Capillary electrophoresis and capillary electrophoresis-ion trap multiple-stage mass spectrometry for the differentiation and identification of oxycodone and its major metabolites in human urine

摘要

Oxycodone (OCOD) and its metabolites, including oxymorphone (OMOR), noroxycodone (NOCOD) and noroxymorphone (NOMOR), are opioids that carry an OH group at position 14. Using capillary electrophoresis (CE) with a binary phosphate buffer containing 60% ethylene glycol (pH 7.9), the migration order of OCOD and OMOR with respect to their N-demethylated analogs was found to be reversed compared to that observed for codeine, dihydrocodeine, morphine and dihydromorphine, compounds that do not have an OH group at position 14. OCOD and structurally related compounds can also be distinguished from these opioids by their absorbance spectra at low wavelengths and via a characteristic neutral H_2O loss at the MS~2 level. Using the binary phosphate buffer, CE with UV detection is shown to be capable of monitoring OCOD, NOCOD, OMOR (after hydrolysis only) and NOMOR (after hydrolysis and in patient urine only) in alkaline liquid-liquid extracts of urines that were collected after ingestion of 10 mg OCOD hydrochloride and in a patient urine collected at steady state (80 mg OCOD hydrochloride daily). Using an aqueous pH 9 ammonium acetate buffer, these results were confirmed by CE-MS. Based on CE-MS~3. MS~2 and MS~3 data, the absorbance spectra measured across the CE peaks and the relative position within the electropherogram, two peaks monitored in the UV absorbance electropherograms could be assigned to the two keto-reduced metabolites 6oxycodol (6OCOL) and nor6oxycodol, for which no standards were available. Comparison of data obtained with urines pretreated with two different enzyme products (β-glucuronidase and β-glucuronidase/arylsulfatase) suggest that OCOD, NOCOD and 6OCOL are mainly glucuronidated, whereas OMOR mainly forms other conjugates. Furthermore, in a first attempt to directly measure conjugates of the compounds of interest, solid-phase extracts were analyzed by CE-MS~4, which revealed the presence of the acyl glucuronides of 6OCOL and OMOR and an unidentified OMOR conjugate. The quantitation of free OCOD and NOCOD by CE-MS using deuterated internal standards is also discussed briefly.