Analysis of Codeine and its Metabolites in Human Plasma by HPLC with Electrochemical Detection

Abstract

The resolving power of high pressure liquid chromatography has been combined with the sensitivity of electrochemical oxidation to develop a method for determination of codeine and its metabolites, morphine and norcodeine, in plasma. Plasma samples containing Internal Standard (Dihydromorphinone) are extracted at pH 8.9 into a 2/98 v/v/ Butanol/methyl tertiary butyl ether organic solvent system and back extracted into 25 mM phosphate buffer pH 2.8. The optimal recovery is greater than 90% for codeine and 70% for morphine and norcodeine. Reverse phase chromatography (5 micrometer phenyl column) with detection by electrochemical oxidation at + 1.2 V vs. Ag/AgCl is utilized. The method is sensitive, specific and precise. This method is used to establish a concentration - time profile for plasma codeine and morphine from a human volunteer after a 60 mg oral dose of codeine phosphate. No measurable concentration of norcodeine was found in the plasma. Codeine has long been used as an analgesic and antitussive agent In pharmaceutical preparations. The hypothesis that codeine exerts Its analgesic action after metabolic conversion to morphine was first suggested by Sanfilippo in 1948¹. Since then several attempts to study the disposition and metabolism of codeine in several animal species have resulted in somewhat contradictory results ^2,3 Since many drugs give rise to pharmacologically active metabolites, it is necessary to monitor simultaneously all the active compounds in the body in order to elucidate the proper relationship between their pharmacokinetic profiles and the kinetics of pharmacological effect. There is no reported method to simultaneously quantitate codeine and its O -Demethylated and N -Demethylated metabolites in plasma. Radioimmunoassay (RIA) can be used for quantisation of codeine and morphine but the assay is not specific due to cross - reaction with the other opiates. Procedures such as gas chromatography and thin - layer chromatography have also been reported to be used in forensic toxicology studies. None of the reported high performance liquid chromatography (HPLC) methods ^4,5 allow simultaneous measurement of codeine and its metabolites. This report describes a specific and sensitive high performance liquid chromatography method utilizing electrochemical oxidation for codeine, morphine and norcodeine in human plasma.