Poster demonstrating the results from development/verification project for the quantitation of clonzapine and n-desmethylclozapine in human serum.

1. © 2008 Mayo Foundation for Medical Education and Research
Measurement Of Clozapine And Desmethylclozapine In Human Serum By High-Throughput
Liquid Chromatography-Tandem Mass Spectrometry
D. M. Garby, R. DelRosso, L. A. Cheryk
Mayo Medical Laboratories, Wilmington, MA
Conclusions
We have developed a reliable assay for the analysis of clozapine and des-
methylclozapine in serum using liquid chromatography-tandem mass
spectrometry. The assay offers a wide analytical range and acceptable
low end sensitivity with excellent accuracy and precision.
References
(1) Worrel JA, Marken PA, Beckman SE, Ruehter VL. Atypical antipsychotic agents: a critical
review. Am J Health Syst Pharm 2000;57:238-258.
(2) Raggi MA. Therapeutic drug monitoring: chemical-clinical correlations of atypical antipsy-
chotic drugs. Curr Med Chem 2002;9:1397-1409.
(3) Physicians’ Desk Reference, 61st ed. Montvale, NJ: Thompson PDR, 2007.
(4) Khan AY, Preskorn SH. Examining concentration-dependent toxicity of clozapine: role of
therapeutic drug monitoring. J Psychiatr Pract 2005;11:289-301.
(5) Aravagiri M, Marder SR. Simultaneous determination of clozapine and its N-desmethyl and
N-oxide metabolites in plasma by liquid chromatography/electrospray tandem mass spec-
trometry and its application to plasma level monitoring in schizophrenic patients. J Pharm
Biomed Anal 2001;26:301-311.
Abstract
Background
Clozapine (Clozaril) is primarily used for the treatment of schizo-
phrenia and related psychotic disorders in patients that have en-
countered non-response or adverse intolerable side effects with
more classical antipsychotics. Clozapine is metabolized to desm-
ethylclozapine and clozapine-N-oxide. The desmethyl metabolite
has only limited activity and the oxide metabolite is inactive. Serum
concentrations are determined in treated patients to monitor patient
compliance as well as to achieve desired levels. Therapeutic serum
concentrations of clozapine can typically range from 100 to 1000 ng/
mL. We have developed an assay for the analysis of clozapine and
desmethylclozapine in serum using high-throughput liquid chro-
matography followed by traditional liquid chromatography-tandem
mass spectrometry (LC-MS/MS).
Methodology
A deuterated stable isotope (clozapine-d4) was added to 50 μL
of serum as an internal standard. Protein was precipitated from the
mixture by the addition of acetonitrile. Clozapine, desmethylclozap-
ine and the internal standard were then extracted from the resulting
supernatant via an on-line extraction utilizing high-throughput liq-
uid chromatography (TLX2, Cohesive Technologies, Franklin, Mas-
sachusetts). This was followed by conventional liquid chromatogra-
phy and analysis on a tandem mass spectrometer (API 5000, Applied
Biosystems, Toronto, Canada) equipped with a heated nebulizer ion
source. Ion transitions monitored in the multiple reaction monitor-
ing (MRM) mode were m/z 327.0 → m/z 270.0 for clozapine, m/z
313.2 → m/z 192.1 for desmethylclozapine, and m/z 331.0 → m/z
272.0 for clozapine-d4. Calibrators consisted of five standard solu-
tions ranging from 0 to 2000 ng/mL.
Results
Method performance was assessed using precision, linearity,
recovery and specimen stability. Precision and linearity studies
were performed using bovine serum spiked with either a clozapine
or desmethylclozapine standard. Intra-run precision (N=14) coeffi-
cients of variation (CVs) ranged from 3.6% to 4.2% for clozapine and
5.2% to 10.3% for desmethylclozapine. Clozapine inter-run preci-
sion (N=20) CVs ranged from 2.5% to 2.9%, and desmethylclozap-
ine inter-run precision CVs ranged from 3.0% to 4.8%. The method
demonstrated linearity over the assay range (25 to 2000 ng/mL) for
each analyte, yielding the following equations: observed clozap-
ine value = 1.0036*(expected value) – 7.1034, R2
= 0.9996; observed
desmethylclozapine value = 0.9840*(expected value) – 8.8886, R2
=
0.9984. Recovery was demonstrated for each analyte by mixing high
and low serum samples. Recovery averaged 99% across the assay
range for both analytes. A stability study demonstrated that speci-
mens are stable at ambient, refrigerate and frozen (-20ºC or lower)
temperatures for up to 7 days.
Conclusion
This method provides for the simultaneous rapid and reliable
analysis of clozapine and desmethylclozapine in serum.
Introduction
First developed over thirty years ago, clozapine had promising initial results,
however, its use was soon discontinued after several fatal cases of agranulocy-
tosis.1
More recently, there has been regained interest in the use of clozapine
for several reasons. Patients who previously did not respond to treatment with
other antipsychotics demonstrated improvement when clozapine was admin-
istered. Also, the agranulocytosis that occurs in approximately 1-2% of the
patients can be screened for with regularly scheduled hematological evalua-
tions.1,2
Currently, clozapine is used primarily in the treatment of schizophren-
ic patients that have encountered non-response or adverse intolerable extra-
pyramidal side effects with more classical antipsychotics (chlorpromazine,
haloperidol). 1,2,3
Structurally clozapine is a tricyclic dibenzodiazepine derivative, 8-chloro-11-
(4-methyl-1-piperazinyl)-5H-dibenzo[b,e] [1,4] diazepine. Clozapine binds to
the dopamine receptors D1 , D2 , D3 , D4 and D5 with highest binding affinity for
D4. In addition to binding to dopamine receptors, clozapine is an antagonist
at adrenergic, cholinergic, histaminergic, and serotonergic receptors. Clozap-
ine is metabolized to desmethylclozapine and clozapine-N-oxide. The desm-
ethyl metabolite has only limited activity and the oxide metabolite is inactive.3
Treatment is usually started with dosages of 25 to 75 mg/day with a gradual
increase of 25 to 50 mg/day to reach a final dose of 300 to 450 mg/day.3
It is
generally accepted that the therapeutic effect is associated with serum clozap-
ine levels of greater than 350 ng/mL.1,2,4
Monitoring of serum clozapine levels
gives clinicians the ability to evaluate patient compliance and adjust dosing to
limit possible side effects.
Assay Principle
A deuterated stable isotope (clozapine-d4) is added to 50 μL of serum as an in-
ternal standard. Protein is precipitated from the mixture by the addition of ac-
etonitrile. Clozapine, desmethylclozapine and internal standard are extracted
from the resulting supernatant by an on-line extraction utilizing high-through-
put liquid chromatography (HTLC) (TLX2, Cohesive Technologies, Franklin,
Massachusetts), followed by conventional liquid chromatography and analy-
sis on a tandem mass spectrometer (API 5000, Applied Biosystems, Toronto,
Canada) equipped with a heated nebulizer ion source.
The mobile phases for the online extraction consisted of an aqueous phase
(98/2 v/v deionized water/acetonitrile, 0.005% formic acid), organic phase
(methanol, 0.005% formic acid), and a cleaning solvent (45/45/10 v/v/v ace-
tonitrile/isopropyl alcohol/acetone).5
ACyclone HTLC column (ThermoFisher
Scientific) was used for the on-line extraction. The mobile phases for the
analytical chromatography consisted of an aqueous phase (98/2 deionized
water/acetonitrile 0.005% formic acid) and an analytical phase (50/45/5 v/v/v
acetonitrile/methanol/60 mM ammonium acetate). Hypersil GOLD columns
(ThermoFisher Scientific) were used as the analytical column.
The operating conditions are as follows: source temperature of 450 °C, nebu-
lizer gas (GS1) at 30 psi, and curtain gas at 35 psi. Ion transitions monitored
in the multiple reaction monitoring (MRM) mode were m/z 327.0 → m/z
270.0 for clozapine, m/z 313.2 → m/z 192.1 for desmethylclozapine, and m/z
331.0 → m/z 272.0 for clozapine-d4. The assay is calibrated with five standard
clozapine and desmethylclozapine solutions ranging from 0 to 2000 ng/mL.
Experimental Design
Reproducibility
Intra-assay and inter-assay precision for clozapine and
desmethylclozapine was demonstrated using tri-level
control material produced by Quality Assurance Service
Corporation, Augusta, GA. Intra-assay precision was
determined from 20 serial measurements of each sample.
Inter-assay precision was calculated from 20 replicate
determinations of each sample. The mean, coefficient of
variation (CV) and standard deviation (SD) were calcu-
lated at each level for each method.
Recovery & Linearity
Linearity over the clozapine and desmethylclozapine
reportable range was assessed using serial dilutions of
bovine serum spiked with clozapine and desmethylclo-
zapine standard. Each level was run in triplicate and
plotted versus the expected concentrations. Percentage
recovery was also calculated from each of the determina-
tions.
Specimen Stability
Specimen stability was assessed under ambient, refrig-
erated and frozen storage conditions and the effect of
multiple freeze thaw cycles was also evaluated.
Method Comparison
The LC-MS/MS method was compared to the current in-
house HPLC method (N=50) and an LC-MS/MS method
(N=40) performed by an external reference laboratory.
Interference Studies
The potential for method interference was assessed by
spiking a known concentration of clozapine and desm-
ethylclozapine with various substances. Multi-analyte
material used included BioRad Liquicheck Immunoassay
Plus Control and BioRad Liquicheck Therapeutic Drug
Monitoring Control. Specific interference studies were
also performed to evaluate interference from other anti-
psychotic medications that may be administered as part
of a multi-pharmaceutical treatment plan. Thioridazine
hydrochloride, haloperidol, olanzapine, quetiapine, and
risperidone were spiked into a known concentration of
clozapine and desmethylclozapine and assayed.
Ion Suppression
Ion suppression was assessed by infusing a constant
concentration of clozapine and desmethylclozapine
while running patient serum samples that had no clozap-
ine, desmethylclozapine, or internal standard. Data was
collected for the expected retention time of clozapine and
desmethylclozapine. Ion suppression would be detected
with this technique through visualization of an inverse
peak.
Results
Reproducibility
Clozapine Intra-assay (n = 20) Clozapine Inter-assay (n = 20)
Mean ± SD
(ng/mL)
%CV
Mean ± SD
(ng/mL)
%CV
189 ± 5 2.7 170 ± 8 4.8
825 ± 12 1.4 722 ± 34 4.7
1598 ± 44 2.7 1483 ± 52 3.5
Desmethylclozapine Intra-assay (n = 20) Desmethylclozapine Inter-assay (n = 20)
Mean ± SD
(ng/mL)
%CV
Mean ± SD
(ng/mL)
%CV
191 ± 6 3.2 181 ± 17 9.2
781 ± 22 2.8 730 ± 49 6.8
1589 ± 49 3.1 1526 ± 120 7.8
Specimen Stability
Clozapine Effect of Storage
% change
Day 1
% change
Day 3
% change
Day 8
% change
Day 14
20 – 25 °C 1.7 3.7 4.6 6.7
2 – 8 °C 2.3 -0.1 -1.3 3.6
– 20 °C -0.5 -0.9 -3.3 0.8
Effect of Multiple Freeze-Thaw Cycles
% change
1 cycle
% change
2 cycles
% change
3 cycles
% change
4 cycles
0.7 3.1 0.2 1.5
Desmethylclozapine Effect of Storage
% change
Day 1
% change
Day 3
% change
Day 8
% change
Day 14
20 – 25 °C 8.0 5.6 6.3 5.6
2 – 8 °C 3.8 3.3 0.8 10.6
– 20 °C 5.9 3.6 2.1 7.7
Effect of Multiple Freeze-Thaw Cycles
% change
1 cycle
% change
2 cycles
% change
3 cycles
% change
4 cycles
6.0 8.4 -0.6 11.0
Recovery & Linearity
Clozapine Spiked Serum Study
Expected Concentration
(ng/mL)
Measured Concentration
(ng/mL)
% Recovery
15.6 16.0 103
31.3 32.8 105
62.5 66.1 106
125 134 107
250 267 107
500 505 101
1000 1008 101
2000 2047 102
Desmethylclozapine Spiked Serum Study
Expected Concentration
(ng/mL)
Measured Concentration
(ng/mL)
% Recovery
15.6 13.9 89
31.3 32.6 104
62.5 59.2 95
125 143 114
250 265 106
500 506 101
1000 974 97
2000 1913 96
Linearity was demonstrated across the assay range yielding the following equations: observed clozapine
value = 1.0192*(expected value) + 1.8408, R2
= 0.9984; observed desmethylclozapine value = 0.9642
*(expected value) + 0.0, R2
= 0.9865.
Interference Studies
None of the following substances were found
to affect the chromatography or integration of
clozapine or desmethylclozapine when assessed at
the following levels:
Immunoassay Plus Control
Acetaminophen 56 ug/mL
Amikacin 13 ug/mL
Carbamazepine 7.55 ug/mL
Chloramphenicol 14.5 ug/mL
Cyclosporine 259 ng/mL
Desipramine 137 ug/mL
Digoxin 1.5 ng/mL
Disopyramide 3.15 ug/mL
Gentamicin 4.2 ug/mL
Ibuprofen 24 ug/mL
Lidocaine 3.4 ug/mL
Lithium 1.0 mEq/L
NAPA 4.65 ug/mL
Nortriptyline 125.5 ng/mL
Phenobarbital 26.5 ug/mL
Phenytoin 12.5 ug/mL
Primidone 6 ug/mL
Procainamide 14.6 ug/mL
Propranolol 100.5 ng/mL
Quinidine 2.4 ng/mL
Salicylate 19.5 mg/dL
Theophylline 14.5 ug/mL
Tobramycin 3.25 ug/mL
Valproic Acid 70 ug/mL
Therapeutic Drug Monitoring Control
Acetaminophen 76.5 ug/mL
Amikacin 16.5 ug/mL
Caffeine 9.5 ug/mL
Carbamazepine 8.35 ug/mL
Chloramphenicol 16 ug/mL
Clonazepam 228 ng/mL
Desipramine 228 ng/mL
Diazepam 2560 ng/mL
Digoxin 1.3 ng/mL
Gentamicin 3.2 ug/mL
Lithium 1.15 mEq/L
NAPA 5.75 ug/mL
Phenobarbital 26 ug/mL
Phenytoin 14 ug/mL
Primidone 7.5 ug/mL
Procainamide 5.8 ug/mL
Quinidine 178 ng/mL
Salicylate 21 mg/dL
Theophylline 14 ug/mL
Tobramycin 3.9 ug/mL
Valproic Acid 60.5 ug/mL
Additional Therapeutic Drugs
Thioridazine 833 ng/mL
Quetiapine 833 ng/mL
Olanzapine 83 ng/mL
Haloperidol 33 ng/mL
Risperidone 83 ng/mL
Method Comparison
vs. Clozapine by HPLC vs. Clozapine by LC-MS/MS
vs. Desmethylclozapine by LC-MS/MS
Investigation of the presence of ion
suppression demonstrated no interference
with either clozapine or demethylclozapine
when each drug was infused into drug
negative serum samples.
The new LC-MS/MS method
demonstrates a high degree of
correlation to both the existing
HPLC method and the reference
LC-MS/MS method. The
advantages of the new LC-MS/
MS assay include the elimination
of interferences currently observed
with the HPLC method as well
as the added quantitation of the
desmethyl metabolite.
R = 0.98 R = 0.98
R = 0.98