A synthesis of carcass decomposition studies conducted at a tropical (Aw) taphonomy facility: 2013-2022.

Decomposition studies have been conducted in several regions of the world, but relatively few have investigated taphonomy in tropical environments. Even fewer have explored carcass decomposition during multiple tropical seasons, leaving the relationships between season and decomposition in tropical environments poorly understood. Ten decomposition studies using 30 carcasses were conducted in Honolulu, Hawaii, USA to start addressing this knowledge gap. These studies show that some postmortem processes were observed regardless of season. Carcass temperature and chemistry were spatiotemporally variable. Fly larval masses were consistently observed within 3 days (∼75 ADD) postmortem and carcasses lost 60%-90% of mass by 10 days (∼250 ADD) postmortem (Total Body Score ∼26). Season had a significant effect on decomposition, yet the warmest and most humid seasons did not always result in the most rapid and extensive decomposition. Seasonal variation appears to be less pronounced than at other tropical decomposition sites.

Fisher ratio feature selection by manual peak area calculations on comprehensive two-dimensional gas chromatography data using standard mixtures with variable composition, storage, and interferences.

Comprehensive two-dimensional gas chromatography (GC×GC) is becoming increasingly more common for non-targeted characterization of complex volatile mixtures. The information gained with higher peak capacity and sensitivity provides additional sample composition information when one-dimensional GC is not adequate. GC×GC generates complex multivariate data sets when using non-targeted analysis to discover analytes. Fisher ratio (FR) analysis is applied to discern class markers, limiting complex GC×GC profiles to the most discriminating compounds between classes. While many approaches for feature selection using FR analysis exist, FR can be calculated relatively easily directly on peak areas after any native software has performed peak detection. This study evaluated the success rates of manual FR calculation and comparison to a critical F-value for samples analyzed by GC×GC with defined concentration differences. Long-term storage of samples and other spiked interferences were also investigated to examine their impact on analyzing mixtures using this FR feature selection strategy. Success rates were generally high with mostly 90-100% success rates and some instances of percentages between 80 and 90%. There were rare cases of false positives present and a low occurrence of false negatives. When errors were made in the selection of a compound, it was typically due to chromatographic artifacts present in chromatograms and not from the FR approach itself. This work provides foundational experimental data on the use of manual FR calculations for feature selection from GC×GC data.

Investigating volatiles as the secondary metabolome of Piper methysticum from root powder and water extracts using comprehensive two-dimensional gas chromatography.

ETHNOPHARMACOLOGICAL RELEVANCE: Kava (Piper methysticum G. Forst) is a plant grown in the Pacific that is used in traditional medicines. The roots are macerated and powdered for consumption as a beverage in social settings as well as in ceremonies. Other types of preparations can also be used as traditional medicines. There has been an increase in demand for kava as there is continued traditional use and as it is becoming utilized more both socially and medicinally outside of Oceania. Currently, most research of this plant has focused on bioactive kavalactones and flavokawains, and there are few studies focusing on the other compounds that kava contains, such as volatile and semivolatile components. AIM OF THE STUDY: This study investigated the kava volatile organic compound (VOC) profile from nine different commercially available samples of dried, powdered kava root sourced across the Pacific region. MATERIALS AND METHODS: The headspace above the kava samples was analyzed, both from the root powder as originally purchased and by performing a scaled-down extraction into water mimicking traditional preparation of the beverage. The headspace of each sample was extracted using solid-phase microextraction arrow (SPME Arrow), followed by analysis using comprehensive two-dimensional gas chromatography - quadrupole mass spectrometry/flame ionization detection (GC×GC-qMS/FID). The superior peak capacity of GC×GC was invaluable in effectively separating the complex mixture of compounds found in all samples, which enabled improved monitoring of minor differences between batches. RESULTS: Dry root powder samples contained high levels of ß-caryophyllene while water extracted samples showed high levels of camphene. Many alcohols, aldehydes, ketones, terpenes, terpenoids, and aromatics were also characterized from both types of samples. All water extracted samples from the different brands followed similar trends in terms of compounds being detected or not. Additional major compounds found in water extracts included benzaldehyde, hexanal, methoxyphenyloxime, camphor, limonene, 1-hexanol, endoborneol, and copaene. While some samples could be differentiated based on brand, samples did not group by purported geographic origin. CONCLUSIONS: This study provides foundational data about a different subset of compounds within kava than previous research has studied, and also informs the community of the compounds that transfer into the consumed beverage during the traditional means of preparing kava.

Pilot Study on Exhaled Breath Analysis for a Healthy Adult Population in Hawaii.

Fast diagnostic results using breath analysis are an anticipated possibility for disease diagnosis or general health screenings. Tests that do not require sending specimens to medical laboratories possess capabilities to speed patient diagnosis and protect both patient and healthcare staff from unnecessary prolonged exposure. The objective of this work was to develop testing procedures on an initial healthy subject cohort in Hawaii to act as a range-finding pilot study for characterizing the baseline of exhaled breath prior to further research. Using comprehensive two-dimensional gas chromatography (GC×GC), this study analyzed exhaled breath from a healthy adult population in Hawaii to profile the range of different volatile organic compounds (VOCs) and survey Hawaii-specific differences. The most consistently reported compounds in the breath profile of individuals were acetic acid, dimethoxymethane, benzoic acid methyl ester, and n-hexane. In comparison to other breathprinting studies, the list of compounds discovered was representative of control cohorts. This must be considered when implementing proposed breath diagnostics in new locations with increased interpersonal variation due to diversity. Further studies on larger numbers of subjects over longer periods of time will provide additional foundational data on baseline breath VOC profiles of control populations for comparison to disease-positive cohorts.

A non-targeted data processing workflow for volatile organic compound data acquired using comprehensive two-dimensional gas chromatography with dual channel detection.

There has been an influx of technology for comprehensive two-dimensional gas chromatography analyses in recent years, calling for development of guided workflows and rigorous reporting of processes. This research focuses on the processing method for data collected on a dual channel detection system using flame ionization detection (FID) and quadrupole mass spectrometry (qMS) for the analysis of volatile organic compounds (VOCs). The samples analyzed were kava (Piper methysticum), which has a rich VOC profile that benefits substantially from a multidimensional approach due to enhanced peak capacity. The procedure which was customized here was the data processing workflow from a manual single-sample analysis to an integrated batch workflow that can be applied across studies.•Parameter choice for baseline correction and peak detection were defined when handling batch data.•Elution regions were defined using qMS data to automate compound identification.•Stencils were transformed onto FID data and sequenced for quantitative information.This dataset can be used as a training tool, as all details, methods and results for the workflow have been provided for users to compare with. The focus on data workflow reproducibility in the field of multidimensional chromatography will assist in adoption by users in new application areas.

Translation of a One-Dimensional to a Comprehensive Two-Dimensional Gas Chromatography Method with Dual-Channel Detection for Volatile Organic Compound Measurement in Forensic Applications.

After its introduction in the early 1990s, comprehensive two-dimensional gas chromatography (GC×GC) has evolved from a separation science research tool to the central component of many industries. Despite the maturity of the technique, some fields remain reluctant to its use in routine applications. In the case of forensic science, some constraints are the strict requirements enforced in forensic laboratories and the time and effort that must be invested for intralaboratory method validation. Concerns may also arise about whether information could be lost when transitioning to a new technique. This study reports on a method translation from conventional one-dimensional (1D) GC to GC×GC, ensuring the integrity of data as conversion is made. The GC was retrofitted with a reverse fill/flush (RFF) flow modulator and equipped with dual-channel detection using a quadrupole mass spectrometer (qMS) and a flame ionization detector (FID). The parallel use of two detectors, where qMS was applied for qualitative identification and FID for quantification, allowed higher flows and slightly wider peaks to be exploited for the analysis of a volatile organic compound (VOC) reference mixture relevant to forensic VOC profiling. Peak quality assessment and calibration curves using GC-qMS and GC×GC-qMS/FID document the transfer and adaptation of the original method without a loss in data quality. Furthermore, the preprocessing and the data analysis processing steps, including calibration and peak quality assessment for each of the three data sets, are explained in detail. This information provides benchmark data for routine laboratories that want to implement a GC×GC approach into routine workflows.

Volatile Organic Compound Profiling from Postmortem Microbes using Gas Chromatography-Mass Spectrometry.

Volatile organic compounds (VOCs) are by-products of cadaveric decomposition and are responsible for the odor associated with decomposing remains. The direct link between VOC production and individual postmortem microbes has not been well characterized experimentally. The purpose of this study was to profile VOCs released from three postmortem bacterial isolates (Bacillus subtilis, Ignatzschineria indica, I. ureiclastica) using solid-phase microextraction arrow (SPME Arrow) and gas chromatography-mass spectrometry (GC-MS). Species were inoculated in headspace vials on Standard Nutrient Agar and monitored over 5 days at 24°C. Each species exhibited a different VOC profile that included common decomposition VOCs. VOCs exhibited upward or downward temporal trends over time. Ignatzschineria indica produced a large amount of dimethyldisulfide. Other compounds of interest included alcohols, aldehydes, aromatics, and ketones. This provides foundational data to link decomposition odor with specific postmortem microbes to improve understanding of underlying mechanisms for decomposition VOC production.

A minimally-invasive method for profiling volatile organic compounds within postmortem internal gas reservoirs.

In forensic casework, non-invasive and minimally-invasive methods for postmortem examinations are extremely valuable. Whole body postmortem computed tomography (PMCT) is often used to provide visualization of the internal characteristics of a body prior to more invasive procedures and has also been used to locate gas reservoirs inside the body to assist in determining cause of death. Preliminary studies have demonstrated that exploiting the volatile organic compounds (VOCs) located in these gas reservoirs by comprehensive two-dimensional gas chromatography-high-resolution time-of-flight mass spectrometry (GC×GC-HRTOF-MS) may assist in providing information regarding the postmortem interval. The aim of the current study was to further develop the procedures related to solid-phase microextraction (SPME) and GC×GC-HRTOF-MS analysis of gas reservoirs collected from deceased individuals. SPME fiber extraction parameters, internal standard approach, and sample stability were investigated. Altering the SPME parameters increased the selectivity and sensitivity for the VOC profile, and the use of a mixed deuterated internal standard contributed to data quality. Samples were found to be stable up to 6 weeks but were recommended to be analyzed within 4 weeks due to higher variation observed beyond this point. In addition, 29 VOC markers of interest were identified, and heart and/or abdominal cavity samples were suggested as a possible standardized sampling location for future studies. The data presented in this study will contribute to the long-term goal of producing a routine, accredited method for minimally-invasive VOC analysis in postmortem examinations.

Advanced method optimization for volatile aroma profiling of beer using two-dimensional gas chromatography time-of-flight mass spectrometry.

The complex mixture of volatile organic compounds (VOCs) present in the headspace of Trappist and craft beers was studied to illustrate the efficiency of thermal desorption (TD) comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS) for highlighting subtle differences between highly complex mixtures of VOCs. Headspace solid-phase microextraction (HS-SPME), multiple (and classical) stir bar sorptive extraction (mSBSE), static headspace (SHS), and dynamic headspace (DHS) were compared for the extraction of a set of 21 representative flavor compounds of beer aroma. A Box-Behnken surface response methodology experimental design optimization (DOE) was used for convex hull calculation (Delaunay's triangulation algorithms) of peak dispersion in the chromatographic space. The predicted value of 0.5 for the ratio between the convex hull and the available space was 10% higher than the experimental value, demonstrating the usefulness of the approach to improve optimization of the GC×GC separation. Chemical variations amongst aligned chromatograms were studied by means of Fisher Ratio (FR) determination and F-distribution threshold filtration at different significance levels (α=0.05 and 0.01) and based on z-score normalized area for data reduction. Statistically significant compounds were highlighted following principal component analysis (PCA) and hierarchical cluster analysis (HCA). The dendrogram structure not only provided clear visual information about similarities between products but also permitted direct identification of the chemicals and their relative weight in clustering. The effective coupling of DHS-TD-GC×GC-TOFMS with PCA and HCA was able to highlight the differences and common typical VOC patterns among 24 samples of different Trappist and selected Canadian craft beers.

Thermal desorption comprehensive two-dimensional gas chromatography coupled to variable-energy electron ionization time-of-flight mass spectrometry for monitoring subtle changes in volatile organic compound profiles of human blood.

Blood is a matrix with high potential for forensic investigations and human rescue. Its volatile signature can be used in search exercises to locate injured or deceased individuals. Little is known, however, about the volatile organic compound (VOC) profile of blood, except that it is complex and varies while blood ages. In the present study, we used thermal desorption (TD) and comprehensive two-dimensional gas chromatography (GCxGC) coupled to variable-energy electron ionization time-of-flight mass spectrometry (TOFMS) to monitor VOC signatures of human blood. A highly complex reference standard (Century Mix) containing 108 compounds of various chemical functionalities and several homologue series of compounds was used for the purpose of transposing our previously developed cryogenically modulated GCxGC-TOFMS methods into the use of a reverse fill/flush (RFF) flow modulator. The average peak width at half height was 340ms and the average tailing factor was 1.16. Light VOCs (down to C4) were effectively flow modulated and exhibited minimal breakthrough over a large dynamic range spanning four orders of magnitude. Mass spectrometric detection was performed using electron impact ionization (EI) carried out at 70eV and lower energies (12, 14, and 16eV). The use of variable-energy (ve) EI allowed mass spectra to be produced with less fragmentation and an increased presence of structurally significant ions and the molecular ion. This provided additional confidence in peak assignments, especially for closely eluting isomers often observed in the profiling of the headspace of blood. Variable-energy EI TD-GCxGC-TOFMS blood data sets were statistically processed using principal component analyses (PCA) and hierarchical cluster analyses (HCA). These techniques demonstrated that the effect of aging was greater than the inter-individual variation on the blood VOC profile. The combination of retention indices, low and high EI MS spectra served as a strong basis to gain more confidence in analytical identification by excluding identities proposed by mass spectral databases (70eV) for compounds contributing to the separation of blood of different ages.

Detection of decomposition volatile organic compounds in soil following removal of remains from a surface deposition site.

PURPOSE: Cadaver-detection dogs use volatile organic compounds (VOCs) to search for human remains including those deposited on or beneath soil. Soil can act as a sink for VOCs, causing loading of decomposition VOCs in the soil following soft tissue decomposition. The objective of this study was to chemically profile decomposition VOCs from surface decomposition sites after remains were removed from their primary location. METHODS: Pig carcasses were used as human analogues and were deposited on a soil surface to decompose for 3 months. The remains were then removed from each site and VOCs were collected from the soil for 7 months thereafter and analyzed by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS). RESULTS: Decomposition VOCs diminished within 6 weeks and hydrocarbons were the most persistent compound class. Decomposition VOCs could still be detected in the soil after 7 months using Principal Component Analysis. CONCLUSIONS: This study demonstrated that the decomposition VOC profile, while detectable by GC×GC-TOFMS in the soil, was considerably reduced and altered in composition upon removal of remains. Chemical reference data is provided by this study for future investigations of canine alert behavior in scenarios involving scattered or scavenged remains.

GC × GC-TOFMS and supervised multivariate approaches to study human cadaveric decomposition olfactive signatures.

In forensic thanato-chemistry, the understanding of the process of soft tissue decomposition is still limited. A better understanding of the decomposition process and the characterization of the associated volatile organic compounds (VOC) can help to improve the training of victim recovery (VR) canines, which are used to search for trapped victims in natural disasters or to locate corpses during criminal investigations. The complexity of matrices and the dynamic nature of this process require the use of comprehensive analytical methods for investigation. Moreover, the variability of the environment and between individuals creates additional difficulties in terms of normalization. The resolution of the complex mixture of VOCs emitted by a decaying corpse can be improved using comprehensive two-dimensional gas chromatography (GC × GC), compared to classical single-dimensional gas chromatography (1DGC). This study combines the analytical advantages of GC × GC coupled to time-of-flight mass spectrometry (TOFMS) with the data handling robustness of supervised multivariate statistics to investigate the VOC profile of human remains during early stages of decomposition. Various supervised multivariate approaches are compared to interpret the large data set. Moreover, early decomposition stages of pig carcasses (typically used as human surrogates in field studies) are also monitored to obtain a direct comparison of the two VOC profiles and estimate the robustness of this human decomposition analog model. In this research, we demonstrate that pig and human decomposition processes can be described by the same trends for the major compounds produced during the early stages of soft tissue decomposition.

Reducing variation in decomposition odour profiling using comprehensive two-dimensional gas chromatography.

Challenges in decomposition odour profiling have led to variation in the documented odour profile by different research groups worldwide. Background subtraction and use of controls are important considerations given the variation introduced by decomposition studies conducted in different geographical environments. The collection of volatile organic compounds (VOCs) from soil beneath decomposing remains is challenging due to the high levels of inherent soil VOCs, further confounded by the use of highly sensitive instrumentation. This study presents a method that provides suitable chromatographic resolution for profiling decomposition odour in soil by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry using appropriate controls and field blanks. Logarithmic transformation and t-testing of compounds permitted the generation of a compound list of decomposition VOCs in soil. Principal component analysis demonstrated the improved discrimination between experimental and control soil, verifying the value of the data handling method. Data handling procedures have not been well documented in this field and standardisation would thereby reduce misidentification of VOCs present in the surrounding environment as decomposition byproducts. Uniformity of data handling and instrumental procedures will reduce analytical variation, increasing confidence in the future when investigating the effect of taphonomic variables on the decomposition VOC profile.

Comparison of the decomposition VOC profile during winter and summer in a moist, mid-latitude (Cfb) climate.

The investigation of volatile organic compounds (VOCs) associated with decomposition is an emerging field in forensic taphonomy due to their importance in locating human remains using biological detectors such as insects and canines. A consistent decomposition VOC profile has not yet been elucidated due to the intrinsic impact of the environment on the decomposition process in different climatic zones. The study of decomposition VOCs has typically occurred during the warmer months to enable chemical profiling of all decomposition stages. The present study investigated the decomposition VOC profile in air during both warmer and cooler months in a moist, mid-latitude (Cfb) climate as decomposition occurs year-round in this environment. Pig carcasses (Sus scrofa domesticus L.) were placed on a soil surface to decompose naturally and their VOC profile was monitored during the winter and summer months. Corresponding control sites were also monitored to determine the natural VOC profile of the surrounding soil and vegetation. VOC samples were collected onto sorbent tubes and analyzed using comprehensive two-dimensional gas chromatography--time-of-flight mass spectrometry (GC × GC-TOFMS). The summer months were characterized by higher temperatures and solar radiation, greater rainfall accumulation, and comparable humidity when compared to the winter months. The rate of decomposition was faster and the number and abundance of VOCs was proportionally higher in summer. However, a similar trend was observed in winter and summer demonstrating a rapid increase in VOC abundance during active decay with a second increase in abundance occurring later in the decomposition process. Sulfur-containing compounds, alcohols and ketones represented the most abundant classes of compounds in both seasons, although almost all 10 compound classes identified contributed to discriminating the stages of decomposition throughout both seasons. The advantages of GC × GC-TOFMS were demonstrated for detecting and identifying trace levels of VOCs, particularly ethers, which are rarely reported as decomposition VOCs.

Effect of age and storage conditions on the volatile organic compound profile of blood.

Cadaver-detection dogs are used by the police to locate missing persons, victims of homicide, and human remains following mass disasters. Training is conducted using a variety of training aids including blood which can be hours, weeks or months old and stored under variable conditions. The aim of this study was to chemically profile human blood using solid-phase microextraction coupled with gas chromatography-mass spectrometry to determine how the volatile organic compound (VOC) profile changed over time and under variable storage conditions. The VOC profiles of fresh and degraded blood were analyzed as well as blood stored at room temperature, refrigerated, and frozen. Fresh and degraded blood samples produced distinctive VOC patterns with VOC profiles becoming more complex over time. Freezing the blood produced a complex VOC profile that was clearly discriminated from the VOC profile for blood stored at room temperature or in a refrigerator. This study highlights the importance of standardizing the age and storage conditions when using blood as a training aid to ensure cadaver-detection dogs are exposed to an accurate representation of the blood VOCs they may encounter at a scene.

Decomposition odour profiling in the air and soil surrounding vertebrate carrion.

Chemical profiling of decomposition odour is conducted in the environmental sciences to detect malodourous target sources in air, water or soil. More recently decomposition odour profiling has been employed in the forensic sciences to generate a profile of the volatile organic compounds (VOCs) produced by decomposed remains. The chemical profile of decomposition odour is still being debated with variations in the VOC profile attributed to the sample collection technique, method of chemical analysis, and environment in which decomposition occurred. To date, little consideration has been given to the partitioning of odour between different matrices and the impact this has on developing an accurate VOC profile. The purpose of this research was to investigate the decomposition odour profile surrounding vertebrate carrion to determine how VOCs partition between soil and air. Four pig carcasses (Sus scrofa domesticus L.) were placed on a soil surface to decompose naturally and their odour profile monitored over a period of two months. Corresponding control sites were also monitored to determine the VOC profile of the surrounding environment. Samples were collected from the soil below and the air (headspace) above the decomposed remains using sorbent tubes and analysed using gas chromatography-mass spectrometry. A total of 249 compounds were identified but only 58 compounds were common to both air and soil samples. This study has demonstrated that soil and air samples produce distinct subsets of VOCs that contribute to the overall decomposition odour. Sample collection from only one matrix will reduce the likelihood of detecting the complete spectrum of VOCs, which further confounds the issue of determining a complete and accurate decomposition odour profile. Confirmation of this profile will enhance the performance of cadaver-detection dogs that are tasked with detecting decomposition odour in both soil and air to locate victim remains.