Automated Analysis of Clozapine and Norclozapine in Human Plasma Using Novel Extraction Plate Technology and Flow-Injection Tandem Mass Spectrometry.
BACKGROUND
Analysis of plasma clozapine and N-desmethylclozapine (norclozapine) for therapeutic drug monitoring purposes is well established. To minimize analysis times and facilitate rapid reporting of results, we have fully automated sample preparation using novel AC Extraction Plates and a Tecan Freedom EVO 100 liquid handling platform, and minimized extract analysis times using flow-injection tandem mass spectrometry (FIA-MS/MS).


METHODS
Analytes and deuterium-labeled internal standards were extracted from plasma (100 μL) at pH 10.6 and extracts analyzed directly using tandem mass spectrometry [20 μL injection, 0.7 mL/min methanol carrier flow, analysis time (injection-to-injection) approximately 60 seconds].


RESULTS
Validation data showed excellent intraplate and interplate accuracy (95%-104% nominal concentrations). Interbatch precision (% RSD) at the limit of quantitation (0.01 mg/L) was 3.5% and 5.5% for clozapine and norclozapine, respectively. Matrix effects were observed for both clozapine and norclozapine, but were compensated for by the internal standards. Overall process efficiency was 56%-70% and 66%-77% for clozapine and norclozapine, respectively. Mean relative process efficiency was 98% and 99% for clozapine and norclozapine, respectively. Comparison of results from patient samples (n = 81) analyzed using (1) manual liquid-liquid extraction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and (2) automated extraction with FIA-MS/MS gave y = 1.01x - 0.002, R(2) = 0.9943 and y = 1.01x + 0.009, R(2) = 0.9957 for clozapine and norclozapine, respectively. Bland-Altman plots revealed a [mean (95% limits of agreement) bias of 0.0074 (-0.04 to 0.06) mg/L and of 0.015 (-0.02 to 0.05) mg/L for clozapine and norclozapine, respectively].


CONCLUSIONS
FIA-MS/MS used with automated extraction offers a rapid, simple, cost-effective alternative to manual liquid-liquid extraction and conventional LC analysis for clozapine therapeutic drug monitoring.
Viapath Analytics, Toxicology Unit, Department of Clinical Biochemistry, King's College Hospital NHS Foundation Trust, London, United Kingdom.
Lewis
L
Couchman
Krithika
K
Subramaniam
Danielle S
DS
Fisher
Sarah L
SL
Belsey
Simon A
SA
Handley
Robert J
RJ
Flanagan
D014150:Antipsychotic Agents / Q000097:blood; D001331:Automation; D002853:Chromatography, Liquid / Q000379:methods*; D003024:Clozapine / Q000031:analogs & derivatives* / Q000097:blood; D016903:Drug Monitoring / Q000379:methods*; D006801:Humans; D059625:Liquid-Liquid Extraction; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
D014150:Antipsychotic Agents; C058272:norclozapine; D003024:Clozapine