Monitoring of the interconversion of gamma‐butyrolactone (GBL) to gamma hydroxybutyric acid (GHB) by Raman spectroscopy

Gamma‐hydroxybutyric acid (GHB) is a drug‐of‐abuse that has recently become associated with drug‐facilitated sexual assault, known as date rape. For this reason the drug is commonly found ‘spiked’ in alcoholic beverages. When GHB is in solution it may undergo conversion into the corresponding lactone, Gamma‐butyrolactone (GBL). Studies have been carried out to determine the detection limits of GHB and GBL in various solutions by Raman spectroscopy and to monitor the interconversion of GHB and GBL in solution with different pH conditions and temperature. In this study, a portable Raman spectrometer was used to study the interconversion of GHB and GBL in water and ethanol solutions as a function of pH, time, and temperature. The aim of this was to determine the optimum pH range for conversion in order to relate this to the pH ranges that the drug is likely to be subjected to, first in spiked beverages and secondly after ingestion in the digestive system. The aim was also to identify a timescale for this conversion in relation to possible scenarios, for example if GHB takes a number of hours to convert to GBL, it is likely for the beverage to be ingested before esterification can take place. GHB and GBL were then spiked into a selection of beverages of known pH in order to study the stability of GHB and GBL in real systems. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of the date‐rape drug GHB and its precursor GBL by Raman spectroscopy

Gamma hydroxybutyric acid (GHB), also known as ‘liquid ecstasy’, has recently become associated with drug‐facilitated sexual assaults, known colloquially as ‘date rape’, due to the ability of the drug to cause loss of consciousness. The drug is commonly found ‘spiked’ into alcoholic beverages, as alcohol increases its sedative effects. Gamma hydroxybutyric acid and the corresponding lactone gamma‐butyrolactone (GBL) will reach an equilibrium in solution which favours the lactone in basic conditions and GHB in acidic conditions (less than pH 4). Therefore, we have studied both GHB and GBL, as a mildly acidic beverage ‘spiked’ with GHB will contain both GHB and GBL. We report the analysis of GHB as a sodium salt and GBL, its precursor, using bench‐top and portable Raman spectroscopy. It has been demonstrated that we are able to detect GHB and GBL in a variety of containers including colourless and amber glass vials, plastic vials and polythene bags. We have also demonstrated the ability to detect both GBL and GHB in a range of liquid matrices simulating ‘spiked’ beverages. Copyright © 2009 John Wiley & Sons, Ltd.     

Systematic review: the effects of carbonated beverages on gastro‐oesophageal reflux disease

Aliment Pharmacol Ther 31 , 607–614

Summary

Background Carbonated beverages have unique properties that may potentially exacerbate gastro‐oesophageal reflux disease (GERD), such as high acidity and carbonation. Cessation of carbonated beverage consumption is commonly recommended as part of lifestyle modifications for patients with GERD. Aims To evaluate the relationship of carbonated beverages with oesophageal pH, oesophageal motility, oesophageal damage, GERD symptoms and GERD complications. Methods A systematic review. Results Carbonated beverage consumption results in a very short decline in intra‐oesophageal pH. In addition, carbonated beverages may lead to a transient reduction in lower oesophageal sphincter basal pressure. There is no evidence that carbonated beverages directly cause oesophageal damage. Carbonated beverages have not been consistently shown to cause GERD‐related symptoms. Furthermore, there is no evidence that these popular drinks lead to GERD complications or oesophageal cancer. Conclusions Based on the currently available literature, it appears that there is no direct evidence that carbonated beverages promote or exacerbate GERD.     

A study of the effectiveness of commercially available drink test coasters for the detection of "date rape" drugs in beverages

The use of illicit substances for the purpose of drug-facilitated sexual assault (DFSA) poses a significant problem. There has been an increase in public awareness of this problem, and a recent invention in the form of a drink coaster claims to detect whether or not a beverage has been spiked with a so-called date rape drug. A person is instructed to place a drop of the suspect beverage onto two spots of the test, smear gently, and wait until dry. If either spot turns to a darker blue color, then a possible date rape drug has been detected by the coaster test. In an effort to determine the effectiveness of the coasters, various drugs that have been associated with drug-facilitated sexual assault were tested at different concentrations in a variety of common alcoholic and non-alcoholic beverages. It was found that although the coasters do detect the presence of GHB and ketamine, two drugs that have been associated with DFSA, there are limiting factors such as the high concentration of the drugs required, hindrance of the reaction due to beverage matrix, and extensive time requirements for ketamine analysis.     

Detecting ketamine in beverage residues: Application in date rape detection

Ketamine can be used to facilitate date-rape when unknowingly spiked into a victim's beverage. If a biological sample is not available from the victim, the beverage container might be the only remaining source of forensic evidence. We present a rapid, simple analysis method for the detection of ketamine in wet or dry beverage residues based on liquid chromatography-mass spectrometry (LC-MS). Wet residues consist of the final few drops (<1 ml) in a container while dry residues are the remains once all liquid has evaporated. By using LC-MS, which readily handles aqueous samples, often no derivatization or sample extraction is needed, thus reducing analysis time and lab technician involvement. Tandem mass spectrometry (MS/MS) provides an enhancement in both selectivity and sensitivity. We have studied a range of beverages and determined limits of detection between 1.2 × 10-3 and 1.3 × 10-4 mg/ml, compared to 0.21-0.85 mg/ml used in most date-rape scenarios. This paper represents the first published report of using LC-MS/MS for the analysis of beverage residues for the presence of a date-rape drug. This method could replace the current gas chromatography-mass spectrometry (GC-MS) methods and provide a faster, more selective method for the analysis of date-rape drugs, requiring virtually no sample preparation.     

The effects of beverage type on homicide rates in Russia, 1970-2005

Introduction and Aims. Previous research from Western Europe and North America has suggested that consuming different types of alcoholic beverage may have differing effects on homicide rates both within and between countries. The aim of this study was to examine the relation between the consumption of different beverage types and homicide rates in Russia across the later‐Soviet and post‐Soviet periods. Design and Methods. Age‐standardised male and female homicide data for the period 1970–2005 and data on beverage‐specific alcohol sales were obtained from the Russian State Statistical Committee (Rosstat). Time series analysis (autoregressive integrated moving average modelling) was used to examine the relation between the sale (consumption) of different alcoholic beverages and homicide rates. Results. Total alcohol consumption and vodka consumption as measured by sales were significantly associated with both male and female homicide rates: a 1 L increase in overall alcohol sales would result in a 5.9% increase in the male homicide rate and a 5.1% increase in the female homicide rate. The respective figures for vodka were 16.4% and 14.3%. The consumption of beer and wine was not associated with changes in homicide rates. Discussion and Conclusions. Our findings suggest that the consumption of distilled spirits has had an especially detrimental impact on lethal violence in Russia from at least 1970 onwards. In order to reduce homicide rates in this context, alcohol policy should focus on reducing overall consumption as well as attempting to shift the beverage preference away from distilled spirits.[Stickley A, Razvodovsky Y. The effects of beverage type on homicide rates in Russia, 1970–2005. Drug Alcohol Rev 2012;31:257–262]     

A naturalistic investigation of the effects of day-long consumption of tea, coffee and water on alertness, sleep onset and sleep quality

Rationale: The effects of caffeine, especially caffeinated coffee, on human performance have been extensively studied. However, few studies have been naturalistic representations of how tea/coffee is normally consumed in terms of dose and time of consumption. Objectives: This study investigated the effects of day-long consumption of tea, coffee and water on cognitive and psychomotor performance, and sleep quality at night. Methods: Thirty healthy volunteers received equal volume drinks equivalent to either 1 or 2 cups of tea (containing 37.5 mg or 75 mg caffeine), or coffee (75 mg or 150 mg caffeine), or water, in a randomised five-way crossover design. Drinks were administered on four occasions during the day (0900, 1300, 1700 and 2300 hours). A psychometric battery consisting of critical flicker fusion (CFF), choice reaction time (CRT) and subjective sedation (LARS) tests, was administered pre-dose and at frequent time points post-dose. The Leeds Sleep Evaluation Questionnaire (LSEQ) was completed each morning and a wrist actigraph was worn for the duration of the study. Results: Caffeinated beverages maintained CFF threshold over the whole day (P<0.05), independent of caffeine dose or beverage type. During the acute phase of beverage ingestion, caffeine significantly sustained performance compared to water after the first beverage for CFF and subjective sedation (P<0.05), and after the second beverage for the Recognition component of the CRT task (P<0.05). Additionally, there were significant differences between tea and coffee at 75 mg caffeine after the first drink. Compared to coffee, tea produced a significant increase in CFF threshold between 30 and 90 min post-consumption (P<0.01). However, following the second beverage caffeinated coffee at 75 mg significantly improved reaction time (P<0.05), compared to tea at the same dose, for the Recognition component of the CRT task. Caffeinated beverages had a dose dependent negative effect on sleep onset (P<0.001), sleep time (P<0.001) and sleep quality (P<0.001). Conclusions: These results indicate that ingestion of caffeinated beverages may maintain aspects of cognitive and psychomotor performance throughout the day and evening when caffeinated beverages are administered repeatedly. This study also demonstrates that day-long tea consumption produces similar alerting effects to coffee, despite lower caffeine levels, but is less likely to disrupt sleep. Other differences between tea and coffee were more subtle, and require further investigation. Received: 16 February 1999 / Final version: 20 December 1999     

Principles and applications of Raman spectroscopy in pharmaceutical drug discovery and development

Introduction: In recent years, Raman spectroscopy has become increasingly important as an analytical technique in various scientific areas of research and development. This is partly due to the technological advancements in Raman instrumentation and partly due to detailed fingerprinting that can be derived from Raman spectra. Its versatility of applications, rapidness of collection and easy analysis have made Raman spectroscopy an attractive analytical tool.

Areas covered: The following review describes Raman spectroscopy and its application within the pharmaceutical industry. The authors explain the theory of Raman scattering and its variations in Raman spectroscopy. The authors also highlight how Raman spectra are interpreted, providing examples.

Expert opinion: Raman spectroscopy has a number of potential applications within drug discovery and development. It can be used to estimate the molecular activity of drugs and to establish a drug’s physicochemical properties such as its partition coefficient. It can also be used in compatibility studies during the drug formulation process. Raman spectroscopy’s immense potential should be further investigated in future.     

Radiotracer‐based method for determining water solubility of highly insoluble compounds

Ascertaining the aqueous solubility of compounds is important in the selection of drug candidates. We describe a radiotracer method for estimating water solubility of compounds that are soluble in dimethyl sulfoxide (DMSO). Various volumes of DMSO, saturated with ¹²⁷I‐labeled compound and spiked with the corresponding ¹²⁵I‐labeled derivative, are mixed in de‐ionized water and the tubes centrifuged to remove insoluble material. Since (i) the iodine‐127 and iodine‐125 compounds have the same solubilities and are equally distributed in the DMSO–water solution, and (ii) the nonradioactive compound is accurately weighed, dissolved in a known volume of DMSO, and then further diluted as required, the concentration of compound in solution can be calculated and plotted as a function of the DMSO‐to‐water ratio. Water solubility of the compound is then determined by extrapolation of the linear fit of data points to zero DMSO. As proof of the methodology, 5‐iodo‐2′‐deoxyuridine (IUdR) and 2‐iodo‐8‐methyl‐8H‐quino[4,3,2‐kl]acridine (IMAc), water‐soluble compounds, were assessed using ¹²⁵IUdR and ¹²⁵IMAc, respectively. The solubility values obtained by the radiotracer method were similar to those acquired by spectrophotometry. Values calculated for several water‐insoluble compounds indicate that the radiotracer method can accurately quantify the solubility of low‐molecular‐weight compounds (1000–2000 Da) in the pg–ng/ml range. Copyright © 2006 John Wiley & Sons, Ltd.     

Trends in the use of alcohol and other drugs in cases of sexual assault

Recent media coverage has raised awareness of the involvement of drugs, both licit and illicit, in the crime of ‘date’ or ‘acquaintance’ rape. The term ‘date rape drug’ has been coined and has been used to label a few specific drugs because of their alleged properties. These include flunitrazepam (Rohypnol), gamma hydroxybutyrate (GHB) and ketamine. Concerns over reports of flunitrazepam in connection with ‘date rape’ led to action by the manufacturer of Rohypnol (flunitrazepam), F. Hoffman‐La Roche Ltd. who undertook an educational campaign warning of the dangers of drug‐assisted sexual assault, and changed their drug's formulation internationally to prevent its clandestine use. Despite the media interest, there has been little evidence and no systematic investigation to date of the incidence of drug use in the crime of sexual assault. The present study, from the USA, was therefore instigated to assess the extent to which different drugs were present in the urinalysis of samples supplied by victims of rape where drugs were allegedly involved. The study was so designed as to examine any changes over time in the patterns of drug use in sexual assault. Between June 1996 and May 1998, 1033 samples were tested. Nearly 20 different substances were detected among the 611 specimens that tested positive. Of these, 382 (37 per cent of all specimens) were positive for alcohol and 191 (18.5 per cent) were positive for cannabinoids. GHB was detected in 4.4 per cent of samples. Only six (<0.6 per cent) specimens showed evidence of flunitrazepam, and four of those contained other substances including cocaine, alcohol and/or morphine. Thus, only 0.2 per cent of all samples were positive for flunitrazepam alone. These data clearly indicate that there is no evidence of widespread misuse of flunitrazepam in sexual assault. Alcohol remains the substance most frequently associated with this type of crime. Copyright © 1999 John Wiley & Sons, Ltd.     

Targeted and non‐targeted drug screening in whole blood by UHPLC‐TOF‐MS with data‐independent acquisition

High‐resolution mass spectrometry (HRMS) is widely used for the drug screening of biological samples in clinical and forensic laboratories. With the continuous addition of new psychoactive substances (NPS), keeping such methods updated is challenging. HRMS allows for combined targeted and non‐targeted screening. First, peaks are identified by software algorithms, and identifications are based on reference standard data. Attempts are made to identify the remaining unknown peaks with in silico and literature data. However, several thousand peaks remain where most are unidentifiable or uninteresting in drug screening. The aims of the study were to apply a combined targeted and non‐targeted screening approach to authentic driving‐under‐the‐influence‐of‐drugs (DUID) samples (n = 44) and further validate the approach using whole‐blood samples spiked with 11 low‐dose synthetic benzodiazepine analogues (SBAs). Analytical data were acquired using ultra‐high‐performance liquid chromatography coupled with a time‐of‐flight mass spectrometer (UHPLC‐TOF‐MS) with data‐independent acquisition (DIA). We present a combined targeted and non‐targeted screening, where peak deconvolution and filtering reduced the number of peaks to inspect by three orders of magnitude, down to four peaks per DUID sample. The screening allowed for tentative identification of metabolites and drugs not included in the initial screening; 3 drugs and 14 metabolites were tentatively identified in the authentic DUID samples. Running targeted‐screening true‐positive identifications through the filters retained 73% of identifications. In the non‐targeted screening, nine of the spiked SBAs were identified in the concentration range of 0.005–0.1 mg/kg, of which three were tentatively identified at concentrations below those reported in the literature. Copyright © 2016 John Wiley & Sons, Ltd.     

Alcohol content variation in the assessment of alcohol consumption.

Most investigators have not adequately accounted for the alcohol content of different beverages when assessing alcohol consumption. Considerable research has assessed consumption in terms of the number of standard drinks. A problem with standard drink measures is that different distilled spirits, wines, and malt beverages vary considerably in alcohol content. State-to-state and brand-to-brand variations in the strength of different malt beverage brands are provided, as malt beverage alcohol contents are not contained on labels due to federal and state regulations. Ignoring alcohol content variation when estimating consumption can produce a large amount of error. Alcohol consumption should be assessed in terms of the number, size, and alcohol content of beverages.     

The importance of alcoholic beverage type for suicide in Japan: a time-series analysis, 1963-2007

Background. Japan has one of the highest suicide rates in the world. Cohort analysis has suggested that alcohol consumption is a risk factor for suicide in Japan. However, this relationship has not been observed at the population level when a measure of per capita total alcohol consumption has been analysed. The present study employed a time‐series analysis to examine whether these contradictory findings may be due to the existence of beverage‐specific effects on suicide. Methods. An autoregressive integrated moving average model was used to assess the relationship between the consumption of different types of alcohol and suicide rates from 1963 to 2007. The data comprised age‐adjusted suicide rates for the ages 15–69, and information on beverage‐specific alcohol consumption per capita (15+). The unemployment rate was included as a control variable. Results. During 1963–2007, male suicide rates increased substantially whereas female rates decreased slightly. Consumption of distilled spirits was significantly related to male suicide rates (but not in women) with a 1 L increase in consumption associated with a 21.4% (95% confidence interval: 3.2–42.9) increase in male suicide rates. There was no statistically significant relationship between suicide and any other form of alcohol consumption (beer, wine, other alcohol). Conclusion. This is the first study that has shown an association between spirits consumption and male suicide in Japan. Potentially beneficial policy changes include increasing spirits prices through taxation, reducing the physical availability of alcohol and discouraging the practice of heavy drinking.[Norström T, Stickley A, Shibuya K. The importance of alcoholic beverage type for suicide in Japan: A time‐series analysis, 1963–2007. Drug Alcohol Rev 2012;31:251–256]     

Heightened aggression after chronic flunitrazepam in male rats: potential links to cortical and caudate–putamen-binding sites

Rationale Higher doses of benzodiazepines induce sedation. However, in low to moderate doses, benzodiazepines can increase aggressive behavior both after acute and chronic administration. The determinants for increasing aggression after chronic intake of flunitrazepam, a so-called date rape drug, in violence-prone individuals are incompletely understood. Objectives The aim of this study is to assess the effects of acute and chronic treatment with flunitrazepam on male aggression in resident rats. We also examined possible changes in binding to benzodiazepine receptors throughout the brain of rats that display aggressive behavior after repeated flunitrazepam treatment using quantitative receptor autoradiography. Materials and methods The behaviors of the male Wistar resident rats (n = 35) toward a male intruder were recorded for 10 min twice a week. The salient aggressive and non-aggressive elements in the resident rat’s behavior were analyzed. Initially, the dose-dependent effects of flunitrazepam (0.01, 0.03, 0.1, 0.18, and 0.3 mg/kg) or vehicle were determined in all rats; subsequently, 0.3 mg/kg per day flunitrazepam was administered for 42 days (n = 15), and a parallel group was treated with vehicle (n = 20). After the chronic treatment, the flunitrazepam (0, 0.01, 0.03, 0.1, 0.18, and 0.3 mg/kg) effects were again assessed. Results The most significant finding is the escalation of aggression after chronic treatment with flunitrazepam. A previously sedative 0.3 mg/kg dose of flunitrazepam engendered very high levels of attack bites, sideways threats, and aggressive postures (total aggression) after 6 weeks of daily administration. Individual differences emerged, and these were associated with decreased binding to benzodiazepine receptors, mainly in the limbic structures such as the cingulate cortex (cingulate areas 1 and 2) and caudate–putamen (posterior part) of aggressive animals, suggesting that these areas are pivotal in the control of emotional and aggressive behavior. Conclusions Chronic flunitrazepam produces changes in receptor binding in discrete areas of the cingulate cortex and caudate–putamen that are proposed to be part of the mechanisms for increased expression of aggressive behavior.     

Development of a quantitative method for the analysis of cocaine analogue impregnated into textiles by Raman spectroscopy

Cocaine trafficking in the form of textile impregnation is routinely encountered as a concealment method. Raman spectroscopy has been a popular and successful testing method used for in situ screening of cocaine in textiles and other matrices. Quantitative analysis of cocaine in these matrices using Raman spectroscopy has not been reported to date. This study aimed to develop a simple Raman method for quantifying cocaine using atropine as the model analogue in various types of textiles. Textiles were impregnated with solutions of atropine in methanol. The impregnated atropine was extracted using less hazardous acidified water with the addition of potassium thiocyanate (KSCN) as an internal standard for Raman analysis. Despite the presence of background matrix signals arising from the textiles, the cocaine analogue could easily be identified by its characteristic Raman bands. The successful use of KSCN normalised the analyte signal response due to different textile matrix background interferences and thus removed the need for a matrix‐matched calibration. The method was linear over a concentration range of 6.25–37.5 mg/cm² with a coefficient of determination (R²) at 0.975 and acceptable precision and accuracy. A simple and accurate Raman spectroscopy method for the analysis and quantification of a cocaine analogue impregnated in textiles has been developed and validated for the first time. This proof‐of‐concept study has demonstrated that atropine can act as an ideal model compound to study the problem of cocaine impregnation in textile. The method has the potential to be further developed and implemented in real world forensic cases.     

Recognizing drug‐facilitated crimes: Detection and quantification of benzodiazepines in beverages using fast liquid–liquid extraction with low temperature partitioning and paper spray mass spectrometry

A novel methodology using liquid–liquid extraction with low temperature partitioning (LLE–LTP) and paper spray mass spectrometry (PS–MS) was developed to identify and quantify benzodiazepines in beverages. Four types of alcoholic beverages usually consumed in parties and bars were spiked with 5 distinct benzodiazepines (diazepam, alprazolam, bromazepam, clonazepam, and cloxazolam) simulating a drug‐facilitated crime occurrence. The direct PS–MS analysis of the spiked beverages revealed a remarkable matrix effect with an unclear detection of protonated benzodiazepines. However, by the application of the LLE–LTP using liquid nitrogen, a prompt and doubtless detection of such compounds was achieved. The quantification potential of the LLE–LTP/PS–MS methodology was demonstrated by using beer as matrix, diazepam as target analyte and cloxazolam as an internal standard. Figures of merit (linearity, limit of detection, linear dynamic range, relative standard deviation, and recovery) were determined and adequate values were obtained. In conclusion, we demonstrated herein that the LLE–LTP/PS–MS methodology has potential to be applied directly at the crime scene through of a portable mass spectrometer and a thermal container for the transport of liquid nitrogen.     

In search of novel protein drug targets for treatment of Enterococcus faecalis infections

Enterococcus faecalis (Ef) is one of the major pathogens involved in hospital‐acquired infections. It can cause nosocomial bacteremia, surgical wound infection, and urinary tract infection. It is important to mention here that Ef is developing resistance against many commonly occurring antibiotics. The occurrence of multidrug resistance (MDR) and extensive‐drug resistance (XDR) is now posing a major challenge to the medical community. In this regard, to combat the infections caused by Ef, we have to look for an alternative. Rational structure‐based drug design exploits the three‐dimensional structure of the target protein, which can be unraveled by various techniques such as X‐ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. In this review, we have discussed the complete picture of Ef infections, the possible treatment available at present, and the alternative treatment options to be explored. This study will help in better understanding of novel biological targets against Ef and the compounds, which are likely to bind with these targets. Using these detailed structural informations, rational structure‐based drug design is achievable and tight inhibitors against Ef can be prepared.     

Solid-phase microextraction and GC-ECD of benzophenones for detection of benzodiazepines in urine

Benzodiazepines are common drugs that cause intoxication. Benzodiazepines and their metabolites can be converted by hydrolysis in acid to the corresponding benzophenones, which are easier to be separated from matrices because of their hydrophobic properties. In this study, a new separation technique called solid-phase microextraction (SPME), which can integrate extraction, concentration, sampling and sample introduction into one single procedure, has been employed to extract the products of benzodiazepines from urine after acid hydrolysis. The extracts were determined by gas chromatography with electron-capture detection (GC-ECD). The hydrolysis conditions were optimized by a statistic orthogonal design. Factors influencing direct-immersion (DI)-SPME process were also checked and chosen experimentally. The method was evaluated with spiked human urine samples. The recoveries of nine benzodiazepines ranged from 1 to 25%, with the highest for oxazolam and the lowest for bromazepam. The calibration curves were linear from 10 to 500 ng/mL for oxazolam, haloxazolam, flunitrazepam, nimetazepam, and clonazepam and from 20 to 1000 ng/mL for the others except bromazepam. The detection limits were 2-20 ng/mL for most drugs tested. The intraday and interday coefficients of variation of the developed method were within 10 and 17%, respectively. In addition, the utility of the method was confirmed by determining two ingested benzodiazepines (flunitrazepam and oxazolam) in a volunteer's urine; urine flunitrazepam was still detectable 32 h after a therapeutic dose (1.2 mg) of the drug. Finally, the DI-SPME was compared with the conventional liquid-liquid extraction with regard to detection limits and extraction efficiency of the analytes. By DI-SPME, more amounts of analytes could be introduced into GC column than by conventional liquid-liquid extraction, and thus lower detection limits of the analytes were reached, although benzophenone recoveries by DI-SPME were rather low.     

Production of human metabolites by gastrointestinal bacteria as a potential source of post‐mortem alteration of antemortem drug/metabolite concentrations

Previous studies have demonstrated that bacterial species are capable of transforming complex chemical substances. Several of these species, native to the human gastrointestinal tract, are active in postmortem decomposition. They have potential to cause biotransformations affecting compound‐to‐metabolite ratios within the human body, especially after death. Investigation of postmortem effects could supply valuable information, especially concerning compound identification and confirmation. The purpose of this research was to investigate the effects of Escherichia coli, Bacteroides fragilis, and Clostridium perfringens on diazepam and flunitrazepam in Reinforced Clostridial Medium, and to compare bacterial biotransformation products to those of human metabolism. A decrease in diazepam concentration between pre‐ and post‐incubation was observed for samples inoculated with Escherichia coli (14.7–20.2%) as well as Bacteroides fragilis (13.9–25.7%); however there was no corresponding increase in concentration for the monitored human metabolites. Flunitrazepam demonstrated a greater concentration loss when incubated with individual bacterial species as well as mixed culture (79.2–100.0%). Samples incubated with Bacteroides fragilis, Clostridium perfringens, and mixed culture resulted in nearly complete conversion of flunitrazepam. Increased 7‐aminoflunitrazepam concentrations accounted for the majority of the conversion; however discrepancies in the mass balance of the reaction suggested the possibility of a minor metabolite that was not monitored in the current analysis. These experiments served as a pilot study and proof of concept that can be adapted and applied to a realm of possibilities. Ultimately, this methodology would be ideal to study compounds that are too toxic or lethal for animal and human metabolic investigations. Copyright © 2014 John Wiley & Sons, Ltd.     

Rapid method for the solid-phase extraction and GC-MS analysis of flunitrazepam and its major metabolites in urine

Recently, flunitrazepam (Rohypnol) has become an increasingly popular drug of abuse among young adults, who take it for its euphoric effects. In other cases, the drug has been used by rapists for its sedative and hypnotic effects that can induce a catatonialike trance and memory loss in potential victims; as a result, it has been nicknamed the "date-rape drug". For these reasons, the Drug Enforcement Administration recently considered adding the drug (a.k.a. "Roofies") to the same category as heroin and LSD. A selective and sensitive technique has been developed for extracting, detecting, and identifying flunitrazepam and its two major metabolites (7-aminoflunitrazepam and N-desmethylflunitrazepam) in human urine. Using a solid-phase extraction cartridge containing a "mixed-mode" bonded silica gel (Bond Elut Certify), flunitrazepam and its metabolites were selectively isolated from other urine components and quantitated and identified by gas chromatography-tandem mass spectrometry with a benchtop ion mass spectrometer. The extraction method is rapid, reproducible, and precise, and it has a broad linear working range. The overall extraction efficiency was found to be more than 90% for the parent drug as well as the two major metabolites.