Post

erRE

PRIN

T

INTRODUCTION

Over the years, reactive metabolites are believed tohave played an important role in the safety profile ofpharmaceuticals. Chemically reactive metabolites of adrug can bind to proteins or DNA which is postulatedto result in various direct and idiosynchratic toxicities.Therefore, this represents a key target for earlyscreening to help prevent compound failure at a laterstage. In order to address this issue, an assay hasbeen developed to detect the formation of reactivemetabolites by using LC/MS/MS with exact massmeasurement to search for metabolites conjugatedwith glutathione from in-vitro incubations (Figure 1).Glutathione conjugates when fragmented by collisioninduced decomposition give a common losscorresponding to the pyroglutamic acid moiety(Figure 2), which can be easily monitored. Untilrecently, this work has been carried out with triplequadrupole technology using nominal mass. Theadvantage of the hybrid quadrupole time of flightmass spectrometer is the selectivity and sensitivity thatcan be achieved with it (Figure 3). Exact neutral lossdetection is achieved via sequential low and highenergy MS acquisitions (Figures 4, 5). When the lossof the pyroglutamic acid moiety is detected on thehigh energy scan with a window of ±20 mDa, thenMS/MS is carried out on the parent mass of interestto confirm the common neutral loss. In thispresentation, we will present data to substantiate thisapproach and also how exact mass measurementcan help to identify and postulate putativemetabolites.The test substrates used herein wereacetaminophen, troglitazone and raloxifene.

Figure 1. Strategy for detection and identification ofPhase I and II metabolites including specific searchfor exact mass neutral loss

Figure 2. Displays an example for the loss ofpyroglutamic acid used in GSH screening

Figure 3. Exact mass neutral loss on a Q-Tof™

Jose Castro Perez1, Steve Preece1, Li Liang2 , Eric. Y. Yang2

1Waters Corporation, Manchester, UK2Worldwide Bioanalysis, DMPK, GlaxoSmithkline Pharm., King of Prussia, PA 19406, US

A HIGH THROUGHPUT LC/MS/MS METHOD FORSCREENING GLUTATHIONE CONJUGATIONS USING

EXACT MASS NEUTRAL LOSS

In-vivo or in-vitro sample

Scan1-LC/MS with Low CD identify Phase I and II metabolites

LC/MS/MS Exact Mass Neutral Loss Scan2- LC/Pseudo

MS/MS with High CE search for

specific exact mass loss

Scan3-LC/MS/MS trigger for specificexact mass loss

to confirm conjugation

The Quadrupole Q1 is set to operatein "Wide-Band" transmission mode

with a low mass cutoff

∆m Exact MassHigh

Energy

"Scan" 1

CellCellQ1Q1 T OFT OFSS DD

"wide-band"

CellCellQ1Q1 T OFT OFSS DD

CellCellQ1Q1 T OFT OFSS DD

"wide-band"

CellCellQ1Q1 T OFT OFSS DD

Low Energy

*

**

"Scan" 2

Post

erRE

PRIN

T

Figure 4. Exact neutral Loss BPI for Acetominophen-GSH1

Figure 5. Single Scans to show exact mass NL on-the-flyfor Acetominophen-GSH

METHOD

MS Conditions

Mass Spectrometer: Waters® Micromass®

Q-Tof Ultima™

Ionization Mode: Positive ion ESI, mass range 50-850 amu

Capillary Voltage: 3.1 kV

Cone Voltage: 35 V

Source Temperature: 100 °C

Desolvation Temperature: 250 °C

Lock Mass: Leucine Enkephalin , M+H @ 556.2771 1 ng/µL concentration

LC Conditions

Solvent Delivery System: Waters 2795 Separations Module

Mobile Phase A : Water +0.1% Formic acid

Mobile Phase B : MeCN +0.1% Formic acid

Gradient:

Time A% B% C% D% Flow Curve

0.00 100.0 0.0 0.0 0.0 0.200 1

0.50 100.0 0.0 0.0 0.0 0.200 6

4.00 80.0 20.0 0.0 0.0 0.200 6

8.00 5.0 95.0 0.0 0.0 0.200 6

11.00 5.0 95.0 0.0 0.0 0.200 6

11.10 100.0 0.0 0.0 0.0 0.200 6

15.00 100.0 0.0 0.0 0.0 0.200 6

Column: Waters Atlantis™

2.1 mm x150 dC18

Injection Volume: 20-40 µL

SamplesIncubations were performed with human livermicrosomes (1 mg/mL) and GSH at 10 mM. Thesubstrates used were Acetominophen,Troglitazoneand Raloxifene at different concentrations: 100, 50,1 µM. The co-factors were: 2 mM NADPH and 3 mM MgCl. The samples were incubated at 37 ºCfor 90 minutes.

The reaction was stopped by adding acetonitrilewith acetic acid, then the samples were centrifugedat 15,000 rpm for 20 minutes and the supernatantcollected for LC/MS/MS analysis.

Post

erRE

PRIN

T

RESULTS

• From the low CE energy scan we were able todetect the Phase I and Phase II metabolites(Figures 6,7).

Figure 6. Hydroxylation metabolite for Troglitazone

Figure 7. Reduction metabolite for Troglitazone

• Mass Accuracies were better than 3 ppm for thedetected Glutathione metabolites (Table 1).

Table 1. Showing mass accuracies for detected GSH-conjugates

• Moreover, all the GSH incubates were spikedtogether in one vial and when analyzed by thismethod it showed the specificity of exact massonly switching on the exact peaks correspondingto the conjugates of interest (Figures 8,9).

Figure 8. Sample pooling for Acetaminophen 50 µM, Troglitazone 50 µM and Raloxifene 100 µM

Compound GSH conjugate m/z found Error ± mDa Error ± ppm

Acetoaminophen 457.1407 0.87 1.9

Troglitazone 747.2360 0.97 1.3

Raloxifene 779.2399 2.20 2.8

NH

OOH

CH3

O

CH3

CH3

OH

CH3

CH3

O

S NH

O

O

S

O

OH

OH

O

N

Post

erRE

PRIN

T

Figure 9. Exact mass MS/MS for sample pooling forAcetaminophen 50 µM, Troglitazone 50 µM andRaloxifene 100 µM

• Sensitivity is also important and we showed anexample where Troglitazone was incubated at1 µM and it was successfully detected (Figure 10).

Figure 10. Exact mass Neutral Loss for Troglitazoneat 1µM incubation2

CONCLUSIONS

• This is a highly selective and sensitive method forscreening of specific conjugations such as GSHby exact mass

• Exact mass provides good selectivity andgenerated rich structural information

• Information from Phase I xenobiotics is kept becausethe data is aqcuired in full scan exact mass

• With this approach there are no limitations to thenumber of exact mass neutral loss acquisitionswhich may be carried out on a single scanbecause the experiments are based upon DDA™

(Data Dependant Analysis)

ACKNOWLEDGEMENT

We would like to thank Abdul Conteh, Liangfu Chenand Harma Ellens for providing the in vitro samples.

REFERENCES

1. (Acetaminophen) Yohannes Teffera and FredAbramson Biological Mass Spectrometry,Vol.23, 776-783 (1994)

2. (Troglitazone) Justice N. Tettey, James L. Maggs,W. Garth Rapeport, Munir Pirmohamed and B.Kevin Park Chem. Res. Toxicol. Vol.14, 965-974 (2001)

Post

erRE

PRIN

T

Sales Offices:AUSTRIA AND EXPORT (CENTRAL EUROPE, CIS, MIDDLE EAST,INDIA AND INDIA SUBCONTINENT) 43 1 877 18 07

AUSTRALIA 61 2 9933 1777

BELGIUM AND LUXEMBOURG 32 2 726 1000

BRAZIL 55 11 5543 7788

CANADA 800 252 4752 X2205

CZECH REPUBLIC 420 2 617 11384

DENMARK 45 46 59 8080

FINLAND 358 09 506 4140

FRANCE 33 1 3048 7200

GERMANY 49 6196 400600

HONG KONG 852 29 64 1800

HUNGARY 36 1 350 5086

INDIA 91 80 837 1900

IRELAND 353 1 448 1500

ITALY 39 02 27 4211

JAPAN 81 3 3471 7191

KOREA 82 2 3284 1300

MEXICO 52 55 5524 7636

THE NETHERLANDS 31 76 508 7200

NORWAY 47 6 384 6050

PEOPLE’S REPUBLIC OF CHINA86 10 8451 8918

POLAND 48 22 833 4400

PUERTO RICO 787 747 8445

RUSSIA/CIS 7 095 931 9193

SINGAPORE 65 6278 7997

SPAIN 34 93 600 9300

SWEDEN 46 8 555 11 500

SWITZERLAND 41 62 889 2030

TAIWAN 886 2 2543 1898

UK 44 208 238 6100

U.S.A. AND ALL OTHER COUNTRIES: WATERS CORPORATION34 Maple St.Milford, MA 01757 U.S.A.T: 508 478 2000F: 508 872 1990www.waters.com

Waters, Micromass, Q-Tof, Q-Tof Ultima, Atlantis and DDA are trademarks of Waters Corporation.All other trademarks are the property of their respective owners.©2003 Waters Corporation Produced in the U.K. October 2003 720000770EN CH-PDF

WATERS CORPORATION34 Maple St.Milford, MA 01757 U.S.A.T: 508 478 2000F: 508 872 1990www.waters.com