Validated method for the simultaneous determination of Δ-THC and Δ-THC-COOH in oral fluid, urine and whole blood using solid-phase extraction and liquid chromatography–mass spectrometry with electrospray ionization

A fully validated, sensitive and specific method for the extraction and quantification of Δ⁹-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-Δ⁹-THC (THC-COOH) and for the detection of 11-hydroxy-Δ⁹-THC (11-OH THC) in oral fluid, urine and whole blood is presented. Solid-phase extraction and liquid chromatography–mass spectrometry (LC–MS) technique were used, with electrospray ionization. Three ions were monitored for THC and THC-COOH and two for 11-OH THC. The compounds were quantified by selected ion recording of m/z 315.31, 329.18 and 343.16 for THC, 11-OH THC and THC-COOH, respectively, and m/z 318.27 and 346.26 for the deuterated internal standards, THC-d₃ and THC-COOH-d₃, respectively. The method proved to be precise for THC and THC-COOH both in terms of intra-day and inter-day analysis, with intra-day coefficients of variation (CV) less than 6.3, 6.6 and 6.5% for THC in saliva, urine and blood, respectively, and 6.8 and 7.7% for THC-COOH in urine and blood, respectively. Day-to-day CVs were less than 3.5, 4.9 and 11.3% for THC in saliva, urine and blood, respectively, and 6.2 and 6.4% for THC-COOH in urine and blood, respectively. Limits of detection (LOD) were 2 ng/mL for THC in oral fluid and 0.5 ng/mL for THC and THC-COOH and 20 ng/mL for 11-OH THC, in urine and blood. Calibration curves showed a linear relationship for THC and THC-COOH in all samples (r² > 0.999) within the range investigated.The procedure presented here has high specificity, selectivity and sensitivity. It can be regarded as an alternative method to GC–MS for the confirmation of positive immunoassay test results, and can be used as a suitable analytical tool for the quantification of THC and THC-COOH in oral fluid, urine and/or blood samples.     

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.     

Drugs in oral fluid in randomly selected drivers

There were 13,176 roadside drug tests performed in the first year of the random drug-testing program conducted in the state of Victoria. Drugs targeted in the testing were methamphetamines and Δ⁹-tetrahydrocannabinol (THC). On-site screening was conducted by the police using DrugWipe^®, while the driver was still in the vehicle and if positive, a second test on collected oral fluid, using the Rapiscan^®, was performed in a specially outfitted “drug bus” located adjacent to the testing area. Oral fluid on presumptive positive cases was sent to the laboratory for confirmation with limits of quantification of 5, 5, and 2 ng/mL for methamphetamine (MA), methylenedioxy-methamphetamine (MDMA), and THC, respectively. Recovery experiments conducted in the laboratory showed quantitative recovery of analytes from the collector. When oral fluid could not be collected, blood was taken from the driver and sent to the laboratory for confirmation. These roadside tests gave 313 positive cases following GC–MS confirmation. These comprised 269, 118, and 87 cases positive to MA, MDMA, and THC, respectively. The median oral concentrations (undiluted) of MA, MDMA, and THC was 1136, 2724, and 81 ng/mL. The overall drug positive rate was 2.4% of the screened population. This rate was highest in drivers of cars (2.8%). The average age of drivers detected with a positive drug reading was 28 years. Large vehicle (trucks over 4.5 t) drivers were older; on average at 38 years. Females accounted for 19% of all positives, although none of the positive truck drivers were female. There was one false positive to cannabis when the results of both on-site devices were considered and four to methamphetamines.     

Evaluation of the Cozart RapiScan drug test system for opiates and cocaine in oral fluid

The purpose of this study was to evaluate the efficiency of the Cozart^® RapiScan (CRS) drug test system for detecting opiates and cocaine in oral fluid. Oral fluid samples were collected using the Cozart^® RapiScan collection system from 358 donors who were receiving treatment for their addiction and were monitored for drug misuse. A further 103 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory using the two-panel Cozart^® RapiScan cartridge for opiates and cocaine and confirmed using gas chromatography–mass spectrometry (GC–MS). The samples were stored frozen at −20 °C until analysis by GC–MS. The overall accuracy of the CRS for both opiates and cocaine was 100%. Samples spiked at 50% above and below the cut-off consistently gave negative and positive results respectively. A total of 88 samples were positive for various opiates and 111 samples were positive for cocaine and/or its metabolites. The CRS for opiates and cocaine in oral fluid, using a cut-off of 30 ng/mL morphine or benzoylecgonine equivalents in neat oral fluid, had overall efficiencies of 98% and 99%, respectively, versus GC–MS. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.     

Development and validation of the Cozart DDS oral fluid collection device

The testing of oral fluid for drugs of abuse has increased significantly over recent years and is now commonplace in drug rehabilitation clinics, the workplace, prisons and custody suites. The global problem of identifying drugged drivers has also led to an increase in oral fluid testing at the roadside. The main requirements for the implementation of roadside drug testing are a rapid sample collection time, collection of a known sample volume and recovery of drugs from the collection device. We report here the development of the Cozart^® DDS oral fluid collector, an oral fluid collector that combines rapid and adequate sample collection with satisfactory drug recovery. Oral fluid was collected from drug users (n = 134) and drug-free individuals (n = 137), using the Cozart^® DDS oral fluid collector. The mean time for the completion of collection (full coloration of the sample presence indicator) was 34 s for drug-free individuals and 44 s for drug users. The average volume collected was 0.34 mL (n = 271). No chemical stimulant (to induce salivation) was used to achieve the collection times observed in either the drug-free or the drug-taking sample populations. Drugs were extracted from the collector using the Cozart^® DDS buffer and drug recovery was determined by Cozart^® enzyme immunoassays. The recovery studies showed that for amphetamine, Δ⁹THC, cocaine, methadone, methamphetamine, morphine and temazepam over 90% of the drug in the sample was eluted from the collector. The Cozart^® DDS oral fluid collector provides a reliable mechanism for the collection of oral fluid at the roadside that achieves the rapid collection times required.     

Quantitative analysis of multiple illicit drugs in preserved oral fluid by solid-phase extraction and liquid chromatography–tandem mass spectrometry

We present a validated method for the simultaneous analysis of basic drugs which comprises a sample clean-up step, using mixed-mode solid-phase extraction (SPE), followed by LC–MS/MS analysis. Deuterated analogues for all of the analytes of interest were used for quantitation. The applied HPLC gradient ensured the elution of all the drugs examined within 14 min and produced chromatographic peaks of acceptable symmetry. Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions for the non-deuterated analogues. Oral fluid was collected with the Intercept^®, a FDA approved sampling device that is used on a large scale in the US for workplace drug testing. However, this collection system contains some ingredients (stabilizers and preservatives) that can cause substantial interferences, e.g. ion suppression or enhancement during LC–MS/MS analysis, in the absence of suitable sample pre-treatment. The use of the SPE was demonstrated to be highly effective and led to significant decreases in the interferences. Extraction was found to be both reproducible and efficient with recoveries >76% for all of the analytes. Furthermore, the processed samples were demonstrated to be stable for 48 h, except for cocaine and benzoylecgonine, where a slight negative trend was observed, but did not compromise the quantitation. In all cases the method was linear over the range investigated (2–200 μg/L) with an excellent intra-assay and inter-assay precision (coefficients of variation <10% in most cases) for QC samples spiked at a concentration of 4, 12 and 100 μg/L. Limits of quantitation were estimated to be at 2 μg/L with limits of detection ranging from 0.2 to 0.5 μg/L, which meets the requirements of SAMHSA for oral fluid testing in the workplace. The method was subsequently applied to the analysis of Intercept^® samples collected at the roadside by the police, and to determine MDMA and MDA levels in oral fluid samples from a controlled study.     

Correlation of Delta9-tetrahydrocannabinol concentrations determined by LC-MS-MS in oral fluid and plasma from impaired drivers and evaluation of the on-site Dräger DrugTest

Oral fluid (collected with the Intercept((R)) device) and plasma samples were obtained from 139 individuals suspected of driving under the influence of drugs and analyzed for Delta(9)-tetrahydrocannabinol (THC), the major psychoactive constituent of cannabis, using a validated quantitative LC-MS-MS method. The first aim of the study was to investigate the correlation between the analytical data obtained in the plasma and oral fluid samples, to evaluate the use of oral fluid as a 'predictor' of actual cannabis influence. The results of the study indicated a good accuracy when comparing THC detection in oral fluid and plasma (84.9-95.7% depending on the cut-off used for plasma analysis). ROC curve analysis was subsequently used to determine the optimal cut-off value for THC in oral fluid with plasma as reference sample, in order to 'predict' a positive plasma result for THC. When using the LOQ of the method for plasma (0.5 ng/mL), the optimal cut-off was 1.2 ng/mL THC in oral fluid (sensitivity, 94.7%; specificity, 92.0%). When using the legal cut-off in Belgium for driving under the influence in plasma (2 ng/mL), an optimal cut-off value of 5.2 ng/mL THC in oral fluid (sensitivity, 91.6%; specificity, 88.6%) was observed. In the second part of the study, the performance of the on-site Dr?ger DrugTest for the screening of THC in oral fluid during roadside controls was assessed by comparison with the corresponding LC-MS-MS results in plasma and oral fluid. Since the accuracy was always less than 66%, we do not recommend this Dr?ger DrugTest system for the on-site screening of THC in oral fluid.     

Analysis of cannabis in oral fluid specimens by GC-MS with automatic SPE

Methamphetamine (MA) is the most commonly abused drug in Korea, followed by cannabis. Traditionally, MA analysis is carried out on both urine and hair samples and cannabis analysis in urine samples only. Despite the fact that oral fluid has become increasingly popular as an alternative specimen in the field of driving under the influence of drugs (DUID) and work place drug testing, its application has not been expanded to drug analysis in Korea. Oral fluid is easy to collect and handle and can provide an indication of recent drug abuse.In this study, we present an analytical method using GC–MS to determine tetrahydrocannabinol (THC) and its main metabolite 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in oral fluid. The validated method was applied to oral fluid samples collected from drug abuse suspects and the results were compared with those in urine. The stability of THC and THC-COOH in oral fluid stored in different containers was also investigated.Oral fluid specimens from 12 drug abuse suspects, submitted by the police, were collected by direct expectoration. The samples were screened with microplate ELISA. For confirmation they were extracted using automated SPE with mixed-mode cation exchange cartridge, derivatized and analyzed by GC-MS using selective ion monitoring (SIM).The concentrations of THC and THC-COOH in oral fluid showed a large variation and the results from oral fluid and urine samples from cannabis abusers did not show any correlation. Thus, detailed information about time interval between drug use and sample collection is needed to interpret the oral fluid results properly. In addition, further investigation about the detection time window of THC and THC-COOH in oral fluid is required to substitute oral fluid for urine in drug testing.     

Validated method for the simultaneous determination of Delta9-THC and Delta9-THC-COOH in oral fluid, urine and whole blood using solid-phase extraction and liquid chromatography-mass spectrometry with electrospray ionization

A fully validated, sensitive and specific method for the extraction and quantification of Delta(9)-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-Delta(9)-THC (THC-COOH) and for the detection of 11-hydroxy-Delta(9)-THC (11-OH THC) in oral fluid, urine and whole blood is presented. Solid-phase extraction and liquid chromatography-mass spectrometry (LC-MS) technique were used, with electrospray ionization. Three ions were monitored for THC and THC-COOH and two for 11-OH THC. The compounds were quantified by selected ion recording of m/z 315.31, 329.18 and 343.16 for THC, 11-OH THC and THC-COOH, respectively, and m/z 318.27 and 346.26 for the deuterated internal standards, THC-d(3) and THC-COOH-d(3), respectively. The method proved to be precise for THC and THC-COOH both in terms of intra-day and inter-day analysis, with intra-day coefficients of variation (CV) less than 6.3, 6.6 and 6.5% for THC in saliva, urine and blood, respectively, and 6.8 and 7.7% for THC-COOH in urine and blood, respectively. Day-to-day CVs were less than 3.5, 4.9 and 11.3% for THC in saliva, urine and blood, respectively, and 6.2 and 6.4% for THC-COOH in urine and blood, respectively. Limits of detection (LOD) were 2 ng/mL for THC in oral fluid and 0.5 ng/mL for THC and THC-COOH and 20 ng/mL for 11-OH THC, in urine and blood. Calibration curves showed a linear relationship for THC and THC-COOH in all samples (r(2)>0.999) within the range investigated. The procedure presented here has high specificity, selectivity and sensitivity. It can be regarded as an alternative method to GC-MS for the confirmation of positive immunoassay test results, and can be used as a suitable analytical tool for the quantification of THC and THC-COOH in oral fluid, urine and/or blood samples.     

Testing human hair for cannabis

To validate information on cannabis use, we investigated human hair and pubic hair for cannabinoids (THC and THC-COOH) by gas chromatography/mass spectrometry. Samples (100 mg approximately) were decontaminated with methylene chloride, then pulverized and dissolved in 1 ml 1 N NaOH for 10 min at 95 °C in the presence of 200 ng of deuterated standards. After cooling, samples were extracted by n-hexane/ethyl acetate after acidification with acetic acid. After derivatization of the dry extract by PFPA/PFP-OH, the drugs were separated on a 30-m capillary column and detected using selected-ion monitoring (m/z 377 and 459 for THC and THC-COOH, respectively). Forty-three hair samples were obtained from fatal heroin overdose cases. Among them, 35% tested positive for cannabinoids. Hair concentrations ranged from 0.26 to 2.17 ng/mg (mean, 0.74 ng/mg) and 0.07 to 0.33 ng/mg (mean, 0.16 ng/mg) of THC and THC-COOH, respectively. As is generally the case for other drugs detected in hair, metabolite concentration was always lower when compared to the parent drug concentration. In pubic hair, THC concentrations ranged from 0.34 to 3.91 ng/mg (mean, 1.35 ng/mg) and THC-COOH concentrations from 0.07 to 0.83 ng/mg (mean, 0.28 ng/mg). In most cases, the highest cannabinoid concentration was found in pubic hair, suggesting that this sample may be the more suitable for cannabis testing.     

Rapid enantiomeric analysis of zopiclone in serum by supercritical fluid chromatography–tandem mass spectrometry

Zopiclone (ZOP) is a hypnotic drug prescribed to treat insomnia. Due to the chiral nature of ZOP, the psychologically active S-form and inactive R-form need to be determined enantiomerically in a forensic drug analysis. In the present study, a supercritical fluid chromatography (SFC) method was designed with a faster analysis ability than that of previously reported techniques. The SFC–tandem mass spectrometry (SFC–MS/MS) method was optimized using a column with a chiral polysaccharide stationary phase (Trefoil CEL2). ZOP was extracted from pooled human serum using solid-phase extraction (Oasis HLB) and analyzed. The developed SFC–MS/MS method achieved the baseline separation of S-ZOP and R-ZOP within 2 min. The fit-for-purpose method validation indicated that the optimized solid-phase extraction achieved near complete recovery and approximately 70% of the matrix effect. Both the retention time and peak area showed sufficient precision. The lower and upper limits of quantification (LOQ) were 5.7 × 10⁻² ng/mL and 25 ng/mL for R-ZOP, and 5.2 × 10⁻² ng/mL and 25 ng/mL for S-ZOP. The calibration line was linear in the range from lower LOQ to upper LOQ. The stability test indicated that ZOP in serum stored in a refrigerator (4°C) degraded and about 55% remained in 31 days. The quick analysis of the SFC–MS/MS method makes it a valid option for the enantiomeric analysis of ZOP.     

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.     

Confirmation by LC–MS of drugs in oral fluid obtained from roadside testing

The aim of this study was to assess the effectiveness of two current on-site oral fluid (OF) drug detection devices (OraLab and Dräger), as part of the Spanish participation in the Roadside Testing Assessment Project (ROSITA Project). The study was done in collaboration with the Spanish Traffic Police, in Galicia (NW Spain), during 2004 and 2005. A total of 468 drivers selected at the police controls agreed to participate through informed consent. In addition, saliva samples were collected and sent to the laboratory to confirm the on-site results. For this purpose, two different analytical liquid chromatography–mass spectrometry (LC–MS) methods were used to detect 11 drugs or metabolites in a 300 μL sample. Simultaneous analysis of morphine, 6-acetylmorphine, amphetamine, methamphetamine, MDA, MDMA, MDEA, MBDB, cocaine and benzoylecgonine was carried out using 100 μL of oral fluid, after an automated solid phase extraction. A different LC–MS method was performed to detect Δ⁹-THC in 200 μL of oral fluid using liquid–liquid extraction with hexane at pH 6. Both methods were fully validated, including linearity (1–250 ng/mL, 2–250 ng/mL) recovery (>50%), within-day and between-day precision (CV < 15%), accuracy (mean relative error < 15%), limit of detection (0.5 and 1 ng/mL), quantitation (1 and 2 ng/mL) and matrix effect. All of the positive cases and a random selection of 30% of the negatives were analyzed for confirmation analysis. Good results (sensitivity, specificity, accuracy, positive predictive value and negative predictive value > 90%) were obtained for cocaine and opiates by OraLab, and for cocaine by Dräger. However, the results for the other compounds could be improved for both detection devices. Differences in the ease of use and in the interpretation mode (visual or instrumental) were observed.     

Fatal intoxication with tianeptine (Stablon)

Tianeptine (Stablon^®), although structurally similar to tricyclic antidepressants, acts by enhancing the reuptake of serotonin. A fatal case is presented involving a 26-year-old man, found lying in bed with a “mushroom of foam” around his mouth. Empty blister packs of Stablon^® and a suicide note were found next to the body. A liquid–liquid extraction procedure with n-hexane: ethyl acetate and n-hexane: 2-propanol, followed by LC-DAD-MS analysis, using positive mode electrospray ionization was performed. The detection limit was 0.001 μg/mL. The toxicological results revealed the following tianeptine concentrations in the post-mortem samples: blood 5.1 μg/mL; urine 2.0 μg/mL; liver 23 μg/g; stomach contents 22 mg. Femoral blood analyses also revealed an ethanol concentration of 0.53 g/L. The present method was also developed and validated for the other post-mortem specimens, since no previous published data had confirmed the post-mortem distribution of tianeptine. The absence of other suitable direct causes of death (macroscopic or histological) and the positive results achieved with the toxicological analysis led the pathologist to rule that death was due to an intoxication caused by the suicidal ingestion of tianeptine in combination with alcohol.     

Proficiency testing (external quality assessment) of drug detection in oral fluid

Eighteen external quality assessment (proficiency testing) samples were prepared from client specimens collected with the Intercept^® oral fluid collection device and by spiking drug-free oral fluid. Samples were circulated in pairs at quarterly intervals to 13 UK and USA based laboratories for analysis by a panel of OraSure micro-plate Intercept^® enzyme immunoassay kits and hyphenated mass spectrophotometric techniques. During the survey, there was a single case of non-specificity in a false report for methadone. The major errors were of lack of sensitivity relative to the concentration thresholds specified for the immunoassays. The sensitivity for overall ‘present’/‘not found’ reports calculated as true positives/(true positives + false negatives) were for the amfetamine specific assay 50%, methyl-amfetamines 93%, barbiturates 64%, cannabinoids 73%, cocaine and metabolites 100%, benzodiazepines 69%, methadone 95%, opiates 79% (opiates excluding oxycodone 93%), phencyclidine 93% and human gamma-globulin 97%. A small number of the sensitivity errors were attributable to errors in chromatographic confirmation techniques.     

Cannabinoids determination in oral fluid by SPME–GC/MS and UHPLC–MS/MS and its application on suspected drivers

The confirmation of Δ9-tetrahydrocannabinol (THC) in oral fluid (OF) is an important issue for assessing Driving Under the Influence of Drugs (DUID). The aim of this research was to develop a highly sensitive method with minimal sample pre-treatment suitable for the analysis of small OF volumes (100 μL) for the confirmation of cannabinoids in DUID cases. Two methods were compared for the confirmation of THC in residual OF samples, obtained from a preliminary on-site screening with commercial devices. An ultra high performance LC–MS (UHPLC–MS/MS) method and an SPME–GC/MS method were hence developed. 100 μL of the residual mixture OF/preservative buffer or neat OF was simply added to 10 μL of THC-D3 (1 μg/mL) and submitted to the two different analyses: A — direct injection of 10 μL in UHPLC–MS/MS in positive electrospray ionisation (ESI) mode and B — sampling for 30 min with SPME (100 μm polydimethylsiloxane or PDMS fibre) and direct injection by desorption of the fibre in the GC injection port.The lowest limit of detection (LLOD) of THC was 2 ng/mL in UHPLC–MS/MS and 0.5 ng/mL in SPME–GC/MS. In addition, cannabidiol (CBD) and cannabinol (CBN) could be detected in GC/MS equipment at 2 ng/mL, whilst in UHPLC–MS/MS the LLOD was 20 ng/mL.Both methods were applied to 70 samples coming from roadside tests. By SPME–GC/MS analysis, THC was confirmed in 42 samples, whilst CBD was detected in 21 of them, along with CBN in 14 samples. THC concentrations ranged from traces below the lowest limit of quantification or LLOQ (2 ng/mL) up to 690 ng/mL.     

Distribution of ∆9‐Tetrahydrocannabinol and 11‐Nor‐9‐Carboxy‐∆9‐Tetrahydrocannabinol Acid in Postmortem Biological Fluids and Tissues From Pilots Fatally Injured in Aviation Accidents

Little is known of the postmortem distribution of ?⁹‐tetrahydrocannabinol (THC) and its major metabolite, 11‐nor‐9‐carboxy‐?⁹‐tetrahydrocannabinol (THCCOOH). Data from 55 pilots involved in fatal aviation accidents are presented in this study. Gas chromatography/mass spectrometry analysis obtained mean THC concentrations in blood from multiple sites, liver, lung, and kidney of 15.6 ng/mL, 92.4 ng/g, 766.0 ng/g, 44.1 ng/g and mean THCCOOH concentrations of 35.9 ng/mL, 322.4 ng/g, 42.6 ng/g, 138.5 ng/g, respectively. Heart THC concentrations (two cases) were 184.4 and 759.3 ng/g, and corresponding THCCOOH measured 11.0 and 95.9 ng/g, respectively. Muscle concentrations for THC (two cases) were 16.6 and 2.5 ng/g; corresponding THCCOOH, “confirmed positive” and 1.4 ng/g. The only brain tested in this study showed no THC detected and 2.9 ng/g THCCOOH, low concentrations that correlated with low values in other specimens from this case. This research emphasizes the need for postmortem cannabinoid testing and demonstrates the usefulness of a number of tissues, most notably lung, for these analyses.     

Generating DNA profiles from immunochromatographic cards using LCN methodology

The aim of this research was to obtain DNA profiles from immunochromatographic test devices which have already yielded positive results with body fluids obtained from fourteen volunteers. Three different immunochromatographic cards for the identification of human blood and one for the identification of human saliva were used for this research. Each body fluid was detected using the appropriate immunochromatographic card. The used cards were kept at room temperature for various lengths of time. The membranes were removed at the end of the designated times and the entire strip was extracted using low copy number (LCN) extraction procedure. The extracted DNA was amplified using reduced amplification volume and higher PCR cycle numbers. Autosomal STR profiles were detected using AmpFℓSTR^® Identifiler™ PCR Amplification Kit from Applied Biosystems (AB). Additionally, DNA extracted from the male volunteers was amplified using the AB AmpFℓSTR^® Yfiler™ PCR Amplification Kit. Analysis of the amplified products was carried out by capillary electrophoresis injection on the AB 3130xl Genetic Analyzer. The generated DNA data was analyzed using the SoftGenetics GeneMarker^® HID Version 1.7 software.Autosomal and Y-STR DNA profiles were obtained from most of the cards which were stored at room temperature for up to three months. DNA profile was obtained from all four types of the immunochromatographic cards used in this study. These profiles were concordant with the profiles obtained from the donors’ reference samples.     

Discriminating Hodgdon Pyrodex® and Triple Seven® Using Gas Chromatography–Mass Spectrometry

Abstract: Pyrodex ^® and Triple Seven ^® are black powder substitutes that often find use as fillers in improvised explosive devices, such as pipe bombs. These propellants have essentially the same overall appearance and oxidizers, but different fuels. For example, Pyrodex ^® contains sulfur, sodium benzoate, and dicyandiamide (DCDA), whereas Triple Seven ^® lacks sulfur but also contains 3‐nitrobenzoic acid. In this method, intact particles and postblast solid residues were reacted with bis(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane in acetonitrile for 30 min at 60°C. The resultant trimethylsilyl derivatives of the organic fuels were then analyzed by gas chromatography–mass spectrometry. Each derivative was clearly resolved from other components, and high‐quality mass spectra were obtained. In addition, characteristic fragments resulting from loss of a methyl radical from the molecular ion (m/z 163 for sulfur, m/z 171 for DCDA, m/z 179 for benzoic acid, and m/z 224 for nitrobenzoic acid) were able to be monitored.     

The NucleoSpin DNA Clean-up XS kit for the concentration and purification of genomic DNA extracts: An alternative to microdialysis filtration

Traditionally, DNA extracts from biological evidence items have been concentrated and rinsed using microdialysis filtration units, including the Centricon^® and Microcon^® centrifugal filter devices. As an alternative to microdialysis filtration, we present an optimized method for using NucleoSpin^® XS silica columns to concentrate and clean-up aqueous extracts from the organic extraction of DNA from biological samples. The method can be used with standard organic extraction and dithiothreitol (DTT)-based differential extraction methods with no modifications to these methods prior to the concentration and clean-up step. Extracts from laboratory-prepared bloodstains, saliva and semen stains have been successfully amplified with both qPCR and STR assays. Finally, the total time to process a set of samples with the NucleoSpin^® XS column is approximately 30 min vs. approximately 1.5 h with the Centricon^® YM-100 filter device.