In vitro conversion of morphine to 6-acetylmorphine in urine samples during enzymatic hydrolysis.

Heroin is an illegal drug and monitoring heroin use is important in rehabilitation, pain management services, and workplace employment testing. In humans, heroin is rapidly converted to 6-acetylmorphine (6AM), which is quickly hydrolyzed to morphine. Morphine glucuronide is the major metabolite of heroin in urine (1 ). However, 6AM is a unique metabolite of heroin and is considered the definitive indicator of heroin intake. 6AM in urine is measured by GC-MS, LCMS, immunoassay, or thin-layer chromatography methods. For measuring opioids in urine, hydrolysis using -glucuronidase is frequently employed to improve consistency because of large variable glucuronide conjugation rates between and within individuals. Acetate buffer is the primary choice for preparing enzymatic hydrolysis solution due to the favorable pH (4.5–5.0). In our routine LC-MS analysis (2, 3 ), a number of lowconcentration 6AM results were observed in patient urine samples with high concentrations of morphine. A Medical Review Officers’ advisory cites possible in vitro synthesis of 6AM in high-morphine samples during derivatization in GC-MS analyses (4 ). To the best of our knowledge, there are no reports on in vitro conversion of morphine to 6AM in LC-MS analyses. We hypothesized that the acetate buffer used for enzymatic hydrolysis may act as an acetylating agent to chemically convert morphine to 6AM. In the first study, morphine and the major morphine metabolite, morphine-3-glucuronide (M3G), were spiked into morphine and 6AM-negative urine samples (n 3) to create 2 sets of samples: 5 concentrations of morphine samples (10 000, 50 000, 100 000, 150 000, and 200 000 g/L) in 1 set and 5 concentrations of M3G samples (16 250, 81 250, 162 500, 243 750, and 325 000 g/L) in the other set. Enzymatic hydrolysis was performed at 60 °C using a glucuronidase from Patella vulgate in 1.0 mol/L sodium acetate buffer (pH 4.5). Aliquots were taken at 0, 2, 4, 6, and 18 h. In a similar experiment, the samples spiked with morphine (100 000, 150 000, and 200 000 g/L) (n 3) and M3G (162 500, 243 750, and 325 000 g/L) (n 3) were incubated for 0 and 12 h under the same conditions. In the second study, patient urine samples (n 4) with increased concentrations (97 000 – 110 000 g/L) of morphine were incubated at 60 °C using glucuronidase in acetate buffer for 0 and 18 h. In the third study, 2 samples spiked with 200 000 g/L morphine, 2 samples spiked with 325 000 g/L M3G, and the 4 patient urine samples used in the second study were incubated at 60 °C using -glucuronidase in 1.0 mol/L citrate buffer (pH 4.5) for 0 and 18 h. In the fourth study, 3 concentrations of spiked morphine samples (100 000, 150 000, and 200 000 g/L) (n 3) and 3 separate concentrations of spiked M3G samples (162 500, 243 750, and 325 000 g/L) (n 3) were incubated in the acetate buffer without -glucuronidase for 0 and 18 h. Examining patient urine samples in this study after clinical testing was approved by the Cleveland Clinic Institutional Review Board. In the LC-MS method, the analytical measurement ranges for morphine and 6AM were 5–5365 g/L and 5–4800 g/L, respectively. The total imprecision was 3%–9% for morphine and 4%–13% for 6AM. After incubation for 18 h using -glucuronidase in acetate buffer, all urine samples with increased morphine ( 100 000 g/L) and 78% of the samples with increased M3G ( 162 500 g/L) formed measurable 6AM ( 5 g/L). One of the 9 samples with 100 000 g/L morphine and 2 of the 9 samples with 162 500 g/L M3G incubated for 12 h using -glucuronidase in acetate buffer formed measurable 6AM (Fig. 1). All samples with 100 000 g/L morphine or 162 500 g/L M3G, and all samples incubated for 12 h using -glucuronidase in acetate buffer, did not form measurable 6AM. All 4 patient urine samples with increased morphine formed 6AM (5–20 g/L) when incubated for 18 h using -glucuronidase in acetate buffer. No samples incubated using citrate buffer formed measureable 6AM, whereas 67% of the samples incubated for 18 h in the acetate buffer without -glucuronidase formed measurable 6AM (5– 8 g/L). Our results show that falsepositive identification of heroin is possible when using acetate buffer to hydrolyze urine samples with extremely high morphine ( 100 000 g/L). After 18 h of incubation, 16 of the 18 samples with extremely high morphine or M3G formed 6AM 5 g/L, and 2 samples formed 6AM 10 g/L (the common cutoff concentration for 6AM in workplace programs to identify heroin). A retrospective analysis of 2 years’ worth of patient data in our laboratory identified 1116 morphine-positive samples, among which approximately 2% had morphine concentrations 100 000 g/L. Caution should be taken in result interpretation. An alternate buffer with no acetate, for example citrate buffer, should be considered for this application.