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

Introduction TurboFlow™ automated sample preparation technology is

Key Words Clozapine (Figure 1) is a tricyclic dibenzodiazepine drug being investigated to simplify sample preparation, reduce
used in the treatment of schizophrenia. It is uniquely the risk of operator error, improve sample throughput,
• TurboFlow
effective in patients resistant to therapy with other and gain further selectivity by utilizing tandem mass
Technology
antipsychotics. In addition to mandatory hematological spectrometry.
• TSQ Quantum monitoring to minimize the risk of agranulocytosis, there
Ultra are large variations (50-fold) among patients’ clozapine Goal
• Clinical Research dose requirements. Moreover, changes in smoking habits To assess Thermo Scientific TurboFlow automated sample
can have a large effect on the clozapine dose requirement preparation technology with tandem mass spectrometry
(on average, the clozapine dose for non-smokers is half for the research analysis of clozapine and norclozapine
that required for smokers) levels in plasma samples.
due to the induction of
cytochrome P450 (CYP) Experimental
enzymes in smokers.1 Studies
Sample Preparation
have indicated that accurate
quantification of clozapine Calibration standards (n=6) were prepared in the range
levels may help researchers 0.05 mg/L to 2 mg/L by addition of clozapine and
better understand, and norclozapine to newborn calf serum. Similarly, both
conduct analysis of, issues analytes were added to drug-free human plasma to give
Figure 1: Structure of clozapine
related to dose optimization internal quality control (IQC) solutions at 0.15, 0.40,
and adherence.2 and 1.20 mg/L. After centrifugation at 11,000 g for
Clozapine is metabolized via N-demethylation, 2 min, 10 µL plasma was injected directly onto the
N-oxidation, and aromatic hydroxylation, amongst other Aria™ TLX-1 system.
pathways. A few drugs, notably fluvoxamine, block all The eluent gradients for both pumps are displayed in Table 1.
four CYP enzymes that can metabolise clozapine.
Measurement of N-desmethylclozapine (norclozapine), TurboFlow LC
which accumulates in plasma to concentrations similar to Column: TurboFlow Cyclone 50 x 0.5 mm
that of clozapine, can give useful information regarding Mobile phase A: 0.05% (v/v) aqueous formic acid
adherence with medication, sample timing in relation to Mobile phase B: 0.05% (v/v) formic acid in methanol
the last dose of clozapine and drug-drug interactions, such Mobile phase D: 45/45/10 Propan-2-ol/acetonitrile/acetone
as that with fluvoxamine.
Analytical LC
Current research methodology in our laboratory for
clozapine and norclozapine involves off-line liquid-liquid Column: Thermo Scientific Hypersil GOLD C18 50 x 2.1 mm, 3 µm
extraction with manual transfer to a high pressure liquid Mobile phase A: 0.05% (v/v) aqueous formic acid
chromatography-ultra violet (HPLC-UV) system. The Mobile phase B: 0.05% (v/v) formic acid in methanol
Thermo Scientific Aria TLX-1 System powered by

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 The selective reaction monitoring (SRM) transitions used
Thermo Scientific TSQ Quantum Ultra triple stage quadrupole mass spectrometer are presented in Table 2.
Ion Source & Polarity: APCI, positive ion mode Scan Collision Tube
Discharge Current: 4.0 µA Analyte Parent Product Time Energy Lens
Vaporizer Temperature: 325 °C
Clozapine 327.20 192 25 ms 60 47
Sheath Gas: 60 units
270 25 ms 21 47
Ion Sweep Gas: 0 units
Auxillary Gas: 10 units Norclozapine 313.20 164 25 ms 67 113
Capillary Temperature: 275 °C 192 25 ms 41 113
Collision Gas Pressure: 1.5 mTorr Table 2: SRM transitions monitored in the experiment

Figure 2: Extracted ion chromatogram of the plasma blank

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 carry-
over. 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 Figure 3: Clozapine and Norcloxapine lowest calibration from plasma, 0.05 mg/L
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 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 one-
tenth 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, offices, Thermo Fisher
Lennard MS, Tucker GT, Flanagan RJ, Scientific maintains
J Clin Psychopharmacol 2004; 24 (1): 1-9
a network of represen-
2. Flanagan RJ, CPD Clin Biochem 2006; 7 (1): 3-18
tative organizations
throughout the world.

Africa-Other
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Denmark
Figure 6: Norclozapine calibration curve, 0.05 – 2 mg/L +45 70 23 62 60
Europe-Other
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Clozapine Norclozapine Finland / Norway /
Sweden
Specified Calculated Specified Calculated
Concentration Response Conc Conc % CV Concentration Response Conc Conc % CV +46 8 556 468 00
France
0.05 mg/L 784733 0.05 0.03 1.2 0.05 mg/L 905801 0.05 0.04 0.4 +33 1 60 92 48 00
0.05 mg/L 797712 0.05 0.03 1.2 0.05 mg/L 897792 0.05 0.04 0.4 Germany
0.05 mg/L 780137 0.05 0.03 1.2 0.05 mg/L 900825 0.05 0.04 0.4 +49 6103 408 1014
0.10 mg/L 1415271 0.10 0.08 1.7 0.10 mg/L 1555897 0.10 0.10 1.1 India
+91 22 6742 9434
0.10 mg/L 1456027 0.10 0.09 1.7 0.10 mg/L 1554377 0.10 0.10 1.1
Italy
0.10 mg/L 1411624 0.10 0.08 1.7 0.10 mg/L 1525338 0.10 0.10 1.1 +39 02 950 591
0.20 mg/L 2745962 0.20 0.20 1.8 0.20 mg/L 2847998 0.20 0.21 3.1 Japan
+81 45 453 9100
0.20 mg/L 2743289 0.20 0.20 1.8 0.20 mg/L 2859029 0.20 0.21 3.1
Latin America
0.20 mg/L 2832044 0.20 0.20 1.8 0.20 mg/L 3006773 0.20 0.22 3.1 +1 608 276 5659
0.50 mg/L 6889405 0.50 0.55 2.6 0.50 mg/L 7099512 0.50 0.57 5.0 Middle East
0.50 mg/L 6682781 0.50 0.54 2.6 0.50 mg/L 6741516 0.50 0.54 5.0 +43 1 333 50 34 0
0.50 mg/L 6549395 0.50 0.52 2.6 0.50 mg/L 6420812 0.50 0.51 5.0 Netherlands
+31 76 579 55 55
1.00 mg/L 12624439 1.00 1.05 2.3 1.00 mg/L 12521697 1.00 1.04 3.6 South Africa
1.00 mg/L 12261014 1.00 1.02 2.3 1.00 mg/L 12383684 1.00 1.02 3.6 +27 11 570 1840
1.00 mg/L 12054848 1.00 1.00 2.3 1.00 mg/L 11695815 1.00 0.97 3.6 Spain
+34 914 845 965
2.00 mg/L 24055429 2.00 2.03 2.5 2.00 mg/L 23888229 2.00 2.01 3.5
Switzerland
2.00 mg/L 22868295 2.00 1.93 2.5 2.00 mg/L 22259134 2.00 1.87 3.5 +41 61 716 77 00
2.00 mg/L 23457123 2.00 1.98 2.5 2.00 mg/L 23241437 2.00 1.95 3.5 UK
+44 1442 233555
Figure 7: Clozapine/Norclozapine reproducibility and variance USA
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