Optimizing the hydrolysis of codeine and morphine glucuronides in urine.

Quality assurance data show that there is very significant inter-laboratory variation of the quantitation of codeine, especially in patient samples. The authors have examined hydrolysis procedures for codeine glucuronide (C6G) and morphine-3- and -6-glucuronides (M3G, M6G) because these are often present together in urine samples. Comparisons of hydrolysis using two different sources of beta-glucuronidases and various concentrations of hydrochloric acid were made. Samples were concentrated using solid phase extraction, derivatized and quantified by selective ion monitoring using gas chromatography-mass spectrometry (GC-MS). and beta-glucuronidase efficiently hydrolyzed M3G (90-95%), while hydrolysis of M6G was lower (60-85%) and that of C6G was very poor (45-58%). These findings were confirmed on examination of urine samples containing codeine and morphine from subjects who had taken codeine, morphine, or heroin. Erratic inter-laboratory quality assurance results for codeine are most probably a result of incomplete C6G hydrolysis. The authors' optimized hydrolysis method using 50% HCl for 1.5 hours at 120 degrees C gave reproducible results that approached the spiked concentration.