Quantitation of hexahydrocannabinol (HHC) and metabolites in blood from DUID cases.

Hexahydrocannabinol (HHC) was first reported in the EU in May 2022. HHC has three chiral carbon atoms, but only (6aR,9R,10aR)-HHC (9R-HHC) and (6aR,9S,10aR)-HHC (9S-HHC) have been encountered in HHC products. The aim of this study was to develop and validate a method for the quantitative analysis of 9R-HHC, 9S-HHC, 11-OH-9R-HHC, 9R-HHC-COOH, 9S-HHC-COOH and 8-OH-9R-HHC. In addition, an objective was to investigate the immunochemical cross-reactivity. Blood samples from driving under the influence of drugs (DUID) cases screened positive for cannabis using enzyme-linked immunoadsorbent assay (ELISA) and confirmed negative for tetrahydrocannabinol (THC), 11-hydroxy-THC and THC-COOH were reanalyzed with a newly validated HHC method to investigate the presence of HHC and metabolites. The LC-MS-MS method was validated for matrix effects, lower limit of quantification (LLOQ), calibration model, precision, bias and autosampler stability. Cross-reactivity on an ELISA method was investigated separately for 9R-HHC-COOH and 9S-HHC-COOH at a concentration range between 5 and 200 ng/mL. The cross-reactivity was found to be 120% for 9R-HHC-COOH and 48% for 9S-HHC-COOH. In the LC-MS-MS method, 9R-HHC-COOH, 9S-HHC-COOH and 11-OH-9R-HHC showed matrix effects <25% at both concentrations, while 8-OH-9R-HHC, 9R-HHC and 9S-HHC matrix effects exceeded 25% at both concentrations but showed good precision (<10% for both inter and intra day) and low bias (<6%) in the further validation. The LLOQ was investigated and established at 0.2 ng/mL for all analytes except the carboxylated metabolites that had an LLOQ of 2.0 ng/mL. The upper LOQ was 20 and 200 ng/mL, respectively. Reanalysis of cases (n = 145) confirmed HHC and metabolites in 32 cases (22%). It was determined that the major metabolite in blood after administration of HHC was 9R-HHC-COOH followed by 11-OH-9R-HHC and that presumptive positive cases are caught by the routine ELISA screening for cannabis.

A quantitative LC-MS/MS analysis of Xylazine, p-Fluorofentanyl, Fentanyl and Fentanyl-Related compounds in postmortem blood.

The purpose of this study was to develop and validate a method to quantitate the veterinary sedative xylazine as well as 4-anilino-N-phenethylpiperidine (4-ANPP), acetyl fentanyl, fentanyl, norfentanyl, and p-fluorofentanyl in blood utilizing liquid chromatography tandem mass spectrometry. This method also qualitatively monitors for the presence of o-fluorofentanyl and m-fluorofentanyl isomers. UCT Clean Screen® DAU extraction columns were utilized to isolate the analytes in postmortem blood samples. The extracts were eluted, evaporated, reconstituted, and then analyzed using a Waters Acquity™ UPLC coupled a triple quadrupole mass spectrometer. The lower limit of quantitation was determined to be 0.1 ng/mL for all analytes, except for xylazine (0.2 ng/mL). The upper limit of quantitation for all analytes was 100 ng/mL. No interferences from matrix, internal standard, or common drug analytes were observed. Bias (-13.1-4.6 %) and precision (-13.14-10.3 %) fell within the acceptable ± 20 % criteria range. Dilution integrity at x2, x10, and x100 was evaluated and all results were within ± 20 % of the target concentration. Processed extract stability was evaluated after 72 h and all results were within ± 20 % of the analyte initial concentration. Matrix effects were the most prominent with xylazine, but deemed acceptable as the deuterated internal standard also observed comparable enhancement. Analysis of 89 postmortem blood samples by this method resulted in positive results for fentanyl (0.27-66 ng/mL, n = 82), xylazine (0.24-958 ng/mL, n = 21), 4-ANPP (0.10-38 ng/mL, n = 72), acetyl fentanyl (0.18-1.5 ng/mL, n = 3), p-fluorofentanyl (0.11-33 ng/mL, n = 30), and norfentanyl (0.10-98 ng/mL, n = 73).

Presumptive identification of nitrite by Griess reagent test strips-Case reports of fatal poisoning with sodium nitrite.

The intentional ingestion of sodium nitrite causes toxicity by inducing methemoglobinemia, which can lead to cyanosis, hypotension and death. The number of reported suicide cases has significantly increased in the past 10 years as sodium nitrite is readily available online. The traditional tests for nitrite and nitrate require specialized detection methods which are not typically available in a postmortem toxicology laboratory. This rise in sodium nitrite overdose cases indicates the need for a simple, quick test for suspected nitrite toxicity. In this study, a common Griess reagent color test (MQuant™ Nitrite Test Strips) was used as a presumptive method in cases where the ingestion of sodium nitrite was suspected. The test results were consistent between specimens in all cases, and vitreous humor was identified as a reliable matrix to be used in the cases of suspected sodium nitrite poisonings. Case reports of five patients who died of suicide by sodium nitrite in a 6-month span are presented.

N,N-Dimethylpentylone (dipentylone)-A new synthetic cathinone identified in a postmortem forensic toxicology case series.

Synthetic cathinones emerged on the novel psychoactive substance (NPS) drug market as alternatives to controlled stimulants and entactogens such as methamphetamine and 3,4-methylenedioxymethamphetamine. The majority of synthetic cathinones can be subclassified into two groups: beta-keto amphetamines (i.e., NPS with the suffix "drone") and beta-keto methylenedioxyamphetamines (i.e., NPS with the suffix "lone"). Although a significant number of beta-keto amphetamines have been identified, beta-keto methylenedioxyamphetamines have dominated the NPS market, including notable drugs like methylone, butylone, N-ethyl pentylone (ephylone), eutylone and now N,N-dimethylpentylone. N,N-Dimethylpentylone, also known as dipentylone or beta-keto-dimethylbenzodioxolylpentanamine, emerged into the illicit drug supply <2 months of the international control of eutylone (September 2021). A novel standard addition method was developed and validated for N,N-dimethylpentylone, pentylone and eutylone, and 18 postmortem cases were quantitated using the method described in this manuscript. The resulting blood concentration range for N,N-dimethylpentylone in this case series was 3.3 to 970 ng/mL (median: 145 ng/mL, mean: 277 ± 283 ng/mL). Pentylone, a metabolite of N,N-dimethylpentylone, was detected in all cases (range: 1.3-420 ng/mL, median: 31 ng/mL and mean: 88 ± 127 ng/mL). Due to the rise in identifications of N,N-dimethylpentylone in postmortem investigations as well as the potential misidentification of N,N-dimethylpentylone as N-ethyl pentylone, samples testing positive for pentylone should be additionally confirmed for the presence of N,N-dimethylpentylone. Based on prior trends of new synthetic cathinones, it can be theorized that N,N-dimethylpentylone may predominate the US synthetic stimulant market for the next 1-2 years; however, given the emergence of additional closely related isomeric compounds, it is important to utilize methodology capable of differentiating N,N-dimethylpentylone from its isomers (N-isopropylbutylone, N-ethyl pentylone, N-ethyl N-methyl butylone, hexylone, N-propylbutylone, diethylone and tertylone).

Fatal intoxication involving 2-methyl AP-237.

Novel synthetic opioids contribute considerably to the opioid epidemic, especially with the frequent emergence of structurally similar compounds. This case report describes a fatal intoxication involving 2-methyl AP-237. A 54-year-old Caucasian male was found deceased from an apparent drug overdose. A plastic container labeled "2MAP" and a cut straw were found in the decedent's backpack at the scene. A white substance found in the container tested positive for fentanyl by field testing. According to his medical history, the decedent was treated for a drug overdose 3 years prior to his death. With no diagnostic findings at autopsy, the case was submitted for toxicological analysis. An unknown substance was detected in peripheral blood and urine using gas chromatography with nitrogen phosphorous detection (GC-NPD). Further testing was conducted using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) which confirmed the presence of 2-methyl AP-237 and potential metabolites in blood and urine. Quantitation by GC-NPD revealed concentrations of 2-methyl AP-237 in blood and urine at 480 ng/mL and 4200 ng/mL, respectively. The toxicological analysis also identified and quantitated alprazolam in the blood at 55 ng/mL. Additionally, the metabolism of 2-methyl AP-237 was investigated and three hydroxylated metabolites were identified in peripheral blood and urine. Limited literature is available for the detection and quantitation of 2-methyl AP-237 in postmortem specimens. Given the toxicological findings with unremarkable autopsy findings, this case is an example of a fatal intoxication involving 2-methyl AP-237.

Rilmazafone: A designer benzodiazepine pro-drug involved in fatal intoxications.

Rilmazafone is a pro-drug that can be prescribed in Japan to treat insomnia. Rilmazafone metabolizes into active compounds by a ring closure resulting in a triazolo benzodiazepine structure similar to alprazolam. In mid-2022, the National Board of Forensic Medicine in Sweden were requested to investigate two separate deaths with the suspected use of pagoclone. Packages labeled "Pagoclone" were found at each scene that was suspected to contain rilmazafone based on website information. During screening by high resolution mass spectrometry, rilmazafone metabolites were presumptively identified. Due to the lack of reference material for the active metabolites, the metabolites were synthesized in house and quantification of the compounds identified in the two autopsy cases was prompted. In Case 1, femoral blood concentrations of 7.9, 65 and 170 ng/g of the metabolites rilmazolam, N-desmethyl rilmazolam and di-desmethyl rilmazolam, respectively, were detected. Additional toxicological findings included the medications haloperidol, alimemazine, fluoxetine, olanzapine and acetaminophen. In Case 2, femoral blood concentrations of 1.7, 1.4 and 70 ng/g of rimazolam, N-desmethyl rilmazolam and di-desmethyl rilmazolam, respectively, were detected. Additional toxicological findings included loperamide, alimemazine and pregabalin. The intake of rilmazafone was determined as the cause of death in Case 1 and contributed in the Case 2.

Review of analytical methods for screening and quantification of fentanyl analogs and novel synthetic opioids in biological specimens.

Fentanyl, fentanyl analogs, and other novel synthetic opioids (NSO), including nitazene analogs, prevail in forensic toxicology casework. Analytical methods for identifying these drugs in biological specimens need to be robust, sensitive, and specific. Isomers, new analogs, and slight differences in structural modifications necessitate the use of high-resolution mass spectrometry (HRMS), especially as a non-targeted screening method designed to detect newly emerging drugs. Traditional forensic toxicology workflows, such as immunoassay and gas chromatography mass spectrometry (GC-MS), are generally not sensitive enough for detection of NSOs due to observed low (sub-µg/L) concentrations. For this review, the authors tabulated, reviewed, and summarized analytical methods from 2010-2022 for screening and quantification of fentanyl analogs and other NSOs in biological specimens using a variety of different instruments and sample preparation approaches. Limits of detection or quantification for 105 methods were included and compared to published standards and guidelines for suggested scope and sensitivity in forensic toxicology casework. Methods were summarized by instrument for screening and quantitative methods for fentanyl analogs and for nitazenes and other NSO. Toxicological testing for fentanyl analogs and NSOs is increasingly and most commonly being conducted using a variety of liquid chromatography mass spectrometry (LC-MS)-based techniques. Most of the recent analytical methods reviewed exhibited limits of detection well below 1 µg/L to detect low concentrations of increasingly potent drugs. In addition, it was observed that most newly developed methods are now using smaller sample volumes which is achievable due to the sensitivity increase gained by new technology and new instrumentation.

Identification of 11-nor-∆8-Tetrahydrocannabinol-9-Carboxylic Acid in Postmortem Urine.

Laws concerning the growing, selling and consuming of cannabis and its related products have been changing considerably over the last few years. The legalization of hemp in 2018 sparked an interest in ∆9-tetrahydrocannabinol (∆9-THC) isomers and analogs that are derived from hemp and sold with minimal oversight. One example is ∆8-tetrahydrocannabinol (∆8-THC). Although less potent than ∆9-THC, ∆8-THC is gaining popularity and can easily be found where cannabis-related products are sold. The Forensic Toxicology Laboratory at the University of Florida routinely tested decedents for 11-nor-∆9-tetrahydrocannabinol-9-carboxylic acid (∆9-THC-acid), the primary metabolite of ∆9-THC. Urine samples from ∼900 decedents were received by the laboratory between mid-November 2021 and mid-March 2022 and subjected to CEDIA™ immunoassay testing. Subsequent confirmation of 194 presumptive positive samples was performed by gas chromatography--mass spectrometry. A peak eluting immediately after ∆9-THC-acid was identified as 11-nor-∆8-tetrahydrocannabinol-9-carboxylic acid (∆8-THC-acid), a metabolite of ∆8-THC, in 26 of those samples (13%). Six of the samples were positive for ∆8-THC-acid only. Other toxicological findings were consistent with poly-drug use including fentanyl/fentanyl analogs, ethanol, cocaine and methamphetamine. There has been an emergence of ∆8-THC use as indicated by the presence of ∆8-THC-acid in 26 of 194 presumptive positive cases during a four-month period. The majority of individuals were White males with a history of drug and/or alcohol use. ∆9-THC-acid, as well as other drugs, was often present. Given the psychoactive potential and availability of ∆8-THC, monitoring ∆8-THC-acid in decedents is important to characterize the risk and prevalence of ∆8-THC use.

Urinary Pharmacokinetics of Immediate and Controlled Release Oxycodone and its Phase I and II Metabolites Using LC-MS-MS.

Oxycodone (OC) is a schedule II semisynthetic opioid in the USA that is prescribed for its analgesic effects and has a high potential for abuse. Prescriptions for OC vary based on the dosage and formulation, immediate release (IR) and controlled release (CR). Monitoring OC metabolites is beneficial for forensic casework. The limited studies that involve pharmacokinetics of the urinary excretion of OC metabolites leave a knowledge gap regarding the excretion of conjugated and minor metabolites, pharmacokinetic differences by formulation, and the impact of CYP2D6 activity on the metabolism and excretion of OC. The objectives of this study were to compare urinary excretion of phase I and II metabolites by formulation and investigate if ratio changes over time could be used to predict the time of intake. Subjects (n = 7) received a single 10 mg IR tablet of Oxycodone Actavis. A few weeks later the same subjects received a single 10 mg CR tablet of Oxycodone Actavis. During each setting, urine was collected at 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 9, 10, 12, 14, 24, 48 and 72 h. Urine samples (100 µL) were diluted with 900 µL internal standard mixture and analyzed on an Acquity UPLC® I-class coupled to a Waters Xevo TQD using a previously validated method. The CYP2D6 phenotypes were categorized as poor metabolizers (PM), intermediate metabolizers (IM), extensive metabolizers (EM) and ultrarapid metabolizers (UM). Comparisons between IR and CR were performed using two-tailed paired t-test at a significance level of P = 0.05. The metabolite ratios showed a general increase over time. Four metabolite to parent ratios were used to predict the time of intake showing that predictions were best at the early time points.

Toxicological Analysis of Fluorofentanyl Isomers in Postmortem Blood.

The opioid epidemic continues to evolve in the USA, with fentanyl the most prevalent synthetic opioid in fatal drug overdoses. Following the scheduling of fentanyl's core structure in 2018, there was a notable decline in the prevalence of fentanyl analogs in decedents; however, fluorofentanyl began being reported in casework in the winter of 2020. Fluorofentanyl has three positional isomers (para-fluorofentanyl (p-FF), ortho-fluorofentanyl (o-FF) and meta-fluorofentanyl (m-FF)), with the most predominant isomer that has recently emerged in the USA being p-FF. The goal of this study was to identify p-FF in postmortem cases between October 2020 and April 2021. Urine and blood were extracted using UCT Clean Screen® extraction columns and then screened using an Agilent 1290 Infinity liquid chromatograph (LC) coupled to an Agilent 6545 accurate mass time-of-flight mass spectrometer (TOF-MS) and quantified using an Agilent 6890N GC system coupled with an Agilent 5973 MS. The limit of quantitation (LOQ) for fentanyl, acetyl fentanyl, butyryl fentanyl, p-FF, o-FF and m-FF was 2.5 ng/mL. The screening method could not differentiate the three positional isomers of fluorofentanyl. Suspected overdose cases (n = 370) received from October 2020 through April 2021 from four Medical Examiner Districts in the state of Florida were analyzed for the presence of fluorofentanyl. The LC-QTOF-MS screen yielded 27 decedents positive for fluorofentanyl, with a majority being Caucasian (93%) and male (70%) with ages ranging from 27 to 63 years. Analysis of the blood and urine by GC-MS yielded 14 decedents positive for p-FF, nine of which were positive in the blood. The blood concentrations (n = 9) for p-FF ranged from

The Quantification of Oxycodone and Its Phase I and II Metabolites in Urine.

The purpose of this research was to develop and validate an analytical method for the detection and quantification of noroxymorphone-3ß-D-glucuronide (NOMG), oxymorphone-3ß-D-glucuronide (NOMG), noroxymorphone (NOM), oxymorphone (OM), 6α-oxycodol (αOCL), 6ß-oxycodol (ßOCL), noroxycodone (NOC) and oxycodone (OC) in urine by liquid chromatography tandem mass spectrometry to be used in a human study. The method was validated according to the Academy Standards Board Standard Practices for Method Development in Forensic Toxicology. The method was then applied to a single-dose pilot study of a subject. Urine samples were collected from the subject after ingesting 10-mg OC as an immediate-release tablet. Additionally, urine specimens (n = 15) that had previously been confirmed positive for OC were analyzed using the validated method. The calibration range for NOMG and OMG was 0.05-10 µg/mL; for all other analytes, it was 0.015-10 µg/mL. Validation parameters such as bias, precision, carryover and dilution integrity, all met the validation criteria. After the method was validated, urine samples from the first subject in the controlled dose study were analyzed. It was observed that OC, NOC and OMG contained the highest concentrations and were present in either the 0.5 or 1 h void. NOC and OMG were detected until the 48 h collection, while OC was detectable till the 24 h collection. Time to reach maximum concentration (Tmax) in the urine was achieved within 1.5 h for OC and within 3 h for NOC and OMG. Maximum concentration (Cmax) in the urine for OC, NOC and OMG was 3.15, 2.0 and 1.56 µg/mg, respectively. OC concentrations in authentic urines ranged from 0.015 to 12 µg/mL. Ranges for NOMG and OMG were 0.054-9.7 µg/mL and 0.14-67 µg/mL, respectively. A comprehensive method for the quantification of NOMG, OMG, NOM, OM, αOCL, ßOCL, NOC and OC in urine was optimized and met the validation criteria. The concentrations of NOMG and OMG presented in this study provide the details needed in the forensic community to better comprehend OC pharmacokinetics.

A sensitive LC-MS/MS method for the quantitation of oxycodone, noroxycodone, 6α-oxycodol, 6ß-oxycodol, oxymorphone, and noroxymorphone in human blood.

The objective of this study was to develop and validate a highly sensitive method for the detection of oxycodone, noroxycodone, 6ß-oxycodol, 6α-oxycodol, oxymorphone, and noroxymorphone in blood by liquid chromatography tandem mass spectrometry. The analytes were extracted from blood (0.5 mL) using Bond Elut Certify Solid Phase Extraction columns, evaporated to dryness and reconstituted before analysis was performed on an Acquity UPLC® I-class coupled to a Waters Xevo TQD. Academy Standards Board Standard Practices for Method Development in Forensic Toxicology were used for the validation of this method. The limit of quantitation for all analytes was established at 0.5 ng/mL. Calibration range for noroxymorphone, oxymorphone, 6α-oxycodol and 6ß-oxycodol was 0.5-25 ng/mL and 0.5-100 ng/mL for noroxycodone and oxycodone. Precision (2.90-17.3%) and bias studies resulted in a ±15% deviation. There were no interferences observed from internal standard, matrix, or common drugs of abuse. Stability of all analytes at two concentrations at 24, 48, and 72 h in the autosampler did not exceed ±20% difference from the initial T0. Dilution integrity at a ten-fold dilution was acceptable as analyte concentrations ranged between (±18%) of the target concentration. Once validated, the method was used in a pilot dosing study of one male subject after taking a 10 mg immediate release tablet of oxycodone. Blood samples were collected at 0.25, 0.50, 0.75, 1.0, 1.5, 2, 3, 4, 5, 6, 8, 9, and 24 h after ingestion. Oxycodone and noroxycodone both reached Tmax at 1.5 h and had Cmax values of 25.9 and 12.8 ng/mL, respectively. Oxycodone, 6α-oxycodol, and 6ß-oxycodol were detectable up to 9 h, while noroxymorphone and noroxycodone were still detected at 24 h.

Heroin-Related Compounds and Metabolic Ratios in Postmortem Samples Using LC-MS-MS.

Analysis of postmortem samples with the presence of morphine can sometimes be challenging to interpret. Tolerance complicates interpretation of intoxications and causes of death due to overlap in therapeutic and fatal concentrations. Determination of metabolites and metabolic ratios can potentially differentiate between abstinence, continuous administration, and perhaps time of administration. The purpose of this study was to (a) develop and validate a method for quantitation of morphine-3ß-D-glucuronide, morphine-6ß-D-glucuronide, normorphine, codeine-6ß-D-glucuronide, norcodeine, codeine, 6-acetylmorphine, and ethylmorphine in urine using liquid chromatography-tandem mass spectrometry; (b) apply the method to opiate related deaths; (c) compare metabolic ratios in urine in different causes of death (CoD) and after different drug intakes and (d) compare heroin intoxications in rapid and delayed deaths. Validation parameters such as precision, bias, matrix effects, stability, process efficiency, and dilution integrity were assessed and deemed acceptable. Lower limits of quantitation ranged from 0.01-0.2 µg/mL for all analytes. Autopsy cases (n=135) with paired blood and urine samples were analyzed. Cases were divided into three groups based on CoD; opiate intoxication, intoxication with other drugs than opiates, and other CoD. The cases were classified by intake: codeine (n=42), heroin (n=36), morphine (n=49), and ethylmorphine (n=3). Five cases were classified as mixed intakes and excluded. Heroin intoxications (n=35) were divided into rapid (n=15) or delayed (n=20) deaths. Parent drug groups were compared using metabolic ratio morphine-3ß-D-glucuronide/morphine and significant differences were observed between codeine vs morphine (p=0.005) and codeine vs heroin (p≤0.0001). Urine and blood concentrations, and metabolic ratios in rapid and delayed heroin intoxications were compared and determined a significant difference for morphine (p=0.001), codeine (p=0.009), 6-acetylmorphine (p=0.02) in urine, and morphine (p=0.02) in blood, but there was no significant difference (p=0.9) between metabolic ratios. Morphine-3ß-D-glucuronide results suggested a period of abstinence prior to death in 25% of the heroin intoxications.

Pharmacodynamics and pharmacokinetics of the novel synthetic opioid, U-47700, in male rats.

Novel synthetic opioids are appearing in recreational drug markets worldwide as adulterants in heroin or ingredients in counterfeit analgesic medications. Trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methyl-benzamide (U-47700) is an example of a non-fentanyl synthetic opioid linked to overdose deaths. Here, we examined the pharmacodynamics and pharmacokinetics of U-47700 in rats. Male Sprague-Dawley rats were fitted with intravenous (i.v.) catheters and subcutaneous (s.c.) temperature transponders under ketamine/xylazine anesthesia. One week later, rats received s.c. injections of U-47700 HCl (0.3, 1.0 or 3.0 mg/kg) or saline, and blood samples (0.3 mL) were withdrawn via i.v. catheters at 15, 30, 60, 120, 240, 480 min post-injection. Pharmacodynamic effects were assessed at each blood withdrawal, and plasma was assayed for U-47700 and its metabolites by liquid chromatography tandem mass spectrometry. U-47700 induced dose-related increases in hot plate latency (ED50 = 0.5 mg/kg) and catalepsy (ED50 = 1.7 mg/kg), while the 3.0 mg/kg dose also caused hypothermia. Plasma levels of U-47700 rose linearly as dose increased, with maximal concentration (Cmax) achieved by 15-38 min. Cmax values for N-desmethyl-U-47700 and N,N-didesmethyl-U-47700 were delayed but reached levels in the same range as the parent compound. Pharmacodynamic effects were correlated with plasma U-47700 and its N-desmethyl metabolite. Using radioligand binding assays, U-47700 displayed high affinity for µ-opioid receptors (Ki = 11.1 nM) whereas metabolites were more than 18-fold weaker. Our data reveal that U-47700 induces typical µ-opioid effects which are related to plasma concentrations of the parent compound. Given its high potency, U-47700 poses substantial risk to humans who are inadvertently exposed to the drug.

Long-term stability of novel synthetic opioids in blood.

Recently, there has been an increase in overdose deaths due to novel synthetic opioids (NSO). Due to backlogs experienced by many forensic laboratories, it is important to understand drug stability in a variety of storage conditions. The objective of this study was to investigate the stability of AH-7921, U-47700, U-49900, U-50488, MT-45, W-15, and W-18 in blood at various temperatures over a 36-week period. NSO were generally stable over the 36-week period (66%-118%) at low and high concentrations when blood samples were stored in the refrigerator or freezer. Most analytes were stable for at least 2 weeks at room temperature (77%-120%). At the elevated temperature (35°C), analytes were generally stable for at least 14 days (75%-109%). This study has determined the stability of several NSO at various temperatures over a 36-week period. These results reflect the forensic significance of keeping samples stored at proper temperatures. Blood samples suspected to contain synthetic opioids should be stored refrigerated or frozen, when possible, in order to preserve analyte stability, especially at low concentrations.

5F-MDMB-PICA metabolite identification and cannabinoid receptor activity.

According to the European Monitoring Center for Drugs and Drug Addiction (EMCDDA), there were 179 different synthetic cannabinoids reported as of 2017. In the USA, 5F-MDMB-PINACA, or 5F-ADB, accounted for 28% of cannabinoid seizures 2016-2018. The synthetic cannabinoid, 5F-MDMB-PICA, is structurally similar to 5F-MDMB-PINACA with an indole group replacing the indazole. Limited data exist from in vivo or in vitro metabolic studies of these synthetic cannabinoids, so potential metabolites to identify use may be missed. The goals of this study were to (a) investigate 5F-MDMB-PICA and 5F-MDMB-PINACA in vitro metabolism utilizing human hepatocytes; (b) to verify in vitro metabolites by analyzing authentic case specimens; and (c) to identify the potency and efficacy of 5F-MDMB-PICA and 5F-MDMB-PINACA by examining activity at the CB1 receptor. Biotransformations found in this study included phase I transformations and phase II transformations. A total of 22 5F-MDMB-PICA metabolites (A1 to A22) were identified. From hepatocyte incubations and urine samples, 21 metabolites (B1 to B21) were identified with 3 compounds unique to urine specimens for 5F-MDMB-PINACA. Phase II glucuronides were identified in 5F-MDMB-PICA (n = 3) and 5F-MDMB-PINACA (n = 5). For both compounds, ester hydrolysis and ester hydrolysis in combination with oxidative defluorination were the most prevalent metabolites produced in vitro. Additionally, the conversion of ester hydrolysis with oxidative defluorination to pentanoic acid for the first time was identified for 5F-MDMB-PICA. Therefore, these metabolites would be potentially good biomarkers for screening urine of suspected intoxication of 5F-MDMB-PICA or 5F-MDMB-PINACA. Both 5F-MDMB-PICA and 5F-MDMB-PINACA were acting as full agonists at the CB1 receptor with higher efficacy and similar potency as JWH-018.

Oral Fluid and Drug Impairment: Pairing Toxicology with Drug Recognition Expert Observations.

According to the Governors Highway Safety Association, drugs are detected more frequently in fatally injured drivers than alcohol. Due to the variety of drugs (prescribed and/or illicit) and their various physiological effects on the body, it is difficult for law enforcement to detect/prosecute drug impairment. While blood and urine are typical biological specimens used to test for drugs, oral fluid is an attractive alternative matrix. Drugs are incorporated into oral fluid by oral contamination (chewing or smoking) or from the bloodstream. Oral fluid is non-invasive and easy to collect without the need for a trained professional to obtain the sample, unlike urine or blood. This study analyzes paired oral fluid and urine with drug recognition expert (DRE) observations. Authentic oral fluid samples (n = 20) were collected via Quantisal™ devices from arrestees under an institutional review board-approved protocol. Urine samples (n = 18) were collected with EZ-SCREEN® cups that presumptively screened for Δ9-tetrahydrocannabinol (cannabinoids), opiates, methamphetamine, cocaine, methadone, phencyclidine, amphetamine, benzodiazepines and oxycodone. Impairment observations (n = 18) were recorded from officers undergoing DRE certification. Oral fluid samples were screened using an Agilent Technologies 1290 Infinity liquid chromatograph (LC) coupled to an Agilent Technologies 6530 Accurate Mass Time-of-Flight mass spectrometer (MS). Personal compound and database libraries were produced in-house containing 64 drugs of abuse. An Agilent 1290 Infinity LC system equipped with an Agilent 6470 Triple Quadrupole MS was used for quantification of buprenorphine, heroin markers (6-acetylmorphine, morphine) and synthetic opioids. Subjects were 23-54 years old; 11 (55%) were male and 9 (45%) were female. Evaluator opinion of drug class was confirmed in oral fluid 90% of time and in urine 85% of the time in reference to scope of testing by the LC-MS methods employed (excludes cannabis and central nervous system depressants). Data indicate that oral fluid may be a viable source for confirming driving under the influence of drugs.

Quantification of U-47700 and its metabolites in plasma by LC-MS/MS.

Novel Synthetic Opioids (NSO) have caused a recent epidemic both nationally and globally. NSO have gained popularity in the illicit drug market and have brought about an increase in fentanyl and its derivatives, as well as other chemically unrelated opioid agonists. U-47700, a non-fentanyl analog analgesic opioid, was first developed by The Upjohn Company and has a reported potency of 7.5 times that of morphine. Like many NSO, U-47700 is usually sold as a research chemical that can be purchased online but can also be found in "Gray Death" which is a mixture of fentanyl(s), heroin, and U-47700. With the emergence of these NSO, there is a need for laboratories to be able to detect these drugs in various matrices. In this study, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was optimized and validated to detect and quantify U-47700 and its metabolites, N-desmethyl-U-47700 and N,N-didesmethyl-U-47700, in 100 µL human plasma using an optimized solid phase extraction procedure. A small sample size (100 µL) was utilized for a future pharmacokinetic study in rats. The method was validated according to SWGTOX guidelines, including: precision and bias, linearity, carryover, interferences, matrix effects, limit of detection (LOD), limit of quantification (LOQ), dilution integrity, and stability. The LOD were 0.05 ng/mL for U-47700 and N-desmethyl-U-47700 and 0.1 ng/mL for N,N-didesmethyl-U-47700. Linear ranges for U-47700 and N-desmethyl-U-47700 were 0.1-100 ng/mL and 0.5-100 ng/mL for N,N-didesmethyl-U-47700. Matrix effects were analyzed following the post-extraction addition approach and were <5%, indicating little ion suppression or enhancement. Extraction recovery was >79%. Analytes were stable in all conditions and no endogenous or exogenous interferences were detected. This method was cross-validated in rat plasma with acceptable bias (2.1-6.2%) and precision (-14.7-15.7%) within acceptable limits. Matrix effects and extraction efficiency was comparable to human plasma validation. Postmortem whole blood samples (n = 15) were analyzed with the validated method. U-47700, N-desmethyl-U-47700 and N,N-didesmethyl-U-47700 concentration ranges were 1.1-1367 ng/mL, 4.0-1400 ng/mL and 0.7-658 ng/mL, respectively.

Quantitative Analysis of Novel Synthetic Opioids, Morphine and Buprenorphine in Oral Fluid by LC-MS-MS.

The opioid epidemic has become a national health emergency in the USA. While heroin and prescription opioid abuse is not uncommon, synthetic opioid use has risen dramatically, creating a public safety concern. Like traditional opioids, novel synthetic opioids are abused due to their analgesic and euphoric effects. Some adverse side effects include respiratory distress, nausea and decreased consciousness. Synthetic opioids have emerged into the illicit and online drug market, including AH-7921, MT-45, U-series and W-series. Though originally developed by pharmaceutical companies, these substances are not well studied in humans and comprehensive analytical methods for detecting and quantifying these opioids are limited. Oral fluid is a useful biological matrix for determining recent drug use, does not require a trained medical professional, and can be collected under direct observation, deterring adulteration. The purpose of this research was to develop and validate a comprehensive analytical method for the detection and quantification of morphine, 6-acetylmorphine, buprenorphine, U-47700, U-49900, U-50488, AH-7921, MT-45, W-18 and W-15 in oral fluid collected via Quantisal. This was achieved by solid-phase extraction followed by liquid chromatography-tandem mass spectrometry. The limits of detection and quantitation were 5 ng/mL and 10 ng/mL, respectively. Linearity was observed between 10 and 500 ng/mL (R2 ≥ 0.9959). Bias and imprecision were <±11.1%. Matrix effects ranged from -21.1 to 13.7%. No carryover was detected following injection of the highest calibrator. All analytes were stable (within ±15% change from baseline) under all tested conditions (24 h at room temperature, 72 h at 4°C, and in the autosampler for 60 h at 4°C).