Chiral analysis of methadone and its main metabolite EDDP in postmortem blood by liquid chromatography-mass spectrometry

Abstract

A chiral liquid chromatography-tandem mass spectrometry method was developed for the measurement of methadone and 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium (EDDP) enantiomers in postmortem blood. Blood (0.100 g) was mixed with water and aqueous methadone-d(3) and EDDP-d(3) solutions. After addition of NaOH, extraction was carried out with butyl acetate. Chromatographic separation was performed on a chiral-AGP analytical column (100 mm x 4.0-mm i.d., 5 microm) with a mobile phase of acetonitrile/ammonium acetate buffer (10 mM, pH 7.0, 22:78, v/v). A Quattro micro mass spectrometer was operated in the positive ion mode with an electrospray source. Multiple reaction monitoring was used with two transitions for each compound. The calibration curves were linear over the range from 0.001 to 2.5 mg/kg blood per enantiomer, and the limit of detection was 0.001 mg/kg, and the limit of quantitation was 0.003 mg/kg for all compounds. The precision (coefficient of variation%) was below 6.3%, and the trueness was 88-104% for all compounds. R- and S-methadone were measured in femoral blood from 10 postmortem cases. The R-methadone concentrations extended from 0.006 to 1.235 mg/kg with a median of 0.41 mg/kg, and the S-methadone concentrations ranged from 0 to 0.794 mg/kg (median 0.33 mg/kg). The median R/S-ratio was 1.46 (total range from 1.00 to 2.62), which tends to be higher than that reported in plasma of living subjects.