Quantification of the O- and N-demethylated metabolites of hydrocodone and oxycodone in human liver microsomes using liquid chromatography with ultraviolet absorbance detection.
High-performance liquid chromatographic assays for the O- and N-demethylated oxidative metabolites of hydrocodone and oxycodone formed in human liver microsomes are described. A solvent-solvent extraction/re-extraction procedure followed by reversed-phase HPLC with UV detection at 210 nm allows for the quantification of hydromorphone, norhydrocodone, oxymorphone and noroxycodone. Calibration curve concentration ranges were 0.63-400 microM (0.18-114 microg/ml) and 1.25-400 microM (0.36-114 microg/ml) for hydromorphone and norhydrocodone, respectively and 0.13-20 microM (0.04-6.03 microg/ml) and 1-200 microM (0.30-60 microg/ml) for oxymorphone and noroxycodone, respectively. Assay performance was determined by intra- and inter-assay precision and inaccuracies for quality control samples and was <15% for all metabolites at each quality control concentration. These methods provide good precision, accuracy and sensitivity for use in in vitro kinetic studies investigating the oxidative metabolism of hydrocodone and oxycodone in human liver microsomes.
Menelaou
Andrew
A
Department of Clinical and Experimental Pharmacology, University of Adelaide, Australia. andrew.menelaou@adelaide.edu.au
Hutchinson
Mark R
MR
Quinn
Ingvild
I
Christensen
Anders
A
Somogyi
Andrew A
AA
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D006853:Hydrocodone / Q000378:metabolism*; D008745:Methylation; D008862:Microsomes, Liver / Q000378:metabolism*; D010098:Oxycodone / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D006853:Hydrocodone; D010098:Oxycodone