2012 Corporate Capabilities - Spectroscopy

APPLICATION NOTES – DECEMBER 2011 Mass Spectrometry 93
Simultaneous Qualitative and
Quantitative Analysis of
Buspirone and Its Metabolites
with the Agilent 6550 iFunnel
Q-TOF LC–MS System
Yuqin Dai, Michael Flanagan, and Keith Waddell,
Agilent Technologies, Inc.
Timely and rapid assessment of metabolic stability, metabolite
identification, and metabolite profiling is critical for accelerating
lead optimization and enhancing the success rate of drug
candidates entering into development. Traditionally, qualitative
and quantitative analyses are often performed on different types
of LC–MS instruments and in multiple runs. Te ability to obtain
quantitation and identification (qual/quan) in a single analysis
makes metabolic stability, metabolite identification, and metabolite
profiling studies much more efficient. Tis note describes an
integrated qual/quan workflow that is enabled by the sensitivity
enhancement of iFunnel technology implementation on a quadrupole
time-of-flight (Q-TOF) instrument. It demonstrates how Agilent
6550 iFunnel Q-TOF permits high sensitivity, simultaneous
determination of metabolic stability, metabolite identification, and
metabolite profiling in an in vitro buspirone (1 μM) metabolism
study in rat liver microsomes.
An Integrated Qualitative/Quantitative Workflow
Te qual/quan workflow starts with the LC–MS injection of a biological
sample. 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 identification and
structure elucidation are facilitated by MassHunter Metabolite ID
software. Metabolic stability and metabolite profiling are established
from the same set of data, which are processed in batch
mode by MassHunter Quantitative Analysis software.
Qual/Quan Analysis
Figure 1 illustrates the metabolic stability of buspirone and the
profiles of its metabolites, illustrating broad coverage of the high
and low abundance metabolites across the entire 60-min time
course.
Figure 2 demonstrates the MS and MS-MS spectra of a buspirone
monohydroxyl metabolite from a 10-min incubation
sample. Te sub-ppm mass accuracy of the precursor and fragment
ions, along with the excellent isotopic fidelity (overall score
>99%), provided highly confident metabolite identification and
structure elucidation.
Normalized peak area (%)
100 Buspirone (parent)
90
80
70
60
50
40
30
20
10
dihydroxy
N-oxide
N, N’-desethyl + O
Incubation time (min)
monohydroxy
trihydroxy
N, N’-desethyl
1-Pyrimidinlypiperazine
0
0 10 20 30 40 50 60
Figure 1: Metabolic stability of buspirone and its metabolite
profiles.
x10 5
2.2 123.0918
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
x10 4 122.0713
C6 H8 N3
4
3.5
3
2.5
2
1.5
1
0.5
0
0.07 ppm
155.1539
O
224.1280
150.1023
C8 H12 N3 178.1212 238.1439 281.1859
-2.01 ppm
C9 H14 N4 C13 H20 N O3 C15 H25 N2 O3
-0.39ppm 0.68 ppm -0.41 ppm
Counts vs. Mass-to-Charge (m/z)
MS spectrum
MS/MS spectrum
402.2499
C21 H32 N5 O3
402.2499
120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420
Figure 2: Excellent mass accuracy in both MS and MS-MS spectra
and isotopic fidelity.
Conclusions
An efficient qual/quan workflow 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. Te 6550 iFunnel Q-TOF LC–MS system enables
the simultaneous quantitation and metabolite identification from
1 μM buspirone incubations with sufficient sensitivity, throughput
(run time of 3.5 min), and high analytical confidence. Tis single
analytical platform also allows rapid generic LC–MS method development
and eliminates the time-consuming MRM optimization
for each analyte that is required on a triple quadrupole instrument.
Agilent Technologies Inc.
2315 Stevens Creek Blvd., Santa Clara, CA 95052
tel. (800) 227-9770; Fax (302) 633-8901
Website: www.agilent.com