Simultaneous analysis of codeine, morphine, and heroin after B-glucuronidase hydrolysis

Analysis of the opiates, morphine and codeine, often proceeds by way of acid hydrolysis for release of the parent morphine from its glucuronide formed during metabolism. Following use, heroin is rapidly deacetylated to 6-monoacetylmorphine (6-MAM), which can be detected in the urine for a short time following injection of heroin. Only a small amount of 6-MAM may be further metabolized to morphine glucuronide. Thus, in general, the urine specimen has not been hydrolyzed prior to analysis for heroin, using a separate procedure from morphine and codeine. Simultaneous analysis of morphine, codeine, 6-MAM and heroin would be complicated by loss of identity between morphine and heroin when heroin converts to morphine following acid hydrolysis for removal of the glucuronide moiety from morphine glucuronide. Another significant problem in simultaneous analysis is the relative disparity in concentration between morphine/codeine and 6-MAM/heroin (which might be present in the urine specimen). In the proposed method of analysis, free morphine resulting from B-glucuronidase rather than acid hydrolysis of morphine glucuronide is derivatized with propionic anhydride to form dipropionylmorphine. Heroin that does not react with B-glucuronidase remains unhydrolyzed as the diacetylmorphine derivative. Some of the more exacting steps for the acid procedure are eliminated altogether making overall costs for the enzyme procedure comparable to those of the acid hydrolysis method. The enzyme reaction mixture is purified through a solid phase column system. The optimal conditions for concentration of enzyme, temperature of hydrolysis and pH are individually characterized for B-glucuronidase hydrolysis and the ions which identify the propionyl derivatives are characterized for the simultaneous analysis of morphine, codeine, 6-MAM and heroin.