1. health and fitness
2. therapy

Application
Note: 472
Research Analysis of Clozapine and
Norclozapine in Plasma Using Automated
Sample Preparation and LC-MS/MS
Phillip Morgan, Kings College Hospital NHS Foundation Trust, London, UK
Shane McDonnell, Sarah Robinson, Thermo Fisher Scientific, Hemel Hempstead, UK
TurboFlow™ automated sample preparation technology is
being investigated to simplify sample preparation, reduce
the risk of operator error, improve sample throughput,
and gain further selectivity by utilizing tandem mass
spectrometry.
Introduction
Key Words
• TurboFlow
Technology
• TSQ Quantum
Ultra
• Clinical Research
Clozapine (Figure 1) is a tricyclic dibenzodiazepine drug
used in the treatment of schizophrenia. It is uniquely
effective in patients resistant to therapy with other
antipsychotics. In addition to mandatory hematological
monitoring to minimize the risk of agranulocytosis, there
are large variations (50-fold) among patients’ clozapine
dose requirements. Moreover, changes in smoking habits
can have a large effect on the clozapine dose requirement
(on average, the clozapine dose for non-smokers is half
that required for smokers)
due to the induction of
cytochrome P450 (CYP)
enzymes in smokers.1 Studies
have indicated that accurate
quantification of clozapine
levels may help researchers
better understand, and
conduct analysis of, issues
Figure 1: Structure of clozapine
related to dose optimization
and adherence.2
Clozapine is metabolized via N-demethylation,
N-oxidation, and aromatic hydroxylation, amongst other
pathways. A few drugs, notably fluvoxamine, block all
four CYP enzymes that can metabolise clozapine.
Measurement of N-desmethylclozapine (norclozapine),
which accumulates in plasma to concentrations similar to
that of clozapine, can give useful information regarding
adherence with medication, sample timing in relation to
the last dose of clozapine and drug-drug interactions, such
as that with fluvoxamine.
Current research methodology in our laboratory for
clozapine and norclozapine involves off-line liquid-liquid
extraction with manual transfer to a high pressure liquid
chromatography-ultra violet (HPLC-UV) system. The
Thermo Scientific Aria TLX-1 System powered by
Goal
To assess Thermo Scientific TurboFlow automated sample
preparation technology with tandem mass spectrometry
for the research analysis of clozapine and norclozapine
levels in plasma samples.
Experimental
Sample Preparation
Calibration standards (n=6) were prepared in the range
0.05 mg/L to 2 mg/L by addition of clozapine and
norclozapine to newborn calf serum. Similarly, both
analytes were added to drug-free human plasma to give
internal quality control (IQC) solutions at 0.15, 0.40,
and 1.20 mg/L. After centrifugation at 11,000 g for
2 min, 10 µL plasma was injected directly onto the
Aria™ TLX-1 system.
The eluent gradients for both pumps are displayed in Table 1.
TurboFlow LC
Column:
Mobile phase A:
Mobile phase B:
Mobile phase D:
TurboFlow Cyclone 50 x 0.5 mm
0.05% (v/v) aqueous formic acid
0.05% (v/v) formic acid in methanol
45/45/10 Propan-2-ol/acetonitrile/acetone
Analytical LC
Column:
Mobile phase A:
Mobile phase B:
Thermo Scientific Hypersil GOLD C18 50 x 2.1 mm, 3 µm
0.05% (v/v) aqueous formic acid
0.05% (v/v) formic acid in methanol
TurboFlow Method
Analytical
Step
Start
Sec
Flow
Grad
%A
%B
%C
%D
Tee
Loop
Flow
Grad
%A
%B
1
00:00
30
1.50
Step
100
-
-
-
====
out
0.50
Step
100
0
2
00:30
60
0.25
Step
100
-
-
-
T
in
0.25
Step
100
0
3
01:30
60
1.50
Step
-
-
-
100
====
in
0.50
Ramp
5
95
4
02:30
60
1.50
Step
70
30
-
-
====
in
0.50
Step
5
95
5
03:30
60
1.50
Step
100
-
-
-
====
out
0.50
Step
100
0
Table 1: Gradient programs for both TurboFlow and analytical methods (flow rate is mL/min)
Mass Spectrometry
Thermo Scientific TSQ Quantum Ultra triple stage quadrupole mass spectrometer
Ion Source & Polarity:
APCI, positive ion mode
Discharge Current:
4.0 µA
Vaporizer Temperature:
325 °C
Sheath Gas:
60 units
Ion Sweep Gas:
0 units
Auxillary Gas:
10 units
Capillary Temperature:
275 °C
Collision Gas Pressure:
1.5 mTorr
Figure 2: Extracted ion chromatogram of the plasma blank
The selective reaction monitoring (SRM) transitions used
are presented in Table 2.
Analyte
Parent
Product
Scan
Time
Collision
Energy
Tube
Lens
Clozapine
327.20
192
270
25 ms
25 ms
60
21
47
47
Norclozapine
313.20
164
192
25 ms
25 ms
67
41
113
113
Table 2: SRM transitions monitored in the experiment
Results and Discussion
Plasma was centrifuged prior to
analysis. Calibration standards were
analyzed from low to high concentration
followed by IQCs. An injection of
solvent after the highest concentration
IQC was used for evaluation of carryover. The volume of plasma injected
was 10 µL, and all plasma analyses
were in triplicate.
The extracted ion chromatograms
of the plasma blank and lowest and
highest concentration calibrators
are presented in Figures 2, 3, and 4,
respectively. The calibration curves for
clozapine and norclozapine covered the
range 0.05- 2.00 mg/L (Figure 5 and 6).
No internal standard was used, and
thus, this work demonstrates the
reproducibility of the system using
external standard calibration.
Reproducibility and variance of
the calibrators are shown in Figure 7.
Carry-over was calculated by
comparing the response for clozapine
and for norclozapine with that of a
solvent blank injected immediately
after a 1.2 mg/L IQC sample. This
was shown to be ~0.1% for both
clozapine and norclozapine.
Additional clozapine metabolites were
not investigated as part of this
evaluation.
Figure 3: Clozapine and Norcloxapine lowest calibration from plasma, 0.05 mg/L
Figure 4: Clozapine and norcloxapine lowest calibration from plasma, 2 mg/L
Conclusion
The research use of TurboFlow
technology for automated sample
preparation and tandem MS detection
allowed the selective analysis of
clozapine and norclozapine in
plasma. The only sample preparation
was the centrifugation of plasma. The
sample volume required was onetenth that used by the existing
method – liquid-liquid extraction
(LLE) followed by HPLC-UV – and
provided lower limits of detection and
quantitation. The calibration curves
for all analytes were linear over the
concentration range and carry-over
was minimal. Use of the automated
TurboFlow method has effectively
eliminated two hours of sample
preparation time for a 100-sample
batch.
Figure 5: Clozapine calibration curve, 0.05 – 2 mg/L
References
In addition to these
1. Rostami-Hodjegan A, Amin AM, Spencer EP,
Lennard MS, Tucker GT, Flanagan RJ,
J Clin Psychopharmacol 2004; 24 (1): 1-9
offices, Thermo Fisher
2. Flanagan RJ, CPD Clin Biochem 2006; 7 (1): 3-18
Scientific maintains
a network of representative organizations
throughout the world.
Figure 6: Norclozapine calibration curve, 0.05 – 2 mg/L
Clozapine
Concentration
Response
Norclozapine
Specified
Conc
Calculated
Conc
% CV
0.05 mg/L
0.05 mg/L
0.05 mg/L
0.10 mg/L
0.10 mg/L
0.10 mg/L
0.20 mg/L
0.20 mg/L
0.20 mg/L
0.50 mg/L
0.50 mg/L
0.50 mg/L
1.00 mg/L
784733
797712
780137
1415271
1456027
1411624
2745962
2743289
2832044
6889405
6682781
6549395
12624439
0.05
0.05
0.05
0.10
0.10
0.10
0.20
0.20
0.20
0.50
0.50
0.50
1.00
0.03
0.03
0.03
0.08
0.09
0.08
0.20
0.20
0.20
0.55
0.54
0.52
1.05
1.2
1.2
1.2
1.7
1.7
1.7
1.8
1.8
1.8
2.6
2.6
2.6
2.3
1.00 mg/L
1.00 mg/L
2.00 mg/L
2.00 mg/L
2.00 mg/L
12261014
12054848
24055429
22868295
23457123
1.00
1.00
2.00
2.00
2.00
1.02
1.00
2.03
1.93
1.98
2.3
2.3
2.5
2.5
2.5
Concentration
0.05 mg/L
0.05 mg/L
0.05 mg/L
0.10 mg/L
0.10 mg/L
0.10 mg/L
0.20 mg/L
0.20 mg/L
0.20 mg/L
0.50 mg/L
0.50 mg/L
0.50 mg/L
1.00 mg/L
1.00 mg/L
1.00 mg/L
2.00 mg/L
2.00 mg/L
2.00 mg/L
Response
905801
897792
900825
1555897
1554377
1525338
2847998
2859029
3006773
7099512
6741516
6420812
12521697
12383684
11695815
23888229
22259134
23241437
Specified
Conc
Calculated
Conc
% CV
0.05
0.05
0.05
0.10
0.10
0.10
0.20
0.20
0.20
0.50
0.50
0.50
1.00
1.00
1.00
2.00
2.00
2.00
0.04
0.04
0.04
0.10
0.10
0.10
0.21
0.21
0.22
0.57
0.54
0.51
1.04
1.02
0.97
2.01
1.87
1.95
0.4
0.4
0.4
1.1
1.1
1.1
3.1
3.1
3.1
5.0
5.0
5.0
3.6
3.6
3.6
3.5
3.5
3.5
Figure 7: Clozapine/Norclozapine reproducibility and variance
Africa-Other
+27 11 570 1840
Australia
+61 2 8844 9500
Austria
+43 1 333 50 34 0
Belgium
+32 2 482 30 30
Canada
+1 800 530 8447
China
+86 10 8419 3588
Denmark
+45 70 23 62 60
Europe-Other
+43 1 333 50 34 0
Finland / Norway /
Sweden
+46 8 556 468 00
France
+33 1 60 92 48 00
Germany
+49 6103 408 1014
India
+91 22 6742 9434
Italy
+39 02 950 591
Japan
+81 45 453 9100
Latin America
+1 608 276 5659
Middle East
+43 1 333 50 34 0
Netherlands
+31 76 579 55 55
South Africa
+27 11 570 1840
Spain
+34 914 845 965
Switzerland
+41 61 716 77 00
UK
+44 1442 233555
USA
+1 800 532 4752
www.thermo.com
For Research Use Only. Not for use in diagnostic procedures.
Legal Notices
©2009 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.
This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these
products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change.
Not all products are available in all countries. Please consult your local sales representative for details.
View additional Thermo Scientific LC/MS application notes at: www.thermo.com/appnotes
Thermo Fisher Scientific,
San Jose, CA USA is ISO Certified.
AN63132_E 11/09M
Part of Thermo Fisher Scientific