Forensic Toxicology Investigations Research Articles

Development of an LC-MS/MS method for the determination of five psychoactive drugs in postmortem urine by optimization of enzymatic hydrolysis of glucuronide conjugates.

Toxicological analyses of biological samples play important roles in forensic and clinical investigations. Ingested drugs are excreted in urine as conjugates with endogenous substances such as glucuronic acid; hydrolyzing these conjugates improves the determination of target drugs by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we sought to improve the enzymatic hydrolysis of glucuronide conjugates of five psychoactive drugs (11-nor-9-carboxy-Δ9-tetrahydrocannabinol, oxazepam, lorazepam, temazepam, and amitriptyline). The efficiency of enzymatic hydrolysis of glucuronide conjugates in urine was optimized by varying temperature, enzyme volume, and reaction time. The hydrolysis was performed directly on extraction columns. This analysis method using LC-MS/MS was applied to forensic autopsy samples after thorough validation. We found that the recombinant β-glucuronidase B-One® quantitatively hydrolyzed these conjugates within 3min at room temperature directly on extraction columns. This on-column method saved time and eliminated the loss of valuable samples during transfer to the extraction column. LC-MS/MS-based calibration curves processed with this method showed good linearity, with r2 values exceeding 0.998. The intra- and inter-day accuracies and precisions of the method were 93.0-109.7% and 0.8-8.8%, respectively. The recovery efficiencies were in the range of 56.1-104.5%. Matrix effects were between 78.9 and 126.9%. We have established an LC-MS/MS method for five psychoactive drugs in urine after enzymatic hydrolysis of glucuronide conjugates directly on extraction columns. The method was successfully applied to forensic autopsy samples. The established method will have broad applications, including forensic and clinical toxicological investigations.

An LC-MS/MS method for the determination of drugs of abuse included THC-COOH, EtG, and NPS, using a single hair extraction sample.

Hair analysis plays an important role in the determination of drugs of abuse in both forensic and clinical toxicology investigations. The analysis of different substances often requires the use of different sample preparation methods, thereby increasing the amount of hair sample and time required. In the present study, a fast method involving a combination of a single 25 mg hair extraction procedure and four liquid chromatography-tandem mass spectrometry methods using the same chromatographic phases and column was developed and validated. The target was the identification and quantification of various commonly abused drugs and their metabolites, including amphetamines, cocaine, opioids, cannabinoids, THC-COOH and EtG, and more than 140 new psychoactive substances, including synthetic cannabinoids, phenethylamines, synthetic opioids, methylphenidate, cathinone, piperidine, and tryptamines.

Visceral Microscopic Pattern From Suicidal Ingestion of Professional Lysoform® With Delayed Death.

Lysoform® in the formulation of professional detergent is widely used in several fields, whereas its suicidal ingestion is an unusual occurrence. Therefore, the biological signs of this fatal poisoning remain unclear and elusive, similarly to the histological lesions induced by its main constituent, which is benzalkonium chloride (BZK). Furthermore, since all the deaths that has been reported in the literature occurred immediately, microscopic pictures of BZK lethal toxicity in subjects with prolonged survival have never been reported to date. Specifically, this brief communication reports the unique case of a woman who ingested professional Lysoform® to commit suicide, for which she died two weeks later. The autopsy examination showed either local or systemic signs of caustic ingestion; moreover, the histological analysis showed clear cellular damage of lungs, heart, and kidneys. In our case, toxicological investigations were not authorized as they were no longer considered significant. In this framework, the histological examination has therefore assumed a fundamental role in investigating and demonstrating the lethal effects caused by the systemic dissemination of BZK, which would otherwise no longer be investigable. Therefore, in cases of substance intoxication with prolonged survival where forensic toxicological investigations may be no longer possible or feasible, the histological examination may be the only resource to successfully observe and demonstrate its lethal effects.

Simultaneous quantification of 106 drugs or their metabolites in nail samples by UPLC-MS/MS with high-throughput sample preparation: Application to 294 real cases

The nail is an alternative matrix to complement hair analysis in proving drug intake over several months in forensic toxicology investigations. However, because of the high hardness and toughness of nails, the existing pretreatment procedures for nails have the disadvantages of either a high degree of time consumption (from hours to days), or low extraction recoveries. This study aims to propose a high-throughput nail sample preparation method and provide a quantitative analytical method for 106 drugs and their metabolites present in nail. We developed cryogenic grinding, coupled with high-speed grinding in the extraction solvent method, which could improve the extraction recovery by thoroughly destroying the nail keratin for approximately 18 min. Subsequently, an ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed for the identification and quantification of 34 synthetic cannabinoids, 26 fentanyls, 18 synthetic cathinones, 10 phenylethylamines, eight opioids, three phencyclidine, two tryptamines, two piperazine, cocaine, benzoylecgonine, and tetrahydrocannabinol (THC). Nail samples were collected from people with a history of drug abuse from five different regions of China. The analysis of 294 authentic samples resulted in 213 detected samples, and showed a broad concentration range including 5.04–67.26 pg/mg for nine synthetic cannabinoids, 109.29–250.29 pg/mg for a synthetic cathinone, 5.06–434291 pg/mg for four phenylethylamines, 5.06–464278 pg/mg for three phencyclidine, 5.50–192195 pg/mg for six opioids, 19.44–36.11 pg/mg for cocaine, and 50.53 pg/mg for THC in nail. Furthermore, up to 10 different compounds were detected in a single nail sample. This nail analysis method serves as a useful tool for the large-scale surveillance of illicit drugs abuse.

[Analysis of the feasibility of a forensic chemical investigation in clobazam poisoning].

The study objective was to determine the relevance of methods to detect clobazam poisoning during the forensic and chemical toxicological examination. The lack of a systematic approach to clobazam identification and assay as a part of forensic chemical and chemical toxicological analyses was demonstrated. The published data on the study of clobazam in biological objects are inconsistent and incomplete, precluding the conduction of focused forensic chemical and toxicological investigations on biological objects with required validity. Various methods of forensic chemical and toxicological investigation of clobazam in biological objects were studied and presented. Data are presented on various physicochemical test methods (TLC, HPLC, UV spectrometry, GC, and GC-MS) for clobazam detection.

Novel extraction method using an ISOLUTE PLD+ protein and phospholipid removal column for liquid chromatography-tandem mass spectrometry analysis of 20 psychoactive drugs in postmortem whole blood samples

A novel sample extraction method using an ISOLUTE PLD+ protein and phospholipid removal column was developed for simultaneous quantification of 20 psychoactive drugs, including antidepressants, antipsychotics, sedative-hypnotics, and amphetamines, in postmortem whole blood samples by liquid chromatography-tandem mass spectrometry. The method showed improvement in extract cleanliness compared with traditional protein precipitation and the QuEChERS extraction method. The method was validated for all analytes; the calibration curves showed good linearity, with r2 values exceeding 0.991. The intra- and interday accuracies and precisions were 87.6–117.5% and 1.0–18.6%, respectively. The recovery efficiencies were in the range of 64.6–96.8%. Matrix effects were observed in the range of 82.6–116.0%. All analytes were stable under different storage conditions. This method was successfully applied in postmortem forensic sample analysis to quantify psychoactive drugs. The method described in the current study will be useful for forensic toxicological investigations.

UPLC-MS/MS method for the simultaneous quantification of caffeine and illicit psychoactive drugs in hair using a single-step high-speed grinding extraction – Insights into a cut-off value for caffeine abuse

Caffeine is a commonly consumed psychoactive substance whose addictive potential has long been reported. Excessive caffeine intake may lead to severe health damage or drug addiction problems; however, studies on the surveillance of caffeine abuse by the Chinese population are lacking. This study aimed to propose a concentration value for caffeine based on hair analysis to distinguish excessive intake from normal consumption, and provide an analytical tool for forensic toxicology investigations of caffeine and other frequently abused drugs. A sensitive and accurate ultra-high performance liquid chromatography-tandem mass spectrometry method was developed to detect caffeine and 13 illicit drugs and their metabolites in hair. Thereafter, this method was employed to test 479 real samples. Briefly, the hair samples were washed with water and acetone, and subsequently extracted by one-step high-speed grinding with acetonitrile-buffer solution. The lower limit of quantifications of 0.05 ng/mg for caffeine and THC, and 0.005 ng/mg for others, were achieved for all substances. The results revealed a mean caffeine concentration of 0.78 (range 0.008–3.5 ng/mg) based on 24 healthy volunteers, 55.0 (range 3.07–292.2 ng/mg) based on 52 self-reported caffeine abuse participants, and 5.78 (range 0–140.34 ng/mg) based on 403 drug addicts. The mean caffeine concentration in hair from self-reported caffeine abusers was 70-fold higher than that in hair from healthy volunteers. A tentative cut-off level of 5.5 ng/mg as an indicator of excessive caffeine consumption was developed based on receiver operating characteristic analysis. Additionally, the assessment of 403 hair samples from drug addicts indicated that illicit drug abusers had potential for caffeine abuse, especially polydrug users. This hair analysis method serves as a useful tool for the large-scale surveillance of caffeine and illicit drug abuse.

The use of fly artifacts in a crime scene: Is there any application for forensic toxicology?

Fly artifacts (FA) are bloodstains resulting from insect activity at a crime scene, usually by feeding on human blood. Whether these artifactual stains might be useful for forensic toxicological investigations in cases of absence of conventional and unconventional matrices, for example, in cases concealment of the body or of extensive putrefaction, has not yet been investigated. The purpose of this study is to understand if FA trace evidence permits toxicological analysis when traditional matrices are not available. To this aim, FA experimentally produced by Calliphora vomitoria feeding on human blood of a cocaine and heroin user were collected from absorptive and non-absorptive material. FA material was analyzed by a new simple and fast LC-MS/MS method. Results were evaluated in terms of presence of the drug and relative amount of the detected molecules. From a qualitative point of view, the analysis of FA revealed all the substances originally detected in post-mortem blood in both cases. The ratios of cocaine/benzoylecgonine, codeine/morphine, and 6-monoacetylmorphine/morphine recovered in FA from cotton-textile materials and from non-absorptive surfaces were consistent with data resulted from original post-mortem blood. The preliminary study herein reported demonstrated that FA are extremely informative in case of cocaine and heroin users and merit further research in order to be applied in real caseworks.

Application of Lithium Assay kit LS for quantification of lithium in whole blood and urine.

A commercially available kit for the quantitation of lithium, the Lithium Assay kit LS, was originally developed to measure lithium in serum or plasma using a conventional microplate reader. We investigated whether use of the kit could be extended to quantify lithium in whole blood and urine samples collected at autopsy. The calibration curve for whole blood showed good linearity ranging from 0.5 to 20µg/mL with a coefficient of determination of 0.998 when samples were pretreated with methanol followed by acetonitrile. Moreover, for urine, we obtained excellent linearity with a coefficient of determination of 0.999 without any pretreatment. The accuracies and precisions were 106.3-174.7% and 1.9-18.1% for whole blood and 83.3-118.8% and 5.7-33.8% for urine. The values in the lower concentration range (0.5-1µg/mL) were not satisfactory, whereas those in the higher range (2-20µg/mL) were acceptable. The Lithium Assay kit LS was successfully applied to the measurement of lithium in whole blood and urine samples collected at autopsies. This method appears to be useful for forensic toxicological investigations because of its simplicity and speed.

LC-MS/MS method for rapid and accurate detection of caffeine in a suspected overdose case

Excessive intake of caffeine, otherwise known to be a safe and mild central nervous system stimulant, causes nausea, vomiting, convulsions, tachycardia, and eventually fatal arrhythmias and death. Caffeine intoxication, a global problem, has been increasing in Japan since 2013. Thus, there is a need for rapid and accurate diagnosis of caffeine poisoning in forensic and clinical toxicology investigations. Herein, we demonstrate rapid and accurate caffeine quantitation by liquid chromatography tandem mass spectrometry using the standard addition method in a fatal case. Biological samples were diluted 500–100,000-fold and subjected to a simple pretreatment (adding caffeine standard and internal standard and passing through a lipid removal cartridge). The multiple reaction monitoring transitions were 195 → 138 for quantitation, 195 → 110 for the qualifier ion, and 204 → 144 for the internal standard (caffeine-d9). The standard plots were linear over 0–900 ng/mL (r2 = 0.9994–0.9999) for biological samples, and the reproducibility (%RSD) of the method was 1.53–6.97% (intraday) and 1.59–10.4% (interday). Fatal levels of caffeine (332 μg/mL) and toxic to fatal levels of olanzapine (625 ng/mL), along with other pharmaceuticals were detected in the external iliac venous blood. The cause of death was determined to be multi-drug poisoning, predominantly caused by caffeine. Our method is useful for not only forensic cases but also the rapid diagnosis of caffeine overdose in emergency clinical settings.

Determination of Illicit Drugs in Dried Blood Spots by LC–MS/MS Method: Validation and Application to Real Samples

Drugs of abuse can be investigated in dried blood spot (DBS) samples to evaluate the intake of psychoactive drugs. We have developed the validated liquid chromatography–tandem mass spectrometry method to determine drug of abuse in whole blood with DBS. The mass spectrometer was performed using multiple reaction monitoring via positive electro-spray ionization. The analysis procedure was applied to real positive blood samples of the 59 cases brought to Forensic Toxicology Laboratory in Faculty of Medicine of Cukurova University by law enforcement agencies. The method based on liquid–liquid extraction and liquid chromatography tandem mass spectrometry was fully validated according to international guidelines and then applied to authentic positive blood samples. Overall, polydrug usage was determined in 24 real samples and one or more psychoactive drugs were found positive in 51 of 59 real samples, which were found to be positive via performing routine whole blood analysis and selected for DBS analysis. Our method developed could applicate as an alternative to conventional analytical and sampling techniques and respond to some of the present issues concerning the different forensic toxicological investigations.

CYP450-Mediated metabolism of suvorexant and investigation of metabolites in forensic case specimens

Suvorexant (Belsomra®) is a sedative hypnotic that was approved for use in 2015. It has a novel mechanism of action and was the first dual orexin receptor antagonist (DORA) to be approved for the treatment of sleep disorders. Sedative hypnotics often feature prominently in forensic investigations such as impaired driving and drug-facilitated sexual assault (DFSA) cases. As such, suvorexant is a drug of interest and its identification in forensic toxicology investigations is of significance. However, limited studies have been published to date and the disposition or importance of its metabolites has been largely uninvestigated. In this report, we investigate the enzymes responsible for metabolism and explore the prevalence of metabolites in blood from a series of thirteen forensic investigations. Recombinant cytochrome P450 enzymes (rCYPs) were used to generate phase I metabolites for suvorexant in vitro, and metabolites were identified using liquid chromatography-quadrupole/time-of-flight-mass spectrometry (LC-Q/TOF-MS). Four rCYP isoenzymes (3A4, 2C19, 2D6, and 2C9) were found to contribute to suvorexant metabolism. The only metabolite identified in blood or plasma arose from hydroxylation of the benzyl triazole moiety (M9). This metabolite was identified in seventeen blood and plasma specimens from twelve medicolegal death investigations and one impaired driving investigation. In the absence of a commercially available reference material, the metabolite was confirmed using rCYP-generated in vitro controls using high resolution mass spectrometry.

Toxicological Analysis of Warfarin in an Adult Developing Diffuse Alveolar Hemorrhage: A Case Report and Literature Review

Development and validation of analytical method for the identification and quantification of any drug in blood is vital significance to study drug metabolism and, link clinical results. A 39-year-old woman who arrived in the emergency unit due to bleeding on her mouth twice. In first arriving the hospital, the patient regularly has ingested warfarin for 12 years and has had never an event related-INR until now. There were radiological lesions of the patient, and the INR value was 12. After the treatment, it was suggested that the patient discontinue warfarin, and she was discharged. After 1.5 month, the patient developed severe hypoxia and the INR value was 9 in second visiting the hospital when she said that she didn’t ingest the drug for ten days. After the patient entered dialysis for three days and isolated from outside, the INR and the other values was normal. A blood specimen was sent to our forensic toxicology laboratory with the suspicion of suicidal or homicidal warfarin poisoning. Warfarin concentration determined using validated method by liquid chromatography-tandem mass spectrometer was 2.16 μg/ml. Clinicians should be aware the importance of analytical toxicological analyses to assess a series of forensic and clinical toxicological investigations in analysis of post-mortem and ante-mortem blood samples. Keywords: toxicology; analysis; warfarin; clinical; suicide; homicide. Special Issue of Health Sciences DOI: 10.7176/JSTR/6-03-04

Commentary. Fentanyl-related death and the underreporting risk

Novel synthetic opioid overdose deaths have been rising largely worldwide as a result of fentanyl adulteration in the illegal drug supply. Interpretation of post mortem analytical results concerning fentanyl can be challenging in particular due to redistribution phenomena. Lacking of resources, infrastructures and expertise to perform forensic toxicological investigation when an unknown drug or complex mixture of drugs is suspected can affect failure in exactly reporting cause in drug related death. Public safety and public health entities are called working together to enhance the timeliness and accuracy of the analytical characterization and toxicology testing of novel synthetic opioids.

Quantification of suvorexant in blood using liquid chromatography-quadrupole/time of flight (LC-Q/TOF) mass spectrometry

Suvorexant is a novel drug for the treatment of insomnia that is marketed under the trade name Belsomra®. Unlike other hypnotics, suvorexant is a dual orexin receptor antagonist that is believed to have a lower abuse potential compared to other therapeutics. Although sedative hypnotics feature prominently in forensic toxicology investigations, there have been limited reports that describe the analysis of suvorexant in biological samples. Following a 10-mg oral dose, peak concentrations are typically <200 ng/mL. A highly sensitive assay is required because forensic toxicology laboratories are often required to identify a drug several hours after a single dose. A new analytical procedure for the quantification of suvorexant in whole blood was developed that will aid in the identification of this new drug in forensic toxicology casework. A simple acidic/neutral liquid-liquid extraction (LLE) was used to isolate suvorexant from whole blood followed by liquid chromatography-quadrupole/time of flight (LC-Q/TOF) mass spectrometry analysis using positive electrospray ionization (ESI). The extraction efficiencies of various solvents in blood were evaluated in addition to limit of detection, limit of quantitation, precision, accuracy and bias, calibration model, matrix effects, interferences, and carryover. The recovery of suvorexant was evaluated using four different extraction solvents (N-butyl chloride, ether/toluene (1:1), hexane/ethyl acetate (9:1), and methyl tert-butyl ether (MTBE). Although no significant differences in analytical recovery were observed, N-butyl chloride demonstrated improved reproducibility, efficiency and convenience. A weighted (1/x) quadratic calibration model was selected over a range of 2–200 ng/mL (R2 = 0.995). Using only 0.5 mL whole blood, limits of detection and quantification were 0.5 ng/mL. Intra-assay (n = 5) and inter-assay (n = 15) precision (% CV) were ≤ 13% and bias ranged from −5 to 2% at concentrations of 5, 50, and 160 ng/mL. Matrix effects were 16% (9% CV) and 15% (8% CV) for 20 ng/mL and 100 ng/mL (n = 20), respectively. No qualitative interferences or carryover were observed; however, a quantitative interference with the internal standard (estazolam-D5) could be attributed to sertraline when present at a 10-fold higher concentration. In the absence of a commercially available deuterated internal standard, the potential for quantitative interferences using LC-based methods are discussed.

Alcohol and drugs use among drivers injured in road accidents in Campania (Italy): A 8-years retrospective analysis

A recent update of the Italian Road Traffic Law (RTL 41/2016), established severe penal sanctions when drivers, driving under the influence of alcohol (DUI) or drugs (DUID), are involved in road accident that results in death or injuries. A study was carried out to assess the trends of consumption of alcohol, illicit drugs or pharmaceutical among injured drivers suspected for DUI or DUID from 2009 to 2016 in the region of Campania (Italy). Confirmation toxicological analyses were performed on 780 blood samples and 1017 urine samples collected from 1797 injured drivers. These drivers all tested positive for alcohol or drug use through immunoassay screening applied at hospital emergency units and their biological samples transferred to the Forensic Reference Laboratory (FRL) for confirmation analysis. The GC/HS-FID methodology was used to test Blood Alcohol Concentration (BAC). Qualitative and quantitative analyses for drugs were performed using the GC/MS or LC–MS/MS methodology.The BAC >0.5g/L was confirmed in 91.5% of drivers suspected for DUI cases and in 93% of DUID respectively. In DUI cases, results show an increasing incidence of road accidents involving drivers with BAC above 1.5g/L while at concentrations above 0.8g/L alcohol and drugs are both used. Among the suspected DUID cases, the intake of alcohol in association with drugs has consistently increased over time and positive results on blood samples was confirmed for multiple drugs (20%) or cannabis and cocaine alone (18%) followed by benzodiazepines (6%) and methadone (3.5%) respectively. The majority of injured drivers suspected for DUID (1017 cases) did not authorize blood sampling, therefore only urine was analyzed showing the prevalent use of cannabis, followed by multiple drug>cocaine>benzodiazepines>opiates.Among 1797 drivers, suspected at screening for DUI or DUID, 15.4% of cases (64 blood and 213 urine samples) were not confirmed by GC/HS, GC/MS or LC–MS/MS analysis. In forensic toxicological investigations, it is mandatory to satisfy the best quality standards, which is not achievable if immunochemical screening is only performed on urine. Therefore, only confirmed positive results of alcohol or drugs on blood samples can represent conclusive evidence to demonstrate the DUI or DUID related offences. An improvement of the protocols currently applied in Italy for the assessment of DUI or DUID crimes is needed and the confirmation analysis on blood should be considered mandatory.

Cathinone stability in authentic urine specimens

PurposeSynthetic cathinones are encountered in a variety of antemortem and postmortem forensic toxicology investigations. Earlier experimental studies using fortified urine have evaluated analyte, temperature and pH-dependent variables associated with their stability. The purpose of this study was to compare experimental findings with those obtained using authentic urine from cathinone users. MethodsIn this report we compare cathinone concentrations in 180 authentic unpreserved urine specimens, following known periods of refrigerated storage. These findings are compared with previously published experimental data using fortified drug-free urine. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS) was used to target 22 cathinones. Quantitative results were compared in urine specimens (pH 4.5–10) following 5–17 months of storage. ResultsThe 180 specimens resulted in 164 quantitative findings involving α-PVP, ethylone, methylone, MDPV and pentylone. Initial drug concentrations ranged from 25ng/mL to over 100,000ng/mL. Upon reanalysis, the percentage of drug remaining (0–119%) was correlated with storage time and specimen pH. The ability to reconfirm original results was not correlated with storage time. Instead, specimen pH was far more predictive. The relationship between initial and final drug concentration was highly pH-dependent, yielding significant correlations for α-PVP, ethylone and methylone, particularly under acidic conditions. ConclusionsThese results are in good agreement with experimental findings and highlight the critical importance of specimen pH, rather than conventional time dependent variables, when considering cathinone stability in biological samples. The potential for pre-analytical changes in cathinone concentrations must be carefully considered when interpreting their results.

Quantification of suvorexant in urine using gas chromatography/mass spectrometry

Suvorexant (Belsomra®) is a novel sedative hypnotic drug that is prescribed to promote sleep in patients with insomnia. It is the first of a new class of drugs classified as dual orexin receptor antagonists (DORAs). Sedative hypnotics with central nervous system depressant effects feature prominently in forensic toxicology investigations. For this reason, a new analytical method was developed to identify suvorexant in urine using liquid-liquid extraction (LLE) and gas chromatography/mass spectrometry (GC/MS). Due to the absence of a commercially available isotopically labeled internal standard, estazolam-D5 was used due to its azepine, triazole and chlorinated functionality. The limit of detection and limit of quantitation was 10ng/mL and the linear range of the assay was 10-1000ng/mL. Accuracy and precision (%CV) were 98-101% and <11% at 30, 250 and 800ng/mL. Interferences from matrix and fifty common drugs were not present and processed samples were stable for 24h at room temperature. Suvorexant is a new drug of significant forensic interest due to its hypnotic and central nervous system depressant effects. The absence of commercially available metabolites and its chromatographic properties present some challenges in terms of identification. Nevertheless, a robust, reliable and sensitive assay was developed to identify suvorexant using GC/MS analysis.

Ocular disposition of diazepam in rabbits: understanding its level in vitreous humour for forensic applications

The disadvantages associated with routine toxicological samples, such as blood samples, have encouraged the forensic toxicologist to use vitreous humour as an alternative matrix. However, its use is still limited. The reason for this is the lack of a systematic study in this domain. Moreover, the phenomena of drug disposition in ocular tissues are mostly unexplored. This paper presents a controlled study to investigate the distribution of diazepam in post-mortem blood, vitreous humour and ocular tissues using rabbit as an animal model. This study reveals that diazepam levels in vitreous and aqueous humour increase with their decrease in whole blood at post-mortem. A further study on the distribution of diazepam in ocular tissues reveals that the anterior and posterior segment tissues might be responsible for the increase in diazepam levels in aqueous humour and vitreous humour respectively. The present study strengthens the possibility of using vitreous humour as a complementary sample to blood in forensic toxicological investigations.

Robust Bayesian Algorithm for Targeted Compound Screening in Forensic Toxicology.

As part of forensic toxicological investigation of cases involving unexpected death of an individual, targeted or untargeted xenobiotic screening of post-mortem samples is normally conducted. To this end, liquid chromatography (LC) coupled to high-resolution mass spectrometry (MS) is typically employed. For data analysis, almost all commonly applied algorithms are threshold-based (frequentist). These algorithms examine the value of a certain measurement (e.g., peak height) to decide whether a certain xenobiotic of interest (XOI) is present/absent, yielding a binary output. Frequentist methods pose a problem when several sources of information [e.g., shape of the chromatographic peak, isotopic distribution, estimated mass-to-charge ratio (m/z), adduct, etc.] need to be combined, requiring the approach to make arbitrary decisions at substep levels of data analysis. We hereby introduce a novel Bayesian probabilistic algorithm for toxicological screening. The method tackles the problem with a different strategy. It is not aimed at reaching a final conclusion regarding the presence of the XOI, but it estimates its probability. The algorithm effectively and efficiently combines all possible pieces of evidence from the chromatogram and calculates the posterior probability of the presence/absence of XOI features. This way, the model can accommodate more information by updating the probability if extra evidence is acquired. The final probabilistic result assists the end user to make a final decision with respect to the presence/absence of the xenobiotic. The Bayesian method was validated and found to perform better (in terms of false positives and false negatives) than the vendor-supplied software package.