[Adequate assessment of linear function of calibration according to GOST R ISO 11095-2007 for ethanol mass concentration measurement]. / Otsenka adekvatnosti lineinoi funktsii kalibrovki po GOST R ISO 11095-2007 dlya izmereniya massovoi kontsentratsii etanola.

OBJECTIVE: To assess the adequacy of linear function of calibration according to GOST R ISO 11095-2007 for ethanol mass concentration measurement using internal reference materials (RMs). MATERIAL AND METHODS: An experiment on calibration in accordance with the GOST R ISO 11095-2007 National standard of the RF was carried out using internal RMs, namely aqueous solutions of ethanol at different concentrations. Measurements were performed for two subbands of ethanol concentrations at RMs: 0.15-1.05 and 1.0-7.0 mg/ml - according to the certified methodology. RESULTS: The graphs of the calibration's functions based on experimental data are consistent with the assumption of the calibration function's linearity, as well as the assumption of the standard deviation's constance of residues is equitable for two subbands of RMs. CONCLUSION: Proven linear models in the calibration experiment may be recommended for use in the ethanol mass concentration measurement.

Drug-mediated ion suppression and mitigation of interferences using liquid chromatography-quadrupole/time of flight mass spectrometry (LC-Q/TOF-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS).

Liquid-chromatography mass spectrometry (LC-MS) is a powerful bioanalytical tool that is gaining widespread use in operational forensic toxicology laboratories. However, changes in ionization efficiency caused by endogenous or exogenous species must be carefully considered. While different modes of ionization can be used, electrospray ionization (ESI) can be especially prone to this phenomenon due to capacity-limited ionization. This decreased ionization efficiency can influence the accuracy and sensitivity of analytical methods. While quantitative matrix effects are evaluated routinely during method development and validation, drug-mediated ion suppression is not always assessed quantitatively, or in sufficient depth. Although stable isotope labeled internal standards (SIL-IS) can mitigate this issue, they are not always commercially available, particularly for new or emerging substances. In this study, the hypnotic drug suvorexant was used as a model compound for the investigation of such interferences. The potential for significant bias in quantitative analysis was demonstrated using this previously validated assay. In this study, quantitative biases due to ionization suppression are discussed, and techniques to overcome this challenge are presented. Decreases in specimen and injection volume were shown to significantly reduce quantitative bias due to drug-mediated suppression. This straight-forward approach can improve the robustness of analytical methodology, which is particularly important when quantitative measurements are relied upon for medicolegal and other purposes.

Position Paper: Recommendations for the Investigation, Diagnosis, and Certification of Deaths Related to Opioid and Other Drugs.

The National Association of Medical Examiners convened an expert panel to update the association's evidence-based recommendations for investigating and certifying deaths associated with opioids and other misused substances to improve death certificate and mortality data for public health surveillance. The recommendations are as follows:1. Autopsy provides the best information on a decedent's medical condition for optimal interpretation of toxicology results, circumstances surrounding death, medical history, and scene findings. The panel considers autopsy an essential component of investigating apparent overdose deaths.2. Scene investigation includes reconciling prescription information and medication counts. Investigators should note drug paraphernalia or other evidence of using intoxicating substances.3. Retain blood, urine, and vitreous humor whenever available. Blood from the iliofemoral vein is preferable to blood from more central sites.4. A toxicological panel should be comprehensive, including potent depressant, stimulant, and antidepressant medications. Detecting novel substances present in the community may require special testing.5. When death is attributed to a drug or combination of drugs (as cause or contributing factor), the certifier should list the drugs by generic name in the autopsy report and death certificate.6. The best classification for manner of death in an overdose without any apparent intent of self-harm is "accident."

11-OH-THC in hair as marker of active cannabis consumption: Estimating a reliable cut-off by evaluation of 672 THC-positive hair samples.

Hair analysis for the assessment of cannabis active use from passive consumption may be failed when performed by the sole detection of compounds present in plant material as well as in cannabis smoke like Δ-9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN). For this reason, the determination of 11-nor-9-carboxy-Δ-9-tetrahydrocannabinol (THC-COOH) has been proposed by the Society of Hair Testing (SoHT) in order to prove active cannabis consumption. The identification of THC-COOH in hair will continue to be complicated by its acidic nature and the critical low concentration due to the preferential incorporation of basic compounds into hair shaft. Alternatively, 11-OH-THC may be considered as a complementary marker for THC administration. Our recent study reported an accurate validated procedure for THC, CBD, CBN and 11-OH-THC in hair, based on a GC/MS-MS method in electron ionization mode. However, unlike THC-COOH, a cut-off level for 11-OH-THC in hair has not been fixed yet. For this reason, the aim of this study is to propose a concentration value for 11-OH-THC in hair analysis in order to discriminate between chronic use and external contamination. Receiver operating characteristics (ROC) analysis was applied for cut-off evaluation after 11-OH-THC quantification in a pool of 672 THC-positive hair samples. Results have shown a concentration range between 0.01-5.34 ng/mg for THC (mean 0.34 ng/mg, median 0.12), 0.00-19.2 pg/mg for THC-COOH (mean 0.72 pg/mg, median 0.19 pg/mg) and 0.01-13.33 ng/mg for 11-OH-THC (mean 1.09 ng/mg, median 0.51 ng/mg) for scalp hair and between 0.03-6.32 ng/mg for THC (mean 0.82 ng/mg, median 0.30), 0.00-42.1 pg/mg for THC-COOH (mean 2.70 pg/mg, median 1.08 pg/mg) and 0.00-7.88 ng/mg for 11-OH-THC (mean 1.70 ng/mg, median 0.89 ng/mg) for body hair. Considering these experimental data collected in our laboratory, we propose a cut-off level of 0.5 for scalp and body hair, as indicative of cannabis active consumption. The ROC curve AUCs for 11-OH-THC were 0.873 and 0.884 in 590 scalp hair and 82 body hair samples, respectively. The comparison of the results for THC-COOH (control method) and 11-OH-THC (test method) was also made by means of the Cohen's kappa statistics providing a good agreement according to both Landis & Koch and Fleiss scales. Additionally, we suggest that the detection of both THC-COOH and 11-OH-THC should be mandatory in order to prove active intake and exclude false positive results from external contamination.

LC-QTOF-MS Identification of Major Urinary Cyclopropylfentanyl Metabolites Using Synthesized Standards.

Cyclopropylfentanyl is a fentanyl analog implicated in 78 deaths in Europe and over 100 deaths in the United States, but toxicological information including metabolism data about this drug is scarce. The aim of this study was to provide the exact structure of abundant and unique metabolites of cyclopropylfentanyl along with synthesis routes. In this study, metabolites were identified in 13 post-mortem urine samples using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Samples were analyzed with and without enzymatic hydrolysis, and seven potential metabolites were synthesized in-house to provide the identity of major metabolites. Cyclopropylfentanyl was detected in all samples, and the most abundant metabolite was norcyclopropylfentanyl (M1) that was detected in 12 out of 13 samples. Reference materials were synthesized (synthesis routes provided) to identify the exact structure of the major metabolites 4-hydroxyphenethyl cyclopropylfentanyl (M8), 3,4-dihydroxyphenethyl cyclopropylfentanyl (M5) and 4-hydroxy-3-methoxyphenethyl cyclopropylfentanyl (M9). These metabolites are suitable urinary markers of cyclopropylfentanyl intake as they are unique and detected in a majority of hydrolyzed urine samples. Minor metabolites included two quinone metabolites (M6 and M7), not previously reported for fentanyl analogs. Interestingly, with the exception of norcyclopropylfentanyl (M1), the metabolites appeared to be between 40% and 90% conjugated in urine. In total, 11 metabolites of cyclopropylfentanyl were identified, including most metabolites previously reported after hepatocyte incubation.

Implementation of a Blind Quality Control Program in Blood Alcohol Analysis.

Declared proficiency tests are limited in their use for testing the performance of the entire system, because analysts are aware that they are being tested. A blind quality control (BQC) is intended to appear as a real case to the analyst to remove any intentional or subconscious bias. A BQC program allows a real-time assessment of the laboratory's policies and procedures and monitors reliability of casework. In September 2015, the Houston Forensic Science Center (HFSC) began a BQC program in blood alcohol analysis. Between September 2015 and July 2018, HFSC submitted 317 blind cases: 89 negative samples and 228 positive samples at five target concentrations (0.08, 0.15, 0.16, 0.20 and 0.25 g/100 mL; theoretical targets). These blood samples were analyzed by a headspace gas chromatograph interfaced with dual-flame ionization detectors (HS-GC-FID). All negative samples produced `no ethanol detected' results. The mean (range) of reported blood alcohol concentrations (BACs) for the aforementioned target concentrations was 0.075 (0.073-0.078), 0.144 (0.140-0.148), 0.157 (0.155-0.160), 0.195 (0.192-0.200) and 0.249 (0.242-0.258) g/100 mL, respectively. The average BAC percent differences from the target for the positive blind cases ranged from -0.4 to -6.3%, within our uncertainty of measurement (8.95-9.18%). The rate of alcohol evaporation/degradation was determined negligible. A multiple linear regression analysis was performed to compare the % difference in BAC among five target concentrations, eight analysts, three HS-GC-FID instruments and two pipettes. The variables other than target concentrations showed no significant difference (P > 0.2). While the 0.08 g/100 mL target showed a significantly larger % difference than higher target concentrations (0.15-0.25 g/100 mL), the % differences among the higher targets were not concentration-dependent. Despite difficulties like gaining buy-in from stakeholders and mimicking evidence samples, the implementation of a BQC program has improved processes, shown methods are reliable and added confidence to staff's testimony in court.

Validation of the Neogen ELISA Benzodiazepine Kit using Clonazepam as the Target Molecule for Blood and Urine.

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.

Inter-laboratory proficiency results of blood alcohol determinations at clinical and forensic laboratories in Italy.

BACKGROUND: Inter-laboratory proficiency schemes are widely used to control the performance of clinical and forensic toxicology laboratories. In 2016 the Laboratory of Environmental Hygiene and Forensic Toxicology - Venice (Italy) initiated an inter-laboratory proficiency test of blood-alcohol analysis. The number of participating laboratories gradually increased from 26 to 36. Furthermore, a few clinical laboratories were included if gas chromatographic (GC) methods were used for blood alcohol analysis. PROCEDURE: Whole blood was obtained from the Blood Transfusion Centre of the Venice Hospital and a mixture of sodium fluoride and potassium oxalate was added as a preservative and anticoagulant, respectively. Aliquots of the blood were spiked with certified pure ethanol to obtain target blood-alcohol concentrations (BACs) ranging from 0 to 5.0g/L. Two blood samples (4mL each) were included in each shipment to the participating laboratories. The laboratories were asked to provide information about number of replicate BAC determinations they made, the types of ethanol reference standards used, and inherent measurement uncertainty. The aim of the testing was to obtain a mean consensus value for the target BAC and to assess inter-laboratory imprecision. All procedures for registration and submission of results were done on-line. A confidential report and statistical evaluations were returned to the participants one week later. ANALYTICAL METHODS: All participants used head-space GC (HS-GC) for the analysis of ethanol in blood. More than 85% of participants used HS-GC with flame-ionization detection, whereas the others used mass spectrometry (MS) as a detector. More than 40% of the participating laboratories kept the blood samples frozen (-20°C) prior to analysis, whereas the others used refrigeration (+4°C). The preliminary validation tests showed that there were no statistically significant differences between BAC in frozen or refrigerated samples for a period of 20 days. RESULTS AND CONCLUSION: The statistical evaluation of results was done using an iterative procedure known as Algorithm A (ISO 13528:2015, C.3.1). This provides robust estimates for mean and standard deviation between laboratories and these were used as consensus values. More than 85% of participants provided satisfactory results (z-score <1) and 94% of laboratories were within z-score <2, based on five control samples. When a blood sample without any alcohol (blank) was sent for analysis, laboratories reported this as zero, 0.00g/L, below limit of detection (LOD) or not detected. Some type of consensus should be reached for reporting blank samples.

Validation and Cross-Reactivity Data for Fentanyl Analogs With the Immunalysis Fentanyl ELISA.

Every year new fentanyl analog compounds, or fentanyls, appear on the drug scene. Development of immunoassays dedicated for screening individual molecules is challenging due to the short-lived presence of these compounds on the recreational drug market. Therefore, we investigated the detecting capabilities of the immunalysis fentanyl direct enzyme-linked immunosorbent assay (ELISA) kit against fentanyl in whole blood, and determined the cross-reactivity of nine fentanyl analogs (2-fluorofentanyl, acetylfentanyl, acrylfentanyl, carfentanil, cyclopropylfentanyl, tetrahydrofuranylfentanyl, furanylfentanyl, ocfentanil, valerylfentanyl) to confirm its validity for the general screening of fentanyls. Immunalysis ELISA assay was used to test whole blood samples fortified with fentanyl on a TECAN Freedom EVOlyzer platform, according to manufacturer specifications. The kit successfully was validated for fentanyl screening with a cutoff set at 0.5 ng/mL, and all tested analogs, with the exclusion of carfentanil, were detected. The lowest cross-reactivity with the kit was obtained with furanylfentanyl (20% ± 1, 95% confidence intervals (CI)) and 4-fluoroisobutyrfentanyl (25% ± 1, 95% CI), while the highest was recorded using acetylfentanyl (99% ± 11, 95% CI) and acrylfentanyl (94% ± 10, 95% CI). Post-mortem samples containing fentanyl, acrylfentanyl, cyclopropylfentanyl, THF-fentanyl and 4-fluoroisobutyrfentanyl were screened, and sensitivity and specificity of each analog were calculated. Positive screening results were generated by all post-mortem cases containing fentanyl (n = 14), acrylfentanyl (n = 11), cyclopropylfentanyl (n = 14), tetrahydrofuranylfentanyl (n = 13) and 4-fluoroisobutyrfentanyl (n = 10). Concentration of post-mortem fentanyl samples ranged from 0.5 ng/mL (cutoff) to 230 ng/mL, while the range for analogs was 3.4-36 ng/mL (cyclopentylfentanyl), 0.76-370 ng/mL (4-fluoroisobutyrfentanyl), 0.02-12 ng/mL (acrylfentanyl) and 2-26 ng/mL (tetrahydrofuranylfentanyl). The immunalysis fentanyl direct ELISA kit was successfully validated and showed significant cross-reactivity for all tested fentanyls, except carfentanil, making it a suitable technique for fentanyl and fentanyl analogs screening.

Child Fatalities Due to Heroin/Fentanyl Exposure: What the Case History Missed.

This case report presents three unrelated children found to have heroin and/or fentanyl in their systems after general unknown systematic toxicological analysis (STA). The first case involves an 11-month-old male found unresponsive at their residence. The scene response suggested a potentially unsafe sleeping condition or a sudden unexplained infant death. The second case is a 14-month-old female found unresponsive after eating soft candies, suggesting that a choking related death may have occurred. The third case is a 12-year-old male found unresponsive in bed and foaming from the mouth. Gum was removed from the child's airway, suggesting another choking related death. The STA included a 14-drug category enzyme linked immunosorbant assay (ELISA) screening in whole blood. Cases 1 and 3 were presumptively positive for fentanyl, while Case 2 was presumptively positive for opiates and fentanyl. Reflex confirmation was performed in blood, urine and gastric contents, by solid-phase extraction (SPE) for 12 opiates including morphine and 6-monoacetylmorphine (6MAM) by gas chromatography-mass spectrometry (GC-MS) and for fentanyl, norfentanyl, and novel analogs, by liquid chromatography tandem mass spectrometry (LC-MS-MS). High concentrations of fentanyl and 6MAM in the gastric contents of Case 1, along with the presence of diacetylmorphine, suggested probable enteral ingestion of heroin and fentanyl, separately or in a combined formulation. Interpretation of the toxicology results could not determine a probable route of exposure to heroin/fentanyl in Case 2, however, the cause of death was clearly related to this drug mixture. In Case 3, the presence of acetylfentanyl suggested an illicit fentanyl exposure. The intention of this case report is to demonstrate the need for a STA approach for all non-trauma postmortem cases regardless of case circumstances, age or suspicion of drug use.

The United Kingdom and Ireland association of forensic toxicologists forensic toxicology laboratory guidelines (2018).

In 2010, the United Kingdom and Ireland Association of Forensic Toxicologists (UKIAFT) created forensic toxicology laboratory guidelines. This represents a revision of those guidelines as a result of the changing toxicological and technical landscape.

Building Bridges between Clinical and Forensic Toxicology Laboratories.

BACKGROUND: Clinical and forensic toxicology can be defined as two disciplines involving the detection, identification and measurement of xenobiotics in biological and non-biological samples to assist in the diagnosis, treatment, prognosis and prevention of poisonings and to disclose causes and contributory causes of fatal intoxications, respectively. OBJECTIVE: This article explores the close connections between clinical and forensic toxicology in overlapping areas of interest. METHODS: An update has been carried out of the following seven areas of interest in analytical toxicology: doping control, Sudden Cardiac Death (SCD), brain death, Sudden Infant Death Syndrome (SIDS) and Munchausen Syndrome by Proxy (MSBP), prenatal exposure to drugs and Fetal Alcohol Syndrome (FAS), Drug-Facilitated Crimes (DFC) and intoxications by new psychoactive substances (NPS). RESULTS: While issues such as SCD, SIDS or doping control are investigated mainly in forensic laboratories, others such as prenatal exposure to drugs or FAS are mainly treated in clinical laboratories. On the other hand, areas such MSBP, DFC or the intoxications by NPS are of interest in both laboratories. Some of these topics are initially treated in hospital emergency departments, involving clinical laboratories and sometimes lately derived to forensic laboratories. Conversely, cases with initial medicallegal implications and fatalities are directly handled by forensic toxicology, but may trigger further studies in the clinical setting. CONCLUSION: Many areas of common interest between clinical and forensic laboratories are building bridges between them. The increasing relationships are improving the growth, the reliability and the robustness of both kinds of laboratories.

A toolbox for microbore liquid chromatography tandem-high-resolution mass spectrometry analysis of albumin-adducts as novel biomarkers of organophosphorus pesticide poisoning.

Exposure to toxic organophosphorus pesticides (OPP) represents a serious problem in the public healthcare sector and might be forced in terroristic attacks. Therefore, reliable verification procedures for OPP-intoxications are required for forensic, toxicological and clinical reasons. We developed and optimized a toolbox of methods to detect adducts of human serum albumin (HSA) with OPP considered as long-term biomarkers. Human serum was incubated with diethyl-oxono and diethyl-thiono pesticides for adduct formation used as reference. Afterwards serum was subjected to proteolysis using three proteases separately thus yielding phosphorylated tyrosine residues (Y*) detected as single amino acid (pronase), as hexadecapeptide LVRY*411TKKVPQVSTPTL (pepsin) and as the tripeptide Y*411TK (trypsin), respectively. Adducts were analyzed via microbore liquid chromatography coupled to electrospray ionization (µLC-ESI) and tandem-high-resolution mass spectrometry (MS/HR MS). Using paraoxon-ethyl as model OPP for adduct formation, methods were optimized with respect to MS/HR MS-parameters, protease concentrations and incubation time for proteolysis. HSA-adducts were found to be stable in serum in vitro at +37 °C and -30 °C for at least 27 days and resulting biomarkers were stable in the autosampler at 15 °C for at least 24 h. Limits of identification of adducts varied between 0.25 µM and 4.0 µM with respect to the corresponding pesticide concentrations in serum. Applicability of the methods was proven by successful detection of the adducts in samples of OPP-poisoned patients thus demonstrating the methods as a reliable toolbox for forensic and toxicological analysis.

Comparison of dispensed medications and forensic-toxicological findings to assess pharmacotherapy in the Swedish population 2006 to 2013.

PURPOSE: To investigate person-level agreement between medication exposure as predicted using the PRE2DUP (a prescription-based design to estimate continuous drug use) method and postmortem toxicological findings, in the Swedish population during the years 2006 to 2013. METHODS: Using the Swedish National Board of Forensic Medicine's toxicology database and the Swedish National Board of Health and Welfare's registries on causes of death, dispensed medications, and in-patient care, forensic-toxicological findings were compared with prescription-based estimates of drug use for 27 medications. We modeled expected drug-use periods with the PRE2DUP using an algorithm of demonstrated high validity that evaluates personal drug-purchasing patterns with consideration to possible stockpiling of drugs and package information. Excluding criteria included self-inflicted death and recent in-patient care. RESULTS: In data from 18 627 performed autopsies, as well as 10 160 instances of dispensed drug use, the agreement between PRE2DUP drug-use periods and forensic toxicology was, overall, moderate (Cohen's kappa: 0.56 [95% confidence interval {CI}: 0.55-0.57]) with a positive predictive value, or predicted adherence rate, of 46.0%. The group-level predicted adherence and agreement were highest for antidepressants, at 71.0% (Cohen's kappa: 0.74 [CI: 0.73-0.76]), and lowest for cardiovascular drugs, at 21.5% (Cohen's kappa: 0.33 [CI: 0.31-0.36]). Predicted recreational use (negative predictive value) was low for all investigated drugs (0.0%-1.4%). The biological half-life explained 29% (P = 0.003) of the variability of the false-positive rate. CONCLUSIONS: Measured agreement between PRE2DUP-based drug-use estimates and forensic-toxicological findings is dependent upon a number of factors, including true continuous drug use and postmortem detectability of the investigated drugs, as well as the occurrence of unconventional dosing and true non-adherence.

Urine specimen validity test for drug abuse testing in workplace and court settings.

In recent decades, urine drug testing in the workplace has become common in many countries in the world. There have been several studies concerning the use of the urine specimen validity test (SVT) for drug abuse testing administered in the workplace. However, very little data exists concerning the urine SVT on drug abuse tests from court specimens, including dilute, substituted, adulterated, and invalid tests. We investigated 21,696 submitted urine drug test samples for SVT from workplace and court settings in southern Taiwan over 5 years. All immunoassay screen-positive urine specimen drug tests were confirmed by gas chromatography/mass spectrometry. We found that the mean 5-year prevalence of tampering (dilute, substituted, or invalid tests) in urine specimens from the workplace and court settings were 1.09% and 3.81%, respectively. The mean 5-year percentage of dilute, substituted, and invalid urine specimens from the workplace were 89.2%, 6.8%, and 4.1%, respectively. The mean 5-year percentage of dilute, substituted, and invalid urine specimens from the court were 94.8%, 1.4%, and 3.8%, respectively. No adulterated cases were found among the workplace or court samples. The most common drug identified from the workplace specimens was amphetamine, followed by opiates. The most common drug identified from the court specimens was ketamine, followed by amphetamine. We suggest that all urine specimens taken for drug testing from both the workplace and court settings need to be tested for validity.

Considerations regarding the validation of chromatographic mass spectrometric methods for the quantification of endogenous substances in forensics.

The requirement for correct evaluation of forensic toxicological results in daily routine work and scientific studies is reliable analytical data based on validated methods. Validation of a method gives the analyst tools to estimate the efficacy and reliability of the analytical method. Without validation, data might be contested in court and lead to unjustified legal consequences for a defendant. Therefore, new analytical methods to be used in forensic toxicology require careful method development and validation of the final method. Until now, there are no publications on the validation of chromatographic mass spectrometric methods for the detection of endogenous substances although endogenous analytes can be important in Forensic Toxicology (alcohol consumption marker, congener alcohols, gamma hydroxy butyric acid, human insulin and C-peptide, creatinine, postmortal clinical parameters). For these analytes, conventional validation instructions cannot be followed completely. In this paper, important practical considerations in analytical method validation for endogenous substances will be discussed which may be used as guidance for scientists wishing to develop and validate analytical methods for analytes produced naturally in the human body. Especially the validation parameters calibration model, analytical limits, accuracy (bias and precision) and matrix effects and recovery have to be approached differently. Highest attention should be paid to selectivity experiments.

Dried blood spots combined to an UPLC-MS/MS method for the simultaneous determination of drugs of abuse in forensic toxicology.

A method for the simultaneous determination of 11 illicit drugs, using the dried blood spot (DBS) sampling technique combined with the UPLC-MS/MS technology was developed to study its applicability within the forensic toxicology. The DBS samples, prepared from a blood volume of 50µL and using the Whatman® BFC 180 bloodstain cards, were extracted with a methanol/acetonitrile mixture. The chromatographic separation was performed using an Acquity UPLC® HSS T3 column (100mm×2.1mm, 1.8µm) and an acetonitrile/2mM ammonium formate (0.1% formic acid) gradient. The detection was accomplished with a TQ Detector, operating in the ESI+ and MRM modes. The method was validated in terms of selectivity, matrix effect, extraction recovery (42%-91%), carryover, LOD and LOQ (0.5-1ng/mL and 1-5ng/mL, respectively), linearity (LOQ to 500ng/mL), intraday and interday precision (3.8-14% and 5.3-13%, respectively), accuracy (-9.3% to 7.9%) and dilution integrity. An eight months stability study at room temperature, 2-8°C and -10°C, was also performed, with the best results obtained at -10°C. The procedure was applied to 64 real samples (92 positive results for substances included in this study). The results were compared with the methodologies routinely applied in the laboratory and the statistical analysis allowed to establish an acceptable correlation. This study permitted to determine that the DBS can represent an alternative or a complement to conventional analytical and sampling techniques, responding to some of the present issues concerning the different forensic toxicology applications.

Reference Brain/Blood Concentrations of Citalopram, Duloxetine, Mirtazapine and Sertraline.

Postmortem blood samples may not accurately reflect antemortem drug concentrations, as the levels of some drugs increase due to postmortem redistribution (PMR). The brain has been suggested as an alternative sampling site. The anatomically secluded site of the brain limits redistribution and prolongs the detection window, thereby enabling sampling from deceased individuals where blood is no longer suitable for analysis. We report concentrations in brain tissue and blood from 91 cases for the four antidepressants citalopram, duloxetine, mirtazapine and sertraline. The cases were classified according to their role in the cause of death, as follows: (A) concentrations where the drug was the sole cause of fatal intoxication; (B) concentrations where the drug contributed to a fatal outcome; and (C) concentrations where the drug was not related to the cause of death. The analytical method was successfully validated in brain tissue in terms of linearity, process efficiency, precision and accuracy. Quantification of analytes was performed by ultra-performance liquid chromatography with tandem mass spectrometry. Correlations between blood and brain concentrations were achieved with R2-values between 0.67 and 0.91. The following median brain-blood ratios were obtained: 3.71 for citalopram (range: 1.4-5.9), 11.0 for duloxetine (range: 5.0-21.6), 1.53 for mirtazapine (range: 1.02-4.71) and 7.38 for sertraline (range: 3.2-14.2). The S/R ratio of racemic citalopram was the same in brain (0.80) and blood (0.85), whereas the median citalopram/N-desmethylcitalopram ratio was higher in brain (9.1) than blood (4.1). The results of this study may serve as reference concentrations in brain for forensic cases.