Simultaneous qualitative and quantitative analysis using the Agilent 6540 Accurate-Mass Q-TOF

Technical Overview

Abstract

The utility of the Agilent 6540 Accurate-Mass Q-TOF LC/MS System for the

determination of metabolic stability, profi ling, and identifi cation in a single

experiment has been demonstrated in a buspirone metabolic study, thus maxi-

mizing the quantitative and qualitative information that can be obtained from one

experiment. The validity of the quantitative data was confi rmed by analysis using

the Agilent 6460 Triple Quadrupole LC/MS System, operating in multiple reaction

monitoring (MRM) mode.

Introduction

Determining metabolic stability is an important aspect of early drug discovery, due

to its role in drug half-life and bioavailability. Metabolite identifi cation is crucial in

order to understand drug safety and effi cacy. In early drug discovery, samples are

typically analyzed by a variety of different LC/MS methods in order to generate

both metabolic stability (quantitative) and metabolite identifi cation (qualitative)

data. The ability to obtain both quantitation and identifi cation in a single analysis

makes metabolic stability, profi ling, and identifi cation studies much more effi cient.

The study described here demonstrates the use of the high resolution and accurate

mass determination capabilities of the Agilent 6540 QTOF LC/MS to perform both

quantitation and identifi cation of metabolites in the same experiment.

Authors

Pat Perkins

Anabel Fandino

Lester Taylor

Agilent Technologies, Inc.

Santa Clara, CA USA

2

Experimental

Instruments

Metabolic stability, profi ling, and

identifi cation were performed using

an Agilent 1290 Infi nity LC System

coupled to an Agilent 6540 Accurate-

Mass Q-TOF System. The 6540 QTOF

was operated in two modes: an initial

MS-only mode at 5 Hz acquisition

rate, and an automatic MS/MS mode,

with the masses of the expected

metabolites on the inclusion list.

Quantifi cation results were confi rmed

using the same LC system and MRM

on the Agilent 6460 Triple Quadrupole

LC/MS System. The same conditions

(e.g. collision energy) were used for

both instruments because they use the

same hardware design from ion source

to collision cell. The instrument condi-

tions are given in Tables 1 and 2.

Sample preparation

Buspirone was chosen as a model

compound for the metabolic stability

study. This drug was incubated at

1 μM in a rat liver S9 preparation,

using an NADPH regenerating system.

The incubation was carried out at 37°C

and 100 μL aliquots were taken at 0, 5,

10, 15, and 20 minutes. The reaction

was stopped by adding acetonitrile;

then the sample was centrifuged.

The supernatant was evaporated

to dryness using a gentle stream of

nitrogen and reconstituted in the

mobile phase for combined ultra high

performance liquid chromatography

(UHPLC) and MS.

Table 1. Ultra High Performance Liquid Chromatography (UHPLC) Conditions

Table 2. 6540 QTOF and 6460 Triple Quadrupole LC/MS mass spectrometer conditions

UHPLC Run Conditions

Column Agilent ZORBAX Eclipse Plus C18, 2.1 mm x 100 mm, 1.8 μm

Injection volume 1 µL

Needle wash Flush port (75:25 MeOH:H2O, 0.1% formic acid ,10 sec)

Mobile phase A = H2O + 0.1% formic acid

B = acetonitrile + 0.1% formic acid

Flow rate 1.0 mL/min

Gradient 5% B to 95% B

Stop time 5.5 min

QTOF and Triple Quadrupole MS Conditions

Ion Source Settings for Both Instruments

Ion mode Positive, ESI

Drying gas temperature 350°C

Drying gas fl ow 10 L/min (nitrogen)

Sheath gas temperature 400°C

Sheath gas fl ow 12 L/min

Capillary voltage 4000 V

Nozzle voltage 0 V (+)

QTOF MS/MS Settings

Acquisition type Auto MS/MS (data-dependent)

Mass range 100–1000 m/z for both MS and MS/MS

Transients 1557/spectrum for both MS and MS/MS

Acquisition rate 5 spectra/sec (combined MS and MS/MS)

Precursors/cycle 1

Active exclusion Exclude after 2 spectra, release after 0.05 min

Collision energy 28 EV (same for Triple Quad)

Charge state 1+ and unknown

Preferred list All expected metabolites

Mode Extended dynamic range (2 GHz)

Triple Quadrupole MS/MS Settings

MS1/MS2 resolution Unit

Delta EMV 200 V

Time fi ltering 0.03 min

MRM transitions 386.3 → 122.1 (Buspirone)

609.3 → 195.1 (Reserpine, internal standard)

Dwell time 100 ms per transition

Cycle time 203.5 ms

3

0.01

0.1

1

10

100

0 5 10 15 20 25

Incubation time (min)

% P

aren

t re

mai

nin

g

QQQ

Q-TOF

Results and Discussion

Reliable metabolic stability results

Metabolic stability results were

plotted using the data from the QTOF

MS only analysis and the Triple Quad

MRM analysis. As shown in Figure 1,

there was good correlation of the

metabolic stability data obtained using

the 6540 QTOF full-spectrum analysis,

with the same data obtained using the

6460 Triple Quadrupole MRM analysis.

The semi-log plot of percent parent

versus time is a typical means used to

determine and compare metabolite half

lives. The 6540 QTOF is an extremely

sensitive instrument capable of good

quantitative measurements.

Simultaneous profi ling of metabolites

The MS analysis using the 6540 QTOF

provided full spectra through the entire

run, allowing profi ling of all metabo-

lites, while the triple quadrupole anal-

ysis using the MRM mode provided

analysis only of the parent drug and

known, selected metabolites. Figure 2

shows extracted ion chromatograms of

the expected metabolites of buspirone,

detected in the QTOF analysis. This

sample was from a 5 min incubation

time, and the QTOF was operated in

high resolution MS scan mode.

x105

0

1

2

3

4

5

6

Counts vs. Acquisition Time (min)

0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6

Buspirone (parent)

N-oxide

Mono-

hydroxy

Dihydroxy

-C2H

2 +O

-C2H

2

Mono-

hydroxy

Buspirone

Dihydroxy

QTOF delivers accurate metabolic stability data

Simultaneous profi ling of metabolites

Figure 1. The 6540 QTOF gave metabolic stability results for buspirone that were in good

agreement with those obtained using the 6460 Triple Quadrupole system. Results are

plotted using a semi-log scale (log %Parent remaining) to provide a linear relationship to

incubation time.

Figure 2. 6540 QTOF analysis EICs revealed the expected metabolites of buspirone.

Time 0 min 5 min 10 min 15 min 20 min

QTOF (%) 100 4.88 0.57 0.100 0.020

QQQ (%) 100 4.95 0.47 0.098 0.017

4

Figure 3 shows representative spectra

from the QTOF analysis, acquired

in MS mode with a 5 Hz acquisition

rate. The spectra were taken from a

sample at a 10 minute incubation time,

when less than 1 percent of the parent

drug remained. The QTOF spectra

for buspirone and its monohydroxy

and dihydroxy metabolites showed

mass errors of less than 1 ppm, which

enabled confi dent identifi cations.

Monohydroxy metabolite

x104

0

1

2

124.0869418.2448

C21 H32 N5 O4 922.0098

972.0061

Counts vs. Mass-to-Charge (m/z)100 200 300 400 500 600 700 800 900 1000

x104

0

2.5

5

7.5 402.2503C21 H32 N5 O3

124.0869

922.0098

Counts vs. Mass-to-Charge (m/z)100 200 300 400 500 600 650 700 750

650 750

800 900 1000

x104

0

2

922.0098121.0509 386.2548C21 H32 N5 O2

273.1665

100 200 300 400 500 600 700 800 900 1000

Counts vs. Mass-to-Charge (m/z)

Buspirone

Dihydroxy metabolite

386.2548C21 H32 N5 O2

387.2589

C21 H32 N5 O2

388.2533

C21 H32 N5 O2

-0.62 ppm

x104

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Counts vs. Mass-to-Charge (m/z)386 387 388 389

402.2503

C21 H32 N5 O3

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

x104

Counts vs. Mass-to-Charge (m/z)

-0.90 ppm

402 403 404 405

403.2531

C21 H32 N5 O3

404.2497

C21 H32 N5 O3

418.2448

C21 H32 N5 O4

0

0.2

0.4

0.6

0.8

1

1.2

1.4

419.2485

C21 H32 N5 O4

420.2444

C21 H32 N5 O4

-0.10 ppm

Counts vs. Mass-to-CHarge (m/z)418 419 420

x104

Reliable identifi cation of metabolites

Figure 3. Metabolite spectra taken with the 6540 QTOF demonstrated excellent mass accuracy and isotopic fi delity, which provided assurance of

correct identifi cations.

Isotope ratios provide additional

information that further increases

confi dence of identifi cation. The

insets in Figure 3 show the measured

isotope patterns versus theoretical

(the latter shown in rectangles), based

on the correct molecular formulae. The

isotopic fi delity is excellent, due in

part to the analog-to-digital converter

(ADC) detector employed in Agilent

TOF and QTOF systems. Time-to-digital

converter (TDC) detectors used in

some other TOF and QTOF systems are

known to have problems with isotope

ratios, which means that analysts

cannot rely on this information for

compound identifi cation.

From these full scan MS mode

analyses, abundances of buspirone

and its metabolites were plotted as a

function of incubation time (Figure 4).

The graph demonstrates that the

6540 QTOF is capable of obtaining

both metabolic stability and metabolic

profi ling in a single MS experiment.

5

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20

Rel

ativ

e am

ount

(%)

Incubation time (min)

MS data (2.5 Hz) from combined

MS and MS/MS analysis

buspirone (parent)

monohydroxy

dihydroxy

N-oxide

-C2H2

-C2H2 +O

QTOF MS/MS mode enables more comprehensive metabolite

identifi cation and structural characterization

Figure 5. Metabolic stability and metabolic profi ling from the same

experiment (combined MS and MS/MS, MS at 2.5 Hz acquisition rate)

with the Agilent 6540 QTOF. Graph assumes all response factors were

the same as for the parent buspirone.

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20

Incubation time (min)

Rel

ativ

e am

ount

(%)

MS-only analysis, 5 Hz

buspirone (parent)

monohydroxy

dihydroxy

trihydroxy

N-oxide

-C2H2

-C2H2 +O

Metabolic stability and profi ling in a single MS experiment

Figure 4. Metabolic stability and metabolic profi ling from the same

experiment (MS-only, 5 Hz acquisition rate) with the Agilent 6540

QTOF. Graph assumes all response factors were the same as for the

parent buspirone.

Augmented metabolite identifi cation

with QTOF MS/MS mode analysis

After successfully determining meta-

bolic stability and metabolic profi ling

using the 6540 QTOF, the possibility

of including MS/MS for more compre-

hensive metabolite identifi cation

and structural characterization was

investigated. For this targeted MS/MS

analysis, the masses of the expected

metabolites were specifi ed on an

inclusion list, to ensure that they

would be analyzed.

With the combined MS and MS/MS

acquisition, the MS acquisition rate

was 2.5 spectra/second and provided

suffi cient data points across the peaks

to generate semi-quantitative results.

In fact, the resulting graph of drug and

metabolite concentrations over time,

shown in Figure 5, is almost identical

to that in Figure 4. When operated

in this mode, the Agilent 6540 QTOF

used half of the time to carry out full

scan MS data acquisition, and half of

the time for MS/MS data acquisition,

which was used for metabolite identifi -

cation, thus providing quantitative

and qualitative results in a single

analytical experiment.

6

In the MS/MS spectrum at the bottom

of Figure 6, the masses of the frag-

ment ions agree very well with the

theoretical values. The collision cell in

the Agilent 6540 QTOF has a unique

design feature that utilizes high

pressure cooling, which means both

product and precursor ions have the

same relative energy. Therefore, the

same tuning parameters can be used

for both MS and MS/MS analysis,

resulting in excellent MS/MS mass

accuracy. Given the quality of the MS

and MS/MS data in Figure 6, the iden-

tifi cation of a metabolite can be made

with a high degree of confi dence.

and the isotope abundances show

excellent agreement with the theo-

retical values (rectangles). Other

QTOF instruments that use a TDC

detector have diffi culty achieving

these accurate isotope ratios, and

due to TDC dead time, may suffer from

poor mass accuracy for chromato-

graphic peaks with high abundance.

The larger mass errors in TDC-based

instruments negatively affect both

identifi cation and quantitation.

The ADC detector in the 6540 QTOF

does not have these problems.

The quality of the MS and MS/MS

spectra for metabolite identifi ca-

tion was excellent, as shown in the

example for the buspirone mono-

hydroxy metabolite (Figure 6). The

spectra are labeled with the results

produced by molecular formula genera-

tion (MFG), which show an excellent

match between the experimental data

and the calculated formula.

In the MS spectrum at the top of

Figure 6, the measured mass matches

the calculated mass within 0.07 ppm,

x102

0

0.2

0.4

0.6

0.8

1121.0509

135.0443 194.1171109.0762 327.2009224.1282

x102

0

0.2

0.4

0.6

0.8

1

1.2

122.0705C6 H8 N3

402.2493150.1022

C8 H12 N3 238.1424C11 H18 N4 O2

178.1217C9 H14 N4

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420

219.1604C12 H19 N4-3.32 ppm

2.07 ppm

-0.54 ppm

-0.28 ppm-2.36 ppm

Buspirone monohydroxy metabolite

MS spectrum

MS/MS spectrum

Counts (%) vs. Mass-to-Charge (m/z)

402.2500

C21 H32 N5 O3

x101

0

1

2

3

4

5

6

7402.2500

C21 H32 N5 O3

403.2530C21 H32 N5 O3

404.2532

Counts (%) vs. Mass-to-Charge (m/z)402 403 404

0.07 ppm

Figure 6. The molecular formula generation algorithm identifi ed the metabolites by taking advantage of both the excellent match of the precursor

isotope pattern and the mass agreement for precursor and product ions.

High quality spectra enable accurate metabolite identifi cation

7

When operated in targeted MS/MS

mode, the 6540 QTOF enabled metabo-

lite identifi cation, a semi-quantitative

metabolic stability study, and meta-

bolic profi ling—all within the same

experiment. The 6540 QTOF produced

very high quality accurate mass MS

and MS/MS spectra for confi dent

metabolite identifi cation, including

accurate isotope ratios. The Agilent

QTOF provided excellent data quality

in all dimensions, simultaneously,

for combined quantitative and

qualitative analyses.

Conclusions

This study clearly demonstrated the

feasibility of using the 6540 QTOF

for metabolic stability, profi ling, and

identifi cation in a single experiment.

It gave quantitation results that were

comparable to those obtained with

the triple-quadrupole instrument. The

Agilent Triple Quad and QTOF employ

common hardware from source to

collision cell, which makes it easy to

transfer methods and compare results.

While the QTOF does not provide the

ultimate detection limits of the most

advanced triple-quadrupole systems,

the sensitivity is more than suffi cient

for early compound assessment in

drug discovery.

Identifi cation of unexpected

metabolites with QTOF

MS/MS analysis

A compelling reason to use an accu-

rate-mass MS/MS instrument for this

workfl ow is to identify un-targeted

metabolites in addition to the expected

metabolites. This may be done using

the standard data dependent acquisi-

tion parameters in combination with

appropriate data mining tools. The

Find by Auto MS/MS algorithm in

the Agilent MassHunter Qualitative

Analysis software was used to auto-

matically discover metabolites which

may be present in the sample. This

processing feature revealed all the

known phase I metabolites detected in

this sample, which were automatically

extracted for verifi cation, without prior

specifi cation. This experiment demon-

strates the feasibility of discovering

unexpected metabolites using this

QTOF MS/MS procedure.

www.agilent.com/chem/qtof

This item is intended for Research Use Only. Not for use in diagnostic procedures.

Agilent Technologies shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, perform-ance or use of this material.

© Agilent Technologies, Inc. 2010Printed in the U.S.A. December 6, 20105990-6867EN