Simultaneous Determination of Hydrocodone, and Its Two Metabolites in Human Plasma by HPLC–MS–MS

A rapid, sensitive and specific assay method has been developed to simultaneously determine human plasma concentrations of hydrocodone and its metabolites, norhydrocodone, hydromorphone, using high-performance liquid chromatography with an electrospray ionization (ESI) tandem mass spectrometry (HPLC–MS–MS). Hydrocodone, its metabolites, and internal standard, hydrocodone-d3, norhydrocodone-d3, hydromorphone-d3, were separated from human plasma using solid-phase extraction (Empore MPC-SD Solid Phase Extraction Disk). The eluate was dried, reconstituted and injected into the LC–MS–MS system. Chromatographic separation was performed on a Kromasil 100-5SIL-Dimensions C18 column (100 × 2.1 mm, 5.0 μm, Thermo Hypersil-Keystone, USA) using a gradient mobile phase with 20 mmol L−1 ammonium formate in water with 0.2% formic acid and 0.1% formic acid in acetonitrile. Detection and quantitation were performed by MS/MS using electrospray ionization and multiple reactions monitoring in the positive ion mode. The calibration curves were linear over the concentration ranges 0.05–50 ng mL−1 for hydrocodone (r2 = 0.9991) and norhydrocodone (r2 = 0.9990), and 0.01–10 ng mL−1 for hydromorphone (r2 = 0.9990). The limit of quantification was 0.05 ng mL−1 for hydrocodone and norhydrocodone, and 0.01 ng mL−1 for hydromorphone. The extraction recovery was above 64.36, 68.51 and 71.78% for hydrocodone, norhydrocodone and hydromorphone. The accuracy was higher than 99.06, 97.70 and 100.07% for hydrocodone, norhydrocodone and hydromorphone. The intra- and inter-day precisions were <5.80, 5.90 and 3.02% for hydrocodone, norhydrocodone and hydromorphone. The method was accurate, sensitive and simple and was successfully applied to a pharmacokinetic study after a single oral administration of hydrocodone bitartrate at a dose of 5 mg in 12 healthy Chinese volunteers.