Mitragynine (MG) is the primary psychoactive component of the kratom plant (Mitragyna speciosa). 7-Hydroxy mitragynine (7OH-MG) is found naturally in the plant but in small amounts (<2%). MG is metabolized to 7OH-MG and further to mitragynine pseudoindoxyl (MP), with the latter documented via in vitro enzymatic activity. Lack of scheduling has caused unregulated markets to proliferate with gas stations, smoke shops, and online retailers selling semi-synthetic formulations of these alkaloids under the umbrella term "kratom," though distinct from botanical kratom. Products containing 7OH-MG, MP, and synthetically derived dihydro-7-hydroxy mitragynine (DH-7OH-MG, aka MGM-15) exhibit greater potencies and activities at the µ-opioid receptor compared to MG and are now highly implicated in intoxications and deaths. These alkaloids present complex analytical and interpretive challenges due to their isomeric forms, incompatibility with certain extraction and instrumental techniques promoting analyte interconversion, instability in blood, and lack of published concentration ranges. A quantitative high-resolution mass spectrometry method for MG, 7OH-MG, MP, and DH-7OH-MG, which prevents interconversion and separates isomers, is presented herein along with stability data and concentrations from more than 200 authentic cases. MG, MP, and DH-7OH-MG were relatively stable in blood, whereas 7OH-MG decreased quickly and by 89% over 112 days with concurrent increase in MP. In a case subset (n = 139), mean ± SD concentrations in blood were 146 ± 477 ng/mL for 7OH-MG (median: 58 ng/mL, range: 0-5,000 ng/mL) and 194 ± 239 ng/mL for MP (median: 120 ng/mL, range: 4.8-1,900 ng/mL). In a second subset (n = 17), the mean ± SD DH-7OH-MG concentration was 324 ± 669 ng/mL (median: 170 ng/mL, range: 50-2,900 ng/mL). Testing for kratom alkaloids is currently extremely limited and/or subject to potential inaccuracy due to interconversion, leaving adverse events and fatalities under-investigated. Continued vigilance is needed as the kratom alkaloid product landscape rapidly evolves.

Chang et al. report substantial variability in fentanyl blood-to-brain relationships and suggest that brain testing may be beneficial in suspected overdose cases with low peripheral blood concentrations. We suggest that this implication would be strengthened by distinguishing analytical confirmation from incremental forensic interpretation. Demonstrating fentanyl in a second matrix does not itself establish that the brain result changes attribution of fentanyl's role in death. A focused analysis of low-blood cases in which brain findings altered interpretation, supported by scene, autopsy, and co-intoxicant data, would better define the practical value of brain testing.

The analysis and determination of blood alcohol concentration (BAC) is a cornerstone test in forensic toxicology that is well researched. The purpose of this study was to determine the post analytical stability of blood ethanol obtained from re-injected vials via Agilent dual column headspace-GC-FID. Over 6 months and 22 individual batch analyses, 303 samples were prepared and analyzed using Headspace-GC-FID for the presence of blood ethanol using 0.02% n-propanol as an internal standard. Each individual batch was re-injected after storage at room temperature for 72 hours to mimic a typical weekend absence. Ethanol signal response, n-propanol signal response and measured blood ethanol concentration were evaluated for stability in terms of percent change. Sample population consisted of negative human blood fortified with known concentrations of ethanol via certified reference material purchased from accredited vendors (n = 187) in the form of calibrators and controls, as well as unknown post-mortem and ante-mortem blood samples submitted to the Nassau County Medical Examiner's Office Department of Forensic Toxicology (n = 116). All samples were tested using similar conditions and internal standard. Significant differences were observed in the signal response for both n-propanol (p<.001) and ethanol (p<.001) between the two test groups however, there was a non-significant difference in measured BAC between the two groups t(302) = 0.3, p = 0.780, d = 0.016. Results suggest very little effect of re-injection after 72-hour storage at room temperature on measured blood ethanol concentration using headspace GC-FID. While there was significant loss of ethanol and internal standard on reinjection, the calculated ethanol concentration was unaffected due to the similar properties of both volatile measurands as well as the consistency of the response ratio of ethanol:n-propanol in reinjected vials. In addition, the observed differences in measured BAC on re-injection were well within the NCME's measurement uncertainty of 11% at k = 3.

Paraphenylenediamine (PPD), the principal ingredient of oxidative hair dyes, is a growing cause of acute poisoning in developing nations. This paper provides a comprehensive overview of published case reports, observational studies of PPD poisoning, clinical manifestations, current management strategies, analytical methods for PPD detection, and offers key clinical and post-mortem toxicology considerations. A literature search related to PPD poisoning was conducted from 1991 to 2025 across PubMed, Scopus, Web of Science, and Google Scholar. Publications encompassing 6,843 individual cases were reviewed, and data on demographics, dose, route, clinical features, laboratory parameters, treatment modalities, outcomes, and analytical approaches were analyzed. Angioedema, rhabdomyolysis, and acute renal failure form a characteristic clinical triad that is almost universal in moderate-to-severe cases, with mortality rates ranging up to 55.5% in hospital cases. Cardiac and hepatic injury are also common. PPD has no specific antidote, and treatment is completely supportive. Analytical methods ranging from colorimetric screening to confirmatory tandem mass spectrometry for PPD detection in biological and non-biological matrices are discussed. This review strongly suggests that PPD poisoning represents a significant public health concern in the developing world. Young women with low socioeconomic and rural backgrounds predominantly ingested PPD with suicidal intent. The highest fatality rates were observed in cases where treatment was delayed due to late diagnosis, primarily because no point-of-care test is currently available for the rapid detection of PPD. The combination of lethality, availability, and accessibility makes PPD an ideal agent for suicidal poisoning in populations with high rates of impulsive suicidal behavior. Early diagnosis can improve survival rates in PPD poisoning cases.

Toxicological analysis is an integral part of forensic death investigations. However, analysis is sometimes complicated by postmortem changes, and blood might not always be useable or available due to postmortem redistribution or degradation. In these cases, brain tissue can be useful as the brain is more protected against trauma, putrefaction, postmortem redistribution, skeletonization and severe blood loss. However, reference concentrations of drugs in brain tissue are still scarce. This study aimed to report brain concentrations and brain-blood ratios for 122 compounds including drugs of abuse, pharmaceuticals, and metabolites frequently detected in forensic autopsy cases. In total, 2264 cases were collected over an 11-year period and retrospectively categorized as having either toxic or therapeutic levels of detected compounds based on blood concentrations. Toxic and therapeutic concentrations showed considerable overlap in both blood and brain due to drug tolerance and multi-drug combinations. Brain-blood ratios were highest for compounds with direct function on the central nervous system, especially antidepressants and sedative-hypnotics. The reported brain concentrations and brain-blood ratios can serve as reference values for interpretation of drug concentrations in brain tissue and the evaluation of cause of death in future forensic investigations.

Novel psychoactive phenethylamines and phenidates are synthetically designed analogs of conventional substances such as 3,4-methylenedioxymethamphetamine (MDMA), amphetamine, and methylphenidate, developed to circumvent existing drug regulations. In Korea, the abuse of these substances has increased, particularly among habitual users and newly addicted younger individuals. To address this issue, new substances are regulated through the analog legislation and the temporary narcotic designation system. In this study, liquid chromatography-tandem mass spectrometry method using solid-phase extraction was developed and validated for the simultaneous determination of 13 substituted phenethylamines and 5 substituted phenidates in human urine. The target analytes included 5-MAPDB, BDB, 4-FEA, PMEA, 3F-Phenetrazine, 25iP-NBOMe, 2C-N, 2C-TFM, 30C-NBOMe, 3C-P, 4-MMA-NBOMe, DOI, and 2C-H, as well as 4-MeTMP, 4F-EPH, 4F-MPH, IPPH, and HDEP-28. No interfering peaks were observed at the retention times of analytes and internal standards. Linearity was confirmed over analyte-specific concentration within the overall range of 0.1-100 ng/ml, with coefficient of determination (r2) exceeding 0.995. The limits of detection and quantification were 0.001-0.1 and 0.1-0.2 ng/ml, respectively. Accuracy and precision, expressed as bias and coefficient of variation (CV), were within ±10% and <10%, respectively. The CVs for matrix effects and extraction efficiencies were approximately 10%, and the extraction efficiencies exceeded 70%, confirming the reliability of the method for forensic applications. The validated method enables simultaneous determination of substituted phenethylamines and phenidates in urine and facilitates prompt positive identification in forensic casework. In addition, the structural information, multiple reaction monitoring conditions, and analytical method validation results presented in this study can serve as a basis for expanding research on substances with similar core structures, which is expected to strengthen regulatory responses to evolving drug trends.

While oral fluid (OF) is increasingly used in forensic and roadside toxicology, measured drug concentrations remain highly dependent on pre-analytical conditions. This study investigated the impact of buccal-cell content and sample fractionation on concentrations of delta 9-tetrahydrocannabinol (THC), cocaine, benzoylecgonine (BZE), and ecgonine methyl ester (EME). Oral-fluid specimens were collected using FLOQSwabs®. Following elution, sonication, and vortex mixing, various fractions (homogenate, pellet, and expressed swab) were analyzed via liquid chromatography-tandem mass spectrometry. For THC (n = 102), median buccal epithelial cell counts and THC concentrations were significantly higher in the cell-rich pellet than in the homogenate (P < .001), with median enrichment ratios of 4.40 and 3.93, respectively. The association between cellular and THC enrichment was strong to very strong (r = 0.77-0.88) for concentrations above 5 ng/mL. Notably, four specimens were below the limit of detection in the homogenate but detectable in the pellet. In contrast, cocaine and its metabolites showed comparable concentrations between the homogenate and pellet, though the expressed swab fraction consistently yielded the highest levels. In 52 specimens, median expressed swab-to-homogenate ratios were 1.16 for cocaine, 1.28 for BZE, and 1.14 for EME. These findings demonstrate that pre-analytical effects are analyte-dependent: buccal cells act as a reservoir for THC, whereas cocaine-related analytes are primarily affected by incomplete release from the swab. These results identify analyte-dependent pre-analytical sources of variability that may affect oral-fluid concentration measurements obtained with this collection device.

Recent studies have demonstrated that dermal exposure to anabolic androgenic steroids (AAS) can result in adverse analytical findings (AAFs) in doping controls with a comparably long detectability of the parent compound and/or their metabolites in urine samples. Transdermal absorption is influenced by multiple factors, including physicochemical properties of the substance and solubility; but is largely driven by the degree of exposure, i.e. amount of substance applied. Therefore, this study aimed to investigate the detectability and urinary excretion profiles of trenbolone (TREN) and metenolone (MET) after dermal exposure to microgram quantities of these frequently abused AAS. Controlled excretion studies were conducted with healthy male volunteers, and each participant received a single dose of 50 µg TREN or MET via dermal application to the forearm. Urine samples were collected and analyzed for the parent compounds and their metabolites by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-HRMS/MS). To enhance sensitivity and product ion formation, derivatization with Girard's reagent T was employed for MET and its metabolites. Following administration of transdermal microdoses, both substances and/or their metabolites were detectable in the urine samples of all participants. For TREN, predominantly the established main metabolite epitrenbolone was identified, with maximum concentrations up to 2.6 ng/ml and a detection window of up to 3 days post-application. Maximum concentrations of MET ranged from 0.1 to 1.2 ng/ml. Notably, particularly the sulfoconjugated metabolite of MET remained detectable for over a week in one volunteer. Metabolite profiles were compared with those of urine samples obtained after oral administration of the steroids to assess potential differences. Considering the increasing number of alleged transdermal contamination cases, the results provide important analytical data to better assess the plausibility of such claims.

Xylazine, a popular compound found independently and in polydrug mixtures throughout the United States, is intended for use in veterinary medicine and not for human consumption. When xylazine is ingested by humans, life-threatening central nervous system depression responses could potentially occur. With its increase in prevalence, several methods of detection were developed within forensic toxicology. Since xylazine is not approved for human clinical use, little is known about its behavior, such as its stability in stored biological matrices. Knowledge of drug stability in biological matrices can provide critical insight on the limitations surrounding specimen storage conditions, which is of great importance in forensic toxicology. This study assessed the stability of xylazine in preserved antemortem and postmortem blood under three storage conditions (23 °C, 2-8 °C, and -20 °C) at two concentrations (3 ng/mL and 160 ng/mL). Once prepared, samples were extracted using a validated protein precipitation procedure and analyzed by liquid chromatography-tandem mass spectrometry. Xylazine was considered stable if the deviation in calculated concentration was within ±20% of the Day 0 concentration. When the results of each individual pool prepared were averaged for reporting, the following results were obtained. When stored at 23 °C in both matrices, xylazine was unstable by Day 7 at 3 ng/mL and unstable by Day 10 at 160 ng/mL. When stored between 2-8 °C in antemortem blood, xylazine was unstable by Day 10 at 3 ng/mL and unstable by Day 24 at 160 ng/mL. When stored between 2-8 °C in postmortem blood, xylazine was unstable by Day 29 at both 3 and 160 ng/mL. When stored at -20 °C in antemortem blood, xylazine was unstable by Day 3 at both 3 ng/mL and 160 ng/mL. When stored at -20 °C in postmortem blood, xylazine was unstable by Day 21 at 3 ng/mL and unstable by Day 10 at 160 ng/mL.

The authors present two cases of sexual assaults where the abuse of anabolic steroids by the perpetrator has been documented. The first perpetrator confessed the use of anabolic steroids for 3 years. He indicated that the abuse of steroids strongly enhanced his libido. In a second case, a specialist of knockout sport assaulted five women and raped another. He claimed his libido was modified due to long-term steroid abuse. Because both subjects were bald, pubic hair, and beard hair were collected, respectively. Anabolic steroids were tested in body hair by chromatography coupled to tandem mass spectrometry. The following steroids were identified and measured in Case 1: testosterone (0.9 pg/mg), testosterone enanthate (102 pg/mg), trenbolone (9 pg/mg), trenbolone enanthate (0.8 pg/mg), and metandienone (4 pg/mg). In Case 2, the following steroids were identified and measured: testosterone (14 pg/mg), testosterone enanthate (48 pg/mg), trenbolone (0.5 pg/mg), and boldenone (0.3 pg/mg). Although literature data are inconclusive concerning the effects of anabolic steroids on libido, the body hair test results support the claim of both subjects about their use. This test should be considered as a piece of evidence to resolve forensic cases where steroids are mentioned.

This report describes an adolescent with severe venlafaxine intoxication in whom serial therapeutic drug monitoring (TDM) was performed, enabling detailed toxicokinetic characterization throughout the clinical course. The initial plasma concentration of the active venlafaxine moiety was 7800 ng/mL, nearly 20 times the upper therapeutic reference limit. On admission, the patient presented with tachycardia, bilateral mydriasis, and generalized tonic-clonic seizures, which are typical clinical manifestations of severe venlafaxine intoxication. Due to progressive impairment of consciousness and increasing oxygen requirements, the patient underwent orotracheal intubation and remained on mechanical ventilation for nearly 120 hours. Serial measurements of venlafaxine and its active metabolite, O-desmethylvenlafaxine (ODV), provided valuable information on the toxicokinetic progression and supported clinical decision-making regarding the timing of extubation. Despite the extremely elevated venlafaxine concentration, cardiotoxicity was limited to transient QTc prolongation without ventricular arrhythmias and moderate elevations of high-sensitivity troponin I (hs-troponin I) and N-terminal pro B-type natriuretic peptide (NT-proBNP). Marked elevations of creatine kinase and myoglobin concentrations were most likely attributable to rhabdomyolysis following general tonic-clonic seizures. TDM demonstrated a progressive increase in the ODV/venlafaxine metabolite-to-parent ratio from 0.30 to 4.50, reflecting the transition from the acute intoxication phase to the elimination phase. The apparent elimination half-life was prolonged to 14.4 hours for venlafaxine and 17.7 hours for ODV. Genotyping revealed the COMT rs4680 Met/Met genotype, which may theoretically influence catecholamine metabolism; however, its contribution to the clinical presentation remains uncertain.

Synthetic cathinones represent an expanding class of novel psychoactive substances (NPS), with their proliferation largely driven by international drug control measures and the rapid cycling of structural analogues designed to circumvent legislative scheduling. Following the international control of N, N-dimethylpentylone (DMP) in 2024, N-isopropyl butylone (NiPB) was increasingly detected in forensic toxicological casework as the prevalence of DMP declined. However, limited data currently exist regarding NiPB's concentration ranges in forensic investigations. This report describes a validated liquid chromatography-tandem quadrupole mass spectrometry (LC-QQQ-MS) method for the quantitation of NiPB in biological specimens, which was applied to the analysis of drugged driving (n = 1) and postmortem (n = 15) cases. The driving under the influence of drugs case yielded a NiPB blood concentration of 16 ng/mL, whereas the remaining 15 cases demonstrated blood concentrations ranging from <1 to 1400 ng/mL. For one case, NiPB was quantitated in blood, vitreous fluid, and urine, with concentrations of 18, 17, and 2200 ng/mL, respectively. Polydrug use was observed for all cases and frequently involved fentanyl (75%), cocaine and/or methamphetamine (69%), other synthetic cathinones (25%), ethanol (31%), and additional NPS such as bromazolam (19%) and MDMB-4en-PINACA (13%). Concentrations overlapped between antemortem and postmortem cases and were comparable to those reported for structurally related beta-keto methylenedioxyamphetamines, suggesting that members of this analogue class may share similar patterns of use and toxicological profiles. Although NiPB was identified as a contributing factor in several fatalities, the lack of pharmacological and toxicological data limits further interpretation of measured concentrations and the extent of its contribution to these fatal outcomes. Additional studies incorporating paired central and peripheral blood specimens are warranted to better characterize potential postmortem redistribution and its impact on NiPB blood concentration interpretation. Overall, this case series addresses a critical literature gap on NiPB by providing analytical and interpretive data to support forensic laboratories as NiPB identifications increase within the evolving NPS stimulant market.

The validation of a semi-quantitative method for the rapid screening of whole blood and urine specimens using the Neogen® Gabapentin kit is presented. Decision points were validated at 1000 ng/mL for whole blood and urine. The validation design was based on ANSI/ASB 036 and internal laboratory requirements. Experiments included the evaluation of sensitivity, precision, specificity, carryover, hook effect, drift, ruggedness/robustness, interference, and an authentic case sample comparison. The experimental limit of detection (LOD) for gabapentin was determined to be at least 500 ng/mL in whole blood and 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. The assay was reliably able to detect gabapentin without interference from matrix components or other substances routinely detected in authentic case samples with the exception of levamisole in urine if present at unusually high concentrations. Urine samples with 50 µg/mL or more of levamisole yielded a false positive screening result. Other case sample results were comparable to those obtained with liquid chromatography tandem high-resolution mass spectrometry or liquid chromatography tandem mass spectrometry for blood and urine, respectively. The Neogen® Gabapentin kit exhibited minimal cross-reactivity for pregabalin and demonstrated excellent precision and appropriate sensitivity in both whole blood and urine, making it an efficient and reliable method to screen for gabapentin.

Carbon monoxide (CO) is a gas that is readily absorbed by the lungs when inhaled. Once in the blood, it forms a complex with hemoglobin (Hb) in red blood cells known as carboxyhemoglobin (COHb), reducing the oxygen-carrying capacity of Hb and therefore disrupting the transport and delivery of oxygen throughout the body. The resulting lack of oxygen can be harmful and even lethal, making it important to be able to detect COHb in postmortem samples of relevant cases for determining a cause of death. Blood and spleen are the preferred specimens for the detection of CO because they are Hb rich, but these specimens may not always be available for collection during autopsy. This study aims to determine if liver tissue can be a reliable source for the detection and/or quantification of CO in postmortem specimens in the absence of blood or spleen. Liver homogenates and blood specimens from 25 postmortem cases with a positive CO result in blood (%COHb ≥10%) and 10 postmortem cases testing negative for CO in blood (%COHb <10%) were analyzed for COHb via headspace gas chromatography/mass spectrometry (HS-GC/MS) and compared. Cases for which the blood produced a positive result for CO did not always result in a positive finding in the liver (%COHb ≥10%), even in cases with high levels of %COHb saturation (>50%). However, all cases that had a liver result ≥10% COHb had a corresponding blood result ≥10% COHb. If blood or spleen is not available for CO testing, liver may be tested as a potential indicator of CO exposure. A positive %COHb finding in the liver is a reliable indicator of a positive %COHb result in blood, but negative results in liver are inconclusive and cannot rule out CO exposure.