A Single Method for 127 Recommended and Additional DUID Drugs in Blood and Urine by LC-MS-MS.

Driving under the influence of drug (DUID) cases continue to challenge forensic toxicologists as both the volume and complexity of casework increases. Comprehensive DUID testing should also meet the drafted Academy Standards Board (ASB)/ American National Standard Institute (ANSI) standard and the National Safety Council's Alcohol, Drugs and Impairment Division (NSC-ADID) recommendations. A simple method using protein precipitation followed by filtration extraction with an 8 minute run time by liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed, and a comprehensive ASB/ANSI validation was performed. Target drugs and metabolites were quantitatively assessed in blood and qualitatively assessed in urine. Included were 127 target analytes including cannabinoids (12), amphetamines (11), cocaine and metabolites (6), benzodiazepines (36), Z-drugs (5), opioids (27), anticonvulsants (3), first-generation antihistamines (6), muscle relaxants (2), dissociatives and hallucinogens (6), barbiturates (10), and miscellaneous substances (3). Limits of detection are appropriate for DUID and other forensic casework such as drug-facilitated crime (DFC) and postmortem investigations. To demonstrate applicability, 78 proficiency test blood and urine samples and 1,645 blood and urine samples from authentic cases samples demonstrated effective detection of target analytes in forensic casework. By increasing the analytical scope of multiple drug classes via a single method, this technique detects drugs that may have previously gone undetected, such as flualprazolam, etizolam, mitragynine, gamma-hydroxybutyric acid and psilocin and improves laboratory efficiency by reducing the number of tests required. The described method is, to the authors' best knowledge, the only published single procedure to meet all drugs listed in the drafted ASB/ANSI standard and recommended Tier 1 and traditional drugs from Tier 2 for DUID screening, while also achieving many drug scope and sensitivity recommendations for DFC and postmortem testing.

Inferences and Legal Considerations Following a Blood Collection Tube Recall.

In mid-2019, medical, forensic and legal communities were notified that a certain shipment of evacuated blood sampling tubes were recalled by the manufacturer. This recall order described that the preservative sodium fluoride (100 mg) and anticoagulant potassium oxalate (20 mg) were missing from a small batch of 10-mL evacuated tubes. This gave cause for concern for possible implications in criminal justice (e.g., in drink-driving offenses) when blood-alcohol concentrations are interpreted. In reality, the lack of an anticoagulant would have been immediately obvious during sample preparation, owing to the formation of a large clot in the tube when received. Certain impairing drugs (e.g., cocaine and 6-acetylmorphine) are unstable in blood and tend to degrade without an enzyme inhibitor, such as sodium fluoride, present. In reviewing available literature related to current practices and the stability of ethanol in stored blood samples, there does not appear to be a clear consensus regarding the amount of sodium fluoride preservative necessary, if any at all, when blood is taken from living subjects under sterile conditions for typical forensic ethanol analysis.

A Case Series of Etizolam in Opioid-Related Deaths.

Etizolam is a novel psychoactive substance and novel benzodiazepine of the thienotriazolodiazepine class, which has recently seen an increasing trend in use worldwide. We report a case series of 10 decedents with etizolam and opioids in their systems. Death investigation, expanded toxicology and medical investigation information were included for contextualization of etizolam in death. Etizolam was detected and confirmed within peripheral and cardiac blood, urine, vitreous humor and, in one case, gastric fluid, by liquid chromatography-tandem mass spectrometry and liquid chromatography-quadrupole time of flight mass spectrometry methodologies. Death investigation indicated nonmedical use of most drugs. Medical investigation commonly noted pulmonary edema, cardiomegaly and cerebral swelling. The majority of the decedents appeared to be unaware of the presence of etizolam and succumbed to the mixed drug toxicity of their routine depressant and narcotic analgesic drug of abuse in combination with etizolam. Etizolam use continues to be observed and poses as a potentially lethal contribution to multiple drug toxicity, especially in the age of the opioid crisis. Assessment of analytes like etizolam requires up-to-date methodologies and vigilance in testing to better characterize the toxicology and interpret the contribution to death.

Evaluation and applicability of Alere iCup DX 14 for rapid postmortem urine drug screening at autopsy.

Performing point-of-care urine drug screen testing at autopsy by a forensic pathologist may provide an early indication of the presence of analytes of interest during autopsy. An evaluation for the screening of 14 classes of common drugs of abuse in postmortem urine by the point-of-care screening device, Alere iCup DX 14, is presented. One hundred ninety postmortem urine samples were screened with the iCup occurring at autopsy by the forensic pathologist. Positive and negative results obtained from the screening kit were evaluated against confirmatory test results obtained using routine forensic toxicology analyses that employed LC-MS/MS and GC-MS to detect a combination of over 85 common drugs of abuse and medications. Sensitivity for each respective iCup drug class ranged from 66% (buprenorphine) to 100% (methadone, tricyclic antidepressants). Specificity for each respective iCup drug class ranged from 89% (benzodiazepines) to 100% (amphetamines, barbiturates, buprenorphine, 3,4-methylenedioxymethamphetamine, methadone). Positive predictive values ranged from 44% (benzodiazepines) to 100% (amphetamines, barbiturates, buprenorphine, methylenedioxymethamphetamine, methadone), while negative predictive values ranged from 96% (methamphetamine) to 100% (barbiturates, methadone, tricyclic antidepressants). A high false-positive rate was yielded by the benzodiazepine class. The lack of fentanyl screening in the point-of-care device is a significant limitation considering its prolific prevalence in forensic casework. The results obtained in the study should be acknowledged when considering the use of the Alere iCup DX 14 in the context of postmortem casework to help indicate potential drug use contemporaneously with autopsy and when requiring such preliminary results prior to the release of a final forensic toxicology report.

Method Consolidation to Improve Scope and Efficiency in Postmortem Toxicology.

Systematic toxicological approaches that employ both ideology changes and improvements in instrumentation and sample extraction allow for improved toxicology testing efficiency through lower sensitivities, higher specificity and minimized resource use. Historically, the San Francisco Office of the Chief Medical Examiner relied heavily on a gas chromatography mass spectrometry (GC-MS) testing regime, comprised of individual drug-class confirmation and quantitation assays. Traditional methods utilizing GC-MS typically require iterations of testing, exhausting sample volume, and hindering productivity and turnaround times, particularly for polypharmacy cases frequently seen in modern postmortem toxicology. The method described here consolidated the scope of seven legacy methods into a single liquid chromatography tandem mass spectrometry (LC-MS/MS) method for better sensitivity, higher throughput, minimal sample consumption for the quantitation of drugs of abuse and improved quality assurance with the incorporation of smart, automated processing. About 100 µL of blood or urine were rapidly extracted using a simple acetonitrile protein crash and subsequent in-vial filtration and injected on to an LC-MS-MS system. The developed method was fully validated to SWGTOX and international guidelines and incorporated 55 analytes along with a customized query that facilitates rapid and consistent application of acceptability criteria for data processing and review. Applicability was demonstrated with the analysis of 1,389 samples (858 blood and 531 urine) where at least 41% of positive results may have been missed due to their decreased sensitivity and 11% of results were not within the scope of the previous analytical methods estimated. On average, cases in this study would have previously required three distinct GC-MS assays, 3 mL of blood, and upwards of 30 h of active staff time. The described LC-MS-MS analytical approach has mitigated the need to perform multiple assays, utilized only 0.1 mL of sample, significantly reduced analyst work time, incorporated 10 additional analytes and allowed for a more comprehensive testing regime to better inform cause of death determinations.

Validation of the i-STAT®1 Analyzer for Postmortem Vitreous Humor Electrolytes and Glucose Analysis.

The analytical value of vitreous humor as a specimen in postmortem forensic toxicology has been known for some time. Numerous medical examiner laboratories outsource the analysis of this important specimen for electrolyte and glucose measurements. This can be both time-consuming and costly. The utility of the i-STAT®1 medical device to measure electrolytes and glucose in whole blood samples has been demonstrated for over two decades in a clinical setting through single-use disposable cartridges that introduce samples to the i-STAT®1. Different cartridge types allow for the analysis of various analytes including sodium, potassium, chloride, creatinine, urea nitrogen and glucose. With only 100 µL of sample, results are obtained in under 4 min. In this study, we utilized the i-STAT®1 using an alternative specimen matrix, postmortem vitreous humor and quantitatively determined the validity and reliability of the instrument for this purpose. Acceptable criterion was used for each test as suggested by the Scientific Working Group for Forensic Toxicology. All analytes of interest, except creatinine, demonstrated a percent error < ±10% for both accuracy and precision studies. Drug interference and stability studies were performed with many of the analytes demonstrating a percent error < ±20%. Throughout drug interference and stability studies, all analytes of interest were detectable except for potassium, which gave inconclusive results. Significant interference with commonly found drugs were shown for creatinine and chloride but must be evaluated carefully. Volume additions to ethanol spiked samples caused significant interference for all analytes and is considered a limitation for this method of analysis that requires additional studies. As vitreous humor continues to be used in forensic medicine to aid in diagnostic interpretation, the i-STAT®1 has the potential to give accurate results in a timely and cost-effective manner.