Drug-facilitated sexual assault in youth nightlife contexts in Spain.

OBJECTIVES: This study aimed to provide epidemiological information on drug-facilitated sexual assault in Spanish youth partying, with a focus on prevalence rates and associated sociodemographic factors. STUDY DESIGN: Cross-sectional study. METHODS: Quota sampling was used to recruit 1601 young people aged 18-35 years in Spain from a digital panel. A validated questionnaire on drug-facilitated sexual assault was used to assess five types of lifetime victimisation experiences while partying. Chi-square and the exact Fisher tests were used to describe the prevalence of victimisation, drug use patterns, and perpetrator profiles. Generalised ordered logistic regression was used to explore factors associated with victimisation, analysed by gender. RESULTS: Half of young women and one-quarter of young men had experienced drug-facilitated sexual assault in their lifetime. Female victimisation due to touching and kissing was notably high, whereas men comprised almost half of the victims of more invasive DFSA experiences involving masturbation, penetration, and oral sex. Opportunism prevailed as the assault tactic, consisting of taking advantage of the victims' incapacity derived from voluntary alcohol use. Among women, risk of victimisation was associated with a lower education level, foreign-born status, and being non-heterosexual. Male victimisation risk was highest among non-heterosexual men. CONCLUSIONS: Drug-facilitated sexual violence in youth nightlife contexts is a serious public health issue in Spain, which requires urgent action. Most assaults involve taking advantage of victims who are incapacitated by the effects of voluntary alcohol consumption. This sexual violence primarily affects women with lower educational levels or those who are foreign-born and non-heterosexual men and women.

Victims of drug facilitated sexual assault aged 13-24: a cross sectional study on the pool of users of a sexual violence relief centre in Northern Italy.

This cross-sectional study aimed to assess the association between drugs and alcohol intake and sexual abuse in adolescents, otherwise defined as Drug Facilitated Sexual Assault (DFSA). We considered the survivors who accessed care at the Centre "Soccorso Violenza Sessuale" (SVS - Sexual Violence Relief Centre) in Turin (Italy), between May 2003 and May 2022. We found that 973 patients aged 13-24 among which 228 were victims of DFSA. Epidemiological and anamnestic aspects of the episode of sexual violence were examined, with a specific focus on investigating the alcohol and/or drug intake as reported by the victim, along with the results of the toxicological analysis. the study further accounts for the variations caused by the COVID-19 pandemic on DFSA-related accesses. Our findings show that 23% of adolescents accessing care at SVS were subjected to DFSA. Six out ten adolescents knew their aggressor, at times a partner (10%) oran acquaintance (43%). In 12% of cases violence was perpetrated by a group of people (12%). Almost 90% of young victims described alcohol consumption, while 37% reported drug use at the time of the assault. Alcohol taken alone or in combination with other substances was the most detected drug in our sample throughout the period considered. Given the large use of psychoactive substances among adolescents, it is imperative to implement harm reduction strategies alongside educational activities aimed at fostering awareness about consent. Health personnel should be trained to manage the needs of victims of DFSA clinically and forensically.

Pro-active drug-facilitated crimes (DFC): a study in the Department of Forensic Medicine of Paris, France.

INTRODUCTION: Proactive drug facilitated crime (DFC) is the administration of psychoactive substances (PAS) for criminal purposes without the victim's knowledge or by force. In Paris, France, patients who report suspected proactive DFC to the police are examined at the Department of Forensic Medicine (DFM) of the Hôtel-Dieu Hospital. Preventively blood and urine samples are collected but not systematically analyzed by the judicial authority. We aimed to assess the proportion of probable proactive DFC in patients examined at the Hôtel-Dieu DFM following a police report for suspected proactive DFC. METHOD: Blood and urine samples were collected from 100 patients. Toxicological analyses were performed by the toxicology laboratory of the Lariboisière Hospital. The results were correlated with the clinical data collected at the initial and follow-up consultations. RESULTS: At least one PAS was detected in 86% of the cases (voluntary or involuntary intake). After correlation with clinical data, 32% of the cases were classified as probable proactive DFC. In these cases, 49% of the substances identified were illicit substances (amphetamines, MDMA, etc.); 16% were benzodiazepines and related substances; 16% were antihistamines and sedatives; 14% were opioids; and 5% were antidepressants and anti-epileptics. In 90% of the cases, patients reported a voluntary ethanol consumption in the hours prior to the suspected proactive DFC. CONCLUSION: Toxicological analyses revealed a high proportion of both probable proactive DFC and probable opportunistic DFC. Our results indicate the need to perform systematical toxicological analysis in cases of suspected DFC.

Drug-facilitated sexual assault followed by femicidal chloroform poisoning and suffocation: A case-report of criminal responsibility.

INTRODUCTION: Criminal responsibility evaluation represents one of the most controversial and debated issues in forensic psychiatry. Although clear procedures have been recommended, little research exists on decision-making process by forensic psychiatrists. We present a case assessing the criminal responsibility of a murderer who committed femicide as a result of chloroform poisoning and suffocation after a drug-facilitated sexual assault. MATERIALS AND METHODS: A.S., a 30-year-old female, was found dead in the home of S.P., a 50-year-old male. S.P. recounted killing A.S. by forced inhalation of chloroform, when the woman had experienced sensory clouding following unintentional ingestion of Zolpidem, a hypnotic agent. A multidisciplinary approach was taken to resolve the case. Autopsy, histological, genetic, and toxicological examinations were performed by a forensic pathologist, while a digital forensic examiner analysed electronic devices. A pool of three forensic psychiatrists and two psychologists was asked to assess the mental state of S.P. at the time of the crime. RESULTS AND CONCLUSIONS: The cause of death of A.S. was identified as a lethal chloroform intoxication in altered consciousness caused by Zolpidem, while homicidal suffocation was also described. Mobile forensics demonstrated that S.P. had videotaped the crime scene, clearly revealing that A.S. had been sexually assaulted by S.P. before dying. Criminal responsibility of S.P. was evaluated through various psychological tests and seven interviews with the accused, each lasting an average of 180 min. Specialists concluded that S.P. could not be exempted from being responsible for the homicide.

The Impact On Eye Movement And Desensitization Reprocessing Of Incomplete Memory In A Drug-Facilitated Rape: A Single Case Study.

In a drug-facilitated sexual assault (DFSA), the person's level of intoxication may result in incomplete memory. This paper describes eye movement and desensitization reprocessing (EMDR) with client-centered adaptations to address an incomplete trauma memory in a 26-year-old woman. The client was experiencing PTSD, characterized by nightmares and derealization. Therapy followed standard EMDR procedures with three minor modifications to help the client maintain current awareness. Although the memory remained incomplete, the client-centered adaptations promoted working through of the clients' trauma responses (e.g. disorientation, physical sensations) and a sense of competence and self-confidence were restored. At the end of reprocessing, and at follow-up, the client was no longer experiencing nightmares or derealization and her wellbeing had improved.

Combining surface-enhanced Raman spectroscopy and paper spray mass spectrometry for the identification and confirmation of psychotropic substances in alcoholic beverages.

Criminal practices in which an individual becomes vulnerable and prone to sexual assault after ingesting drinks spiked with doping substances have become a social concern globally. As forensic protocols require a multi-tiered strategy for chemical evidentiary analysis, the backlog of evidence has become a significant problem in the community. Herein, a fast, sensible, and complementary dual analytical methodology was developed using a single commercial paper substrate for surface-enhanced Raman spectroscopy (SERS) and paper spray mass spectrometry (PS-MS) analysis to identify psychotropic substances added to alcoholic beverages irrefutably. To study and investigate this criminal practice, pharmaceutical formulations containing distinct psychotropic substances (zolpidem, clonazepam, diazepam, and ketamine) were added to drinks typically consumed at parties and festivals (Pilsen beer, açaí Catuaba®, gin tonic, and vodka mixed with Coca-Cola Zero®). A simple liquid-liquid extraction with a low-temperature partitioning (LLE-LTP) procedure was applied to the drinks and effectively minimized matrix effects. As a preliminary analysis, SERS spectra combined with Hierarchical Clustering Analysis (HCA) provided sufficient information to investigate the samples further. The presence of the protonated species for the psychotropic substances in the spiked drinks was readily verified in the mass spectra and confirmed by tandem mass spectrometry. Finally, the results demonstrate the potential of this methodology to be easily implemented into the routine of forensic laboratories and to be further employed at harm reduction tends at parties and festivals to detect contaminated beverages promptly and irrefutably as an efficient tool to prevent such crimes.

Development and validation of presumptive spot test for the identification of z-drugs used in drug-facilitated crimes.

The significance and desire for preliminary testing approaches that are straightforward, quick, selective, affordable, and practical for use in the field are highlighted by the increasing enormous amounts of potentially illegal samples being seized worldwide. The "z-drugs," which include zolpidem, zopiclone, and eszopiclone, are non-benzodiazepine medications used to treat insomnia. z-drugs are short-term solutions for sleeplessness and anxiety but have a long history of abuse and misuse. The extensive list is primarily utilized for drug-facilitated crimes and drug dependence. The presumptive color spot test for z-drugs, such as zolpidem, zopiclone, and eszopiclone, has been created and validated in this study. In the preliminary identification of zolpidem, zopiclone, and eszopiclone, no color spot test has been documented as per the literature. The color spot test is the most essential and routinely used technique for identifying any unknown sample substance. The color test method was proven to provide high-quality, dependable presumptive test findings and satisfy standards for preliminary screening usage. Validation experiments demonstrate that, at room temperature, the color change is specific to the zolpidem, zopiclone, and eszopiclone classes and unaffected by the common cutting agent's presence. It was discovered that 5, 10, and 6 ppm were the operational limit of detection of the sample present against the reagents 0.1% diphenyl carbazone, aqueous potassium iodoplatinate, and modified cobalt thiocyanate reagent, respectively. The color test is immediate and validated with other substances of a similar category and 10 ppm was the operational limit of detection.

On-site drug detection coasters: An inadequate tool to screen for GHB and ketamine in beverages.

With drug facilitated sexual assault (DFSA) being alleged in 15-20 % of sexual assault cases, drink spiking is a serious concern for several people, casting doubts over the expected safety at events in public spaces. On-site drug testing material is often touted as a solution, allowing attendees to test their drinks for the presence of certain so-called "date-rape drugs". In this manuscript, we aim to evaluate the efficiency of such a coaster device, manufactured by Drink Safe Technologies (Tallahassee, Florida, United States) and sold by Alco Prevention Canada (Laval, Québec, Canada), in detecting drink spiking by GHB and ketamine. From the onset, several generic arguments call into question the practicality of the test: limitations set by the manufacturer on drinks that can be tested, cost, waiting time, interpretation in suboptimal lighting and elevated limits of detection (LODs) compared to a standard recreational or impairing dose. More importantly, the test simply isn't effective at detecting the targeted drugs. The GHB test reagent was identified as bromocresol green using surface-enhanced Raman spectroscopy (SERS). Therefore, it does not detect GHB, but any matrix with a pH higher than 5.5. The ketamine test reagent was identified as cobalt thiocyanate, a non-specific chemical commonly used in colorimetric drug testing. Performance tests were carried with more than 22 drug-free and drug-spiked (≥125 % of the LOD) matrices, including solvent solutions (water, methanol), fixed pH solutions, and an array of popular drinks (including wine, beer, cocktails and spirits). While specificity in drug-free drinks was 100 % for both GHB and ketamine, provided that the manufacturer's limitations on drinks were respected, sensitivity in drug spiked drinks (at 150 % of the LOD) was 0 % for ketamine and between 31 % and 69 % for GHB, depending on whether one classifies inconclusive results as negatives or positives. We conclude that these coasters are an inadequate tool to screen for GHB and ketamine in beverages.

Toxicological analysis in victims of sexual and domestic violence: A retrospective study of a 3-year period (2018-2020).

This manuscript presents an epidemiological investigation carried out on abuse victims who accessed the Sexual and Domestic Violence Service (SVS&D) of IRCCS Ca' Granda in Milan, Italy. The focal point of this research was the detection of alcohol, prescription medications, and illicit substances in victims who solicited help from the SVS&D center between 2018 and 2020. Over this three-year span, biological samples of blood and urine were procured from 207 victims, out of a patient pool of 2470. All collected samples were analyzed via High Performance Liquid Chromatography - Tandem Mass Spectrometry (HPLC-MS/MS) and Gas Chromatography - Mass Spectrometry (GC-MS). Toxicological examination results demonstrated that 43% of the cases tested positive for substances in 2018, 39% in 2019 and 60% of the cases in 2020. Overall, 45% of the victims tested resulted positive to some substance over a 3-year period, equivalent to 3.6% of the overall cases (2470 victims). Substances of toxicological interest were detected in 104 samples (out of 377, corresponding to 27.6%) belonging to 94 patients. The most detected classes of drugs were stimulants, antidepressants, benzodiazepines and antipsychotics. Moreover, BAC (Blood Alcohol Concentration) indicated positivity in 25 cases (out of 184 cases analyzed - 14% of positive cases). Based on this study's findings, we recommend broadening the range of substances evaluated in drug-facilitated sexual assaults and establishing standardized protocols for both national and international implementation. Implementing procedures would significantly enhance forensic support provided to victims of abuse seeking healthcare services post-incident.

Effect of Repeated Administration of ɣ-Valerolactone (GVL) and GHB in the Mouse: Neuroadaptive Changes of the GHB and GABAergic System.

BACKGROUND: Gamma-hydroxybutyric acid (GHB) at low dosages has anxiolytic effects and promotes REM sleep and low-wave deep sleep. In the U.S., the legal form of GHB is prescribed to adults suffering from narcolepsy-associated cataplexy; the sodium salt of GHB is reserved for alcohol-addiction treatment. GHB is also a molecule of abuse and recreational use, it is a controlled substance in several countries, so gamma-valerolactone (GVL) has frequently been used as a legal substitute for it. GHB's abuse profile is most likely attributable to its anxiolytic, hypnotic, and euphoric properties, as well as its widespread availability and inexpensive/low cost on the illicit market. METHODS: Our study is focused on evaluating the potential effects on the mouse brain after repeated/prolonged administration of GHB and GVL at a pharmacologically active dose (100 mg/kg) through behavioral study and immunohistochemical analysis using the markers tetraspanin 17 (TSPAN17), aldehyde dehydrogenase 5 (ALDH5A1), Gamma-aminobutyric acid type A receptor (GABA-A), and Gamma-aminobutyric acid type B receptor (GABA-B). RESULTS: Our findings revealed that prolonged administration of GHB and GVL at a pharmacologically active dose (100 mg/kg) can have effects on a component of the mouse brain, the intensity of which can be assessed using immunohistochemistry. The findings revealed that long-term GHB administration causes a significant plastic alteration of the GHB signaling system, with downregulation of the putative binding site (TSPAN17) and overexpression of ALDH5A1, especially in hippocampal neurons. Our findings further revealed that GABA-A and GABA-B receptors are downregulated in these brain locations, resulting in a greater decrease in GABA-B expression. CONCLUSIONS: The goal of this study, from the point of view of forensic pathology, is to provide a new methodological strategy for better understanding the properties of this controversial substance, which could help us better grasp the unknown mechanism underlying its abuse profile.

Ultrasound assisted membrane-assisted solvent extraction for the simultaneous assessment of some drugs involved in drug-facilitated sexual assaults by liquid chromatography-tandem mass spectrometry.

A simple and highly efficient ultrasound assisted membrane-assisted solvent extraction (MASE) pre-treatment method for urine has been developed and validated for the simultaneous determination of twenty-two drugs involved in drug-facilitated sexual assaults (DFSAs) by liquid chromatography-tandem mass spectrometry. MASE was performed with 4.0 mL of urine (pH adjusted at 12), 400 µL of hexane as an organic solvent inside the polypropylene membrane, and ultrasonication (45 kHz, 120 W) for 10 min. A pre-concentration factor of 40 was achieved after evaporation (N2 stream) and re-dissolution in 100 µL of methanol. Analytes were separated using a Zorbax Eclipse Plus C18 column under gradient elution with aqueous 10 mM NH4HCO3 (pH 8.0) and methanol as mobile phases. Matrix-matched calibrations allowed the assessment of DFSA drugs of quite different octanol-water partition coefficients (Ko/w), from 1.32 101 for pregabalin to 2.45 105 for clomipramine (Log P values from 1.12 (pregabalin) to 5.39 (clomipramine)). The limit of detection (LOD) was between 0.0075 to 0.37 µg L-1, with analytical recoveries ranging from 73 to 103%, and relative standard deviations (RSDs) within the 2-20% range. The applicability of the method was demonstrated after analysing urine samples under forensic investigation.

Drug-Facilitated Sexual Assault: A Systematic Review.

Since the last two decades, the scientific community has made an effort to analyze drug-facilitated sexual assault (DFSA). However, a lack of understanding remains about the DFSA problem, particularly concerning the opportunistic variant. Facing this situation, a systematic review of the term DFSA is carried out from its first appearance in the scientific databases consulted (Web of Science, Scopus, and PubMed) to the current day. The search resulted in 773 publications, reduced to a final study sample composed of 19 articles. Eligible studies for this review had to meet certain inclusion criteria, in addition to providing information on DFSA prevalence, DFSA victim profile, DFSA offender profile, involved drugs, or contextual information about the assault. The results demonstrated that the assailants are men, who mostly know victims before the assault. The victims are young women under 30 years old. Alcohol is the drug involved in most DFSA cases, prevailing a voluntary use. Most assaults occur in private spaces, particularly the aggressors' own homes. Furthermore, there is a detected need for a standard definition of DFSA to allow the different actors involved in dealing with sexual violence to work effectively together, and, at the same time, it is detected that the available studies overrepresent proactive DFSA and underestimate opportunism, the most common modus operandi involved in DFSA cases.

Application of Paper-Based Microfluidic Analytical Devices (µPAD) in Forensic and Clinical Toxicology: A Review.

The need for providing rapid and, possibly, on-the-spot analytical results in the case of intoxication has prompted researchers to develop rapid, sensitive, and cost-effective methods and analytical devices suitable for use in nonspecialized laboratories and at the point of need (PON). In recent years, the technology of paper-based microfluidic analytical devices (µPADs) has undergone rapid development and now provides a feasible, low-cost alternative to traditional rapid tests for detecting harmful compounds. In fact, µPADs have been developed to detect toxic molecules (arsenic, cyanide, ethanol, and nitrite), drugs, and drugs of abuse (benzodiazepines, cathinones, cocaine, fentanyl, ketamine, MDMA, morphine, synthetic cannabinoids, tetrahydrocannabinol, and xylazine), and also psychoactive substances used for drug-facilitated crimes (flunitrazepam, gamma-hydroxybutyric acid (GHB), ketamine, metamizole, midazolam, and scopolamine). The present report critically evaluates the recent developments in paper-based devices, particularly in detection methods, and how these new analytical tools have been tested in forensic and clinical toxicology, also including future perspectives on their application, such as multisensing paper-based devices, microfluidic paper-based separation, and wearable paper-based sensors.

Examination of acetone findings in suspected drug-facilitated sexual assaults: A case series.

Acetone presence in human biological specimens can result from exogenous administration or endogenous production, resulting from diabetes, dietary composition, alcoholism, and stress response. Victims of drug-facilitated sexual assaults (DFSA) are understood to experience enhanced stress. At the Harris County Institute of Forensic Sciences (HCIFS), DFSA drug testing includes analysis of volatile compounds, ethanol, methanol, isopropanol, and acetone, by headspace gas chromatography/flame ionization detection. The prevalence of acetone-positive specimens in DFSA casework has been observed to exceed that of other human performance case types. In this report, DFSA cases received between 2019 and 2021 (n = 393) were reviewed and 41 acetone-positive cases were detailed. Overall, nearly 11% of the DFSA cases had acetone-positive blood or urine specimens, where 3% identified acetone only, 6% identified acetone and other drug(s), and 2% identified acetone, ethanol, and other drug(s). Acetone concentrations ranged from 0.010 to 0.147 g/100 mL in urine. Other drugs such as nor-carboxy-Δ9 -tetrahydrocannabinol, amphetamine, methamphetamine, ethanol, and benzoylecgonine were commonly detected. Elevated stress response encountered during DFSAs may facilitate the mechanism behind enhanced acetone production leading to increased identification. Limited availability of victim medical history precludes understanding the contribution of other disease states or physiological conditions. Nonetheless, the identification of acetone in DFSA specimens supports its potential as a biomarker of trauma in forensic toxicology casework and warrants future research within the community.

Gamma-hydroxybutyrate (GHB), 1,4-butanediol (1,4BD), and gamma-butyrolactone (GBL) intoxication: A state-of-the-art review.

γ-hydroxybutyrate (GHB) is synthesized endogenously from γ-aminobutyric acid (GABA) or exogenously from 1,4-butanediol (butane-1,4-diol; 1,4-BD) or γ-butyrolactone (GBL). GBL, and 1,4-BD are rapidly converted to GHB. The gastric absorption time, volume of distribution, and half-life of GHB are between 5 and 45 min, 0.49 ± 0.9 L/kg, and between 20 and 60 min, respectively. GHB and its analogues have a dose-dependent effect on the activation of GHB receptor, GABA-B, and GABA localized to the central nervous system. After ingestion, most patients present transient neurological disorders (lethal dose: 60 mg/kg). Chronic GHB consumption is associated with disorders of use and a withdrawal syndrome when the consumption is discontinued. GHB, GBL, and 1,4-BD are classified as narcotics but only the use of GHB is controlled internationally. They are used for drug facilitated (sexual) assault, recreational purposes, slamsex, and chemsex. To confirm an exogenous intake or administration of GHB, GBL, or 1-4-BD, the pre-analytical conservation is crucial. The antemortem cutoff doses for detection are 5 and 5-15 mg/L, with detection windows of 6 and 10 h in the blood and urine, respectively Control of GHB is essential to limit the number of users, abuse, associated risks, and death related to their consumption.

Analysis of Forty-Two Psychoactive Substances in a Single Hair by Micro-Segmental Technique.

OBJECTIVES: To establish an LC-MS/MS method based on single hair micro-segmental technique, and verify the detection of 42 psychoactive substances in 0.4 mm hair segments. METHODS: Each piece of single hair was cut into 0.4 mm segments and extracted by sonication and the segments were immersed in dithiothreitol-containing extraction medium. Mobile phase A was the aqueous solution containing 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile. Mobile phase B was acetonitrile. An electrospray ionization source in positive ion mode was used for data acquisition in multiple reaction monitoring (MRM) mode. RESULTS: The 42 psychoactive substances in hair had a good linear relationship within their respective linear ranges (r>0.99), the limits of detection were 0.2-10 pg/mm, the limits of quantification were 0.5-20 pg/mm, the intra-day and inter-day precisions were 1.5%-12.7%, the intra-day and inter-day accuracies were 86.5%-109.2%, the recovery rates were 68.1%-98.2%, and the matrix effects were 71.3%-111.7%. The method was applied to hair samples collected from one volunteer at 28 d after a single dose of zolpidem, with zolpidem detected in 5 hairs was 1.08-1.60 cm near the root tip, and the concentration range was 0.62-20.5 pg/mm. CONCLUSIONS: The micro-segmental technique of single hair analysis can be applied to the investigation of drug-facilitated sexual assault cases.

The Use of Volatile Substances in Drug-Facilitated Sexual Assault: A Systematic Review.

Drug-facilitated sexual assault (DFSA) is a significant crime that is increasing in incidence. The employment of volatile substances such as chloroform and aromatic petroleum hydrocarbons in DFSAs is quite an unusual choice. The objective of this review is to explore the use of volatile substances in DFSAs. Using the PubMed database, a systematic review of the literature was conducted. Thereafter, citation searching was carried out within the included studies from the primary search. A total of five studies were eligible for inclusion. Chloroform was the drug used in the DFSA in three of the included studies, and aromatic hydrocarbons in the remaining two. Two of the offenders who employed chloroform possessed a unique way to access the drug: their degrees. The evidence found in the DFSA cases included a chloroform-scented scarf and a solvent-immersed cloth. Headspace gas chromatography-mass spectrometry, liquid chromatography-electrospray coupled tandem mass spectrometry, toxicology assays of blood and urine, and solvent or hydrocarbon gas chromatography flame-ionization detection followed by gas chromatography-mass spectrometry were among the investigations performed to detect the volatile substances. The implementation of stricter regulations on chloroform for employees in chemical industries and laboratories is recommended. In cases where the autopsy is unclear and there are conspicuous facial and airway injuries, it is prudent to collect an early sample for volatile substance analysis.

Green nanostructured liquids for the analysis of urine in drug-facilitated sexual assault cases.

In this work, we optimize and validate a simple, time-saving, and environmentally friendly sample preparation method based on supramolecular solvents (SUPRAS), green nanostructured liquids, for the extraction of selected drug-facilitated sexual assault (DFSA) substances from human urine. The methodology was fast and simple (stirring, centrifugation, and dilution). Cubosomic SUPRAS were formed by the addition of 1,2-hexanediol (200 µL) to 1.0 mL of human urine containing 1 M Na2SO4. SUPRAS extracts were analyzed by LC-MS/MS. The method was fully validated for 23 DFSA compounds including 10 benzodiazepines, 1 z-hypnotic drug, 5 amphetamine derivatives, 3 cocaine metabolites, and 4 miscellaneous compounds. Extraction efficiency varied between 79 and 119%, and matrix effects were acceptable (-14.3/+21.5) for 87% of the compounds. Method detection and quantification limits ranged from 0.003 to 0.75 ng/mL and from 0.01 to 2.50 ng/mL, respectively. These values were low enough for the established minimum required performance limits (MRPL) of these substances. This simple and green method has a great potential to be implemented for the monitoring of illegal drugs involved in DFSA cases by forensic laboratories.

Two DFSA cases involving midazolam clarified by the micro-segmental hair analyses.

PURPOSE: In this study, an analytical procedure to identify trace amounts of drug in hair based on micro-segmental hair analysis was presented. The method also can be used to estimate the time of drug ingestion at daily precision by cutting a single hair into sub-millimeter segments which correspond to daily hair growth. METHODS: A method was established for efficient extraction of midazolam, one of the most frequently detected compound in drug-facilitated sexual assault (DFSA) cases, from each 0.4-mm hair segment and validated by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Moreover, two DFSA cases were used to compare the micro-segmental hair analysis with the 1- cm segmental analysis method. RESULTS: The validation showed a lower limit of quantification of 0.5 pg/mm for midazolam, with intraday and interday accuracies (bias) from - 5.2 to 0.9%. The micro-segmental hair analysis method was applied to proximal 1-cm hair segment including hair bulbs in two DFSA cases. The micro-segmental hair analysis results in case 1 showed midazolam in the S15-S17 (5.6-6.8 mm from hair bulb) in a concentration range from 0.5 to 0.9 pg/mm, and the concentrations of midazolam in all hair micro-segments (0-1 cm from the scalp) in case 2 were from 0.5 to 2.0 pg/mm. CONCLUSIONS: Comparison with the conventional method revealed that micro-segmental hair analysis may enhance the utility of hair drug testing and strengthen probative force in DFSA cases.

Time course of estazolam in single-strand hair based on micro-segmental analysis after controlled oral administration.

The mechanism of estazolam incorporation into hair was investigated by studying the time course of estazolam along single-strand hair after two oral administration of estazolam at 28 days interval. Estazolam in single hair segments 0.4 mm in length was verified and quantified by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). The distributions of estazolam within a strand of hair (collected at 12 h, 28 days, and 56 days post-administration) were visualized by micro-segmental analysis. The highest estazolam concentration (1.5-9.9 pg/mm) was detected in the hair bulb region (S1), and it then decreased through the hair shaft to the distal end, with a small fluctuation (0.3-3 pg/mm) near the junction of the hair roots and shafts (S4-S7) 12 h after drug intake. These findings suggested that the incorporation of estazolam occurred in two regions, mainly in the hair bulb and to a lesser extent in the upper dermis zone. Models using internal temporal markers (TIMs) and temporal intervals (TIs) were constructed to estimate the day of estazolam ingestion. The estimation accuracy was within an average error of 1.7 mm and 3.0 mm between the calculated and actual positions, based on the TIMs and TIs 56 days after estazolam intake. These findings can help in further elucidation of the drug incorporation mechanism, which is crucial for interpreting hair analysis results used to reveal individual drug-use history.