Refining Stable Oxygen and Hydrogen Isoscapes for the Identification of Human Remains in Mississippi,

Isoscape refinement is an essential component for accurately predicting region‐of‐origin in forensic investigations involving isotope analysis of unidentified human remains. Stable oxygen (δ¹⁸O) and hydrogen (δ²H) isotopes were measured from 57 tap water samples collected across Mississippi to model refined isoscapes for the state. A tap water conversion equation, δ¹⁸O_tw=1.64 δ¹⁸Op?31.35, was developed for the southeastern USA to test the prediction accuracy of the δ¹⁸O_tw isoscape using individuals with known residential histories. A local Mississippi resident (USAFA‐134) was assigned with 90% probability to the correct region‐of‐origin reported by the participant. Assignments for Georgia residents (USAFA‐118 and USAFA‐205) had variable results, predicting USAFA‐118 from Mississippi and USAFA‐205 as a nonlocal resident. Stable isotope values often overlap geographically and a multi‐isotope approach should be used when narrowing region(s)‐of‐origin(s). This study demonstrates the utility of refining isoscapes and the importance of tissue calibration in prediction assignments of human remains.     

Fatal Overdose of Gamma‐hydroxybutyrate Acid After Ingestion of 1,4‐Butanediol

We report a case of fatal intoxication from 1,4‐butanediol (1,4‐BD), which was ingested by a young and “naïve” gamma‐hydroxybutyrate (GHB) consumer during a party with the co‐ingestion of alcohol, cannabis, and methylene‐dioxy‐methamphetamine. The following drug concentrations were found using gas chromatography coupled with mass spectrometry on autopsy samples and on a cup and a glass found at the scene: 20,350 mg/L (bottle) for 1,4‐BD; 1020 mg/L (femoral blood), 3380 mg/L (cardiac blood), 47,280 mg/L (gastric content), and 570 mg/L (vitreous humor) for GHB. The concentration of GHB is difficult to interpret in forensic cases due to the possibility of an endogenous production of GHB. The variable tolerance of the user may also modify the peri‐ and postmortem GHB concentrations. This case underscores the need to have many different sources of toxicology samples analyzed to avoid the hypothesis of endogenous production of GHB.     

Evaluation of the Role of Toxicological Data in Discriminating Between H2S Femoral Blood Concentration Secondary to Lethal poisoning and Endogenous H2S Putrefactive Production

Hydrogen sulfide is a colorless gas and has a strong odor of rotten eggs. It is absorbed by the upper respiratory tract mucosa, and it causes histotoxic hypoxemia and respiratory depression by exerting an inhibitory effect on cytochrome oxidase. To evaluate the role of toxicological data in distinguishing between the H₂S blood concentration secondary to lethal poisoning and the endogenous H₂S produced during putrefaction, we compared the postmortem H₂S concentrations of six fatal H₂S poisoning cases (8.7–28.6 mg/L) with the postmortem concentrations of endogenous H₂S of 12 subjects who died from other causes (traffic‐related deaths) (2.2–32.7 mg/L). These results will be of interest to the forensic community as it underlines the importance of considering circumstantial evidence along with the toxicological and pathological findings in the identification of H₂S lethal poisoning.     

Stability of Morphine,Codeine, and 6‐Acetylmorphine in Blood at Different Sampling and Storage Conditions

The stability of drugs in biological specimens is a major concern during the evaluation of the toxicological results. The stability of morphine, codeine, and 6‐acetyl‐morphine in blood was studied after different sampling conditions: (i) in glass, polypropylene or polystyrene tubes, (ii) with addition of dipotassium ethylene diamine tetraacetic acid (K₂EDTA) or sodium oxalate (Na₂C₂O₄), and (iii) with or without the addition of sodium fluoride (NaF). Spiked blood samples were stored at two different temperatures (4 and ?20^°C), analyzed after different storage times and after three freeze–thaw cycles. Opiate concentrations were decreased in all conditions, but the most unstable was 6‐acetyl‐morphine. The addition of NaF as preservative improved the stability of opiates at all conditions studied, whereas the type of anticoagulant did not affect the stability of opiates. It was concluded that blood samples should be stored at ?20^°C in glass tubes containing oxalate and NaF for maximum stability.     

Butalbital and Driving Impairment

Butalbital (Fiorinal^®), used in the treatment of migraines and muscle pain, is the most commonly encountered barbiturate in impaired driving cases. It has central nervous system (CNS) depressant properties, including sedation, drowsiness, and feelings of intoxication, which can contribute to driving impairment. Twenty‐six driving under the influence cases are reviewed including results from field sobriety tests and toxicology testing. Blood samples were screened using enzyme multiplied immunoassay technique immunoassay, and the presence of butalbital was confirmed and quantified using gas chromatography/mass spectrometry, gas chromatography with flame ionization detection, or gas chromatography nitrogen/phosphorus detection. Butalbital concentrations ranged from 1.0 to 30.2 mg/L, with a mean and median of 16.0 mg/L. General impairment indicators in these cases included horizontal and vertical nystagmus, lack of convergence, poor motor coordination, and balance and speech problems, which are common to CNS depressant intoxication, similar to that associated with alcohol. These findings indicate the importance of toxicological testing for butalbital in cases where CNS depressants are indicated.     

Investigating Residential History Using Stable Hydrogen and Oxygen Isotopes of Human Hair and Drinking Water

The relationship between isotopic signals in human hair and geographic region has potential forensic applications for identifying unknown individuals' place of recent residence. This study analyzes δ²H and δ¹⁸O isotopes in residential tap water and bulk hair samples from 17 volunteers representing 12 locations in Ontario, Canada. There is a strong correlation (R² = 0.9) between δ²H and δ¹⁸O values of the water samples. In contrast, the δ²H and δ¹⁸O values of the hair samples are weakly correlated (R² = 0.3), and the greater variability in the data is linked to dietary factors. This study demonstrates that the δ²H and δ¹⁸O values of hair and drinking water can be used to help identify potential place of residence in forensic cases, particularly in relation to proximity to large bodies of water such as the Great Lakes, but interpretations are complicated by the contribution of both water and diet to δ²H and δ¹⁸O values in hair.     

Estimating Time Since Death from Postmortem Human Gut Microbial Communities

Postmortem succession of human‐associated microbial communities (“human microbiome”) has been suggested as a possible method for estimating postmortem interval (PMI) for forensic analyses. Here we evaluate human gut bacterial populations to determine quantifiable, time‐dependent changes postmortem. Gut microflora were repeatedly sampled from the proximal large intestine of 12 deceased human individuals as they decayed under environmental conditions. Three intestinal bacterial genera were quantified by quantitative PCR (qPCR) using group‐specific primers targeting 16S rRNA genes. Bacteroides and Lactobacillus relative abundances declined exponentially with increasing PMI at rates of N_t = 0.977e^?0.0144t (r² = 0.537, p < 0.001) and N_t = 0.019e^?0.0087t (r² = 0.396, p < 0.001), respectively, where N_t is relative abundance at time (t) in cumulative degree hours. Bifidobacterium relative abundances did not change significantly: N_t = 0.003e^?0.002t (r² = 0.033, p = 0.284). Therefore, Bacteroides and Lactobacillus abundances could be used as quantitative indicators of PMI.     

Validation of an LC–MS Method for the Detection and Quantification of BZP and TFMPP and their Hydroxylated Metabolites in Human Plasma and its Application to the Pharmacokinetic Study of TFMPP in Humans*

Abstract: An LC–MS method was developed for benzylpiperazine (BZP) and trifluoromethylphenylpiperazine (TFMPP), constituents of “party pills” or “legal herbal highs,” and their metabolites in human blood plasma. Compounds were resolved using a mixture of ammonium formate (pH 4.5, 0.01 M) and acetonitrile (flow rate of 1.0 mL/min) with a C18 column. Calibration curves were linear from 1 to 50 ng/mL (R² > 0.99); the lower limit of quantification (LLOQ) was 5 ng/mL; the accuracy was >90%; the intra‐ and interday relative standard deviations (R.S.D) were <5% and <10%, respectively. Human plasma concentrations of TFMPP were measured in blood samples taken from healthy adults (n = 6) over 24 h following a 60‐mg oral dose of TFMPP: these peaked at 24.10 ng/mL (±1.8 ng/mL) (C_max) after 90 min (T_max). Plasma concentrations of 1‐(3‐trifluoromethyl‐4‐hydroxyphenyl) piperazine peaked at 20.2 ng/mL (±4.6 ng/mL) after 90 min. TFMPP had two disposition phases (t_½ = 2.04 h (±0.19 h) and 5.95 h (±1.63 h). Apparent clearance (Cl/F) was 384 L/h (±45 L/h).     

A novel method for cyanide quantification in human whole blood using ion chromatography with amperometric detection and its application to cyanide intoxication cases

Cyanide is a highly toxic agent that has been frequently used for suicide in South Korea. It is also used in various industrial fields, such as metal plating, in which many accidental cyanide intoxications have occurred. To overcome the disadvantages of conventional cyanide analysis methods, a simple and fast method for the analysis of cyanide in whole blood using ion chromatography (IC) with amperometric detection was developed in this study. Whole blood samples were deproteinized, diluted, and analyzed using an IC–amperometric detection system. The limits of detection and quantitation were 0.1 and 0.2 mg/L, respectively. The method showed good linearity in the range of 0.2 to 50 mg/L with R² > 0.99. The intra- and inter-assay precision and accuracy values were <10%. The established method was successfully applied to analyze whole blood samples from three cyanide intoxication cases.     

A City‐wide Investigation of the Isotopic Distribution and Source of Tap Waters for Forensic Human Geolocation Ground‐truthing

Human geolocation is prefaced on the accuracy of the geographic precision of mapped isotopic values for drinking water. As most people live in cities, it becomes important to understand city water supplies and how the isotopic values uniquely reflect that city. This study investigated the isotopic distribution of δ²H and δ¹⁸O from sourced tap waters that were collected from across the Metro Vancouver (MV) area (n = 135). The results revealed that the isotopic values reflect their water sources with a range of 5.3‰ for δ¹⁸O_tap and 29.3‰ for δ²H_tap for MV. The results indicate that individual cities need higher resolution studies to determine their tap water isotopic ranges, and a good understanding of the water supply network itself for human geolocation work. With an extended high‐resolution understanding of each city, human tissue may be compared with more certainty for geolocation.     

Comparative analysis of 1-phenyl-2-propanone (P2P), an amphetamine-type stimulant precursor, using stable isotope ratio mass spectrometry: Presented in part as a poster at the 2nd meeting of the Joint European Stable Isotope User Meeting (JESIUM), Giens, France, September 2008

The isotope ratios of amphetamine type stimulants (ATS) depend as well on the precursor as the synthetic pathway. For clandestine production of amphetamine and methamphetamine, 1-phenyl-2-propanone (P2P, benzylmethylketone) is a commonly used precursor.Our aim was to determine the variation of the isotope ratios within precursor samples of one manufacturer and to compare seized samples of unknown sources to these values. δ¹³C_V-PDB, δ²H_V-SMOW and δ¹⁸O_V-SMOW isotope ratios were determined using elemental analysis (EA) and gas chromatography (GC) coupled to an isotope ratio mass spectrometer (IRMS). The comparison of all seized samples to the data of the samples of one manufacturer revealed considerable differences. The results show that IRMS provides a high potential in differentiating between precursors from different manufacturers for the clandestine production of ATS and identifying corresponding sources.     

The Potential of Isotope Ratio Mass Spectrometry (IRMS) and Gas Chromatography‐IRMS Analysis of Triacetone Triperoxide in Forensic Explosives Investigations

Studying links between triacetone triperoxide (TATP) samples from crime scenes and suspects can assist in criminal investigations. Isotope ratio mass spectrometry (IRMS) and gas chromatography (GC)‐IRMS were used to measure the isotopic compositions of TATP and its precursors acetone and hydrogen peroxide. In total, 31 TATP samples were synthesized with different raw material combinations and reaction conditions. For carbon, a good differentiation and a linear relationship were observed for acetone–TATP combinations. The extent of negative (δ¹³C) fractionation depended on the reaction yield. Limited enrichment was observed for the hydrogen isotope (δ²H) values of the TATP samples probably due to a constant exchange of hydrogen atoms in aqueous solution. For oxygen (δ¹⁸O), the small isotopic range and excess of water in hydrogen peroxide resulted in poor differentiation. GC‐IRMS and IRMS data were comparable except for one TATP sample prepared with high acid concentration demonstrating the potential of compound‐specific isotope analysis. Carbon IRMS has practical use in forensic TATP investigations.     

Determination of Herbicides Paraquat,Glyphosate, and Aminomethylphosphonic Acid in Marijuana Samples by Capillary Electrophoresis

In this work, two methods were developed to determine herbicides paraquat, glyphosate, and aminomethylphosphonic acid (AMPA) in marijuana samples by capillary electrophoresis. For paraquat analysis, sample was extracted with aqueous acetic acid solution and analyzed by capillary zone electrophoresis with direct UV detection. The running electrolyte was 50 mmol/L phosphate buffer (pH 2.50). For glyphosate and AMPA, indirect UV/VIS detection was used, as these substances do not present chromophoric groups. Samples were extracted with 5 mmol/L hydrochloric acid. The running electrolyte was 10 mmol/L gallic acid, 6 mmol/L TRIS, and 0.1 mmol/L CTAB (pH = 4.7). The methods presented good linearity, precision, accuracy, and recovery. Paraquat was detected in 12 samples (n = 130), ranging from 0.01 to 25.1 mg/g. Three samples were positive for glyphosate (0.15–0.75 mg/g), and one sample presented AMPA as well. Experimental studies are suggested to evaluate the risks of these concentrations to marijuana user.     

A Hydrologic Retention System and Water Quality Monitoring Program for a Human Decomposition Research Facility: Concept and Design

Forensic taphonomy is an essential research field; however, the decomposition of human cadavers at forensic science facilities may lead to nutrient loading and the introduction of unique biological compounds to adjacent areas. The infrastructure of a water retention system may provide a mechanism for the biogeochemical processing and retention of nutrients and compounds, ensuring the control of runoff from forensic facilities. This work provides a proof of concept for a hydrologic retention system and an autonomous water quality monitoring program designed to mitigate runoff from The Southeast Texas Applied Forensic Science (STAFS) Facility. Water samples collected along a sample transect were analyzed for total phosphorous, total nitrogen, , , NH₄, F^?, and Cl^?. Preliminary water quality analyses confirm the overall effectiveness of the water retention system. These results are discussed with relation to how this infrastructure can be expanded upon to monitor additional, more novel, byproducts of forensic science research facilities.     

Monitoring of Levamisole Concentration in Serum of Traffic Participants after Cocaine Consumption

This study highlights the problem of levamisole‐adulterated cocaine in context of active traffic participation. For the purposes of levamisole concentration monitoring in human serum, an analytical method based on LC‐MS/MS and solid‐phase extraction was applied. A Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm column and a mobile phase consisting of A (H₂O/methanol = 95/5, v/v) and B (H₂O/methanol = 3/97, v/v), both with 10 mM ammonium acetate and with 0.1% acetic acid (pH = 3.2), were used. The validation experiments demonstrated that the method applied was appropriate for levamisole quantification in human serum. For 23% of levamisole‐positive samples, the concentrations exceeded 20 ng/mL. Therefore, the interaction of this drug with cocaine has to be considered as important for active traffic participation. As a consequence, monitoring of levamisole concentration in human serum is recommended, as long as it is used as cocaine adulterant.     

The Effects of Different Maceration Techniques on Nuclear DNA Amplification Using Human Bone

Abstract: Forensic anthropologists routinely macerate human bone for the purposes of identity and trauma analysis, but the heat and chemical treatments used can destroy genetic evidence. As a follow‐up to a previous study on nuclear DNA recovery that used pig ribs, this study utilizes human skeletal remains treated with various bone maceration techniques for nuclear DNA amplification using the standard Combined DNA Index System (CODIS) markers. DNA was extracted from 18 samples of human lower leg bones subjected to nine chemical and heat maceration techniques. Genotyping was carried out using the AmpF?STR^® COfiler^® and AmpF?STR^® Profiler Plus^® ID kits. Results showed that heat treatments via microwave or Biz/Na₂CO₃ in sub‐boiling water efficiently macerate bone and produce amplifiable nuclear DNA for genetic analysis. Long‐term use of chemicals such as hydrogen peroxide is discouraged as it results in poor bone quality and has deleterious effects on DNA amplification.     

Mineralogical and elemental data for soil discriminating and geolocation tracing

One of the key tasks of soil analysis in forensic sciences is to provide information about its diversities and geolocation. In fact, soil analysis is relevant for forensic geologists. In this study, a total of 80 soil samples were collected from eight Chinese cities (10 samples per city). Different minerals and their relative percentages were analyzed by the X-ray diffraction (XRD) method. In addition, the relative amounts of montmorillonite, kaolinite, amphibole, feldspar, calcite, and dolomite provided information about the origin of a soil, either if it came from a northern or southern city of China. The oxide weight percentages of 10 elements of Al₂O₃, SiO_2, Fe₂O₃, K₂O, Na₂O, MgO, CaO, P₂O₅, MnO, and TiO₂ were also obtained by using X-ray fluorescence (XRF) from the 80 soil samples. Moreover, principal component analysis (PCA) and hierarchical clustering analysis (HCA) methods were performed for dimensionality reduction, elemental marker identification and soils classification to the city they came from purposes. The eighty soils analyzed in this study could be tracked correctly to their city of origin. The K-Nearest Neighbors (KNN) model was done to evaluate the prediction ability based on the soil elemental composition, and it was confirmed by cross validation methods. The results demonstrated that mineralogical and elemental composition can provide powerful information for soil discrimination and source tracing.     

Development of Rapid and Economical Colorimetric Screening Method for p‐Phenylenediamine in Variety of Biological Matrices and its Application to Eleven Fatal Cases of p‐Phenylenediamine Poisoning

A rapid colorimetric method for detection of p‐phenylenediamine (PPD) in various biological samples is developed. The o‐cresol test for acetaminophen detection has been modified to detect PPD in blood, urine, gastric contents, and liver. After precipitating protein with trichloroacetic acid solution (2 mL, 10% w/v), biological specimens were required to convert PPD metabolites to PPD by acid hydrolysis. Finally, o‐cresol solution (1 mL, 1% w/v), hydrogen peroxide (200 μL, 3%v/v), and concentrated ammonium hydroxide (0.5 mL) were added in the biological samples. The presence of PPD was indicated by formation of violet color which was turned to bluish green color within 10–15 min. The limit of detection was found to be 2 mg/L in blood, urine, and gastric contents and 2 mg/Kg in liver. This method is also free from any potential interference by p‐aminophenol, acetaminophen, and other amine drugs under test conditions. This method was successfully employed to thirteen fatal cases of PPD poisoning.     

Developmental Validation of the PrepFiler™ Forensic DNA Extraction Kit for Extraction of Genomic DNA from Biological Samples*

Abstract: The PrepFiler? Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 μL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA^® paper), and touch evidence‐type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol–chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.