Identification of Crystals Forming on Porcine Articular Cartilage: A New Method for the Estimation of the Postmortem Interval

Articular cartilage was examined to determine its decomposition sequence and its potential for assessing the postmortem interval. Scanning electron microscopy of articular cartilage from buried porcine trotters showed the presence of microcrystals on the synovial surface. These orthorhombic pyramidal or “coffin”‐shaped crystals, appeared at 3 weeks (22 days) after interment and disappeared after 6 weeks. The disappearance of these crystals was linked to decompositional changes to the integrity of the synovial joint. The formation and disappearance of these crystals was associated with a pH change at the cartilage surface. Scanning electron microscopy–energy‐dispersive X‐ray (SEM‐EDX) analysis showed that the five main elements contained within these crystals were carbon, nitrogen, oxygen, magnesium, and phosphorous. Such elemental analysis suggested the crystals may be struvite (MgNH₄PO₄6(H₂O)). Bacteria cultured from the cartilage synovial surface produced struvite crystals when grown in suitable media and were identified by DNA analysis to be Comamonas sp.     

Analysis of Ecstasy Tablets Using Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection

A method for the identification of 3,4‐methylenedioxymethamphetamine (MDMA) and meta‐chlorophenylpiperazine (mCPP) was developed employing capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D). Sample extraction, separation, and detection of “Ecstasy” tablets were performed in <10 min without sample derivatization. The separation electrolyte was 20 mm TAPS/Lithium, pH 8.7. Average minimal detectable amounts for MDMA and mCPP were 0.04 mg/tablet, several orders of magnitude lower than the minimum amount encountered in a tablet. Seven different Ecstasy tablets seized in Rio de Janeiro, Brazil, were analyzed by CE‐C⁴D and compared against routine gas chromatography‐mass spectrometry (GC‐MS). The CE method demonstrated sufficient selectivity to discriminate the two target drugs, MDMA and mCPP, from the other drugs present in seizures, namely amphepramone, fenproporex, caffeine, lidocaine, and cocaine. Separation was performed in <90 sec. The advantages of using C⁴D instead of traditional CE‐UV methods for in‐field analysis are also discussed.     

Voltammetric determination of cocaine in confiscated samples using a cobalt hexacyanoferrate film-modified electrode

In this work, a fast, non destructive voltammetric method for cocaine detection in acetonitrile medium using a platinum disk electrode chemically modified with cobalt-hexacyanoferrate (CoHCFe) film is described. The deposition of CoHCFe film at platinum disk (working electrode) was carried out in aqueous solution containing NaClO₄ at 0.1 mol L⁻¹ as supporting electrolite. Stability studies of the film and subsequent voltammetric analysis of cocaine were made in acetonitrile medium with NaClO₄ at 0.1 mol L⁻¹ as supporting electrolite. A reversible interaction between cocaine and CoHCFe at the film produces a proportional decrease of original peak current, due to the formation of a complex between cocaine and cobalt íons, with subsequent partial passivation of the film surface, being the intensity of current decrease used as analytical signal for cocaine. A linear dependence of cocaine detection was carried out in the range from 2.4 × 10⁻⁴ to 1.5 × 10⁻³ mol L⁻¹, with a linear correlation coefficient of 0.994 and a detection limit of 1.4 × 10⁻⁴ mol L⁻¹. The analysis of confiscated samples by the proposed method indicated cocaine levels from 37% to 95% (m/m) and these results were validated by comparison to HPLC technique, being obtained good correlation between both methods.     

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.     

Characterization and Differentiation of Geometric Isomers of 3‐methylfentanyl Analogs by Gas Chromatography/Mass Spectrometry,Liquid Chromatography/Mass Spectrometry,and Nuclear Magnetic Resonance Spectroscopy

The cis and trans isomers of 3‐methylfentanyl and its three analogs were chemically synthesized, and these compounds were characterized and differentiated by gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), and nuclear magnetic resonance (NMR) spectroscopy. The cis and trans isomers of the 3‐methylfentanyl analogs were completely separated by GC/MS. Although the high temperature of the GC injection port caused thermal degradation of β‐hydroxy‐3‐methylfentanyl, the degradation was completely suppressed by trimethylsilyl derivatization. The isomers were also well separated by LC/MS on an octadecylsilyl column with 10 mM ammonium acetate and methanol as the mobile phase. The proton NMR signals were split when the hydrochloride salts of the 3‐methylfentanyl analogs were dissolved in deuterated chloroform because stereoisomers were formed by the coordination of the hydrochloride proton to the nitrogen of the piperidine ring of the 3‐methylfentanyl analogs.     

Nondestructive Differentiation of Polyester Single White Fibers Using Synchrotron Radiation Microbeam X-ray Fluorescence Spectrometry with Vertical Focusing

In this study, the nondestructive differentiation of individual white polyester clothing fibers was accomplished via synchrotron radiation microbeam X-ray fluorescence (SR-μ-XRF) analysis. SR-μ-XRF with vertical focusing is a useful nondestructive method for the analysis of a single polyester clothing fiber. Kirkpatrick–Baez (KB) mirror was used to vertically focus 20 keV X-rays for the analysis of 22 individual white polyester fibers taken from clothing commonly sold in Japan. SR-μ-XRF with a vertical focused 2 μm (V) × 300 μm (H) beam was approximately 12.8 times more sensitive than SR-XRF with an unfocused 300 μm (V) × 300 μm (H) beam for the detection of elements in single fibers. The minimum detection limits (MDLs) of the SR-μ-XRF method were 8.15 ppm for Cl and 0.06 ppm for Br. In addition to Ti in TiO₂ delustering agents, Zr and Nb impurities in the delustering agents were detected in individual fibers. Sb from a polymerization catalyst and Co from a transesterification catalyst were also detected in individual fibers. Comparing the Ti K_β/Sb L_α,β and Zr K_α/Nb K_α X-ray intensity ratios was a useful way to distinguish individual clothing fibers, and 98% of the fibers were differentiated when additional trace elements were used as discrimination indicators.     

Piercing the surface: A mechanical analysis of stabbing with household tools

Estimating the applied power during a stabbing incident, or estimating the minimal force necessary to penetrate the body with a certain weapon is a challenging task in forensic practice. A thorough forensic evaluation of stabbing forces needs objective numerical experimental data. Stabbing tests of 12 different weapons – including knives, a pair of scissors, a fork, screwdrivers, a rasp, a corkscrew, and a utility knife blade – were performed with a Mecmesin MultiTest-dV material tester on pork loin and ballistic gel to estimate the stabbing forces and dynamics. Penetration force (F_p) and maximal force (F_max) were recorded, and the registered force curves were analyzed. F_max was 159.8–212.07 Newton (N), 30.56–30.58 N, 168.9–185.48 N for various knives; 171.39–190.43 N for the pair of scissors, 233.6 N for the fork; 532.65–562.65 N, 370.31–367.19 N and 314.51–432.89 N for various screwdrivers, 44.14–56.62 N for utility knife during pork loin stabbing. The butter knife, corkscrew and rasp were not able to penetrate the pork loin, and the curved fork bent during stabbing. The results prove that weapon characteristics greatly influence the force necessary for penetration. Maximal stabbing force depends mostly on tip sharpness, and the force sharply decreases after penetration occurs, which indicates that edge sharpness is not as important as tip characteristics during stabbing perpendicular to skin surface. The penetration force during stabbing with a pair of scissors is comparable to the penetration force of knives. Stabbing with screwdrivers generally needs larger force than average knives but depends greatly on screwdriver size.     

Identifying reliable ions for the statistical differentiation of structurally similar fentanyl analogs

Definitive identification of fentanyl analogs based on mass spectral comparison is challenging given the high degree of structural and, hence, spectral similarity. To address this, a statistical method was previously developed in which two electron-ionization (EI) mass spectra are compared using the unequal variance t-test. Normalized intensities of corresponding ions are compared, testing the null hypothesis (H₀) that the difference in intensity is equal to zero. If H₀ is accepted at all m/z values, the two spectra are statistically equivalent at the specified confidence level. If H₀ is not accepted at any m/z value, then there is a significant difference in intensity at that m/z value between the two spectra. In this work, the statistical comparison method is applied to distinguish EI spectra of valeryl fentanyl, isovaleryl fentanyl, and pivaloyl fentanyl. Spectra of the three analogs were collected over a 9-month period and at different concentrations. At the 99.9% confidence level, the spectra of corresponding isomers were statistically associated. Spectra of different isomers were statistically distinct, and ions responsible for discrimination were identified in each comparison. To account for inherent instrument variations, discriminating ions for each pairwise comparison were ranked based on the magnitude of the calculated t-statistic (t_calc) value. For a given comparison, ions with higher t_calc values are those with the greatest difference in intensity between the two spectra and, therefore, are considered more reliable for discrimination. Using these methods, objective discrimination among the spectra was achieved and ions considered most reliable for discrimination of these isomers were identified.     

Analysis of Δ-tetrahydrocannabinol in oral fluid samples using solid-phase extraction and high-performance liquid chromatography–electrospray ionization mass spectrometry

An analytical method using solid-phase extraction (SPE) and high-performance liquid chromatography–mass spectrometry (LC–MS) has been developed and validated for the confirmation of Δ⁹-tetrahydrocannabinol (THC) in oral fluid samples. Oral fluid was extracted using Bond Elut LRC-Certify solid-phase extraction columns (10 cm³, 300 mg) and elution performed with n-hexane/ethyl acetate. Quantitation made use of the selected ion-recording mode (SIR) using the most abundant characteristic ion [THC + H⁺], m/z 315.31 and the fragment ion, m/z 193.13 for confirmation, and m/z 318.00 for the protonated internal standard, [d₃-THC + H⁺]. The method proved to be precise for THC, in terms of both intra-day and inter-day analyses, with coefficients of variation less than 10%, and the calculated extraction efficiencies for THC ranged from 76 to 83%. Calibration standards spiked with THC between 2 and 100 ng/mL showed a linear relationship (r² = 0.999). The method presented was applied to the oral fluid samples taken from the volunteers during the largest music event in Portugal, named Rock in Rio-Lisboa. Oral fluid was collected from 40 persons by expectoration and with Salivette^®. In 55% of the samples obtained by expectorating, THC was detected with concentration ranges from 1033 to 6552 ng/mL and in 45% of cases THC was detected at concentrations between 51 and 937 ng/mL. However, using Salivette^® collection, 26 of the 40 cases had an undetectable THC.     

Molecular Assay for Screening and Quantifying DNA in Biological Evidence: The Modified Q‐TAT Assay*

Abstract: A method is described for the quantitation of total human and male DNA. Q‐TAT utilizes end‐point, multiplex polymerase chain reaction (PCR) amplification of the amelogenin and SRY loci to quantify DNA and incorporates a cloned nonhuman template to detect PCR inhibition. Standard curves of fluorescence from amelogenin or SRY amplicons were generated from amplification of known amounts of NIST traceable SRM‐female or SRM‐male DNA. Curves showed good linearity up to 500 pg of SRM‐template (R² > 0.99) and reliably estimated total and male DNA content in casework samples. The nonhuman pRL_null template included in each PCR was a sensitive indicator of known PCR inhibitors including EDTA, hemin, blue denim dye, and humic acid. Finally, the SRY amplicon was a sensitive indicator of male DNA and, in mixtures, could reliably estimate male DNA present in an excess of female DNA. The Q‐TAT multiplex is a reliable quantitation method for forensic DNA typing.     

Counterfeit Adderall Containing Aceclofenac from Internet Pharmacies

A nontargeted approach based on liquid chromatography equipped with a quadrupole time‐of‐flight mass detector (LC‐MS Q‐TOF) joined to nuclear magnetic resonance (NMR) analysis allowed rapid identification and quantification of the anti‐inflammatory drug aceclofenac in illegal Adderall tablets. The largest chromatographic peak had m/z = 354.030 and m/z = 376.012 matching, respectively, the ionic structures (M + H)⁺ and (M + Na)⁺ of a molecule M. The accurate mass data generated the molecular formula C₁₆H₁₃Cl₂NO₄. A screening of the pharmaceutical active substances having that molecular formula together with the MS/MS fragmentation pattern suggested aceclofenac. Aceclofenac structure was unambiguously confirmed by ¹H and ¹³C NMR experiments. The aceclofenac content was 90 mg/tablet (RSD 2%) as detected by quantitative NMR. Information on the identity and content of illegal drugs is required for legal purposes; it supports in evaluating the effective impact on users safety, and it is useful for control laboratories using a targeted approach in their analytical activities.     

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.     

Evaluation of the composition of street cocaine seized in two regions of Brazil

This work evaluates cocaine purity and the concentration ranges of adulterants and inorganic constituents for 31 street cocaine samples seized in two different regions of Brazil from July 2008 to May 2010. Cocaine and adulterants, such as caffeine, lidocaine and benzocaine, were quantified by Gas chromatography–mass spectrometry (GC–MS), and the inorganic constituents were determined by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) and ion chromatography (IC). The cocaine concentrations in the samples seized in the Amazonas state (AM samples) ranged from 154 to 978 mg g^? 1, and these samples did not contain any of the adulterants studied. The cocaine concentrations in the samples seized in the Minas Gerais state (MG samples) ranged from 63.9 to 753 mg g^? 1. Caffeine was the main adulterant found in 76% of the MG samples, ranging in concentration from 5.5 to 645.3 mg g^? 1. Lidocaine was found in 66.7% of the MG samples, with concentrations ranging from 16.3 to 576.7 mg g^? 1. Benzocaine was found in only one MG sample, at a concentration of 84.8 mg g^? 1. Fourteen elements were identified by ICP-OES, and a wide variation was observed in the concentrations of Ca, Mg, Na, P, Al, Fe, Mn and Zn. Pearson Product–moment Correlations between the analytes allowed the constituents to be associated with the chemicals used in the manufacturing of cocaine and with some common diluents. The study of the purity of cocaine and the presence and concentration of adulterants and inorganic constituents is important because the latter can have deleterious effects on health.     

Estimating postmortem interval for human cadavers in a sub‐tropical climate using UV‐Vis‐near‐infrared Spectroscopy

Estimating postmortem interval (PMI) of surface found skeletal remains is challenging. This novel study used UV‐Vis‐NIR spectroscopy to scan soil collected from cadaver decomposition islands (CDIs) ranging from 15‐ to 963‐d postmortem and control soils. A decomposition product spectra model (DPS model) was constructed by deducting the control soil spectra from the CDI soil spectra for the estimation of postmortem indices: PMI (d), ADD₄, ADD₁₀, and ADD₂₀. The DPS model (n = 55) was calibrated and subjected to a full cross‐validation. Calibration R² and RPD for the DPS model ranged from 0.97 to 0.99 and from 6.1 to 9.9, respectively, for the four postmortem interval indices. Validation R² and RPD for the DPS model ranged from 0.73 to 0.80 and from 1.9 to 2.2, respectively. The DPS model estimated postmortem intervals for three test CDIs in a clay soil under perennial grassland (test set 1; n = 3) and six CDIs in a sandy soil under a loblolly pine forest (test set 2; n = 6). Test set 1 had PMI prediction ranges from ?69 to ?117 days, ?796 to +832 ADD₄, +552 to +2672 ADD₁₀, and ?478 to ?20 ADD₂₀ of observed PMI. Test set 2 PMI prediction ranged from ?198 to ?65 days, ?9923 to +2629 ADD₄, ?6724 to +1321 ADD₁₀, and ?2850 to +540 ADD₂₀ of observed PMI. Test set 2 had poor predictions for two CDIs, for all measures of postmortem indices resulting in discussion of sampling depth, effect of body mass index (BMI), and scavenging.     

Constructing STR Multiplexes for Individual Identification of Hungarian Red Deer

Red deer is the most valuable game of the fauna in Hungary, and there is a strong need for genetic identification of individuals. For this purpose, 10 tetranucleotide STR markers were developed and amplified in two 5‐plex systems. The study presented here includes the flanking region sequence analysis and the allele nomenclature of the 10 loci as well as the PCR optimization of the DeerPlex I and II. LD pairwise tests and cross‐species similarity analyses showed the 10 loci to be independently inherited. Considerable levels of genetic differences between two subpopulations were recorded, and F_ST was 0.034 using AMOVA. The average probability of identity (PI_ave) was at the value of 2.6736 × 10^?15. This low value for PI_ave nearly eliminates false identification. An illegal hunting case solved by DeerPlex is described herein. The calculated likelihood ratio (LR) illustrates the potential of the 10 red deer microsatellite markers for forensic investigations.     

Physical Matching of Metals: Grain Orientation Association at Fracture Edge

The objective of this study was to examine whether surface crystal orientation can be used to associate or differentiate metal fracture fragments. The orientations of individual crystals and crystals across the fracture plane were measured on polished steel and iron alloy surfaces using Electron Backscattered Diffraction/ Orientation Imaging Microscopy (EBSD/OIM). This investigation examined crystallographic characteristics within a metal. This study showed that for transgranular fracture, it is feasible that pieces of grains could be associated across the fracture surface with the difference in orientation between grains (misorientation) along a length sequence of grain orientations on one side of the fracture surface to associate the other side of a fracture surface. Regarding pair comparisons of crystals on fracture surfaces, it was estimated that the probability for an ordered sequence of six distinct oriented grains along a fracture surface to occur again is about 1 in 4.82 (10)³⁰ or 2.07 (10)⁻³¹.     

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.     

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.     

Discrimination Between Infant and Adult Bloodstains Using Micro‐Raman Spectroscopy: A Preliminary Study

In the present study, we used micro‐Raman spectroscopy with high‐resolution analysis to discriminate between bloodstains from infants and bloodstains from adults. Raman peaks were detected at 674, 754, 976, 1002, 1105, 1127, 1176, 1248, 1340, 1368, 1390, 1560, and 1611 cm^?1; these peaks were derived from hemoglobin, albumin, and glucose. However, a peak was obtained at 1105 cm^?1, which was assigned to histidine; this peak was observed only for bloodstains from adults. Human adult hemoglobin (HbA) is composed of an α₂β₂ tetramer structure, whereas human fetal hemoglobin (HbF) is composed of an α₂γ₂. Therefore, the lack of a Raman peak at 1105 cm^?1 in bloodstains from infants indicates the possibility of two histidine substitutions (His116Ile and His143Ser) in the γ chain of HbF. This study discriminates between bloodstains from infants and bloodstains from adults using micro‐Raman spectroscopy, with beneficial implications in forensic science.     

Infrared Spectra of U.S. Automobile Original Finishes (Post – 1989). VIII: In Situ Identification of Bismuth Vanadate Using Extended Range FT‐IR Spectroscopy,Raman Spectroscopy,and X‐Ray Fluorescence Spectrometry

Chrome Yellow (PbCrO₄·xPbSO₄) was a common pigment in U.S. automobile OEM finishes for more than three decades, but in the early 1990s its use was discontinued. One of its main replacements was Bismuth Vanadate (BiVO₄·nBi₂MoO₆, n = 0–2), which was commercially introduced in 1985, as this inorganic pigment also produces a very bright hue and has excellent outdoor durability. This paper describes the in situ identification of Bismuth Vanadate in automotive finishes using FT‐IR and dispersive Raman spectroscopy and XRF spectrometry. Some differentiation of commercial formulations of this pigment is possible based on far‐infrared absorptions, Raman data, and elemental analysis. The spectral differences arise from the presence or absence of molybdenum, the use of two crystal polymorphs of BiVO₄, and differences in pigment stabilizers. Bismuth Vanadate is usually not used alone, and it is typically found with Isoindoline Yellow, hydrous ferric oxide, rutile, Isoindolinone Yellow 3R, or various combinations of these.