Skip to main content
SpringerLink
Log in

A novel approach to quantitative LC-MS/MS: therapeutic drug monitoring of clozapine and norclozapine using isotopic internal calibration

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Therapeutic drug monitoring (TDM) requires timely results in order to be clinically helpful. Such assays, when carried out using mass spectrometry-based methods, typically involve a batched sample approach with multipoint calibration. Isotopic internal calibration offers the possibility of open-access mass spectrometric analysis with consequent shortening of turnaround times. We measured plasma clozapine and N-desmethylclozapine (norclozapine) concentrations in (1) external quality assessment (EQA) samples (N = 22) and (2) patient samples (N = 100) using liquid chromatography-tandem mass spectrometry with isotopic internal calibration (ICAL-LC-MS/MS). Analyte concentrations were calculated from graphs of the response of three internal calibrators (clozapine-D4, norclozapine-D8, and clozapine-D8) against concentration. Precision (% RSD) and accuracy (% nominal concentrations) for the ICAL-LC-MS/MS method were <5 % and 104–112 %, respectively for both analytes. There was excellent agreement with consensus mean and with ‘spiked’ values on analysis of the EQA samples (R 2 = 0.98 and 0.97, respectively, inclusive of clozapine and norclozapine results). In the patient samples, comparison against traditionally calibrated HPLC-UV and LC-MS/MS methods showed excellent agreement (R 2 = 0.97 or better) with small albeit significant mean differences (<0.041 and <0.042 mg/L for clozapine and norclozapine, respectively). These differences probably reflect discrepancies in the in-house preparation of calibrators and/or interference in the UV method. Internal calibration offers a novel and attractive alternative to traditionally calibrated batch analysis in analytical toxicology. The method described has been validated for use in the high-throughput TDM of clozapine and norclozapine, and allows for (1) same-day reporting of results and (2) significant cost savings.

A typical internal calibration curve produced using three different isotopically-labelled analogues of clozapine and norclozapine (clozapine-D4, norclozapine-D8 and clozapine-D8) and interpolation of the clozapine and norclozapine concentrations from an extracted patient sample (calculated clozapine and norclozapine 0.47 and 0.31 mg/L, respectively)

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Couchman L, Morgan PE, Spencer EP, Flanagan RJ (2010) Plasma clozapine, norclozapine, and the clozapine:norclozapine ratio in relation to prescribed dose and other factors: data from a therapeutic drug monitoring service, 1993–2007. Ther Drug Monit 32:438–447

    Article  CAS  Google Scholar 

  2. Hiemke C, Baumann P, Bergemann N, Conca A, Dietmaier O, Egberts K et al (2011) AGNP consensus guidelines for therapeutic drug monitoring in psychiatry: update. Pharmacopsychiatry 44:195–235

    Article  Google Scholar 

  3. FDA/CDER (Food and Drug Administration/Center for Drug Evaluation and Research) (2001) Guidance for industry. Bioanalytical method validation. http://www.fda.gov/cder/guidance/4252fnl.htm. Accessed 10 January 2013

  4. Peters FT, Maurer HH (2007) Systematic comparison of bias and precision data obtained with multiple-point and one-point calibration in six validated multi-analyte assays for quantification of drugs in human plasma. Anal Chem 79:4967–4976

    Article  CAS  Google Scholar 

  5. Crawford M, Rappold B, Grant RP. Clinical calibration: moving away from the line. https://www.msacl.org/2013_Long_Abstracts/crawfm1labcorp_201210101341_6878583.pdf. Accessed 02 March 2013

  6. Kemp GJ (1984) Theoretical aspects of one-point calibration: causes and effects of some potential errors, and their dependence on concentration. Clin Chem 30:1168–1170

    CAS  Google Scholar 

  7. Olson MT, Breaud A, Harlan R, Emezienna N, Schools S, Yergey AL, Clarke W (2013) Alternative calibration strategies for the clinical laboratory: application to nortriptyline therapeutic drug monitoring. Clin Chem. doi:10.1373/clinchem.2012.194639

    Google Scholar 

  8. Grant RP (2013) The march of the masses. Clin Chem. Doi.10.1373/clinchem.2013.205435

  9. Cooper DP, Eastwood MP, Roberts M, Molloy BJ. Internal calibration: toward random access LC-MS analysis. https://www.msacl.org/2013_Long_Abstracts/201212010515_27984.pdf. Accessed 02 March 2013

  10. McCarthy PT, Hughes S, Paton C (1995) Measurement of clozapine and norclozapine in plasma/serum by high performance liquid chromatography with ultraviolet detection. Biomed Chromatogr 9:36–41

    Article  CAS  Google Scholar 

  11. Fisher DS, Partridge SJ, Handley SA, Couchman L, Morgan PE, Flanagan RJ (2013) LC-MS/MS of some atypical antipsychotics in human plasma, serum, oral fluid and haemolysed whole blood. Forensic Sci Int. 229:145–150. doi:10.1016/j.forsciint.2013.02.010

    Google Scholar 

  12. Bonfiglio R, King RC, Olah TV, Merkle K (1999) The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds. Rapid Commun Mass Spectrom 13:1175–1185

    Article  CAS  Google Scholar 

  13. Fisher DS, Partridge SJ, Handley SA, Flanagan RJ (2013) Stability of some atypical antipsychotics in human plasma, haemolysed whole blood, oral fluid, human serum and calf serum. Forensic Sci Int. 229:151–156. doi:10.1016/j.forsciint.2013.03.021

    Google Scholar 

  14. Croes K, McCarthy PT, Flanagan RJ (1995) HPLC of basic drugs and quaternary ammonium compounds on microparticulate strong cation-exchange materials using methanolic or aqueous methanol eluents containing an ionic modifier. J Chromatogr A 693:289–306

    Article  CAS  Google Scholar 

  15. Choong E, Rudaz S, Kottelat A, Guillarme D, Veuthey JL, Eap CB (2009) Therapeutic drug monitoring of seven psychotropic drugs and four metabolites in human plasma by HPLC-MS. J Pharm Biomed Anal 50:1000–1008

    Article  CAS  Google Scholar 

  16. Morgan PE, Couchman L, Robinson S, McDonnell S, Flanagan RJ (2010) Analysis of clozapine and norclozapine in plasma using on-line sample preparation and LC-MS/MS. LCGC: Column 6:10–16

    Google Scholar 

  17. Couchman L, Morgan PE (2011) LC-MS in analytical toxicology: some practical considerations. Biomed Chromatogr 25:100–123

    Article  CAS  Google Scholar 

  18. Couchman L, Morgan PE, Flanagan RJ (2011) Basic drug analysis by strong cation-exchange liquid chromatography-tandem mass spectrometry: simultaneous analysis of amisulpride, and of metamfetamine and amfetamine in serum/plasma. Biomed Chromatogr 25:867–872

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Don Cooper (Waters Corporation, Manchester, UK) for permission to cite early internal calibration work, Novartis for the gift of pure clozapine and norclozapine, and LGC Standards Proficiency Testing for permission to quote EQA data. We also thank Dr Cajetan Moniz (King's College Hospital) for continued support.

Author information

Authors and Affiliations

  1. Toxicology Unit, Department of Clinical Biochemistry, King’s College Hospital NHS Foundation Trust, London, SE5 9RS, UK

    Lewis Couchman, Sarah L. Belsey, Simon A. Handley & Robert J. Flanagan

Authors
  1. Lewis Couchman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sarah L. Belsey
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Simon A. Handley
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert J. Flanagan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lewis Couchman.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Couchman, L., Belsey, S.L., Handley, S.A. et al. A novel approach to quantitative LC-MS/MS: therapeutic drug monitoring of clozapine and norclozapine using isotopic internal calibration. Anal Bioanal Chem 405, 9455–9466 (2013). https://doi.org/10.1007/s00216-013-7361-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-013-7361-8

Keywords

Search

Navigation