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R2 = 0.992
1000 pg/ 0.1 L
Sulfadimethoxine, APCI+
pg/uL
800 600 400 200
R
e
s
p
o
n
s
e
0
10000
20000
30000
R2 = 0.991
pg/ 200 0.01 L
Dapsone, ESI+
pg/uL
180 140 100 60 20
R
e
s
p
o
n
s
e
0
20000
40000
60000
R2 = 0.984
pg/ 100 0.1 L
Ibuprofen, ESI-
pg/uL
800 600 400 200
R
e
s
p
o
n
s
e
0
1000
2000
3000
R2 = 0.991
1000 pg/ 0.1 L
Tolbutamide, APCI-
pg/uL
1000 800 600 400 200
R
e
s
p
o
n
s
e
0
2000
4000
6000
8000
10000
12000
Figure 6. The calibration curves obtained from the mixture analysis. Each mixture contained four
analytes. During each LC injection, the mass spectrometer was switching among the four ionization
modes rapidly. The quantication results are summarized in Table 2.
Copyright # 2007 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2007; 21: 893902
DOI: 10.1002/rcm
ESI-APCI multimode ionization source for UPLC/MS/MS 899
Fig. 8. Linear dynamic range was up to 4 orders of
magnitude. The r
2
value was over 0.99 for all analytes
except nortriptyline (0.980). The LOD was as low as 0.01 ng/
mL. Compared with the results obtained from the four-
compound mixture analysis, we saw better linearity, wider
dynamic range and lower LODs.
UPLC/ESCi-MS/MS applied to a microsome
stability test in drug discovery
The UPLC/MS/MS method showed promise for the further
enhancement of the analytical throughput for the microsome
stability test. Currently typically used for the quantitative
determination of the microsomal stability is a fast HPLC/
Table 2. Summary of the UPLC/ESCi-MS/MS quantication results for the four-compound mixture analysis. During each
injection, the mass spectrometer was switching rapidly among four ionization modes (ESI, ESI, APCI, and APCI) so that
four MRM chromatograms were obtained at the end of each LC run, one chromatogram per ionization mode
Ionization
Mode
Linear
Range (mg/mL) r
2
LOD
(mg/mL)
QC RSD%
20 mg/mL, N18
QC RSD%
200 mg/mL, N18
Daspone ESI 0.01200 0.991 0.01 9.39 8.61
Ibuprofen ESI 0.1100 0.984 0.1 8.26 3.57
Sulfadimethoxine APCI 0.11000 0.992 0.1 7.30 3.44
Tolbutamide APCI 0.11000 0.992 0.1 7.74 4.71
Figure 7. ESCi MRM chromatograms obtained from the single-compound analysis. These four chro-
matograms were obtained from four separate LC injections. During each LC injection, the mass
spectrometer was detecting a single MRM channel at a specic ionization mode of choice with
20 ms dwell time. More than 20 data points per peak were obtained.
Table 3. Summary of the UPLC/ESCi-MS/MS quantication results for the single-analyte protocol. A single MRM channel in a
specic ionization mode was monitored during each injection. For the whole analytical batch, the mass spectrometer could detect
samples with a choice among any of the four ionization modes (ESI, ESI, APCI, and APCI)
Ionization
Mode
Linear
Range (mg/mL) r
2
LOD
(mg/mL)
QC RSD%
20 mg/mL, N18
QC RSD%
200 mg/mL, N18
Acetophenone APCI 5.01000 0.990 10.0 7.61 2.84
Corticosterone APCI 2.01000 0.991 2.0 6.65 4.58
Daspone ESI 0.11000 0.996 0.01 1.74 2.21
Hydroxyprogestrone APCI 0.2200 0.990 0.2 6.52 2.52
Ibuprofen ESI 0.1500 0.990 0.1 3.35 2.58
Nortriptyline ESI 1500 0.980 2.0 2.70 8.11
Sulfadimethoxine APCI 0.01100 0.995 0.01 10.23 6.48
Tolbutamide APCI 0.1100 0.991 0.1 6.92 6.70
Copyright # 2007 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2007; 21: 893902
DOI: 10.1002/rcm
900 K. Yu et al.
MS/MS method with 2.5 min run time. The injection-
to-injection cycle time is approximately 3.5 min.
19
The
UPLC/MS/MS method has a 1 min run time with an
injection-to-injection cycle time of approximately 1.5 min. As
a result, the analysis time can be reduced from 5.6 to 2.4 h for
the analysis of a 96-well plate by switching the separation
protocol from HPLC to UPLC.
The single-analyte UPLC/MS/MS protocol was used for
the microsome stability test. During the microsomal stability
screening, a large number of samples containing wide
chemical polarity and lipophilicity are encountered. For each
individual sample, the target is always an individual parent
analyte, not the metabolites. Each MS method requires a
single MRM channel in an optimized ionization mode.
However, for an entire batch of samples to be analyzed, there
may be a need to analyze compounds in different ionization
modes or polarity from one injection to another. The ESCi
multimode ionization source allowed multiple ionization
experiments to be carried in a single batch. This allows a
large variety of compounds to be analyzed by a single
ionization source regardless of the preferred ionization
mode, hence further increasing the analytical throughput.
It was previously demonstrated that for microsome
stability assays, a single-time-point assay is sufcient at
the screening stage to support drug discovery.
21
A moderate
incubation time (e.g. 20 min) is preferred since a short
incubation time is more suitable to give stability resolution
among unstable compounds, whereas a long incubation time
is more applicable to compounds with longer half-life.
A 96-well plate was used for the incubation and injection.
For each compound, duplicate tests were run, each in a
separate well. Typically, three standards covering a range of
metabolic rates were used as references. The reference percent
remaining values for these three compounds from the HPLC/
MS/MS protocol are: verapamil 39%, loperamide 1015%,
and zolpadem 4459%. Table 4 shows the results obtained
from UPLC/ESCi-MS/MS protocol with the in vitro metabolic
half-life calculated. The percent remaining values for all three
Dapsone, ESI+
R2 = 0.992
1000 pg/ 0.1 L
pg/uL
450 350 250 150 50
R
e
s
p
o
n
s
e
0
50000
150000
250000
R2 = 0.992
500 pg/ 0.1 L
Ibuprofen, ESI-
pg/uL
450 350 250 150 50
R
e
s
p
o
n
s
e
0
500
1000
1500
2000
R2 = 0.992
100 pg/ 0.01 L
Sulfadimethoxine, APCI+
pg/uL
90 70 50 30 10
R
e
s
p
o
n
s
e
0
2000
4000
6000
8000
10000
R2 = 0.995
1000 pg/ 0.1 L
Tolbutamide, APCI-
pg/uL
800 600 400 200
R
e
s
p
o
n
s
e
0
5000
10000
15000
20000
25000
Figure 8. The example calibration curves obtained from the single-compound
analysis. During each LC injection, the mass spectrometer was detecting in a
particular ionization mode. The quantication results of this analysis are summarized
in Table 3.
Table 4. The microsome stability analysis results obtained from the single-compound analysis method. Tolbutamide, verapamil
and zolpadem were used as the reference standards. The reference percent remaining values typically obtained from the HPLC/
MS/MS protocol are: 39% for verapamil, 1015% for loperamide, and 4459% for zolpadem
Compounds MW Ionization mode %Remaining
In vitro metabolic
half-life (min)
Acetophenone 120 APCI 55.60 18
Corticosterone 346 APCI 56.14 18
Daspone 248 ESI 48.19 14
17a-Hydroxyprogesterone 330 APCI 0.54 2
Ibuprofen 206 ESI 74.73 >30
Loperamide 476 ESI 11.21 4
Nortriptyline 263 ESI 0.59 2
Sulfadimethoxine 310 APCI 87.65 >30
Tolbutamide 270 ESI 81.88 >30
Verapamil 454 ESI 3.79 3
Zolpadem 307 ESI 47.12 14
Copyright # 2007 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2007; 21: 893902
DOI: 10.1002/rcm
ESI-APCI multimode ionization source for UPLC/MS/MS 901
reference compounds fall within the reference range. This was
an indication of a good correlation between the UPLC/
ESCi-MS/MS and the HPLC/MS/MS protocols.
CONCLUSIONS
The UPLC/ESCi-MS/MS technique offers time savings in
analysis and therefore can further improve analytical
throughput. The evaluation of the UPLC/MS/MS studies
indicated that 510 data points were obtained for an
eight-compound mixture analysis (insufcient for quanti-
tative analysis), and 15 data points were obtained for a
four-compound mixture analysis (sufcient for quantitative
analysis). In both cases, the mass spectrometer was detecting
in MRM mode and switching rapidly among the four
ionization modes.
An ultra-fast LC/ESCi-MS/MS quantication protocol for
the microsomal stability assay allows a single analyte to be
detected during each LC injection. The mass spectrometer
was detecting in a single MRM channel in a particular
ionization mode. However, for the entire analytical batch, the
mass spectrometer was able to detect compounds in all four
ionization modes of choice.
The test results showed good agreement with the pre-
viously acquired HPLC/MS/MS results. The UPLC separ-
ation was obtained with a 1.5 min injection-to-injection cycle
time. This allowed more than 3 h to be saved for the analysis
of one 96-well plate (compared with the 3.5 min injection-
to-injection cycle time in HPLC). Additional time saving was
also obtained by eliminating sample re-analysis using APCI
mode for the compounds that are not detected by ESI.
Acknowledgements
The authors wish to thank Mr. Michael Balogh from Waters
Corporation (Milford, MA, USA) for many helpful discussions.
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Copyright # 2007 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2007; 21: 893902
DOI: 10.1002/rcm
902 K. Yu et al.