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.

Case Report of Methylone, Oxymorphone and Ethanol in a Fatality Case with Tissue Distribution.

It is reasonable to expect the presence of multiple drugs to present a complicated picture of toxicity. We report a fatal case involving a young man who purchased illicit drugs and knowingly consumed them. After consuming these drugs and going to sleep in his friend's car, he was found unresponsive the next morning with no signs of physical violence. Drugs found in the peripheral blood at autopsy were oxymorphone, methylone and ethanol at concentrations of 0.106, 0.50 and 130 mg/dL, respectively. The levels of oxymorphone and methylone in peripheral blood were comparable to those observed in other reported fatalities. Cocaine and benzoylecgonine were detected in the urine but not in the blood. Measureable concentrations were also observed for oxymorphone and methylone in urine, liver, kidney and bile. The physical findings at autopsy included pulmonary edema. This is the only reported fatal case involving this combination of drugs encountered in our laboratory.

Analysis of Parent Synthetic Cannabinoids in Blood and Urinary Metabolites by Liquid Chromatography Tandem Mass Spectrometry.

Synthetic cannabinoids emerged on the designer drug market in recent years due to their ability to produce cannabis-like effects without the risk of detection by traditional drug testing techniques such as immunoassay and gas chromatography-mass spectrometry. As government agencies work to schedule existing synthetic cannabinoids, new, unregulated and structurally diverse compounds continue to be developed and sold. Synthetic cannabinoids undergo extensive metabolic conversion. Consequently, both blood and urine specimens may play an important role in the forensic analysis of synthetic cannabinoids. It has been observed that structurally similar synthetic cannabinoids follow common metabolic pathways, which often produce metabolites with similar metabolic transformations. Presented are two validated quantitative methods for extracting and identifying 15 parent synthetic cannabinoids in blood, 17 synthetic cannabinoid metabolites in urine and the qualitative identification of 2 additional parent compounds. The linear range for most synthetic cannabinoid compounds monitored was 0.1-10 ng/mL with the limit of detection between 0.01 and 0.5 ng/mL. Selectivity, specificity, accuracy, precision, recovery and matrix effect were also examined and determined to be acceptable for each compound. The validated methods were used to analyze a compilation of synthetic cannabinoid investigative cases where both blood and urine specimens were submitted. The study suggests a strong correlation between the metabolites detected in urine and the parent compounds found in blood.

Multidrug toxicity involving sumatriptan.

A multidrug fatality involving sumatriptan is reported. Sumatriptan is a tryptamine derivative that acts at 5-HT(1B/1D) receptors and is used for the treatment of migraines. The decedent was a 21-year-old white female found dead in bed by her spouse. No signs of physical trauma were observed and a large number of prescription medications were discovered at the scene. Toxicological analysis of the central blood revealed sumatriptan at a concentration of 1.03 mg/L. Following therapeutic dosing guidelines, sumatriptan concentrations do not exceed 0.095 mg/L. Sumatriptan was isolated by solid-phase extraction and analyzed using liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode. A tissue distribution study was completed with the following concentrations measured: 0.61 mg/L in femoral blood, 0.56 mg/L in iliac blood, 5.01 mg/L in urine, 0.51 mg/kg in liver, 3.66 mg/kg in kidney, 0.09 mg/kg in heart, 0.32 mg/kg in spleen, 0.01 mg/kg in brain, 15.99 mg/kg in lung and 78.54 mg/45 mL in the stomach contents. Carisoprodol, meprobamate, fluoxetine, doxylamine, orphenadrine, dextromethorphan and hydroxyzine were also present in the blood at the following concentrations: 3.35, 2.36, 0.63, 0.19, 0.06, 0.55 and 0.16 mg/L. The medical examiner ruled the cause of death as acute mixed drug toxicity and the manner of death as accident.

Evaluation of designer amphetamine interference in GC-MS amine confirmation procedures.

In recent years, a class of new designer drugs commonly referred to as 'bath salts' have made their way to the illicit drug market. The most common drugs encountered are designer amphetamines and cathinones. Many analytical methods for analysis and identification of bath salts have been published, but there has been little reported on their impact on existing gas chromatography-mass spectrometry (GC-MS) amine confirmation methods. Due to structural similarities, the potential exists that designer amphetamines may interfere with methods used for analysis of sympathomimetic amines. Methiopropamine, 4-fluoroamphetamine, 4-fluoromethamphetamine (4-FMA) and 4-methylamphetamine were examined for potential interference with immunoassays and GC-MS confirmation analysis utilizing three derivatization procedures: R(-)-α-methoxy-α-trifluoromethylphenylacetyl chloride (R-MTPAC), heptafluorobutyric anhydride (HFBA) and chlorodifluoroacetic anhydride (ClF(2)AA). Significant cross-reactivity was observed with all the four compounds on the Syva Emit(®) II Plus Amphetamines and Roche KIMS Amphetamines II immunoassays. Laboratories utilizing GC-MS selected-ion-monitoring confirmation methods with R-MTPAC, HFBA or ClF(2)AA derivatives could experience potential chromatographic and mass spectral interferences from 4-fluroamphetamine, 4-FMA and methiopropamine in the form of ion ratio and quantitative failures. Careful ion selection, proper selectivity and specificity studies during method validation and rigid chromatographic and spectral acceptance criteria are required to assure the robustness and accuracy of GC-MS methods.

A fatality involving AH-7921.

A case is presented of a 19-year-old white male who was found dead in bed by a friend. While no anatomic cause of death was observed at autopsy, toxicological analysis of his blood identified AH-7921, a synthetic opioid. AH-7921 was isolated by liquid-liquid extraction into n-butyl chloride from alkalinized samples. Extracts were analyzed and quantified by gas chromatography mass spectrometry in selected ion monitoring mode. The heart blood had an AH-7921 concentration of 3.9 mg/L and the peripheral blood concentration was 9.1 mg/L. In addition to the blood, all submitted postmortem specimens including urine, liver, kidney, spleen, heart, lung, brain, bile and stomach content were quantified. The following concentrations of AH-7921 were reported: 6.0 mg/L in urine, 26 mg/kg in liver, 7.2 mg/kg in kidney, 8.0 mg/kg in spleen, 5.1 mg/kg in heart, 21 mg/kg in lung, 7.7 mg/kg in brain, 17 mg/L in bile and 120 mg/125 mL in the stomach content. The medical examiner reported that the cause of death was opioid intoxication and the manner of death was accident.

Comparison of oxycodone in vitreous humor and blood using EMIT screening and gas chromatographic-mass spectrometric quantitation.

Vitreous humor may serve as a useful alternative specimen for oxycodone analysis in death investigations where blood samples are not available or are of poor quality or limited quantity. The purpose of this study was to investigate the relationship between immunoassay results and gas chromatography-mass spectrometry (GC-MS) quantitation of oxycodone in postmortem vitreous humor and blood. When used with vitreous humor calibrators, the Microgenics DRI Oxycodone (EMIT) Assay was found to be linear from 25 to 500 ng/mL with an limit of detection of 25 ng/mL. Vitreous humor and postmortem blood precipitate immunoassay responses in 57 oxycodone-positive cases were found to be correlated (r(2) = 0.69, p < 0.01). Confirmation and quantitation of oxycodone in vitreous humor by GC-MS was linear from 50 to 1000 ng/mL with a limit of detection of 10 ng/mL and a limit of quantitation of 50 ng/mL. In 30 cases, oxycodone vitreous humor concentrations ranged from less than 50 to 945 ng/mL, and blood concentrations ranged from 103 to 768 ng/mL. The average vitreous humor/blood ratio was 1.16 and ranged from 0.12 to 3.26. Disparities between vitreous fluid and blood oxycodone concentrations were seen in a few cases.