Determination and distribution of clotiapine (Entumine) in human plasma, post-mortem blood and tissue samples from clotiapine-treated patients and from autopsy cases

The antipsychotic drug clotiapine (Entumine^®) has been marketed for more than 35 years, however there is little published data on the therapeutic and toxic concentrations of this drug. To fill this gap, two rapid and sensitive methods were developed for the determination of clotiapine (2-chloro-11-(4-methyl-1-piperazinyl)dibenzo-[b,f][1,4]-thiazepine), in human plasma and post-mortem blood and tissue samples. After simple liquid–liquid extraction at pH 9.5 with n-hexane/dichloromethane (85/15, v/v), clotiapine was quantitated by HPLC-DAD and by GC-NPD. The calibration curve was linear between 10 and 1000 μg/L. The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 2 and 6 μg/L for the GC-NPD method and 5 and 15 μg/L for the HPLC-method, respectively. These methods were applied to 12 plasma samples from patients treated with clotiapine, to seven autopsy cases and to one case of driving under the influence of drugs (DUID). Concentrations ranged for the clotiapine-treated patients between 6 and 155 μg/L (mean 46 μg/L), and for the autopsy cases between 22 and 341 μg/L (mean 123 μg/L).     

Distribution of dimethoate in the body after a fatal organphosphateintoxication

Many organophosphate pesticides (OPs) such as dimethoate are used to eradicate household pests, and those occurring in agriculture and forestry sectors. Combinations of two or more different insecticides have been manufactured to increase their effectiveness. A case of death is presented as suspected organophosphates intoxication. Autopsy was unremarkable except for grayish fluid in the stomach, with garlicky odor. A systematic toxicological analysis on post-mortem specimens revealed high concentrations of dimethoate in blood 38 μg/mL, urine 0.47 μg/mL, brain 2.2 μg/g, myocardial muscle 7.6 μg/g, liver 4.6 μg/g, lung 7.6 μg/g, skeletal muscle 21 μg/g, kidney 55 μg/g and gall bladder 31 μg/g. Blood alcohol was 2.85 g/L, cyclohexanone and cyclohexanol were also detected in the blood but not quantified. The cause of death was determined as organophosphate intoxication.     

Post-injection hybridization of complementary DNA strands on capillary electrophoresis platforms: A novel solution for dsDNA artifacts

Several laboratories have reported the occurrence of a split or n − 1 peak at the vWA locus in PowerPlex^® 16 and PowerPlex^® ES amplification products separated on 4- and 16-capillary electrophoresis instruments. The root cause of this artifact is post-PCR reannealing of the unlabeled, unincorporated vWA primer to the 3′-end of the tetramethylrhodamine (TMR)-labeled strand of the vWA amplicon. This reannealing occurs in the capillary post-electrokinetic injection. The split peak is eliminated by incorporation into the loading cocktail of a sacrificial hybridization sequence (SHS) oligonucleotide that is complementary to the vWA primer. The SHS preferentially anneals to the primer instead of the TMR-labeled strand of the vWA amplicon. In addition, the n − 10/n − 18 artifact that may be seen at the vWA locus was determined to be due to double-stranded amplicon formed post-electrokinetic injection into the capillary. This was also eliminated by adding in two Complementary Oligo Targets (COT1 and COT2) in addition to the SHS oligonucleotide into the loading cocktail. These three oligonucleotides are complementary to the 33 bases at the 5′-end of the unlabeled vWA amplicon strand and the 60 bases at its 3′-end and therefore compete for hybridization to the TMR-labeled amplicon strand. Incorporation of these three oligonucleotides in the Internal Lane Standard 600 (ILS600) eliminate both the split peak and n − 10/n − 18 artifact in PowerPlex^® 16 and PowerPlex^® ES amplification products without affecting sizing of alleles at the vWA locus or any locus in the PowerPlex^® 16, PowerPlex^® Y, PowerPlex^® ES, AmpFlSTR^® Profiler Plus^® ID, AmpFlSTR^® Cofiler^®, and AmpFlSTR^® SGM Plus^® kits.     

Fatal intoxication with tianeptine (Stablon)

Tianeptine (Stablon), although structurally similar to tricyclic antidepressants, acts by enhancing the reuptake of serotonin. A fatal case is presented involving a 26-year-old man, found lying in bed with a "mushroom of foam" around his mouth. Empty blister packs of Stablon and a suicide note were found next to the body. A liquid-liquid extraction procedure with n-hexane: ethyl acetate and n-hexane: 2-propanol, followed by LC-DAD-MS analysis, using positive mode electrospray ionization was performed. The detection limit was 0.001 microg/mL. The toxicological results revealed the following tianeptine concentrations in the post-mortem samples: blood 5.1 microg/mL; urine 2.0 microg/mL; liver 23 microg/g; stomach contents 22 mg. Femoral blood analyses also revealed an ethanol concentration of 0.53 g/L. The present method was also developed and validated for the other post-mortem specimens, since no previous published data had confirmed the post-mortem distribution of tianeptine. The absence of other suitable direct causes of death (macroscopic or histological) and the positive results achieved with the toxicological analysis led the pathologist to rule that death was due to an intoxication caused by the suicidal ingestion of tianeptine in combination with alcohol.     

Analysis of Δ-tetrahydrocannabinol in oral fluid samples using solid-phase extraction and high-performance liquid chromatography–electrospray ionization mass spectrometry

An analytical method using solid-phase extraction (SPE) and high-performance liquid chromatography–mass spectrometry (LC–MS) has been developed and validated for the confirmation of Δ⁹-tetrahydrocannabinol (THC) in oral fluid samples. Oral fluid was extracted using Bond Elut LRC-Certify solid-phase extraction columns (10 cm³, 300 mg) and elution performed with n-hexane/ethyl acetate. Quantitation made use of the selected ion-recording mode (SIR) using the most abundant characteristic ion [THC + H⁺], m/z 315.31 and the fragment ion, m/z 193.13 for confirmation, and m/z 318.00 for the protonated internal standard, [d₃-THC + H⁺]. The method proved to be precise for THC, in terms of both intra-day and inter-day analyses, with coefficients of variation less than 10%, and the calculated extraction efficiencies for THC ranged from 76 to 83%. Calibration standards spiked with THC between 2 and 100 ng/mL showed a linear relationship (r² = 0.999). The method presented was applied to the oral fluid samples taken from the volunteers during the largest music event in Portugal, named Rock in Rio-Lisboa. Oral fluid was collected from 40 persons by expectoration and with Salivette^®. In 55% of the samples obtained by expectorating, THC was detected with concentration ranges from 1033 to 6552 ng/mL and in 45% of cases THC was detected at concentrations between 51 and 937 ng/mL. However, using Salivette^® collection, 26 of the 40 cases had an undetectable THC.     

Development and validation of the Cozart DDS oral fluid collection device

The testing of oral fluid for drugs of abuse has increased significantly over recent years and is now commonplace in drug rehabilitation clinics, the workplace, prisons and custody suites. The global problem of identifying drugged drivers has also led to an increase in oral fluid testing at the roadside. The main requirements for the implementation of roadside drug testing are a rapid sample collection time, collection of a known sample volume and recovery of drugs from the collection device. We report here the development of the Cozart^® DDS oral fluid collector, an oral fluid collector that combines rapid and adequate sample collection with satisfactory drug recovery. Oral fluid was collected from drug users (n = 134) and drug-free individuals (n = 137), using the Cozart^® DDS oral fluid collector. The mean time for the completion of collection (full coloration of the sample presence indicator) was 34 s for drug-free individuals and 44 s for drug users. The average volume collected was 0.34 mL (n = 271). No chemical stimulant (to induce salivation) was used to achieve the collection times observed in either the drug-free or the drug-taking sample populations. Drugs were extracted from the collector using the Cozart^® DDS buffer and drug recovery was determined by Cozart^® enzyme immunoassays. The recovery studies showed that for amphetamine, Δ⁹THC, cocaine, methadone, methamphetamine, morphine and temazepam over 90% of the drug in the sample was eluted from the collector. The Cozart^® DDS oral fluid collector provides a reliable mechanism for the collection of oral fluid at the roadside that achieves the rapid collection times required.     

Simultaneous analysis of six amphetamines and analogues in hair, blood and urine by LC-ESI-MS/MS: Application to the determination of MDMA after low Ecstasy intake

A rapid and sensitive method using LC-MS/MS triple stage quadrupole for the determination of traces of amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”), 3,4-methylenedioxyethamphetamine (MDEA), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair, blood and urine has been developed and validated. Chromatography was carried out on an Uptisphere ODB C₁₈ 5 μm, 2.1 mm × 150 mm column (Interchim, France) with a gradient of acetonitrile and formate 2 mM pH 3.0 buffer. Urine and blood were extracted with Toxitube A^® (Varian, France). Segmented scalp hair was treated by incubation 15 min at 80 °C in NaOH 1 M before liquid–liquid extraction with hexane/ethyl acetate (2/1, v/v). The limits of quantification (LOQ) in blood and urine were at 0.1 ng/mL for all analytes. In hair, LOQ was <5 pg/mg for MA, MDMA, MDEA and MBDB, at 14.7 pg/mg for AP and 15.7 pg/mg for MDA. Calibration curves were linear in the range 0.1–50 ng/mL in blood and urine; in the range 5–500 pg/mg for MA, MDMA, MDEA and MBDB, and 20–500 pg/mg for AP and MDA. Inter-day precisions were <13% for all analytes in all matrices. Accuracy was <20% in blood and urine at 1 and 50 ng/mL and <10% in hair at 20 and 250 pg/mg. This method was applied to the determination of MDMA in a forensic case of single administration of ecstasy to a 16-year-old female without her knowledge during a party. She suffered from hyperactivity, sweating and agitation. A first sample of urine was collected a few hours after (T + 12 h) and tested positive to amphetamines by immunoassay by a clinical laboratory. Blood and urine were sampled for forensic purposes at day 8 (D + 8) and scalp hair at day 60 (D + 60). No MDMA was detected in blood, but urine and hair were tested positive, respectively at 0.42 ng/mL and at 22 pg/mg in hair only in the segment corresponding to the period of the offence, while no MDA was detectable. This method allows the detection of MDMA up to 8 days in urine after single intake.     

Drugs in oral fluid in randomly selected drivers

There were 13,176 roadside drug tests performed in the first year of the random drug-testing program conducted in the state of Victoria. Drugs targeted in the testing were methamphetamines and Δ⁹-tetrahydrocannabinol (THC). On-site screening was conducted by the police using DrugWipe^®, while the driver was still in the vehicle and if positive, a second test on collected oral fluid, using the Rapiscan^®, was performed in a specially outfitted “drug bus” located adjacent to the testing area. Oral fluid on presumptive positive cases was sent to the laboratory for confirmation with limits of quantification of 5, 5, and 2 ng/mL for methamphetamine (MA), methylenedioxy-methamphetamine (MDMA), and THC, respectively. Recovery experiments conducted in the laboratory showed quantitative recovery of analytes from the collector. When oral fluid could not be collected, blood was taken from the driver and sent to the laboratory for confirmation. These roadside tests gave 313 positive cases following GC–MS confirmation. These comprised 269, 118, and 87 cases positive to MA, MDMA, and THC, respectively. The median oral concentrations (undiluted) of MA, MDMA, and THC was 1136, 2724, and 81 ng/mL. The overall drug positive rate was 2.4% of the screened population. This rate was highest in drivers of cars (2.8%). The average age of drivers detected with a positive drug reading was 28 years. Large vehicle (trucks over 4.5 t) drivers were older; on average at 38 years. Females accounted for 19% of all positives, although none of the positive truck drivers were female. There was one false positive to cannabis when the results of both on-site devices were considered and four to methamphetamines.     

Quantitative analysis of multiple illicit drugs in preserved oral fluid by solid-phase extraction and liquid chromatography–tandem mass spectrometry

We present a validated method for the simultaneous analysis of basic drugs which comprises a sample clean-up step, using mixed-mode solid-phase extraction (SPE), followed by LC–MS/MS analysis. Deuterated analogues for all of the analytes of interest were used for quantitation. The applied HPLC gradient ensured the elution of all the drugs examined within 14 min and produced chromatographic peaks of acceptable symmetry. Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions for the non-deuterated analogues. Oral fluid was collected with the Intercept^®, a FDA approved sampling device that is used on a large scale in the US for workplace drug testing. However, this collection system contains some ingredients (stabilizers and preservatives) that can cause substantial interferences, e.g. ion suppression or enhancement during LC–MS/MS analysis, in the absence of suitable sample pre-treatment. The use of the SPE was demonstrated to be highly effective and led to significant decreases in the interferences. Extraction was found to be both reproducible and efficient with recoveries >76% for all of the analytes. Furthermore, the processed samples were demonstrated to be stable for 48 h, except for cocaine and benzoylecgonine, where a slight negative trend was observed, but did not compromise the quantitation. In all cases the method was linear over the range investigated (2–200 μg/L) with an excellent intra-assay and inter-assay precision (coefficients of variation <10% in most cases) for QC samples spiked at a concentration of 4, 12 and 100 μg/L. Limits of quantitation were estimated to be at 2 μg/L with limits of detection ranging from 0.2 to 0.5 μg/L, which meets the requirements of SAMHSA for oral fluid testing in the workplace. The method was subsequently applied to the analysis of Intercept^® samples collected at the roadside by the police, and to determine MDMA and MDA levels in oral fluid samples from a controlled study.     

Post-mortem cases involving amphetamine-based drugs in the Netherlands: Comparison with driving under the influence cases

In this study we reviewed the post-mortem cases in the years 1999–2004 that were presented at the Netherlands Forensic Institute. The concentrations of amphetamine-based drugs in femoral blood from cases of suspected unnatural death were compared with concentrations in whole blood from non-fatal cases of driving under the influence (DUI cases) and with literature. Furthermore, the combinations with other drugs and/or alcohol were investigated. Amphetamine-based drugs were present in 70 post-mortem cases and 467 DUI cases. The most detected amphetamine-based drug was MDMA, followed by amphetamine. The presence of MDA could usually be explained by metabolism of MDMA. Methamphetamine and MDEA were rarely present. Frequently, the amphetamine-based drugs were taken in combination with alcohol and/or other non-amphetamine-based drugs such as cocaine or cannabinoids. The 70 post-mortem cases were divided into 38 amphetamine-based drug caused (i.e. the amphetamine-based drug directly caused or contributed to the death) and 32 amphetamine-based drug related deaths (i.e. death was not directly caused by the amphetamine-based drug). In the latter category, other (poly)drug intoxications and death by violence or drowning were the most frequent causes of death.In 30 cases, MDMA caused death directly. The range in blood concentrations of MDMA in these cases was substantial, i.e. 0.41–84 mg/L with a median concentration of 3.7 mg/L (n = 30). MDMA blood concentrations in the MDMA related deaths (n = 20) and in the DUI cases (n = 360) varied up to 3.7 and 4.0 mg/L, respectively. Seven victims died from the direct effects of amphetamine; the blood concentration of amphetamine ranged from 0.24 to 11.3 mg/L, with a median concentration of 1.7 mg/L (n = 7). The median concentrations of amphetamine in the amphetamine related deaths (n = 13) and the DUI cases (n = 208) were much lower, i.e. 0.28 and 0.22 mg/L, respectively. Amphetamine blood concentrations up to 6.0 and 2.3 mg/L were seen in the drug related deaths and DUI cases, respectively. The most frequently encountered amphetamine-based drugs in the investigated deaths were MDMA and amphetamine. The majority of MDMA- and amphetamine-caused deaths, i.e. 90% of these deaths, occurred with blood concentrations above 1.5 and 0.80 mg/L, respectively. MDMA and amphetamine blood concentrations in drug related deaths and DUI cases, however, overlap the range of fatal concentrations. Therefore, MDMA or amphetamine concentrations should never be used alone to establish the cause of death.     

Carbohydrate-deficient transferrin (CDT): A reliable indicator of the risk of driving under the influence of alcohol when determined by capillary electrophoresis

Carbohydrate-deficient transferrin (CDT) is a marker of chronic alcohol abuse, which has recently been introduced to evaluate the physical fitness for obtaining a driving license. The aim of the present study was to evaluate the prevalence of elevated CDT levels in subjects stopped while driving under the influence of alcohol by using a validated method based on capillary electrophoresis. The study was carried out on a group of 40 drunken drivers (group A) and on a control group (n = 51) of subjects chosen from the general population (group B). CDT was directly determined by capillary electrophoresis in free solution and UV detection at 200 nm. CDT results from both groups were classified as “negative” or “positive” on the basis of the cut-off set at 2.00% (CDT index). The subjects classified as “positive” in group A were 24 (60%), whereas in group B were 2. The subjects classified as “negative” in group A were 16 (40%), whereas in group B was 49 (96.1%). The comparison of the observed percentages, evaluated with the χ²-test, was highly significant (p < 0.001). The present study confirms the high prevalence of chronic alcohol abusers among drunken drivers and the usefulness of CDT as a predictor of the risk of drunk driving.     

Oral fluid drug tests: Effects of adulterants and foodstuffs

An on-site oral fluid drug screen, Oratect^®, was used to investigate the effects of adulterants and foodstuffs on oral fluid test results. Common foods, beverages, food ingredients, cosmetics and hygienic products were demonstrated not to cause false positive results when tested 30 min after their consumption. Evaluations of two commercial oral fluid adulterants, “Clear Choice^® Fizzy Flush™” and “Test’in™ Spit n Kleen Mouthwash” suggest their mechanism of action is the clearing of residual drugs of abuse compounds through rinsing of the oral cavity. They do not directly destroy the drug compounds or change the pH of the oral fluid. It is also suggested that a common mouthwash would perform similar action.     

Vitality and age of conjunctival petechiae: The expression of P-selectin

Confocal laser scanning microscopy (CLSM) results in displaying multiple ruptures of capillaries and venules define conjunctival petechiae as a rhexis haemorrhage. In order to distinguish between petechiae which have arisen before death as opposed to post-mortem artefacts and the establishment of the age of the vessel's damage, we carried out an investigation on a collection of samples from medico-legal cases (positive petechiae: n = 65; post-mortem artefacts: n = 12; control: n = 19) to establish whether the expression of the endothelial adhesion molecule P-selectin as demonstrated by immunohistochemical methods is influenced by trauma before death and possibly by time. Except for cases with massive congestion in those cases where the victim had survived for around minutes, CLSM examination revealed a strong endothelial reaction at the site where the vessel had ruptured. It was impossible to detect a strong P-selectin expression signal in both those vessels which had not suffered any mechanical insult and those which had experienced post-mortem rupture. Therefore, the elevated expression of P-selectin can be used as a criterion to distinguish between those lesions which have occurred in life and those which are post-mortem artefacts.     

Post-mortem investigation of calcium content in liver, heart, and skeletal muscle in accidental hypothermia cases

The identification of hypothermia as cause of the death was always quite problematic in the field of forensic medicine. The aim of the present study was to verify the determination of calcium content in post-mortem liver, heart, and skeletal muscle samples as the biochemical marker defining hypothermia as the cause of death. The study involved 43 autopsy cases in which the circumstances of death indicated the effects of overcooling. The control group consisted of material collected from the corpses of 30 persons who were not exposed to low temperatures but died due to technical injuries (n = 5), asphyxia (n = 6), intoxication with ethanol and other substances (n = 8), and acute myocardial infarction/ischemia (n = 11). The concentration of calcium in autopsy samples was determined applying flame atomic absorption spectroscopy. Our study showed no significant differences of calcium content in tissues of persons who died due to hypothermia, over those who died in normothermic conditions.     

Toxicological analysis after asphyxial suicide with helium and a plastic bag

A 23-year-old man was found on a raised hide in lying position, the head wrapped in a plastic bag connected with a helium gas cylinder by a polypropylene tube. The autopsy did not show any specific findings nor did the routine toxicological analysis reveal significant information regarding the cause of death (BAC 0.9 mg/g, diphenhydramine 0.81 μg/ml in heart serum). For the detection of helium in the lungs, gas samples from both lungs were collected by a method ensuring minimal dilution. Gas analyses were performed using a GC–MS with a split–splitless injector and a headspace syringe. As carrier gas the commonly used helium was replaced by nitrogen. Helium was found in clearly elevated concentrations in gas samples from both lungs. Therefore, suffocation by breathing helium enriched, and thus oxygen deficient atmosphere, can strongly be assumed as the cause of death.     

Evaluation of amylase testing as a tool for saliva screening of crime scene trace swabs

Amylase testing has been used as a presumptive test for crime scene saliva for over three decades, mainly to locate saliva stains on surfaces. We have developed a saliva screening application for crime scene trace swabs, utilising an amylase sensitive paper (Phadebas^® Forensic Press test). Positive results were obtained for all tested dried saliva stains (0.5–32 μL) with high or intermediate amylase activity (840 and 290 kU/L). Results were typically obtained within 5 min, and all samples that produced DNA profiles were positive. However, salivary amylase activities, as well as DNA concentrations, vary significantly between individuals. We show that there is no correlation between amylase activity and amount of DNA in fresh saliva. Even so, a positive amylase result indicates presence of saliva, and thereby presence of DNA. Amylase testing may be useful for screening in investigations where the number of DNA analyses is limited due to cost, e.g., in volume crime.     

Application of solvent microextraction to the analysis of amphetamines and phencyclidine in urine

A fast and simple method to detect some commonly abused illicit drugs, amphetamine, methamphetamine, 3,4-methylendioxy-amphetamine (MDA), 3,4-methylendioxy-methamphetamine (MDMA), 3,4-methylendioxy-N-ethylamphetamine (MDEA) and phencyclidine (PCP) in urine using solvent microextraction (SME) combined with gas chromatography (GC) analysis has been developed. The extraction is conducted by suspending a 2 μl drop of chloroform in a 2 ml urine sample. Following 8 min of extraction, the organic solvent is withdrawn into the syringe and injected into a GC with a pulsed discharge helium ionization detector (PDHID).The effects of different extraction solvents and times, pH and sample preparation were studied. The optimized method was capable of detecting drugs in urine at concentrations below Substance Abuse and Mental Health Services Administration (SAMHSA) established cut-off values for preliminary testing. Good linearity and reproducibility of extraction were obtained. The limits of detection were 0.5 μg/ml for amphetamine, 0.1 μg/ml for methamphetamine and MDA, 0.05 μg/ml for MDMA, 0.025 μg/ml for MDEA and 0.015 μg/ml for PCP. Relative standard deviation (R.S.D.) values ranged between 5 and 20% for the studied drugs.