Cuticular hydrocarbons as a tool for determining the age of Chrysomya rufifacies (Diptera: Calliphoridae) larvae

Calliphoridae are one of the most important insect groups encountered as evidence collected from a crime scene. Age determination of the immature stages of these necrophagous flies is an important step toward estimating the time of colonization and inferring a minimum postmortem interval (PMI_min) in most instances. To determine if the cuticular hydrocarbons could be used to establish whether the development stages yield characteristics profiles, allowing for age estimation, hydrocarbons were extracted from 1st and 2nd, as well as feeding and post‐feeding 3rd instar Chrysomya rufifacies, the hairy maggot blow fly. Extracted hydrocarbons were analyzed using gas chromatography coupled to mass spectrometry with the aim to investigate the changes in chemical profiles of each larval stage. A total of 23 compounds were identified with most of them being alkanes (65%) with carbon chain lengths of 9–33 carbons, alkenes (18%), and methyl‐branched alkanes (17%). All the hydrocarbons except pentadecane (C15), hexadecane (C16), and nonacosane (C29) showed significant differences in their expression throughout larval development. For 1st instars, nonane was the most abundant (17% of the total hydrocarbons content) compound. Accounting for 11% and 10% of the cuticular hydrocarbons, tricosane and pentacosane, respectively, were the notable hydrocarbons in 2nd instars. For post‐feeding 3rd instars, hentriacontane and tritriacontane were present with relative abundances 18% and 15%, respectively. On average, there was a shift from low to high molecular weight hydrocarbons as the larvae aged. These results indicate the change in hydrocarbons makeup as larvae age and could potentially be used to determine the age of immature C. rufifacies and hence aid in PMI_min estimations. However, future research is needed to validate these results under natural conditions in the field.     

Free Fatty Acids Composition in Adipocere of the Kwäday Dän Ts’ìnchí Ancient Remains Found in a Glacier*

Abstract: Adipocere is a postmortem decomposition product consisting of mostly a mixture of free fatty acids (FFAs) that are formed because of the hydrolysis of triglycerides in adipose tissues. This article describes a simple and robust method for the extraction, identification, and quantification of FFA commonly found in adipocere using gas chromatography–mass spectrometry (GC/MS). This method was applied to analyze tissues from Kwäday Dän Ts’ìnchí, ancient remains discovered in a retreating glacier in the Tatshenshini‐Alsek Park, British Columbia, Canada in August 1999. The lyophilized tissues were grinded and extracted with hexane. The trimethylsilyl fatty acid derivatives were analyzed by GC/MS, and the relative abundances of myristic acid, palmitic acid, oleic acid, and stearic acid were determined. Milligram per gram levels of saturated fatty acids were found in the tissues of the ancient remains, while the levels of unsaturated fatty acids, such as palmitoleic acid, were found to be negligible. The results provided further evidence of the existence of adipocere found during forensic examination of the Kwäday Dän Ts’ìnchí ancient remains.     

The Effect of Body Mass on Outdoor Adult Human Decomposition

Forensic taphonomy explores factors impacting human decomposition. This study investigated the effect of body mass on the rate and pattern of adult human decomposition. Nine males and three females aged 49–95 years ranging in mass from 73 to 159 kg who were donated to the Complex for Forensic Anthropology Research between December 2012 and September 2015 were included in this study. Kelvin accumulated degree days (KADD) were used to assess the thermal energy required for subjects to reach several total body score (TBS) thresholds: early decomposition (TBS ≥6.0), TBS ≥12.5, advanced decomposition (TBS ≥19.0), TBS ≥23.0, and skeletonization (TBS ≥27.0). Results indicate no significant correlation between body mass and KADD at any TBS threshold. Body mass accounted for up to 24.0% of variation in decomposition rate depending on stage, and minor differences in decomposition pattern were observed. Body mass likely has a minimal impact on postmortem interval estimation.     

A Study of the Human Decomposition Sequence in Central Texas*

Abstract: Decomposition studies utilizing nonhuman subjects as human analogues are well established, but fewer studies utilizing intact human remains exist. This study provides data from a controlled decomposition study involving human remains in Central Texas. A 63.5‐kg unmodified cadaver was placed in an open‐air site and observed over a 10‐week period from April 11 through June 19, 2008. A wire enclosure restricted scavenger access. State of decomposition and environmental conditions were recorded daily for the first 36 days and then every 2 weeks thereafter. Results indicated a high degree of correlation with other decomposition studies originating in the southwestern United States, although slight deviations for the average duration of early events were noted. The data were also utilized to test a quantitative method for estimating the postmortem interval. Results indicated preliminary support for a quantitative approach. Additional research is encouraged to further establish the human decomposition data set for Central Texas.     

An Aquatic Decomposition Scoring Method to Potentially Predict the Postmortem Submersion Interval of Bodies Recovered from the North Sea

This study aimed to develop an aquatic decomposition scoring (ADS) method and investigated the predictive value of this method in estimating the postmortem submersion interval (PMSI) of bodies recovered from the North Sea. This method, consisting of an ADS item list and a pictorial reference atlas, showed a high interobserver agreement (Krippendorff's alpha ≥ 0.93) and hence proved to be valid. This scoring method was applied to data, collected from closed cases—cases in which the postmortal submersion interval (PMSI) was known—concerning bodies recovered from the North Sea from 1990 to 2013. Thirty‐eight cases met the inclusion criteria and were scored by quantifying the observed total aquatic decomposition score (TADS). Statistical analysis demonstrated that TADS accurately predicts the PMSI (p < 0.001), confirming that the decomposition process in the North Sea is strongly correlated to time.     

A Rare Case of Poisoning with a Volatile Substance: Quantitative Determination of n‐Butane in Postmortem Tissue

Poisoning with volatile substances remains exceptional. Authors report the case of a married couple who were found in a car with a butane gas bottle: the woman was dead and her husband alleged it was an unsuccessful suicide pact. A specific research of volatile substances on postmortem samples with headspace gas chromatography–mass spectrometry following a quantitative determination was performed. The n‐butane concentrations detected were composed of 610 μg/L (cardiac blood), 50 μg/kg (brain), 134 μg/kg (lungs), 285 μg/kg (liver), and 4090 μg/kg (heart) and were compatible with the rare lethal concentrations evoked in the literature. The cause of death was determined to be asphyxiation through n‐butane criminal poisoning. Authors recommendation therefore is to take samples immediately and place them in properly sealed containers and hence analyzing the samples as soon as possible after collecting them or storing them under ?30°C (?22°F) if analyses cannot be performed immediately.     

The Effects of Burning and Mold Growth on the Chemical Composition of Firelog Fuels

Firelogs consist of a cellulosic material, such as sawdust or wood particles, and a combustible binder (fuel). Historically, the fuel typically consisted of a petroleum‐based (paraffin) wax; however, some manufacturers now include vegetable oils in their firelog fuels. To determine fuel composition, fuels from various brands of firelogs were extracted and analyzed by high‐temperature gas chromatography–mass spectrometry (HTGC‐MS) and a GC‐MS with a polar column specific for the analysis of fatty acid methyl esters (FAMEs). Firelogs were also burned, allowed to grow mold, and analyzed by GC‐MS to determine the effects that burning and mold growth have on firelog fuel composition. Mold did not tend to preferentially degrade any of the fatty acids. Burning caused a decrease in the relative amount of all of the fatty acids present in the vegetable oil fuel, with a greater effect on unsaturated fatty acids than saturated ones.     

Potential Use of Hydrocarbons for Aging Lucilia sericata Blowfly Larvae to Establish the Postmortem Interval

Previous studies on Diptera have shown the potential for the use of cuticular hydrocarbons' analysis in the determination of larval age and hence the postmortem interval (PMI) for an associated cadaver. In this work, hydrocarbon compounds, extracted daily until pupation from the cuticle of the blowfly Lucilia sericata (Diptera: Calliphoridae), have been analyzed using gas chromatography–mass spectrometry (GC–MS). The results show distinguishing features within the hydrocarbon profile over the period of the larvae life cycle, with significant chemical changes occurring from the younger larvae to the postfeeding larvae. Further interpretation of the chromatograms using principal component analysis revealed a strong correlation between the magnitudes of particular principal components and time. This outcome suggests that, under the conditions of this study, the cuticular hydrocarbons evolve in a systematic fashion with time, thus supporting the potential for GC–MS analysis as a tool for establishing PMI where such a species is present.     

Experimental adipocere formation: implications for adipocere formation on buried bone

Adipocere, or grave wax (adipo = fat, cere = wax), is a distinctive decomposition product composed primarily of fatty acids (FA) and their alkali salts. FA result from the bacterial enzymatic hydrolysis of body fats. Reactions with ammonia and alkali metals originating from body fluids and pore waters of the depositional environment produce alkali salts of FA (soap). Adipocere formation is generally associated with burial of corpses with ample adipose tissue available. No indications that adipocere can form on defleshed remains have been presented in the literature. At the termination of a long-term bone diagenesis experiment, several samples were found to possess growths of an unknown compound. Gas chromatography-mass spectrometry confirmed that the growths are adipocere. The results herein reveal that adipocere can indeed form on defleshed bones under the right conditions and that even residual adipose and lipids in defleshed bones are sufficient to produce adipocere growth on the surfaces of bone.     

The Effects of Sharp‐Force Thoracic Trauma on the Rate and Pattern of Decomposition

Given the multiple factors that may alter the rate of decomposition, one of the more difficult tasks that anthropologists and pathologists face is determining of the postmortem interval (PMI). While trauma has been widely accepted as being among those factors, recent published studies have contradicted this premise. However, major issues exist with these studies. Consequently, this study was designed to simulate a more realistic setting in an effort to settle this discrepancy in the literature. This study utilized eight porcine remains; three of which received incised wounds penetrating the thoracic cavity, three others received nonpenetrating wounds, and two were designated nontrauma controls. Measurements included evaluating the total body score (TBS) on temporal and accumulated degree days (ADD). Using a two‐way repeated measures ANOVA, the results of this study found that while trauma does have an influence in the pattern of decomposition, it does not influence the rate of decomposition.     

Predicting the Postmortem Submersion Interval for Human Remains Recovered from U.K. Waterways*

Abstract: This article aims to increase accuracy in estimating the postmortem submersion interval (PMSI) for bodies recovered from rivers in the United Kingdom. Data were collected from closed case files, crime scene reports, and autopsy files concerning bodies recovered over a 15‐year period from the River Clyde, Scotland, and the River Mersey and canals in northwest England. One hundred and eighty‐seven cases met the study criteria and were scored by quantifying the overall amount of decomposition observed in each case. Statistical analysis showed that the duration of a body’s submergence in water and the temperatures to which it was exposed, as measured in accumulated degree days (ADD), had a significant effect on the decay process. Further analysis indicated that there were no significant differences in decomposition between the waterways. By combining the data from all study samples, it was possible to produce a single linear regression model for predicting ADD from observed decomposition.     

The Influence of Preburial Insect Access on the Decomposition Rate

Abstract: This study compared total body score (TBS) in buried remains (35 cm depth) with and without insect access prior to burial. Sixty rabbit carcasses were exhumed at 50 accumulated degree day (ADD) intervals. Weight loss, TBS, intra‐abdominal decomposition, carcass/soil interface temperature, and below‐carcass soil pH were recorded and analyzed. Results showed significant differences (p << 0.001) in decomposition rates between carcasses with and without insect access prior to burial. An approximately 30% enhanced decomposition rate with insects was observed. TBS was the most valid tool in postmortem interval (PMI) estimation. All other variables showed only weak relationships to decomposition stages, adding little value to PMI estimation. Although progress in estimating the PMI for surface remains has been made, no previous studies have accomplished this for buried remains. This study builds a framework to which further comparable studies can contribute, to produce predictive models for PMI estimation in buried human remains.     

Comparison of the Volatile Organic Compounds from Different Biological Specimens for Profiling Potential*

Previous work has demonstrated the ability to differentiate individuals based on the analysis of human scent hand odor chemicals. In this paper, a range of forensic biological specimens are shown to also have the ability to differentiate individuals based upon the volatile organic compounds (VOCs) present. Human VOC profiles from hand odor, oral fluid, breath, blood, and urine of 31 individuals were analyzed by solid‐phase microextraction–gas chromatography–mass spectrometry (SPME‐GC‐MS) and combined methods of chromatogram comparison, Spearman rank correlation comparison, and principal component analysis. Intra‐specimen comparisons demonstrated the distinguishability of individuals above 99%. Inter‐specimen VOC profiles from the same individual were found to be too different to be used for scent‐matching purposes, with Spearman rank coefficients below 0.15. A 6‐month VOC profile monitoring of two individuals demonstrated the consistency of VOC profiles over time across specimens.     

The Identification of Spiromesifen,a Recently Introduced Pesticide,Using Approaches to Chemical Unknown Analysis

An unknown, viscous, opaque, white liquid with a strong, unpleasant odor was submitted with a request for identification. It was analyzed using infrared (IR) spectroscopy, gas chromatography–mass spectrometry, and X‐ray fluorescence spectroscopy. It was found to contain glycerin as well as another compound. IR and mass spectral data were readily obtained for this second component, but it was not easily identified as common instrument libraries had no matching spectra. After an extensive literature search, the unknown compound was identified as spiromesifen, a recently introduced pesticide. The IR spectrum and electron impact mass spectrum of spiromesifen are presented here as these are not available in the published literature. This case report also provides useful approaches for searching for and identifying an unknown compound when it is not found in a laboratory's spectral libraries.     

Stereochemical Analysis of Methorphan Using (−)‐Menthyl Chloroformate

Abstract: Forensic laboratories around the world have seen an increase in the number of drug seizures containing methorphan. Levomethorphan is a narcotic and a controlled substance, and its enantiomer dextromethorphan is an antitussive agent used in over‐the‐counter medications. For the forensic analysis of seized drugs containing methorphan, it is important to report the stereochemical composition of the drug. Ideally, a method based on common forensic laboratory instrumentation is desirable. The use of the chiral derivatizing agent (?)‐menthyl chloroformate followed by routine gas chromatography–mass spectrometry analysis of the derivative was shown to successfully determine the stereochemical composition of methorphan. This approach was applied to a street seizure containing methorphan proving that it was pure dextromethorphan. The derivatives of dextro‐ and levomethorphan were subjected to mass spectroscopic and nuclear magnetic resonance analysis, which confirmed that the structure of the derivatives remained unchanged as a result of the derivatization process.     

Determining the Toxicological Significance of Pregabalin in Fatalities

Pregabalin has become more widely prescribed and abused in recent years but is still not always included in laboratory analysis. An LC‐MS‐MS method has been developed and applied to measure pregabalin in 93 postmortem cases, including drug‐related deaths, alternative causes of death, and fatalities where pregabalin was likely to have contributed to death. Other drugs or alcohol was detected, and the most common drug types (in decreasing frequency) were antidepressants, opioids, benzodiazepines, opiates, alcohol, antipsychotics, cocaine, cardiac drugs, amphetamines, cannabis, anticonvulsants, and antihistamines. New psychoactive substances (methoxphenidine and synthetic cannabinoids) were only found in two cases. The results provide further data to assist in evaluating the significance of postmortem pregabalin concentrations and a toxicologically significant concentration of 25 mg/L is proposed. Pregabalin, especially with concomitant use of other CNS depressant drugs, presents a significant toxicological risk and existing laboratory protocols should be reviewed for their suitability to detect pregabalin.     

Growth of Chrysomya megacephala (Fabricius) Maggots in a Morgue Cooler*

Abstract: In estimating the postmortem interval (PMI) using maggots obtained during autopsy, the forensic entomologist makes decisions regarding the effects of low‐temperature storage of the body on the insects. In this case report, a corpse was found in an abandoned house in the residential area of Bukit Mertajam, Penang, Malaysia. The maggots were found to be alive inside the mouth of the deceased although the corpse had been in the morgue cooler for 12 days. The maggots were reared and identified as Chrysomya megacephala (Fabricius). The emerged adult flies were kept as a stock colony, and the duration of development under the indoor fluctuating temperature regime was studied. The total duration of developmental process of this species was 9.5 ± 0.5 days, and the PMI estimated was 3.2 ± 0.6 days. This case report demonstrates the survival of Ch. megacephala maggots for 12 days and their growth inside the morgue cooler.     

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).