Seized drug reporting of NPS helps to guide regional toxicological practice: A 17 month review between 2022 and 2023.

Novel psychoactive substances (NPS) are everchanging and plague forensic laboratories who must identify an unending variety of emerging substances and evolve current methodologies to detect these substances. Identifying potential regional NPS targets and timely examining trends in seized drug data could help mitigate the burden laboratories face. Over 17 months, NPS seized drug data were processed and categorized from three laboratories located across the United States to determine any NPS regional similarities and prevalent NPS drug categories: the South Carolina Law Enforcement Division (SLED), the Sedgwick County Regional Forensic Science Center (SCRFSC), and the Orange County Crime Laboratory (OCCL). Seized drug materials, including pills, powders, and plant material, were primarily analyzed for NPS via gas chromatography-mass spectrometry and Fourier transform infrared spectroscopy. From June 2022 to October 2023, 1940 NPS seized drug identifications were reported by these laboratories with 63 different NPS reported. Novel synthetic opioids (NSO) were the most prevalent NPS class across all three laboratories (55%), with fluorofentanyl accounting for 74% of NSO identifications. This is unsurprising given the fentanyl epidemic in the United States. Furthermore, these data highlighted varying regional NPS seized drug trends: eutylone, a synthetic cathinone, was one of the most frequently identified NPS in SLED, SCRFSC observed the most diverse set of synthetic cannabinoids, and OCCL observed an increased prevalence in the designer benzodiazepine, bromazolam. NPS scope recommendations are a valuable resource for forensic laboratories; however, most focus on a national perspective. Timely analysis and reporting of NPS seized drug data may help to develop regional NPS scope recommendations laboratories may employ.

Mitragynine in the Orange County DUID population.

In recent years, mitragynine has been consistently detected in driving under the influence of drug (DUID) cases. In this paper, we evaluate 3 years (2017-2019) worth of DUID data from arrested drivers in Orange County, CA, USA. From the 25,398 DUID cases received in those 3 years, there were 60 (0.24%) cases with detectable concentrations, >10 ng/mL, of mitragynine. The majority of drivers were male (90%) and were stopped during the week (81%), considered Monday 0000 to Friday 1159. The concentration range for all mitragynine cases was 10.5-960 ng/mL, with a mean of 109 ng/mL and a median of 58 ng/mL. Forty four of the 60 cases were also screened for 7-hydroxymitragynine, and 27 (63%) were positive. The police reports and drug recognition expert evaluations were collected and evaluated. No case contained solely mitragynine, and the most common drugs detected in combination were central nervous system depressants (ethanol, followed by benzodiazepines), stimulants (methamphetamine and cocaine) and opioids (fentanyl and indication of heroin). Two cases containing only one other psychoactive substance are discussed more thoroughly to attempt to identify the contributions of mitragynine to driving impairment. Collected demographic, toxicological and field observations are presented for all cases.

Postmortem Mitragynine Distribution in a Single Drug Fatality Case.

A 32-year-old Caucasian male was found unconscious at his sober-living home and pronounced dead after transportation to the emergency room. The decedent had a documented history of substance-use disorder and past suicide attempts, but according to his family, he was sober for the past year. Significant autopsy findings were cardiomegaly, hepatomegaly, congested lungs, cerebral edema, and obesity. The toxicology examination of blood and tissues using liquid chromatography tandem mass spectrometry detected only mitragynine in the central blood (7.5 mg/L), peripheral blood (3.3 mg/L), liver (42.2 mg/kg), and gastric contents (33.1 mg). The qualitative identification of 7-hydroxymitragynine was performed only on the central blood. The pathologist ruled the cause of death acute mitragynine intoxication combined with cardiomegaly with left ventricular hypertrophy, with severe hepatomegaly and obesity listed as other significant conditions. The mode, or manner, of death was determined to be an accidental overdose. To the authors' knowledge, this is the first reported case where mitragynine was the only drug detected. This case study will contribute to the understanding of mitragynine-only death investigation and provide valuable toxicology information for medical examiners and pathologists.

A Two-Year Evaluation of Flualprazolam Concentrations in Orange County Drivers: Effects on Driving and Field Sobriety Test Performance.

Starting in early 2017, flualprazolam was detected in toxicology and seized drug cases across the USA. Due to the addition of fluorine to alprazolam, flualprazolam's chemistry was enough to bypass targeted toxicology confirmations, and it has become increasingly available for purchase both on the dark web and in counterfeit pills. Flualprazolam was added to the exact mass screening regiment of the Orange County forensic laboratory in December 2018. Through data mining of previously analyzed cases, data were evaluated beginning from August 2018. Flualprazolam was subsequently added to the laboratory's validated quantitative liquid chromatography dual mass spectrometry method in the summer of 2020, and all driving cases from August 2018 to June 2020 were re-analyzed to obtain concentrations. The police and drug recognition evaluation (if available) reports were collected and reviewed for all cases where flualprazolam was detected. Of the 203 cases containing quantifiable drug, the average flualprazolam concentration (median, range) was 22.8 ng/mL (15.4 ng/mL, 4.0-133.3 ng/mL). Only two cases had flualprazolam detected with no other drugs. The other most common drugs detected were cannabinoids (62%), ethanol (20%) and cocaine and/or methamphetamine (32%). The most common reason for the police investigation was an accident/collision (50%), and the most common time of officer contact was evening (18:00 to 23:59). The field sobriety test (FST) results were evaluated and showed a higher frequency of impaired performance in cases of flualprazolam in combination with other drugs. No conclusion could be made regarding the effect of flualprazolam alone on FST performance.

The impact of fentanyl on DUIDs and traffic fatalities: Blood and oral fluid data.

Fentanyl has emerged as the most prolific drug in the ongoing opioid epidemic and has greatly impacted traffic safety in recent years. This study aimed to evaluate fentanyl prevalence and concentrations in blood and oral fluid in driving under the influence of drugs (DUID) cases in three different regions (i.e., Alabama, Orange County, CA, and Houston, TX) from 2017 to 2022. Furthermore, traffic fatalities were evaluated for Alabama and Orange County, CA. Fentanyl positivity in DUID and traffic fatalities increased for most years in this study. In Alabama, the prevalence of fentanyl DUID cases increased 4-fold in 2022 compared to 2017. Orange County's increase from 2017 to 2022 was 14-fold. In Houston, the increase was approximately 2-fold from 2019 to 2022. The greatest increase for all laboratories coincided with the start of the COVID-19 pandemic. In 2022, the median fentanyl DUID blood concentrations were 4.7, 11, and 4.7 ng/mL in Alabama, Orange County, and Houston, respectively. Most fentanyl cases were polydrug cases (≥90%). Methamphetamine, THC, and alprazolam were the most frequently detected drugs in combination with fentanyl. Alabama has collected oral fluid and blood in DUID cases since 2018. The detection of fentanyl in oral fluid was comparable to blood. However, 59% and 8.7% of fentanyl-positive cases had concentrations of >20 ng/mL in oral fluid and blood, respectively. Therefore, oral fluid as an alternative or supplemental specimen to blood is an attractive approach for fentanyl in DUID cases. This study contributes to understanding recent fentanyl trends and their impact on traffic safety.

A Two-Year Review of Fentanyl in Driving under the Influence and Postmortem Cases in Orange County, CA, USA.

In recent years, fentanyl is increasingly detected in overdose cases as well as human performance cases due to the current opioid epidemic. The comparison of fentanyl concentrations in deaths to recreational fentanyl use found in driving under the influence of drugs (DUID) cases is important to show how increased availability affects the historical knowledge of fentanyl. A 2-year review of fentanyl cases from Orange County, CA, USA, is presented to highlight fentanyl concentrations in these two case types. Any non-urine sample with fentanyl detected above the limit of detection of 0.5 ng/mL was reviewed in DUID and postmortem (PM) cases. The mean and median fentanyl concentrations in 199 antemortem (AM) blood samples from DUID cases were 14.2 ng/mL and 5.3 ng/mL, respectively, with a range of 0.5-303 ng/mL. In 285 central blood samples from PM cases, the mean was 29.1 ng/mL, the median was 16.9 ng/mL and the range was 0.6-636 ng/mL. In a total of 58 PM peripheral blood samples, the mean, median and range were 14.0 ng/mL, 10.0 ng/mL and 0.9-78.0 ng/mL, respectively. Of the 55 brain samples analyzed, the fentanyl mean, fentanyl median and range of fentanyl concentrations were 50.0 ng/g, 31.4 ng/g and 1.9-441 ng/g, respectively. Fentanyl concentrations in 16 liver samples had a mean of 82.0 ng/g, a median of 69.4 ng/g and a range of 11.6-226 ng/g. The overlap of fentanyl concentrations from the AM and PM samples highlights the importance of evaluating case circumstances when providing an interpretation in PM cases. Additional information such as age, gender, polydrug use and PM redistribution is presented for the cases included in the study.

Flualprazolam distribution in postmortem samples.

The constant emergence of novel psychoactive substances is troubling to both public health officials and legislators. Additionally, sufficient data collection for each new compound can take months up to years. Flualprazolam, a triazolobenzodiazepine, quickly garnered attention as a sedative drug that likely expresses adverse reactions similarly to alprazolam. This study focuses on the distribution of flualprazolam in multiple common postmortem matrices. Central blood, vitreous humor, liver homogenate, brain homogenate, gastric contents, and urine samples from death investigation cases were quantitated when available. Samples were screened with liquid chromatography quadrupole time-of-flight with limit of detection set at 4 ng/ml and quantitated on liquid chromatography tandem mass spectrometry, with concentration range from 4 to 256 ng/ml. From August 2018 to September 2020, 24 central blood samples were quantitated for flualprazolam. Central bloods of 22 cases had concentrations above the limit of quantitation. The average flualprazolam central blood concentration was 16.3 ng/ml with a median of 9.95 ng/ml (4.24-48.0). Additional analyses for unconjugated flualprazolam were performed on at a total of 15 urine samples ( x ¯  = 14.4, 4.07-36.1 ng/ml), 23 brain homogenates ( x ¯  = 23.2, 3.99-69.3 ng/g), 23 liver homogenates ( x ¯  = 50.7, 13.6-156 ng/g), five vitreous humor samples ( x ¯  = 7.70, 4.03-12 ng/ml), and 12 gastric contents samples ( x ¯  = 0.36, 0.02-2.51 mg). The cause of death for 13 of the 24 cases listed flualprazolam as a contributing factor of death.

Prevalence of Five Commonly Analyzed Cannabinoids in Forensic Toxicology Casework from Orange County CA, USA, from 2016 to 2019.

Over the last 25 years, marijuana laws have been changing throughout the USA. California started legalizing medicinal marijuana in 1996 and has since continued to relax laws. Compared to Washington and Colorado, there are little data on how the changing laws have affected the cannabinoid detection rate in California. This paper looks at the prevalence of five cannabinoids (Δ9-tetrahydrocannabinol (THC), 11-hydroxy-tetrahydrocannabinol (hydroxy-THC), 11-nor-9-carboxy-tetrahydrocannabinol (carboxy-THC), cannabinol and cannabidiol) in Orange County, CA, from 2016 to 2019. From 2016 to 2017, after legalizing recreational marijuana, there was an increase in the presence of THC, carboxy-THC and hydroxy-THC in postmortem and major crime cases, consisting mostly of sexual assaults. However, driving under the influence of drugs (DUID) saw a slight decrease. In 2018, when shops could be licensed to sell marijuana to anyone over 21 years old, there was an increase seen in all five cannabinoids for DUID and postmortem cases. The age group from 21 to 30 years showed the most prevalent cannabinoid use in all case types for all years except in major crime cases in 2019, where <21 year-old age group was the most prevalent. Surprisingly, the >50-year-old group in death investigation cases was a close second in prevalence in all years, which differs from DUID and major crime cases.

Simultaneous quantitative analysis of 39 common toxicological drugs for increased efficiency in an ante- and postmortem laboratory.

BACKGROUND: A novel forensic method was developed to quantitate 39 drugs of toxicological interest for ante-mortem and postmortem analysis. This method was created to combine and replace four existing quantitation methods as well as add three additional compounds of interest and serves to drastically increase the efficiency of the criminalists and reduce the case backlog. The method is currently applied to ante-mortem blood, postmortem blood, urine, liver, brain, and gastric contents. METHODS: The extraction was performed by using a protein precipitation and DPX WAX-S tips with analysis on a Waters® i-class Acquity ultra-performance liquid chromatography with a Phenomenex Kinetex® Column (1.7 µm Biphenyl Å, 2.1 ×100 mm) followed by a Waters® XeVo-TQS tandem mass spectrometer using positive electrospray ionization in multiple reaction monitor mode. The sample volume required for analysis was 0.5 mL, or 0.5 g, an improvement from 4 mL when performing previous methods utilized in the laboratory. RESULTS: The improved method incorporated the 2017 recommended cut-offs for toxicological investigation of driving under the influence of drugs and was validated following the SWGTOX and ANSI/ASB guidelines of method validation. The advantages of analyzing low volume cases and/or detecting drugs previously outside the laboratory's scope of analysis, (such as gabapentin, pregabalin and baclofen) will be presented in two case studies. CONCLUSION: The multi-drug quantitation method allowed for the analysis of 39 drugs including a hydrolysis step, if needed, with only 0.5 mL or 0.5 g of sample. The method condensed two previously un-validated quantitative methods and two additional qualitative methods, which detected many commonly seen drugs, all into a single method. Three additional analytes of interest, gabapentin, pregabalin and baclofen, which it had previously been unable to detect, were added to the new method. The added benefit of these new drugs added both the coroner's investigators in cause of death determination and driving under the influence of drugs investigation especially with the high prevalence of gabapentin.

Unusually High Concentration of Fentanyl in a Driver: A Case Report.

A case of driving under the influence of drugs with an extremely high blood fentanyl concentration and concomitant use of methamphetamine is presented. As the increase of the appearance of fentanyl in drug seizures continues, the likelihood of tolerant users increases as well. Data presented on fentanyl in antemortem cases over time from several regions of North America show geographic differences on whether fentanyl concentrations are increasing. This case is an example of a blood concentration that could have been assumed to be fatal if presented on its own, a reminder that concentrations, especially those of opioids, must be interpreted with care.

Cannabinoid Stability in Antemortem and Postmortem Blood.

In toxicological testing, drug stability is important when providing quantitative results and interpretation of findings, as well as when collecting correlation data. The goal of this study was to expand on previous stability studies and to evaluate other cannabinoids encountered in forensic toxicology. In this 6-month study, the stability of Δ-9-tetrahydrocannabinol (THC), 11-hydroxy-THC, 11-nor-9-carboxy-THC, Cannabinol and Cannabidiol in antemortem and postmortem blood was evaluated in refrigerated (4°C) and frozen (-4°C) storage conditions. Pooled antemortem and postmortem bloods were fortified at low and high concentrations and stored in untreated glassware. Over 6 months, samples were analyzed by automated extraction and liquid chromatography-mass spectrometry/mass spectrometry to evaluate the change in concentration over time. Samples in each storage condition were analyzed in triplicate 12 times over the 6-month period. Cannabinoids in antemortem blood were more stable in the refrigerated condition than in the frozen condition, with 11-hydroxy-THC, 11-nor-9-carboxy-THC and Cannabinol having more than 80% of the original concentration remaining at the end of the study. Cannabinoids in postmortem blood had improved stability in the frozen storage condition with THC, 11-hydroxy-THC, 11-nor-9-carboxy-THC and Cannabinol in the low concentration pool with more than 80% of the original concentration remaining. These data demonstrated that cannabinoids may decrease in concentration over time when stored in untreated glass vials. To ensure the most accurate determination of drug concentration, samples containing cannabinoids should be analyzed as soon as possible.

Endogenous Gamma-Hydroxybutyrate in Postmortem Samples.

Gamma-hydroxybutyrate (GHB) is a naturally occurring molecule present in the human body as a catabolite of the neurotransmitter gamma-aminobutyrate (GABA). In the USA, GHB has a history of being manufactured illicitly and abused, with misguided proposed benefits for the body-building community and a persistent party drug with reported GHB overdoses occurring worldwide. The interpretation of GHB in postmortem biological fluids is complicated by the endogenous nature of the molecule. Analysis often requires more than one biological matrix to detect exogenous exposure, typically in urine. The analysis is further complicated by the endogenous de novo production of GHB in postmortem specimens. This work sought to examine the prevalence of endogenous GHB concentrations in postmortem toxicology samples from Orange County, CA, and to establish suitable in-house secondary matrices to confirm or rule out exogenous GHB exposure. A total of 348 postmortem heart blood samples were randomly selected and analyzed for GHB using liquid-liquid extraction followed by gas chromatography-mass spectrometry with selective ion monitoring and GHB-d6 as an internal standard. Of the 348 cases analyzed, 39 cases resulted in positive GHB detection with the median concentration of 22.45 mg/L (10.4-62.16 mg/L). None of the positive samples had suspected GHB ingestion or usage from the case report. GHB concentrations were then examined in secondary matrices collected at autopsy from the positive cases that included (when available) peripheral blood, urine, vitreous humor, liver homogenate and brain homogenate. Within the secondary matrices, GHB levels in peripheral blood compared to that of heart blood, while liver homogenate levels were variable. Quantifiable GHB levels were not identified in vitreous humor and brain homogenate samples. Our findings reaffirm the importance of multi-matrix analysis in postmortem toxicology and further confirm the utility of vitreous humor and brain tissue to distinguish exogenous GHB exposure from endogenous production.

Stability of 26 Sedative Hypnotics in Six Toxicological Matrices at Different Storage Conditions.

Forensic laboratories are challenged with backlogs that produce turnaround times that vary from days to months. Therefore, drug stability is important for interpretation in both antemortem (blood and urine) and postmortem (blood, brain, liver, stomach contents) cases. In this study, 23 benzodiazepines (2-hydroxyethylflurazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, α-hydroxyalprazolam, α-hydroxytriazolam, alprazolam, bromazepam, chlordiazepoxide, clonazepam, demoxepam, desalkylflurazepam, diazepam, estazolam, flunitrazepam, flurazepam, lorazepam, midazolam, nitrazepam, nordiazepam, oxazepam, phenazepam, temazepam and triazolam) and three sedative hypnotics (zaleplon, zopiclone and zolpidem) were spiked into the six matrices at two different concentrations for each drug. The samples were stored in either a refrigerator (4°C) or freezer (-20°C) and analyzed in triplicate at various time intervals over an 8-month period using an SWGTOX validated method. The concentrations decreased over time regardless of the initial spiked concentration, and the storage conditions had little effect on the decrease of most drugs. Conversion from drug to metabolite was difficult to determine since all 26 drugs were present in each sample. Zopiclone and phenazepam were the least stable drugs; zopiclone was the only drug that completely disappeared in any matrix (both antemortem and postmortem blood). Urine was the most stable matrix with only phenazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, 2-hydroxyethylflurazepam, and zopiclone decreasing >20% over the 8 months in either storage condition. Postmortem blood, the least stable matrix, had only two drugs, zolpidem and bromazepam, decreasing <20% in the 8-month time period. Further experiments on stability of these drugs should be undertaken to remove the freeze-thaw cycle effect and more thoroughly examining drug-metabolite conversion.

Ultra Performance Liquid Chromatography with Tandem Mass Spectrometry for the Quantitation of Seventeen Sedative Hypnotics in Six Common Toxicological Matrices.

An ultra performance liquid chromatography triple quadrupole mass spectrometry (LC-MS-MS) method for the quantification of 14 benzodiazepines and three sedative hypnotics is presented. The fast and inexpensive assay was developed for California's Orange County Crime Lab for use in antemortem (AM) and postmortem casework. The drugs were rapidly cleaned up from AM blood, postmortem blood, urine, liver, brain and stomach contents using DPX(®) Weak Anion Exchange (DPX WAX) tips fitted on a pneumatic extractor, which can process up to 48 samples at one time. Assay performance was determined for validation based on recommendations by the Scientific Working Group for Forensic Toxicology for linearity, limit of quantitation, limit of detection, bias, precision (within run and between run), dilution integrity, carry-over, selectivity, recovery, ion suppression and extracted sample stability. Linearity was verified using the therapeutic and toxic ranges of all 17 analytes. Final verification of the method was confirmed by four analysts using 20 blind matrix matched samples. All results were within 20% of each other and the expected value.