Optimizing screening cutoffs for drugs of abuse in hair using immunoassay for forensic applications.

BACKGROUND: In forensic toxicology, positive immunoassay (IA) test results do not hold forensic validity and need to be confirmed with mass spectrometry (MS). On the other hand, a negative result is a strong indication that the drug and/or the drug metabolites are not present in the sample and that confirmatory analyses are not necessary. Consequently, a negative IA result must have forensic validity since it can be admitted in court during a trial. OBJECTIVES: Screening cutoffs for the analysis of hair samples using immunoassays (IAs) were retrospectively optimized based on the Society of Hair Testing (SoHT) confirmation cutoffs and the utility of the test for forensic applications was discussed. MATERIAL AND METHODS: Hair samples taken from 150 patients with a history of drug addiction were analyzed with ILab 650, Werfen (Milan, Italy) using DRI® reagents. Confirmatory analyses were subsequently performed using the ACQUITY UPLC® System, Waters Corporation (Milford, USA). Screening cutoffs were retrospectively optimized using receiver operating characteristic (ROC) analysis. RESULTS: A total of 162 single positive results were obtained for confirmatory analysis (10 for amphetamines/methamphetamines, 11 for MDMA, 37 for cocaine, 40 for THC, 33 for methadone, and 31 for opiates). The optimized screening cutoffs were 0.27 IA ng/mg for amphetamines, 0.51 IA ng/mg for MDMA, 0.59 IA ng/mg for cocaine, 0.14 IA ng/mg for cannabinoids, 0.63 IA ng/mg for methadone, and 0.26 IA ng/mg for opiates. An area under the curve (AUC) greater than 0.95 was obtained with very high sensitivity and specificity for all drugs. CONCLUSIONS: The presented screening method proved to be a useful technique on hair samples for the classes of drugs most commonly found in Italy and Europe and can be applied to forensic analysis.

External hair contamination from cannabis and "light cannabis" delivered by smoking and vaping: An in vitro study.

External contamination of hair by cannabis smoking requires a careful evaluation in forensic toxicology. Medical and recreational cannabis are increasingly consumed by e-cigarettes, which give rise to side-stream vapor. Moreover, products containing low Δ9-tetrahydrocannabinol (Δ9-THC) and rich in cannabidiol (CBD) started spreading legally. The goal of the present study was to assess whether hair analysis could allow to distinguish the type of delivered product, with low or high Δ9-THC, and the delivering mode, by smoking or vaping. Contamination of blank hair was mimicked by in vitro exposure to low- (0.4%) and high-Δ9-THC (9.7%) products delivered by smoking and vaping within a small confined system. Cannabis vaping extracts were prepared to deliver identical target Δ9-THC doses. Eighty samples were analyzed by ultrahigh-performance liquid chromatography mass spectrometry and quantified for Δ9-THC and CBD. After contamination by cannabis smoking, THC levels were in line with past in vitro and in vivo studies. Samples exposed to cannabis (169.30 ng/mg) showed significantly higher Δ9-THC than hair exposed to "light cannabis" (35.54 ng/mg), and the opposite was seen for the CBD/Δ9-THC ratio. Hair contaminated by vaping or smoking did not show a statistically different Δ9-THC content. Under our in vitro conditions, hair analysis might allow to discriminate whether external contamination is determined by products containing low or high Δ9-THC, but not the delivering mode. More research is needed in real-life conditions, to see whether the same also applies to the interpretation of forensic casework.

Development and validation of a fast UPLC-MS/MS screening method for the detection of 68 psychoactive drugs and metabolites in whole blood and application to post-mortem cases.

We report a rapid and sensitive LC-MS/MS method that allows the simultaneous detection of 68 commonly prescribed antidepressants, benzodiazepines, neuroleptics, and metabolites in whole blood with a small sample volume after a rapid protein precipitation. The method was also tested on post-mortem blood from 85 forensic autopsies. Three sets of commercial serum calibrators containing a mix of prescription drugs of increasing concentration were spiked with red blood cells (RBC) to obtain 6 calibrators (3 "serum calibrators" and 3 "blood calibrators"). Curves obtained from serum calibrators and from blood calibrators were compared using a Spearman correlation test and by analyzing slopes and intercepts, to assess if the points from six calibrators could be plotted together in a single calibration model. The validation plan included interference studies, calibration model, carry-over, bias, within-run and between-run precision, limit of detection (LOD), limit of quantification (LOQ), matrix effect and dilution integrity. Four deuterated Internal Standards (Nordiazepam-D5, Citalopram-D6, Ketamine-D4 and Amphetamine-D5) and two different dilutions were assessed. Analyses were performed using an Acquity UPLC® System coupled with triple quadrupole detector Xevo TQD®. The degree of agreement with a previously validated method was calculated on whole blood samples of 85 post-mortem cases, by performing a Spearman correlation test with a Bland-Altman plot. Percentage error between the two methods was evaluated. Slopes and intercepts of curves obtained from serum calibrators and from blood calibrators showed a good correlation, and the calibration model was built plotting all points together. No interferences were found. The calibration curve appeared to provide a better fit of the data using an unweighted linear model. Negligible carry-over was observed, and very good linearity, precision, bias, matrix effect and dilution integrity were achieved. The LOD and the LOQ were at the lower limits of the therapeutic range for the tested drugs. In a series of 85 forensic cases, 11 antidepressants, 11 benzodiazepines and 8 neuroleptics were detected. For all analytes, a very good agreement between the new method and the validated method was demonstrated. The innovation of our method consists in the use of commercial calibrators, readily available to most forensic toxicology laboratories, for the validation of a fast, inexpensive, wide-panel LC-MS/MS method that can be used as a reliable and accurate screening for psychotropic drug in postmortem samples. As observed in the implementation on real cases, this method could be profitably applied in forensic cases.

Development and validation of a rapid LC-MS/MS method for the detection of 182 novel psychoactive substances in whole blood.

INTRODUCTION: The analysis of novel psychoactive substances (NPS) represents a challenge in forensic toxicology, due to the high number of compounds characterized by different structures and physicochemical properties both among different subclasses and within a single subclass of NPS. The aim of the present work is the development and validation of a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the detection of NPS in whole blood. MATERIALS AND METHODS: A protein-precipitation based LC-MS/MS method for the detection of more than 180 NPS was developed and validated by assessing the following parameters: selectivity, linearity, accuracy, precision, limit of detection (LOD) and of quantification (LOQ) recovery, and matrix effect. Then, the method was applied to real forensic samples. RESULTS: The method allowed the identification of 132 synthetic cannabinoids, 22 synthetic opioids, and 28 substances among synthetic cathinones, stimulants, and other drugs. Validation was successfully achieved for most of the compounds. Linearity was in the range of 0.25-10 ng/ml for synthetic cannabinoids and 0.25-25 ng/ml for other drugs. Accuracy and precision were acceptable according to international guidelines. Three cases tested positive for fentanyl and ketamine, in the setting of emergency room administration. CONCLUSIONS: The present methodology represents a fast, not expensive, wide-panel method for the analysis of more than 180 NPS by LC-MS/MS, which can be profitably applied both in a clinical context and in postmortem toxicology.

"Light cannabis" consumption in a sample of young adults: Preliminary pharmacokinetic data and psychomotor impairment evaluation.

INTRODUCTION: In 2019, the Italian Supreme Court established that hemp cannot be commercialized for human use, when the "psychotropic effect" of the product or its "offensiveness" can be demonstrated. The aim of the present study is to assess Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) blood concentrations after smoking cannabis with a low percentage of Δ9-THC, also referred as "light cannabis", and its effects on young adults' vigilance, cognitive and motor skills. MATERIALS AND METHODS: Eighteen young adults consumed three light cannabis cigarettes containing 400 mg of inflorescences each, with a percentage of 0.41% of Δ9-THC and of 12.41% of CBD. Blood samples were collected before the experiment (t0), after each light cannabis cigarette (t1→t3), 60 (t4) and 120 (t5) minutes after the beginning of the experiment. Five performance tasks and a subjective scale were employed for measuring cognitive and psychomotor performances the day before the experiment (TT0) and after the third cigarette (TT1). RESULTS: Mean (SD) concentrations (ng/ml) were 1.0 (0.8) in t1, 1.2 (0.9) in t2, 1.0 (0.8) in t3, 0.6 (0.4) in t4 and 0.3 (0.3) in t5 for Δ9-THC; 10.5 (10.3) in t1, 10.3 (13.2) in t2, 15.1 (14.8) in t3, 9.9 (9.2) in t4 and 5.7 (5.7) in t5 for CBD. No significant differences were observed between TT0 and TT1 for all performed psychomotor performance task. None of the subjects declared to feel "high" after the experiment. DISCUSSION: All study participants reported that a higher number of cigarettes, corresponding in this study to 1200 mg of herbal product, could hardly be consumed by smoking in a recreational setting. Δ9-THC and CBD concentrations showed a high inter-subject variability, and the average concentrations were lower than those previously reported. Toxicological results showed a decrease of Δ9-THC and CBD after the third light cannabis cigarette, and a Δ9-THC /CBD ratio always<1 was observed. The lack of impairment observed in our participants can be interpreted as a consequence of the very low concentrations detectable in the blood.

Importance of dashboard camera (Dash Cam) analysis in fatal vehicle-pedestrian crash reconstruction.

The reconstruction of dynamic of traffic injuries remains a challenge in forensic pathology and is often based on circumstantial data. Dash Cams are digital video recorders which can be located inside a vehicle and continuously record the view through the windscreen, thus providing objective evidence. Here we present the case of a traffic crash in which a pedestrian was hit by an articulated lorry. The analysis of a video recorded from a Dash Cam retrieved inside the vehicle during the death scene investigation (DSI) was crucial in the reconstruction of the manner of death. Indeed, the death, which was initially assumed to be accidental, was finally deemed as a suicide on the basis of the video recording, which showed an intentional and sudden rush of the victim to the middle of the roadway. Advantages and disadvantages of the use of Dash Cams will be discussed, focusing on the profound differences in the related national and international regulations. Based on the present case, in traffic crashes, the search for Dash Cams during the DSI may be recommended and the video recordings should be analyzed in the setting of a multidisciplinary and multimodal evaluation of the case, for a proper reconstruction of the facts.

Deaths related to nitrogen inhalation: Analytical challenges.

Dinitrogen (N2) has been increasingly connected to suicidal deaths. The analysis of N2 in post-mortem cases still represents a major challenge in forensic toxicology and circumstantial data has so far played a major role for the determination of the cause of death. In this paper, after presenting a review of cases of N2 intoxication described in forensic literature, we report the application of two approaches in order to quantify an excess of N2 in post-mortem whole blood collected from a case of suicide by nitrogen inhalation. N2 analyses were performed by GC-MS on the suicidal case and on controls taken from 10 autopsy cases with similar PMI (5 traumatic deaths and 5 deaths by asphyxia). The percentage of N2 was estimated by building a five-point N2 peak area calibration curve (0, 15.6 %, 62.4 % 78.1 %, 100 %) and through an external QC, assessing linearity, accuracy and precision, LLOQ, specificity and stability of N2 in the sample vial. Percentage of N2 of the case was significantly higher than the post-mortem controls (p<0.05). The N2/O2 ratio of the case and controls was also calculated as an additional indicator, and was significantly higher in the case (p<0.05). The strengths and the limitation of both methods are reported in the paper. Toxicological confirmation for N2 are rarely performed when the cause of death is evident, probably due to the lack of validated methods and the complexity of the interpretation of N2 concentration in biological fluids. The presented methods can be rapidly and profitably applied with instrumentation normally available in forensic laboratories.

Optimization of cloned enzyme donor immunoassay cut-offs for drugs of abuse in post-mortem whole blood.

INTRODUCTION: Immunoassay (IA) tests are not widely applied in post-mortem samples, since they are based on technologies requiring relatively non-viscous specimens, and compounds originating from the degradation of proteins and lipids during the post-mortem interval can alter the efficiency of the test. However, since the extraction techniques for IA tests are normally rapid and low-cost, IA could be used as near-body drug-screening for the classes of drugs most commonly found in Italy and Europe. In this study, semi-quantitative results on post-mortem whole blood samples obtained through CEDIA analysis (cannabinoids, cocaine, amphetamine compounds, opiates and methadone), were compared with results of confirmatory analysis obtained using GC-MS. Screening cut-offs for all drugs were retrospectively optimized. METHODS: Post-mortem whole blood samples from autopsy cases of suspected fatal intoxication were collected over 3 years. Samples were initially analyzed through CEDIA (CEDIA, ILab 650, Werfen). Confirmatory analyses were then performed by GC-MS (QP 2010 Plus, Shimadzu). Screening cut-offs were retrospectively optimized using Receiver Operating Characteristic (ROC) analysis. RESULTS: CEDIA results were available for 125 samples. Two-hundred-eighty-nine (289) positive screening results were found. Among these, 162 positive confirmation results were obtained. Optimized screening cut-offs were as follows: 6.5ng/ml for THC; 4.2ng/ml for THC-COOH; 12.0ng/ml for cocaine; 6.6ng/ml for benzoylecgonine; 6.4ng/ml for opiates; 2.0ng/ml for methadone. Analysis of ROC-curves showed a satisfying degree of separation in all tests except for amphetamine compounds, with areas under the curve (AUC) between 0.915 (THC) and 0.999 (for benzoylecgonine and methadone). DISCUSSION: The results of the study showed that CEDIA screening at the optimized cut-offs exhibits a very high sensitivity and good specificity and positive predictive value (PPV) for cannabinoids, cocaine and metabolites, opiates and methadone. A high number of false positives (n=19) for amphetamine compounds was observed at the optimized cut-off, resulting in a very low PPV, which is also influenced by the very low number of TP (n=4). CONCLUSION: The results of the study show that the CEDIA is a valuable screening test on post-mortem whole blood for cannabinoids, cocaine and metabolites, opiates and methadone, but it is not recommended for amphetamine compounds, due to the high number of false positives. The strengths of the study are the large sample size, the inclusion of post-mortem cases only and the high level of sensitivity and specificity obtained at the optimized cut-offs.

Alcohol and illicit drugs in drivers involved in road traffic crashes in Italy. An 8-year retrospective study.

This study aims to investigate the prevalence of alcohol and drugs of abuse in Italian drivers involved in road traffic crashes between 2011 and 2018. Toxicological analyses were performed on the whole blood of 7593 injured drivers. Alcohol and illicit drugs, namely tetrahydrocannabinol (THC; cut-off 2ng/ml), cocaine (cut-off 10ng/ml), illicit opiates (cut-off 10ng/ml) and amphetamines (amphetamine, methamphetamine, MDMA, MDA; cut-off 20ng/ml) were investigated. The age and gender of the driver, the time of the crash (weekend/weekday and day/night), the road crash year and Blood Alcohol Concentration (BAC) were also considered. The 16.2% of samples tested positive for alcohol, 2.5% for cocaine, followed by opiates (2.0%), cannabinoids (1.5%), and amphetamines (0.5%). The overall prevalence of alcohol and drugs was lower than those reported in previous epidemiological studies of the DRUID project. The year 2011 showed the highest prevalence of drug-positive cases (24.1%), while the lowest prevalence was found in 2016 (16.8%), after the update of the Road Traffic Law (RTL) that increased punishments for driving under the influence. A progressive increase in the number of alcohol-positive female drivers was observed from 2011 to 2018, and the highest prevalence was found in the 26-35-year-old age range. Illicit drugs showed the highest overall prevalence in drivers <26 years of age but, if considering single drugs, cocaine and opiates were mostly found in subjects older than 36 years of age. A higher percentage of drug-positive drivers was found on weekend nights for alcohol and on both weekend and weekday nights for drugs. The types of drugs used by drivers did not change during the studied period.

Prevalence of therapeutic drugs in blood of drivers involved in traffic crashes in the area of Bologna, Italy.

INTRODUCTION: Psychoactive medicines, such as benzodiazepines and Z-drugs (BdZ), antidepressants and antipsychotics (AA) and medical opioids (MO), have an impairing effect on driving ability. In previous epidemiological studies performed on impaired and/or injured drivers, not all relevant psychoactive substances were included in the toxicological assessment, and their prevalence may be underestimated. This study aims to assess the prevalence of a large set of psychoactive substances (n=53) in Italian drivers involved in a road traffic crash and in predefined population subgroups. MATERIALS AND METHODS: The toxicological analyses were performed on the whole blood of 1026 drivers involved in a road traffic crash in the area of Bologna, Italy, from January 2017 to March 2018. Analyses were performed using GC-FID (alcohol), GCMS (illicit drugs) and LC/HRMS (psychoactive drugs). The population was divided into subgroups according to gender, age and crash time. Descriptive statistics were used in order to assess differences among sub-groups. RESULTS: The highest prevalence was found for alcohol (17.3%), followed by medicinal drugs (13.6%) and illicit drugs (5.5%). The prevalence of BdZ, AA and MO were 7.3%, 7.2% and 3.1%, respectively. The frequency of BDZ and AA was significantly higher in female drivers and showed higher prevalence at increasing age. The presence of medicinal drugs was significantly higher during the week and in crashes occurring during the day. CONCLUSION: Results for alcohol and illicit drugs partially overlap with those reported in previous European and Italian studies, but the prevalence of BdZ was much higher. We also found a high prevalence of AA, which are rarely investigated in epidemiological studies performed on drivers, but may cause impairment of the ability to drive, especially when taken in combination with alcohol or other drugs. The pattern of medication use differs from that involving drugs of abuse, since it is mainly observed in female subjects and older drivers and does not follow the same weekly trend observed for alcohol and other illicit drugs.

Validation and preliminary application of a GC-MS method for the determination of putrescine and cadaverine in the human brain: a promising technique for PMI estimation.

INTRODUCTION: Among the several techniques proposed for the estimation of the Post Mortem Interval (PMI), the analysis of odorous amines has been applied in the past, with conflicting results. The aims of this study are: (i) to develop and validate a GC-MS method for the determination of putrescine (PUT) and cadaverine (CAD) in the human brain (validation study) and (ii) to study the relation of PUT and CAD concentration in the human brain and the PMI (decomposition study). MATERIALS AND METHODS: Validation study. Analysis has been performed through GC-MS after a liquid-liquid extraction and a single step-derivatization for the identification and quantification of odorous amines in brain cortex samples. The standard protocol used in forensic toxicology, slightly modified for endogenous compounds according to recent guidelines, was used for validation. Decomposition study. Three uninjured human brains were sampled during the autopsy of three fatal traumatic cases. Along a 120-hour period of decomposition under experimental conditions, each brain was sampled along predetermined time intervals. RESULTS: Validation study. Both PUT and CAD validation parameters were within the acceptable values defined by the Scientific Working Group for Forensic Toxicology (SWGTOX), with better selectivity, linearity, accuracy and precision values for PUT. Decomposition study. A significant relationship between PUT and CAD levels and PMI has been demonstrated through statistical analysis with a correlation coefficient of 0.98 for PUT and 0.93 for CAD (p < 0.0001). CONCLUSION: Although further experimental studies on a wider number of samples are necessary, the results of this study suggest a possible role of polyamine levels in brain cortex for the estimation of PMI.

Optimization of cloned enzyme donor immunoassay cut-offs for drugs of abuse in whole blood of drivers involved in road accidents.

INTRODUCTION: Immunoassay (IA) tests should be able to detect low concentrations of illegal drugs when used for the screening of drugs in drivers. False negatives should be avoided, and false positives should be reduced as far as possible. In this study, semi-quantitative results for blood samples containing illicit drugs (cannabinoids, cocaine, amphetamines/methamphetamines, opiates and methadone) obtained with cloned enzyme donor immunoassay (CEDIA), were compared with results of confirmatory analysis performed through gas chromatography-mass spectrometry (GC-MS). Screening cut-off points for each class of drugs were retrospectively optimized. METHODS: Whole blood samples from drivers involved in road accidents in the period from January 2013-December 2017 were analyzed with CEDIA (4200 samples). Confirmatory analyses were performed through (GC-MS) on: (i) all samples with screening concentrations above 1ng/ml for at least one drug (positive screening results); (ii) 800 samples with screening concentration lower than 1ng/ml (negative screening results). Recommended per se limits in relation to driving under the influence of drugs were set as fixed values. Sensitivity, specificity, positive and negative predictive values were evaluated by contingency tables and compared to ROC-analysis in order to obtain ideal screening cut-offs. RESULTS: CEDIA results were available for 4200 blood samples and 1172 positive screening results were found. Among these, 1008 confirmation analysis were obtained through GC-MS. Optimized screening cut-offs obtained through ROC analysis were as follows: 8.0ng/ml for THC; 5.5ng/ml for THC-COOH; 21.1ng/ml for cocaine; 6.9ng/ml for benzoylecgonine; 33.1ng/ml for opiates; 61.6ng/ml for amphetamines; 5.0ng/ml for methadone. Using these cut-offs, sensitivity was above 97% for THC-COOH, cocaine, benzoylecgonine, amphetamines, opiates and methadone, and 92% for THC; specificity was above 90% for cocaine, benzoylecgonine, amphetamines, opiates and methadone, 80% for THC and 89% for THC-COOH; negative predictive value was above 99% for all drugs and metabolites. CONCLUSION: Previous studies have shown that CEDIA tests are useful for preliminary screening of serum and urine. Its implementation in whole blood is of primary importance for the assessment of impaired driving, since the per se limits of many European countries refer to whole blood, and preparation of the serum and/or the collection of urine is not always possible in the hospital emergency department, where blood samples are withdrawn. Our study shows that CEDIA tests on whole blood permit the definition of cut-off values with optimal sensitivity and negative predictive values for all analytes (near to 100%), including very good specificity.

Medico-legal implications of toluene abuse and toxicity. Review of cases along with blood concentrations.

Toluene, a liquid aromatic hydrocarbon, is one of the most widely used industrial solvents, and is present in numerous paints, paint thinners, glues and other industrial and household products. It has become the most abused solvent in the world due to its rapid effects following inhalation. However, the numerous cases of fatal and non-fatal toluene-related intoxication reported in the literature have not yet been collected and discussed in the forensic setting. In this paper we aim to provide a review of the cases of toluene abuse and intoxication and the state of the art of the forensic toxicological analysis of toluene intoxications in the living and in the dead subject, from the early identification to the medico-legal interpretation of the toxicological result. We have identified a total of 45 papers regarding different aspects of toluene abuse, and divided them into three sections, namely sampling, storage and techniques of analysis, assessment in living subjects and post-mortem assessment. This article reports toluene concentrations in blood from 202 living subjects, 23 fatal toluene intoxications and 85 toluene related deaths. Toxicological results are discussed in relation to the clinical presentation (living subjects, including impaired drivers), and the manner of death according to the medical examiner reports (post-mortem examinations). Finally, we discuss the strengths and limitations of the review.

Safe drugs in drug facilitated crimes and acute intoxications in Northern Italy.

Toxicological analyses are often performed in drug-facilitated sexual assaults (DFSA), when the victim shows or reports impaired consciousness and reduced ability. However, in other crimes or fatalities, especially in cases of concurrent natural disease or when another likely cause of death has been established, the involvement of drugs can be overlooked. The aim of this study is to report a series of cases of (i) victims of drug-facilitated crimes (DFC) other than DFSA and (ii) victims of acute intoxications, in which "licit" psychoactive drugs were found in blood samples, with the aim of understanding in which circumstances and to what extent prescription drugs have been used for non-medical purposes in recent Italian casuistry. Circumstantial, autopsy, and toxicological data were collected through a retrospective analysis performed between 2013 and 2017 in the Forensic Toxicology Unit of the University of Bologna. Cases of "DFC other than DFSA" and "Acute Intoxication" in which "psychoactive drugs" or "prescription drugs" or "licit drugs" were found in the blood samples of the victims were included in the study. Nine cases of DFC other than DFSA, and 11 cases of acute intoxication, were identified. Different categories of "licit" psychoactive drugs (e.g. hypnotics, antipsychotics, antidepressants, anticonvulsants) had been used to facilitate diverse types of crime (homicide, robberies, elder abuse, fatal poisoning) or acute intoxication (suicide, attempted suicide, accidental death). The circumstances of these cases, as well as toxicological findings in blood samples and other relevant forensic elements, are reported, summarized and discussed in this paper. The non-medical use of pharmaceuticals has been identified by recent forensic literature and the present study as a significant and growing phenomenon, and its implication in fatalities should be taken into consideration and accurately investigated through appropriate toxicological analysis. Our study presents an overview of the circumstances of non-medical use of prescription drugs, usually considered "safe drugs", and their involvement in cases of DFC, suicides and accidental intoxication. In order to estimate the real incidence of these medications in DFC and acute intoxication, and thus collect more analytical and contextual data, further studies are needed, along with effective cooperation among police officers, clinicians, forensic pathologists, and toxicologists.

Determination of levamisole and tetramisole in seized cocaine samples by enantioselective high-performance liquid chromatography and circular dichroism detection.

Levamisole, an anthelmintic drug, has been increasingly employed as an adulterant of illicit street cocaine over the last decade; recently, the use of tetramisole, the racemic mixture of levamisole and its enantiomer dexamisole, was also occasionally observed. A new enantioselective high-performance liquid chromatography (HPLC) method, performed on cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phases in normal-phase mode, was validated to determine the enantiomeric composition of tetramisole enantiomers in seized cocaine samples. Furthermore, the hyphenation of the validated HPLC method with a circular dichroism (CD) detection system allowed the direct determination of elution order and a selective monitoring of levamisole and dexamisole in the presence of possible interferences. The method was applied to the identification and quantitation of the two enantiomers of tetramisole in seized street cocaine samples.

Determination of dextromethorphan and levomethorphan in seized heroin samples by enantioselective HPLC and electronic CD.

A new enantioselective HPLC method was developed for the resolution and determination of the enantiomers of methorphan, dextromethorphan (DXM) and levomethorphan (LVM), on a Chiralcel OJ column (250 mm × 4.6mm I.D.). The resolution of DXM and LVM was obtained using a mobile phase consisting of (n-hexane)-(2-propanol)-diethylamine (70:30:0.1, v/v/v) at a flow rate of 0.5 mL min(-1). The enantioselective method was found to be selective (α=1.92) and sensitive (LOD=2.8 µg mL(-1) for both DXM and LVM). The method was coupled with electronic circular dichroism (CD), allowing the determination of the elution order on the basis of the sign of CD signals of the single enantiomers at 285 nm (positive for DXM, negative for LVM). Under the optimized conditions, the validated method was applied to the identification and quantitation of the enantiomers of methorphan in samples of different sources of illicit drugs of abuse (heroin). DXM was found in eight seized samples of street heroin; two of these samples, for which the DXM content was found to be over 5% (w/w) and exceeding a 10% (w/w) ratio with respect to diacetylmorphine, were the cause of two deaths for overdose due to acute narcotism.