Cannabinoids in blood and urine after passive inhalation of Cannabis smoke

To test the possibility that cannabinoids are detectable following passive inhalation of Cannabis smoke the following study was performed. Five healthy volunteers who had previously never used Cannabis, passively inhaled Cannabis smoke for 30 min. Cannabis smoke was provided by other subjects smoking either marijuana or hashish cigarettes in a small closed car, containing approximately 1650 L of air. delta 9-Tetrahydrocannabinol (THC) could be detected in the blood of all passive smokers immediately after exposure in concentrations ranging from 1.3 to 6.3 ng/mL. At the same time total blood cannabinoid levels (assayed by radioimmunoassay [RIA] ) were higher than 13 ng/mL in four of the volunteers. Both THC and cannabinoid blood concentrations fell close to the cutoff limits of the respective assays during the following 2 h. Passive inhalation also resulted in the detection of cannabinoids in the urine by RIA and enzyme multiple immunoassay technique (EMIT) assays (above 13 and 20 ng/mL, respectively). It is concluded that the demonstration of cannabinoids in blood or urine is no unequivocal proof of active Cannabis smoking.     

Passive exposure in detection of low blood and urine cannabinoid concentrations

Whenever small amounts of drugs are present in blood or urine samples, especially of substances that are preferentially smoked such as cannabinoids, the discrimination between active and passive inhalation may cause severe problems. The statement of a passive exposure by marijuana smoke has been scrutinized reviewing the literature. The pharmacokinetics of smoked marijuana as well as experimental data on cannabinoid concentrations in plasma and urine samples following passive exposure are summarized. As a conclusion it seems urgent to enlarge the existing data base.     

Urinary excretion profiles of 11-nor-9-carboxy-Delta9-tetrahydrocannabinol: a Delta9-THC-COOH to creatinine ratio study #2

Subjects with a history of chronic marijuana use were screened for cannabinoids in urine specimens with the EMIT((R)) II Plus cannabinoids assay with a cut-off value of 50 ng/ml. All presumptively positive specimens were submitted for confirmatory analysis for the major urinary cannabinoid metabolite (Delta(9)-THC-COOH) by GC-MS with a cut-off value of 15 ng/ml. Creatinine was analyzed in each specimen as an index of dilution. Huestis and Cone [J. Anal. Toxicol. 22 (1998) 445] reported that serial monitoring of Delta(9)-THC-COOH to creatinine ratios in paired urine specimens collected at least 24h apart could differentiate new drug use from residual Delta(9)-THC-COOH excretion. The best accuracy (85.4%) for predicting new marijuana use was a Delta(9)-THC-COOH/creatinine ratio > or =0.5 (dividing the Delta(9)-THC-COOH to creatinine ratio of specimen 2 by the specimen 1 ratio). In a previous study in this laboratory [J. Anal. Toxicol. 23 (1999) 531], urine specimens were collected from chronic marijuana users at least 24h apart and dilute urine specimens (creatinine values <2.2 micromol/l) were excluded from the data analysis. The objective of the present study was to determine whether creatinine corrected urine specimens positive for cannabinoids could differentiate new marijuana use from the excretion of residual Delta(9)-THC-COOH in chronic users of marijuana based on the Huestis 0.5 ratio. Urine specimens (N=946) were collected from 37 individuals with at least 48h between collections. All urine specimens were included in the data review irrespective of creatinine concentration. The mean urinary Delta(9)-THC-COOH concentration was 302.4 ng/ml, mean Delta(9)-THC-COOH/creatinine ratio (ng/ml Delta(9)-THC-COOH/(mmol/l) creatinine) was 29.3 and the Huestis ratio calculation indicated new drug use in 83% of all sequentially paired urine specimens. The data were sub-divided into three groups (A-C) based on the mean Delta(9)-THC-COOH/creatinine values. Interindividual Delta(9)-THC-COOH/creatinine mean values ranged from 2.2 to 13.8 in group A (264 specimens, N=15 subjects) where 80.7% of paired specimens indicated new drug use. In group B, mean Delta(9)-THC-COOH/creatinine values ranged from 15.3 to 37.8 in 444 specimens (N=14 subjects) and 83.3% of paired specimens indicated new drug use. In group C, individual mean Delta(9)-THC-COOH/creatinine values were >40.1 (41.3-132.5) in 238 urine specimens (N=8 subjects) and 85.3% of paired urine specimens indicated new marijuana use. Correcting Delta(9)-THC-COOH excretion for urinary dilution and comparing Delta(9)-THC-COOH/creatinine concentration ratios of sequentially paired specimens (collected at least 48h apart) provided an objective indicator of new marijuana use in this population.     

Time of drug elimination in chronic drug abusers. Case study of 52 patients in a "low-step" detoxification ward

The elimination time of illicit drugs and their metabolites is of both clinical and forensic interest. In order to determine the elimination time for various drugs and their metabolites we recruited 52 volunteers in a protected, low-step detoxification program. Blood samples were taken from each volunteer for the first 7 days, daily, urine sample for the first 3 weeks, daily. Urine was analyzed using a fluorescence-polarization immunoassay (FPIA) and gas chromatography/mass spectrometry (GC/MS), serum using GC/MS. The elimination times of the drugs and/or their metabolites in urine and serum as well as the tolerance intervals/confidence intervals were determined. Due to the sometimes extremely high initial concentrations and low cut-off values, a few of the volunteers had markedly longer elimination times than those described in the literature. The cut-off values were as follows: barbiturates II (200ng/ml), cannabinoids (20ng/ml), cocaine metabolites (300ng/ml), opiates (200ng/ml). GC/MS detected the following maximum elimination times: total morphine in urine up to 270.3h, total morphine and free morphine in serum up to 121.3h, monoacetylmorphine in urine up to 34.5h, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in urine up to 433.5h, THC-COOH in serum up to 74.3h, total codeine in urine up to 123h, free codeine in urine up to 97.5h, total codeine in serum up to 29h, free codeine in serum up to 6.3h, total dihydrocodeine (DHC) in urine up to 314.8h, free DHC in urine up to 273.3h, total and free DHC in serum up to 50.1h. Cocaine and its metabolites were largely undetectable in the present study.     

Correlations on radioimmunoassay, fluorescence polarization immunoassay, and enzyme immunoassay of cannabis metabolites with gas chromatography/mass spectrometry analysis of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid in urine specimens.

Results obtained from three commercial immunoassay kits, Abuscreen, TDx, and EMIT, commonly used for the initial test of urine cannabinoids (and metabolites) were correlated with the 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (9-THC-COOH) concentration as determined by GC/MS. Correlation coefficients obtained based on 26 (out of 1359 total sample population) highly relevant samples, are 0.601 and 0.438 for Abuscreen and TDx. Correlation coefficients obtained from a parallel study on a different set of 47 (out of 5070 total sample population) highly relevant specimens are 0.658 and 0.575 for Abuscreen and Emit. The immunoassay concentration levels, that correspond to the commonly used 15 ng/ml GC/MS cutoff value for 9-THC-COOH, as calculated from the regression equations are 82 ng/ml and 75 ng/ml for TDx and EMIT and 120 ng/ml and 72 ng/ml for Abuscreen manufactured at two different time periods. The difference of these calculated corresponding concentrations provides quantitative evidence of the reagent specificity differences.     

Urinary excretion profiles of 11-nor-9-carboxy-Delta9-tetrahydrocannabinol. Study III. A Delta9-THC-COOH to creatinine ratio study

Huestis and Cone reported in [J. Anal. Toxicol. 22 (1998) 445] that serial monitoring of Delta9-THC-COOH/creatinine ratios in paired urine specimens collected at least 24h apart could differentiate new drug use from residual Delta(9)-THC-COOH excretion following acute marijuana use in a controlled setting. The best accuracy (85.4%) for predicting new marijuana use was for a Delta(9)-THC-COOH/creatinine ratio > or = 0.5 (dividing the Delta9-THC-COOH/creatinine ratio of specimen no. 2 by the specimen no. 1 ratio). In previous studies in this laboratory [J. Anal. Toxicol. 23 (1999) 531 and Forensic Sci. Int. 133 (2003) 26], urine specimens were collected from chronic marijuana users > or = 24 h or > = 48 h apart in an uncontrolled setting. Subjects with a history of chronic marijuana use were screened for cannabinoids with the EMIT II Plus cannabinoids assay (cut-off 50 ng/ml) followed by confirmation for Delta9-THC-COOH by GC-MS (cut-off 15 ng/ml). Creatinine was analyzed as an index of dilution. The objective of the present study was to evaluate whether creatinine corrected specimens could differentiate new marijuana or hashish use from the excretion of residual Delta(9)-THC-COOH in chronic marijuana users based on the Huestis 0.5 ratio. Urine specimens (N=376) were collected from 29 individuals > or = 96 h between urine collections. The mean urinary Delta9-THC-COOH concentration was 464.4 ng/ml, mean Delta9-THC-COOH/creatinine ratio (ng/(ml Delta9-THC-COOH mmoll creatinine)) was 36.8 and the overall mean Delta9-THC-COOH/creatinine ratio of specimen 2/mean Delta9-THC-COOH/creatinine ratio of specimen 1 was 1.37. The Huestis ratio calculation indicated new drug use in 83% of all sequentially paired urine specimens. The data were sub-divided into three groups (Groups A-C) based on mean Delta9-THC-COOH/creatinine values. Interindividual mean Delta9-THC-COOH/creatinine values ranged from 4.7 to 13.4 in Group A where 80% of paired specimens indicated new drug use (N=10) and 20.4-39.6 in Group B where 83.6% of paired specimens indicated new drug use (N=7). Individual mean Delta9-THC-COOH/creatinine values ranged from 44.2 to 120.2 in Group C where 84.5% of paired urine specimens indicated new marijuana use (N=12). Correcting Delta9-THC-COOH excretion for urinary dilution and comparing Delta9-THC-COOH/creatinine concentration ratios of sequentially paired specimens (collected > or = 96 h apart) may provide an objective indicator of ongoing marijuana or hashish use in this population.     

Comparison of nonradioactive microtiter plate enzyme immunoassays for the sensitive detection of fentanyl

Fentanyl is a very strong opioid with analgesic properties that are approximately 80 times stronger than those of morphine and therefore is used in major surgery and treatment of pain in tumor patients. Cases of fentanyl abuse by intravenous injection, inhalation, oral or nasal application have been reported especially in the USA. Therapeutic levels of fentanyl are as low as 1 ng/ml of serum and therefore a screening test must have a detection limit below that concentration. Recently three non-radioactive enzyme immunoassays (EIAs) have become commercially available from COZART, STC and DIAGNOSTIX, all of them supplied by MAHSAN Diagnostika for evaluation with serum samples from forensic and clinical cases. A calibration curve is obtained with samples that contain 0, 0.1, 0.5, 1 and 5 ng fentanyl per ml of negative serum. The calibration curve of COZART is especially in the low range, steeper than those of STC and DIAGNOSTIX. The cut-off for all these EIAs, however, can be set at 0.5 ng/ml. After the administration of therapeutic doses, fentanyl concentrations were between 3 and more than 5 ng/ml as determined with the EIAs. The presence of the typical drugs of abuse, e.g. heroin, methadone, cocaine, cannabinoids and amphetamines including the derivatives of methylenedioxyamphetamine, don't generate false-positive results. No cross-reactivity was also observed at toxic levels of benzodiazepines and paracetamol and therapeutic levels of barbiturates, phenothiazines, antidepressants and analgesics. The EIAs tested so far appear to be suitable for the detection of fentanyl at therapeutic levels. False-positive results or cross-reactivity towards other compounds have not been observed.     

Direct ELISA kits as a sensitive and selective screening method for abstinence control in urine

In 2009 cutoff values of assessment criteria to testify abstinence control in order to estimate driving ability were standardized in Germany. The cutoff values are lower than required in existing guidelines like SAMHSA and there is critical discussion about detection of low concentrations by using immunoassay, especially concerning amphetamines in urine (50 ng/ml). In this study Direct ELISA kits were tested for their applicability to identify the absence of amphetamines, cannabinoids, opiates, cocaine, methadone and benzodiazepines in urine. Results were confirmed by LC/MS or GC/MS analyses. Sensitivity, specificity, predictive values (positive as well as negative) and overall misclassification rates were evaluated by contingency tables and were compared to ROC-analyses. Sensitivity results as well as specificity results were satisfying showing sensitivity values higher than 96% for each analyte. The amphetamine test we used showed sensitivity and specificity of 100% and 88%, respectively, even if amphetamine tests usually react with high cross-reactivity. Our study results include high discrimination at required cutoff values between positives and negatives for each drug group and demonstrate that immunological tests complying with requirements of current decreased urine cutoff values for assessment of driving ability do exist.     

Concentrations of unconjugated morphine, codeine and 6-acetylmorphine in urine specimens from suspected drugged drivers

Concentrations of unconjugated morphine, codeine and 6-acetylmorphine (6-AM), the specific metabolite of heroin, were determined in urine specimens from 339 individuals apprehended for driving under the influence of drugs (DUID) in Sweden. After an initial screening analysis by immunoassay for 5-classes of abused drugs (opiates, cannabinoids, amphetamine analogs, cocaine metabolite and benzodiazepines), all positive specimens were verified by more specific methods. Opiates and other illicit drugs were analyzed by isotope-dilution gas chromatography-mass spectrometry (GC-MS). The limits of quantitation for morphine, codeine and 6-AM in urine were 20 ng/mL. Calibration plots included an upper concentration limit of 1000 ng/mL for each opiate. We identified the heroin metabolite 6-AM in 212 urine specimens (62%) at concentrations ranging from 20 ng/mL to > 1000 ng/mL. The concentration of 6-AM exceeded 1000 ng/mL in 79 cases (37%) and 31 cases (15%) were between 20 and 99 ng/mL. When 6-AM was present in urine the concentration of morphine was above 1000 ng/mL in 196 cases (92%). The concentrations of codeine in these same urine specimens were more evenly distributed with 35% being above 1000 ng/mL and 21% below 100 ng/mL. These results give a clear picture of the concentrations of unconjugated morphine, codeine and 6-acetylmorphine that can be expected in opiate-positive urine specimens from individuals apprehended for DUID after taking heroin.     

Urinary excretion profiles of 11-nor-9-carboxy-delta9-tetrahydrocannabinol and 11-hydroxy-delta9-THC: cannabinoid metabolites to creatinine ratio study IV

The objective of this study was to compare urinary excretion patterns of two cannabinoid metabolites in subjects with a history of chronic marijuana use. The first metabolite analyzed was nor-9-carboxy-delta9-tetrahydrocannabinol (delta9-THC-COOH), the major urinary cannabinoid metabolite that is pharmacologically inactive. The second metabolite 11-OH-delta9-THC is an active cannabinoid metabolite and is not routinely measured. Urine specimens were collected from four subjects on 12-20 occasions > or = 96 h apart in an uncontrolled clinical setting. Creatinine was analyzed in each urine specimen by the colorimetric modified Jaffé reaction on a SYVA 30R biochemical analyzer. All urine specimens analyzed for 11-OH-delta9-THC had screened positive for cannabinoids with the EMIT II Plus cannabinoids assay (cut-off 50 ng/mL) on a SYVA 30R analyzer and submitted for delta9-THC-COOH confirmation by GC-MS (cut-off concentration 15 ng/mL). Eleven-OH-delta9-THC was measured by GC-MS with a cut-off concentration of 3 ng/mL. Both GC-MS methods for cannabinoid metabolites used deuterated internal standards for quantitative analysis. The mean (range) of urinary delta9-THC-COOH concentration was 1153 ng/mL (78.7-2634) with a cut-off of 15 ng/mL. The mean (range) of delta9-THC-COOH/creatinine ratios (ng/mL delta9-THC-COOH/mmol/L creatinine) was 84.1 (8.1-122.1). The mean (range) urinary of 11-OH-delta9-THC concentration was 387.6 ng/mL (11.9-783) with a cut-off of 3 ng/mL, and the mean (range) of 11-OH-delta9-THC/creatinine ratio (ng/mL 11-OH-delta9-THC/mmol/L creatinine) was 29.7 (1.2-40.7). Of the 63 urine specimens submitted for delta9-THC-COOH confirmation by GC-MS, 59/63 urine specimens (94%) were positive for delta9 -THC-COOH and 51/63 (81%) were positive for 11-OH-delta9-THC. Overall, the concentrations of 11-OH-delta9-THC in urine specimens collected > or = 96 h apart were lower than delta9-THC-COOH concentrations in 50/51 of the urine specimens in this population. Further urinary cannabinoid excretion studies are needed to assess whether 11-OH-delta9-THC analyses have a role when assessing previous marijuana or hashish use in chronic users whose urine specimens remain positive for delta9-THC-COOH for an extended period of time after last drug use.     

Deaths after intravenous misuse of transdermal fentanyl

Fentanyl is a potent synthetic opioid used as a general anesthetic and analgetic. Fatal outcome from intravenous misuse of transdermal fentanyl is rare, and there are few such reports in literature. Here we report two cases of fatal intravenous injection of the content from fentanyl patches. Both were male drug addicts, found dead within a one week interval in the same apartment. Post-mortem femoral blood was screened for amphetamines, cannabinoids, cocaine, and opioids with immunological methods (EMIT II) and further with headspace gas chromatography for alcohol and with liquid chromatography mass spectrometry (LC-MS) for different drugs, including fentanyl. Confirmatory analysis of fentanyl and morphine was performed by gas chromatography-mass spectrometry (GC-MS). In the first case, the toxicological analysis revealed fentanyl (2.7 ng/mL), morphine (31.4 ng/mL), and ethanol (1.1 g/L) in postmortem blood and amphetamine, cannabinoids, morphine, and ethanol (1.4 g/L) in postmortem urine. In the second case, the analysis revealed fentanyl (13.8 ng/mL), 7-aminoclonazepam (57.1 ng/mL), and sertralin (91.9 ng/mL) in postmortem blood and a small amount of ethanol (0.1 g/L) in postmortem urine. Police investigation revealed that both the deceased had bought the patches from the same source. The present cases demonstrate the possibility of intravenous misuse of transdermal patches and the risk of fatal outcome.     

用气相色谱法/电子捕获检测器测定尿液中的三唑仑

建立了用气相色谱／电子捕获检测器测定尿液中三唑仑含量的方法。2ml尿样在破性条件下用2ml×2氯仿提取净化后,60℃水浴下用空气吹干,残留物用环己烷定容溶解后,进气相色谱仪分析,三唑仑的保留时间为10．74min。最低检测限为0．5ng／ml,回收率为95．98％,变异系数为7．85％（n＝5）。在2～50ng／ml浓度范围内有良好的线性关系：A＝－67．9＋570．IC,r＝0．9939。     

The determination of clozapine in blood and urine by gas chromatography-mass spectrometry]

A method for measuring closapine in the blood and urine by gas chromatography-mass spectrometry as a trifluoroacetic derivative is proposed. The threshold level for closapine detection is 25 ng/ml in the blood and 30 ng/ml in the urine. Calibration curves are linear in the range 0.025-5 mcg/ml for the blood and 0.03-50 mcg/ml for the urine. The method can be used in forensic chemical and clinical toxicological analysis.     

Optimization and validation of CEDIA drugs of abuse immunoassay tests in serum on Hitachi 912

Due to sensitive limits of detection of chromatographic methods and low limit values regarding the screening of drugs under the terms of impairment in safe driving (§ 24a StVG, Street Traffic Law in Germany), preliminary immunoassay (IA) tests should be able to detect also low concentrations of legal and illegal drugs in serum in forensic cases. False-negatives should be avoided, the rate of false-positive samples should be low due to cost and time. An optimization of IA cutoff values and a validation of the assay is required for each laboratory. In a retrospective study results for serum samples containing amphetamine, methylenedioxy derivatives, cannabinoids, benzodiazepines, cocaine (metabolites), methadone and opiates obtained with CEDIA drugs of abuse reagents on a Hitachi 912 autoanalyzer were compared with quantitative results of chromatographic methods (gas or liquid chromatography coupled with mass spectrometry (GC/MS or LC/MS)). Firstly sensitivity, specificity, positive and negative predictive values and overall misclassification rates were evaluated by contingency tables and compared to ROC-analyses and Youden-Indices. Secondly ideal cutoffs were statistically calculated on the basis of sensitivity and specificity as decisive statistical criteria with focus on a high sensitivity (low rates of false-negatives), i.e. using the Youden-Index. Immunoassay (IA) and confirmatory results were available for 3014 blood samples. Sensitivity was 90% or more for nearly all analytes: amphetamines (IA cutoff 9.5 ng/ml), methylenedioxy derivatives (IA cutoff 5.5 ng/ml), cannabinoids (IA cutoff 14.5 ng/ml), benzodiazepines (IA cutoff >0 ng/ml). Test of opiates showed a sensitivity of 86% for a IA cutoff value of >0 ng/ml. Values for specificity ranged between 33% (methadone, IA cutoff 10 ng/ml) and 90% (cocaine, IA cutoff 20 ng/ml). Lower cutoff values as recommended by ROC analyses were chosen for most tests to decrease the rate of false-negatives. Analyses enabled the definition of cutoff values with good values for sensitivity. Small rates of false-positives can be accepted in forensic cases.     

Near‐fatal Intoxication with the “New” Synthetic Opioid U‐47700: The First Reported Case in the Czech Republic

Recreational use of the potent synthetic opioid 3,4‐ dichloro‐N‐(2‐(dimethylamino)cyclohexyl)‐N‐methylbenzamide (U‐47700) is rising, accompanied by increasingly frequent cases of serious intoxication. This article reports a case of near‐fatal U‐47700 intoxication. A man was found unconscious (with drug powder residues). After 40 h in hospital (including 12 h of supported ventilation), he recovered and was discharged. Liquid chromatography/high‐resolution mass spectrometry (LC/HRMS) or gas chromatography/mass spectrometry (GC/MS) were used to detect and quantify substances in powders, serum and urine. Powders contained U‐47700 and two synthetic cannabinoids. Serum and urine were positive for U‐47700 (351.0 ng/mL), citalopram (<LOQ), tetrahydrocannabinol (THC: 3.3 ng/mL), midazolam (<LOQ) and a novel benzodiazepine, clonazolam (6.8 ng/mL) and their metabolites but negative for synthetic cannabinoids. If potent synthetic opioids become cheaper and more easily obtainable than their classical counterparts (e.g., heroin), they will inevitably replace them and users may be exposed to elevated risks of addiction and overdose.     

固相萃取同时提取尿中的39种药毒物

目的建立固相萃取方法同时提取尿中的39种药毒物并用高效液相色谱法进行分析。方法以多沙普仑为内标,1ml尿样经Oasis小柱固相萃取,用HPLC进行分析。结果39种药毒物可同时从尿中提出,内源性物质不干扰测定。其绝对回收率除吗啡外均大于70％;天内及天间精密度均小于10％;检测限1-15ng／ml;线性相关系数在0.9977以上。结论本法快速、简便、重现性好,空白干扰小,可用于实际案例的药物毒物筛选。     

A fentanyl fatality involving midazolam

A case is presented of a 35-year-old black African male anesthesiology resident, found dead in his apartment. At the scene a syringe, butterfly intravenous line and a bottle of Versed (Midazolam) were recovered. A comprehensive screen for common drugs of abuse and therapeutic agents failed to detect any drugs in blood and urine. The blood ethanol concentration was 0.06 g/dl. A GC/MS SIM assay for midazolam was developed. A sub-therapeutic midazolam blood concentration of 7.5 ng/ml was detected and concentrations (ng/ml or ng/g) in bile, urine, and liver were 3.3, 7.5, and 96, respectively. The syringe fluid was then analyzed and found to contain only fentanyl, midazolam was absent. The blood fentanyl concentration was 4.9 ng/ml which is consistent with those reported in fentanyl fatalities. Fentanyl concentrations (ng/ml or ng/g) in bile, urine, and liver were 8.8, 5.0, and 5.9, respectively. The cause of death was ruled to be fentanyl intoxication and the manner of death undetermined.     

The detection of acetylcodeine and 6-acetylmorphine in opiate positive urines

Acetylcodeine (AC), an impurity of illicit heroin synthesis, was investigated as a urinary biomarker for detection of illicit heroin use. One hundred criminal justice urine specimens that had been confirmed positive by GC/MS for morphine at concentrations >5000 ng/ml were analyzed for AC, 6-acetylmorphine (6AM), codeine, norcodeine and morphine. The GC/MS analysis was performed by solid phase extraction and derivatization with propionic anhydride. Total codeine and morphine concentrations were determined by acid hydrolysis and liquid/liquid extraction. AC was detected in 37 samples at concentrations ranging from 2 to 290 ng/ml (median, 11 ng/ml). 6AM was also present in these samples at concentrations ranging from 49 to 12 600 ng/ml (median, 740 ng/ml). Of the 63 specimens negative for AC, 36 were positive for 6AM at concentrations ranging from 12 to 4600 ng/ml (median, 124 ng/ml). When detected, the AC concentrations were an average of 2.2% (0.25 to 10.2%) of the 6AM concentrations. There was a positive relationship between AC concentrations and 6AM concentrations (r=0.878). Due to its very low concentration in urine, AC was found to be a much less reliable biomarker for illicit heroin use than 6AM in workplace or criminal justice urine screening programs. However, AC detection could play an important role in determining if addicts in heroin maintenance programs are supplementing their supervised diacetylmorphine doses with illicit heroin.     

Validation of a high performance liquid chromatographic method for alpha amanitin determination in urine

The objective of the present study was to develop and validate a liquid chromatographic method with electrochemical detection to measure alpha amanitin concentrations in urine after sample pretreatment with double mechanism (reversed phase/cation exchange) solid-phase extraction cartridges. The urine samples (10 ml) were purified and concentrated to 1 ml with elimination of matrix contaminants. The extracts were then separated by isocratic reversed-phase chromatography using a C18 column (4.6 mm×25 cm) with a mobile phase composed of 0.005 M phosphate buffer (pH 7.2) and acetonitrile (90:10). Coulometric detection was performed by applying an oxidation potential of +500 mV to a porous graphite electrode in a low-volume analytical cell. The limit of quantitation was 10 ng/ml with a signal-to-noise ratio=25. The linearity studied on spiked urine was satisfactory (r=0.9966) from 10 ng/ml to 200 ng/ml. The average extraction recovery of alpha amanitin was 78%, determined using spiked urine samples ranging from 10–300 ng/ml. The intra-assay precision was checked at 10, 50 and 100 ng/ml levels (n=10) in spiked urine samples, with resulting coefficients of variation of 3.6%, 2% and 1.5%, respectively.     

Mixed fatal poisoning caused by taking L-methadone and chloral hydrate

A 42-year-old female drug user who was enrolled in a methadone maintenance program was found dead in her apartment. Cause of death was an intoxication with chloral hydrate and L-methadone. Trichloroethanol (TCE), the primary metabolite of chloral hydrate, was quantified by solid phase microextraction (SPME) and GC/MS in heartblood (27 micrograms/ml) and urine (338 micrograms/ml). D- and L-methadone were differentiated by chiral HPLC, which showed that only L-methadone had been taken. The quantitation of L-methadone and its metabolite EDDP was carried out by GC/MS from heartblood (1300 ng/ml and 86 ng/ml, respectively), urine (5239 ng/ml and 4960 ng/ml, respectively) and gastric contents (159 ng/ml and 122 ng/ml, respectively). The concentrations of both--trichloroethanol and methadone--were in toxic ranges.