Brain tissue is a potentially valuable postmortem specimen because of its relative anatomical isolation, reduced susceptibility to postmortem degradation, and relevance as the primary site of action for many drugs. Despite the frequent detection of stimulants and synthetic opioids in medicolegal death investigations, limited data exist regarding their regional distribution within the human brain. This study evaluated the distribution of amphetamine (AMP), methamphetamine (MAMP), cocaine, benzoylecgonine, cocaethylene, and fentanyl across seven anatomically distinct brain regions (basal ganglia, cerebellum, medulla, midbrain, occipital lobe, thalamus, and pons) in postmortem human cases. Brain samples were collected from cases screening positive for cocaine/benzoylecgonine or fentanyl using urine drug screening at autopsy. Brain homogenates were extracted in triplicate using solid-phase extraction followed by instrumental analysis using liquid chromatography tandem mass spectrometry. To make comparisons between cases, concentrations were normalized to the occipital lobe. Statistical analysis using one-way ANOVA determined that there were significant differences in concentrations within cases and normalized concentrations between cases. The basal ganglia had the highest concentrations for AMP, cocaethylene, cocaine, fentanyl, and MAMP. Benzoylecgonine concentrations were highest in the cerebellum. Concentrations of metabolite to parent compound showed that there were weak correlations between benzoylecgonine-to-cocaine, cocaethylene-to-cocaine, and AMP-to-MAMP. While there was heterogeneous distribution in the brain, any of the brain regions included in this study could have utility for postmortem toxicology testing as analytes were generally detected in all examined brain regions. The basal ganglia typically had highest concentrations in this study and therefore are recommended for collection.

Interpretation of postmortem toxicology results is complicated by postmortem redistribution (PMR), which can alter drug concentrations between tissues and blood (BL). This study evaluates brain (BR) to BL concentration ratios for amphetamine (AMP), methamphetamine (MAMP), cocaine (COC), benzoylecgonine (BE), cocaethylene (CE), and fentanyl (FENT) using postmortem casework data to evaluate the utility of BR tissue as an alternative matrix. BR:BL ratios varied widely across analytes: AMP (2.2-5.8), BE (0.2-2.1), CE (1.2-5.1), COC (0.1-5.8), MAMP (2.2-7.3), and FENT (1.1-24.8). Lipophilic compounds (AMP, CE, COC, MAMP, and FENT) generally exhibited ratios greater than one, consistent with preferential BR partitioning and susceptibility to PMR, whereas the polar metabolite BE showed ratios less than one, reflecting limited BR accumulation. FENT demonstrated the greatest variability, highlighting challenges in interpreting its postmortem concentrations. Correlations between BR and femoral BL concentrations were slightly positive for all analytes, indicating that BR concentrations may not be reliable proxies for BL levels. Evaluation of select antemortem cases and outliers revealed additional influences on BR:BL ratios, including survival time, drug stability, route of administration, and polysubstance use. COC instability and hydrolysis to BE, as well as incomplete drug distribution in rapid deaths, contributed to atypical ratios. Overall, BR:BL ratios are highly analyte dependent and influenced by both pharmacokinetic properties and postmortem factors. While BR tissue may provide useful complementary information when BL is unavailable, it should not be used as a direct substitute for BL in toxicological interpretation. These findings underscore the need for further research to expand BR:BL datasets across drug classes.

The Harris County Institute of Forensic Sciences recently added brain to its fentanyl analog testing method for 14 analogs (fluoroisobutyryl fentanyl, acetyl fentanyl, acryl fentanyl, alfentanil, butyryl fentanyl, carfentanil, fentanyl, para-fluorofentanyl, furanyl fentanyl, methoxyacetyl fentanyl, norcarfentanil, norfentanyl, sufentanil, and valeryl fentanyl) and 3 U-series drugs (U-47700, U-48800, and U-49900). Brain is a protected and isolated organ with lower metabolic activity than other tissues, which can assist in interpreting results and preserving parent drug. Limited publications testing brain samples for fentanyl and fentanyl analogs exist and none describe homogenate stability for these analytes. Validation of the solid phase extraction and liquid chromatography tandem mass spectrometry method followed the ASB 036 Standard Practices for Method Validation in Forensic Toxicology and included limit of detection, limit of quantification, calibration model, bias and precision, ionization suppression/enhancement, interferences, carryover, processed sample stability, and dilution integrity. Carfentanil, fentanyl, furanyl fentanyl and methoxyacetyl fentanyl met quantitative bias and precision acceptance criteria in brain. To assess homogenate stability, brain homogenates (both unpreserved and preserved with 1% sodium fluoride) were fortified with 50 ng/mL of analyte, stored at room temperature (∼20°C), refrigerated (2-8°C), or frozen (∼-20°C), and analyzed in triplicate over a 90-day period. Analytes were considered stable if analyte/internal standard response ratio was within ± 20% of Day 0 and chromatographic peaks met qualitative acceptance criteria. Frozen brain homogenates could be stored for up to 90 days and withstood three freeze/thaw cycles for acetyl fentanyl, alfentanil, fentanyl, para-fluorofentanyl, FIBF, methoxyacetyl fentanyl, and norfentanyl. Brain homogenate stability was improved when frozen and was not impacted by the addition of 1% sodium fluoride. The study herein provides insight into the feasibility of testing brain for fentanyl analogs and their stability under various storage conditions, contributing valuable data to the limited literature on brain toxicology testing.

Brain can be a useful specimen for toxicology testing as it is a protected and isolated organ with lower metabolic activity than other tissues, but there is currently no published data supporting the stability of stimulant drugs in prepared brain homogenates. Brain homogenates were evaluated to determine the stability of the following stimulant drugs: amphetamine, benzoylecgonine, bupropion, cocaethylene, cocaine, ephedrine, methylenedioxyamphetamine, methylenedioxymethamphetamine, methamphetamine, and phentermine. Four different homogenates were prepared at a 1:4 dilution with deionized water and fortified at 500 ng/mL of: cocaine without sodium fluoride, cocaine with 1% sodium fluoride, stimulant drugs other than cocaine without sodium fluoride, and stimulant drugs other than cocaine with 1% sodium fluoride. The fortified homogenates were aliquoted into 13 x 100-mm screw cap tubes and stored at room temperature (similar to 20 degrees C), refrigerated (2-8 degrees C), or frozen (<-5 degrees C) and analyzed in triplicate on Days 0, 1, 3, 7, 14, 30, 60, and 90. Analytes were considered stable as long as the difference in analyte/internal standard response ratio from Day 0 was less than 20% and the peaks met qualitative acceptance criteria. All analytes were stable for up to 90 days when stored frozen with or without sodium fluoride and had variable stability at all other evaluated conditions.

Since the opioid epidemic was declared in 2017, postmortem fentanyl cases and the need for interpretation of their results have increased. Postmortem redistribution (PMR) is one of the factors to consider when interpreting cases. There have been several previous studies regarding fentanyl PMR; however, these studies either have small sample sizes or were conducted prior to the declaration of the opioid epidemic, which may cause conflicting results and not be reflective of current trends. This study includes fentanyl central/peripheral (C/P) blood ratios from 748 cases from both Harris County, TX, and Orange County, TX, spanning from January 2009 to June 2022. Because the data set was determined to be non-normally distributed, a Kruskal-Wallis test was used for statistical comparisons. There were statistically significant differences between epidemic cases from the Harris County Institute of Forensic Sciences and the Orange County Crime Laboratory, C/P ratios from pre-epidemic to epidemic years, and in cases where medically related administration of fentanyl was documented when compared to cases where there was no documentation of licit fentanyl use. Various factors that could impact PMR were evaluated (age, gender, polydrug use, etc.), and no clear trend or observation was made from the data. Based on the results of this study, there is still no clear indication as to what caused the increase in C/P ratios, but it may be related to an increase in illicit fentanyl use.

This study demonstrates the validation of a semi-quantitative method for the rapid screening of whole blood and urine specimens using clonazepam as the target molecule for the Neogen® Benzodiazepine kit. Decision points were validated at 10.0 ng/mL for whole blood and 25.0 ng/mL for urine. The validation design was based on the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation and included the evaluation of sensitivity, precision, specificity, carryover, hook effect, drift, ruggedness/robustness and a case sample evaluation. The experimental limit of detection for clonazepam was determined to be at least 5.0 ng/mL in whole blood and at least 10.0 ng/mL in urine. Excellent precision was demonstrated when the assay was evaluated using the mean of three replicates from five separate runs (n = 15) at the decision point and at concentration levels ±50% and +100% of the decision point. Although the method was optimized and exceptional precision was demonstrated at each level, the current SWGTOX validation requirements for a valid decision point were not fulfilled. However, both the blood and urine matrix did meet the proposed revision of the SWGTOX requirements for determining a valid decision point promulgated by the American Academy of Forensic Sciences Standards Board and the assay was reliably able to detect benzodiazepines without interference from matrix components or other compounds routinely detected in authentic case samples. Case sample results were comparable with those obtained when the samples were initially screened using oxazepam as the target molecule. The Neogen® Benzodiazepine kit using clonazepam as the target molecule exhibited cross-reactivity for 29 different benzodiazepines and demonstrated excellent precision and sensitivity in both whole blood and urine, making it an efficient and reliable method to screen for benzodiazepines, even though the validation did not fulfill current SWGTOX requirements for a valid decision point.

Solid-state nuclear magnetic resonance (SS-NMR) spectroscopy has become a common technique to study polymorphism in pharmaceutical solids at high-resolution. However, high-throughput application of high resolution SS-NMR spectroscopy is severely limited by the long 1H spin-lattice relaxation (T1) that is common to solid phase compounds. Here, we demonstrate the use of paramagnetic relaxation reagents such as chromium (III) acetylacetonate (Cr(acac)3) and nickel (II) acetylacetonate (Ni(acac)2) for fast data acquisition by significantly reducing the T1 value for carbamazepine Forms I, II, III, and dihydrate, cimetidine Forms A and B, nabumetone Form I, and acetaminophen Form I polymorphs. High resolution 13C cross-polarization and magic angle spinning were used to measure T1 values for each polymorph. In order to confirm the absence of polymorphic transitions during SS-NMR experiments, powder x-ray diffraction was implemented. The amount of chromium ions incorporated by the recrystallization process was quantified by using inductively coupled plasma optical emission spectroscopy. Our results suggest that the paramagnetic ions added to the polymorphs do not affect the polymorphic transformation or the quality of NMR spectra. We believe that this successful demonstration of fast data collection will enable high-throughput utilization of SS-NMR techniques to study polymorphic solids and could set the groundwork for NMR crystallography studies.

Nitinol (NiTi) nanoparticles are a valuable metal alloy due to many unique properties that allow for medical applications. NiTi nanoparticles have the potential to form nanofluids, which can advance the thermal conductivity of fluids by controlling the surface functionalization through chemical attachment of organic acids to the surface to form self-assembled alkylphosphonate films. In this study, phosphonic functional head groups such as 16-phosphonohexadecanoic acid, octadecylphosphonic acid, and 12-aminododecylphosphonic acid were used to form an ordered and strongly chemically bounded film on the NiTi nanopowder. The surface of the NiTi nanoparticles was modified in order to tailor the chemical and physical properties to the desired application. The modified NiTi nanoparticles were characterized using infrared spectroscopy, powder X-ray diffraction, X-ray photoelectron spectroscopy, and 31P solid-state nuclear magnetic resonance. The interfacial bonding was identified by spectroscopic data suggesting the phosphonic head group adsorbs in a mixed bidentate/monodentate binding motif on the NiTi nanoparticles. Dynamic light scattering and scanning electron microscopy-energy dispersive X-ray spectroscopy revealed the particle sizes. Differential scanning calorimetry was used to examine the phase transitions. Zeta potential determination as a function of pH was examined to investigate the surface properties of charged nanoparticles. The influence of environmental stability of the surface modifications was also assessed.