver/heart tissue extracts

Correlation between PFPP and ion pairing Methods We have previously reported simultaneous analysis of 55 method described above and the aliquots were measured metabolites which were related to central carbon by the simultaneous method using either ion pairing metabolic pathway by using ion pairing chromatography at chromatography or PFPP separation system. As a result, we ASMS conference 2013. To evaluate the accuracy of this could see the similar trend of elevation/decrease of peak simultaneous method using PFPP column, we compared area in metabolites of 20/25 between nine samples. The the resulting peak area of 25 metabolites, which were peak areas between 9 samples of representative covered as targets in both methods. The 25 metabolites metabolites are shown in Figure 3. This result shows that a are Lysine, Arginine, Histidine, Glycine, Serine, Asparagine, ratio of areas between 9 samples is kept in both methods. Alanine, Glutamine, Threonine, Methionine, Tyrosine, The four metabolites (TMP, cGMP, cAMP and Cysteine) Glutamate, Aspartae, Phenylalanine, Tryptophan, Cysteine, could be hardly detected on simultaneous analysis by CMP, NAD, GMP, TMP, AMP, cGMP, cAMP, MES and alternately ion-paring chromatography or PFPP column. L-Methionine sulfone as internal standards. Heart tissue Tryptophan had a faint peak in this experiment and led to extracts were prepared from mice (n=9) according to the the low similarity.

6 Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta uorophenylpropyl column

MES L-Methionine sulfone Threonine Serine 1.5E+06 5.0E+05 5.0E+05 2.0E+06 4.0E+05 4.0E+05 1.5E+06 1.0E+06 3.0E+05 3.0E+05 PFPP 1.0E+06 2.0E+05 2.0E+05 5.0E+05 1.0E+05 1.0E+05 5.0E+05 0.0E+00 0.0E+00 0.0E+00 0.0E+00 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

MES L-Methionine sulfone Threonine Serine 1.0E+06 1.0E+06 4.0E+04 2.5E+04 8.0E+05 8.0E+05 3.0E+04 2.0E+04 6.0E+05 6.0E+05 1.5E+04 Ion pairing 2.0E+04 4.0E+05 4.0E+05 1.0E+04 2.0E+05 2.0E+05 1.0E+04 5.0E+03 0.0E+00 0.0E+00 0.0E+00 0.0E+00 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

Aspartate Phenylalanine AMP GMP 8.0E+06 4.0E+06 1.5E+07 3.0E+05 2.5E+05 6.0E+06 3.0E+06 1.0E+07 2.0E+05 PFPP 4.0E+06 2.0E+06 1.5E+05 5.0E+06 1.0E+05 2.0E+06 1.0E+06 5.0E+04 0.0E+00 0.0E+00 0.0E+00 0.0E+00 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

Aspartate Phenylalanine AMP GMP 5.0E+05 4.0E+04 6.0E+05 6.0E+04 5.0E+05 5.0E+04 4.0E+05 3.0E+04 4.0E+05 4.0E+04 3.0E+05 Ion pairing 2.0E+04 3.0E+05 3.0E+04 2.0E+05 2.0E+05 2.0E+04 1.0E+04 1.0E+05 1.0E+05 1.0E+04 0.0E+00 0.0E+00 0.0E+00 0.0E+00 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

Figure 3 Correlation of peak areas between PFPP and ion-pairing method

Conclusions • The 97 metabolites were separated by PFPP column with high resolution and this method was applied to biological samples. • The utility of this simultaneous analysis using PFPP column was con rmed by comparing between PFPP and ion paring chromatography.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 Life Science • Page 222 Surface analysis of permanent wave processing hair using DART-MS

• Page 229 Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromato- graph Mass Spectrometer) PO-CON1454E

Surface analysis of permanent wave processing hair using DART-MS

ASMS 2014 MP 476

Shoji Takigami1, Erika Ikeda1, Yuta Takagi1, Jun Watanabe2, Teruhisa Shiota3 1 Gunma University, Kiryu, Japan; 2 Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan; 3 AMR Inc., Meguro-ku, Tokyo, Japan Surface analysis of permanent wave processing hair using DART-MS

Introduction Permanent wave processing of hair is carried out at two After hair is applying permanent wave processing and processes as follows; coloring repeatedly, the chemical structure of a keratin (A) Reducing agent (permanent wave 1 agent) makes the molecule and ne structure in the hair have been damaged bridge construction between the keratin protein molecular and it resulted as damage hair. It is thought that hair chains of hair, especially disul de (S-S) bond of cystine becomes dryness and twining if the cuticle which covers residue cleaved to thiol (-SH) group and hear results a wave hair is damaged, so it is important to investigate the and curl. surface structure of hair and its chemical structure (B) Oxidizing agent (permanent wave 2 agent) makes -SH changing. group oxidized to be reproduced S-S bond. As reducing DART (Direct Analysis in Real Time), a direct atmospheric agents used for permanent wave 1 agent, the thing of pressure ionization source, is capable of analyzing samples cosmetics approval, such as cysteamine hydrochloride and directly with little or no sample preparation. Here, analysis a butyrolactone thiol (brand name Spiera, other than quasi of the ingredient which has deposited on the permanent drugs, such as ammonium thioglycolate, acetyl cystein, and wave processing hair surface was tried using this DART thiolactic acid, are used. combined with a mass spectrometer.

High Speed Mass Spectrometer

Ufswitching High-Speed Polarity Switching 15msec Ufscanning High-Speed Scanning 15,000u/sec

Figure 1 DART-OS ion source & LCMS-2020

TGA CA BLT (thioglycolate) (cysteamine hydrochloride) (butyrolactone thiol) O O HCl

H N SH SH 2 O HO SH

Fw 92 Fw 113 Fw 118

Wave ef ciency is good in a Wave ef ciency is good in a Wave ef ciency is good in a weak alkaline (pH 8 - 9.5) weak alkaline (pH 8 - 9.5) weak acid (pH 6)

The chemical state and property were investigated in the surface of the hair which repeated permanent wave processing with these reducing agents.

2 Surface analysis of permanent wave processing hair using DART-MS

Methods and Materials The Chinese virgin hair purchased from the market was agent. After hair sample was reduced for 15 minutes at washed with the 0.5% non-ionic surfactant containing 35°C using each solvent, it was carried out oxidation saturated EDTA solution, and then it was considered as treatment at 35°C by being immersed in 8% sodium untreated hair sample. Permanent wave processing of hair bromate solution (pH7.2) for 15 minutes. was prepared as following; the 0.6M TGA solution and LCMS-2020 (Shimadzu) was coupled with DART-OS ion 0.6M CA solution which were adjusted to pH8.5 with source (IonSense) and hear samples were held onto DART aqueous ammonia and the 0.6M BLT solution adjusted to gas ow directly, then their surface analyzed. pH6.0 with arginine water, which were used as a reducing

MS condition (LCMS-2020; Shimadzu Corporation)

Ionization : DART (Direct Analysis in Real Time) Heater Temperature (DART) : 350°C Measuring mode (MS) : Positive/Negative scanning simultaneously

Chinese Virgin Hair

0.5% Laureth - 9 solution - EDTA saturated 35°C 1h Water washing and air drying Permanent wave processing Untreated by agent 1 & 2 at 0.6M each

permanent wave 1 agent : TGA or CA (pH 8.5; aqueous ammonium) BLT (pH 6; arginine) 35°C 15min Water washing Repeat 6 times permanent wave 2 agent : 8% NaBrO3 solution (pH 7.2) 35°C 15min Water washing Britton - Robinson buffer (pH 4.6) 35°C 15min Water washing

Air drying

Analyzed by DART-MS

3 Surface analysis of permanent wave processing hair using DART-MS

Result After repeating operation of permanent wave processing was performed. 1-6 times using TGA (thioglycollic acid), CA (cysteamine), DART-MS analysis was conducted in order of #1 Untreated and BLT (Butyrolactonethiol), hair was immersed for 15 (woman hair), #2 control; ammonia treatment (pH 8.5), #3 minutes at 35°C and with a ush and air-drying, then 0.6M thioglycolic acid (TGA) processing, #4 0.6M permanent wave processing hair was prepared. In order to butyrolactone thiol (BLT) processing, #5 0.6M cysteamine investigate the ingredient which has deposited on the hydrochloride (CA) processing and #6 control; arginine permanent wave processing hair surface, DART-MS analysis processing (pH 6).

#1 #2 #3 #4 #5 #6 2:TIC(+) 75000000 Positive TIC m/z 30-2000

50000000

25000000

0 4:TIC(-) 15000000 Negative TIC m/z 30-2000

10000000

5000000

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min

Figure 2 TIC chromatogram of each sample analyzing with DART

In the DART mass spectra of #1 untreated and #6 control, 231 were detected. They were considered the signal many signals considered as triglyceride and diglyceride equivalent to [M-H]- and [2M-H]- of BLT oxide compound were detected in both positive and negative spectra (C4H4O2S, molecular weight 116) in which two hydrogen obtained by DART-MS. In #3 0.6M thioglycolic acid (TGA) atoms were removed from BLT. Carrying out permanent processing spectra, the signal in particular of TGA origin wave processing by BLT, it was found that the dimer of BLT was not detected. accumulated on the cuticle surface. In #4 BLT processing spectra (Figure 3), the signals In #5 CA processing spectrum (Figure 5), the signal considered to be oxidized BLT (3, 3'-dithiobis considered to be the dimer (Fw152) origin in which CA (tetrahydrofuran2-one), molecular weight 234) were carried out S-S bond in the positive mode was detected at detected at m/z 235 and 252 in the positive mode. The m/z 153. signal m/z 235 is equivalent to [M+H]+ and m/z 252, This is equivalent to [M+H]+. [M+NH4]+. In the negative mode, the signals, m/z 115,

4 Surface analysis of permanent wave processing hair using DART-MS

Inten. (x10,000,000) 252 1.50 Positive [M+NH4]+ 1.25 [M+H]+ 1.00 235

0.75

0.50

0.25 282 486 368 424 516 0.00 100 200 300 400 500 600 700 800 900 1000 1100 m/z

Inten. (x100,000) 5.0 179 Negative 4.0 [M-H]- 115 3.0 [2M-H]- 231 2.0

1.0 321 347 411 501 579 0.0 100 200 300 400 500 600 700 800 900 1000 1100 m/z

Figure 3 DART-MS spectra of #4 BLT processing The BLT-related signals were detected from the positive and the negative spectra.

Inten. (x1,000,000) 124 282 1.50 Positive

1.25 [2M+H]+ 391

1.00 252 0.75 153 0.50 468 424 563 0.25 184 102 600 644 691 769 851 922 0.00 100 200 300 400 500 600 700 800 900 1000 1100 m/z

Figure 4 DART-MS spectra of #5 CA processing The CA-related signal was detected from the positive spectrum

5 Surface analysis of permanent wave processing hair using DART-MS

4:325.15(-) 100000 #1 #2 #3 #4 #5 #6

50000 Negative XIC m/z 325

0

10000000 2:234.70(+)

5000000 Positive XIC m/z 235

0

2:251.75(+) 10000000

5000000 Positive XIC m/z 252

0

4:114.95(-)

250000 Negative XIC m/z 115

0 4:230.90(-)

500000 Negative XIC m/z 231

0

1500000 2:123.85(+) 1000000 Positive XIC m/z 124 500000

0

1000000 2:152.85(+)

500000 Positive XIC m/z 153

0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min

Figure 5 XIC chromatorgam of each sample analyzing with DART

In order to indicate clearly the signals specifically methyl eicosanoic acid (18MEA, molecular weight 326). detected in each sample, the extraction chromatograms 18MEA is one of lipid components which protect a (XIC) were shown (Figure 5). It turned out that cuticle. There is no significant difference of this signal in BLT-related signals were detected only in #4 and the the hair between treated hair and untreated hair. We CA-related signal in #5. would like to inquire so that intensity difference can be Moreover, although the signal intensity was weak, the found out by further verifying the detection technique in signal at negative m/z 325 was detected from all the future. samples. Negative m/z 325 is equivalent to [M-H]- of 18

6 Surface analysis of permanent wave processing hair using DART-MS

Conclusions By direct analysis of the hair by DART-MS, the chemical structure change in the surfaces of hair, such as permanent wave processing, was able to be observed.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1469E

Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)

ASMS 2014 TP761

Sanket Chiplunkar, Prashant Hase, Dheeraj Handique, Ankush Bhone, Durvesh Sawant, Ajit Datar, Jitendra Kelkar, Pratap Rasam Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh Chambers, Makwana Road, Marol, Andheri (E), Mumbai-400059, Maharashtra, India. Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)

Introduction Cosmetics, fragrances and toiletries (Figure 1) are used European Directive (EU) 2003/15/EC and International safely by millions of people worldwide. Although many Fragrance Association (IFRA)[1] labeled on cosmetics. people have no problems, irritant and allergic reactions Shimadzu MDGC-GCMS technology facilitates the may occur. Irritant and allergic skin reactions are the types identi cation and quanti cation of these allergens to of contact dermatitis. Essential oils present in fragrance comply with the threshold limits of 100 ppm for rinse-off contain some natural and synthetic compounds, which products. may cause allergic reactions to the end user after Co-eluting peaks were resolved completely with the help application. There are 26 potential allergens listed by of MDGC-GCMS heart-cut technique.

Figure 1. Cosmetics, fragrances and toiletries

Method of Analysis Extraction of allergens from shampoo sample Shampoo samples were collected from local market. polarities. In MDGC-GCMS, 1st instrument was GC-2010 Standard solutions of 23 allergens were procured from Plus equipped with FID as a detector and 2nd instrument ACCU Standard and dilutions were carried out in was GCMS-QP2010 Ultra with MS as a detector. Columns Ethanol/Acetonitrile to yield 1000 ppm concentration. in both the instruments were connected with Deans Further dilutions were made in methanol. switch. Allergens in shampoo samples were determined by MDGC-GCMS technique was effectively used to minimize using this technique. For sample preparation, following matrix effect. Co-eluting peaks were resolved with methodology was adopted. heart-cut technique using two columns of different

1) Blank Solution : 10 mL of methanol was transferred in 20 mL centrifuge tube and vortexed for 5 minutes. The mixture was then centrifuged for 5 minutes at 3000 rpm. This solution was filtered through 0.2 µm nylon syringe filter. Initial 2 mL was discarded and remaining filtrate was collected. 2) Sample Solution : 1 g of shampoo sample was weighed in 10 mL volumetric flask and diluted up to the mark with methanol. Above mixture was transferred in 20 mL centrifuge tube. Further processing was done as mentioned in blank solution. 3) Spike Sample Solution : For recovery study, 1 g of sample was spiked with different volumes of standard stock solution. The above procedure was repeated for preparing different concentration levels of allergens in samples. These spiked samples were treated as mentioned in sample solution.

Part method validation was carried out by performing were prepared using 40 ppm (actual concentration) system precision, sample precision, linearity and recovery standard stock solution mixture of allergens. study. For validation, solutions of different concentrations

2 Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)

Table 1. Method validation parameters

Parameter Concentration System Precision 10 ppm Sample Precision 10 % in Methanol Linearity 2.5, 5, 7.5, 10, 15 (ppm) Accuracy / Recovery 5, 10, 15 (ppm)

MDGC-GCMS Analytical Conditions The instrument con guration used is shown in Figure 2. Samples were analyzed using Multi-Dimensional GC/GCMS as per the conditions given below.

Figure 2. Multi-Dimensional GC/GCMS System by Shimadzu

Figure 3. Schematic diagram of multi-Deans switch in MDGC-GCMS

3 Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)

MDGC-GCMS analytical parameters

Chromatographic parameters (1st GC : GC-2010 Plus)

• Column : Stabilwax (30 m L x 0.25 mm I.D.; 0.25 μm) • Injection Mode : Split • Split Ratio : 5.0 • Carrier Gas : Helium • Column Flow : 2.27 mL/min • Detector : FID • APC Pressure : 200 kPa (For switching) • Column Oven Temp. : Rate (ºC /min) Temperature (ºC) Hold time (min) 50.0 0.00 15.00 100.0 0.00 5.00 240.0 43.67

Chromatographic parameters (2nd GCMS : GCMS-QP2010 Ultra)

• Column : Rxi-1ms (30 m L x 0.25 mm I.D.; 0.25 μm) • Detector : Mass spectrometer • Ion Source Temp. : 200 ºC • Interface Temp. : 240 ºC • Ionization Mode : EI • Event Time : 0.30 sec • Mode : SIM and SCAN • Column Oven Temp. : Rate (ºC /min) Temperature (ºC) Hold time (min) 80.0 13.00 3.00 180.0 0.00 10.00 260.0 20.67 • Total Program Time : 75.00 min

Results Sample analysis using MDGC-GCMS MDGC-GCMS technique was used to avoid matrix interference from sample. Using multi-Deans switch and heart-cut technique (Figure 3), co-eluted components from the 1st column were transferred to the 2nd column with different polarity.

4 Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)

uV (x100,000) Chromatogram uV (x10,000) uV (x10,000) Chromatogram Chromatogram 5.0 10.0 9.5

1.1 4.5 9.0 Fernesol - 2 8.5 4.0 8.0 7.5 1.0 3.5 7.0 6.5 3.0 6.0 Benzyl Alcohol 5.5 2.5 5.0 Geraniol Citronellol 0.9 Fernesol - 2 4.5 2.0 Methyl heptine carbonate 4.0 3.5 Fernesol - 2

1.5 Hexyl cinnam aldehyde 3.0 Cinnamyl alcohol Citral - 2 2.5 Amyl cinnamal Citral - 1 Anisyl alcohol Isoeugenol

0.8 1.0 Sample - 2 2.0 Fernesol - 1

Sample - 3 1.5 0.5 1.0

25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 min 0.7 12.0 13.0 14.0 15.0 16.0 17.0 min

0.6 Sample - 2 Anisyl alcohol 0.5 Isoeugenol

0.4 Linalool Limonene Fernesol - 2 Hexyl cinnam aldehyde Cinnamyl alcohol Benzyl benzoate Amylcin namyl alcohol

0.3 Benzyl Alcohol Amyl cinnamal Sample - 6 Coumarin Eugenol Geraniol Citronellol Cinnamal Sample - 5 Benzyl salicylate Benzyl Cinnamate Methyl heptine carbonate Citral - 1

0.2 Fernesol - 1 Hydroxy-citronellal Citral - 2 0.1 Sample - 1 Sample - 3

5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 min

Figure 4. Chromatogram of spiked sample solution before switching

uV (x10,000) (x100,000) 3.50 Chromatogram 164.00 (100.00) 149.00 (100.00) Target compound - Isoeugenol 3.25 Target compound - Isoeugenol 6.0 103.00 (100.00) 3.00 138.00 (100.00)

28.105 109.00 (100.00) 2.75 5.0 137.00 (100.00) 92.00 (100.00) 2.50 115.00 (100.00) 134.00 (100.00) 2.25 4.0 2.00 3.0 1.75

1.50 2.0 1.25

1.00 26.491 1.0 26.256 0.75 0.0 0.50

0.25 -1.0 26.5 27.0 27.5 28.0 min 27.0 27.5 28.0 28.5 29.0 29.5 Figure 5. Chromatogram with 1st column (FID) Figure 6. SIM chromatogram with 2nd column (MS)

Summary of results

Table 2. Summary of results for precision on GC and GCMS

Sr. No. Type of sample Sample name Concentration Result 1 Standard 23 Allergens mixture 10 ppm % RSD for area (n=6) < 2.0 2 Cosmetic Shampoo Unknown % RSD for area (n=6) < 2.0

5 Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)

Table 3. Linearity by GC Area (x10,000) Sr. No. Name of allergen Linearity (R2) 7.0 1 Linalool 0.9945 6.0 2 Methyl heptine carbonate 0.9949 3 Citronellol 0.9965 5.0

4 Geraniol 0.9962 4.0 5 Hydroxy citronellal 0.9973 3.0 6 Cinnamal 0.9959 7 Amyl Cinnamal 0.9976 2.0

8 Coumarin 0.9971 1.0

9 Amylcin namyl alcohol 0.9983 0.0 10 Benzyl benzoate 0.9979 0.0 2.5 5.0 7.5 10.0 12.5 Conc. Figure 7. Linearity graph for linalool

Table 4. Linearity by GCMS Area(x10,000) Sr. No. Name of allergen Linearity (R2) 1.75 1 Limonene 0.9945

2 Benzyl alcohol 0.9871 1.50 3 Citral - 1 0.9889 1.25 4 Citral - 2 0.9902

5 Eugenol 0.9894 1.00 6 Anisyl alcohol 0.9916 0.75 7 Cinnamyl alcohol 0.9937

8 Isoeugenol 0.9902 0.50 9 Farnesol - 1 0.9919 10 Farnesol - 2 0.9929 0.25 11 Hexyl cinnam aldehyde 0.9932 0.00 12 Benzyl salicylate 0.9853 0.0 2.5 5.0 7.5 10.0 12.5 Conc. 13 Benzyl cinnamate 0.9927 Figure 8. Linearity graph for benzyl cinnamate

Quantitation of allergens in shampoo sample For the quantitation studies, the shampoo sample was In below recovery study, some allergens had recovery value spiked with allergens standard to achieve 5, 10 and 15 out side the acceptance limit (70-130 %). Optimization can ppm concentrations. Recovery studies were performed on be done by means of change in sample clean up procedure 13 allergens, having co-elution or matrix interference, using and filtration study. heart-cut technique. The quantitation of these allergens was carried out using 2nd detector (MS) in SIM mode.

6 Analysis of allergens found in cosmetics using MDGC-GCMS (Multi-Dimensional Gas Chromatograph Mass Spectrometer)

Table 5. Quantitation of allergens – Recovery Study (x1,000) m/z : 69.00 % Recovery 3.00 Sr. No. Name of allergen Level -1 Level -2 Level -3 2.75 Farnesol-1 5 ppm 10 ppm 15 ppm Farnesol-2 2.50 1 Limonene 127 126 129 2 Benzyl alcohol 114 114 123 2.25 3 Citral - 1 101 106 114 2.00

4 Citral - 2 97 103 112 1.75

5 Eugenol 96 105 116 1.50 6 Anisyl alcohol 94 105 116 1.25 7 Cinnamyl alcohol 98 106 115 1.00 8 Isoeugenol 103 108 118 Spiked 0.75 9 Farnesol - 1 83 95 107 Unspiked 10 Farnesol - 2 84 95 106 0.50 11 Hexyl cinnam aldehyde 121 122 130 0.25 12 Benzyl salicylate 63 47 32 25.0 27.5 30.0 32.5 min 13 Benzyl cinnamate 66 61 56 Figure 9. Overlay SIM chromatogram of unspiked and spiked sample Conclusion • MDGC-GCMS method was developed for quantitation of allergens present in cosmetics. Part method validation was performed as per ICH guidelines.[2] Results obtained for reproducibility, linearity and recovery studies were well within acceptable limits. • Simultaneous SCAN/SIM and high-speed scan rate 20,000 u/sec are the characteristic features of GCMS-QP2010 Ultra, which enables quantitation of allergens at very low concentration level. • Matrix effect from cosmetics was selectively eliminated using MDGC-GCMS with multi-Deans switching unit and heart-cut technique. • MDGC-GCMS was found to be very useful technique for simultaneous identi cation and quantitation of components from complex matrix.

Reference [1] IFRA guidelines (International Fragrance Association), GC/MS Quanti cation of potential fragrance allergens, Version 2, (2006), 6. [2] ICH guidelines, Validation of Analytical Procedures: Text And Methodology Q2(R1), Version 4, (2005).

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 Technical Applications • Page238 Applicationsofdesorptioncoronabeam ionization-massspectrometry

• Page243 Rapidanalysisofcarbonfiberreinforcedplastic usingDART-MS

•Page249 Analysisofstyreneleachedfrompolystyrene cupsusingGCMScoupledwithHeadspace(HS) sampler PO-CON1474E

Applications of Desorption Corona Beam Ionization-Mass Spectrometry

ASMS 2014 WP 393

Yuki Hashi1, Shin-ichi Kawano1, Changkun Li1, Qian Sun1, Taohong Huang1, Tomoomi Hoshi2, Wenjian Sun3 1Shimadzu (China) Co., Ltd., Shanghai, China 2Shimadzu Corporation, Kyoto, Japan 3Shimadzu Research Laboratory (Shanghai) Co., Ltd., Shanghai, China Applications of Desorption Corona Beam Ionization-Mass Spectrometry

Introduction Numerous ambient ionization mass spectrometric desorption. A visible thin corona beam is formed by using techniques have been developed for high throughput hollow needle/ring electrode structure. This feature analysis of various compounds with minimum sample facilitates localizing sampling areas and obtaining good pretreatment.(1) Desorption corona beam ionization (DCBI) reproducibility of data. Details of DCBI hardware are is a more recent technique.(2) In DCBI, helium is used as shown in Figs. 1 and 2. In this study, DCBI was applied for discharge gas and heating of the gas is required for sample analysis of various samples.

Helium ow HVDC

Heated thin wall tubing - LVDC + Counter Discharge electrode needle Sampling capillary

MS inlet Sample and stage

Figure 1 Schematic diagram of DCBI

DCBI probe

Corona beam

MS Inlet

Manual liquid sampler

Figure 2 DCBI interface

2 Applications of Desorption Corona Beam Ionization-Mass Spectrometry

Method Sample Preparation Samples (melamine, saturated hydrocarbon mixture, polyaromatic hydrocarbon mixture, testosterone, pirimicarb, and methomyl) were dissolved in methanol or acetonitrile.

DCBI-MS Analysis Samples were analyzed using a DCBI system coupled to a LCMS-2020 quadrupole mass spectrometer (Shimadzu Corporation, Japan). The system was operated with the DCBI control software and LabSolutions for LCMS version 5.42.

Analytical Conditions

DCBI

Flow rate : 0.6 L/min HV discharge : +2.0-3.0 kV He gas temperature : 350 ºC Sample volume : 1, or 2 µL

MS (LCMS-2020 quadrupole mass spectrometer)

Polarity : Positive DL temperature : 250 ºC BH temperature : 400 ºC Mass range : m/z 100-500

Results and Discussion In this experiment, all compounds with variety of polarity cleavage at methylcarbamoyl group, while fragment ions from non- to high-polar gave protonated molecules (Figs. with signi cant intensity were not observed for other 3-8). Methomyl gave also fragment ions (m/z 106) by compounds. Analysis time was less than 1 minute.

Inten. (x1,000) 127.1 2.25

2.00

1.75

1.50

1.25

1.00

0.75

0.50

0.25 136.0 148.6 0.00 100.0 105.0 110.0 115.0 120.0 125.0 130.0 135.0 140.0 145.0 m/z

Figure 3 Mass spectrum of melamine (0.5 mg/mL)

3 Applications of Desorption Corona Beam Ionization-Mass Spectrometry

Inten. (x100,000) 1.50 Compound MW 241.3 213.2 C10H22 142 255.3 C11H24 156 1.25 269.3 C12H26 170 C H 184 199.2 13 28 C H 198 1.00 14 30 283.3 C15H32 212 C16H34 226 0.75 185.2 297.3 C17H36 240 C18H38 254 311.3 C19H40 268 0.50 C20H42 282 C H 296 171.2 325.3 21 44 C22H46 310 0.25 339.3 C23H48 324 157.2 C H 338 115.1 24 50 143.2 367.4 C H 352 0.00 25 52 100 150 200 250 300 350 m/z

Figure 4 Mass spectrum of saturated hydrocarbon mixture (1 mg/mL)

Inten. (x10,000) 6.5 153.1 Compound MW 6.0 Naphthalene 128 Acenaphthylene 152 5.5 Acenaphthene 154 5.0 155.2 Fluorene 166 4.5 Anthracene 178 4.0 179.1 Phenanthrene 178 Pyrene 202 3.5 Fluoranthene 202 3.0 Chrysene 228 2.5 203.1 Benzo[a]anthracene 228 167.2 2.0

1.5 209.1 1.0 129.1 195.1 0.5 141.2 115.1 235.1 0.0 276.2 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z

Figure 5 Mass spectrum of polyaromatic hydrocarbon mixture (2 mg/mL)

Inten. (x10,000) 289.2 7.0

6.0

5.0

4.0

3.0

2.0

1.0 112.1 331.2 424.5 461.4 0.0 150 200 250 300 350 400 450 m/z

Figure 6 Mass spectrum of testosterone (1 mg/mL)

4 Applications of Desorption Corona Beam Ionization-Mass Spectrometry

Inten. (x100,000) Inten. (x100,000) 163.0 9.0 239.2 1.2

1.1 8.0 105.9 1.0 7.0 0.9

6.0 0.8

0.7 5.0 0.6 4.0 0.5

3.0 0.4

0.3 2.0 0.2 194.0 1.0 182.2 0.1 121.9 252.0 208.0 354.1 394.3 0.0 0.0 100 150 200 250 300 350 400 450 m/z 100 150 200 250 300 350 400 450 m/z

Figure 7 Mass spectrum of pirimicarb (0.5 mg/mL) Figure 8 Mass spectrum of methomyl (0.5 mg/mL)

Conclusion The DCBI system was successfully applied for analysis of samples with various polarity. Mass spectra were quickly obtained after sample introduction to the DCBI probe. The method is useful for fast identi cation of various compounds.

References (1) Monge ME et al, Chem. Rev. 113 (2013), 2269-2308 (2) Hua W et al, Analyst 135 (2010), 688-695

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1456E

Rapid analysis of carbon ber reinforced plastic using DART-MS

ASMS 2014 TP 782

Hideaki Kusano1, Jun Watanabe1, Yuki Kudo2, Teruhisa Shiota3 1 Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan; 2 Bio Chromato, Inc., Fujisawa, Japan; 3 AMR Inc., Meguro-ku, Tokyo, Japan Rapid analysis of carbon ber reinforced plastic using DART-MS

Introduction DART (Direct Analysis in Real Time) can ionize and analyze carbon in many cases. An epoxy resin is mainly used for a samples directly under atmospheric pressure, independent base material in CFRP. While CFRP is widely used taking of the sample forms. Then it is also possible to measure in advantage of strength and lightness, most approaches form as it is, without sample preparation. Qualitative which measure CFRP with analytical instruments were not analysis of target compounds can be conducted very fast tried, triggered by the dif culty of the preparation. and easily by combining DART with LCMS-2020/8030 DART (Direct Analysis in Real Time), a direct atmospheric which have ultra high-speed scanning and ultra high-speed pressure ionization source, is capable of analyzing samples polarity switching. with little or no sample preparation. Here, rapid analysis of Carbon- ber-reinforced plastics, CFRP is the carbon ber reinforced plastic was carried out using DART ber-reinforced plastic which used carbon ber for the combined with a mass spectrometer. reinforced material, which is only called carbon resin or

Figure 1 CFRP：carbon- ber-reinforced plastic

Methods and Materials Thermosetting polyimide (carbon- ber-reinforced plastics) Kyoto Japan). Ultra-fast polarity switching was utilized on and thermoplastic polyimide (control sample) were the mass spectrometer to collect full scan data. privately manufactured. After cutting a sample in a suitable LCMS-8030 can achieve the polarity switching time of size, it applied DART-MS analysis. They were introduced to 15msec and the scanning speed of up to 15,000u/sec, the DART gas using tweezers. The DART-OS ion source therefore the loop time can be set at less than 1 second (IonSense, MA, USA) was interfaced onto the single despite the relatively large scanning range of 50-1,000u. quadrupole mass spectrometer LCMS-8030 (Shimadzu,

MS condition (LCMS-8030; Shimadzu Corporation)

Ionization : DART (Direct Analysis in Real Time)

2 Rapid analysis of carbon ber reinforced plastic using DART-MS

High Speed Mass Spectrometer

UFswitching High-Speed Polarity Switching 15msec UFscanning High-Speed Scanning 15,000u/sec

Figure 2 DART-OS ion source (IonSense) & triple quadrupole LCMS (Shimadzu)

Result 3 CFRP samples were analyzed by DART-MS. Mass chromatograms of each sample were shown in Figure 3 and mass spectra in Figure 4.

Sample

#1 thermoplastic polyimide (control) #2 thermosetting polyimide (molded; dried) #3 thermosetting polyimide (immediately after molded; wet state with solvent)

Analytical Condition

Heater Temperature (DART) : 300ºC Measuring mode (MS) : Positive/Negative scanning simultaneously

1:MIC1(+) Positive TIC m/z 50-500 50000000

25000000

0 6000000 2:MIC1(-) 5000000 Negative TIC m/z 50-500 4000000 3000000 2000000 1000000 0 #1 #2 #3 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 min

Figure 3 TIC chromatogram of CFRP samples #1, #2, #3

3 Rapid analysis of carbon ber reinforced plastic using DART-MS

Inten. (x1,000,000) 7.5 Positive, m/z 50-300 #1

5.0

2.5

100.1 199.1 0.0 172.1 228.3 282.2 50 100 150 200 250 m/z Inten. (x1,000,000) 7.5 Positive, m/z 50-300 #2 N-methyl pyrrolidone C5H9NO 5.0 Mw 99

2.5 [M+H]+ [2M+H]+ 100.1 199.1 0.0 172.2 282.3 50 100 150 200 250 m/z Inten. (x1,000,000)

7.5 100.1 199.1 Positive, m/z 50-300 5.0 #3

2.5

0.0 50 100 150 200 250 m/z

Figure 4 DART-MS spectra of each sample

Since the thermosetting polyimide used for this it raised the heating gas temperature of DART to high measurement was molded using the organic solvent temperature (up to 500°C), MS signal considered to (N-methyl pyrrolidone, C5H9NO, molecular weight 99), originate in the structural information of CFRP was not molecular related ions of N-methyl pyrrolidone, [M+H]+ able to be obtained. (m/z 100) and [2M+H]+ (m/z 199), were detected very Then, the optional heating mechanism, ionRocket (Bio strongly in the mass spectrum of #1. The mass spectrum Chromato, Inc.; Figure 5), in which a sample could be of #2 also showed the same ions that intensity was heated directly was developed to the sample stage of intentionally detected strongly compared with #3 DART, and analysis of CFRP was verified by heating the although intensity was weak compared with #1. Even if sample directly up to 600°C.

Sample

#4 thermosetting polyimide (molded; dried) #5 thermoplastic polyimide (control)

Analytical Condition

Heater Temperature (DART) : 400°C Temperature control (ionRocket) : 0-1min room temp., 4min 600°C Measuring mode (MS) : Positive scanning

4 Rapid analysis of carbon ber reinforced plastic using DART-MS

600°C

r.t.

1 4 time[min]

evaporated ingredient

excitation helium

MS DART ion source spectrometer

sample pot

small heating furnace heater

Figure 5 DART-MS system integrated with ionRocket

When heating temperature was set to 600ºC, the rudder high temperature, it was considered that the thermal shape signals of 28u (C2H4) interval was appeared decomposition of resin started, the thermal around m/z 900. This signal was more notably detected decomposition ingredient of polyimide clustered, and with the thermosetting polyimide sample than the possibly the structures of the rudder signals of equal thermoplastic sample. Since the sample was heated at interval were generated.

5 Rapid analysis of carbon ber reinforced plastic using DART-MS

#4

Zoom

#5

#4 thermosetting polyimide

#5 thermoplastic polyimide

Figure 6 DART-MS with ionRocket spectra of each sample Conclusions The result of having analyzed the carbon fiber plastic CFRP (thermosetting polyimide and thermoplastic polyimide) using DART-MS, a. residue of the solvent used in fabrication was able to be checked by direct analysis of CFRP by DART. b. analyzing CFRP by DART and the heating option ionRocket, the difference between thermosetting polyimide and thermoplastic polyimide was able to be found out.

Acknowledgment We are deeply grateful to Mr. Yuichi Ishida, Japan Aerospace Exploration Agency (JAXA), offered the CFRP sample used for this experiment.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014 PO-CON1464E

Analysis of styrene leached from polystyrene cups using GCMS coupled with Headspace (HS) sampler

ASMS 2014 TP763

Ankush Bhone(1), Dheeraj Handique(1), Prashant Hase(1), Sanket Chiplunkar(1), Durvesh Sawant(1), Ajit Datar(1), Jitendra Kelkar(1), Pratap Rasam(1), Nivedita Subhedar(2) (1) Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh Chambers, Makwana Road, Marol, Andheri (E), Mumbai-400059, Maharashtra, India. (2) Ramnarain Ruia College, L. Nappo Road, Matunga (E), Mumbai-400019, Maharashtra, India. Analysis of styrene leached from polystyrene cups using GCMS coupled with Headspace (HS) sampler

Introduction Worldwide studies have revealed the negative impacts of body and can therefore disrupt normal hormonal household disposable polystyrene cups (Figure 1) on functions. This could also lead to breast and prostate human health and environment. cancer. Molecular structure of styrene is shown in Figure 2. Styrene The objective of this study is to develop a sensitive, is considered as a possible human carcinogen by the WHO selective, accurate and reliable method for styrene and International Agency for Research on Cancer (IARC).[1] determination using low carryover headspace sampler, Migration of styrene from polystyrene cups containing HS-20 coupled with Ultra Fast Scan Speed 20,000 u/sec, beverages has been observed.[2] Styrene enters into our GCMS-QP2010 Ultra to assess the risk involved in using body through the food we take, mimics estrogens in the polystyrene cups.

Figure 1. Polystyrene cup Figure 2. Structure of styrene

Method of Analysis Extraction of styrene from polystyrene cups This study was carried out by extracting styrene from commercially available polystyrene cups and recoveries were established by spiking polystyrene cups with standard solution of styrene. Solutions were prepared as follows,

1) Standard Stock Solution: 1000 ppm of styrene standard stock solution in DMF: Water-50:50 (v/v) was prepared. It was further diluted with water to make 100 ppm and 1 ppm of standard styrene solutions. 2) Calibration Curve: Calibration curve was plotted using standard styrene solutions in the concentration range of 1 to 50 ppb with water as a diluent. 5 mL of each standard styrene solution was transferred in separate 20 mL headspace vials and crimped with automated crimper. 3) Sample Preparation: 150 mL of boiling water (around 100 ºC)[1] was poured into polystyrene cups. The cup was covered with aluminium foil and kept at room temperature for 1 hour. After an hour, 5 mL of sample from the cup was transferred into the 20 mL headspace vial and crimped with automated crimper.

Method was partly validated to support the findings by performing reproducibility, linearity, LOD, LOQ and recovery studies. For validation, solutions of different concentrations were prepared using standard stock solution of styrene (1000 ppm) as mentioned in Table 1.

2 Analysis of styrene leached from polystyrene cups using GCMS coupled with Headspace (HS) sampler

Table 1. Method validation parameters

Parameter Concentration (ppb) Linearity 1, 2.5, 5, 10, 20, 50 Accuracy / Recovery 2.5, 10, 50 Precision at LOQ level 1 Reproducibility 50

HS-GCMS Analytical Conditions Figure 3 shows the analytical instrument, HS-20 coupled with GCMS-QP2010 Ultra on which samples were analyzed with following instrument parameter.

Figure 3. HS-20 coupled with GCMS-QP2010 Ultra by Shimadzu

HS-GCMS analytical parameters

Headspace parameters

• Sampling Mode : Loop • Oven Temp. : 80.0 ºC • Sample Line Temp. : 130.0 ºC • Transfer Line Temp. : 140.0 ºC • Equilibrating Time : 20.00 min • Pressurizing Time : 0.50 min • Pressure Equilib. Time : 0.10 min • Load Time : 0.50 min • Load Equilib. Time : 0.10 min • Injection Time : 1.00 min • Needle Flush Time : 10.00 min • GC Cycle Time : 23.00 min

3 Analysis of styrene leached from polystyrene cups using GCMS coupled with Headspace (HS) sampler

Chromatographic parameters

• Column : Rxi-5Sil MS (30 m L x 0.25 mm I.D., 0.25 μm) • Injection Mode : Split • Split Ratio : 10.0 • Carrier Gas : Helium • Flow Control Mode : Linear Velocity • Linear Velocity : 36.3 cm/sec • Pressure : 53.5 kPa • Column Flow : 1.00 mL/min • Total Flow : 14.0 mL/min • Total Program Time : 12.42 min • Column Oven Temp. : Rate (ºC /min) Temperature (ºC) Hold time (min) 50.0 0.00 40.00 200.0 1.00 30.00 280.0 5.00

Mass Spectrometry parameters

• Ion Source Temp. : 200 ºC • Interface Temp. : 230 ºC • Ionization Mode : EI • Event Time : 0.20 sec • Mode : SIM • m/z : 104,103 and 78 • Start Time : 1.00 min • End Time : 5.00 min

Results Fragmentation of styrene Mass spectrum of styrene is shown in Figure 4. From the with m/z 104, 103 and 78 is shown in Figure 5. mass spectrum, base peak of m/z 104 was used for Method validation data is summarized in Table 2. Figures 6 quantitation where as m/z 103 and 78 were used as and 7 show overlay of SIM chromatograms for m/z 104 at reference ions. linearity levels and calibration curve respectively. SIM chromatogram of 50 ppb standard styrene solution

4 Analysis of styrene leached from polystyrene cups using GCMS coupled with Headspace (HS) sampler

Inten. 104 100

75

50 103

78 25 51

44 52 63 58 65 74 85 89 98 0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.0 95.0 100.0 105.0 m/z

Figure 4. Mass spectrum of styrene

(x1,000,000) 104.00 (10.00) 7.5 103.00 (10.00) 78.00 (10.00)

5.0

2.5

0.0

2.325 2.350 2.375 2.400 2.425 2.450 2.475 2.500 2.525 min

Figure 5. SIM chromatogram of 50 ppb standard styrene solution

Summary of validation results

Table 2. Validation summary

Sr. No. Compound Name Parameter Concentration in ppb Result 1 Reproducibility (% RSD) 50 % RSD : 1.74 (n=6) 2 Linearity* (R2) 1 – 50 R2 : 0.9996 3 LOD LOD : 0.2 ppb Styrene 1 – 50 4 LOQ LOQ : 1 ppb S/N ratio : 38 (n=6) 5 Precision at LOQ 1 % RSD : 3.2 (n=6) * Linearity levels – 1, 2.5, 5, 10, 20 and 50 ppb.

5 Analysis of styrene leached from polystyrene cups using GCMS coupled with Headspace (HS) sampler

(x1,000,000) Area

m/z : 104.00 2 2.00 1250000 R = 0.9996

1.75 50 ppb 1000000 1.50 20 ppb 1.25 10 ppb 750000 1.00 5 ppb 0.75 2.5 ppb 500000 0.50 1 ppb 0.25 250000

0.00 0 2.2 2.3 2.4 2.5 2.6 min 0 10 20 30 40 Conc.

Figure 6. Overlay of SIM chromatograms for m/z 104 at linearity levels Figure 7. Calibration curve for Styrene

Quantitation of styrene in polystyrene cup sample Analysis of leachable styrene from polystyrene cups was styrene solutions in polystyrene cups. Figure 8 shows done as per method described earlier. Recovery studies overlay SIM chromatogram of spiked and unspiked were carried out by spiking 2.5, 10 and 50 ppb of standard samples. Table 3 shows the summary of results.

(x100,000) m/z : 104.00

7.5

5.0 Spiked

2.5 Unspiked

0.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 min

Figure 8. Overlay SIM chromatograms of spiked and unspiked samples

Table 3. Summary of results for sample analysis

Observed Spiked Sr. No. Sample Name Parameter Concentration Concentration % Recovery in ppb in ppb 1 Unspiked sample Precision 9.8 NA NA 12.0 2.5 88.0 2 Spiked polystyrene cups Recovery 18.5 10 87.0 55.9 50 92.2

6 Analysis of styrene leached from polystyrene cups using GCMS coupled with Headspace (HS) sampler

Conclusion • HS-GCMS method was developed for quantitation of styrene leached from polystyrene cup. Part method validation was performed. Results obtained for reproducibility, linearity, LOQ and recovery studies were within acceptable criteria. • With low carryover, the characteristic feature of HS-20 headspace, reproducibility even at very low concentration level could be achieved easily. • Ultra Fast Scan Speed 20,000 u/sec is the characteristic feature of GCMS-QP2010 Ultra mass spectrometer, useful for quantitation of styrene at very low level (ppb level) with high sensitivity.

References [1] Maqbool Ahmad, Ahmad S. Bajahlan, Journal of Environmental Sciences, Volume 19, (2007), 422, 424. [2] M. S. Tawka; A. Huyghebaerta, Journal of Food Additives and Contaminants, Volume 15, (1998), 595.

First Edition: June, 2014

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. www.shimadzu.com/an/ © Shimadzu Corporation, 2014