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.     

High performance liquid chromatography-immunoassay of delta 9-tetrahydrocannabinol and its metabolites in urine

High performance liquid chromatographic-immunoassay (HPLC-IA) profiles of cannabinoid metabolites in urine samples were obtained using four different antisera. The urines were chromatographed on a reverse phase system using a gradient of acetonitrile in water (pH 3.3) and fractions collected every 30 s. Some urine samples were hydrolyzed with methanolic sodium hydroxide before fractionation. Peaks of immunoreactivity were detected at a fraction corresponding to 11-nor-9-carboxy-delta 9-tetrahydrocannabinol (COOH-THC) and at an early eluting fraction; however, the profiles depended upon the specificity of the antisera used.     

Gas chromatographic method of quantitative determination of carbon monoxide in cadaver blood

A gas chromatographic method has been developed for measurement of carbon monoxide in cadaveric blood on a chromatographer with thermal conductivity detector and air oxygen as the internal standard. The error of the method is no more than 4% with the mean quadratic deviation +/- 0.18-0.33 min. The method was studied on 10 model and 30 expert blood samples. Putrefactive products did not influence the accuracy of analysis.     

Excretion of Delta9-tetrahydrocannabinol in sweat

The aims of this study were: first, to determine the accuracy of the Cameriere method for assessing chronological age in children based on the relationship between age and measurement of open apices in teeth and, second, to compare the accuracy of this method with the widely used Demirjian et al. method and with the method proposed by Willems et al. Orthopantomographs taken from white Italian, Spain and Croatian children (401 girls, 355 boys) aged between 5 and 15 years were analysed following the Cameriere, Demirjian and Willems methods. The difference between chronological and dental age was calculated for each individual and each method (residual). The accuracy of each method was assessed using the mean of the absolute values of the residuals (mean prediction error). Results showed that the Cameriere method slightly underestimated the real age of children. The median of the residuals was 0.081 years (interquartile range, IQR=0.668 years) for girls and 0.036 years for boys (interquartile range, IQR=0.732 years). The Willems method showed an overestimation of the real age of boys, with a median residual error of -0.247 years and an underestimation of the real age of girls (median residual error=0.073 years). Lastly, the Demirjian method overestimated the real age of both boys and girls, with a median residual error of -0.750 years for girls and -0.611 years for boys. The Cameriere method yielded a mean prediction error of 0.407 years for girls and 0.380 years for boys. Although the accuracy of this method was better for boys than for girls, the difference between the two mean prediction errors was not statistically significant (p=0.19). The Demirjian method was found to overestimate age for both boys and girls but the mean prediction error for girls was significantly greater than that for boys (p=0.024), and was significantly less accurate than the Cameriere method (p<0.001). The Willems method was better than that of Demirjian (p=0.0032), but was significantly less accurate than that of Cameriere (p<0.001).     

Profiles of delta 9-tetrahydrocannabinol metabolites in urine of marijuana users: preliminary observations by high performance liquid chromatography-radioimmunoassay

Metabolic profiles of 11-nor-9-carboxylic acid-delta 9-tetrahydrocannabinol (COOH-THC) and other THC metabolites were determined in an infrequent and a frequent marijuana user by high performance liquid chromatography-radioimmunoassay (HPLC-RIA). In the infrequent user, no unconjugated COOH-THC was detected in urine samples for the first 8 h following smoking, whereas this metabolite was detected in the urine samples from a frequent user. A metabolite was also detected in the frequent user, which was not present in the urine sample from the infrequent user.     

Screening for cannabinoids in blood using EMIT: concentrations of delta-9-tetrahydrocannabinol in relation to EMIT results.

The EMIT d.a.u. cannabinoid assay of methanolic extracts of blood was found to be usable as a screening method in cases of suspected impairment by cannabis, when delta-9-tetrahydrocannabinol (THC) was analysed in the subsequent assay. A prerequisite is that the blood sample is taken some time after cannabis smoking. When a cut-off limit corresponding to 50 nM delta-9-tetrahydrocannabinol carboxylic acid (17 ng/ml) was used, 86% of the EMIT positive blood samples contained THC concentrations above the cut-off limit of 1 nM (0.3 ng/ml). A high EMIT result gave a high probability of finding a high THC concentration in the subsequent confirmation analysis.     

Method of determining methemoglobin in cadaver blood

Poisoning with methemoglobin toxins are not rare in forensic medical practice, but there is still no reliable method for measuring methemoglobin in cadaveric biological fluids and tissues. The author investigated factors affecting evaluation of natural amounts of methemoglobin in the blood, compared several methods for methemoglobin measurements, and developed qualitative characteristics indicating the presence of methemoglobin in cadaveric blood. A rational method for measuring methemoglobin in the blood, blood clots, and tissues is offered for forensic chemical and biochemical evaluations.     

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.     

Use of thin-layer chromatography for detection of 11-nor-9-carboxy-delta9-tetrahydrocannabinol in the urine

A simple method of thin-layer chromatography for detection of 11-nor-9-carboxy-delta9-tetrahydrocannabinol in biological material (urine) can be used in chemicotoxicological laboratories and chemical departments of forensic-medical expert examination without special facilities.     

Quantitative determination of strychnine in blood and urine by gas chromatography with mass-selective detector

A method for the quantitative determination of strychnine in biological fluids by gas chromatography--mass spectrometry is proposed. The preparation of samples for the analysis included extraction of strychnine from blood and urine with the use of AccuBond(II) EVIDEX cartridges for solid-phase extraction and SPEC MP3 disks respectively. The efficiency of extraction was estimated at 0.05 mg/l for blood and 0.02 mg/l for urine. The detection limit was 0.10 mg/l in blood and 0.05 mg/l in urine.     

A gas chromatographic method for determining acetaldehyde in cadaver blood

Gas-chromatographic method of acetaldehyde detection in blood of subjects who died of alcoholic intoxication is suggested. Method is simple, does not require additional expenses, can be readily used in medicolegal practice and in difficult cases it may help the expert to make an objective conclusion on the cause of death.     

Simultaneous quantitation of delta-9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in serum by GC/MS using deuterated internal standards and its application to a smoking study and forensic cases.

A new procedure for the simultaneous detection of delta-9-tetrahydrocannabinol (THC) and its major metabolite, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in serum has been evaluated. The method combines rapid, efficient, solid-phase extraction and simple derivatization by methylation. Analysis and quantitation is performed by gas chromatography/mass spectrometry (GC/MS) using deuterated cannabinoids as internal standards (IS). Reproducibility and sensitivity of the method are good. The procedure is applied to serum specimens collected from a smoking study with 24 volunteers and 212 forensic cases. Results are interpreted based upon the current knowledge about THC metabolism and pharmacokinetics.     

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.     

Hemolyzed blood and serum levels of delta 9-THC: effects on the performance of roadside sobriety tests

A pilot study was conducted to ascertain the range of induced hemolyzed blood/serum delta 9-tetrahydrocannabinol (delta 9-THC) concentrations in 58 human subjects. Subjects were tested within 5 min of smoking a delta 9-THC cigarette and then at half-hour intervals to 150 min. The subjects initially demonstrated a broad range of delta 9-THC hemolyzed blood levels, which settled within an hour to levels comparable to those measured in California drivers who had been stopped for impaired driving, arrested, and tested for delta 9-THC. Serum levels, when correlated with performance or roadside sobriety tests, demonstrated a broad range (5 to 183 ng/mL) of delta 9-THC levels and an "adaptation" effect in the subjects' perception of their own impairment. Although this preliminary study was not a double-blind placebo experiment, the overall performance of human subjects demonstrated the "adaptation" effect, which may be a significant factor in making judgments while performing such complex tasks as driving. Also, the effects of the drug extended beyond the period of elevated delta 9-THC blood levels, perhaps because of THC metabolites that may contribute to impairment or the persistence of THC in the central nervous system. This pilot study will lay the groundwork for a program designed to determine the epidemiology and behavior correlates of marijuana use in motorists.     

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.     

Practical experiences with the determination of cadaver age by evaluation of bacterial metabolic products

In an experiment a corpse had been kept at room temperature (16 degrees-23 degrees C) for 1163 h. At regular intervals brain samples were taken and the content of free amino acids and related compounds was determined by column chromatography. It could be demonstrated that in a period of 4-20 days postmortem the age of the corpse could be calculated from the concentrations of alpha-aminobutyrate (ABU), gamma-aminobutyrate (GABA), and glutamic acid (GLU) in brain employing the previously [5] presented formula: (formula; see text) T is the postmortem time lapse (days). ABU, GABA, and GLU are the concentrations (mumol/g) wet tissue of the corresponding amino acids. During this postmortem interval there is nearly a linear correlation; from this interval a correct assignment between concentrations and time cannot be given. The determination method is in the range of about 15 degrees-25 degrees C (guarantees bacterial growth and metabolism) independent of ambient temperature. The brain samples (cortex or parts of the putrified brain mush) can be taken without any special precaution during normal autopsy. The results of the experiment (in Fig. 1) were supported by some practical cases where the time lapse since death was well (in Fig. 1) or reasonably (in Fig. 1) known. These results suggest that this method allows in many cases the determination of the age of a corpse found in a warm environment (approximately 15 degrees-25 degrees C) approximately 4-20 days after death.     

A specific G. C. method for the determination of carbamazepine in blood.

Carbamazepine is extracted with dichloromethane from whole blood. After a rinsing procedure the residue is gaschromatographed partly in methanol, where two peaks occur, and partly in a methylating reagent, where the same two peaks are seen, the heights of these now being reversed. This reversal is used as a means of identification. Recovery was found to be 94 +/- 12 %. Blood analysis from 12 patients being treated with carbamazepine gave values from 0.6 - 6.5 mg/kg.