Cross-Reactivity of 24 Cannabinoids and Metabolites in blood using the immunalysis cannabinoids direct enzyme-linked immunosorbent assay kit.

With a wider availability of synthetic and semi-synthetic cannabinoids in the consumer space, there is a growing impact on public health and safety. Forensic toxicology laboratories should keep these compounds in mind as they attempt to remain effective in screening for potential sources of human performance impairment. Enzyme-linked immunosorbent assay (ELISA) is a commonly utilized tool in forensic toxicology, as its efficiency and sensitivity make it useful for rapid and easy screening for a large number of drugs. This screening technique has lower specificity, which allows for broad cross-reactivity among structurally-similar compounds. In this study, the Cannabinoids Direct ELISA kit from Immunalysis was utilized to assess the cross-reactivities of 24 cannabinoids and metabolites in whole blood. The assay was calibrated with 5 ng/mL of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol and the analytes of interest were evaluated at concentrations ranging from 5 to 500 ng/mL. Most parent compounds demonstrated cross-reactivity ≥ 20 ng/mL, with increasing alkyl side chain length relative to Δ9-tetrahydrocannabinol resulting in decreased cross-reactivity. Of the 24 analytes, only the carboxylic acid metabolites, 11-nor-9-carboxy-Δ8-tetrahydrocannabinol, 11-nor-9(R)-carboxy-hexahydrocannabinol, and 11-nor-9(S)-carboxy-hexahydrocannabinol, were cross-reactive at levels ≤ 10 ng/mL. Interestingly, 11-nor-9(R)-carboxy-hexahydrocannabinol demonstrated cross-reactivity at 5 ng/mL, where its stereoisomer 11-nor-9(S)-carboxy-hexahydrocannabinol, did not. As more information emerges about the prevalence of these analytes in blood specimens, it is important to understand and characterize their impact on current testing paradigms.

Screening and confirmation of psilocin, mitragynine, phencyclidine, ketamine and ketamine metabolites by liquid chromatography-tandem mass spectrometry.

A safe and productive workplace requires a sober workforce, free from substances that impair judgment and concentration. Although drug monitoring programs already exist, the scope and loopholes of standard workplace testing panels are well known, allowing other substances to remain a source of risk. Therefore, a high-throughput urine screening method for psilocin, mitragynine, phencyclidine, ketamine, norketamine and dehydronorketamine was developed and validated in conjunction with a urine and blood confirmation method. There are analytical challenges to overcome with psilocin and mitragynine, particularly when it comes to drug stability and unambiguous identification in authentic specimens. Screening and confirmation methods were validated according to the American National Standards Institute/Academy Standards Board (ANSI/ASB) Standard 036, Standard Practices for Method Validation in Forensic Toxicology. An automated liquid handling system equipped with dispersive pipette extraction tips was utilized for preparing screening samples, whereas an offline solid-phase extraction method was used for confirmation sample preparation. Both methods utilized liquid chromatography-tandem mass spectrometry to achieve limits of detection between 1-5 ng/mL for the screening method and 1 ng/mL for the confirmation method. Automation allows for faster throughput and enhanced quality assurance, which improves turnaround time. Compared to previous in-house methods, specimen volumes were substantially decreased for both blood and urine, which is an advantage when volume is limited. This screening technique is well suited for evaluating large numbers of specimens from those employed in safety-sensitive workforce positions. This method can be utilized by workplace drug testing, human performance and postmortem laboratories seeking robust qualitative screening and confirmation methods for analytes that have traditionally been challenging to routinely analyze.

An Enhanced LC-MS-MS Technique for Distinguishing Δ8- and Δ9-Tetrahydrocannabinol Isomers in Blood and Urine Specimens.

Among the abundance of cannabinoids identified in cannabis, the active parent drug, Δ9-tetrahydrocannabinol (Δ9-THC), and its oxidized metabolite, 11-nor-9-carboxy-Δ9-THC (Δ9-THCCOOH), are attractive analytical targets to detect cannabis use. More recently, confirmation of these analytes may be hindered by a related interfering compound. Forensic toxicology laboratories attribute this phenomenon to an increase in cases containing Δ8-tetrahydrocannabinol (Δ8-THC) and 11-nor-9-carboxy-Δ8-THC (Δ8-THCCOOH). It is technically challenging to chromatographically resolve and accurately quantify Δ8- and Δ9-THC and THCCOOH in toxicology specimens due to their structural resemblance. This study describes a validated method to resolve and quantify active Δ8-THC and Δ9-THC in blood while qualitatively confirming the inactive metabolites Δ8-THCCOOH and Δ9-THCCOOH in blood and urine. Analytes are extracted and concentrated by solid-phase extraction and analyzed by liquid chromatography--electrospray ionization tandem mass spectrometry, which is amenable to modern toxicology laboratory routine workflows. This procedure offers a clear solution to untangling mixtures of these isomers, particularly in cases where Δ8-THC and its metabolite are the sole or dominant form.

Screening and confirmation methods for the qualitative identification of nine phytocannabinoids in urine by LC-MS/MS.

Qualitative liquid chromatography tandem mass spectrometry (LC-MS/MS) methods were developed and validated to screen and confirm the presence of nine phytocannabinoids in urine. The nine phytocannabinoids targeted in the methods included Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC, 11-nor-9-carboxy-THC, cannabidiol, 7-carboxy cannabidiol, cannabinol, cannabigerol, Δ9-tetrahydrocannabivarin (THCV), and 11-nor-9-carboxy-THCV. The methods presented use a rapid, single-step enzymatic hydrolysis followed by solid-phase extraction and LC-MS/MS analysis. Limits of detection were established at 1 µg/L for non-carboxylated analytes and 5 µg/L for carboxylated analytes. The screening and confirmation methods were validated and implemented in the analysis of authentic case samples. These methods can assist forensic, medicolegal, or medical compliance investigations as the presence of phytocannabinoids, or lack there-of, may be used to help differentiate cannabis (hemp, marijuana) use from synthetic THC (dronabinol) exposure.

Identification and Quantification of 5-Fluoro ADB and the 5-Fluoro ADB Ester Hydrolysis Metabolite in Postmortem Blood Samples by LC-MS/MS.

5-Fluoro ADB, also known as 5-fluoro MDMB-PINACA, is a potent synthetic cannabinoid that is an agonist to the human cannabinoid CB1 and CB2 receptors. Adverse physiological and psychological effects that have resulted in hospitalization and/or death have been associated with 5-Fluoro ADB use. In addition, analytical confirmation of 5-Fluoro ADB use has been reported in both forensic human performance toxicology and postmortem cases. An analytical method for the identification and quantification of 5-fluoro ADB and the 5-fluoro ADB ester hydrolysis metabolite in human blood samples by liquid chromatography-tandem mass spectrometry was created and validated. The linear range of this assay was determined to be 0.01-10 ng/mL for 5-fluoro ADB and 10-500 ng/mL for the 5-fluoro ADB ester hydrolysis metabolite. The method met both precision and accuracy requirements. Endogenous and exogenous interferences were not observed. Ion suppression exceeding 25% was observed for 5-fluoro ADB. However, additional experiments were performed to ensure that the observed suppression did not affect other method validation parameters such as limit of detection and accuracy. Blood samples from 36 postmortem cases were analyzed utilizing this methodology. The average blood concentration of 5-fluoro ADB was 0.29 ng/mL in central blood specimens and 0.05 ng/mL in peripheral blood specimens. The average blood concentration of the 5-fluoro ADB ester hydrolysis metabolite was 49 ng/mL in central blood specimens and 21 ng/mL in peripheral blood specimens. A serum sample was also analyzed and had a serum concentration of 0.12 ng/mL for 5-fluoro ADB and 42 ng/mL for the 5-fluoro ADB ester hydrolysis metabolite. As the concentration of the 5-fluoro ADB ester hydrolysis metabolite was found at a greater concentration than that of 5-fluoro ADB, this metabolite may be a useful marker to monitor in an attempt to confirm 5-fluoro ADB use in toxicological investigations.

LC-MS-MS vs ELISA: Validation of a Comprehensive Urine Toxicology Screen by LC-MS-MS and a Comparison of 100 Forensic Specimens.

Toxicology laboratories commonly employ immunoassay methodologies to perform an initial drug screen on urine specimens to direct confirmatory testing. Due to limitations of immunoassay testing and the need to screen for a broader range of drugs with lower limits of detection at a lower cost, mass spectrometry screening techniques have gained favor in the toxicology field. A liquid chromatography-tandem mass spectrometry (LC-MS-MS) urine screening panel was developed and validated for 52 drugs and metabolites. A simple dilute-and-shoot with enzymatic hydrolysis technique was utilized to prepare the urine specimens for analysis. Limit of detection, interference, ionization suppression/enhancement, carryover and stability of processed specimens were assessed during validation. To evaluate the toxicological results obtained from utilizing the LC-MS-MS in comparison with the laboratory's current enzyme-linked immunosorbent assay (ELISA) panel, 100 authentic urine specimens from suspected driving under the influence and drug-facilitated crime cases were analyzed using both methodologies and the results were compared. In addition, the cost of each methodology was evaluated and compared. The validated LC-MS-MS method had limits of detection that were equal to or lower than the concentrations validated for ELISA cutoffs, had fewer exogenous interferences, and the cost of screening per specimen was reduced by ~70% when compared to ELISA. Comparing the toxicology results of forensic urine specimens demonstrated that by only using ELISA, the laboratory was unable to detect benzoylecgonine in 26%, lorazepam in 33% and oxymorphone in 60% of the positive specimens. Additional analytes detected using the LC-MS-MS method were zolpidem and/or metabolite, gabapentin, tramadol and metabolite, methadone and metabolite, meprobamate and phentermine. The results of the validation, the toxicological result comparison and the cost comparison showed that the LC-MS-MS screening method is a simple, sensitive and cost-effective alternative to ELISA screening methods for urine specimens.