Validation of a HPLC/MS method for simultaneous quantification of clonidine, morphine and its metabolites in human plasma

Fei Tang, Henrietta Bada, Chee M. Ng, Markos Leggas

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

A high-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous quantification of morphine, morphine's major metabolites morphine-3-glucuronide and morphine-6-glucuronide, and clonidine, to support the pharmacokinetic analysis of an ongoing double-blinded randomized clinical trial that compares the use of morphine and clonidine in infants diagnosed with neonatal abstinence syndrome. Plasma samples were processed by solid-phase extraction and separated on an Inertsil ODS-3 (4 μm) column using an 0.1% formic acid in water–0.1% formic acid in methanol gradient. Detection of the analytes was conducted in the positive multiple reaction monitoring mode. The range of quantitation was 1–1000 ng/mL for morphine, morphine-3-glucuronide and morphine-6-glucuronide, and 0.25–100 ng/mL for clonidine. Intra-day and inter-day accuracy and precision were ≤15% for all analytes across the quantitation range. Extraction recovery rates were ≥94% for morphine, ≥90% for M3G, ≥87% for M6G and ≥ 79% for clonidine. Matrix effect ranged from 85–94% for clonidine to 101–106% for M3G. The method fulfilled all predetermined acceptance criteria and required only 100 μL of starting plasma volume. Furthermore, it was successfully applied to 30 clinical trial plasma samples.

Original languageEnglish
Article numbere4527
JournalBiomedical Chromatography
Volume33
Issue number7
DOIs
StatePublished - Jul 2019

Bibliographical note

Funding Information:
This work was supported by a National Institutes of Health MPI grant

Funding Information:
This work was supported by a National Institutes of Health MPI grant award (R01DA043519) to M. Leggas and H. Bada.

Publisher Copyright:
© 2019 John Wiley & Sons, Ltd.

Keywords

  • LC–MS/MS
  • clonidine
  • method validation
  • morphine
  • neonatal abstinence syndrome
  • solid-phase extraction

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

Fingerprint

Dive into the research topics of 'Validation of a HPLC/MS method for simultaneous quantification of clonidine, morphine and its metabolites in human plasma'. Together they form a unique fingerprint.

Cite this