Drug discovery & development solutions

FOR DRUG METABOLISM

DRUG METABOLISM

Fast and efficient metabolite identification is critical in today’s

drug discovery pipeline. The goal is to achieve rapid structural

identification and complete characterization of major and

low-level metabolites in a single analysis. AB SCIEX triple

quadrupole-linear ion trap mass spectrometers and accurate

mass approaches make that goal a reality.

– Qual & Quant for the answers you need: Get high-sensitivity

quantification and new qualitative capabilities on a single

platform—the AB SCIEX TripleTOF™ 5600 System and

MetabolitePilot™ Software.

– Fast and efficient low-level metabolite identification:

Acquire predictive MRM (pMRM) and multiple precursor ion

and/or neutral loss survey scans in a single analysis, including

polarity switching, with the AB SCIEX QTRAP® 5500 and

LightSight® Software.

– From early discovery to late stage development: Get the

information you need to take products from the lab to the

clinic with integrated workflows.

The AB SCIEX TripleTOF™ 5600 System and

MetabolitePilot™ Software deliver high

sensitivity quantification and new qualitative

capabilities on a single platform.

The AB SCIEX QTRAP® 5500 and LightSight®

Software use predictive MRM (pMRM) and

multiple precursor ion and/or neutral loss survey

scans in a single analysis, including polarity

switching.

AB SCIEX TripleTOF™ 5600 System

Accurate mass at the speed and sensitivity of a triple quadrupole:

• Fast MS and MS/MS acquisition speeds compatible with

fast chromatography

• Resolution over 30,000

• External mass accuracy ~1ppm

• 4 orders of Linear Dynamic Range

MetabolitePilot™ Software

Accurate mass data processing and interrogation:

• Generate cleaner, more relevant data with Multiple Mass

Defect Filtering.

• Store and retrieve critical information in Compound Library

& Results Database.

• Quickly process multiple sample sets in batches.

• Increase confidence in your results with intelligent scoring

and easy-to-visualize color-coding.

• Predict formulae with a high level of chemical intelligence.

Simple, clear metabolism workflows

AB SCIEX QTRAP® 5500 System

A single platform for drug metabolism and bioanalytical

quantification workflows:

• Targeted and non-targeted workflows

• Increased sensitivity

• Increased speed

• Full quantitative capabilities

LightSight® Software

Exploit the full functionality of QTRAP® technology and processing

strategies for multiple MetID workflows

• pMRM – high sensitivity targeted approach for really low

level detection and confirmation

• PI/NL (+/-ve polarity switching) – structure based filtering

approach ideal for reactive metabolite screening

• Multiple Ion Monitoring (MIM) & Q3 single MS strategies

for a non-targeted approach

AB SCIEX TripleTOF™ 5600 System MetabolitePilot™ Software AB SCIEX QTRAP® 5500 System Lightsight® Software

Drug discovery & development pipelineMetabolite identification is central to many of the activities in the discovery and development

pipeline. From rapid structural identification during the discovery process to provide an early

perspective on the metabolically labile sites, or soft spots of a drug candidate, to a more complete

characterization of the metabolic clearance process to maximize safety and efficacy, there’s an

ever-increasing demand on throughput.

The ability to find, identify and confirm metabolites as fast as possible is critical; not only

for the major metabolites, but the very low-level metabolites as well. Being able to do this in a

single analysis is a highly sought after goal. With improved strategies for metabolite ID from hybrid

triple quadrupole-linear ion trap mass spectrometers and an ever increasing use of accurate mass

approaches, these different but complementary mass spectrometric techniques have brought

the goal to reality.

DRUG DISCOVERY PRE-CLINICAL CLINICAL TRIALS FDA REVIEW

Average time in stage

Phase I20 To 100 volunteers

Phase III1,000 To 5,000 volunteers

Phase II100 to 500 volunteers

5 YEARS 1.5 YEARS 6 YEARS 2 YEARS 2 YEARS

LARGE-SCALE MANUFACTURING

PHASE IV

IND

SUB

MIT

TE

D

ND

A SU

BM

ITT

ED

FDAAPPROVED

DRUG

10,000COMPOUNDS 5 COMPOUNDS250 COMPOUNDS

Application snapshots within the drug discovery & development pipeline

1. EARLY DISCOVERY

Metabolic Stability

GSH Detection

3. DEVELOPMENT

Definitive Identification

Integrated Qual/Quant workflows

2. LATE STAGE DISCOVERY

In-vivo Metabolite ID

Low-level Metabolite ID

Early discovery:metabolic stability screening

C The MS/MS spectrum of Imipramine obtained automatically in IDA mode. The high

mass accuracy and resolution greatly simplify structure elucidation.

B A wide range of Imipramine phase I metabolites detected at t=5 minutes by

TOF MS scan.

A Using a generic IDA method and fast chromatography, metabolic stability profiles were

obtained in rat liver microsomes at a substrate concentration of 1 μM at t=0.

Metabolic stability assessment

Take soft-spot analysis to new levels of speed, efficiency and depth

of data with the TripleTOF™ 5600 and MetabolitePilot™ Software.

Apply completely generic methodology to acquire high quality

quantitative and qualitative data simultaneously for soft-spot

analysis. The AB SCIEX TripleTOF™ 5600 System makes it possible,

with TOF MS and MS/MS scanning at rates suitable for UPLC.

• Generate quantitative data on the parent compound from the

TOF MS scan.

• Automatically acquire MS/MS data on the major metabolites

using high-speed IDA logic.

• Use accurate mass MS/MS for structural elucidation.

t=5 min

Metabolic Stability – Rat Liver Microsomes

Detect low-level reactive metabolites in complex samples using

glutathione (GSH) detection with the AB SCIEX QTRAP® 5500

System and LightSight® Software.

The formation of undetected reactive metabolite species can

potentially cause idiosyncratic adverse drug reactions (IADRs),

which can lead to Black Box warnings and withdrawal of drugs

from the market. GSH detection is one of the most sensitive ways

of detecting low-level, transient reactive metabolites in complex

biological samples.

H3C

OO

O

OO

OH2N

NH

NH

HN

OHS

HO

H3C

OO

O

OO

OH2N

NH

NH

HN

OHS

HO

Neutral Loss m/z 129 Precursor Ion m/z 272

Early discovery: reactive metabolite detection

High sensitivity GSH adduct screening: Fast scanning and polarity switching with the QTRAP® 5500 System enable sensitive detection of GSH-trapped reactive

metabolites with a combined precursor ion (negative ion mode, 272 m/z) and neutral loss (positive ion mode, m/z 129) in a single injection. The survey scans are

specific to the GSH moiety. They do not require knowledge of parent MS/MS fragmentation, or prediction of metabolites. This provides for comprehensive detection

of GSH conjugated metabolites – whether predicted or not.

GSH Adduct Screening2 Survey scans with polarity switching in 1 injection

Total cycle time1.3 sec

+ CNL of 129

- Precursor m/z 272

+EPI m/z 632 +EPI m/z 648

at 20,000 amu/sec

Both triggered via ER scan

- Prec + ER + EPI + CNL + ER + EPI

Identify metabolites of lead drug candidates for structural

elucidation using selective detection techniques such as mass

defect filtering with the TripleTOF™ 5600 System. For the first time,

multiple mass defect filters can be applied in real time to select

peaks for MS/MS data acquisition. There is no need to schedule a

second injection to obtain MS/MS information. With the TripleTOF™

5600 you can detect and characterize major metabolites in vivo

with a single injection.

Late stage discovery: In vivo metabolite identification

A Minor oxidative metabolite (2.59 minutes) of Carbamazepine detected in protein

precipitated plasma. Although eluting just before a major metabolite, multiple mass

defect filtering enabled acquisition of MS/MS spectra of both metabolites in the

presence of strong matrix interferences.

B TOF MS scan of peak at 2.59 min in above chromatogram. Large endogenous

interferences are present in the scan. Real time mass defect filtering ensures successful

acquisition of MS/MS data for the ion of interest in the presence of high background.

C The accurate mass MS/MS spectrum of the minor oxidative metabolite obtained in

IDA mode using real time mass defect filtering.

A

B C

In vivo detection in complex matrices

Detect low levels of metabolites at clinically relevant concentrations

in vivo—even in matrices such as bile, plasma, urine and fecal

extracts—using the QTRAP® 5500 System. The predictive MRM

(pMRM) survey method on the QTRAP® 5500 System is the most

sensitive and selective single-injection strategy for identifying

metabolites in challenging matrices.

Low level in vivo metabolite

detection in protein precipitated

plasma: extracted ion

chromatograms of Carbamazepine

metabolites from a 4 mg/Kg IV

administration in rat, detected by

pMRM (176 transitions monitored

simultaneously) with the QTRAP

5500 System.

Late stage discovery: low-level metabolite ID

Metabolite characterization in lead compounds

Acquire accurate mass data for both parent and fragments in lead

compounds with the TripleTOF™ 5600 System and MetabolitePilot™

Software. An advanced collision cell design and collision energy

spread function produce rich MS/MS fragmentation in the low mass

range for structure elucidation. Rich fragmentation, high resolution

and mass accuracy enable definitive metabolite profiling.

Hydroxymidazolam in rat liver microsomes. Excellent accuracy

and resolution at the low mass range in MS/MS enables

unambiguous assignment of elemental composition.

Development: definitive in vivo metabolite ID

Metabolite quantification

Estimate metabolite concentrations, without a reference standard,

using mass extracted ion chromatograms from high-resolution

experiments using the TripleTOF™ 5600 System, MetabolitePilot™

and MultiQuant™ Software.

Quantification of metabolites and cross-species quantitative

comparisons have received greater emphasis due to the Metabolites

in Safety Testing (MIST) Guidelines. Estimating metabolite

concentrations without a reference standard, however, is challenging.

TOF MS extracted ion chromatograms of OH-Midazolam metabolites and the Midazolam

parent. All peaks are automatically integrated and labeled with area counts.Corresponding TOF MS scans for the metabolite and parent peaks in Figure 6A.

High resolution enables relative quantitation in TOF MS mode while reducing or

eliminating matrix interference.

Development: integrated qual/quant workflows

TripleTOF™ 5600 System, MetabolitePilot™ and MultiQuant™

Software meets the challenge by enabling you to compare the

response in the extracted ion chromatogram (XIC) of the metabolite

to the parent drug, with high resolution, high resolution and mass

accuracy at the low mass range, and more selectivity. Thanks to fast

scanning, you can perform these studies under UPLC.

Biotransformation studies

Acquire and process mass spectrometric and UV or

radiochromatographic data in the same experiment with the

QTRAP® 5500 System and LightSight® Software—and without

time-consuming manual procedures.

All together now: Correlation of UV and MS

data of rat liver microsomal metabolites of

Bromocriptine. The Processing Workspace of

LightSight® Software makes the correlation of UV

absorbance, pMRM, and EPI-MS/MS data easy

and intuitive.

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

© 2011 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.

0041610-02 01/11

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