Drug-facilitated sexual assault (DFSA) involves acts of sexual violence towards an individual who is incapacitated or otherwise unable to provide consent due to the influence of intoxicating substances. Whether the victim has consumed any substances knowingly or unknowingly determines the assault as opportunistic or proactive in approach. The longer it takes the victim to recover and report the assault, the higher the risk is of not finding any forensic evidence of the drug used to facilitate the assault. In these cases, urine samples are advantageous because they provide a broader window of detection compared to blood. Drug metabolism often involves a series of conjugation reactions that allow for renal excretion. Consequently, analysis of urine enables detection of conjugated metabolites of the parent drug. In DFSA cases, sensitive and broad analytical methods are needed to find relevant substances at low single dose concentrations. Finding evidence of the victim being under the influence of intoxicating substances during the assault is essential for judicial proceedings and may provide some closure for the victim.

Study I reviewed the toxicological results published from DFSA cases on an international level. The included 22 publications covered findings from 10680 cases from 16 countries during the period 1996–2018. Five publications estimated that 2–22% of DFSA cases were of a proactive approach. As such, the opportunistic approach appears to be more prevalent with the most commonly observed substances being ethanol, drugs of abuse, analgesics, and benzodiazepines. Results were comparable across countries but with minor differences in Africa and Asia where methaqualone, diphenhydramine, dexmedetomidine and some new psychoactive substances (NPSs) were detected.

Study II investigated which drugs were detected in samples from police reported DFSA cases in Eastern Denmark from 2015–2022. During the period, samples from 369 sexual assault cases were analyzed. Of these, 268 cases were marked as suspected DFSA cases. The victims were female in 98% of cases and the mean age was 26 years. Ethanol was the most frequently detected substance followed by drugs of abuse, antidepressants, antihistamines, benzodiazepines, and opioids. Hypnotics and antipsychotics were found to a lesser extent. As such, findings suggested that recreational alcohol and drug consumption is a risk factor for opportunistic DFSA.

Study III investigated the efficiency of different enzymes on the hydrolysis of 11 chosen glucuronides relevant to DFSA. The experiment indicated that recombinant enzymes were most efficient with a rapid glucuronide hydrolysis at room temperature. Consequently, treatment with a recombinant enzyme could improve workflow for the pretreatment of urine samples from DFSA cases.

Study IV involved method development, validation, and incorporation of an automated enzymatic hydrolysis step in sample preparation of urine. The use of a recombinant enzyme in the automated sample preparation enabled an efficient and simplified workflow for an improved systematic toxicological analysis strategy for DFSA cases.

This PhD thesis demonstrated that toxicological findings in Denmark are very similar to findings across the world. Alcohol and recreational drug consumption appears to be a risk factor for DFSA by rendering the victim vulnerable to opportunistic DFSA. Additionally, the use of recombinant enzymes combined with an automated sample preparation results in efficient hydrolysis of urine samples and improved systematic toxicological analysis of samples from DFSA cases.