Simultaneous determination of dihydrocodeine and its metabolites in dog plasma by high-performance liquid chromatography with electrochemical and ultraviolet detection.

An HPLC method with electrochemical and UV detection was established for the simultaneous determination of dihydrocodeine and its metabolites, dihydronorcodeine, dihydromorphine, and dihydrocodeine glucuronide, in dog plasma using N-ethylnormorphine as the internal standard. The method involved sample pretreatment with a C18-bonded disposable column, and the injected fraction was separated and detected on the C18-bonded column with serially coupled UV and coulometric detectors. Dihydromorphine was detected with the coulometric detector at 0.4 V, and dihydrocodeine and dihydronorcodeine at 0.8 V. Dihydrocodeine glucuronide was detected with UV at 210 nm. Recoveries of the studied compounds were quantitative at the individual assay ranges, and validation of the assay gave results that were satisfactory in terms of within-run or between-run precision and accuracy. Lower limits of quantitation were 2 ng/ml for dihydrocodeine and dihydronorcodeine, 0.5 ng/ml for dihydromorphine, and 200 ng/ml for dihydrocodeine glucuronide.