Determination of perhexiline and hydroxyperhexiline in plasma by liquid chromatography–mass spectrometry

Abstract

A method for the quantitative determination of perhexiline and its main hydroxylated metabolites in human plasma, based on liquid chromatography–mass spectrometry (LC–MS), was developed. The method used protein precipitation with acetonitrile followed by dilution with water and subsequent direct injection of the extract into the LC–MS system. Hexadiline was used as internal standard and the intra-assay coefficients of variation were ≤5% for perhexiline and cis-hydroxyperhexiline over the target concentration range in patients. The lower limits of quantification were 0.005 mg/l for perhexiline and 0.015 mg/l for cis-hydroxyperhexiline, and the measuring ranges were from 0.05 to 3.0 and from 0.2 to 6.0 mg/l, respectively. The method was compared with an established HPLC method with fluorescence detection and the correlation between the methods was close to 1 for both compounds. The predominant form of hydroxyperhexiline in 87% of the patient samples was found to be one of the diastereomeric pairs of cis-hydroxyperhexiline. In patients not forming this metabolite, trans-hydroxyperhexiline could be detected. We conclude that the present LC–MS method is suitable for use in a clinical routine laboratory.