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.     

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.     

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.     

Hair analysis for delta(9)-THC,delta(9)-THC-COOH,CBN and CBD,by GC/MS-EI. Comparison with GC/MS-NCI for delta(9)-THC-COOH

A sensitive analytical method was developed for quantitative analysis of delta(9)-tetrahydrocannabinol (delta(9)-THC), 11-nor-delta(9)-tetrahydrocannabinol-carboxylic acid (delta(9)-THC-COOH), cannabinol (CBN) and cannabidiol (CBD) in human hair. The identification of delta(9)-THC-COOH in hair would document Cannabis use more effectively than the detection of parent drug (delta(9)-THC) which might have come from environmental exposure. Ketamine was added to hair samples as internal standard for CBN and CBD. Ketoprofen was added to hair samples as internal standard for the other compounds. Samples were hydrolyzed with beta-glucuronidase/arylsulfatase for 2h at 40 degrees C. After cooling, samples were extracted with a liquid-liquid extraction procedure (with chloroform/isopropyl alcohol, after alkalinization, and n-hexane/ethyl acetate, after acidification), which was developed in our laboratory. The extracts were analysed before and after derivatization with pentafluoropropionic anhydride (PFPA) and pentafluoropropanol (PFPOH) using a Hewlett Packard gas chromatographer/mass spectrometer detector, in electron impact mode (GC/MS-EI). Derivatized delta(9)-THC-COOH was also analysed using a Hewlett Packard gas chromatographer/mass spectrometer detector, in negative ion chemical ionization mode (GC/MS-NCI) using methane as the reagent gas. Responses were linear ranging from 0.10 to 5.00 ng/mg hair for delta(9)-THC and CBN, 0.10-10.00 ng/mg hair for CBD, 0.01-5.00 ng/mg for delta(9)-THC-COOH (r(2)>0.99). The intra-assay precisions ranged from <0.01 to 12.40%. Extraction recoveries ranged from 80.9 to 104.0% for delta(9)-THC, 85.9-100.0% for delta(9)-THC-COOH, 76.7-95.8% for CBN and 71.0-94.0% for CBD. The analytical method was applied to 87 human hair samples, obtained from individuals who testified in court of having committed drug related crimes. Quantification of delta(9)-THC-COOH using GC/MS-NCI was found to be more convenient than GC/MS-EI. The latter may give rise to false negatives due to the detection limit.     

Creatinine analysis in single collection urine specimens.

Controlled and uncontrolled fluid intake studies were conducted on series of volunteers over the 6 or 12 h of the study periods. Urine specimens were obtained from each subject randomly or at specified times relative to fluid ingestion. Creatinine analysis performed by a modification of the Abbott TDx procedure demonstrates that the values obtained from single collection specimens fall almost in the same range as the values from 24 h pooled collection specimens. The creatinine concentration can be used to indicate possible adulteration of urine specimens by dilution as a means of avoiding detection of use of drugs of abuse. Between 4 and 7 h are required for a decrease in creatinine concentration to about 100 mg/dL from an initial mean of about 170 mg/dL. A minimum of 6 h is needed for any creatinine value to fall to 50 mg/dL or less. Thus, it appears that creatinine output is sensitive to the amount of fluid ingested, but the relationship is neither linear nor immediate. The absence of a significant creatinine concentration in a specimen can be used as an indication of direct or indirect adulteration of the urine specimen by dilution or replacement with water. At NDSL-Great Lakes, a decline of the creatinine concentration to 30 mg/dL is used as a cutoff for differentiating between urine specimens that might have been tampered with to avoid detection of drug use and those specimens that are dilute for other reasons. Values at 10 mg/dL or less are suggestive of replacement by water. The information is provided to local commands for investigation prior to initiation of punitive action by the command.     

High prevalence of 6-acetylmorphine in morphine-positive oral fluid specimens

Identification of 6-acetylmorphine, a specific metabolite of heroin, is considered to be definitive evidence of heroin use. Although 6-acetylmorphine has been identified in oral fluid following controlled heroin administration, no prevalence data is available for oral fluid specimens collected in the workplace. We evaluated the prevalence of positive test results for 6-acetylmorphine in 77,218 oral fluid specimens collected over a 10-month period (January-October 2001) from private workplace testing programs. Specimens were analyzed by Intercept immunoassay (cutoff concentration=30 ng/ml) and confirmed by GC-MS-MS (cutoff concentrations=30 ng/ml for morphine and codeine, and 3 ng/ml for 6-acetylmorphine). Only morphine-positive oral fluid specimens were tested by GC-MS-MS for 6-acetylmorphine. A total of 48 confirmed positive morphine results were identified. An additional 107 specimens were confirmed for codeine only. Of the 48 morphine-positive specimens, 32 (66.7%) specimens were positive for 6-acetylmorphine. Mean concentrations (+/-S.E.M.) of morphine, 6-acetylmorphine and codeine in the 32 specimens were 755+/-201, 416+/-168 and 196+/-36 ng/ml, respectively. Concentrations of 6-acetylmorphine in oral fluid ranged from 3 to 4095 ng/ml. The mean ratio (+/-S.E.M.) of 6-acetylmorphine/morphine was 0.33+/-0.06. It is suggested that, based on controlled dose studies of heroin administration, ratios >1 of 6-acetylmorphine/morphine in oral fluid are consistent with heroin use within the last hour before specimen collection. The confirmation of 6-acetylmorphine in 66.7% of morphine-positive oral fluid specimens indicates that oral fluid testing for opioids may offer advantages over urine in workplace drug testing programs and in testing drugged drivers for recent heroin use.     

Ethyl glucuronide concentrations in two successive urinary voids from drinking drivers: relationship to creatinine content and blood and urine ethanol concentrations

The concentrations of alcohol in blood (BAC) and two successive urine voids (UAC) from 100 drunk drivers were compared with the concentration of ethyl glucuronide (EtG), a minor metabolite of ethanol in urine, and the urinary creatinine content as an indicator of dilution. The subjects consisted of 87 men with mean age 42.2+/-14.2 years (+/-standard deviation, S.D.) and 13 women with mean age 42.5+/-14.4 years. Ethanol was measured in blood and urine by headspace gas chromatography (GC) and EtG was determined in urine by liquid chromatography-mass spectrometry (LC-MS). The mean UAC was 2.53+/-1.15g/l for first void compared with 2.35+/-1.17g/l for second void, decreasing by 0.18+/-0.24g/l on average (P<0.001 in paired t-test). The ratios of UAC/BAC were 1.35+/-0.25 for first void and 1.20+/-0.16 for second void and the difference of 0.15+/-0.27 was statistically significant (P<0.001). The UAC/BAC ratio was not correlated with creatinine content of the urine specimens, whereas the concentration of urinary EtG was positively correlated with creatinine (r=0.64 for first void and r=0.62 for second void). The UAC was not correlated with urinary EtG directly (r=-0.03 for first void and r=0.08 for second void) but after adjusting for the relative dilution of the specimens (EtG/creatinine ratio) statistically significant positive correlations were obtained (r=0.58 for first void and r=0.57 for second void). The dilution of the urine, as reflected in creatinine content, is important to consider when EtG measurements are interpreted. The excretion of EtG in urine, like glucuronide conjugates of other drugs, is influenced by diuresis. EtG represents a sensitive and specific marker of acute alcohol ingestion with applications in clinical and forensic medicine.     

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.     

Analysis of 11-nor-9-carboxy-delta(9)-tetrahydrocannabinol in biological samples by gas chromatography tandem mass spectrometry (GC/MS-MS)

Gas chromatography tandem mass spectrometry (GC/MS-MS) analysis of 11-nor-carboxy-delta(9)-tetrahydrocannabinol (delta(9)-THC-COOH), the major metabolite of delta(9)-tetrahydrocannabinol, in biological samples is reported. The proposed method, using deuterated delta(9)-THC-COOH as an internal standard, is able to detect the major metabolite of cannabis derivatives at very low levels (picograms/millilitre) with high specificity. These characteristics render the proposed analytical procedure suitable for confirmatory analysis in drug testing for cannabis use.     

Comparison of daily urine,sweat, and skin swabs among cocaine users

This study (1) compares urine, skin swabs, and PharmChek sweat patches for monitoring drug use; (2) measures possible environmental contamination in recent cocaine (COC) users; and (3) evaluates various immunoassays (IA) for screening COC in diverse matrices. Unique aspects include daily urine monitoring of 10 participants for 4 weeks, multiple monitoring methods, analysis for all specimens by IA and gas chromatography (GC)/mass spectrometry (MS), and the potential for continued illicit drug use by participants. Urine served as the "gold standard" specimen for determining drug use. Only cocaine and related substances were detected.Trace amounts of drugs were found on the skin (<50 ng per swab) of urine-negative participants' hands or forehead. In contrast, larger quantities of COC were found on the skin of individuals with BE-positive urines or individuals living with drug users (up to 20 microg per swab). Patch COC amounts among the three regular users (250-9000, 0-240, 160-22,000 ng per patch) exceeded BE (50-950, none, 30-2200 ng per patch). Pre-swabs, valuable for interpreting the source or time frame of positive patch results, contained substantial COC (38-1160, 0-152, 34-762 ng per swab) prior to patch application; therefore, patch results may represent current use, prior use, contamination, or a combination. In three individuals with no indication of cocaine use, false positives (defined as sweat patch positive when urine specimens were <300ng BE/ml) occurred at a 7% rate. Proposed cut-off concentrations of 75 ng cocaine per patch and 300 ng BE/ml urine curtail the incidence of false positives in this limited population. Three immunoassays were compared to screen specimens for cocaine: a modified, manual Microgenics CEDIA; a Cozart ELISA; and an OraSure ELISA. CEDIA's limit of detection (LOD) was 81ng/ml, compared with LODs of 4 ng/ml for the Cozart ELISA and 1.5 ng/ml for the OraSure ELISA. Cozart correlated with OraSure results for COC concentrations <2000 ng per swab (n=117), r(2)=0.79.     

Delta(9)-THC, 11-OH-Delta(9)-THC and Delta(9)-THCCOOH plasma or serum to whole blood concentrations distribution ratios in blood samples taken from living and dead people.

The recreational use and abuse of Cannabis is continuously increasing in Switzerland. Cannabinoids are very often detected alone or in combination with other drugs in biological samples taken from drivers suspected of driving under the influence of drugs. Moreover, they are also frequently found in blood specimens from people involved in various medico-legal events, e.g. muggings, murders, rapes and working accidents as well. In order to assess the influence of Cannabis exposure on man behavior and performances, it is often needed to estimate the time of Cannabis use. For that purpose two mathematical models have been set up by Huestis and coworkers. These models are based on cannabinoids concentrations in plasma. Because plasma samples are rarely available for forensic determinations in our laboratory, it could be useful to assess the time-laps since Cannabis use through these models from whole blood values. One prerequisite to the use of these models from whole blood values is the knowledge of the plasma to whole blood concentrations distribution ratios of cannabinoids. In this respect, the Delta(9)-THC, 11-OH-Delta(9)-THC and Delta(9)-THCCOOH concentrations were measured in plasma and whole blood taken from eight volunteers who smoke Cannabis on a regular basis. Cannabinoids levels were also determined in "serum" and whole blood samples taken from six corpses. The values of the plasma to whole blood distribution ratios were found to be very similar and their individual coefficient of variation relatively low suggesting that plasma levels could be calculated from whole blood concentrations taken into account a multiplying factor of 1.6. The data obtained postmortem suggest that the distribution of cannabinoids between whole blood and "serum" is scattered over a larger range of values than those determined from living people and that more cannabinoids (mean value of the serum/whole blood concentrations ratios=2.4) can be recovered from the "serum" fraction. The successful use of the mathematical models of Huestis and coworkers may, therefore, rely in part upon the selection of the appropriate blood sample, i.e. plasma. When plasma is not available, whole blood values could be considered with some caution taken into account a multiplying factor of 1.6 to calculate plasma concentrations from blood values. In the case of blood samples taken after death, the use of these models to assess the time of Cannabis use is not recommended.     

Opiate concentrations following the ingestion of poppy seed products – evidence for `the poppy seed defence'

The universally accepted 300 ng/ml cut-off limit for opiate assays stated to be mandatory for all drug screening laboratories by the Substance Abuse and Mental Health Services Administration, has been questioned recently due to positive results being obtained following the ingestion of poppy seed containing food products. To establish the plausibility of the `the poppy seed defence' the concentrations of codeine, norcodeine, morphine, normorphine and thebaine (a potential marker for seed ingestion) in several varieties of poppy seeds from different countries were quantified by GC–MS. The country of origin of the seed specimen analysed and the preparation of the seeds prior to their culinary use was found to influence the alkaloid concentration determined. The maximum morphine and codeine concentrations determined in the seeds were found to be 33.2 and 13.7 μg/g seed respectively. In addition, thebaine concentrations were found to vary with each seed sample analysed. Following the consumption of bread rolls (mean 0.76 g seed covering per roll) by four subjects, all urine specimens analysed produced negative results (using the Dade Bebring EMIT II opiate screening assay) with the exception of one subject (body weight 63.0 kg) who consumed two poppy seed rolls. In this subject opiate positive screening results were obtained for up to 6 h post ingestion with maximum urinary morphine and codeine concentrations of 832.0 ng/ml (@ 2–4 h post ingestion) and 47.9 ng/ml (@ 0–2 h post ingestion) respectively being achieved. Following the ingestion of poppy seed cake containing an average of 4.69 g of seed per slice by four individuals, opiate positive screening results were obtained for up to 24 h. In one subject (dose equivalent to 0.07 g poppy seed/kg body weight) maximum urinary morphine and codeine concentrations of 302.1 ng/ml (@ 0–2 h) and 83.8 ng/ml (@ 2–4 h) respectively were recorded. The elimination of thebaine was found to vary widely between individuals, therefore suggesting that its absence from a specimen is not necessarily indicative of opiate abuse. These findings demonstrate that the poppy seed defence could be used as an argument in medico-legal and employment medical cases. Great care should therefore be taken when interpreting the data produced when screening for opiates.     

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.     

Oral fluid testing for cannabis: on-site OraLine IV s.a.t. device versus GC/MS

Saliva or "oral fluid" has been presented as an alternative matrix to document drug use. The non-invasive collection of a saliva sample, which is relatively easy to perform and can be achieved under close supervision, is one of the most important benefits in a driving under the influence situation. Moreover, the presence of Delta9-tetrahydrocannabinol (THC) in oral fluid is a better indication of recent use than when 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) is detected in urine, so there is a higher probability that the subject is experiencing pharmacological effects at the time of sampling. In the first part of the study, 27 drug addicts were tested for the presence of THC using the OraLine IV s.a.t. device to establish the potential of this new on-site DOA detection technique. In parallel, oral fluid was collected with the Intercept DOA Oral Specimen Collection device and tested for THC by gas chromatography mass spectrometry (GC/MS) after methylation for THC (limit of quantification: 1 ng/mL). The OraLine device correctly identified nine saliva specimens positive for cannabis with THC concentrations ranging from 3 to 265 ng/mL, but remained negative in four other samples where low THC concentrations were detected by GC/MS (1-13 ng/mL). One false positive was noted. Secondly, two male subjects were screened in saliva using the OraLine and Intercept devices after consumption of a single cannabis cigarette containing 25mg of THC. Saliva was first tested with the OraLine device and then collected with the Intercept device for GC/MS confirmation. In one subject, the OraLine on-site test was positive for THC for 2 h following drug intake with THC concentrations decreasing from 196 to 16 ng/mL, while the test remained positive for 1.5 h for the second subject (THC concentrations ranging from 199 to 11 ng/mL). These preliminary results obtained with the OraLine IV s.a.t. device indicate more encouraging data for the detection of THC using on-site tests than previous evaluations.     

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.     

Validity testing of the TDx Cocaine Metabolite Assay with human specimens obtained after intravenous cocaine administration

Clinical specimens obtained from human subjects after intravenous cocaine administration were analyzed by the TDx Cocaine Metabolite Assay (TDx) and by GC/MS for benzoylecgonine. The TDx results were significantly correlated with results by GC/MS assay with no evidence of bias in the TDx assay. All cocaine metabolite positive specimens (greater than or equal to 300 ng/ml) were confirmed by GC/MS. Detection times to the last positive specimen by TDx assay and GC/MS assay of four subjects after a 20-mg intravenous dose of cocaine ranged from 29.3 to 39.1 h and 27.9 and 36.6 h, respectively. Overall, the TDx assay was found to be highly specific and accurate for the detection and measurement of benzoylecgonine in urine.     

The U.S. Mandatory Guidelines for Federal Workplace Drug Testing Programs: current status and future considerations

The U.S. Department of Health and Human Services (HHS) drug testing standards were published in 1988 and revised in 1994, 1998, and 2004. In 2004, significant revisions defining, standardizing, and requiring specimen validity testing on Federal employee donor urine specimens were included. In a separate notice, HHS proposed to establish scientific and technical guidelines for the Federal Workplace Drug Testing Program to: (1) permit laboratory testing of hair, oral fluid, and sweat patch specimens in addition to urine specimens for marijuana, cocaine, phencyclidine, opiates (with focus on heroin), and amphetamines [including methylenedioxymethamphetamine (MDMA), methylenedioxyethamphetamine (MDEA), methylenedioxyamphetamine (MDA)]; (2) permit use of on-site point of collection test (POCT) devices to test urine and oral fluid at collection sites; (3) permit use of instrumented initial test (screening only) facilities [IITF] to quickly identify negative specimens; and (4) add training requirement for collectors, on-site testers, and MROs. This proposal was published in the Federal Register on 13 April 2004, with a 90-day public comment period. The Substance Abuse and Mental Health Services Administration, HHS, reviewed those comments and is preparing the Final Notice that will define the requirements for such testing, including: specimen collection procedures, custody and control procedures that ensure donor specimen identity and integrity, testing facility, initial and confirmatory test cutoff concentrations, analytical testing methods, result review and reporting, evaluation of alternative medical explanations for presence of drug or metabolite in the donor's specimen, and laboratory certification issues. Voluntary pilot performance testing (PT) programs for each specimen type are on-going since April 2000 to determine how to prepare PT materials for specimens other than urine to evaluate laboratories' ability to routinely achieve accuracy and precision required. Certification programs will be developed using the current urine drug testing National Laboratory Certification Program model. The addition of accurate and reliable workplace drug testing using hair, oral fluid, and sweat patch specimens will complement urine drug testing, and aid in combating industries devoted to suborning drug testing through adulteration, substitution, and dilution. For example, hair testing may detect chronic drug use for up to 90 days and be useful in pre-employment situations; oral fluid testing may detect drug use in past hours and be useful in post-accident situations; sweat patch testing may be useful in follow-up drug testing and treatment programs; POCTs and IITFs may be most useful for quickly identifying specimens that are negative for drugs and indicate that the specimen is valid.     

Ethanol distribution ratios between urine and capillary blood in controlled experiments and in apprehended drinking drivers.

Healthy men drank 0.51, 0.68, and 0.85 g of ethanol per kilogram of body weight as neat whisky in the morning after an overnight fast. During 6 to 8 h after the whisky was consumed, nearly simultaneous specimens of fingertip blood and pooled bladder urine were obtained for analysis of ethanol using an enzymatic method. The mean ratios of ethanol concentration [urine alcohol concentration (UAC)/blood alcohol concentration (BAC)] were mostly less than unity during the absorption phase. The UAC exceeded the BAC in the postpeak phase. The mean UAC/BAC ratios varied between 1.4 and 1.7 when the BAC exceeded 0.50 mg/mL. When the BAC decreased below 0.40 mg/mL, the UAC/BAC ratios increased appreciably. The mean UAC/BAC ratios of ethanol were not dependent on the person's age between the ages of 20 and 60 years old, but there were large variations within the age groups. In apprehended drinking drivers (N = 654) with a mean BAC of 1.55 mg/mL, the UAC/BAC ratio of ethanol varied widely, with a mean value of 1.49. In 12 subjects (3.2%), the ratio was less than or equal to unity. In a second specimen of urine obtained approximately 60 min after an initial void (N = 135), the mean UAC/BAC ratio was 1.35 (standard deviation = 0.17). The magnitude of the UAC/BAC ratio of ethanol can help to establish whether the BAC curve was rising or falling at or near the time of voiding. The status of alcohol absorption needs to be documented if drinking drivers claim ingestion of alcohol after the offence or when back-estimation of the BAC from the time of sampling to the time of driving is required by statute.     

Metal and metalloid multi-elementary ICP-MS validation in whole blood, plasma, urine and hair. Reference values

Four multi-elementary metal and metalloid quantification methods using inductively coupled plasma mass spectrometry (ICP-MS) were developed and validated in human whole blood, plasma, urine and hair by means of a single preparation procedure for each sample. The ICP-MS measurements were performed using a Thermo Elemental X7CCT series and PlasmaLab software without a dynamic reaction cell. With this procedure 27-32 elements can be simultaneously quantified in biological matrices: Li, Be, B, Al, V, Cr, Mn, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Rb, Sr, Mo, Pd, Ag, Cd, Sn, Sb, Te, Ba, W, Pt, Hg, Tl, Pb, Bi, U. Whole blood, plasma and urine samples (0.4 ml each) were diluted with purified water, acid, triton X100 and butanol. Rhodium was used as internal standard. The urine sample results were corrected for enzymatic creatinine determination. Twenty-five milligrams hair samples were acid mineralized after a decontamination procedure and diluted as previously described for biological fluids. To be validated, each element had to show linearity with a correlation coefficient higher than 0.99. The intra-assay and inter-assay inaccuracy, measured as the variation coefficient, were below 5 and 10% respectively. Global performance was assessed by a quality control program. Our laboratory is a registered participant of the Institut National de Santé Publique du Québec (Sainte-Foy, Canada) inter-laboratory comparison program for whole blood, urine, and beard hair of non-occupationally exposed individuals spiked with selected elements. In our study multi-element metal and metalloid analysis was assessed for 27 elements in whole blood, 27 elements in plasma, 30 elements in urine and 32 elements in hair, from 0 to 25, or 250 to 1000 ng/ml, depending on the element. Quantification limits ranged from 0.002 ng/ml (U) to 8.1 ng/ml (Al) for whole blood, from 0.002 ng/ml (U) to 7.7 ng/ml (Al) for plasma, from 0.001 ng/ml (U) to 2.2 ng/ml (Se) for urine, and from 0.2 pg/mg (Tl) to 0.5 ng/mg (B) for hair. Normal values were determined in whole blood (n=100), plasma (n=100), urine (n=100), and hair (n=45) of healthy volunteers, leading to approximately 10,000 analyses. All results are presented and discussed. Clinical toxicology and forensic toxicology applications are also reported. ICP-MS has made significant advances in the field of clinical biology, particularly in toxicological analysis. This is due to the use of extremely effective equipment that permits better clinical and forensic toxicological analysis of metal and metalloid status of each individual patient.     

Validity testing of commercial urine cocaine metabolite assays: I. Assay detection times, individual excretion patterns, and kinetics after cocaine administration to humans

A validity study of eight commercial urine assays for detection of cocaine metabolite was performed on clinical specimens collected from human subjects who received single 20-mg intravenous doses of cocaine hydrochloride. The specimens were collected under controlled conditions and analyzed in random order under blind conditions. Benzoylecgonine concentration in each specimen also was determined by gas chromatography/mass spectrometry (GC/MS). Mean times of detection of the last positive specimen (greater than or equal to 300 ng/mL of benzoylecgonine equivalents) after cocaine administration varied among seven of the commercial tests from 16.9 to 52.9 h in the following ascending order: Toxi-Lab less than TDx = EMIT dau = EMIT st less than Abuscreen less than Coat-A-Count = Double Antibody. In contrast, a commercial spot test (KDI Quik Test) which was evaluated for detection of cocaine metabolite produced both false positives and false negatives for benzoylecgonine and was not considered to be a valid test for detection of cocaine metabolite. Half-lives of excretion of benzoylecgonine among four subjects varied from 5.9 to 7.9 h, and overall recovery of benzoylecgonine varied from 15.0 to 34.3% of the administered dose of cocaine.