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Quantitation of Seven Designer Cathinones in Urine Using Q Exactive Mass Spectrometer
Metoptolol
Ticlopidine
Compound A
Erythromycin
Clomipramin
Bendamustine
omparison
QE SIM
QE Full
Exactive Plus
Triple
Conclusion
83% of compounds analyzed met the assay calibration curve LOQ of 5nM.
92% of the compounds provided sufficient signal in the assay for calculation of
the % Free in all scan modes evaluated.
Full scan analysis using high resolution accurate mass provided adequate signal
response and linear dynamic range to accurately measure 92% compounds
analyzed in the PPB assay.
Additional sensitivity and linear dynamic range may be achieved through further
method optimization.
References
1. Zhang J, Shou WZ, Vath M, Kieltyka K, Maloney J, Elvebak L, Stewart J, Herbst
J, Weller HN., Rapid Commun Mass Spectrom. 2010 Dec 30;24(24):3593
Acknowledgements
We would like to thank Dr. Jun Zhang and Jennifer Maloney of Bristol-Myers Squibb,
Wallingford, CT for sample preparation and assistance with data processing.
GMSU Gubbs™ Mass Spec Utilities is a trademark of Gubbs Inc. Microsoft and Excel are registered trademarks
of Microsoft Corporation. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the
intellectual property rights of others.
Twenty-two of the twenty-four compounds analyzed in the protein binding assay
provide a %CV of less than 25% across the various scan modes while providing
adequate sensitivity for analyte analysis in the binding assay. Although four
compounds did not provide enough signal in the calibration curve analysis only two
did not provide enough signal for % Free calculation in the PPB assay itself. 92% of
the compounds analyzed provided sufficient signal in both full scan and SIM mode
with a %CV of less than 25%. The two compounds that did not provide enough
sensitivity to generate a % Free value in the binding assay were challenging in full
scan on the Exactive Plus only and not on the Q Exactive. One explanation for this
observation maybe due to the generic mass spec and chromatographic conditions
used for data analysis. Although both instruments collected data in full scan mode, the
Q Exactive filters all ions outside of the specified full scan mass range. While the
Exactive Plus does filter some ions at the s-lens, additional ions outside the specified
mass range are also collected and injected into the Orbitrap Mass Analyzer. Further
optimization of the ion target amount collected per scan in the mass spec method
along with optimized chromatographic clean up of the assay samples in the generic
method may improve signal response in full scan mode in the absence of true ion
filtering with a quadrupole and will be evaluated in future work.
Triple
Free
Avg(%)
StdDev(%)
% CV
30.00
30.87
1.44
4.66
27.70
27.64
1.54
5.58
13.10
13.15
0.75
5.69
9.50
9.97
1.56
5.70
26.40
28.29
1.81
6.40
27.00
25.06
6.26
7.00
33.80
37.36
2.79
7.46
23.30
20.12
2.73
7.71
16.20
16.64
1.32
7.92
16.10
17.14
1.40
8.18
6.70
6.88
0.58
8.42
65.10
73.41
6.98
9.51
2.00
1.58
0.36
11.01
0.50
0.64
0.13
12.93
0.90
0.86
0.12
13.73
88.10
79.71
11.19
14.03
28.60
21.97
7.32
14.57
1.20
1.51
0.31
14.64
79.00
70.64
11.66
16.51
0.60
0.75
0.13
16.74
1.50
1.24
0.25
20.22
57.10
54.18
10.99
20.28
6.70
5.66
1.99
20.38
0.20
0.26
0.13
5.41
ided adequate sensitivity for all
ovement for some compounds
protein binding assay was
he coefficient of variation of the %
h compound and the results were
%CV (Table 1).
e that did not provide sufficient
value and were excluded from
scan mode and %CV across
n modes with no results due to
re plotted in a bar chart to
h scan mode for the PPB
s each scan mode used for
ounds analyzed demonstrate a
odes while providing adequate
es.