Chapter Metabolism

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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 automated 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

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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 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

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

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 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 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