Aim
In drug-facilitated sexual assaults (DFSA) the victim is incapacitated by involuntary or voluntary substance consumption. Unfortunately, drug analysis of samples from DFSA is often complicated by late reporting and rapid drug metabolism. Thus, it can be challenging to detect relevant drugs in samples collected more than 24 hours after the assault and sensitive analytical methods are therefore essential. Urine samples provides a longer detection window compared to blood samples. However, many drugs are excreted in urine as conjugated metabolites and sample pretreatment commonly includes an overnight enzymatic hydrolysis under heating conditions. Investigations on the pretreatment conditions and the effects on drug stability provides valuable knowledge in these settings. The aim of this study was to improve enzymatic pretreatment of urine samples and investigate efficiency of a β-glucuronidase/arylsulfatase enzyme using morphine-6-glucuronide as test compound. Additionally, we investigated the stability of 110 drugs and metabolites relevant to DFSA under the optimal pretreatment conditions.

Method
To test enzyme efficiency, drug-free urine samples were spiked with morphine-6-glucuronide (M6G) and enzymatically hydrolyzed with β-glucuronidase/arylsulfatase (Helix pomatia, Roche) for 2, 4, 6 and 18 hours at 37 °C. To test drug stability, additional samples spiked with morphine were incubated for 18 hours at 37 °C without enzymatic treatment. After incubation, the enzymatic hydrolysis was stopped by protein precipitation before the extracts were analyzed by UHPLC–MS/MS. To test drug stability for the optimal enzyme incubation time, drug-free urine was collected and spiked with 110 DFSA relevant drugs and metabolites. Samples were either 1) enzymatically hydrolyzed with a β-glucuronidase/arylsulfatase and incubated overnight at 37 °C, 2) incubated overnight at 37 °C without enzymatical hydrolysis or 3) not treated with enzyme or heat. Following overnight incubation, samples were centrifuged twice and diluted with methanol and acetonitrile (50:50) before analysis by UHPLC–MS/MS.

Results
Initial results showed that urine samples spiked with morphine and M6G had decreasing concentration of M6G following incubation at 2 h (91% of spiked concentration), 4 h (56%), 6 h (44%), and 18 h (18%). Juxtaposed, morphine concentration increased with incubation at 2 h (19% of theoretical concentration), 4 h (38%), 6 h (47%), and 18 h (92%). This indicated an expected M6G to morphine turnover where the optimal enzyme efficiency was at incubation for 18 h. Results further showed that morphine stability was not affected (recovery: 102%) by heating conditions. In the multi-drug experiment, most drugs were stable under the heating conditions with recoveries above 80%. For some drugs, a decline in concentration was observed after incubation at 37 °C for 18 h, indicating drug instability. For the drug clozapine, the recovery decreased by ¼ while the recovery of the metabolite N-demethylclozapine increased. This demonstrates the importance of including both parent drug and metabolites in the analytical method of urine samples. To prevent this drug instability during pretreatment of urine samples, further experiments will be conducted to examine the use of other enzymes that requires shorter incubation and lower temperatures.

Conclusion
The preliminary results showed that the efficiency of a β-glucuronidase/arylsulfatase improved with increasing incubation time. Furthermore, the enzymatical hydrolysis and incubation at 37 °C did not affect the stability of most drugs (75%) relevant to DFSA investigations. This work is financially supported by the Danish Victims Fund (grant number 20-610-0092).