Simultaneous Qualitative and Quantitative Analysis of Buspirone and its Metabolites with the Agilent 6550 iFunnel Q-TOF LC/MS System

Application Note

Introduction

Timely and rapid assessment of metabolic stability, metabolite identifi cation,

and metabolite profi ling is critical for accelerating lead optimization and

enhancing the success rate of drug candidates entering into development. Triple

quadrupole LC/MS instruments with multiple reaction monitoring (MRM) have

been the workhorse for quantitative analyses, such as metabolic stability and

metabolite profi ling. However, this platform is optimized for high sensitivity target

quantitation and is not well suited for non-targeted qualitative analysis. For this

reason, metabolite identifi cation (qualitative analysis) is often performed in a

separate analysis on a different type of mass spectrometer. Due to limitations

in the sensitivity of traditional tandem mass spectrometers, a relatively high

substrate concentration (i.e.10-20 µM) is often required in order to identify

metabolites with broad coverage.

The ability to obtain quantitation and identifi cation (Qual/Quan) in a single

analysis makes metabolic stability, metabolite identifi cation, and metabolite

profi ling studies much more effi cient. It also has the potential to increase assay

productivity and decrease costs in drug discovery and development. This note

describes an integrated Qual/Quan workfl ow. It also demonstrates the utility of

the Agilent 6550 iFunnel Q-TOF LC/MS system for the determination of metabolic

stability, metabolite identifi cation, and metabolite profi ling in an in vitro buspirone

(1µM) metabolism study in rat liver microsomes.

Authors

Yuqin Dai, Michael Flanagan, and

Keith Waddell

Agilent Technologies, Inc.

Santa Clara, CA USA

2

An Integrated Qualitative/Quantitative Workflow

The Qual/Quan workfl ow starts

with the LC/MS injection of a

biological sample (Figure 1). Using

the Agilent 6550 iFunnel Q-TOF

LC/MS system, data acquisition

includes a full MS scan followed by

three data dependent auto MS/MS

scans. Metabolite identifi cation and

structure elucidation are facilitated by

MassHunter Metabolite ID software.

Metabolic stability and metabolite

profi ling are established from the

same set of data, which are processed

in batch mode by MassHunter

Quantitative Analysis software.

Key Features and Benefits

• The 6550 iFunnel Q-TOF enables

simultaneous qualitative and

quantitative analysis of metabolic

stability, metabolite identifi cation,

and metabolite profi ling, without

compromising the quality of data or

the speed of analysis.

• High sensitivity at low pg/mL

permits simultaneous quantitation

and identifi cation of buspirone and

its metabolites over a time course

of up to 60 minutes with 1 µM

buspirone in rat liver microsomes.

• Sub-ppm mass accuracy in both

MS and MS/MS combined with

the excellent isotopic fi delity

provide high confi dence in

metabolite identifi cation and

structure elucidation.

• Fast scan speed of up to

50 spectra/second allows rigorous

quantitation in a three and a half

minute run time.

• MassHunter Metabolite ID software

greatly facilitates metabolite

identifi cation by using a wide range

of feature identifi cation and Novatia

Autoshift algorithms.

• MassHunter Quantitative Analysis

software provides quick and effi cient

data processing and reporting in a

batch mode.

Figure 2. Metabolic stability of buspirone and the profi le of its metabolites.

Figure 1. Integrated Qual/Quan workfl ow.

Biological Sample

Single injection

Data acquisition by 6550 QTOF

MS followed by 3 auto MS/MS

Metabolite ID Software

ID and structure elucidation

MassHunter Quantitative Software

Parent and metabolite quantitation

Qual/Quan Analysis

Figure 2 shows the metabolic stability

of buspirone and the profi les of

its metabolites, illustrating broad

coverage of the high and low

abundance metabolites across the

entire 60 minute time course.

3

Figure 3 shows the MS and MS/MS

spectra of a buspirone monohydroxy

metabolite from a 10 minute incubation

sample. The sub-ppm mass accuracy of

the precursor and fragment ions, along

with the excellent isotopic fi delity

Fi gure 3. Excellent mass accuracy in MS and MS/MS spectra, as well as isotopic fi delity.

Figure 4. Extracted ion chromatograms of buspirone and its phase I metabolites from a 10 minute incubation sample.

(overall score > 99 %), provided highly

confi dent metabolite identifi cation

and structure elucidation. Excellent

separation and broad metabolite

coverage was achieved in three and a

half minutes (Figure 4).

www.agilent.com/chem/

metabolomics

Information, descriptions, and specifi cations in this publication are subject to change without notice.

© Agilent Technologies, Inc., 2011Published in the USA, October 11, 20115990-9209EN

Conclusions

An effi cient Qual/Quan workfl ow

has been developed using the Agilent

6550 iFunnel Q-TOF LC/MS system

combined with two powerful software

tools: MassHunter Metabolite ID

and Quantitative Analysis. The 6550

iFunnel Q-TOF LC/MS system enables

the simultaneous quantitation and

metabolite identifi cation from 1µM

buspirone incubations with suffi cient

sensitivity, throughput, and high

analytical confi dence. This single

analytical platform also allows rapid

generic LC/MS method development

and eliminates the time-consuming

MRM optimization for each analyte.