Serum cannabinoid levels 24 to 48 hours after cannabis smoking

Low concentrations of THC and 11-hydroxy-THC in serum samples are often claimed not to result from recent cannabis use. Prediction of time of exposure is difficult, especially if distinctive features of drug use could not be observed. Therefore, the aim of the study was to investigate the presence of THC and 11-hydroxy-THC in serum samples as well as to obtain preliminary data on the analyte profile for a time window of 24-48 hours after discontinuation of cannabis smoking. Serum samples from heavy (n = 12, > 1 joint/day), moderate (n = 11, < or = 1 joint/day) and light (n = 6, < 1 joint/week) smokers of cannabis were analyzed for THC, 11-hydroxy-THC and free THC-COOH by GC/MS as well as for glucuronidated THC-COOH by LC/MS-MS. The blood samples were collected 24-48 hours after abstaining from cannabis use. Additionally, 8 specimens were obtained from persons after discontinuation of the drug for more than 48 hours. During collection of the blood samples, distinctive effects due to drug use could not be observed. For heavy users of cannabis, THC was detectable in 8 samples, and in 5 cases both biologically active compounds, THC and 11-hydroxy-THC, were present (1.3-6.4 ng THC/mL serum, 0.5-2.4 ng 11-hydroxy-THC/mL serum). Among moderate users, in 1 sample 1.8 ng THC/mL serum and 1.3 ng 11-hydroxy-THC/mL serum were determined, and another sample was tested positive with low concentrations close to the limit of detection. In serum samples of light users both analytes could not be detected, indicating that in those persons a positive finding of THC and 11-hydroxy-THC may rather result from recent consumption than from cannabis use 1 or 2 days prior to blood sampling. The concentrations of THC-COOH and its glucuronide covered a wide range in all groups of cannabis users. However, there was a trend to higher concentrations in heavy users compared to moderate users, and the mean concentration was smaller in light smokers than in moderate smokers. Overall, the findings indicated that data from pharmacokinetic studies should be supplemented by data obtained from "real-life" samples.     

Evaluation of the One-Step ELISA kit for the detection of buprenorphine in urine, blood, and hair specimens

A solid-phase enzyme immunoassay involving microtiter plates was recently proposed by International Diagnostic Systems corporation (IDS) to screen for buprenorphine in human serum. The performance of the kit led us to investigate its applicability in other biological matrices such as urine or blood, and also hair specimens. Low concentrations of buprenorphine were detected with the ELISA test and confirmed by HPLC/MS (buprenorphine concentrations measured by HPLC/MS: 0.3 ng/mL in urine, 0.2 ng/mL in blood, and 40 pg/mg in hair). The intra-assay precision values were 8.7% at 1 ng/mL of urine (n = 8), 11.5% at 2 ng/mL in serum (n = 8), and 11.5% at 250 pg/mg of hair (n = 8), respectively. The immunoassay had no cross-reactivity with dihydrocodeine, ethylmorphine, 6-monoacetylmorphine, pholcodine, propoxyphene, dextromoramide, dextrometorphan at 1 and 10 mg/L, or codeine, morphine, methadone, and its metabolite EDDP. A 1% cross-reactivity was measured for a norbuprenorphine concentration of 50 ng/mL. Finally, the immunoassay was validated by comparing authentic specimens results with those of a validated HPLC/MS method. From the 136 urine samples tested, 93 were positive (68.4%) after the ELISA screening test (cutoff: 0.5 ng/mL) and confirmed by HPLC/MS (buprenorphine concentrations: 0.3-2036 ng/mL). From the 108 blood or serum samples screened, 27 were positive (25%) after the ELISA test with a cutoff value of 0.5 ng/mL (buprenorphine concentrations: 0.2-13.3 ng/mL). Eighteen hair specimens were positive (72%) after the screening (cutoff: 10 pg/mg) and confirmed by LC/MS (buprenorphine concentrations: 40-360 pg/mg). The ELISA method produced false positive results in less than 21% of the cases, but no false negative results were observed with the immunological test. Four potential adulterants (hypochloride 50 mL/L, sodium nitrite 50 g/L, liquid soap 50 mL/L, and sodium chloride 50 g/L) that were added to 10 positive urine specimens (buprenorphine concentrations in the range 5.3-15.6 ng/mL), did not cause a false negative response by the immunoassay.     

In vitro production of GHB in blood and serum samples under various storage conditions

The in vitro production of GHB was observed in freshly collected, untreated whole blood samples using glass BD-Vacutainers and polypropylene S-monovettes. GHB concentrations were determined daily over a period of one week and after 3, 6 and 9 weeks again. Furthermore, the GHB concentration in 40 untreated random whole blood samples stored at 4°C for a longer period of time (10 samples 12 month, 10 samples 24 month and 20 samples 36 month) was also determined. For comparison, the in vitro production of GHB in freshly collected and prepared serum samples was observed. GHB serum concentrations were determined three times over a period of one week and once again after six weeks. Sample preparation was performed by means of methanolic extraction following the precipitation of whole blood and serum samples. A methanolic standard calibration was done in a low range of 0.005-0.1 μg/mL (LOD: 0.004, LLOQ: 0.013). For quantification a spiked blood bank serum with a determined GHB concentration of 0.09 μg/mL was used. Corrected calibrations in the range of 0.09-5.09 μg/mL were used (LOD: 0.08 μg/mL, LLOQ: 0.30 μg/mL), recovery: 91.3% (high level: 4.09 μg/mL) 50.5% (low level: 0.19 μg/mL). RESULTS: Relevant elevation of GHB was observed in all whole blood samples stored in liquid form (4°C or room temperature). In two of the 40 whole blood samples stored over a longer period of time at 4°C, GHB concentrations in the range of 13 μg/mL were even determined. These findings constitute grounds for caution. Even a GHB cut-off level of 5 μg/mL cannot be considered as "absolutely positive" proof of a case of exogenous administration, at least in untreated liquid blood samples in long time storage. However, no significant elevations of GHB were otherwise observed in any of the serum samples independently of storage temperature nor in the whole blood samples that were frozen for storage. CONCLUSIONS: The results suggest that the cut-off for exogenous GHB of 5 μg/mL could be lowered significantly, with the consequence of winning valuable time for the potential victim, but only if serum is collected for GHB determination or if the whole blood sample is frozen immediately after collection and the procedure well documented.     

Analysis of LSD in human body fluids and hair samples applying ImmunElute columns

Immunoaffinity extraction units (LSD ImmunElute) are commercially available for the analysis of lysergic acid diethylamide (LSD) in urine. The ImmunElute resin contains immobilized monoclonal antibodies to LSD. We applied the ImmunElute procedure to serum and also to human hair samples. For hair analysis the samples were first extracted with methanol under sonication. The extracts were then purified using the ImmunElute resin. LSD analysis was carried out with HPLC and fluorescence detection. The immunoaffinity extraction provides highly purified extracts for chromatographic analysis. The limit of detection (signal-to-noise ratio = 3) has been determined to be < 50 pg regardless of which sample material was used. The procedure was applied to authentic hair samples from drug abusers (n = 11). One of these samples tested positive with an amount of 110 pg LSD in 112 mg extracted hair corresponding to a concentration of 1 pg/mg.     

Digoxin, magnesium, and potassium levels in a forensic autopsy material of sudden death from ischemic heart disease

In 91 cases where the cause of death was heart disease, digoxin, Mg and K concentrations in serum and ventricular myocardium were measured post mortem. Forty per cent were positive for digoxin in both serum and myocardium. The mean serum level was 5.1 +/- 2.4 nmol/l and the mean myocardial level was 42.6 +/- 27.5 ng/g. Correlation could be established between serum and myocardial concentrations of digoxin. There were statistically significant differences in serum as well as in myocardial digoxin levels in persons on 0.13 mg and 0.25 mg per day, respectively. Myocardial levels of Mg and K were low as generally found in persons with ischemic heart disease. There was no correlation between these levels and myocardial digoxin concentrations. Caution must be exercised in the assessment of digoxin results from cadaver samples because of the postmortem rise of digoxin serum concentrations. Considering this fact, the results still indicate that the prevalence of toxic digoxin concentrations might be more common than previously thought.     

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.     

A study on the concentrations of 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in hair root and whole hair

In this study, we investigated the patterns of cannabis users (n=412) according to their sex, age, and the results of urinalysis and hair analysis, and classified the concentrations of THCCOOH in hair into three categories to examine the levels of cannabis use. We also compared the concentrations of THCCOOH in hair root, hair without the hair root and whole hair and examined the relationship among them according to the results of urinalysis. The hair samples were washed, digested with 1ml of 1M NaOH at 85°C for 30min and extracted with 2ml of n-hexane:ethyl acetate (9:1) two times after adding 1ml of 0.1N sodium acetate buffer (pH 4.5) and 200μl of acetic acid. The final mixture was derivatized with 50μl of PFPA and 25μl of PFPOH for 30min at 70°C. The solution was evaporated, and the residue was reconstituted in 40μl of ethyl acetate and transferred to an autosampler vial. One microlitre was injected into the GC/MS/MS-NCI system. The concentrations of THCCOOH ranged from 0.06 to 33.44pg/mg (mean 2.96; median 1.32) in hair from cannabis users who had positive urine results and ranged from 0.05 to 7.24pg/mg (mean 1.35; median 0.37) in hair from cannabis users who had negative urine results. The average concentration of THCCOOH in hair from cannabis users who had positive urine results was higher than that from cannabis users who had negative urine results. Male cannabis users in their forties were predominant. We classified the concentrations of THCCOOH in hair into three groups (low, medium and high), and could use the grouping of THCCOOH in hair as a guide for determining the level of use. The low, medium and high concentration ranges for THCCOOH in hair were 0.05-0.24, 0.25-2.60 and 2.63-33.44pg/mg, respectively. We also investigated 28 hair samples with the root. The highest concentrations of THCCOOH were seen in the hair root from 18 out of the 28 hair samples. The average concentrations of THCCOOH in hair root, hair without hair root and whole hair from cannabis users who had positive urine results were higher than those who had negative urine results.     

Driving under the influence of drugs -- evaluation of analytical data of drugs in oral fluid, serum and urine, and correlation with impairment symptoms

A study was performed to acquire urine, serum and oral fluid samples in cases of suspected driving under the influence of drugs of abuse. Oral fluid was collected using a novel sampling/testing device (Dr?ger DrugTest System). The aim of the study was to evaluate oral fluid and urine as a predictor of blood samples positive for drugs and impairment symptoms. Analysis for cannabinoids, amphetamine and its derivatives, opiates and cocaine was performed in urine using the Mahsan Kombi/DOA4-test, in serum using immunoassay and gas chromatography-mass spectrometry (GC-MS) confirmation and in oral fluid by GC-MS. Police and medical officer observations of impairment symptoms were rated and evaluated using a threshold value for the classification of driving inability. Accuracy in correlating drug detection in oral fluid and serum were >90% for all substances and also >90% in urine and serum except for THC (71.0%). Of the cases with oral fluid positive for any drug 97.1% of corresponding serum samples were also positive for at least one drug; of drug-positive urine samples this were only 82.4%. In 119 of 146 cases, impairment symptoms above threshold were observed (81.5%). Of the cases with drugs detected in serum, 19.1% appeared not impaired which were the same with drug-positive oral fluid while more persons with drug-positive urine samples appeared uninfluenced (32.7%). The data demonstrate that oral fluid is superior to urine in correlating with serum analytical data and impairment symptoms of drivers under the influence of drugs of abuse.     

Determination of ethyl glucuronide in human hair by SPE and LC-MS/MS

A method for the sensitive and selective determination of ethyl glucuronide (EtG) in hair has been developed using solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Washed and cut hair segments were extracted by ultrasonication (3h, 50 degrees C) and the extracts were cleaned-up with aminopropyl SPE columns. LC-MS/MS analysis was performed using a polar-endcapped phenyl-hexyl-RP-phase with negative mode electrospray ionisation (ESI) using a triple quadrupole mass spectrometer (Sciex API 365) with a turboionspray source and post-column addition of acetonitrile for enhanced sensitivity. The MS/MS transitions monitored were m/z 221 -->75 for EtG and 226 -->75 for D(5)-EtG as an internal standard. The method was selective and sensitive, with a detection limit of 51 pg/mg hair at a signal-to-noise ratio of 3:1. The mean recovery was 96%, with an intra- and inter-day precision of less than 11.7% at a concentration of 200 pg/mg. The linearity was assessed in the range of 25-2000 pg/mg hair, with a correlation coefficient of 0.997. The method was successfully applied to 97 human hair samples which were taken at autopsies from persons with known alcoholism or were obtained from alcoholics who were hospitalized for ethanol withdrawal, from social drinkers and from children having not consumed any alcohol. Although, approximately two-third of the alcoholics showed EtG concentrations in hair of higher than 51 pg/mg (up to >4000 pg/mg), in one-third the EtG concentration was below the detection limit. However, only in one of five hair samples of "social drinkers", the EtG concentration was above the detection limit (51 pg/mg). No EtG has been detected in the hair of children. These investigations demonstrate that heavy alcohol consumption may be but not necessarily has to be detectable by EtG analysis in hair.     

Levels of cocaine and its metabolites in washed hair of demonstrated cocaine users and workplace subjects

In a study of subjects in drug rehabilitation programs, cocaine and cocaine metabolite levels were determined in the hair of 75 subjects who had produced cocaine-positive urine results. The hair was analyzed after being washed with the 3.75 h wash procedure developed by this laboratory. In addition, results of testing 73 non-users are presented, as well as levels of cocaine, benzoylecgonine (BE), cocaethylene, and norcocaine from workplace population samples. The data support a recommendation of reporting as positive a sample with cocaine of 500 pg/mg hair and either a 5% ratio of benzoylecgonine (BE) to cocaine in samples, or the presence of cocaethylene at 50 pg/mg hair, or norcocaine at 50 pg/mg hair for samples < or =2000 pg cocaine/mg hair. For samples with cocaine present at >2000 pg/mg hair, the data indicate that a ratio of 5% BE may be an overly conservative approach. In appropriately washed hair samples, cocaine users can produce hair levels of <5% BE and thus a minimum BE cutoff in lieu of a ratio could be considered.     

Hair analysis of seven bodybuilders for anabolic steroids,ephedrine, and clenbuterol

Several bodybuilders, all winners of international competitions, were arrested for trafficking of a number of doping agents including anabolic steroids, ephedrine, beta-adrenergics, human chorionic gonadotropin, antidepressants, and diuretics. In accordance with the recent French law against doping, the judge asked to test seven bodybuilders to identify doping practices. Hair and urine specimens were collected for analysis. After decontamination, a 100 mg hair strand was pulverized in a ball mill, hydrolyzed, extracted, and derivatized to be tested by GC/MS for anabolic steroids, beta-adrenergic compounds, ephedrine, and other doping agents. Urine was analyzed for anabolic steroids and metabolites, beta-adrenergic compounds, ephedrine, and human chorionic gonadotropin, in addition to a broad spectrum screening with GC/MS. The following compounds were detected in urine: ephedrine (29 and 36 ng/mL, n = 2), clenbuterol (0.2 to 0.3 ng/mL, n = 3), norandrosterone (4.7 to 100.7 ng/mL, n = 7), norethiocholanolone (0.9 to 161.8 ng/mL, n = 6), stanozolol (1 to 25.8 ng/mL, n = 4), methenolone (2.5 to 29.7 ng/mL, n = 4), testosterone (3 to 59.6 ng/mL, n = 7), epitestosterone (1 to 20.4 ng/mL, n = 7) and ratio testosterone/epitestosterone >6 for four subjects (18.5 to 59.6). The following drugs were detected in hair: ephedrine (0.67 and 10.70 ng/mg, n = 2), salbutamol (15 to 31 pg/mg, n = 3), clenbuterol (15 to 122 pg/mg, n = 6), nandrolone (1 to 7.5 pg/mg, n = 3), stanozolol (2 to 84 pg/mg, n = 4), methenolone (17 and 34 ng/ml, n = 2), testosterone enanthate (0.6 to 18.8 ng/mg, n = 5), and testosterone cypionate (3.3 to 4.8 ng/mg, n = 2). These results document the doping practice and demonstrate repetitive exposure to anabolic compounds and confirm the value of hair analysis as a complement to urinalysis in the control of doping practice.     

Site-dependent production of gamma-hydroxybutyric acid in the early postmortem period

This study compared endogenous gamma-hydroxybutyric acid (GHB) concentrations in various postmortem fluid samples of 25 autopsy cases. All bodies were stored between 10-20 degrees C until autopsy, and the intervals between death and autopsy were less than 2 days (6-48 h). GHB concentrations were measured by headspace gas chromatography after GHB was converted to gamma-butyrolactone. Endogenous GHB concentrations were significantly higher in femoral venous blood (4.6+/-3.4 microg/ml, n=23) than in cerebrospinal fluid (1.8+/-1.5 microg/ml, n=9), vitreous humor (0.9+/-1.7 microg/ml, n=8), bile (1.0+/-1.1 microg/ml, n=9) and urine (0.6+/-1.2 microg/ml, n=12). GHB concentrations were similar in blood samples taken from different sites. Cut-off limits of 30 and 10 microg/ml are proposed for blood and urine, respectively, to discriminate between exogenous and endogenous GHB in decedents showing no or little putrefaction (postmortem intervals usually 48 h or less). The criterion established for endogenous GHB in postmortem urine may also be applicable to analytical results in cerebrospinal fluid, vitreous humor and bile from deceased persons.     

Target screening and confirmation of 35 licit and illicit drugs and metabolites in hair by LC-MSMS

A liquid chromatography-tandem mass spectrometry (LC-MSMS) target screening in 50mg hair was developed and fully validated for 35 analytes (Δ9-tetrahidrocannabinol (THC), morphine, 6-acetylmorphine, codeine, methadone, fentanyl, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, benzoylecgonine, cocaine, lysergic acid diethylamide, ketamine, scopolamine, alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, 7-aminoflunitrazepam, lorazepam, lormetazepam, nordiazepam, oxazepam, tetrazepam, triazolam, zolpidem, zopiclone, amitriptyline, citalopram, clomipramine, fluoxetine, paroxetine and venlafaxine). Hair decontamination was performed with dichloromethane, and incubation in 2 mL of acetonitrile at 50°C overnight. Extraction procedure was performed in 2 steps, first liquid-liquid extraction, hexane:ethyl acetate (55:45, v:v) at pH 9, followed by solid-phase extraction (Strata-X cartridges). Chromatographic separation was performed in AtlantisT3 (2.1 mm × 100 mm, 3 μm) column, acetonitrile and ammonium formate pH 3 as mobile phase, and 32 min total run time. One transition per analyte was monitored in MRM mode. To confirm a positive result, a second injection monitoring 2 transitions was performed. The method was specific (no endogenous interferences, n=9); LOD was 0.2-50 pg/mg and LOQ 0.5-100 pg/mg; linearity ranged from 0.5-100 to 2000-20,000 pg/mg; imprecision <15%; analytical recovery 85-115%; extraction efficiency 4.1-85.6%; and process efficiency 2.5-207.7%; 27 analytes showed ion suppression (up to -86.2%), 4 ion enhancement (up to 647.1%), and 4 no matrix effect; compounds showed good stability 24-48 h in autosampler. The method was applied to 17 forensic cases. In conclusion, a sensitive and specific target screening of 35 analytes in 50mg hair, including drugs of abuse (THC, cocaine, opiates, amphetamines) and medicines (benzodiazepines, antidepressants) was developed and validated, achieving lower cut-offs than Society of Hair Testing recommendations.     

Benzoylecgonine (cocaine metabolite) detection in hair samples of jail detainees using radioimmunoassay (RIA) and gas chromatography/mass spectrometry (GC/MS)

Benzoylecgonine (BE) was detected in hair samples using nonproprietary extraction methodology and modifications of well-established radioimmunoassay (RIA) screening/quantitative gas chromatography/mass spectrometry (GC/MS) confirmation procedures. Samples collected anonymously from a population of 48 jail detainees weighed between 5.3 and 61.2 mg. All of the 22 hair samples which had RIA results indicating the presence of BE or immunologically similar substances above a cutoff amount of 1.25 ng/sample (50 ng/mL) were confirmed by GC/MS. Several varieties of hair color and texture were tested, although in each general category there were samples which contained BE as well as other samples which did not reveal detectable amounts of BE. The range of concentrations in 22 hair extracts that screened positive were 0.26 to 18 ng/mg hair as determined by GC/MS. In comparison with other reports of cocaine-related substances in hair, these data show consistent concentrations.     

流动注射—化学方光法检测人体血清内枸橼酸芬太尼

目的建立灵敏测定生物样品中皮克级枸橼酸芬太尼的方法。方法使用流动注射-化学发光法、鲁米诺-溶解氧体系检测芬太尼,蠕动泵流速2.0mL/min,鲁米诺与NaOH混合后被六通阀定量注入系统直至形成稳定基线,同时将样品推进送入混合管。碱性混合液被送入化学发光流通池产生化学发光,光电倍增管和光度计即检测到发光信号强度。结果在1.0～700pg/mL线性范围内,化学发光强度与芬太尼浓度呈线性关系,且发光强度随浓度增大呈增敏趋势,检出限低至0.3pg/mL。相对标准偏差在3.0%以内。分析过程可在30s内完成,采样频率120次/h。回收率98.9%～103.1%。结论该方法已经成功应用于检测人体血清中的枸橼酸芬太尼。     

Analysis of pain management drugs, specifically fentanyl, in hair: application to forensic specimens

This article discusses the immunoassay screening of pain management drugs, and the mass spectrometric confirmation of fentanyl in human hair. Hair specimens were screened for fentanyl, opiates (including oxycodone), tramadol, propoxyphene, carisoprodol, methadone, and benzodiazepines and any positive results were confirmed using gas chromatography or liquid chromatography with mass spectral detection. The specific focus of the work was the determination of fentanyl in hair, since autopsy specimens were also available for comparison with hair concentrations. Using two-dimensional gas chromatography with electron impact mass spectrometric detection, fentanyl was confirmed in four of nine hair specimens collected at autopsy. The accuracy of the assay at 10 pg/mg was 95.17% and the inter-day and intra-day precision was 5.04 and 13.24%, respectively (n=5). The assay was linear over the range 5-200 pg/mg with a correlation of r(2)>0.99. The equation of the calibration curve forced through the origin was y=0.0053x and the limit of quantitation of the assay was 5 pg/mg. The fentanyl concentrations detected were 12, 17, 490, and 1930 pg/mg and the results were compared with toxicology from routine post-mortem analysis. The screening of pain management drugs in hair is useful in cases where other matrices may not be available, and in routine testing of hair for abused drugs.     

Olanzapine concentrations in clinical serum and postmortem blood specimens--when does therapeutic become toxic?

The concentration of olanzapine (Zyprexa) was determined in 1653 clinical serum specimens during routine drug monitoring, and in 58 postmortem whole blood specimens as part of routine toxicological analysis. The analysis of olanzapine was performed by the solid-phase extraction of 1.0 mL of buffered serum or blood, followed by gas chromatography separation with nitrogen-phosphorus detection. The analysis of the clinical serum samples showed that 86% of positive serum values were within the range of 5 to 75 ng/mL, with a mean and median of 36 and 26 ng/mL, respectively. These data suggest that the concentrations of olanzapine expected during therapy may be higher than those previously reported. In 58 postmortem whole blood specimens the mean olanzapine concentration was 358 ng/mL with a range of 10 to 5200 ng/mL. Further, investigation of deaths involving olanzapine suggest that potential toxicity should be considered at concentrations above 100 ng/mL. Although the majority of the olanzapine-related deaths were associated with many other drugs, death primarily due to olanzapine toxicity was determined at concentrations in post-mortem blood as low as 160 ng/mL.     

Analysis of Delta9-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 Delta(9)-tetrahydrocannabinol (THC) in oral fluid samples. Oral fluid was extracted using Bond Elut LRC-Certify solid-phase extraction columns (10 cm(3), 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, [d(3)-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 (r(2)=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.     

Poppy seed consumption and toxicological analysis of blood and urine samples

Poppy seeds contain morphine in different amounts. Reported concentrations are up to 294 mg morphine/kg poppy seeds. Since penalties based on Street Traffic Law (parapgraph 24a StVG) in Germany (administrative offence) require definitive proof of morphine in blood samples, and the "Grenzwertkommission" in consultation with the Ministry of Transportation recommended a threshold of free morphine of 10 ng/mL, the question arose whether the consumption of poppy seeds can lead to a blood concentrations equal or higher than 10 ng/mL of free morphine. Therefore, five volunteers ate poppy seed products (50 mg morphine/kg poppy seeds). In urine, all on-site tests were enzyme immunologically positive for opiates and were positive to morphine by GC/MS. All the blood samples were negative to morphine by EIA and to free morphine by GC/MS. However, after hydrolysis, morphine was detected by GC/MS in all cases. Accordingly, in Germany, penalties based on parapgraph 24a StVG are not likely to cause road users any concerns should they have consumed poppy seeds. Driver Licensing Authorities, however, should be advised of this problem to avoid unjustified legal measures.     

A comparative study on the concentrations of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in head and pubic hair

In this study, the concentrations of 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in pubic, axillary and beard hair were measured and the correlation between the concentrations of THCCOOH in head and pubic hair from same cannabis users were evaluated. The papers on body hair analysis for THCCOOH were rarely found although police officers submit body hair as a complimentary specimen to forensic laboratories in case cannabis users had no hair. Head, pubic, axillary, and beard hair were collected. All hair samples were cut into 0.5mm segments and decontaminated with methanol, digested with 1 mL of 1M NaOH at 85 °C for 30 min and extracted in 2 mL of n-hexane:ethyl acetate (9:1) two times after adding 1 mL of 0.1N sodium acetate buffer (pH = 4.5) and 200 μL of acetic acid followed by derivatization with 50 μL of PFPA and 25 μL of PFPOH for 30 min at 70 °C. The extracts were analyzed using gas chromatography tandem mass spectrometry operating in negative chemical ionization mode (GC/MS/MS-NCI). We determined the concentrations of THCCOOH in both pubic and head hair. The concentrations of THCCOOH in pubic hair were higher than those in head hair. We also evaluated the concentrations of THCCOOH in body hair (pubic, axillary and beard hair) and head hair according to the positive/negative urine test results. There was no statistically significant difference in the concentrations of THCCOOH in head and body hair according to urine results.