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

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 mu m) 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 mu L of starting plasma volume. Furthermore, it was successfully applied to 30 clinical trial plasma samples.