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Cyanide (in the form of cyanide anion (CN − ) or hydrogen cyanide (HCN), inclusively represented as CN) can be a rapidly acting and deadly poison, but it is also a common chemical component of a variety of natural and anthropogenic substances. The main mechanism of acute CN toxicity is based on blocking terminal electron transfer by inhibiting cytochrome c oxidase, resulting in cellular hypoxia, cytotoxic anoxia, and potential death. Due to the well-established link between blood CN concentrations and the manifestation of symptoms, the determination of blood concentration of CN, along with the major metabolite, thiocyanate (SCN – ), is critical. Because currently there is no method of analysis available for the simultaneous detection of CN and SCN – from blood, a sensitive method for the simultaneous analysis of CN and SCN – from human ante- and postmortem blood via liquid chromatography–tandem MS analysis was developed. For this method, sample preparation for CN involved active microdiffusion with subsequent chemical modification using naphthalene-2,3-dicarboxaldehyde (NDA) and taurine (i.e., the capture solution). Preparation for SCN – was accomplished via protein precipitation and monobromobimane (MBB) modification. The method produced good sensitivity for CN with antemortem limit of detection (LODs) of 219 nM and 605 nM for CN and SCN – , respectively, and postmortem LODs of 352 nM and 509 nM. The dynamic ranges of the method were 5–500 µM and 10–500 µM in ante- and postmortem blood, respectively. In addition, the method produced good accuracy (100 ± 15%) and precision (≤ 15.2% relative standard deviation). The method was able to detect elevated levels of CN and SCN – in both antemortem ( N  = 5) and postmortem ( N  = 4) blood samples from CN-exposed swine compared to nonexposed swine.

[Display omitted] •This study presents a method to quantify antidepressants in postmortem blood.•Applied an optimization of QuEChERS method using design of experiments (DOE).•The complete method was validated and successful applied in postmortem real samples. In this study, a comprehensively optimization of QuEChERS (quick, easy, cheap, effective, rugged and safe) method using design of experiments (DOE) was conducted to evaluate the best conditions to obtain the most effective extraction. Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) analysis was performed to identify and quantify the antidepressants, with electrospray ionization acquired in positive mode. The method was validated for all analytes; the calibration curves were linear from 10–1000ng/mL, with R2>0.98, and with LOD and LOQ defined as 10ng/mL. Method imprecision and bias were less than 14.3% and 18.9%, respectively. Neither carryover nor interferences were observed. Overall, the optimized method was applied in postmortem real sample analysis to quantify the antidepressants. This study showed a viable method that can be applied for routine forensic analysis, with a quick and easy sample preparation and a rapid total run time of 8min for each analysis.

Cocaine and antidepressants rank high globally in substance consumption, emphasizing their impact on public health. The determination of these compounds and related substances in biological samples is crucial for forensic toxicology. This study focused on developing an innovative analytical method for the determination of cocaine, antidepressants, and their related metabolites in postmortem blood samples, using unmodified commercial Fe3O4 nanoparticles as a sorbent for dispersive magnetic solid-phase extraction (m-d-SPE), coupled with liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) analysis. An aliquot of 100 µL of whole blood and 5 µL of the internal standard pool were added to 30 mg of nanoparticles. The nanoparticles were separated from the sample using a neodymium magnet inserted into a 3D-printed microtube rack. The liquid was then discarded, followed by desorption with 300 µL of 1/1/1 acetonitrile/methanol/ethyl acetate. The sample was vortexed and separated, and 1.5 µL of the organic supernatant was injected into the LC–MS/MS. The method was acceptably validated and successfully applied to 263 postmortem blood samples. All samples evaluated in this study were positive for at least one substance. The most frequent analyte was benzoylecgonine, followed by cocaine and cocaethylene. The most common antidepressants encountered in the analyzed samples were citalopram and fluoxetine, followed by fluoxetine’s metabolite norfluoxetine. This study describes the first report of this sorbent in postmortem blood analysis, demonstrating satisfactory results for linearity, precision, accuracy, and selectivity for all compounds. The method’s applicability was confirmed, establishing it as an efficient and sustainable alternative to traditional techniques for forensic casework.

A simple and sensitive analytical method was developed for qualitative and quantitative analysis of Δ9-tetrahydrocannabinol (Δ9-THC) and its metabolite 11-nor-Δ9-tetrahydrocannabinol-carboxylic acid (Δ9-THC-COOH) in human postmortem blood using gas chromatography/mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode. The method involved a liquid-liquid extraction in two steps, one for Δ9-THC and a second one for Δ9-THC-COOH. The first extract was analyzed using Δ9-THC-D3 as internal standard. The second extract was derivatized and analyzed using Δ9-THC-COOH-D3 as internal standard. The method was shown to be very simple, rapid, and sensitive. The method was validated for the two compounds, including linearity (range 0.05–1.5 µg/mL for Δ9-THC and 0.08–1.5 µg/mL for Δ9-THC-COOH), and the main precision parameters. It was linear for both analytes, with quadratic regression of calibration curves always higher than 0.99. The coefficients of variation were less than 15%. Extraction recoveries were superior to 80% for both compounds. The developed method was used to analyze 41 real plasma samples obtained from the Forensic Toxicology Service of the Institute of Forensic Sciences of Santiago de Compostela (Spain) from cases in which the use of cannabis was involved, demonstrating the usefulness of the proposed method.

•Data independent screening method for fentanyl analogs in blood and oral fluid.•Limits of detection 0.1–0.25 ng/mL in blood and oral fluid, respectively.•Screening results of authentic blood and oral fluid samples presented. Recently, fentanyl analogs account for significant number of opioid deaths in the United States. Routine forensic analyses are often unable to detect and differentiate these analogs due to low concentrations and presence of structural isomers. A data-independent screening method for 14 fentanyl analogs in whole blood and oral fluid was developed and validated using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Data were acquired using Time of Flight (TOF) and All Ions Fragmentation (AIF) modes. The limits of detection (LOD) in blood were 0.1–1.0 ng/mL and 0.1–1.0 ng/mL in TOF and AIF modes, respectively. In oral fluid, the LODs were 0.25 ng/mL and 0.25–2.5 ng/mL in TOF and AIF modes, respectively. Matrix effects in blood were acceptable for most analytes (1–14.4%), while the nor-metabolites exhibited ion suppression >25%. Matrix effects in oral fluid were −11.7 to 13.3%. Stability was assessed after 24 h in the autosampler (4 °C) and refrigerator (4 °C). Processed blood and oral fluid samples were considered stable with −14.6 to 4.6% and −10.1 to 2.3% bias, respectively. For refrigerated stability, bias was −23.3 to 8.2% (blood) and −20.1 to 20.0% (oral fluid). Remifentanil exhibited >20% loss in both matrices. For proof of applicability, postmortem blood (n = 30) and oral fluid samples (n = 20) were analyzed. As a result, six fentanyl analogs were detected in the blood samples with furanyl fentanyl and 4-ANPP being the most prevalent. No fentanyl analogs were detected in the oral fluid samples. This study presents a validated screening technique for fentanyl analogs in whole blood and oral fluid using LC-QTOF-MS with low limits of detection.

•A GC–MS method was optimized for 14 chemicals in human postmortem blood.•Samples were extracted with d-SPE, LOQs were 0.02 or 0.03μg/mL.•Ten forensic samples were analyzed, and 6 contained cocaine (0.06–3.1μg/mL).•Carbofuran was found at the highest concentration (27.3μg/mL). A d-SPE protocol followed by gas chromatography–mass spectrometry (GC–MS) analysis using large volume injection–programmed temperature vaporization (LVI–PTV) was optimized for simultaneous quantification of 14 pesticides, drugs of abuse, prescription drugs and metabolites in human postmortem blood without derivatization. The validated method showed good repeatability, linearity, intermediate precision, and recovery. LOQs were 0.02 or 0.03μg/mL. The method showed to be fast and easy-to-implement in a forensic laboratory and was satisfactorily applied for the analysis of 10 postmortem blood real samples. Six samples contained cocaine (0.04–3.13μg/mL), two 3,4-methylenedioxymethamphetamine hydrochloride (MDMA, 0.04–0.09μg/mL) and two carbamazepine (0.08–0.98μg/mL). Other analytes found were carbofuran (27.3μg/mL), the metabolite 7-aminoflunitrazepam (1.12μg/mL), amitriptyline (0.21μg/mL) and diazepam (0.03μg/mL).

At crime scenes, apart from the detection of blood, it may be important to determine whether a person was alive at the time of blood deposition. Based on the rapid onset of fibrinolysis after death, this pathway could be considered to identify potential biomarkers for postmortem blood. Fibrinolysis is the natural process that breaks down blood clots after healing a vascular injury. One of its products, D-dimer, could be a potential biomarker for postmortem blood. SERATEC® (SERATEC® GmbH, Göttingen, Germany) has developed the PMB immunochromatographic assay to simultaneously detect human hemoglobin and D-dimer. The main goals of this study were to assess the possibility of using this test to detect postmortem blood, evaluate D-dimer levels in antemortem, menstrual, and postmortem blood, and assess the ability to obtain STR profiles from postmortem blood. Except for one degraded sample, all postmortem blood samples reacted positively for the presence of D-dimer using the SERATEC® PMB test. All antemortem blood samples from living individuals showed negative results for D-dimer detection, except for one liquid sample with a weak positive result, probably due to pre-existing health conditions. Menstrual blood samples gave variable results for D-dimer. The DIMERTEST® Latex assay was used for semi-quantitative measurement of D-dimer concentrations, with postmortem and menstrual blood yielding higher D-dimer concentrations compared to antemortem blood. Full STR profiles were developed for all postmortem samples tested except for one degraded sample, pointing to the possibility of not only detecting postmortem blood at the crime scene but also the potential identification of the victim.

Precision (or imprecision) is one of the central performance parameters of all analytical methods. It is often evaluated during method validation in spiked samples, with a relatively low number of measurements typically during a short period. Validation results are well documented in the literature; however, evaluation of imprecision in authentic cases compared with long-term imprecision from quality control samples has not often been reported on. The aim of this study was to investigate imprecision estimated from duplicate measurements of ante- and postmortem blood samples and long-term imprecision estimates from quality control samples and to compare variations between them. Data were analyzed by using robust statistics, where results for the 29 analytes most frequently quantified by liquid chromatography–tandem mass spectrometry in ante- and postmortem blood samples were included. A total of 41,460 positive findings in authentic whole blood and 51,522 measurements from quality controls were investigated. Analysis was performed in duplicate on independent batches on two separate days. Overall, the imprecision estimated in quality control and the authentic samples were akin for most analytes. Ante- and postmortem blood samples showed similar imprecision for the majority of the analytes and were approximately the same level as long-term imprecision estimated from the quality control samples at low level, whereas relative imprecision of the quality control samples at high level were slightly lower than ante- and postmortem blood samples. The methods we evaluated showed satisfactory reproducibility and robustness for the investigated analytes. •Examination of the 29 most frequently quantified analytes in authentic samples.•Imprecision estimated using median absolute deviation of the median.•Independent duplicate measurements of antemortem and postmortem blood samples.•Long term measurements of quality control samples.•Satisfactory reproducibility and robustness of multi-analyte methods.

•5F-ADB is regarded as one of the most dangerous synthetic cannabinoids.•5F-ADB is currently the most frequently consumed SC in Turkey.•Seventy 5F-ADB intoxication case reports are presented.•5F-ADB and its metabolite are detected and quantified in blood and urine.•Bucket method is used for inhalation in Turkey. 5F-ADB (methyl 2-{[1-(5-fluoropentyl)-1H-indazole-3-carbonyl]amino}-3,3-dimethylbutanoate) is a frequently abused new synthetic cannabinoid that has been sold since at least the end of 2014 on the drug market. It has been classified as an illicit drug in most European countries, and also in Turkey, Japan, and the United States. In this study, 5F-ADB and its methyl ester metabolite were determined in the blood and urine samples taken from fatal cases using liquid chromatography–highresolution mass spectrometry (LC–HRMS). The extraction of samples was performed using a solid-phase extraction method, followed by LC–HRMS analysis. The method was fully validated for linearities, limits of detection (LODs), limits of quantification (LOQs), recoveries, matrix effects, process efficiencies, accuracies, precisions, and stabilities and was applied to 70 blood and 36 urine samples from fatal cases where 5F-ADB was the only drug detected. The LODs were between 0.08 and 0.10ng/mL, and LOQs were between 0.10 and 0.12ng/mL for both blood and urine samples. 5F-ADB and its methyl ester hydrolysis metabolite were found at the blood concentrations ranging from 0.10 to 1.55ng/mL (mean=0.40ng/mL) and 0.15 to 23.4ng/mL (mean=2.69ng/mL), respectively. 5F-ADB was not detected in any urine samples. 5F-ADBmethyl ester hydrolysis metabolite was detected in 35 urine samples with a detection range of 0.28–72.2ng/mL and a mean of 9.02ng/mL. The synthetic cannabinoid 5F-ADB and its methyl ester metabolite were identified and quantified in authentic human blood and/or urine specimens obtained from 70 fatal cases. The method was successfully applied to postmortem blood and urine samples.

A quite intriguing subject being intensively researched in the forensic toxicology field is the source of postmortem determined blood ethanol concentration: antemortem ingestion or postmortem microbial production. Our previous research on microbial ethanol production has reported a quantitative relationship between the ethanol and the higher alcohols and 1-butanol produced by Escherichia coli, Clostridium perfrigens, and Clostridium sporogenes. In this contribution, we continue our research reporting on the following: (i) the patterns of ethanol, higher alcohols, and 1-butanol production by the microbes Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis (all being aerobic/facultative anaerobic species, common corpse’s colonizers, and ethanol producers), under controlled laboratory conditions, (ii) the mathematical modeling, with simple mathematical equations, of the correlation between ethanol concentration and the other studied alcohols’ concentrations, by performing multiple linear regression analysis of the results, and (iii) the applicability of the constructed models in microbial cultures developed under different temperature than that used to build the models, in denatured blood cultures and in real postmortem cases. The aforementioned alcohols were proved to be all indicators of ethanol production, both in qualitative and quantitative terms. 1-Propanol was the most significant alcohol in modeling microbial ethanol production, followed by methyl-butanol. The K. pneumoniae’s models achieved the best scoring in applicability (E < 40%) compared to the S. aureus and E. faecalis models, both at laboratory microbial cultures at 37 °C and real postmortem cases. Overall, a noteworthy accuracy in estimating the microbial ethanol in cultures and autopsy blood is achieved by the employed simple linear models.