Confirmation by LC-MS of drugs in oral fluid obtained from roadside testing

The aim of this study was to assess the effectiveness of two current on-site oral fluid (OF) drug detection devices (OraLab and Dr?ger), as part of the Spanish participation in the Roadside Testing Assessment Project (ROSITA Project). The study was done in collaboration with the Spanish Traffic Police, in Galicia (NW Spain), during 2004 and 2005. A total of 468 drivers selected at the police controls agreed to participate through informed consent. In addition, saliva samples were collected and sent to the laboratory to confirm the on-site results. For this purpose, two different analytical liquid chromatography-mass spectrometry (LC-MS) methods were used to detect 11 drugs or metabolites in a 300 microL sample. Simultaneous analysis of morphine, 6-acetylmorphine, amphetamine, methamphetamine, MDA, MDMA, MDEA, MBDB, cocaine and benzoylecgonine was carried out using 100 microL of oral fluid, after an automated solid phase extraction. A different LC-MS method was performed to detect Delta(9)-THC in 200 microL of oral fluid using liquid-liquid extraction with hexane at pH 6. Both methods were fully validated, including linearity (1-250 ng/mL, 2-250 ng/mL) recovery (>50%), within-day and between-day precision (CV<15%), accuracy (mean relative error<15%), limit of detection (0.5 and 1 ng/mL), quantitation (1 and 2 ng/mL) and matrix effect. All of the positive cases and a random selection of 30% of the negatives were analyzed for confirmation analysis. Good results (sensitivity, specificity, accuracy, positive predictive value and negative predictive value>90%) were obtained for cocaine and opiates by OraLab, and for cocaine by Dr?ger. However, the results for the other compounds could be improved for both detection devices. Differences in the ease of use and in the interpretation mode (visual or instrumental) were observed.     

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.     

Evaluation of the IDS One-Step™ ELISA kits for the detection of illicit drugs in hair

This work presents the validation of a new immunological assay, the One-Step™ enzyme-linked immunosorbent assay (ELISA) tests from International Diagnostic Systems Corp. for the screening of drugs of abuse (cannabis, amphetamines, opiates, and cocaine) in human hair, with subsequent GC–MS confirmation. After decontamination and segmentation into small pieces, 50 mg of hair sample were incubated in 1 ml of methanol during 16 h at 40 °C. A 100 μL aliquot was collected and evaporated to dryness in presence of 100 μL of methanol/hydrochloric acid (99:1, v/v) to avoid amphetamines loss. The dried extract was dissolved in 100 μL of the “sample and standard diluent” solution included in the kit. This solution was submitted to analysis according to the recommended instructions of the manufacturer. During the validation phase, GC–MS confirmations were conducted according to our fully validated and published methods for opiates, cocaine, cannabis, and amphetamines determinations in hair. In a last development step, these procedures were slightly modified to directly confirm ELISA results by GC–MS using the methanolic extract. Ninety-three specimens were simultaneously screened by the ELISA tests (103 for tetrahydrocannabinol (THC)) and confirmed by GC–MS. Twenty were found positive for cannabis (THC: 0.10–6.50 ng/mg), 21 for cocaine (0.50–55.20 ng/mg), 24 for opiates (6-acetylmorphine (6-AM): 0.20–11.60 ng/mg, MOR: 0.20–8.90 ng/mg, codeine (COD): 0.20–5.90 ng/mg), and 13 for amphetamines (AP: 0.20 and 0.27 ng/mg, methamphetamine (MAP): 0.30 and 1.10 ng/mg, methylenedioxymethamphetamine (MDMA): 0.22–17.80 ng/mg). No false negative results were observed according to the Society of Hair Testing's (SoHT) cutoffs (0.5 ng/mg for cocaine, 0.2 ng/mg for opiates and amphetamines, and 0.1 ng/mg for THC). The One-Step™ ELISA kits appear suitable due to their sensitivity and specificity for drug of abuse screening in hair. This technology should find interest in workplace drug testing or driving license regranting, especially when many samples have to be tested with a high rate of negative samples, as ELISA is an easy and high-throughput method.     

Correlation of Delta9-tetrahydrocannabinol concentrations determined by LC-MS-MS in oral fluid and plasma from impaired drivers and evaluation of the on-site Dräger DrugTest

Oral fluid (collected with the Intercept((R)) device) and plasma samples were obtained from 139 individuals suspected of driving under the influence of drugs and analyzed for Delta(9)-tetrahydrocannabinol (THC), the major psychoactive constituent of cannabis, using a validated quantitative LC-MS-MS method. The first aim of the study was to investigate the correlation between the analytical data obtained in the plasma and oral fluid samples, to evaluate the use of oral fluid as a 'predictor' of actual cannabis influence. The results of the study indicated a good accuracy when comparing THC detection in oral fluid and plasma (84.9-95.7% depending on the cut-off used for plasma analysis). ROC curve analysis was subsequently used to determine the optimal cut-off value for THC in oral fluid with plasma as reference sample, in order to 'predict' a positive plasma result for THC. When using the LOQ of the method for plasma (0.5 ng/mL), the optimal cut-off was 1.2 ng/mL THC in oral fluid (sensitivity, 94.7%; specificity, 92.0%). When using the legal cut-off in Belgium for driving under the influence in plasma (2 ng/mL), an optimal cut-off value of 5.2 ng/mL THC in oral fluid (sensitivity, 91.6%; specificity, 88.6%) was observed. In the second part of the study, the performance of the on-site Dr?ger DrugTest for the screening of THC in oral fluid during roadside controls was assessed by comparison with the corresponding LC-MS-MS results in plasma and oral fluid. Since the accuracy was always less than 66%, we do not recommend this Dr?ger DrugTest system for the on-site screening of THC in oral fluid.     

Evaluation of the Cozart RapiScan drug test system for opiates and cocaine in oral fluid

The purpose of this study was to evaluate the efficiency of the Cozart^® RapiScan (CRS) drug test system for detecting opiates and cocaine in oral fluid. Oral fluid samples were collected using the Cozart^® RapiScan collection system from 358 donors who were receiving treatment for their addiction and were monitored for drug misuse. A further 103 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory using the two-panel Cozart^® RapiScan cartridge for opiates and cocaine and confirmed using gas chromatography–mass spectrometry (GC–MS). The samples were stored frozen at −20 °C until analysis by GC–MS. The overall accuracy of the CRS for both opiates and cocaine was 100%. Samples spiked at 50% above and below the cut-off consistently gave negative and positive results respectively. A total of 88 samples were positive for various opiates and 111 samples were positive for cocaine and/or its metabolites. The CRS for opiates and cocaine in oral fluid, using a cut-off of 30 ng/mL morphine or benzoylecgonine equivalents in neat oral fluid, had overall efficiencies of 98% and 99%, respectively, versus GC–MS. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.     

Validated method for the simultaneous determination of Δ-THC and Δ-THC-COOH in oral fluid, urine and whole blood using solid-phase extraction and liquid chromatography–mass spectrometry with electrospray ionization

A fully validated, sensitive and specific method for the extraction and quantification of Δ⁹-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-Δ⁹-THC (THC-COOH) and for the detection of 11-hydroxy-Δ⁹-THC (11-OH THC) in oral fluid, urine and whole blood is presented. Solid-phase extraction and liquid chromatography–mass spectrometry (LC–MS) technique were used, with electrospray ionization. Three ions were monitored for THC and THC-COOH and two for 11-OH THC. The compounds were quantified by selected ion recording of m/z 315.31, 329.18 and 343.16 for THC, 11-OH THC and THC-COOH, respectively, and m/z 318.27 and 346.26 for the deuterated internal standards, THC-d₃ and THC-COOH-d₃, respectively. The method proved to be precise for THC and THC-COOH both in terms of intra-day and inter-day analysis, with intra-day coefficients of variation (CV) less than 6.3, 6.6 and 6.5% for THC in saliva, urine and blood, respectively, and 6.8 and 7.7% for THC-COOH in urine and blood, respectively. Day-to-day CVs were less than 3.5, 4.9 and 11.3% for THC in saliva, urine and blood, respectively, and 6.2 and 6.4% for THC-COOH in urine and blood, respectively. Limits of detection (LOD) were 2 ng/mL for THC in oral fluid and 0.5 ng/mL for THC and THC-COOH and 20 ng/mL for 11-OH THC, in urine and blood. Calibration curves showed a linear relationship for THC and THC-COOH in all samples (r² > 0.999) within the range investigated.The procedure presented here has high specificity, selectivity and sensitivity. It can be regarded as an alternative method to GC–MS for the confirmation of positive immunoassay test results, and can be used as a suitable analytical tool for the quantification of THC and THC-COOH in oral fluid, urine and/or blood samples.     

Review: Pharmacokinetics of illicit drugs in oral fluid

This article reviews studies that have measured drug concentrations in oral fluid following controlled dosing regimens. A total of 23 studies have been identified over the last 15 years. These show that the amphetamines including designer amphetamines, cocaine, cannabis and cocaine are quickly found in oral fluid following dosing and usually have similar time-courses to that in plasma. Following common doses peak oral fluid concentrations exceed 0.1 μg/mL and often even 1 μg/mL. The drug concentration will depend on whether a dilution step occurs with buffer as part of the sampling procedure. The uses of collectors that stimulate oral fluid usually reduce the drug concentration compared to a non-stimulated manner. This reduction will not disadvantage the recipient since it will potentially reduce the detectablity of drug in oral fluid compared to non-stimulated collections. Only one recent study has been reported for a benzodiazepine. This showed nanogram per milliliter concentrations for flunitrazepam. More studies are required for benzodiazepines and indeed for other drugs, particularly in multiple drug situations and where disease may affect the pharmacokinetics of drugs.     

Determination of MDMA, MDA, MDEA and MBDB in oral fluid using high performance liquid chromatography with native fluorescence detection

This paper describes the analytical methodology for the determination of MDMA, MDA, MDEA and MBDB in oral fluid. After a liquid–liquid extraction, the analysis was carried out by high performance liquid chromatography (HPLC), with fluorescence detection. The detector wavelength was fixed at 285 nm for excitation and 320 nm for emission. The mobile phase, a mixture of phosphate buffer (pH = 5) and acetonitrile (75:25), and the column, Kromasil 100 C8 5 μm 250 mm × 4.6 mm, allowed good separation of the compounds in an isocratic mode in only 10 min. The method was validated and showed good limits of detection (2 ng/mL) and quantitation (10 ng/mL) for all the amphetamine derivatives. No interfering substances were detected. A stability study of these compounds in oral fluid stored at three different temperatures (−18, 4 and 20 °C) over 10 weeks was conducted, showing a time-dependent degradation of the four compounds.     

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.     

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.     

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.     

Oral fluid collection: The neglected variable in oral fluid testing

The potential to use oral fluid as a drug-testing specimen has been the subject of considerable scientific interest. The ease with which specimens can be collected and the potential for oral fluid (OF) drug concentrations to reflect blood–drug concentrations make it a potentially valuable specimen in clinical as well as forensic settings. However, the possible effects of the OF collection process on drug detection and quantification has often been over looked. Several studies have documented that drug-contamination of the oral cavity may skew oral fluid/blood drug ratios and confound interpretation when drugs are smoked, insufflated or ingested orally. OF pH is predicted to have an effect on the concentration of drugs in OF. However, in a controlled clinical study, the effect of pH was less than that of collection technique. Mean codeine OF concentrations in specimens collected a non-stimulating control method were 3.6 times higher than those in OF collected after acidic stimulation. Mean codeine concentrations were 50% lower than control using mechanical stimulation and 77% of control using commercial collection devices.Several factors should be considered if a commercial OF collection device is used. In vitro collection experiments demonstrated that the mean collection volume varied between devices from 0.82 to 1.86 mL. The percentage of the collected volume that could be recovered from the device varied from 18% to 83%. In vitro experiments demonstrated considerable variation in the recovery of amphetamines (16–59%), opiates (33–50%), cocaine and benzoylecgonine (61–97%), carboxy-THC (0–53%) and PCP (9–56%). Less variation in collection volume, volume recovered and drug recovery was observed intra-device. The THC stability was evaluated in a common commercial collection protocol. Samples in the collection buffer were relatively stable for 6 weeks when stored frozen. However, stability was marginal under refrigerated conditions and poor at room temperature. Very little has been published on the efficacy of using IgG concentration, or any other endogenous marker, as a measure of OF specimen validity. Preliminary rinsing experiments with moderate (50 mL and 2 × 50 mL) volumes of water did not reduce the OF IgG concentration below proposed specimen validity criteria. In summary, obvious and more subtle variables in the OF collection may have pronounced effects on OF–drug concentrations. This has rarely been acknowledged in the literature, but should to be considered in OF drug testing, interpretation of OF–drug results and future research studies.     

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.     

Development and Validation of a Method Using Dried Oral Fluid Spot to Determine Drugs of Abuse

A liquid chromatography–mass spectrometry method using dried oral fluid spots was developed and validated for the simultaneous quantification of cocaine, benzoylecgonine, cocaethylene, amphetamine, and 3,4‐methylenedioxymethamphetamine. The oral fluid was applied to a Whatman 903 grade paper and submitted to a drying time of 2.5 h. The extraction procedure was optimized by chemometric approach using simplex centroid design. Spots were extracted with a mixture of acetonitrile, buffer, and methanol. Calibration curves covered a linear concentration range of 40–500 ng/mL. Validation parameters of linearity, precision, accuracy, selectivity, carryover, matrix effects, and stability were evaluated and showed satisfactory results. Spot homogeneity was also satisfactory, with less than 15% of deviation from nominal concentration. Spot volume did not influence accuracy when less than 100 μL of the sample was applied to the spot. The validation of the proposed method suggests a potential application in different scenarios in toxicology.     

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.     

The impurity characteristics of methamphetamine synthesized by Emde and Nagai method

Impurity profiling and classification of seized methamphetamine may play an important role in the interpretation of analytical results, the determination of the synthetic method employed, and the criminal investigations of drug traffic routes. Our study is focused on classifying seized methamphetamine samples according to the groups sorted by the types and quantities of impurities present in illicit methamphetamine samples. The samples (100 mg) were dissolved in 2 mL of potassium phosphate buffer (pH 7.0), extracted with 200 μL of ethyl acetate under basic condition, and then analyzed by gas chromatography-mass spectrometry (GC–MS) with a DB-1 capillary column (30 m × 0.25 mm i.d., 0.25 μm). Five impurities are used as criteria for the classification of seized methamphetamine samples by Emde and Nagai method. A total of fifty-two samples of seized methamphetamine were analyzed by GC–MS and classified by five organic impurities, and then sorted into four groups, which are Nagai type, Emde Type, Undetermined I type, and Undetermined II type.     

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 μg/mL. The toxicological results revealed the following tianeptine concentrations in the post-mortem samples: blood 5.1 μg/mL; urine 2.0 μg/mL; liver 23 μg/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.     

Cannabinoids determination in oral fluid by SPME–GC/MS and UHPLC–MS/MS and its application on suspected drivers

The confirmation of Δ9-tetrahydrocannabinol (THC) in oral fluid (OF) is an important issue for assessing Driving Under the Influence of Drugs (DUID). The aim of this research was to develop a highly sensitive method with minimal sample pre-treatment suitable for the analysis of small OF volumes (100 μL) for the confirmation of cannabinoids in DUID cases. Two methods were compared for the confirmation of THC in residual OF samples, obtained from a preliminary on-site screening with commercial devices. An ultra high performance LC–MS (UHPLC–MS/MS) method and an SPME–GC/MS method were hence developed. 100 μL of the residual mixture OF/preservative buffer or neat OF was simply added to 10 μL of THC-D3 (1 μg/mL) and submitted to the two different analyses: A — direct injection of 10 μL in UHPLC–MS/MS in positive electrospray ionisation (ESI) mode and B — sampling for 30 min with SPME (100 μm polydimethylsiloxane or PDMS fibre) and direct injection by desorption of the fibre in the GC injection port.The lowest limit of detection (LLOD) of THC was 2 ng/mL in UHPLC–MS/MS and 0.5 ng/mL in SPME–GC/MS. In addition, cannabidiol (CBD) and cannabinol (CBN) could be detected in GC/MS equipment at 2 ng/mL, whilst in UHPLC–MS/MS the LLOD was 20 ng/mL.Both methods were applied to 70 samples coming from roadside tests. By SPME–GC/MS analysis, THC was confirmed in 42 samples, whilst CBD was detected in 21 of them, along with CBN in 14 samples. THC concentrations ranged from traces below the lowest limit of quantification or LLOQ (2 ng/mL) up to 690 ng/mL.     

Distribution profile of 2,5-dimethoxy-4-bromoamphetamine (DOB) in rats after oral and subcutaneous doses

2,5-Dimethoxy-4-bromoamphetamine (DOB) is one of the potent hallucinogenic phenylalkylamines, whose ingestion has already caused several deaths reported all over the world. However, there is unsufficient information on DOB properties based on controlled pharmacokinetic studies available. The aim of this study was to clarify the distribution profile of DOB and its phenolic metabolite 2-methoxy-5-hydroxy-4-bromoamphetamine (2M5H4BA) in blood and biological tissues of experimental rats. The rats were administered a 20 mg/kg dose of DOB·HCl by oral ingestion or subcutaneous injection. Plasma and brain, liver and lung tissues were collected at 0.5, 1, 2, 4, 8, 16, and 32 h after dosing (three animals per time point). The samples were prepared by a liquid–liquid extraction procedure and the extracts were assayed by GC–MS. After per oral application, DOB peak plasma level of 320 ng/mL was reached after one-hour post dosing as well as 2M5H4BA peak concentration of 203 ng/mL. A rapid phase of DOB absorption, 2M5H4BA formation and their tissue distribution during the first two hours after application were followed by a slow decrease rate of the elimination process until 32 h. After subcutaneous application, high plasma levels of the unchanged parent drug and relatively reduced formation of its metabolite 2M5H4BA were observed. DOB maximum plasma concentration of 1143 ng/mL was reached after one-hour post application, whereas its metabolite peak level after 8 h was 213 ng/mL. The concentration profiles of both compounds in plasma after per oral and subcutaneous administration revealed the existence of significant first pass effect after per oral administration that significantly affected DOB bioavailability. DOB tissue concentrations exceeded plasma and the highest values were found in the lungs, where drug accumulation occurred with prolonged retention till 32 h after subcutaneous dose. Although the plasma/tissue transfer was more effective for the lipophilic parent drug than for its hydroxylated metabolite 2M5H4BA, the metabolite tissue levels were significant. The hallucinogenic potential of 2M5H4BA appearing in brain remains unclear as nothing is known about its pharmacological activity at present.     

Evaluation of the Cozart RapiScan drug test system for opiates and cocaine in oral fluid

The purpose of this study was to evaluate the efficiency of the Cozart RapiScan (CRS) drug test system for detecting opiates and cocaine in oral fluid. Oral fluid samples were collected using the Cozart RapiScan collection system from 358 donors who were receiving treatment for their addiction and were monitored for drug misuse. A further 103 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory using the two-panel Cozart RapiScan cartridge for opiates and cocaine and confirmed using gas chromatography-mass spectrometry (GC-MS). The samples were stored frozen at -20 degrees C until analysis by GC-MS. The overall accuracy of the CRS for both opiates and cocaine was 100%. Samples spiked at 50% above and below the cut-off consistently gave negative and positive results respectively. A total of 88 samples were positive for various opiates and 111 samples were positive for cocaine and/or its metabolites. The CRS for opiates and cocaine in oral fluid, using a cut-off of 30 ng/mL morphine or benzoylecgonine equivalents in neat oral fluid, had overall efficiencies of 98% and 99%, respectively, versus GC-MS. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.