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

• Published in: Chromatographia 2011-10, Vol.74 (7-8), p.567-574
• Main Authors: Hao, Guang-Tao, Chen, Yan, Dong, Rui-Hua, Qu, Heng-Yan, Liang, Yu-Guang, Li, Yuan-Yuan, Zheng, Zhuan-Jie, Gao, Hong-Zhi, Liu, Ze-Yuan
• Format: Article
• Language: English
• Subjects: Analysis; Analytical Chemistry; Biological and medical sciences; Chemistry; Chemistry and Materials Science; Chromatography; General pharmacology; Laboratory Medicine; Medical sciences; Original; Pharmacology. Drug treatments; Pharmacy; Proteomics
• ISSN: 0009-5893; 1612-1112
• DOI: 10.1007/s10337-011-2118-z

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- d 3 , norhydrocodone- d 3 , hydromorphone- d 3 , 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 C 18 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 ( r 2  = 0.9991) and norhydrocodone ( r 2  = 0.9990), and 0.01–10 ng mL −1 for hydromorphone ( r 2  = 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