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). Helix pomatia and Escherichia coli 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 interlaboratory 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 120degreesC gave reproducible results that approached the spiked concentration.