Assessment of ∆9-THC and ∆9-THCCOOH Bias, Precision, and Ionization Suppression/Enhancement between Solid Tissue Homogenate and Supernatant by LC-MS/MS.

LC-MS-MS assays are frequently utilized for screening and confirmatory purposes in the forensic toxicology laboratory. While these techniques are excellent for the targeted identification and quantitation of a wide variety of drug classes, validation and determining fit-for-purpose is a requirement for each method. In the United States, ANSI/ASB Standard 036 currently serves as a primary resource in forensic toxicology method validation, and mandates that laboratories evaluate critical performance characteristics to help ensure the production of forensically defensible results. Due to the variability of specimen quality frequently encountered in the discipline of postmortem toxicology, the [Author Information Removed] Office of the Chief Medical Examiner Forensic Toxicology Laboratory routinely analyzes solid tissue specimens as part of the medicolegal death investigation process and evaluates liver as a representative solid tissue matrix during method validation. Authentic postmortem specimens (e.g., liver, kidney, skeletal muscle, and spleen) were used to investigate the effects of analyzing solid tissue homogenate versus solid tissue supernatant on bias, precision, and ionization suppression/enhancement of ∆9-THC and ∆9-THCCOOH. Bias was <20% for Δ9-THC and ∆9-THCCOOH in liver homogenate and supernatant with a single exception of the low QC concentration for Δ9-THC in liver homogenate (-29%). Within-run and between-run CV was <20% for Δ9-THC and ∆9-THCCOOH in liver homogenate and supernatant. Δ9-THC and Δ9-THC-d3 exhibited significant ion suppression in both liver homogenate and supernatant, while ∆9-THCCOOH and ∆9-THCCOOH-d3 showed both ion suppression and enhancement in these matrices. Noticeable quantitative differences were observed in authentic postmortem solid tissue homogenate and supernatant specimens despite evaluating from identical tissue samplings. A brief discussion of the results is presented using a validated LC-MS-MS method for the confirmation and quantitation of ∆9-THC and ∆9-THCCOOH in postmortem casework.

The relationship between elastin cross linking and alveolar wall rupture in human pulmonary emphysema.

To better define the role of mechanical forces in pulmonary emphysema, we employed methods recently developed in our laboratory to identify microscopic level relationships between airspace size and elastin-specific desmosine and isodesmosine (DID) cross links in normal and emphysematous human lungs. Free DID in wet tissue (a biomarker for elastin degradation) and total DID in formalin-fixed, paraffin-embedded (FFPE) tissue sections were measured using liquid chromatography-tandem mass spectrometry and correlated with alveolar diameter, as determined by the mean linear intercept (MLI) method. There was a positive correlation between free lung DID and MLI (P < 0.0001) in formalin-fixed lungs, and elastin breakdown was greatly accelerated when airspace diameter exceeded 400 µm. In FFPE tissue, DID density was markedly increased beyond 300 µm (P < 0.0001) and leveled off around 400 µm. Elastic fiber surface area similarly peaked at around 400 µm, but to a much lesser extent than DID density, indicating that elastin cross linking is markedly increased in response to early changes in airspace size. These findings support the hypothesis that airspace enlargement is an emergent phenomenon in which initial proliferation of DID cross links to counteract alveolar wall distention is followed by a phase transition involving rapid acceleration of elastin breakdown, alveolar wall rupture, and progression to an active disease state that is less amenable to therapeutic intervention.NEW & NOTEWORTHY The current findings support the hypothesis that airspace enlargement is an emergent phenomenon in which initial proliferation of DID cross links to counteract alveolar wall distention is followed by a phase transition involving rapid acceleration of elastin breakdown, alveolar wall rupture, and progression to an active disease state that is less amenable to therapeutic intervention.

Free lung desmosine: a potential biomarker for elastic fiber injury in pulmonary emphysema.

INTRODUCTION: Desmosine and isodesmosine (DID) are biomarkers for elastic fibre damage in pulmonary emphysema. However, current methods for measuring lung DID involve tissue hydrolysis and lack specificity for those fibres undergoing breakdown. To address this limitation, free (nonpeptide-bound) DID content in unhydrolyzed tissues was evaluated as a more accurate biomarker in an animal model of pulmonary emphysema. METHODS: Hamsters were treated with either cigarette smoke and lipopolysaccharide (LPS), room air and LPS, or room air alone (controls). Free DID levels in fresh and formalin-fixed lungs were measured by LC-MS/MS and correlated with the mean linear intercept (MLI) measure of airspace size. RESULTS: There was no significant difference in free DID between fresh and formalin-fixed lungs. Animals treated with smoke and LPS had significantly higher levels of free DID than the LPS only group (359 vs. 93.1 ng/g wet lung, respectively; p = 0.0012) and room air controls (undetectable levels; p = 0.0002). There was a significant positive correlation between free DID and MLI (p < 0.0001). CONCLUSIONS: The results support the hypothesis that free lung DID is a sensitive indicator of alveolar wall injury that may be used to study the development of pulmonary emphysema in both animal models and post-mortem human lung tissue.

Evaluation of Acetylfentanyl Following Suspected Heroin Overdose When Complicated by the Presence of Toxic Fentanyl and Alprazolam Concentrations.

A 34-year-old male was reported to be snorting a white powder that was believed to contain heroin. Toxicological analysis revealed free morphine (356 µg/L), fentanyl (34.7 µg/L), alprazolam (64.9 µg/L), and acetylfentanyl (32.9 µg/L) in femoral blood and 6-monoacetylmorphine (6-MAM, <10.0 µg/L) in vitreous fluid. Norfentanyl was only detected in stomach contents (<1.00 µg/total). Heroin, fentanyl, and acetylfentanyl were also detected in solid dose evidence submitted by law enforcement. The fentanyl and alprazolam concentrations might normally be associated with a fatal outcome and are supported with the distribution of fentanyl and alprazolam being consistent with an acute intoxication. In addition, the presence of 6-MAM and a free versus total morphine ratio of 67.9% provide supporting evidence of a rapid death following intranasal (IN) administration. However, the presence of illicit acetylfentanyl complicates toxicologic interpretation due to overlapping recreational and fatal concentrations of this compound reported in the literature as well as a potential for postmortem redistribution (PMR). Reported acetylfentanyl concentrations have also varied when presented with significant fentanyl concentrations and underscore the need to consider a wide range of illicit opioid compounds when investigating drug-related deaths. Based on our comprehensive toxicologic analysis, the results suggest an acute intoxication primarily by IN administration of acetylfentanyl and fentanyl. In addition, we suggest the presence of alprazolam, 6-MAM, and a percentage free morphine is also consistent with rapid death. The cause of death was officially attributed to an acute combined intoxication of acetylfentanyl, fentanyl, alprazolam, and heroin, with the manner of death as accidental.

Five Postmortem Case Reports with Qualitative Analysis of Cyclopropylfentanyl by LC-MS-MS.

In January 2018, the Drug Enforcement Agency temporarily designated cyclopropylfentanyl as a Schedule I drug. Over the course of 5 months (December 2017-May 2018), the Nassau County Medical Examiner Toxicology Laboratory qualitatively identified and confirmed cyclopropylfentanyl in specimens obtained from five postmortem cases. We describe the five cases and include pertinent autopsy findings and decedent histories, along with results for cyclopropylfentanyl determined in postmortem cardiac blood. Samples were prepared by an alkaline liquid-liquid extraction, with sample pH adjusted to >9 and utilizing an extraction solvent consisting of 90:10 hexane:ethyl acetate. Instrumental analysis was achieved via liquid chromatography tandem mass spectrometry with a dual jetstream electrospray source operating in positive ion mode. Two ion transitions were monitored for each analyte of interest and the internal standard. The estimated concentration range of cyclopropylfentanyl in the reported cases was 5.6 to 82 ng/mL for five postmortem cardiac blood specimens. All five cases included cyclopropylfentanyl in the established cause of death.

Screening of Novel Psychoactive Substances in Postmortem Matrices by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS-MS).

Novel psychoactive substances (NPS) are newly emerging compounds, natural and synthetic, that are often sold as "legal" alternatives to controlled substances. These substances can include phenethylamines, tryptamines, synthetic cathinones, piperazines and others. Most novel psychoactive substances and similar designer drugs are often based on the chemical backbone of classic drugs of abuse but are not regularly encountered in postmortem casework. These compounds may go undetected in forensic toxicology labs where suitable identification methods for NPS may not exist. In order to keep up with this ever-expanding list of pharmacologically active and toxicologically hazardous substances, there is an increased need for a screening panel suitable for postmortem human samples such as whole blood and urine. In order to address this increased prevalence of NPS, a method was developed and validated to identify 50 NPS in postmortem samples using liquid chromatography triple quadrupole mass spectrometry. The data presented here represents a validated liquid chromatography triple quadrupole mass spectrometry method to qualitatively identify NPS in postmortem samples. The limit of detection for all compounds was set at 2.5 ng/mL. To evaluate the prevalence of NPS in the region, blood and urine specimens from 110 postmortem cases were submitted for analysis based on initial screening results and/or case history. Of those cases, 28% were positive for several NPS, though only a few were included in the cause of death. The robustness of this method proves that it is suitable for the continual addition of newer substances, ensuring up-to-date simultaneous drug detection.