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Mass Spectrometer
Mass spectrometer: Thermo Scientific LCQ Advantage
MAX
Ionization mode:
Positive electrospray ionization (ESI)
Capillary
temperature:
275 °C
Spray voltage:
4.5 kV
Sheath gas:
30 units
Sweep gas:
8 units
Standards
Calibration standards were prepared as follows:
Imipramine, desipramine and clomipramine working
standard solutions were 50 ng/mL
Samples and Internal Standard
Imipramine was administered to the horse and a urine
sample drawn after 0, 2, 4, 8 and 24 hours, post dose.
One mL of the urine sample was spiked with 10 µL of
50 ng/µL clomipramine internal standard.
Sample Preparation
The calibration standard and urine samples were treated
with
`
-glucuronidase to hydrolyze glucuronide conjugates
of desipramine and imipramine, followed by solid phase
extraction.
Results and Discussions
LC-UV-MS/MS analysis of
imipramine and desipramine
Figures 1 and 2 show the analysis
of tricyclic antidepressant imipramine,
its major metabolite desipramine, and
the internal standard clomipramine
by LC with MS/MS and UV detec-
tion, respectively. Figure 1 shows
base peak and extracted ion chromat-
ograms for the three analytes along
with MS and MS/MS spectra. The
MS and MS/MS spectra help in
unambiguous identification of these
analytes and represent the high speci-
ficity that can be obtained from such
an analysis. Further, the MS/MS
spectra can be stored in a library and
used for rapid confirmation of the
drug and its metabolite. Figure 2
shows total spectra obtained from a
PDA detector as well as UV trace at
254 nm and 280 nm. The position
of elution of the three compounds
had to be determined by sequential
injections of individual analytes. As
illustrated in Figure 2, the UV spectra
for these compounds appear almost
identical, making their unambiguous
identification difficult.
Page 2 of 8
Volume of
Volume of
Imipramine and
Clomipramine
Equivalent to
Equivalent to
Desipramine
working
Imipramine
Clomipramine
Calibration
working standard
standard
in the urine
in the urine
level
solution (µL)
solution (µL)
(ng/mL)
(ng/mL)
C1
1:1 Dilution of C2
10
15.6
500
C2
1:1 Dilution of C3
10
31.3
500
C3
1:1 Dilution of C2
10
62.5
500
C4
1:1 Dilution of C2
10
125
500
C5
1:1 Dilution of C2
10
250
500
C6
1:1 Dilution of C2
10
500
500
C7
1:1 Dilution of C2
10
1000
500
C8
1:1 Dilution of C2
10
2000
500
C9
1:1 Dilution of C2
10
4000
500
C10
160
10
8000
500
Quantifying
Isolation
Collision
Scan
MS/MS
Analyte
MH
+
Width
Energy %
Range
Product Ions
Imipramine
281.2
1.5
30
75-285
86
Desipramine
267.2
1.5
30
70-290
236
Clomipramine
(internal standard)
315.2
4
35
85-320
270
Table 1: MS parameters for imipramine, desipramine, and clomipramine (internal standard)