Studies on the stability and detection of cocaine, benzoylecgonine, and 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid in whole blood using Abuscreen radioimmunoassay

A study was undertaken to assess the stability and the radioimmunoassay (RIA) detection of cocaine, benzoylecgonine (BZE), and 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in whole blood while stored in 4 different kinds of blood collection tubes for up to 30 days at refrigeration and room temperatures. At various intervals, the tubes were sampled and analyzed using Abuscreen RIA. Also, semi-quantitative data derived from RIA analysis of forensic blood specimens were compared with quantitative data acquired using gas chromatography (GC) or GC/mass spectrometry (GC/MS) on the same specimens. RIA and chromatographic studies revealed that BZE and THC-COOH were stable in blood under all conditions studied. Cocaine, however, was found not to be stable in blood, especially when stored at room temperatures. Despite cocaine's instability in blood, RIA was able to detect the presence of cocaine and its breakdown products in blood under all conditions studied.     

Measurement of delta 9-tetrahydrocannabinol (THC) in whole blood samples from impaired motorists

The major psychoactive cannabinoid in marihuana, delta 9-tetrahydrocannabinol (THC) was measured in 1792 randomly selected blood specimens from erratic motorists arrested for impairment who submitted to blood alcohol sampling. Of these specimens, 14.4% were positive for THC (greater than or equal to 5.5 ng/mL). In those erratic driver specimens negative for alcohol THC positives rose to 23%. Drivers who used marihuana covered a broad age range. Aliquots of hemolyzed blood (10 microL) were analyzed by a sensitive radioimmunoassay (RIA) not requiring extraction. RIA accuracy and specificity were validated by gas liquid chromatography/mass spectroscopy (GLC/MS) split pair analysis (correlation coefficient = 0.93). This initial experience should facilitate and amplify a program designed to set forth the epidemiology of marihuana use in motorists and possible behavioral correlates.     

Determination of deslanoside in antemortem and postmortem specimens. Unusual case report

One case of the erroneous administration of deslanoside and high level of drug in antemortem plasma and postmortem specimens has been reported owing to the unusual surrounding circumstances. Deslanoside in antemortem plasma was determined by FPIA and the analysis was done by HPLC in the postmortem tissue samples. The analytical results and methods used in the examinations are discussed in the following paper.     

Detection of blood opiates by fluorescence-polarization immunoassay

It was found possible to use fluorescence-polarization immunoassay (FPIA) in screening of opium drugs in blood and blood serum as a preliminary test. The results of the study of blood and serum samples by FPIA and chromato-mass-spectroscopy were compared. As a criterion of assessment of the preliminary study results we propose to use a cutoff point by serial production of the analysed blood samples of the threshold morphine concentrations (20 ng/ml for living subjects and 40 ng/ml--for corpses). The method of opiates screening in blood proposed is characterized by fast conduction, high specificity and precision, small amounts of the expert material.     

The determination of drugs of abuse in whole blood by means of FPIA and EMIT-dau immunoassays--a comparative study

Six groups of common drugs of abuse (cannabinoids, benzoylecgonine, opiates, barbiturates, benzodiazepines and amphetamines) were determined in whole blood after acetone precipitation, using enzyme multiplied immunoassay (EMIT dau) and fluorescence polarisation immunoassay (FPIA--Abbott TDx and ADx) methods. Both methods, designed primarily for urine, allowed the determination of all above mentioned class of drugs but amphetamine. Only 1 ml of a pre- or postmortem blood sample was needed. The sensitivity of cannabinoids determination was higher by FPIA. The FPIA method gave more precise results, particularly in the case of autopsy blood. The method was applied for drug screening in autopsy and police blood samples. The results (both positive and negative) were in agreement with those obtained with chromatographic methods.     

Poppy seed ingestion as a contributing factor to opiate-positive urinalysis results: the Pacific perspective.

The possible contribution of poppy seed foods to positive opiate urinalysis results, especially from foods available in the Pacific Rim area, has recently become an issue for the U.S. Army Forensic Toxicology Drug Testing Laboratory in Hawaii. To assess the likelihood of this possible contribution, seven different poppy seed food products were consumed by male and female volunteers, and urine specimens were collected at time increments up to either 24 or 72 h. Specimens were evaluated for opiates using Roche Abuscreen radioimmunoassay (RIA), and all RIA positive specimens were analyzed for morphine and codeine using gas chromatography/mass spectrometry (GC-MS). Poppy seed cake, bagels, muffins, and rolls did not contain sufficient quantities of poppy seeds to give rise to opiate positive specimens by U.S. Department of Defense (DOD) GC-MS cutoff levels (morphine = 4000 ng/mL, codeine = 2000 ng/mL), although a number of specimens were positive by National Institute on Drug Abuse (NIDA) cutoff levels (morphine and codeine = 300 ng/mL). However, ingestion of poppy seed streusel or Danish pastry led to confirmed morphine and codeine positive specimens, irrespective of the use of DOD or NIDA confirmation cutoff values. In addition, significant amounts of codeine were observed in a number of these specimens. These findings argue against the unqualified application of previously published quantitative guidelines for eliminating poppy seed ingestion as a possible cause for a positive opiate urinalysis result.     

A research note: the outcome of GC/MS/MS confirmation of hair assays on 93 cannabinoid (+) cases

This article reports the outcome of gas chromatography/tandem mass spectrometry confirmations for THC and carboxy-THC on 93 hair samples screened by RIA for cannabinoids. The samples were taken from probationers in Pinellas County, FL, who voluntarily provided the research staff with six hair and six urine specimens, collected at 1-month intervals. There were 40 samples that were RIA (+), urinalysis (−). Samples were selected which had cannabinoid (+) outcomes for hair, urine, or both. The THC and/or the carboxy-THC was (+) on confirmation. Of these 40 samples, 22 were (+) for both THC and carboxy-THC, 15 were (+) for THC but not carboxy-THC, and three were carboxy THC (+), but THC (−). Only one sample had a (+) RIA, but was (−) for both THC and carboxy-THC on confirmation. RIA detection of cannabinoids was confirmed in nearly all cases. Most cases that were RIA (−) but urine (+) were cannabinoid (+) when analyzed by GC/MS/MS.     

Screening of amphetamine/methamphetamine and their derivatives in urine using FPIA and Triage 8 and the Scope and limits of a subsequent identification by means of the REMEDi HS system

This study describes screening and identifying amphetamines, methamphetamines, and their derivatives in urine using immunochemical (Triage, FPIA) and chromatographic techniques (REMEDi HS). Amphetamines, methamphetamines, MDMA (3,4-methylenedioxymethamphetamine), MDA (3,4-methylenedioxyamphetamine), MDE (3,4-methylenedioxyethyl-amphetainine), MBDB (N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine), BDB (3,4-(methylenedioxyphenyl)-2-butanamine), PMA (4-methoxyamphetamine), DOM (2,5-dimethyloxy-4-methylamphetamine), DOB (4-bromo-2,5-dimethyloxyamphetamine), amphetaminil, pholedrine, fenfluramine, and amfepramone were subjected to a comparative study. For this, the substances were analyzed to determine their specific threshold concentration for a positive detection in the Triage test and their limit of detection and positive threshold concentration for the FPIA test and the results compared. Furthermore, the capabilities of a more detailed analysis with the REMEDi system were studied. This HPLC system was able to produce information on the single drugs and main metabolites found in the sample with the danger of false-positive or false-negative screening results greatly minimized.     

Determination of opiates in postmortem bone and bone marrow.

Bone and bone marrow of a fatally poisoned heroin addict were analyzed by FPIA and GC-FID, immediately after death. A piece of the bone from the above case was buried for 1 year and analyzed by the same procedure. Morphine was detected in all specimens at concentrations of 195, 340 and 155 ng/g for bone marrow, bone and buried bone, respectively. A loss of 54.4% of morphine concentration was observed during 1-year burial. Such findings have potential forensic value in cases of skeletonized remains.     

Determination of cross-reactant drugs with a new morphine radioimmunoassay procedure

A study to determine the specificity of a new morphine radioimmunoassay (RIA) procedure was performed on 23 drugs of forensic science interest. Spiked whole blood and urine specimens were analyzed and the apparent morphine concentrations determined. All compounds analyzed showed low cross-reactivity, indicating a high specificity for morphine.     

Screening postmortem blood and tissues for nine classes [correction of cases] of drugs of abuse using automated microplate immunoassay

A commercially available enzyme-linked immunosorbant assay (ELISA) was evaluated as a screening procedure for the detection of nine classes of abused drugs in postmortem blood and tissue specimens. Specifically, postmortem blood, fluid and/or tissue homogenates were screened for amphetamine (AMP), methamphetamine (MET), barbiturates (BARB), benzodiazepines (BZD), cannabinoids (CNB), cocaine (benzoylecgonine; BE), morphine-specific (MOR), opiates (class; OPI), phencyclidine (PCP) and lysergic acid diethylamide (LSD) by ELISA and by coated tube radioimmunoassay (CTR) (BARB, BE, OPI, PCP, LSD) or double-antibody radioimmunoassay (DAR) (AMP/METH, BZD, CNB). Specimens that screened 'positive' by any method were confirmed and quantitated by gas chromatography/mass spectrometry (GC/MS). The only assay that appeared to perform less optimally than RIA was the MOR assay (five false negatives). However, this assay is very specific for free morphine while the GC/MS confirmation method provided a total morphine value. The OPI assay was more sensitive, producing fewer false negatives, and is recommended for broad class opiate screening. EIA is an adequate alternative to RIA for screening postmortem specimens, including blood and tissue, for nine major classes of drugs.     

Confirmation of Syva enzyme multiple immunoassay technique (EMIT) d.a.u. and Roche Abuscreen radioimmunoassay (RIA) (125I) urine cannabinoid immunoassays by gas chromatographic/mass spectrometric (GC/MS) and bonded-phase adsorption/thin-layer chromatographic (BPA-TLC) methods

Thirty human urines screened positive by the Syva enzyme multiple immunoassay technique (EMIT) d.a.u. urine cannabinoid assay were also positive for the major marijuana urinary metabolite 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) when assayed by gas chromatographic/mass spectrometric (GC/MS) and a noninstrumental qualitative bonded-phase adsorption/thin-layer chromatographic (BPA-TLC) technique. The noninstrumental BPA-TLC procedure was the simpler of the two techniques to perform and interpret. Assay of these same samples by the Roche Abuscreen radioimmunoassay (RIA) for cannabinoids (125I) revealed that reliance on the 100-ng/mL equivalent positive calibrator yielded a high incidence of false negative results (10 out of 30). The performance of these same 4 assays on 30 true negatives also was evaluated. All samples were negative for cannabinoids by EMIT and RIA, and for THC-COOH by BPA-TLC. GC/MS assay, however, detected spurious low levels of approximately 5-ng/mL THC-COOH in two instances. Because of this, a reliability level of 10 ng/mL was set for the routine quantitative confirmation of THC-COOH by the GC/MS method.     

Criminal poisoning of commuters in Bangladesh: prospective and retrospective study

Travel-related poisoning is an emerging social and public health emergency in Bangladesh but its cause and significance have not been determined. To investigate this syndrome we performed a prospective clinical study and retrospective analysis of hospital records in a general medicine unit of a public tertiary care teaching hospital in Dhaka, Bangladesh, using toxicological analysis by fluorescence polarization immunoassay (FPIA) and liquid chromatography coupled to time-of-flight mass spectrometry (LC-TOF MS). The participants of the prospective study were 130 consecutive patients aged 16-80 years who were admitted with central nervous system depression (Glasgow Coma Score 3-14) after using public transportation, in the absence of other abnormalities, from January through June 2004, and a convenience sample of 15 such patients admitted during 3 days in May 2006. In 2004-2006, travel-related poisoning increased from 6.1 to 9.5% of all admissions (210-309 of 3266-3843 per year), representing 46.6-55.7% of all admitted poisoning cases. Incidents were associated with bus (76%), taxi, train, and air travel, or local markets; 98% of patients remembered buying or accepting food or drinks before losing consciousness. Direct financial damage (missing property) was diverse and frequently existential. Among 94 urine samples analyzed by FPIA, 74% tested positive for benzodiazepines. Among 15 urine samples analyzed by LC-TOF MS, lorazepam was detected in all; five also contained diazepam or metabolites; nitrazepam was present in three. FPIA results obtained for these 15 samples were below the recommended cut-off in eight (53%; lorazepam only). Our findings show that the massive medicosocial emergency of travel-related poisoning in Bangladesh is the result of drug-facilitated organized crime and that benzodiazepine drugs are used to commit these crimes, suggesting modifications to the local emergency management of the victims of this type of poisoning. They also highlight the need for more research in the neglected field of acute poisoning in Bangladesh, and for criminal investigations of the use of benzodiazepine drugs in this country.     

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.     

The detection of cocaine in hair specimens using micro-plate enzyme immunoassay.

The analysis of hair for drugs of abuse is becoming increasingly popular and is under consideration by the Division of Health and Human Services as a possible alternative or adjunct to urinalysis in workplace programs. The detection of cocaine in human hair using a commercially available micro-plate enzyme immunoassay is described for the first time. Sample size and incubation time were the major variables in the optimization of the method. In order to validate the procedure, the method was applied to 105 consecutive hair samples routinely received into our laboratory. The samples were simultaneously analyzed by the Micro-Plate immunoassay (EIA), as well as our current fluorescence polarization immunoassay (FPIA) procedure and gas chromatography-mass spectrometry (GC/MS). The sensitivity of the EIA and FPIA assays were 75% and 67.8% respectively; specificity 97.4% and 80.5% respectively; and efficiency 91.4 and 77.1% respectively. The Micro-Plate EIA was shown to be a valid alternative to other immunoassay screening methods for the detection of cocaine in hair by demonstrating increased sensitivity, specificity and efficiency over our current technique.     

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.     

Evaluation of cocaine, amphetamines and cannabis use in university students through hair analysis: preliminary results

The evaluation of drug abuse in a defined population was performed through toxicological hair analysis. Hair samples from university students ranging from 18 to 25 years of age were anonymously collected and screened for cocaine, amphetamines and cannabinoids by radioimmunoassay (RIA). Positive results (cut-off values adopted were 2 ng/mg for cocaine and amphetamines and 0.5 ng/mg for cannabinoids) were confirmed by GC/MS. Preliminary results showed 19% of positive results for cocaine on 200 samples analysed. No confirmed positive results were obtained for amphetamine analysis. RIA technique demonstrated its unsuitability for cannabinoids preliminary screening on hair, giving a high percent of false positive results.     

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.     

The detection of 6-monoacetylmorphine in urine, serum and hair by GC/MS and RIA

6-Monoacetylmorphine (6-MAM) is a good indicator for the intake of heroin and can be detected in blood, urine and hair of heroin users. A new radioimmunoassay (RIA) designed specifically for 6-monoacetylmorphine (6-MAM) was tested for its usefulness for the quantitation of the drug in urine, serum and hair. Its cross-reactivity with heroin and its metabolites, and related compounds was also determined. Eighty-nine hair, six serum and 25 urine samples where 6-MAM had been previously identified by GC/MS were analysed for 6-MAM with the new RIA kit. A good correlation existed between the GC/MS and RIA results for the hair samples. However, the amount of 6-MAM found in serum and urine differed considerably between the two methods. This difference could be explained by the cross-reactivity of the antibody with morphine and morphine-6-glucuronide, which are present in much larger amounts in serum and urine, than in hair. To evaluate a new rationalisation procedure, some hair samples were split into two portions after incubation. One part was analyzed for 6-MAM by RIA, and the other portion by GC/MS.     

Adult sudden death caused by aspiration of chewing gum

A case of a fatal foreign material aspiration is presented in the following text. A 24-year-old white male died suddenly. A piece of chewing gum lodged in a pool of frothy fluid was revealed at autopsy. Microscopic examinations revealed atelectasia emphysema, eosinophilic exudate and empty spaces. Blood and urine samples were analyzed, for alcohol and drug use by fluorescence polarization immunoassay (FPIA) on an Abbott AXSYM system. No alcohol or other drugs were detected in blood or urine.