Automated enzymatic hydrolysis of urine samples for improved systematic toxicological analysis of drug-facilitated sexual assault cases.

Drug-facilitated sexual assault (DFSA) is characterized by victim incapacitation due to intoxicating substances. Detection of single drug exposure from DFSA requires a systematic toxicological analysis strategy including sensitive methods covering a broad spectrum of substances. The aim of this study was to develop and validate an UHPLC-MS/MS screening method for analysis of samples from DFSA cases and incorporate an automated enzymatic pre-treatment of urine samples into a robotic sample preparation for an efficient laboratory workflow. The screening method included 144 drugs of abuse, pharmaceuticals, and metabolites relevant to DFSA. The use of a recombinant enzyme showed an efficient glucuronide hydrolysis with an average parent drug recovery of 97%. Investigation of matrix effect showed no pronounced ion enhancement or suppression for most analytes (96%), and extraction recovery was above 80% for 97% of analytes. Process efficiency ranged from 50% to 138% for most analytes. The LODs ranged from 0.0001 mg/L to 2 mg/L depending on analyte, and most analytes met the SOFT recommended minimum performance limits. The validated method was applied to authentic suspected DFSA cases (n = 38). Results showed that drugs of abuse, benzodiazepines, and antidepressants were most commonly found in suspected DFSA cases. Incorporation of an automated enzymatic hydrolysis step during sample preparation enables a fast and simple workflow for simultaneous analysis of blood and urine samples for an improved systematic toxicological analysis strategy for DFSA cases.

Uncovering forensic evidence of drug-facilitated sexual assault: Toxicological findings from Eastern Denmark from 2015-2022.

In drug-facilitated sexual assault (DFSA), the victim is unable to provide consent or resists sexual activity due to substance intoxication by voluntary or covert consumption. Obtaining forensic evidence of the assault is challenged by rapid drug metabolism and late sample collection. The objective of this study was to present toxicological findings and associated demographics from police reported sexual assault cases in Eastern Denmark from 2015 to 2022. A total of 369 sexual assault cases were submitted for analysis and a subgroup of 268 cases were categorized as suspected DFSA cases. The majority of the total sexual assault victims were women at the age 15-25 and the perpetrators were often unknown or an acquaintance. Time from assault to sample collection was slightly longer for suspected DFSA cases (12-24 h) compared to non-DFSA (<12 h). Positive toxicology was observed in 86 % of cases and the most common drug groups included alcohol (45 %), drugs of abuse (38 %), antidepressants (14 %), antihistamines (12 %), and benzodiazepines (11 %). Hypnotics were detected to a smaller extent (7 %). A total of 77 drugs were detected and the most commonly observed were cocaine, tetrahydrocannabinol (THC), cetirizine, amphetamine, diazepam and sertraline. The high level of observed alcohol and drugs of abuse indicated that most DFSA cases in Eastern Denmark were of an opportunistic approach rather than proactive.

Exploring Enzymatic Hydrolysis of Urine Samples for Investigation of Drugs Associated with Drug-Facilitated Sexual Assault.

Analyzing urine is common in drug-facilitated sexual assault cases if the analysis of blood is not optimal. The efficient enzymatic pretreatment of urine is important for cleaving glucuronides and improving the detection of the parent drug. The aim was to investigate the efficiency of three ß-glucuronidases on eleven glucuronides relevant to DFSA at different incubation periods and temperatures. Human drug-free urine was fortified with 11 glucuronides, hydrolyzed with either ß-glucuronidase/arylsulfatase (Helix Pomatia), recombinant ß-glucuronidase B-One™ or recombinant ß-glucuronidase BGTurbo™ and incubated for 5, 10, 60 min, 18 h and 24 h at 20 °C/40 °C/55 °C before UHPLC-MS/MS analysis. The stability of 141 drugs and metabolites relevant to DFSA was investigated by incubating fortified urine under the same hydrolysis conditions. B-One™ showed efficient hydrolysis (>90%) of most glucuronides in 5 min at all temperatures, while BGTurbo™ showed a similar efficiency (>90%), but the optimal temperature (20-55 °C) and incubation time (5-60 min) varied among analytes. The ß-glucuronidase/arylsulfatase had the lowest efficiency and required the longest incubation (24 h) at 40-55 °C. The stability of 99% of 141 drugs and metabolites was not affected by incubation at 20-55 °C for 24 h. Recombinant enzymes show promising results for the simple and efficient hydrolysis of a broad panel of glucuronides relevant for DFSA.

Enzymatic Hydrolysis of a Collagen Hydrolysate Enhances Postprandial Absorption Rate-A Randomized Controlled Trial.

Collagen is characterized by its high content of glycine, proline and hydroxyproline, and is found to exert beneficial effects on joint pain related to activity and osteoarthritis. However, to exert any beneficial effects it is essential that collagen is optimally absorbed. This study aimed to investigate the postprandial absorption of collagen and elucidate the impact of an exogenous enzymatic hydrolysis on absorption rate and bioavailability. A randomized, blinded, cross-over study was conducted where ten healthy male subjects received either 35 g enzymatically hydrolyzed collagen protein (EHC), 35 g non-enzymatically hydrolyzed collagen protein (NC) or placebo (250 mL water) on three nonconsecutive days. Blood samples were drawn before, and up to 240 min following, ingestion and the blood metabolome was characterized by nuclear magnetic resonance (NMR)-based metabolomics. A significant increase in the plasma concentration of nearly all amino acids (AAs) was observed over a 240 min period for both EHC and NC. In addition, the absorption rate and bioavailability of glycine, proline and hydroxyproline were significantly higher for EHC (p < 0.05). In conclusion, ingestion of collagen hydrolysates increases postprandial plasma concentrations of AAs over a period of 240 min, and an enzymatic hydrolysis increases the absorption rate and bioavailability of the collagen-rich AAs glycine, proline and hydroxyproline.