Evaluating opiate metabolism: LC-MS/MS method for the determination of heroin, codeine, and morphine in horse urine

Codeine, heroin, and morphine are opioids used for the management of pain. Morphine is the major metabolite of heroin and codeine, and the metabolism of these drugs is quite complex. Since morphine and/or its metabolites can occur as a result of the use of any of the three drugs, separation of the parent drugs and their metabolites is crucial for correct interpretation of the results of the doping analysis. In this study, a sensitive and specific method has been developed for the determination of heroin, 6-monoacetylmorphine, codeine, norcodeine, hydrocodone, morphine, hydromorphone, morphine 3-glucuronide, morphine 6-glucuronide, and normorphine in horse urine. The samples were prepared with and without enzymatic hydrolysis before solid phase extraction. Separation was achieved on a C18 column (50×2.1 mm, 1.7 µm) maintained at 30°C using gradient elution consisting of 1 mM ammonium formate-0.1% formic acid (A) and methanol (B) with a total run time of 9 min by liquid chromatography tandem mass spectrometry. Because the glucuronide bonds of morphine are difficult to dissociate, the effects of the β-glucuronidase enzyme, incubation temperature and time were also optimized. Morphine d3 was used as an internal standard. The method was validated according to the screening requirements of the 2021/808/EC directive. The CCβ for all compounds were between 0.25 and 2.5 ng/mL. The inter- and intraday precision CV% were between 2.55 and 5.01 for morphine. The applicability of the method was demonstrated with the analyses of previously reported positive post-race samples.