Determination of Oxycodone and Its Metabolite, Noroxycodone, in Human Plasma by HPLC with Post‐column Chemiluminescence Detection Using Electrogenerated Tris(2,2′‐Bipyridyl)ruthenium(III)

Abstract

A sensitive high performance liquid chromatography (HPLC) method with post‐column chemiluminescence detection was developed for the simultaneous determination of oxycodone (OXC) and its metabolite, noroxycodone (NOC), in human plasma. Sample preparation for human plasma was performed by means of solid extraction with Sep‐Pak Vac C18 (100 mg/mL). Separation was performed by reversed‐phase HPLC using a 5‐µm Develosil ODS‐HG‐5 column (150 mm × 4.6 mm) and a mobile phase consisting of a mixture of 0.1 mol/L phosphate buffer (pH 2.6) containing 5 mmol/L sodium dodecylsulfate and methanol (53:47, v/v). Quantitation was performed by the measurement of chemiluminescence using electrogenerated tris(2,2′‐bipyridyl)ruthenium(III). The lower and upper limits of quantitation for OXC and NOC were 0.5–250 ng/mL and 1–500 ng/mL, respectively. The calibration curve for this assay showed good linearity for the concentration range of 0.5–50 ng/mL for OXC and 1–100 ng/mL for NOC. Recoveries of the studied compounds were excellent at the individual assay ranges (102.9–108.7%), and validation of the assay gave results that were satisfactory in terms of within‐run or between‐run precision (1.9–11.9% as RSD) and accuracy (−13.5% to +9.2% as bias). Stability studies showed that OXC and NOC were stable in assay solutions for at least 6 days at 4°C storage in the autosampler. OXC and NOC were also stable in human plasma for at least 48 hr at room temperature, for at least 8 months at −20°C and after five repeated freeze–thaw cycles. Furthermore, OXC and NOC were stable in human whole blood for up to 5 days at 4°C and for up to 5 hr at room temperature. This system was applied to the clinical study of cancer patients after single oral administration doses of controlled‐released tablets of OXC hydrochloride. Consequently, this method should serve as a useful tool for pharmacokinetic studies of OXC and NOC.