Metabolism€¢ Page 197 High sensitivity analysis of metabolites in serum using simultaneous SIM...

Metabolism

• Page 197High sensitivity analysis of metabolites in serum using simultaneous SIM and MRM modes in a triple quadrupole GC/MS/MS

• 02Analysis of D- and L-amino acids using automated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry

• Page 208Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/time-of-flight mass spectrometry

• Page 213Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta- fluorophenylpropyl column

PO-CON1443E

High Sensitivity Analysis ofMetabolites in Serum UsingSimultaneous SIM and MRM Modesin 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

2

High Sensitivity Analysis of Metabolites in Serum Using Simultaneous SIM and MRM Modes in a Triple Quadrupole GC/MS/MS

IntroductionGas 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.

Sample• Human serum

MRM measurement modeSome 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 suggestionSIM, MRM, and simultaneous SIM/MRM modes are evaluated for analysis of metabolites in human serum.

Materials and MethodSample and Sample preparation

Sample Preparation1)

Instrumentation

Freeze-dry

Residue

Sample

Add 40 µL methoxyamine solution (20 mg/mL, pyridine)

Heat at 30 ºC for 90 min

Add 20 µL MSTFA

Heat at 37 ºC for 30 min

1) Nishiumi S et. al. Metabolomics. 2010 Nov;6(4):518-528

Supernatant 250 µL

Add 250 µL water / methanol / chloroform (1 / 2.5 / 1)

Add internal standard (2-Isopropylmalic acid)

Stir, then centrifuge

Extraction solution 225 µL

Add 200 µL Milli-Q water

Stir, then centrifuge

50uL serum

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

3


Simultaneous SIM and MRM modes in GC/MS/MSFigure 1 shows the theory of Simultaneous SIM and MRM modes. This analysis mode can measure SIM and MRM data in a single analysis.

Method Creation using Database and SmartMRMFigure 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.

• 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.

Poor sensitivity of MRM in some compounds because of low CID ef�ciency

Figure 1 The concept of simultaneous SIM and MRM analysis mode.

Figure 3 GC/MS Metabolites Database Ver.2

Figure 2 Mass Spectrum of Precursor (or SIM) and Product ion

SIM

MRMSIM

MRM

Q1 Q3Collision Cell

SIMSIM CID

100 200 300 400 0

25

50

75

100 %

361

73

217 147 437 103 271

243 319 191

100 200 300 0

25

50

75

100

%

169

103 73

243 361

Precursor ion (or SIM) Product ion

CID

4


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.

ResultsComparison of the chromatogram between SIM and MRM in human serum

Detected the peak in MRM because of high selectivity

Peak was not detected in MRM because of low CID ef�ciency.

SIM MRM

SIM MRM

0.5

1.0

1.5

2.0

2.5

3.0

3.5

2.5

5.0

7.5

0.25

0.50

0.75

1.00

1.25

1.50

1.75

(x100,000)333.10160.10

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

(x100,000)238.10218.10

(x10,000)218.10>73.00

(x100)238.10>91.00

238.10>91.00

(x10,000)333.10>143.10333.10>171.10

21.00 21.25 21.00 21.25

21.25 21.50 21.2521.00 21.50

21.2521.00 21.500.250.500.751.001.251.501.75

a) Glucuronic acid-meto-5TMS(2)

b) S-Benzyl-Cysteine-4TMS

Table 1 The number of identi�ed metabolites each analysis mode

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.

Modes

SIM

MRM

SIM/MRM

A

57

131

133

B

51

14

22

C

8

1

1

Total

116

146

156


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.

© Shimadzu Corporation, 2014

First Edition: June, 2014

www.shimadzu.com/an/

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.

Figure 4 Detected metabolites in human serum each analysis mode.

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

The reproducibility(n=6) in MRM and SIM/MRMTable 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

- 4.99%

5 - 9.99%

10 - 14.99%

15 - 19.99%

> 20%

MRM

73

26

8

9

30

146

SIM/MRM

76

30

10

10

30

156

Improvement

+3

+4

+2

+1

0

+10

SIM MRM

10 40 106

Metabolites with low CIDef�ciency in MRM

Metabolites withinterference in SIM

PO-CON1451E

Analysis of D- and L-amino acids usingautomated pre-column derivatizationand liquid chromatography-electrosprayionization 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

2

Analysis of D- and L-amino acids using automated pre-column derivatization and liquid chromatography-electrospray ionization mass spectrometry

IntroductionRecently, several species of D- amino acids have been found in mammals including humans and their physiological functions have been elucidated. Quantitating each enantiomer of amino acids is indispensable for such studies. In order to diagnose diseases, it is desirable that D- and L-amino acid can be separately quantitated and applied to metabolic analysis. Pre-column derivatization with o-phthalaldehyde (OPA) and N-acetyl-L-cysteine(NAC) is widely utilized for the analysis of D- and L- amino acids since the method can be performed with a rapid reversed phase separation on a relatively simple hardware (U)HPLC con�guration with

good reliability. One of the drawbacks of pre-column derivatization is less reproducibility due to the tedious manual procedure and human errors. We have launched an autosampler for a UHPLC system equipped with an automated pretreatment function that allows overlapping injections in which the next derivatization proceeds during the current analysis for saving total analytical time. We have applied this autosampler and its function to fully automate pre-column derivatization for the determination of amino acids. In this study, we developed a methodology which enabled the automated procedure of pre-column chiral derivatization of D- and L- amino acids.

Experimental

The system used was a SHIMADZU UHPLC Nexera pre-column Amino Acids (AAs) system consisting of LC-30AD solvent delivery pump, DGU-20A5R degassing unit, SIL-30AC autosampler, CTO-30A column oven, and SHIMADZU triple quadrupole mass spectrometer LCMS-8040. The software is integrated in the LC/MS/MS

workstation (LabSolutions, Shimadzu Corporation, Japan) so that selected conditions can be seamlessly translated into method �les and registered to a batch queue, ready for instant analysis. A 1.9um YMC-Triart C8 column (2.0 mm x 150 mm L.) was used for the analysis.

Instruments

Derivatizing solutions: 0.1 mol/L boric acid buffer was prepared by dissolving 6.18 g of boric acid and 2.00 g of sodium hydroxide in 1 L of water. 10 mmol/L NAC solution was prepared by dissolving 16.3 mg of N-acetyl-L-cysteine in 10 mL of the 0.1 mol/L boric acid buffer. 10 mmol/L OPA solution was prepared by dissolving 6.7

mg of o-phthalaldehyde in 0.3 mL of ethanol, adding 0.7 mL of the 0.1 mol/L boric acid buffer and 4 mL of water.Fig.1 shows the schematic procedure for amino acids derivatization with the SIL-30AC.Samples, including the derivatized amino acids, were injected onto the UHPLC and separated under the conditions shown in Table 1.

Derivatization Method

3


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

A standard solution containing 27 amino acids was prepared at 1 mmol/L concentration each in 0.1 mol/L HCl solution. The MS conditions such as ESI positive and negative ionization modes were optimized in parallel with the column separation, and compound dependent parameters such as CID and pre-bias voltage were adjusted

using the function for automatic MRM optimization. The transition that provided the highest intensity was used for quanti�cation. Table 2 shows the MRM transition of each derivatized amino acid. The MRM chromatogram is illustrated in Fig.2.

Analysis of Standard Solution

(1)

Take 20 μL of 10 mmol/L NAC solution

Supply 1 μL ofsample solution to the vial for mixing

(3)

Take 20μL of 10 mmol/L OPA solution

Mix the sample solutionand derivatizing solutions

Inject 1μL of the mixed solution to the column

Supply 20 μL of NAC solution to thevial for mixing

Supply 20 μL of10 mmol/L OPA solution to the vial for mixing

(5)

Take 1 μL of sample solution

Wait for 3min untilthe derivatization ends

Take 1μL of the mixedsolution

(2) (4)

(6) (7) (8) (10)(9)

4


Fig. 2 Chromatogram of a 27 amino acid standard solution

Compound

Aspartic acid

Glutamic acid

Serine

Glutamine

Glycine

Histidine

Threonine

Arginine

Tyrosine

Valine

Tryptophan

Isoleucine

Phenylalanine

Polarity

+

+

+

+

+

+

+

+

+

+

+

+

+

Precursor m/z

395.00

409.10

367.00

408.20

337.00

417.10

381.20

436.10

443.00

379.10

466.20

393.00

427.20

Product m/z

130.00

130.05

130.00

130.05

130.00

244.05

130.05

263.10

130.05

250.05

337.10

264.05

298.05

Table 2 Compounds, Ionization polarity and MRM transition

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

0

25000

50000

75000

100000

125000

150000

175000

200000

225000

250000

12

3 45

6

78

9

10

11

12 13

14

1516

17

18 1921

22

20

23

24

2526

27

■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-Glutamine8. D-Glutamine, 9. Glycine, 10. L-Histidine, 11. D-Histidine ,12. D-Threonine, 13. L-Threonine, 14. L-Arginine15. D-Arginine, 16. D-Alanine, 17. L-Alanine, 18. D-tyrosine, 19. L-Tyrosine, 20. L-Valine, 21. D-Valine22. L-Tryptophan, 23. D-Tryptophan, 24. L-Isoleucine, 25. D-Phenylalanine, 26. L-Phenylalanine, 27.D-Isoleucine

5


CompoundRepeatability (%RSD)

D-Aspartic acid

D-Glutamic acid

D-Serine

D-Glutamine

D-Histidine

D-Threonine

D-Arginine

D-Alanine

D-Tyrosine

D-Valine

D-Tryptophan

D-Isoleucine

D-Phenylalanine

5 μmol/L

3.5

3.7

4.8

4.1

4.3

3.8

3.4

4.0

3.2

3.3

3.9

3.1

3.5

25 μmol/L

2.5

3.1

3.0

3.4

1.8

2.6

1.7

2.3

2.9

2.2

3.2

2.9

1.8

Table 3 Reproducibility

Compound

D-Asparic acid

D-Glutamic acid

D-Serine

D-Glutamine

D-Histidine

D-Threonine

D-Arginine

D-Alanine

D-Tyrosine

D-Valine

D-Tryptophan

D-Isoleucine

D-Phenylalanine

Cali.F

Y = (44661.8)X + (1829.61)

Y = (12191.8)X + (10390.7)

Y = (22319.5)X + (-2869.30)

Y = (3458.60)X + (1521.83)

Y = (5778.33)X + (-341.182)

Y = (10800.6)X + (-1874.07)

Y = (10535.7)X + (-1298.12)

Y = (15349.1)X + (-4719.98)

Y = (17098.7)X + (-1812.69)

Y = (23707.7)X + (772.548)

Y = (18089.1)X + (-3620.41)

Y = (44017.1)X + (67903.1)

Y = (22426.0)X + (-736.090)

r2

0.998

0.999

0.999

0.999

0.998

0.999

0.998

0.999

0.999

0.999

0.998

0.999

0.999

Table 4 Linearity

Reproducibility and linearity in this analysis were evaluated with a plasma spiked standard solution. As a result, less than 5% relative standard deviation of peak areas were obtained. Table 3 shows the reproducibility of repeated analysis of spiked sample (n=6). Five different levels of

spiked sample concentration from 1 to 100 μmol/L standard solution were used for the linearity evaluation. The coef�cients of determination (r2) were approximately 0.999. Table 4 shows the summary for the linearity results.

Method Validation






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.

CompoundRecovery (100%)

D-Asparic acid

D-Glutamic acid

D-Serine

D-Glutamine

D-Histidine

D-Threonine

D-Arginine

D-Alanine

D-Tyrosine

D-Valine

D-Tryptophan

D-Isoleucine

D-Phenylalanine

5 μmol/L

100.3

92.8

97.9

103.2

104.8

101.1

102.4

93.5

98.1

101.0

97.8

98.8

104.5

25 μmol/L

107.1

97.8

100.6

104.3

100.4

98.8

99.6

99.5

101.0

99.2

100.4

102.4

100.9

Table 5 Recovery

PO-CON1476E

Characterization of metabolites in microsomal metabolism of aconitineby 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

2

Characterization of metabolites in microsomal metabolism of aconitine by high-performance liquid chromatography/quadrupole ion trap/time-of-�ight mass spectrometry

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

0.0

2.5

5.0

7.5(x1,000,000)

Introduction

Results

Aconitine (AC) is a bioactive alkaloid from plants of the genus Aconitum, some of which have been widely used as medicinal herbs for thousands of years. AC is also well known for its high toxicity that induces severe arrhythmias leading to death. Although numerous studies have raised on its pharmacology and toxicity, data on the identi�cation

metabolites of AC in liver microsomes are limited. The study of metabolic pathways is very important for ef�cacy of therapy and evaluation of toxicity for those with narrow therapy window. The aim of our work was to obtain the metabolic pathways of AC by the human liver microsomes.

Methods and Materials

The typical reaction mixture incubation contained 10 μmol/L aconitine and was preincubated at 37 ºC for 3 min. Reactions were initiated by adding 50 μL of NADPH (20 mmol/L), then incubated at 37 ºC in a waterbath shaker for

60 min. The reactions were terminated by adding 3-volume of ice-cold acetonitrile, then vortexed and centrifuged to remove precipitated protein.

Sample Preparation

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

M11M1

M0

M2M3

M4

M5

M6

M7

M8M9

M10

M12

M13 M14M16

M15

B

Fig.1 TIC chromatogram (A) and mass chromatograms of the metabolites of AC in the microsomal incubation mixture of human (B)

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 35.00.0

2.5

5.0

7.5(x1,000,000)

1:TIC (1.00)

A

3


Fig. 3 Proposed metabolic pro�le of AC in the human liver microsomes

Fig. 2 Proposed fragmentation pathway of AC

OH+

OH

OO

O

O

N

O

OH

H

OH

O

O

C34H48NO11+

Exact Mass: 646.3227C32H44NO9

+

Exact Mass: 586.3016

C31H40NO8+


C25H34NO8+


C29H36NO8+


C25H36NO9+

Exact Mass: 494.2390 C22H26NO4+

Exact Mass 368.1862 C21H25NO4+

Exact Mass 354.1705

O

OH

O

+

O

O

N

O

OH

H

OH

O

OH

O

+

O

O

N

O

OH

H

OH

O

OH

O

+

O

O

HN

O

OH

H

OH

O

OH

O

+

O

O

N

O

OH

H

OH

O

OH

O

+

O

O

N

O

OH

H

OH

OOH

O

O

HN

OH

OHH+

O

O

HN

HOH

OHH+

O

HO

HO

O

O

O

N

O

OH

H

OH

O

O

OH

HO

HO

O

O

O

N

O

OH

H

OH

O

O

OH

HO

O

O

O

OH

N

O

OH

H

OH

O

O

O

HO

O

O

O

O

N

HOH2C

O

OH

H

OH

O

O

O

OH

O

OH

O

O

N

O

OH

H

OH

O

O

OH

O

O

O

O

N

HOH2C

O

OH

H

OH

O

O

O

OH

O

O

O

O

N

O

OH

H

OH

O

O

O

OH

O

O

O

OH

N

O

OH

H

OH

O

O

OH

OH

O

O

O

O

N

O

OH

H

OH

O

O

O

OH

O

O

O

N

O

OH

H

OH

O O

O

O

O

N

O

OH

H

OH

O

O

O

OH

O

O

O

O

HN

O

OH

H

OH

O

OO

O

OH

O

O

N

O

OH

H

OH

O

O

OH

OH

O

O

O

O

N

O

OH

H

OH

O

O

M0

M13

M15

M11

M8M9M2

M10

M7

M16

M12

M3 M1

M5

O

HO

HO

O

O

O

N

O

OH

H

OH

O

O

M6O

HO

O

O

O

O

N

O

OH

H

OH

O

O

M4

O

HO

O

O

O

O

N

HOH2C

O

OH

H

OH

O

O

M14

4


No.

M0

M1

M2

M3

M4

M5

M6

M7

M8

M9

M10

M11

M12

M13

M14

M15

M16

RT(min)

22.3

10.5

11.2

11.3

11.8

12.2

13.3

13.5

13.7

13.8

14.1

15.0

15.1

16.0

17.3

17.6

17.9

Meas.MW(m/z)

646.3230

618.2922

616.2754

604.3140

630.2930

586.3005

616.2769

632.3035

648.3016

618.2935

618.2890

662.3179

602.2948

632.3054

662.3209

632.3068

584.2826

Pred.MW(m/z)

646.3222

618.2909

616.2752

604.3116

630.2909

586.3011

616.2752

632.3065

648.3015

618.2909

618.2909

662.3171

602.2960

632.3065

662.3171

632.3065

584.2854

mDaerror

0.8

1.3

0.2

2.4

2.1

0.6

2.3

3.0

0.1

3.0

1.5

0.8

1.6

1.1

3.8

0.3

2.8

MS2 data

586.3000, 554.2752, 526.2785, 494.2536, 476.2431, 404.2432, 368.1847, 354.1687

558.2710, 498.2469, 480.2378, 436.2093, 354.1725

556.2510, 554.2335, 494.2106, 478.2321, 434.1908, 402.1682

554.2744, 522.2398, 434.1898

570.2686, 552.2576, 510.2457, 492.2381

568.2938, 554.2705, 522.2537, 466.2168, 434.1922

584.2477, 524.2316, 434.1941

572.2866, 512.2638, 494.2468, 480.2283, 462.2214, 290.2236, 354.1652, 340.1871

588.2702, 570.2654, 528.2566, 510.2434, 406.2161

558.2714, 494.2109, 476.2400, 340.1548

558.2722, 494.2127, 476.2009, 354.1635

602.2964, 570.2654, 542.2750, 510.2434, 420.2416

584.2533, 524.2249, 510.2179, 406.1582

572.2853, 512.2661, 480.2368, 476.2445, 436.2082, 368.1812

602.2947, 570.2654, 542.2766, 510.2434, 478.2187

586.2973, 526.2738, 508.2273, 494.2490

552.2669, 492.2111, 460.2063

Formula

C34H47NO11

C32H43NO11

C32H41NO11

C32H45NO10

C33H43NO11

C32H43NO9

C32H41NO11

C33H45NO11

C33H45NO12

C32H43NO11

C32H43NO11

C34H47NO12

C32H43NO10

C33H45NO11

C34H47NO12

C33H45NO11

C32H41NO9

Biotransformation

Parent

deethylation

bidemethylation+dehydrogenation

deacetylation

demethylation+dehydrogenation

deacetylation+dehydration

bidemethylation+dehydrogenation

demethylation

oxidation+demethylation

bidemethylation

bidemethylation

oxidation

deacetylation+dehydrogenation

demethylation

oxidation

demethylation

deacetylation+dehydration+dehydrogenation

ppmerror

1.26

2.10

0.26

3.94

3.35

0.96

3.68

4.81

0.23

4.88

2.43

1.21

2.66

1.80

5.74

0.42

4.82

Table1 Mass data for characterization of metabolites in of AC in the microsomalincubation mixture of human






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.

PO-CON1447E

Simultaneous analysis of primarymetabolites by triple quadrupole LC/MS/MSusing 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

2

Simultaneous analysis of primary metabolites by triple quadrupole LC/MS/MS using penta�uorophenylpropyl column

IntroductionVarious metabolic pathways are controlled to keep a biological function in the cell and to monitor the rapid and slight changes of these metabolism, a simple simultaneous analysis is required for quanti�cation of primary metabolites. A typical LC/MS system with an ODS column is not effective to measure primary metabolites because of low af�nity of ODS column to hydrophilic metabolites. Here we report the

simultaneous measurement of 97 metabolites by triple quadrupole LC/MS/MS using penta�uorophenylpropyl column. In this experiment, MRM transitions of these metabolites were optimized and this method was applied to biological samples. Furthermore, to evaluate the accuracy of developed method for quanti�cation, simultaneous analysis by PFPP column was compared to measurement of ion-paring chromatography.

Commercially available compounds were used as standards to optimize MRM transition and LC condition for separation. Mixed standard solutions were diluted to a range of 10 nM~10000 nM for a calibration curve and an aliquot of 3 µL was subjected to LC/MS/MS measurement.Mice were sacri�ced under anesthesia and the isolated heart/liver tissues were rapidly frozen in liquid nitrogen. Frozen liver or heart tissues (>50 mg) from mice were homogenized in 0.5 mL methanol including L-methionine sulfone and 2-morpholinoethanesulfonic

acid (MES) as internal standards. After a general chloroform/methanol extraction, upper aqueous layer �ltered through 5-kDa cutoff �lter. The �ltrate was dried up and dissolved in 0.1 mL puri�ed water. Further, the solution was diluted to 20-100 folds in puri�ed water. An aliquot of 3 µL was analyzed to measure primary metabolites by LC/MS instrument, Nexera UHPLC system and LCMS-8030/LCMS-8040 triple quadrupole mass spectrometer. The following is detailed conditions of LC/MS mesurement.

Methods and materials

3


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

The MRM transitions for 97 standard compounds were optimized on both positive and negative mode by flow injection analysis (FIA). The MRM transitions of the 97 metabolites were determined as described in Table 1. Subsequently, LC condition was investigated to separate the 97 metabolites with a good resolution. As a consequence, the 97 metabolites were eluted from a PFPP column with a gradient of acetonitrile for <15 min in the

condition described in Figure 1. The linearity of this method was also confirmed by the simultaneous analysis of a serial of diluted calibration curve.

Figure 1 shows the MRM chromatogram of 97 metabolites at a concentration of 5 µM. In this figure, we can see the peak from all metabolites with a good separation.

Optimization of MRM transition

4


Table 1 MRM transition of 97 metabolites

Name Product ion Precursor ion Linearity (R2)

2-Aminobutyrate

Acetylcarnitine

Acetylcholine

Adenine

Adenosine

Adenylsuccinate

ADMA

Ala

AMP

Arg

Argininosuccinate

Asn

Asp

cAMP

Carnitine

Carnosine

cCMP

cGMP

Choline

Citicoline

Citrulline

CMP

Creatine

Creatinine

Cys

Cystathionine

Cysteamine

Cystine

Cytidine

Cytosine

Dimethylglycine

DOPA

Dopamine

Epinephrine

FAD

GABA

gamma-Glu-Cys

Gln

Glu

Gly

GMP

GSH

Guanosine

His

Histamine

Homocysteine

Homocystine

Hydroxyproline

Hypoxanthine

Ile

No.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

104.10

204.10

147.10

136.00

268.10

464.10

203.10

89.90

348.00

175.10

291.00

133.10

134.00

330.00

162.10

227.10

306.00

346.00

104.10

489.10

176.10

324.00

132.10

114.10

122.00

223.00

78.10

241.00

244.10

112.00

104.10

198.10

154.10

184.10

786.15

104.10

251.10

147.10

147.90

75.90

364.00

308.00

284.00

155.90

112.10

136.00

269.00

132.10

137.00

132.10

58.05

85.05

87.05

119.05

136.05

252.10

70.10

44.10

136.05

70.10

70.10

87.15

74.05

136.05

103.05

110.05

112.10

152.05

60.05

184.10

70.05

112.05

44.05

44.05

76.05

88.05

61.05

151.95

112.05

95.10

58.05

152.10

91.05

166.10

136.10

87.05

84.10

84.15

84.10

30.15

152.05

179.10

152.00

110.10

95.05

90.10

136.05

86.05

55.05

86.20

Polarity

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

0.99

0.99

0.99

0.98

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99*

0.99

0.99

0.99*

0.99

0.99

0.99

0.99*

0.99

0.99

0.99

0.99

0.99*

0.99

0.98*

0.99

0.99

0.99

0.99

0.99*

0.99*

0.99

0.99*

0.99

0.99*

0.99

0.99*

0.99*

0.99

0.99*

0.99

0.99

0.99*

0.99*

0.99

0.99

0.98*

0.99

Name Product ion Precursor ion Linearity (R2)

Inosine

Kynurenine

Leu

L-Norepinephrine

Lys

Met

Methionine-sulfoxide

Nicotinamide

Nicotinic acid

Ophthalmic acid

Ornitine

Pantothenate

Phe

Pro

SAH

SAM

SDMA

Ser

Serotonin

Thr/Homoserine

Thymidine

Thymine

TMP

Trp

Tyr

Uracil

Uridine

Val

2-Oxoglutarate

Allantoin

Cholate

cis-Aconitate

Citrate

FMN

Fumarate

GSSG

Guanine

Isocitrate

Lactate

Malate

NAD

Orotic acid

Pyruvate

Succinate

Taurocholate

Uric acid

Xanthine

No.

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

269.10

209.10

132.10

170.10

147.10

149.90

166.00

123.10

124.05

290.10

133.10

220.10

166.10

115.90

385.10

399.10

203.10

105.90

177.10

120.10

243.10

127.10

322.90

205.10

182.10

113.00

245.00

118.10

144.90

157.00

407.20

172.90

191.20

455.00

115.10

611.10

150.00

191.20

89.30

133.10

663.10

155.00

86.90

117.30

514.20

167.10

151.00

137.05

192.05

86.05

152.15

84.10

56.10

74.10

80.05

80.05

58.10

70.10

90.15

120.10

70.10

134.00

250.05

70.15

60.10

160.10

74.15

127.10

54.05

81.10

188.15

136.10

70.00

113.05

72.15

101.10

97.10

343.15

85.05

111.10

97.00

71.00

306.00

133.00

111.10

89.05

114.95

541.05

111.10

87.05

73.00

107.10

123.95

108.00

Polarity

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.99

0.98

0.99*

0.99

0.99*

0.99

0.99

0.99

0.99*

0.99*

0.99

0.99

0.99*

0.99

0.99

0.98*

0.98*

0.99**

0.99

0.99*

0.99

0.99**

0.99*

0.99*

0.99*

0.97*

0.99*

0.99*

0.99

0.99*

0.99*

0.99*

0.99*

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.

5


Figure 1 MRM chromatogram of 97 compounds

Simultaneous analysis of 99 compounds was performed for heart / liver tissue extracts as biological samples. Figure 2 shows MRM chromatograms of 99 compounds from tissue extracts (liver/heart). In this measurement, 83/97 metabolites were detected from liver tissue extracts and 88/97 metabolites were confirmed from heart tissue extracts. These results show this method is also effective to

simultaneous analysis of biological samples. As shown in the resulting MRM chromatogram, some major peaks were derived from the metabolites which were known to be characteristic to each tissue. Furthermore, this characteristic difference in each tissue was also confirmed in some faint peaks (e.g., cholate, cystine and homocysteine).

Application to tissue extracts as biological samples

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

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000


6

We have previously reported simultaneous analysis of 55 metabolites which were related to central carbon metabolic pathway by using ion pairing chromatography at ASMS conference 2013. To evaluate the accuracy of this simultaneous method using PFPP column, we compared the resulting peak area of 25 metabolites, which were covered as targets in both methods. The 25 metabolites are Lysine, Arginine, Histidine, Glycine, Serine, Asparagine, Alanine, Glutamine, Threonine, Methionine, Tyrosine, Glutamate, Aspartae, Phenylalanine, Tryptophan, Cysteine, CMP, NAD, GMP, TMP, AMP, cGMP, cAMP, MES and L-Methionine sulfone as internal standards. Heart tissue extracts were prepared from mice (n=9) according to the

method described above and the aliquots were measured by the simultaneous method using either ion pairing chromatography or PFPP separation system. As a result, we could see the similar trend of elevation/decrease of peak area in metabolites of 20/25 between nine samples. The peak areas between 9 samples of representative metabolites are shown in Figure 3. This result shows that a ratio of areas between 9 samples is kept in both methods. The four metabolites (TMP, cGMP, cAMP and Cysteine) could be hardly detected on simultaneous analysis by alternately ion-paring chromatography or PFPP column. Tryptophan had a faint peak in this experiment and led to the low similarity.

Correlation between PFPP and ion pairing Methods

Figure 2 MRM chromatogram of liver/heart tissue extracts

Liver Tissue

Heart Tissue

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

0

5000000

10000000

15000000

20000000

25000000

30000000

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

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

9000000

10000000

Acetylcarnitine

Creatine

Ophtalmic acid

GSSG

Guanosine

S-Adenosylhomocysteine

GSH

AMP






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.

0.0E+00

5.0E+05

1.0E+06

1.5E+06

1 2 3 4 5 6 7 8 9

MES

0.0E+00

5.0E+05

1.0E+06

1.5E+06

2.0E+06

1 2 3 4 5 6 7 8 9

Serine

0.0E+00

1.0E+05

2.0E+05

3.0E+05

4.0E+05

5.0E+05

1 2 3 4 5 6 7 8 9

Threonine

0.0E+00

1.0E+05

2.0E+05

3.0E+05

4.0E+05

5.0E+05

1 2 3 4 5 6 7 8 9

L-Methionine sulfone

PFPP

0.0E+00

2.0E+05

4.0E+05

6.0E+05

8.0E+05

1.0E+06

1 2 3 4 5 6 7 8 9

MES

0.0E+00

5.0E+03

1.0E+04

1.5E+04

2.0E+04

2.5E+04

1 2 3 4 5 6 7 8 9

Serine

0.0E+00

1.0E+04

2.0E+04

3.0E+04

4.0E+04

1 2 3 4 5 6 7 8 9

Threonine

0.0E+00

2.0E+05

4.0E+05

6.0E+05

8.0E+05

1.0E+06

1 2 3 4 5 6 7 8 9

L-Methionine sulfone

Ion pairing

0.0E+00

2.0E+06

4.0E+06

6.0E+06

8.0E+06

1 2 3 4 5 6 7 8 9

Aspartate

0.0E+005.0E+041.0E+051.5E+052.0E+052.5E+053.0E+05

1 2 3 4 5 6 7 8 9

GMP

0.0E+00

5.0E+06

1.0E+07

1.5E+07

1 2 3 4 5 6 7 8 9

AMP

0.0E+00

1.0E+06

2.0E+06

3.0E+06

4.0E+06

1 2 3 4 5 6 7 8 9

Phenylalanine

PFPP

0.0E+00

1.0E+05

2.0E+05

3.0E+05

4.0E+05

5.0E+05

1 2 3 4 5 6 7 8 9

Aspartate

0.0E+001.0E+042.0E+043.0E+044.0E+045.0E+046.0E+04

1 2 3 4 5 6 7 8 9

GMP

0.0E+001.0E+052.0E+053.0E+054.0E+055.0E+056.0E+05

1 2 3 4 5 6 7 8 9

AMP

0.0E+00

1.0E+04

2.0E+04

3.0E+04

4.0E+04

1 2 3 4 5 6 7 8 9

Phenylalanine

Ion pairing

Figure 3 Correlation of peak areas between PFPP and ion-pairing method

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