Mathematical Method Validation Tools for Application to a Proteomics Approach of Postmortem Metabolic Capacity Estimation

In postmortem cases, forensic toxicologists perform analyses for legal and illegal drugs, volatile substances, poisons and biochemical parameters in order to determine the causes and circumstances of death. Evaluation of the metabolic capacity of an individual might help to achieve this goal. Knowledge that the deceased had a poor metabolic capacity might help differentiate between medical error and accidental overdose, for example. Traditionally, DNA analysis of genes encoding for metabolizing enzymes has been used for this purpose. However, the genotype can be quite a poor predictor of phenotype; intervening factors such as sex, age, presence of inducers or inhibitors act as confouding factors. A proof-of-concept methodology estimating the postmortem metabolic capacity through characterization and quantification of cytochrome P450 (CYP) enzymes in liver tissue is presented here. Combining quantitative proteomics with detection of the peptides bearing mutation sites allowed for a more accurate estimation of the metabolic capacity than genotyping alone. The current regulatory environment, and best practices, requires forensics bioanalytical methods to be validated. Anticipating the validation of this method, several methodological issues were foreseen. In order to properly validate the quantitative part of the CYP analysis method, a simple, analyst-independent, and systematic procedure to choose and validate a calibration model (order, weighting) based on statistical analysis was developed. Additionally, the omnipresence of the target analyte(s) in authentic matrix (human liver) calls for a methodology allowing to deal with endogenous concentration(s) of analytes in matrices used to prepare calibration standards and quality control samples. An automated tool was developed to correct for the endogenous analytes’ concentration. Finally, characterization of the CYP enzymes, via the monitoring of peptides bearing a mutation site, requires validation via a qualitative decision point method. Current guidelines about this type of analysis are ill adapted to deal with the binary nature of the results. A more suitable set of guidelines was developed and tested. These mathematical method validation tools, in combination with the CYP analysis method, provide the necessary framework for metabolic capacity estimation in postmortem cases

One hundred years of forensic sciences in Quebec: the evolution of scientific techniques since 1914

In 2014, Quebec's forensic science laboratory (Laboratoire de sciences judiciaires et de medicine légale) is celebrating its 100th anniversary. Since its foundation by Dr. Derome in 1914, scientific techniques in the various areas of forensic science have greatly evolved. Not only was the Laboratory the first forensic science institution in North America, it has been a pioneer for several analytical methods. The early days of the Laboratory as well as the specific evolution of the departments and divisions of biology, chemistry, arson and explosion, questioned documents, forensic pathology, odontology and anthropology, toxicology and administration will be reviewed here

Interference of fetal hemoglobin in the determination of carboxyhemoglobin by spectrophotometry

Determination of carboxyhemoglobin (HbCO) is routinely performed in suspected cases of carbon monoxide intoxication and unexplained deaths. However, some authors have suggested that measured HbCO may be falsely elevated in infants (0–12 months) due to the presence of fetal hemoglobin (HbF). The purpose of this study was to evaluate the impact of fetal hemoglobin on the spectrophotometric determination of carboxyhemoglobin. The interference of HbF in the determination of HbCO in infants aged from 0 to 12 months was evaluated using 16 ante-mortem and 19 post-mortem blood samples. The %HbCO was quantified spectrophotometrically by calculating the 560 nm/530 nm absorbance ratio, using a dual beam spectrophotometer. The average measured HbCO in infants of 3 months of age or under was 17%, which is abnormally elevated. No significant difference in HbCO measurement was found between ante-mortem and post-mortem samples. These results highlight the fact that care must be taken in interpretation of carboxyhemoglobin measurements in infants when using a spectrophotometric method

Determination of Confidence Intervals in Non-normal Data: Application of the Bootstrap to Cocaine Concentration in Femoral Blood

Calculating the confidence interval is a common procedure in data analysis and is readily obtained from normally distributed populations with the familiar formula. However, when working with non-normally distributed data, determining the confidence interval is not as obvious. For this type of data, there are fewer references in the literature, and they are much less accessible. We describe, in simple language, the percentile and bias-corrected and accelerated variations of the bootstrap method to calculate confidence intervals. This method can be applied to a wide variety of parameters (mean, median, slope of a calibration curve, etc.) and is appropriate for normal and non-normal data sets. As a worked example, the confidence interval around the median concentration of cocaine in femoral blood is calculated using bootstrap techniques. The median of the non-toxic concentrations was 46.7 ng/mL with a 95% confidence interval of 23.9–85.8 ng/mL in the non-normally distributed set of 45 postmortem cases. This method should be used to lead to more statistically sound and accurate confidence intervals for non-normally distributed populations, such as reference values of therapeutic and toxic drug concentration, as well as situations of truncated concentration values near the limit of quantification or cutoff of a method

Cent ans de sciences judiciaires au Québec : l'évolution des techniques scientifiques depuis 1914

En 2014, le Laboratoire de sciences judiciaires et de médicine légale du Québec célèbre son 100ième anniversaire. Depuis sa fondation par Dr Wilfrid Derome en 1914, les techniques scientifiques ont grandement évolué dans les différents domaines des sciences judiciaires. Non seulement le laboratoire fut la première institution de sciences judiciaires en Amérique du Nord, mais plusieurs méthodes analytiques y furent aussi développées. Les débuts du Laboratoire ainsi que l'évolution spécifique des différentes directions et divisions de la biologie, chimie, incendies et explosions, documents, pathologie judiciaire, odontologie et anthropologie, toxicologie et administration seront relatés ici

A model of evaluative opinion to encourage greater transparency and justification of interpretation in post-mortem forensic toxicology

Over the past decades, the calls to improve the robustness of interpretation in forensic science have increased in magnitude. Forensic toxicology has seen limited progress in this regard. In this work, we propose a transparent interpretive pathway for use in postmortem forensic toxicology cases. This process allows the selection of the interpretive methodology based on the amount of previous information that is available for the drug(s) in question. One approach is an assessment of various pharmacological and circumstantial considerations resulting in a toxicological significance score (TSS), which is particularly useful in situations where limited information about a drug is available. When there is a robust amount of case data available, then a probabilistic approach, through the evaluation of likelihood ratios by the forensic toxicologist and of prior probabilities by the fact finder, is utilized. This methodology provides a transparent means of making an interpretive decision on the role of a drug in the cause of death. This will allow the field of forensic toxicology to take a step forward in using best practice in evaluative reporting, a tool already used by many other forensic science disciplines.</p

Cyanide quantification in post-mortem biological matrices by headspace GC–MS

Cyanide is a powerful chemical asphyxiant found in some forensic cases following voluntary (suicide) or involuntary ingestion (fire, accidental exposure). A quantification method for cyanide that is specifically suited to post-mortem forensic purposes was developed. Determination was performed by headspace gas chromatography coupled to mass spectrometry using a GS-GASPRO column on an HP-6890 gas chromatograph with an HP-5973N mass detector. The biological sample was treated with an internal standard, frozen, glacial acetic acid was added and the sample was then incubated at 60 °C for 15 min. The headspace was sampled with a disposable syringe, and analyzed to quantify hydrogen cyanide. Isotopically labeled cyanide (13C15N) was used as the internal standard to minimize matrix effect and sampling error. The method produced an extended linear dynamic range (0.07–50 μg/mL), and a method detection limit of 0.02 μg/mL. Identical calibration curves were obtained when blood, gastric contents and aqueous solutions were used as the calibration standard matrix. This method was also successful in quantitating cyanide in gastric contents, one of the most variable biological fluids. The method has been validated and is being used for current forensic cases such as fire victims and suicides

A case of fatal idiosyncratic reaction to the designer drug 3,4-methylenedioxypyrovalerone (MDPV) and review of the literature

The stimulant designer drug 3,4-methylenedioxypyrovalerone (MDPV) was first synthesized by Boehringer Ingelheim in 1969 and introduced on the black market in 2006. Only a small number of fatal intoxication cases have been reported in the literature, all with significant blood MDPV concentrations. In this report, we describe one fatality attributed to an idiosyncratic reaction to MDPV. The victim displayed agitation, violent behavior and delirium followed by cardiac arrest. Hyperthermia was observed at the hospital. The MDPV cardiac and femoral blood concentrations were 6 ng/mL. The presence of excited delirium syndrome and MDPV, a drug with a pharmacology similar to cocaine, leads to the conclusion that the victim suffered a fatal adverse reaction to MDPV. This is the first published case of idiosyncratic reaction to MDPV

A Tool for Automatic Correction of Endogenous Concentrations: Application to BHB Analysis by LC–MS-MS and GC-MS

Several substances relevant for forensic toxicology purposes have an endogenous presence in biological matrices: beta-hydroxybutyric acid (BHB), gamma-hydroxybutyric acid (GHB), steroids and human insulin, to name only a few. The presence of significant amounts of these endogenous substances in the biological matrix used to prepare calibration standards and quality control samples (QCs) can compromise validation steps and quantitative analyses. Several approaches to overcome this problem have been suggested, including using an analog matrix or analyte, relying entirely on standard addition analyses for these analytes, or simply ignoring the endogenous contribution provided that it is small enough. Although these approaches side-step the issue of endogenous analyte presence in spiked matrix-matched samples, they create serious problems with regards to the accuracy of the analyses or production capacity. We present here a solution that addresses head-on the problem of endogenous concentrations in matrices used for calibration standards and quality control purposes. The endogenous analyte concentration is estimated via a standard-addition type process. This estimated concentration, plus the spiked concentration are then used as the de facto analyte concentration present in the sample. These de facto concentrations are then used in data analysis software (MultiQuant, Mass Hunter, etc.) as the sample’s concentration. This yields an accurate quantification of the analyte, free from interference of the endogenous contribution. This de facto correction has been applied in a production setting on two BHB quantification methods (GC-MS and LC–MS-MS), allowing the rectification of BHB biases of up to 30 μg/mL. The additional error introduced by this correction procedure is minimal, although the exact amount will be highly method-dependent. The endogenous concentration correction process has been automated with an R script. The final procedure is therefore highly efficient, only adding four mouse clicks to the data analysis operations

Interférence de l’hémoglobine foetale dans la quantification de la carboxyhémoglobine par spectrophotométrie

La quantification de la carboxyhémoglobine (HbCO) est régulièrement effectuée lorsqu’une intoxication au monoxyde de carbone est soupçonnée, ainsi que dans les cas de morts inexpliquées. Cependant, certains auteurs ont soulevé la problématique de la fausse élévation de la HbCO mesurée chez les enfants (0 à 12 mois), due à la présence d’hémoglobine foetale (HbF). Le but de cette étude est d’évaluer l’impact de l’hémoglobine foetale sur la quantification de la carboxyhémoglobine par spectrophotométrie. L’interférence de l’HbF dans la quantification de la HbCO chez les enfants âgés de 0 à 12 mois a été évaluée en utilisant 16 échantillons de sang ante-mortem et 19 échantillons de sang post-mortem. Le pourcentage de HbCO (%HbCO) a été quantifié par spectrophotométrie en calculant le ratio d’absorbance 560 nm/530 nm, en utilisant un spectrophotomètre à double faisceau. La moyenne des mesures de HbCO chez les enfants de 3 mois et moins était de 17%, ce qui est anormalement élevé. Aucune différence significative dans la mesure de la HbCO n’a été déterminée entre les échantillons ante-mortem et post-mortem