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ASMS 2014 Poster Collection 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 efcient 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 HN NH O- H N N N OH Cl N O H2N O O NH2 NH2 N HC3 O N Cl O H CH3 O Gabapentin Lamotrigine Levetiracetam Nitrazepam Phenobarbial Phenytoin CH3 CH3 S O O H HC3 CH O N 3 O HC3 N S N OH O O O O NH O O O CH S 3 O S H2C OH HC3 H2NO 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.
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