An exploratory view into allelic drop-out of sequenced autosomal STRs.

As massively parallel sequencing is implemented in forensic genetics, an understanding of sequence data must accompany these advancements, that is, accurate modeling of data for proper statistical analysis. Allelic drop-out, a common stochastic effect seen in genetic data, is often modeled in statistical analysis of STR results. This proof-of-concept study sequenced several serial dilutions of a standard sample ranging from 4 ng to 7.82 pg to evaluate allelic drop-out trends on a select panel of autosomal STRs using the ForenSeq™ DNA Signature Prep Kit, Primer Set A on the Illumina MiSeq FGx. Parameters assessed included locus, profile, and run specific information. A majority of the allelic drop-out occurred in DNA concentrations less than 31.25 pg. Statistical results indicated a need for locus-specific modeling based on STR descriptors, like simple versus compound repeat patterns. No correlation was seen between average read count of scored alleles and allelic drop-out at a locus. A statistical correlation was observed between the amount of allelic drop-out and the starting amount of DNA in a sample, average read count of a sample, and total read count generated on a flow cell. This study supports using common allelic drop-out factors used in fragment length analysis on sequenced STRs while including additional locus, sample, and run specific information. Results demonstrate multiple factors that can be considered when developing probability of allelic drop-out models for sequenced autosomal STRs including locus-specific analysis, total read count of a profile, and total read count sequenced on a flow cell.

Time- and temperature-dependent postmortem ∆9-tetrahydrocannabinol concentration changes in rabbits following controlled inhaled cannabis administration.

ostmortem redistribution (PMR), a well-known phenomenon in forensic toxicology, can result in substantial changes in drug concentrations after death, depending on the chemical characteristics of the drug, blood collection site, storage conditions of the body and postmortem interval (PMI). Limited PMR data are available for ∆9-tetrahydrocannabinol (THC), the primary psychoactive component in Cannabis sativa. PMR was evaluated after controlled cannabis inhalation via a smoking machine and exposure chamber in New Zealand white rabbits. Necropsies were performed on five control rabbits immediately after euthanasia, whereas 27 others were stored at room temperature (21°C) or refrigerated conditions (4°C) until necropsy at 2, 6, 16, 24 or 36 h after death. THC and its Phase I and glucuronidated Phase II metabolites were quantified in blood, vitreous humor, urine, bile and tissues by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Under refrigerated temperature, heart blood THC concentrations significantly increased at PMI 2 h in rabbits, whereas peripheral blood THC concentrations showed a significant increase at PMI 16 h. Central:peripheral blood and liver:peripheral blood ratios for THC ranged from 0.13 to 4.1 and 0.28 to 8.9, respectively. Lung revealed the highest THC concentrations, while brain and liver exhibited the most stable THC concentrations over time. This report contributes much needed data to our understanding of postmortem THC behavior and can aid toxicologists in the interpretation of THC concentrations in medicolegal death investigations.

Characterization of fentanyl HCl powder prior to and after systematic degradation.

Fentanyl HCl is of particular interest in forensic cases but there is a notable gap in literature regarding its analysis. This study utilized a multi-method approach to characterize fentanyl HCl powder, both fresh and following a forced degradation process. Using sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) and direct injection gas chromatography-mass spectrometry (GC-MS), five compounds were identified in fresh fentanyl HCl powder. The identified compounds were: N-phenylpropanamide, 1-phenethyl-4-propionyloxypiperidine (1-P-4-POP), 4-anilino-N-phenethylpiperidine (4-ANPP), acetylfentanyl, and fentanyl; all identified compounds but acetylfentanyl and fentanyl decreased in quantity as the sample was degraded. Fresh headspace samples analyzed with solid phase microextraction (SPME)-GC-MS identified four compounds in common with the powder analyses: N-phenylpropanamide,1-P-4-POP, 4-ANPP, and fentanyl. Acetylfentanyl was not present in the headspace samples, although two additional compounds were: N-phenylacetamide and N-phenethyl-4-piperidinone (NPP). Where direct analysis of degraded fentanyl HCl showed decreased quantities of the identified compounds, headspace samples of the degraded fentanyl HCl resulted in higher quantities, implying that the degradation process drove those compounds to volatilize. Notably, fentanyl was identified in the headspace, implying that this could be an appropriate target for standoff detection. Finally, thermogravimetric analysis (TGA) and differential scanning calorimetry (DCS) confirmed that the forced degradation process had little permanent effect on the powder.

Cannabinoid distribution in fatally-injured pilots' postmortem fluids and tissues.

The primary psychoactive component of cannabis, Δ9-tetrahydrocannabinol (THC) impairs cognitive function and psychomotor performance, particularly for complex tasks like piloting an aircraft. The Federal Aviation Administration's (FAA) Forensic Sciences Section at the Civil Aerospace Medical Institute (Oklahoma City, OK) performs toxicological analyses on pilots fatally injured in general aviation incidents, permitting cannabinoids measurement in a broad array of postmortem biological specimens. Cannabinoid concentrations in postmortem fluids and tissues from 10 pilots involved in airplane crashes are presented. Median (range) THC blood concentration was 1.6 (1.0-13.7) ng/mL. Phase I metabolites, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) and phase II glucuronide metabolite, THCCOOH-glucuronide, had median (range) blood concentrations of 1.4 (0.5-1.8), 9.9 (2.2-72.6) and 36.6 (7.1-160) ng/mL, respectively. Urine analyses revealed positive results for THCCOOH, THC-glucuronide, THCCOOH-glucuronide and 11-nor-9-carboxy-Δ9-tetrahydrocannabivarin (THCVCOOH). THC was readily distributed to lung, brain, kidney, spleen and heart. The psychoactive metabolite, 11-OH-THC, was identified in liver and brain with median (range) concentrations 7.1 (3.5-10.5) and 2.4 (2.0-6.0) ng/g, respectively. Substantial THCCOOH and THCCOOH-glucuronide concentrations were observed in liver, lung, brain, kidney, spleen and heart. These cannabinoid concentrations from multiple types of postmortem specimens add to the limited postmortem cannabinoid research data and suggest useful biological matrices for investigating cannabinoid-related deaths.

Identification and quantification of cannabinoids in postmortem fluids and tissues by liquid chromatography-tandem mass spectrometry.

Cannabis sativa is commonly used worldwide and is frequently detected by forensic laboratories working with biological specimens from potentially impaired drivers or pilots. To address the problem of limited published methods for cannabinoids quantification in postmortem specimens, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), 8ß,11-dihydroxy-THC (8ß-diOH-THC), 8ß-hydroxy-THC (8ß-OH-THC), THC-glucuronide (THC-g), THCCOOH-glucuronide (THCCOOH-g), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), Δ9-tetrahydrocannabivarin (THCV), and 11-nor-9-carboxy-THCV (THCVCOOH). Solid phase extraction concentrated analytes prior to analysis on a biphenyl column coupled to a mass spectrometer in electrospray positive ionization mode using multiple reaction monitoring. Linearity ranged from 0.25-50 ng/mL (THC-g), 0.5-100 ng/mL (CBN), 0.5-250 ng/mL (THC, 11-OH-THC, THCCOOH, CBD, and CBG), 1-100 ng/mL (8ß-diOH-THC, THCVCOOH, 8ß-OH-THC, and THCV) and 1-250 ng/mL (THCCOOH-g). Within-run imprecision was <11.2% CV, between-run imprecision <18.1% CV, and bias was less than ±15.1% of target concentration in blood for all cannabinoids at three concentrations. No carryover or interferences were observed. All cannabinoids were stable in blood at room temperature for 24 h, refrigerated (4°C) for 96 h, and following three freeze/thaw cycles. Matrix effects greater than 25% were observed for most analytes in tissues. The proof of concept for method applicability involved measurement of cannabinoids in a pilot fatally injured in an aviation crash. This new analytical method is robust and sensitive, enabling collection of additional cannabinoid postmortem distribution data to improve interpretation of postmortem cannabinoid results.

Cannabinoids in Oral Fluid: Limiting Potential Sources of Cannabidiol Conversion to Δ9- and Δ8-Tetrahydrocannabinol.

In late 2019, the National Laboratory Certification Program (NLCP) published an article reporting on the potential analytical conversion of 7-carboxy cannabidiol (CBD-COOH) to 11-nor-9-carboxy-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine samples. The same conversion is possible in oral fluid with the parent analyte cannabidiol (CBD) converting to Δ9-tetrahydrocannabinol (Δ9-THC) and Δ8-tetrahydrocannabinol (Δ8-THC) under strong acidic conditions. With the recent rise in states legalizing the use of THC and the availability of products containing only CBD, unless the analytical in vitro conversions are controlled, the detection of Δ9-THC or Δ8-THC in oral fluid may not clarify whether the donor was using a CBD product or licit or illicit THC product. Authentic oral fluid samples submitted for cannabinoid analysis were subjected to multiple sample preparation procedures and extraction methods to determine the conditions that allow CBD to convert to THC. CBD single analyte controls prepared from a certified THC-free source were added to the batch to monitor the rate of conversion. Samples were prepared using a base hydrolysis, solid phase extraction, derivatization and analysis by liquid chromatography with tandem mass spectrometry (LC-MS-MS). The base hydrolysis and derivatization were tested independently and did not contribute to the conversion rate. Adjusting the pH of the sample preparation and extraction from pH 2.0 to pH 5.0 changed the conversion rate from 5 to 1%. A pH of 6.0 was not strong enough to extract the cannabinoids efficiently. Removing the acid component of the preparation and extraction procedure eliminated the conversion to THC; however, this did reduce the analyte recovery depending on which extraction column was used. Processing time also contributed to the conversion rate. With smaller trial runs, conversion was not always seen, but with larger validation batches low-level conversion of 1-2% was observed. A fully validated LC-MS-MS method utilizing solid-phase extraction was developed for CBD, Δ9-THC, Δ8-THC and cannabinol. The method specifically targets those analytes found in oral fluid after CBD administration and those that are seen during in vitro CBD conversion. CBD administration was performed using a certified THC-free CBD control.

An optimized method for sample collection, extraction, and analysis of fentanyl and fentanyl analogs from a non-porous surface.

Illicit use of the potent opioid fentanyl and its analogs (fentanyls) are on the rise in the United States. As use increases, drug production tends to also increase, leading to more locations being contaminated with the potentially lethal substance. Because fentanyl-contaminated locations may present a risk to the general public, a method for sampling, identifying, and quantitating these fentanyls from surfaces is in need. This research developed and optimized a surface-wipe collection and extraction method for 17 fentanyls and 10 common fentanyl adulterants from a non-porous surface and quantitated the amount of each compound collected with liquid chromatography tandem mass spectrometry. The final, optimized surface-wipe method resulted in an average collection and extraction efficiency (±SD) of 62.0 (±14.0)%, with a range of 34.1 (±2.6) - 82.5 (±9.6)%. While legislation has yet to be implemented regarding remediation levels for fentanyl-contaminated locations, when such legislation is drafted, this method can be implemented to determine the safety of these locations prior to and after decontamination has occurred.

Estimating drug consumption during a college sporting event from wastewater using liquid chromatography mass spectrometry.

Consumption of licit and/or illicit compounds during sporting events has traditionally been monitored using population surveys, medical records, and law enforcement seizure data. This pilot study evaluated the temporal and geospatial patterns in drug consumption during a university football game from wastewater using liquid chromatography tandem mass spectrometry (LC-MS/MS). Untreated wastewater samples were collected from three locations within or near the same football stadium every 30 min during a university football game. This analysis leveraged two LCMS/ MS instruments (Waters Acquity TQD and a Shimadzu 8040) to analyze samples for 58 licit or illicit compounds and some of their metabolites. Bayesian multilevel models were implemented to estimate mass load and population-level drug consumption, while accounting for multiple instrument runs and concentrations censored at the lower limit of quantitation. Overall, 29 compounds were detected in at least one wastewater sample collected during the game. The 10 most common compounds included opioids, anorectics, stimulants, and decongestants. For compounds detected in more than 50% of samples, temporal trends in median mass load were correlated with the timing of the game; peak loads for cocaine and tramadol occurred during the first quarter of the game and for phentermine during the third quarter. Stadium-wide estimates of the number of doses of drugs consumed were rank ordered as follows: oxycodone (n = 3246) > hydrocodone (n = 2260) > phentermine (n = 513) > cocaine (n = 415) > amphetamine (n = 372) > tramadol (n = 360) > pseudoephedrine (n = 324). This analysis represents the most comprehensive assessment of drug consumption during a university football game and indicates that wastewater-based epidemiology has potential to inform public health interventions focused on reducing recreational drug consumption during large-scale sporting events.

Surface Contamination Generated by "One-Pot" Methamphetamine Production.

Methamphetamine production is the most common form of illicit drug manufacture in the United States. The "one-pot" method is the most prevalent methamphetamine synthesis method and is a modified Birch reduction, reducing pseudoephedrine with lithium and ammonia gas generated in situ. This research examined the amount of methamphetamine surface contamination generated by one-pot syntheses or "cooks", as well as the effectiveness of hosing with water as a simplified decontamination technique, to assess associated public health and environmental consequences. Concentrations of methamphetamine contamination were examined prior to production, after production, and after decontamination with water. Contamination was qualitatively field screened using lateral flow immunoassays and quantitatively assessed using a fluorescence covalent microbead immunosorbent assay. Following screening, 0 of 23 pre-cook samples, 29 of 41 post-cook samples, and 5 of 27 post-decontamination samples were positive. Quantitatively, one pre-cook sample had a methamphetamine concentration of 1.36 ng/100 cm2. Post-cook and post-decontamination samples had average methamphetamine concentrations of 26.50 ± 63.83 and 6.22 ± 12.17 ng/100 cm2, respectively. While all one-pot methamphetamine laboratories generate different amounts of waste, depending on the amount of precursors used and whether the reaction vessel remained uncompromised, this study examined the surface contamination generated by a popular one-pot method known to law enforcement. By understanding the amount of surface contamination generated by common methods of one-pot methamphetamine production and the effectiveness of decontamination techniques used to remediate them, health risks associated with these production sites can be better understood and environmental contamination can be mitigated.

Postmortem Fluid and Tissue Concentrations of THC, 11-OH-THC and THC-COOH.

Marijuana is the most commonly abused illicit drug worldwide. Marijuana is used for its euphoric and relaxing properties. However, marijuana use has been shown to result in impaired memory, cognitive skills and psychomotor function. The Federal Aviation Administration's Civil Aerospace Medical Institute conducts toxicological analysis on aviation fatalities. Due to severe trauma associated with aviation accidents, blood is not always available; therefore, the laboratory must rely on specimens other than blood for toxicological analysis in ~30-40% of cases. However, the postmortem distribution of cannabinoids has not been well characterized. The purpose of this research is to evaluate the distribution of Δ9-tetrahydrocannabinol (THC), and its metabolites, 11-hydroxy-tetrahydrocannabinol (11-OH-THC) and THC-COOH, in postmortem fluid and tissue specimens from 11 fatal aviation accident cases (2014-2015) previously found positive for cannabinoids. Specimens evaluated, when available, included: blood, urine, vitreous humor, liver, lung, kidney, spleen, muscle, brain, heart and bile. We developed and validated (following SWGTOX guidelines) a sensitive and robust method using solid-phase extraction and liquid chromatography-tandem mass spectrometry to identify and quantify THC, 11-OH-THC and THC-COOH in postmortem fluids and tissues. The method readily identified and quantified these cannabinoids in postmortem fluids and tissues below 1 ng/mL. Qualitative cannabinoid results within each case were comparable between blood and non-blood specimens. However, there was no consistent distribution of the cannabinoids between blood and any other fluids or tissues. Therefore, while quantitative interpretation of non-blood postmortem fluid and tissues samples is not prudent, a majority of the non-blood specimens tested could be suitable alternative/supplemental choices for qualitative cannabinoid detection.

Evaluation of Oral Fluid as a Specimen for DUID.

Drugged driving has become more prevalent than drunk driving and is quickly gaining national attention due to increased prescription drug abuse and recent cannabis legalization. Unlike alcohol, police officers do not generally have access to approved devices to screen for drugs at the roadside. Onsite drug screening devices do exist and are used in other countries, but have not garnered widespread approval for use in the United States in driving under the influence of drugs (DUID) cases. One reason for this is that the devices are designed to test oral fluid, which is not a commonly accepted specimen for DUID. A study was conducted to test the effectiveness of using oral fluid during routine traffic stops in DUID cases in conjunction with drug recognition expert (DRE) officers from the Tulsa Police Department (TPD). Samples were screened at the roadside using an Alere DDS®2 Mobile Test System and Quantisal™ collection devices were used for laboratory based screening by enzyme-linked immunosorbent assay and confirmation by liquid chromatography-tandem mass spectrometry. The results of the DRE observations, alternate specimens like blood and urine, onsite oral fluid screening and laboratory based oral fluid screening and confirmations were used to assess the usefulness of oral fluid as a DUID specimen. Due to the small sample size (N = 9), no significant differences in the measured performance of onsite and laboratory based tests was seen. The results of this study indicate that oral fluid testing is a viable option both at the roadside and in a laboratory setting.

The quantitation of cocaine on U.S. currency: survey and significance of the levels of contamination.

It has long been suspected that the illicit distribution of cocaine in the United States has led to a large-scale contamination of the currency supply. To investigate the extent of contamination, 418 currency samples (4174 bills) were collected from 90 locations around the United States from 1993 to 2009. The extent of their cocaine contamination was quantitated via gas chromatography/mass spectrometry or liquid chromatography/mass spectrometry. The level of cocaine contamination was determined to average 2.34 ng/bill across all denominations ($1, $5, $10, $20, $50, and $100). Levels of cocaine contamination on currency submitted to the Federal Bureau of Investigation Laboratory in criminal cases over the 1993-2001 timeframe had significantly higher contamination than currency in general circulation. A mathematical model was developed based on the background survey that indicates the likelihood of drawing a bill in specific concentration ranges. For example, there is a 0.8349 likelihood that random bill will have contamination less than 20 ng.

Simultaneous quantification of vinblastine and desacetylvinblastine concentrations in canine plasma and urine samples using LC-APCI-MS/MS.

A highly sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS/MS) method has been developed and validated for simultaneous quantification of vinblastine and its metabolite, desacetylvinblastine, in canine plasma and urine samples. Plasma and urine samples were processed by a solid phase extraction procedure. The optimal chromatographic behavior of these analytes was achieved on pentafluorophenyl (PFP) propyl analytical column (5µm, 50×2.1mm) under isocratic elution of 0.75mL/min with a mobile phase of 5mM ammonium acetate and methanol. The samples were analyzed in positive ion, multiple reaction monitoring mode. The calibration curves were linear over 0.125-2ng/mL (lower calibration curve); 2-100ng/mL (higher calibration curve) and 0.125-5ng/mL for vinblastine and desacetylvinblastine in plasma, and over 1-2000ng/mL and 0.5-100ng/mL for vinblastine and desacetylvinblastine in urine samples, respectively. The limits of quantitation of vinblastine and desacetylvinblastine were 0.125ng/mL in both matrices. The intra and interday accuracy was above 89% and precision below 8.6% for both analytes in both matrices. The developed method was successfully applied to ongoing in vivo vinblastine pharmacokinetic studies in dogs.

Oxidative stress and mitochondrial-mediated apoptosis in dopaminergic cells exposed to methylcyclopentadienyl manganese tricarbonyl.

Methylcyclopentadienyl manganese tricarbonyl (MMT), an organic manganese-containing gasoline additive, was investigated to determine whether MMT potentially causes dopaminergic neurotoxic effects. MMT is acutely cytotoxic and dopamine-producing cells (PC-12) seemed to be more susceptible to cytotoxic effects than nondopaminergic cells (striatal gamma-aminobutyric acidergic and cerebellar granule cells). MMT also potently depleted dopamine apparently by cytoplasmic vesicular release to the cytosol, a neurochemical change resembling other dopaminergic neurotoxicants. Generation of reactive oxygen species (ROS), an early effect in toxicant-induced apoptosis, occurred within 15 min of MMT exposure. MMT caused a loss of mitochondrial transmembrane potential (DeltaPsim), a likely source of ROS generation. The ROS signal further activated caspase-3, an important effector caspase, which could be inhibited by antioxidants (Trolox or N-acetyl cysteine). Predepletion of dopamine by using alpha-methyl-p-tyrosine (tyrosine hydroxylase inhibitor) treatment partially prevented caspase-3 activation, denoting a significant dopamine and/or dopamine by-product contribution to initiation of apoptosis. Genomic DNA fragmentation, a terminal hallmark of apoptosis, was induced concentration dependently by MMT but completely prevented by pretreatment with Trolox, deprenyl (monoamine oxidase-B inhibitor), and alpha-methyl-p-tyrosine. A final set of critical experiments was performed to verify the pharmacological studies using a stable Bcl-2-overexpressing PC-12 cell line. Bcl-2-overexpressing cells were significantly refractory to MMT-induced ROS generation, caspase-3 activation, and loss of DeltaPsim and were completely resistant to MMT-induced DNA fragmentation. Taken together, the results presented herein demonstrate that oxidative stress plays an important role in mitochondrial-mediated apoptotic cell death in cultured dopamine-producing cells after exposure to MMT.

Caspase-3-dependent proteolytic cleavage of protein kinase Cdelta is essential for oxidative stress-mediated dopaminergic cell death after exposure to methylcyclopentadienyl manganese tricarbonyl.

In the present study, we characterized oxidative stress-dependent cellular events in dopaminergic cells after exposure to an organic form of manganese compound, methylcyclopentadienyl manganese tricarbonyl (MMT). In pheochromocytoma cells, MMT exposure resulted in rapid increase in generation of reactive oxygen species (ROS) within 5--15 min, followed by release of mitochondrial cytochrome C into cytoplasm and subsequent activation of cysteine proteases, caspase-9 (twofold to threefold) and caspase-3 (15- to 25-fold), but not caspase-8, in a time- and dose-dependent manner. Interestingly, we also found that MMT exposure induces a time- and dose-dependent proteolytic cleavage of native protein kinase Cdelta (PKCdelta, 72-74 kDa) to yield 41 kDa catalytically active and 38 kDa regulatory fragments. Pretreatment with caspase inhibitors (Z-DEVD-FMK or Z-VAD-FMK) blocked MMT-induced proteolytic cleavage of PKCdelta, indicating that cleavage is mediated by caspase-3. Furthermore, inhibition of PKCdelta activity with a specific inhibitor, rottlerin, significantly inhibited caspase-3 activation in a dose-dependent manner along with a reduction in PKCdelta cleavage products, indicating a possible positive feedback activation of caspase-3 activity by PKCdelta. The presence of such a positive feedback loop was also confirmed by delivering the catalytically active PKCdelta fragment. Attenuation of ROS generation, caspase-3 activation, and PKCdelta activity before MMT treatment almost completely suppressed DNA fragmentation. Additionally, overexpression of catalytically inactive PKCdelta(K376R) (dominant-negative mutant) prevented MMT-induced apoptosis in immortalized mesencephalic dopaminergic cells. For the first time, these data demonstrate that caspase-3-dependent proteolytic activation of PKCdelta plays a key role in oxidative stress-mediated apoptosis in dopaminergic cells after exposure to an environmental neurotoxic agent.