Legal and Forensic Sampling

1.23

Legal and Forensic Sampling

A Kabir, H Holness, KG Furton, and JR Almirall, Florida International University, Miami, FL, USA Ó 2012 Elsevier Inc. All rights reserved.

1.23.1 Introduction and Scope 1.23.2 Role of Statistics as Sampling Strategy 1.23.3 Analysis of Ignitable Liquid Residues from Fire Debris 1.23.4 Detection and Analysis of Explosives 1.23.4.1 Pre-Blast Detection 1.23.4.1.1 Portal Sampling 1.23.4.1.2 Standoff Sampling 1.23.4.2 Post-Blast Analysis 1.23.5 Gunshot Residue Analysis 1.23.5.1 Inorganic Gunshot Residues 1.23.5.2 Organic Gunshot Residues 1.23.5.3 GSR Sampling Techniques 1.23.5.3.1 Tape Lift 1.23.5.3.2 Vacuum Lift 1.23.5.3.3 Swabbing 1.23.5.3.4 Glue Lift 1.23.6 Analysis of Controlled Substances and Toxicants from Different Matrices 1.23.6.1 Urine 1.23.6.2 Blood 1.23.6.3 Hair 1.23.6.4 Saliva 1.23.6.5 Sweat 1.23.6.6 Sampling of other Biological Tissues 1.23.6.6.1 Liver, Lungs, Kidney, Spleen, and Brain 1.23.6.6.2 Fingernails and Toenails 1.23.6.6.3 Bone 1.23.7 Forensic Examination of Trace Evidence 1.23.7.1 Hairs, Fibers, Glass, and Paint 1.23.7.2 Ink and Paper Analysis 1.23.7.3 Miscellaneous Trace Evidence 1.23.8 Forensic Environmental Analysis 1.23.8.1 Volatile Hydrocarbon Fingerprinting 1.23.8.1.1 Air 1.23.8.1.2 Soil 1.23.8.1.3 Water 1.23.8.1.4 Nonaqueous Phase Liquid (NAPL) Samples 1.23.8.2 Semi-Volatile Hydrocarbon Fingerprinting 1.23.8.2.1 Water 1.23.8.2.2 Soil and Sediment 1.23.8.2.3 Nonaqueous Phase Liquid (NAPL) Samples 1.23.9 Analysis of Human Odor Profile 1.23.10 Analysis of Human Decomposition Products 1.23.11 Nuclear Forensics 1.23.12 Field Sampling Methods for Analytes of Forensic Interests 1.23.12.1 Field-Sampling Strategies 1.23.12.2 Storage 1.23.13 Conclusion References Relevant Websites

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Comprehensive Sampling and Sample Preparation, Volume 1

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doi:10.1016/B978-0-12-381373-2.10003-1

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Sampling Theory and Methodology

1.23.1

Introduction and Scope

Forensic science is defined as the application of scientific principles and methodologies to resolve legal disputes. In addition to analyzing a wide variety of samples of forensic interest, forensic chemists interpret the extracted information from the analytical data for presenting in civil and/or criminal judicial proceedings. Continual advances in sampling and sample preparation in forensic analysis are necessary as the application of forensic chemistry has serious ramifications in establishing evidential value of the analyzed samples with a high level of confidence. Sampling and sample preparation is an important first step in forensic chemical analysis, especially when dealing with trace and ultra-trace levels of the target analyte(s) present in complex matrices (e.g., biological, soil, environmental, fire debris, explosive residues, etc.), and the volume of the sample available to the investigator, applicable for most cases, is limited. Due to the complex nature of the sample matrix in which the analyte(s) of interest is present, such samples cannot be introduced directly into the analytical instrument for qualitative and/or quantitative analyses. This incompatibility stems from two factors. First, the complex sample matrix may exert a detrimental effect on the performance of the analytical method if introduced directly without prior sample treatment/cleanup procedures. Second, the concentration of the analyte of interest in the sample matrix may be so low that it may fall below the detection limit of the analytical instrument. Finally, every forensic case is different and therefore the analytical approach to sampling and sample preparation is difficult to standardize as is possible with other disciplines. New developments over the last decade in sampling and sample preparation for forensic samples are presented here. In recent years, a variety of new sample preparation techniques has been applied to analyze forensic samples. This chapter primarily focuses on the current best practices and most recent developments in sampling and sample preparation techniques of different analytes of forensic importance. Figure 1 illustrates the major sample preparation techniques currently being used in different areas of forensic chemistry. As forensic science is growing rapidly, the span and scope of forensic sample preparation is also growing in a similar pace. As such, we have classified this chapter into different sections to discuss all the major fields within the discipline of forensic chemistry. Different matrices for a given field are discussed in subsections. It is worthy to mention that the scope of sampling and sample preparation in forensic chemistry is so broad that it is very challenging to cover the entire topic in a single book chapter. Nonetheless, the authors have made their best effort to accommodate as much information as possible and interested readers are encouraged to read the references cited in the chapter for additional and more comprehensive information.

Figure 1

Major sample preparation techniques used in forensic chemical analysis.

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1.23.2

443

Role of Statistics as Sampling Strategy

An important consideration when developing a sampling strategy is to account for all the possible sources of variation (or uncertainty) in the measurements, the most significant being the measurement error and the inherent heterogeneity in the sample matrix. In the case of forensic toxicology, blood and urine samples are expected to be taken as relatively homogeneous samples but the more typical forensic matrix can be heterogeneous in nature. A sampling strategy that can account for both the measurement error and the sample heterogeneity is recommended. A common question in forensic science is: “Could this evidence sample collected at the crime scene have originated from this other source of known origin” in order to determine if an association between the evidence sample and the known source can be made. In order to address this question, the source should be characterized as well. In the example of glass examination and comparison, a multivariate analysis of the trace elemental composition in the glass, adequate amounts of the known source (i.e., the windshield from the vehicle suspected of the hit-and-run accident) are available for a good characterization of the source. A minimum of 3 different fragments, each of which is analyzed a minimum of 3 times, for a total of 9 measurements’ characterization would be required in this example. The evidence glass recovered from the crime scene could be a small, single fragment from the head hair of the victim or multiple fragments from the clothing of the victim. Each of the recovered fragments would then be compared to the known source using hypothesis testing or nonparametric methods already available. The nature of both the known source and the nature and quantity available for the recovered sample would dictate the sampling and analysis scheme, including the number of measurements. Some analytical methods are more amenable to taking large numbers of measurements (i.e., laser-induced breakdown spectroscopy or LIBS) since LIBS measurements are rapid and the data can be processed very quickly. Elemental analysis by LIBS can generate many more data points in the same analysis time as uXRF or LA-ICPMS, e.g., making LIBS more attractive from the point of view of statistical analysis of the data generated and all other figures of merit (sensitivity, precision, accuracy, etc.) being equal.

1.23.3

Analysis of Ignitable Liquid Residues from Fire Debris

Unlike other crime scenes, it is often difficult to obtain direct physical evidence related to the arsonist (e.g., DNA, fingerprint) from the fire scene as they are destroyed by the fire. As such, in majority of the arson cases, the investigators rely on collecting, analyzing, and tracking the potential sources of ignitable liquid residues (ILRs) or accelerants that the arsonist(s) uses to rapidly spread the fire. American Society for Testing and Materials (ASTM) classified ignitable liquids into nine primary classes: gasoline, petroleum distillates, isoparaffinic products, aromatic products, naphthenic products, n-alkanes products, de-aromatized distillates, oxygenated solvents, and others/miscellenous.1 Most common accelerants used by arsonists include gasoline, kerosene, paint thinners, charcoal lighter fluids, alcohols, mineral spirits, fuel oils, and vegetable oils. Detection and identification of ignitable liquid residues obtained from the fire scene provides the investigators valuable information about the type of the accelerants used in the arson case, helping them in tracking down the suspected arsonist(s). Two recent books Analysis and Interpretation of Fire Scene Evidence2 and Fire Debris Analysis3 extensively cover all the important aspects of fire science in forensic perspective. In addition, a number of review articles shed light on different aspects of this important topic.4–6 The highly volatile nature of the ignitable liquid residues present in the fire scene demands utmost care during the sample collection from the crime scene. The fire debris samples, as a source of ILR, are collected from the fire scene in clean, leak-free containers and are immediately transported to the laboratory for analysis. Commercial containers (e.g., metal paint cans, glass mason jars, and copolymer bags) are among the most frequently used as fire debris evidence collection and storage containers. ASTM International has developed a number of standard practices7 for screening, isolating and/concentration of ILR, and archiving of extracts recovered from the fire debris, which include: ASTM E 1388-05 Standard Practice for Sampling of Headspace Vapors from Fire Debris Samples ASTM E 1413-07 Standard Practice for Separation and Concentration of Ignitable Liquid Residues from Fire Debris Samples by Dynamic Headspace Concentration ASTM E 1412-07 Standard Practice for Separation of Ignitable Liquid Residues from Fire Debris Samples by Passive Headspace Concentration with Activated Charcoal ASTM E 2541-08 Standard Practice for Preserving Ignitable Liquid Residue Extracts from Fire Debris Samples ASTM E 1386-10 Standard Practice for Separation of Ignitable Liquid Residues from Fire Debris Samples by Solvent Extraction ASTM E 2154-01(2008) Standard Practice for Separation and Concentration of Ignitable Liquid Residues from Fire Debris Samples by Passive Headspace Concentration with Solid-Phase Microextraction (SPME) ASTM E251-08 Standard Practice for Preserving Ignitable Liquids and Ignitable Liquid Residue Extracts from Fire Debris Samples. Solvent extraction is among the oldest techniques known and used to isolate ignitable liquid residues from fire debris. This method uses selective organic solvent suitable for extracting hydrocarbons (as the common ILR ingredient) to soak the fire debris followed by decanting, filtering, and evaporating the solvent to smaller volumes prior to injecting a small aliquot into the analytical instrument. Common organic solvents used for solvent extraction include carbon tetrachloride, acetone, carbon disulfide, hexane, and methylene chloride, with carbon disulfide being the most desirable for its high solubility and efficiency in displacing ILR from charcoal. In addition to efficiently extracting VOCs, this method can also isolate nonvolatile organic compounds from the fire debris providing additional information. A recent study comparing the relative efficiency of passive headspace concentration and solvent

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extraction for extracting alternative fuels (biodiesel and its blends) from fire debris found solvent extraction being more representative of the liquid residue than passive headspace concentration in terms of chromatographic profiling.8 However, loss of high volatile organic compounds during the evaporation of the solvent, insolubility of certain ILR, and extraction of unwanted components from the debris not originally present in ignitable liquid pose serious limitation to the wide application of this technique. Passive headspace concentration of ignitable liquid residues is the method of choice for many forensic chemists as it is relatively simple, being less laborious yet based on equilibrium extraction technique that offers the possibility of multiple extractions from the same sample without discernible loss in signal intensity. Passive headspace extraction can be performed either by using activated charcoal or by solid-phase microextraction (SPME) and involves natural and passive diffusion of analyte vapors onto the surface of the adsorbent. Passive headspace concentration with activated charcoal utilizes activated charcoal strips (ACSs) to extract residues from a confined sampling container and is recommended to heat the matrix to 60–80
C for 8–24 h in order to achieve higher sensitivity. Extraction into the ACS strip is often followed by desorption using carbon disulfide as the eluting solvent, evaporation to reduce the solvent volume, and injection into the analytical instrument. In forensic perspective, this method has a huge advantage over other methods since the ACS strip can be cut into pieces and a segment of it can be stored for future use if the validity of the test result is challenged in court. In addition to using commercially available ACS strip, passive headspace concentration can be performed using the diffusive flammable liquid extraction (DFLEX) device, which uses an ACS placed inside a metal frame between two permeable Teflon sheets. A new device known as Radiello Passive Air Sampler9 is a new addition to the commercially available passive air sampler using activated carbon as the adsorbent medium. This device is comprised of an adsorbing cartridge containing 530  30 mg of activated carbon, a porous hydrophobic diffusive body with a porosity of 25  5 mm, and a supporting plate that holds the diffusive body. Radiello’s saturation threshold has been claimed to be in the range between 85 and 100 ml, which is significantly higher than other commercial samplers. However, one major setback of this device is its inertness toward highmolecular-weight hydrocarbons (>n-C16). Over the last decade, SPME has emerged as a viable passive headspace extraction technique with many advantages over ACS, e.g., greatly reduced sampling time, higher sensitivity, total elimination of using hazardous and toxic organic solvents such as carbon disulfide, ability to extract from aqueous matrix as well as from the headspace, and finally transferring the extracted analyte by inserting into hot GC inlet. Among the wide range of commercially available SPME fiber chemistries, PDMS (30 mm), PDMS (100 mm) and Carboxen/PDMS (75 mm) fibers are used most frequently for fire debris analysis. A study comparing the selectivity between PDMS and Carboxen/PDMS for headspace sampling found that polydimethylsiloxane (PDMS) and Carboxen/PDMS solid-phase microextraction fibers show preferential extraction of aliphatic or aromatic compounds from the headspace depending on fiber type and temperature. However, the Carboxen/PDMS fiber showed higher selectivity for extracting aromatic hydrocarbons.10 SPME of ILR from the fiber debris has been reported by using direct contact to the matrix, by exposing to the headspace11 or by immersing directly into the liquid sample matrix, with optimized extraction time from 15 to 30 min, matrix heating from 20 to 80
C, and desorbing in the GC inlet for up to 5 min. Oftentimes, the arsonist, while pouring the accelerants onto the object to be burnt, accidentally spills the liquid on his hand, which potentially provides valuable clue to the investigators about the suspect. In response to the high demand of a simple and efficient sampling technique to collect ignitable liquid sample from the arson suspect’s hand, Almirall et al.12 presented a SPME method capable of extracting extremely low quantitites of ILR present on the skin of the arson suspect up to 3.5 h after the exposure. The method utilized a 100-mm PDMS fiber with gentle heating for 5 min followed by 10 min of extraction from a plastic bag enveloping the suspect’s hand. Isabelle et al.13 and Darrer et al.14 independently investigated different collection materials for ILR from the suspect’s hand (e.g., polyethylene gloves, latex gloves, polyvinyl gloves, humidified cotton swab, nitrile gloves, and synthetic elastomers). The collection efficiency of each material was evaluated by passive headspace extraction on activated charcoal strips (ACSs) followed by GC-MS analysis. Nonpowder latex gloves were found to perform best in collecting and retaining ILR from the suspect’s hand. This finding led to the development of a unique sampling kit for the collection of ignitable liquid samples from the suspected arsonist’s hand, which contains three pairs of gloves isolated from each other, one for the sampler, a blank second pair, and the third for the arson suspect. The individual packing of each pair of gloves eliminates the possibility of crosscontamination during sample collection. A novel sampling technique known as thermal desorption cold trap (TCT) extraction process15 utilized a thermal desorption cold trap injector coupled on-line with GC-MS. The desorber was programmed at 120
C to drive off the ILR under helium flow for 4 min, the analytes were cryofocused at 100
C and then introduced into the GC column by increasing the inlet temperature to 250
C. This solvent-less method is fast, does not require any matrix manipulation prior to extraction, and claims to be capable of detecting as low as 100–150 mg of ILR present in the matrix. However, one major shortcoming of this technique is the requirement for the matrix to be absolutely moisture free, otherwise risking the blockage of the cryotrap. In dynamic headspace concentration, air or an inert gas (e.g., nitrogen) is passed over the sample to force high to medium boiling ignitable liquid residues to diffuse through the adsorbents and get extracted onto it. Most common adsorbents include ACS, Porapack Q, Tenax GC, and Chromosorb 102. All these adsorbents possess very high specific surface area and are thermally stable so that analyte(s), after extraction, can be desorbed by thermal desorption process and are often introduced into the gas chromatographic system for separation and identification of individual compounds. Only exception is ACS which requires very high temperature to release extracted analytes; as a result, usually carbon disulfide is used to desorb extracted analytes. One recent development in the sample preparation techniques for fire debris analysis is the introduction of headspace singledrop microextraction (HS-SDME) that uses 2.5 ml benzyl alcohol microdrops exposed to a headspace of a 10 ml aqueous sample

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placed in a 15-ml vial for 20 min with continuous stirring of the aqueous phase at 1500 rpm. Driven by the concentration difference between the acceptor and donor phases, HS-SDME demonstrated a limit of detection of 1.5 ml for kerosene in the simulated fire debris experiment.16

1.23.4

Detection and Analysis of Explosives

An explosive may be defined as a material that can be initiated to undergo very rapid and self-propagating decomposition. Research into the sampling and detection of explosive devices has blossomed to produce a myriad of highly sensitive techniques able to detect explosives before they detonate (pre-blast) and others that can detect explosive debris fragments after they detonate (postblast). Pre-blast detection typically involves the identification of the parent high energetic material (HEM) or detection of the more volatile nonenergetic material of the explosive device. Common target compounds for pre-blast detection have included HEMs such as 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitroperhydro-1,3,5-triazine or research development explosive (RDX), pentaerythritol tetranitrate (PETN), trinitroglycerin (NG), and triacetone triperoxide (TATP). Post-blast detection typically targets inorganic compounds left after the intense heat and shock of detonation itself. These include ions such as chlorides, nitrates, sulfates, and some metal species. Though far greater attention is given to pre-blast detection for obvious reasons, the information gathered from post-blast detection is just as important in the effort to detect and mitigate the future use of these destructive devices.

1.23.4.1

Pre-Blast Detection

Explosives fall into several categories;17 here, we will classify them as either military or commercial. Commercial explosives are typically used for construction and mining efforts to move large volumes of earth quickly and efficiently. These explosives include ammonium nitrate mixtures such as ammonium nitrate fuel oils (ANFOs), detonation cord containing PETN, and dyanamite which is comprised of NG combined with plasticizers. The most commonly used explosives for illicit activity are by far the widely available and uncontrolled smokeless powders which contain NG, diphenylamine (DPA), and ethylcentralite (EC). Also commonly used for illicit activity are homemade explosives (HMEs) such as urea nitrate and TATP made through crude chemical reactions of readily available household products. Military explosives are used during wartime efforts to destroy or disable enemy forces. Military explosives are typically more powerful and commensurately have much more stringent controls on their use and availability. These are used less frequently for illicit activity and include TNT, RDX, HMX, and Tetryl. Recently developed are new military grade high explosive materials, such as I-RDX (intense-RDX), hexanitrohexazaisouwurtzitane (HNIW) more commonly known as CL-20,18 heptanitrocubane,19 and octanitrocubane (ONC).19 Many of these new HEMs currently being developed are considerably more powerful than either RDX or HMX. These new HEMs will undoubtedly shape the sampling and detection schemes of the future, but as of today are expensive to produce and not used on a large scale. There are currently two approaches to sampling pre-blast explosives. These are portal and standoff; portal sampling requires an upclose but noncontact sampling of some characteristic that indicates the presence of the explosive while standoff is performed at a distance from the suspect sample. The advantages and challenges of each sampling technique will be further discussed.

1.23.4.1.1

Portal Sampling

Portal sampling requires that the suspect device be sampled within close proximity to the sampling apparatus, thereby putting the device and possibly the operator at risk if the putative sample detonates. One of the most common portal schemes has been to sample the vapors emitted by explosives. The difficulties with detecting explosive vapors is a direct result of the inherently low vapor pressures of HEMs, for instance, one liter of equilibrated air sampled at standard temperature and pressure only contains the equivalent amount of RDX molecules that would fit on a 5-mm diameter particle. As a result, research has focused on sampling not the HEMs but the more volatile nonenergetic compounds such as 2-ethyl-1-hexanol and diphenylamine. In 1996, the United States Congress passed the Anti-Terrorism and Death Penalty Act that require the addition of detection taggants to plastic explosives.20 These detection taggants are chemicals that have relatively high vapor pressures and therefore easily evaporate from the explosive materials allowing for detection by specialized instrumentation or canine teams. Two commonly used taggants are 2,3-dimethyl2,3-dinitrobutane (DMNB) and ethylene glycol dinitrate (EGDN). Researchers have successfully revealed the capabilities of sampling pre-blast explosives simply by evaluating these volatile headspace vapor components.21 Much research has also been devoted to sample and preconcentrate the minute quantities of explosive vapors available to improve the overall detection limit of a technique. More recently, research has focused on utilizing and improving solid-phase microextraction (SPME) in order to sample the volatile components of pre-blast explosives through the development of planar-SPME (PSPME) technology.22 Other sampling techniques have utilized cavity-enhanced absorption spectroscopy (CEAS).23 Still others have developed disposable colorimetric devices that are sensitive enough to detect 2 parts per billion of explosive vapors from triacetone triperoxide (TATP) in air.24 Sampling of explosives such as dinitrotoluene (DNT) and TNT has also been reported utilizing surface acoustic wave (SAW) containing a novel PDMS copolymer.25 Fluorescent chemosensors have been reported that can detect explosive vapors of TNT as low as 4 parts per billion26 and selective microcantilevers have been developed that respond to vapors of hydrogen peroxide from homemade explosives.27 Canines have traditionally been the tool of choice for vapor detection, as they sequentially sample, detect, and identify the possible sources of pre-blast explosives.28 Organisms as small as nematodes have been studied to track their response to vapors emitted from both homemade and military explosives in an attempt to identify and exploit their chemical

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receptor capabilities. Research has since attempted to mimic these capabilities, with devices such as electronic noses made of metal oxide semiconductors (MOSs) with capabilities to detect 3.34 mg of explosive compounds per liter of air.29 Also developed are molecular imprinted sensing films that can extract explosive compounds such as TNT and DNT, with detection limits being reported as low as 5 parts per billion from air.30 Attempts are still being made to sample nonvolatile energetic particulates of explosives (HEMs) as opposed to the volatile nonenergetic components, utilizing inertial impactor technology to sample explosive particles suspended in air from ULD-3 Cargo containers.31 Miniature mass spectrometers have also been developed that are currently able to sample and detect HEMs such as TNT, Tetryl, and HMX directly from surfaces as large as 75 cm2 in just 22 s.32 This approach, though difficult, is still preferred by some as it provides unambiguous evidence of the presence of the explosive device.

1.23.4.1.2

Standoff Sampling

Standoff sampling allows for sampling at a distance, but has much lower detection capabilities than portal and so while it maintains a relatively safe distance for the equipment and the operator, it risks not detecting a large percentage of the suspect materials (high false negatives) or greater detection of interfering compounds (resulting in possible false positives). Current state-of-the-art research focuses on standoff sampling of explosives. One relatively new form of standoff sampling that has been reported within the last decade is that of using laser-induced breakdown spectroscopy (LIBS). This technique uses a laser with enough energy to break down a miniscule amount of the sample into plasma. The plasma emits a characteristic spectra based on the excited atoms present within the sample that can then be detected by a spectrometer. Standoff explosive sampling by LIBS measures the atomic nitrogen/oxygen levels and ratios these with carbon/hydrogen levels within the sample. Nitrogen/oxygen levels tend to be significantly higher in explosives than other molecules. However, since standoff analyses are conducted through air, interferences with ambient nitrogen/ oxygen levels are problematic; though LIBS sampling has been reported for RDX as far as 25 m, greater distances have proved to be problematic.33 To overcome this obstacle, Lucena et al. have proposed using molecular bands of carbon–nitrogen and carbon– carbon bonds emitted during laser excitation with LIBS.34 Another technique used for standoff sampling and detection of explosives is Raman spectroscopy. Early work in 2004 boasted sampling of explosives using Raman at distances of only 12 m.35 Today, research boasts standoff sampling of explosives at distances as far as 150 m,36 with still further research showing the potential to detect as far as 200 m utilizing narrowband backreflected coherent anti-Stokes Raman scattering (B-CARS).37 A succinct review of standoff sampling and detection utilizing Raman spectroscopy was published by Hobro and Lendl.38 An increasing popular technique for standoff sampling is the use of tetrahertz spectroscopy (THz) (300 GHz to 3 THz). THz has two strong suites; it is able to penetrate most objects giving the ability to ‘see through’ threats and many explosives have a characteristic THz spectra. The technique has been heavily researched within the past decade showing great potential; however, the highly specific nature of THz spectra results in each molecule within the sample exhibiting its own signal within the matrix resulting in wide inter-sample variations.

1.23.4.2

Post-Blast Analysis

The data obtained from post-blast sampling and detection is the evidence that will be directly used for criminal proceedings. As mentioned previously, the typical target analytes for post-blast analysis are inorganic ions such as chlorides, nitrates, sulfates, and some metal species as these are able to survive the intense heat and shock that accompanies an explosion. Though there are many instances where unburned organic explosive material may also be present and detected, the anionic species detected are formed through reduction reactions that occur during the detonation process from chlorates and nitrates within the explosive material. Cations may also be found in the post-blast residue as a result of metals intentionally added to the explosive which increase the temperature of the reaction. The sampling of material in order to perform post-blast analyses often involves extraction and preconcentration commonly with solid-phase extraction (SPE) cartridges followed by aqueous elution into the analytical instrument of choice; very little developments have been made with regard to direct sampling of post-blast material. It is important to note that the simple detection of these inorganic species does not confirm the presence of the explosive device. An investigation into the concentrations of the background ions present in the environment where the post-blast debris is found is crucial.39 The technique used for post-blast detection must be able to distinguish the natural background ion levels from that found in the post-blast residue. Research has developed identification particles (IPs); these are microtaggants developed by Microtrace USA.40 These IPs contain a specific ratio of cations and anions that, when added to an explosive, may be sampled post-blast and used to reveal the origin of that particular explosive debris (Figure 2).

1.23.5

Gunshot Residue Analysis

In any criminal case involving actual or suspected use of firearms, the detection and identification of residues from the firearms’ discharge is of prime importance as they provide valuable information in estimating the firing distances, identifying bullet holes, and in determining whether or not the suspected person was involved in the shooting. Gunshot residues (GSRs), also known as cartridge discharge residue (CDR) or firearm discharge residue (FDR), are composed of unburned or partially burnt propellant powder, particles from the ammunition primer, grease, lubricants, and metals from the cartridge as well as the weapon itself.41–43 Gunshot residue contains both organic and inorganic components. Inorganic residues include nitrates, nitrites, and metallic particles originate from the primer, propellant, and cartridge case. Table 1 provides an abbreviated list of characteristic organic and inorganic gunshot residue components.

Legal and Forensic Sampling

Figure 2

Explosive Sampling Schemes.

1.23.5.1

Inorganic Gunshot Residues

447

The major source of inorganic components of GSR is the primer mixture. Since different manufacturers use different formulations, the primer composition instantaneously provides a clue about the origin of the bullet. In addition to the primer, cartridge case, primer cup, bullet, and barrel are also contributors to inorganic GSR.42 Traditionally, the primers contain compounds of lead, barium, and antimony, which, as heavy metals, pose risk to the health of the shooter as well as the environment. As a result, lead and heavy metal-free primers have been introduced that are considered to be environmentally compatible.

1.23.5.2

Organic Gunshot Residues

The major sources of the organic components in GSR are the propellant powder and the primer mixture. Black powder is known to be the first propellant type used in firearms and is typically composed of potassium nitrate, sulfur, and charcoal.42 However, black powder has been replaced with a new propellant known as the smokeless powder. Smokeless powder can be classified into three different categories: single base powder uses only nitrocellulose (NC) as an explosive, double base powder utilizes both nitrocellulose and nitroglycerine, whereas triple base powder uses nitroglycerine, nitrocellulose, and nitroguandine.42 In addition to the major ingredients, additives, stabilizers, plasticizers, flash inhibitors, coolants, moderants, surface lubricants, and anti-wear additives are also contributors of organic components in GSR.

1.23.5.3

GSR Sampling Techniques

Gunshot residues can be collected from a wide range of locations including skin/hair/bodyparts/clothing of the suspect, vehicles, surroundings of the incident, and any surfaces in the close vicinity of the firearm discharge. As such, different sample collection techniques have been developed and are in use with a common objective of maximizing collection efficiency of GSR and

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Table 1

Characteristic organic and inorganic gunshot residue components

Compound

Abbreviation

Usage

Nitroglycerin Resorcinol 2,4-Dinitrotoluene 2,6-Dinitrotoluene 2,3-Dinitrotoluene Dimethyl phthalate Diethyl phthalate Dibutyl phthalate Diphenylamine Methyl centralite Ethyl centralite Antimony Iron Barium Calcium Magnesium Aluminum Nickel Zinc Lead Copper

NG Rs 24-DNT 26-DNT 23-DNT MF EF BF DPA MC EC Sb Fe Ba Ca Mg Al Ni Zn Pb Cu

Propellent Stabilizer Flash inhibitor Flash inhibitor Flash inhibitor Plasticizer Plasticizer Plasticizer Stabilizer Stabilizer Stabilizer Fuel Bullet material Oxidizing agent Fuel Fuel Fuel Bullet material Bullet material Explosive (lead styphnate) Bullet material

These compounds do not represent a comprehensive list of chemicals that may be found in gunshot residues.43 Reproduced with permission from Morales, E. B.; Vazquez, A. L. R. Simultaneous determination of inorganic and organic gunshot residues by capillary electrophoresis. J. Chromatogr. A 2004, 1061, 225–233 (Copyright 2004, Elsevier Science).

minimizing the matrix interferences. Among the GSR sampling techniques, tape lift, vacuum lift, swabbing, glue lift, and hair combing are most common. It is worthy to mention that although the analytical techniques for the analysis of inorganic and organic GSR are significantly different, their sampling methods are still the same.

1.23.5.3.1

Tape Lift

Tape lift represents the most commonly used sampling technique for inorganic gunshot residues and can be applied to sample collection from skin, hair, and other surfaces.41 Different adhesives used in tape lifting include double-sided tape, adhesive tabs, adhesive liquids, glue sticks, and carbon-conductive cements. A comparative study focused in verifying collection efficiency of different adhesives revealed that Sellotape 404 double-sided tape performs best among all the tested adhesives in the study.42 Another study comparing tape/sticky lifts to swabs for the collection of inorganic GSR demonstrated that tape lifting is much more effective than swabs.42 Tape lifting was also reported to collect organic gunshot residues (OGSRs) followed by extraction and introduction to GC for profiling.44 Collecting GSR from suspects’ clothing by tape lift is challenging as it collects fiber and other debris from the cloth surface as well and may mask the identification process. Carbon/gold coating of the collected samples often helps reducing the matrix effect.

1.23.5.3.2

Vacuum Lift

Vacuum lift is primarily used for the collection of both organic and inorganic GSR from cloth surfaces. Collected samples are cleaned and preconcentrated by solid-phase extraction prior to injecting into the analytical instrument. Teflon filter has been found more effective in collecting organic GSR. Also, methylene chloride is advantageous when extracting propellant compounds.42

1.23.5.3.3

Swabbing

Swabbing can be used for collecting both organic and inorganic GSR. Sample collection by swab is generally carried out using water or organic solvents, e.g., ethanol. Ethanol is preferred as it prevents the growth of micro-organisms that are likely to degrade nitroglycerine. Reardon and MacCrean45 made a comparison between supercritical fluid extraction (SFE) and ultrasonic solvent extraction (USE) for quantitative extraction of smokeless powder. SFE did not perform well for double-base powder, although the performance with regards to single-base powder was satisfactory.

1.23.5.3.4

Glue Lift

Glue lift sampling technique is recommended for collecting GSR from the hand surface. As glue does not contain any highmolecular-weight element that can interfere with GSR particle analysis in SEM, it is preferred over tape lifting when there are options.

Legal and Forensic Sampling

1.23.6

449

Analysis of Controlled Substances and Toxicants from Different Matrices

Controlled substances are those regulated by a government agency and generally include illegal and prescription drugs. Toxins are substances which may or may not be controlled but have extremely high mortality at relatively low doses. Forensic toxicology combines analytical chemistry and the medico legal aspects of these substances. This section discusses the advances made in the techniques used to sample or extract controlled substances found in biological matrices such as urine, blood, hair, saliva, sweat, and other bodily tissues for subsequent analysis.

1.23.6.1

Urine

Of all biological matrices listed, urine is the most commonly sampled in forensic science and is the matrix of choice for drug testing. The kidneys, which produce urine, are the most important organs responsible for the elimination/excretion of drugs and metabolites from the human body. Due to glomerular filtration processes, urine is devoid of suspended proteins, lipids, and large-molecular-weight molecules. This greatly simplifies any additional sample preparation required prior to analysis. However, most biological matrices, including urine, cannot be analyzed directly due to their incompatibility with analytical instruments; this includes high polarity, low volatility, etc. As a result, samples must first be ‘prepared’ in order for analytes to be extracted or isolated from the matrix. Techniques have thus been developed to eliminate or reduce these factors. Headspace-solid-phase microextraction (HS-SPME) has become increasingly popular for the analysis of volatile organic compounds found in urine as it allows solvent-free extraction and preconcentration in a single procedure. Good detection limits have been reported for the extraction of common target analytes such as ethanol from urine, 0.0001 g dl1.46 Direct immersion solid-phase microextraction (DI-SPME) which utilizes SPME to extract analytes by being immersed directly into the liquid sample matrix has been utilized to extract nonvolatile drugs such as barbiturates from urine.47 Extraction efficiencies of 0.01–2.76 % were reported and detection limits of 0.01–0.6 mg ml1 for seven barbiturates, all directly extracted from urine. Electrochemically enhanced solid-phase microextraction (EE-SPME) was developed by applying electrical potentials to the SPME fiber while it is immersed in the sample matrix, which extracted 11 times more analyte than conventional DI-SPME.48 For trace level detection where higher extraction efficiencies are required, stir bar sorptive extraction (SBSE) that utilizes a stir bar coated with polydimethylsiloxane (PDMS) which stirs in the matrix of interest to extract a target analyte is used.49 This method has yielded extraction values of 45.7–99.9% for analytes dissolved in urine giving detetction in the parts per trillion (ppt) range.49 Solid-phase microextraction membrane (SPMEM) was introduced in 2004 to address the limitation of conventional SPME sampling in extracting nonvolatiles.50 The SPMEM device is immersed directly in the matrix of interest; the device is then desorbed using a solvent and sonication. The solvent extract is then subjected to LC analysis, extracting less volatile compounds than HS-SPME and using far less solvent than conventional SPE.50 Other non-SPME-based sampling methods have been developed, such as DESI and MALDI. These are ambient mass spectrometry sampling techniques, where the sample is ionized under atmospheric pressure directly from the matrix and analyzed exclusively by a mass spectrometer. DESI utilizes secondary electrospray ionization to simultaneously extract and ionize analytes from the surface of sample matrices, requiring little sample preparation. Ethanol has been targeted as an analyte of interest in one recent DESI study.51 MALDI requires that the sample be mixed with an organic acid matrix. The sample/matrix mixture is then subjected to a laser, which in turn simultaneously ionizes and extracts the target analytes from the sample. The ionized analyte is then separated and detected by a mass spectrometer. Analytes such as controlled doping agents and drug metabolites such as benzoylecgonine have also been detected by MALDI from urine.

1.23.6.2

Blood

Blood, like urine, is another common matrix analyzed in forensic testing and is often the preferred specimen for testing. Blood has two principal components, plasma and cells. Numerous tests have been developed and optimized for extraction of analytes from serum or plasma, though plasma is preferred as it produces less precipitate of fibrin.52 There are few methods that analyze blood directly, one of the more traditional techniques being static headspace analysis to analyze volatile components found in whole blood. One new sampling technique focuses on sampling blood in vivo. In vivo SPME has been developed by Vuckovic et al. and has been used to detect drugs such as pseudoephedrine and diazepam with extraction efficiencies from 0.28 to 0.80%.53 This novel sampling technique utilizes SPME coupled with a hypodermic needle to sample blood directly from arteries and veins (Figure 3). Most techniques optimized for urine will also extract analytes from plasma. However, urine contains mostly metabolized polar components while blood will contain these as well as the parent unmetabolized species. The parent compounds are often more useful as direct evidence in criminal proceedings. A new format of the traditional solid-phase extraction (SPE) termed microextraction by packed sorbents (MEPSs) has been successfully used for the extraction of analytes from whole blood and plasma utilizing much smaller volumes than traditional SPE. A review highlighting this technique has been previously published by Rehim.54 A high-throughput automated system utilizing SPME has been developed that mimics the automated SPE systems traditionally used. This allowed for the extraction of 96 samples in as little as 100 min.55 Thin film microextraction (TFME) has also been developed to perform direct extraction of analytes from whole blood without requiring any prior sample preparation.55 Recent work by Szultka et al. has led to the development of a polypyrrole solid-phase microextracition fiber coating that is able to be directly immersed in whole blood samples for the extraction of polar analytes.56 Restricted access materials (RAMs) have also been extensively developed and used for the online extraction of plasma samples as

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Figure 3 In vivo SPME used for direct blood sampling. Reproduced with permission from Vuckovic, D.; Shirey, R.; Chen, Y.; Sidisky, L.; Aurand, C.; Stenerson, K.; Pawliszyn, J. In vitro evaluation of new biocompatible coatings for solid-phase microextraction: Implications for drug analysis and in vivo sampling applications. Anal. Chim. Acta 2009, 638, 175–185, Copyright 2009, Elsevier Science.

RAM combines the tedious SPE or LLE and protein precipitation into a single step providing a faster sample purification process for the analysis of blood.

1.23.6.3

Hair

Unlike urine and blood, which are traditional specimens, hair is considered an alternative specimen. Hair is made up of proteins, water, lipids, and minerals and consists of a root which is implanted in the skin and a shaft made of keratin that protrudes from the surface of the skin or scalp. The root consists of an enlargement called the hair bulb within which sits the hair follicle that is surrounded by a dense network of blood capillaries. Through these capillaries, substances are deposited which become ‘locked’ into the hair shaft as it grows at an average rate of 1 cm month1.57 Many research groups have used this relationship to determine long-term exposure levels of a person to a particular controlled substance creating a ‘diary of exposure.’58 Substances within the hair remain for an extended period of time allowing the hair to be stored without refrigeration, pH control, or other preserving agents. This, coupled with the wide detection window, makes testing hair advantageous over testing urine or blood. Hair samples are typically collected from the back of the head, the vertex posterior,58 though studies have shown differences in drug concentrations between hairs collected from different regions of the body.57 Differences have also been observed where pigmented hair will have a 10 to 157-fold higher concentration of basic drugs than nonpigmented hair, although this difference was not observed for neutral drugs. The first step in hair analysis is a decontamination step to remove external contamination followed by extraction to release the entrapped analyte as well as preconcentrate the analyte. Traditional methods are still used such as liquid–liquid extraction and solid-phase extraction. Other techniques such as pressurized liquid extraction, more commonly used on environmental samples, have been applied to extracting analytes from hair.59 Matrix solid-phase dispersion (MSPD) has also been reported as an alternative extraction technique for solid samples such as hair.60 Liquid–liquid–liquid microextraction (LLLME) or suspended drop liquid-phase microextraction was also reported as an extraction technique to remove and analyze methylenedioxy methamphetamine (MDMA) from hair samples,61 giving detection limits of 0.1 ng ml1 using as little as 50 mg of hair and a 10ml drop of the extracting solution.

1.23.6.4

Saliva

Saliva or more appropriately ‘oral fluid’ is another alternative specimen sampled in forensics. Saliva is a colorless liquid discharge into the oral cavity that is produced by three glands: the parotid, submandibular, and sublingual. It is composed of 90% water and other constituents such as digestive enzymes, mucins, and mineral salts all maintained at a pH of 6.8. Once released into the oral cavity, saliva mixes with other substances and is then termed ‘oral fluid’. One disadvantage with the use of oral fluid is that persons are generally unable to produce sufficient quantities for analysis. The rate of oral fluid production changes based on nervous system and hormonal impulses; as a result, there exist several methods to stimulate oral fluid production. These include placing of lemon juice or citric acid in the mouth or chewing paraffin wax, parafilm, teflon, rubber bands, and gum base. Despite low sample collection, oral fluid is still advantageous as a biological specimen due to its noninvasive collection. Typically, only parent drugs and not metabolites are found in the saliva. Substances found in the saliva reflect the concentrations of unbound substances also found in the blood, but at much lower levels. Reports have also been made of detecting strongly bound analytes such as NSAIDs from oral fluid,62 which demonstrates the information-rich character of oral fluid samples. Oral fluid is almost always collected utilizing commercially available devices such as the Drager drug test, StatSure, Cozart RapiScan, Drug Swipe, and Varian OraLab. These devices use a porous applicator placed inside the mouth sublingually that wicks 0.5–1 ml of fluid that is later extracted through centrifugation. The accumulated oral fluid is then further prepared by traditional extraction methods such as liquid–liquid or solidphase extraction to remove target analytes for analysis. Other extraction procedures for collected oral fluid include using protein precipitation,63 stir bar sorptive extraction (SBSE),64 liquid-phase microextraction,65 and solid-phase microextraction.66

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Figure 4 Roller device designed for stir bar surface sampling. Reproduced with permission from Soini, H. A.; Bruce, K. E.; Klouckova, I.; Brereton, R. G.; Penn, D. J.; Novotny, M. V. In situ surface sampling of biological objects and preconcentration of their volatiles for chromatographic analysis. Anal. Chem. 2006, 78, 7161–7168, Copyright 2006, American Chemical Society.

1.23.6.5

Sweat

Sweat is also classified as an alternative specimen for forensic sampling similar to hair and saliva as previously discussed. Sweat composition is similar to that of plasma with the exception that it contains no proteins. Sweat is an excretory product that is released through the estimated 2–3 million pores covering the human body. Its main function is temperature regulation through evaporation, which removes heat from the surface of the body. The average person produces anywhere from 100 ml to 10 l of sweat on a daily basis. Despite the seemingly large volume produced, sweat samples are difficult to collect and vary based on the site of collection on the body. Samples are collected with an absorbent patch worn on the skin over an extended period of time, a few days to weeks. Collection is further complicated by the uneven distribution of sweat over different areas of the body as 50% of sweat volume is produced by torso and the remaining volume distributed equally between the legs and the head. Samples are collected utilizing a sweat patch, which is then subsequently extracted using solid-phase extraction and liquid–liquid extraction. Some recent advances in this area have included the use of stir-bar sorptive extraction (SBSE) in the form of a special device that allows the bar to be rolled over the skin (Figure 4).67 Previous to this, a 2004 study, conducted by Follador et al., utilized DI-SPME to extract analytes in sweat collected by a sweat patch.68

1.23.6.6

Sampling of other Biological Tissues

This section focuses on samplings from the liver, lungs, kidney, spleen, brain, bone, finger, and toenails. These samples are almost exclusively obtained for postmortem analysis as they often cannot be obtained in a noninvasive manner or collected without serious bodily injury. A recent and comprehensive review on the collection of biological samples by Dinis-Oliveria et al. has highlighted the most relevant tissues for this purpose, namely the liver, lungs, kidney, spleen, brain, skeletal, and cardiac muscle.52 Other tissue such as nails from the fingers and toes will also be discussed. After death occurs, the collection of traditional bodily fluids, such as blood, provides little information owing to the severe postmortem hemolysis that occurs due to decomposition, rendering the sample more viscous and extraction of analytes from bodily fluids of little effect. A recent study by Tolliver et al. has highlighted the fact that blood mixtures are ‘inherently contaminated by tissues and organs’ when comparing antemortem and postmortem blood analysis.69 Drugs with volumes of distribution greater than 3 l kg1 are most likely to undergo postmortem redistribution (PMR). The analysis of these solid tissues also presents particular difficulties as they contain high protein contents, ranging from 6% to 50% by weight. Tissue samples must first be homogenized, to allow much of the proteins to be precipitated by a variety of reagents. These reagents include organic solvents such as acetone or acetonitrile, zinc sulfate, 5-sulfosalicyclic acid, perchloric acid, trichloroacetic acid, sodium tungstate, or ammonium sulfate. Once proteins have been precipitated, the liquefied sample may then be separated from the solid precipitate by centrifugation and be subject to further sample preparation, which will extract and isolate the target analyte from the tissue sample.

1.23.6.6.1

Liver, Lungs, Kidney, Spleen, and Brain

The liver is considered the most important tissue sample for postmortem analysis. Higher concentrations of drugs tend to be found in the liver than can be found in the blood. However, for these tests, the tissue samples must still be excised from the body. A 2007 study proposes the use of computer tomography (CT) scans to aid in ‘needle autopsy’ where the relevant tissues are excised by needle incisions as opposed to full body conventional autopsy.70 The study successfully performed needle autopsies on three bodies to sample brain, heart, lung, liver, spleen, kidney, and muscle tissue, boasting minimally invasive sampling in instances where permission for conventional autopsy cannot be obtained, faster sampling time, as well as significant reduction in the risk of infection to the forensic pathologist and the morgue attendant (Figure 5).

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Figure 5 Performance of CT-guided Needle Autopsy. Reproduced with permission from Aghayev, E.; Thali, M. J.; Sonnenschein, M.; Jackowski, C.; Dirnhofer, R.; Vock, P. Post-mortem tissue sampling using computed tomography guidance. Forensic Sci. Int. 2007, 166, 199–203, Copyright 2007, Elsevier Science.

1.23.6.6.2

Fingernails and Toenails

Nails are also considered alternative specimens like hair. It consists of a nail body (plate) and root. Its growth is similar to that of hair but has growth rates of 3–5 mm per month for fingers and 1.1 mm per month for toes. Drugs are also considered to be deposited in the nail through blood capillaries at the nail root but, unlike hair, there have been no studies that indicate an influence on analyte extraction based on pigmentation. Sampling of nails also provides data of exposure over time and like hair must also be washed to remove external contamination prior to extraction. After washing, the nail is cut into small pieces followed by an extraction technique. Common techniques used are acid digestion for elemental analysis in cases where heavy metal toxicity is suspected, or liquid–liquid and solid-phase extraction. Some researchers have concluded that the use of toenails is preferred over finger nails as these are less likely to come in contact with external contamination. Direct analysis of nails is also possible utilizing laser ablation,71 laser induced break down spectroscopy,72 Raman Spectroscopy, and X-ray fluorescence (XRF).73 A fairly recent review of nail analysis can be found in the literature by Trunova et al. and discusses the use of data obtained by sampling nails for the diagnosis of human health.74 Typically, both parent drug and metabolites are found in the nails providing convincing evidence of their ingestion.

1.23.6.6.3

Bone

Bone is the forensic sample of last resort. It is the only material remaining after advanced stages of decay have deteriorated all other specimens that may be relevant to forensics. Within the center of all bones of the human body is the marrow, this fibrous network has been reported to be a reliable alternative specimen in instances where blood is not available. Analytes typically detected in the bone marrow are identical to those that would normally be found in the blood. A review on bone marrow analysis has been published in the literature and cited.75 Sampling of bone marrow involves cutting the bone and removing the marrow; the marrow is then subject to liquid–liquid extractions. Direct sampling techniques such as laser ablation on bone have also been reported in an attempt to discriminate between different bone fragments found in mass graves76 as well as reveal the presence of certain xenobiotics useful to the forensic toxicologist.77

1.23.7

Forensic Examination of Trace Evidence

Sampling and sample preparation of materials prior to forensic analysis is particularly important for forensic samples due to the nature of the samples of interest and the form that they are collected at the crime scene and received into the laboratory. Very small (trace) amounts of natural and man-made materials readily transfer between objects or between people and objects. The examination of glass, fiber, tapes, adhesives, and paint evidence is included in this category and, because the quantity of the material analyzed in other substrates is also very small, paper, ink on paper, and other materials that can also be categorized as trace evidence are therefore also included here.

1.23.7.1

Hairs, Fibers, Glass, and Paint

Several different chromatographic methods have recently been coupled to mass spectrometry (MS) to analyze drugs and other organic compounds of interest to forensic scientists in hair,78 and a paper describing the sample preparation for elemental analysis in hair using ICP-OES79 was also reported as was the direct solid sampling of hair for elemental analysis using capillary electrophoresis coupled to a chemiluminescence detector80 and ICP-MS.81 An FTIR-ATR sampling was recently reported for the

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examination of hair-keratin fibers82 that could be of interest to hair and fiber examiners and the characterization and interpretation of dyed hair was reported using two-dimensional infrared correlation spectroscopy.83 The use of laser-scanning confocal microscopy for the examination of hairs and textile fibers84 was also recently reported and the chemical analysis of Azo and methane fiber dyes was possible using microextraction followed by CE-MS.85 The characterization of wood and vegetable fibers was reported by first using thermally assisted hydrolysis and methylation with pyrolysis-GC-MS86 while resonance Raman and UV-visible spectroscopy was used to analyze black textile dyes.87 A more general fiber characterization using UV-visible microspectrophotometry was reported88 and the sampling and analysis of a single PET fiber for the elemental composition in the fiber was reported using laser ablation (LA) coupled to an ICP-MS.89 LA-ICP-MS and laser-induced breakdown spectroscopy (LIBS) were also used for the elemental analysis of white cotton fibers.90,91 LA-ICP-MS has been used for the elemental analysis of glass for quite some time, and a recent application of LA sampling of ancient glass92 was reported to produce useful information when coupled to ICP-MS. LIBS is also a relatively new sampling/analysis method reported as providing excellent discrimination between glass samples thought to have originated from different manufacturing sources.93 ATR-FTIR imaging of paint cross-sections was reported94 as a useful method for paint characterization, and laser desorption MS was used to analyze synthetic organic pigments in works of art,95 and the novel application of Fourier-transform photoacoustic infrared (PAIR) spectroscopy was used in the forensic analysis of inorganic pigments.96 Microspectrophotometry in the visible range was used to differentiate car paints97 and pyrolysis-GC-MS was used to analyze spray paints98 and plasticizer content in polyvinyl acetate polymer (PVA) binders in paint medium.99

1.23.7.2

Ink and Paper Analysis

Acid digestion followed by ICP-MS100 procedures were reported for the elemental analysis of paper within the field of document examination in order to discriminate between and associate paper sources. A comparison of different methods (XRF, LA-ICP-MS, and IRMS) for the analysis of paper was also recently reported.101 Direct solid sampling of printer toners for chemical characterization was reported using LA-ICP-ToF-MS.102 The organic dye analytes in inks were sampled and analyzed with the use of a variety of techniques including laser desorption ionization MS103 and direct analysis in real time (DART) MS.104 ESI-MS of both dyes and the vehicles in extracted ink from a document was reported105 as was the use of LDI-MS106 for the sampling and characterization of inks. The characterization of naturally and artificially aged inks and papers was conducted using pyrolysis-GCMS.107 The Raman and SERS analysis of synthetic dyes found in ballpoint pen inks108 and LDI-MS analysis of pigmented inkjet printer inks and printed documents109 were also recently reported. The novel application of micro-attenuated total reflectance sampling coupled to FTIR was also reported for the study of documents containing red seal inks,110 and the use of ToF-SIMS for the simultaneous analysis of organic and inorganic components from ballpoint pen inks111 was also reported. The elemental analysis of paper and gel inks was studied using the micro-spectrochemical analysis techniques of LA-ICP-MS and LIBS112 and the elemental composition of blue ballpoint pen ink was determined by LA-ICP-MS113 as was the use of TXRF for the elemental characterization of ink samples.

1.23.7.3

Miscellaneous Trace Evidence

The analysis of capsicum extracts from self-defense devices114 was carried out with carbon nanotube sensors,115 direct analysis in real time (DART) coupled to MS.116 Laser desorption MS was used to characterize the inorganic components in costemics.117 The comparison of electrical tapes and the characterization of acrylic and rubber-based adhesives were analyzed by ATR FT-IR118 in order to better discriminate between different sources of adhesive tapes.

1.23.8

Forensic Environmental Analysis

With the growing concern about environmental pollution and its detrimental effect on the environment as well as on the well-being of human health, environmental forensics has drawn enormous interest among scientists in recent years. Environmental forensics involves identification of a contaminant’s release, determination of the possible sources of the contaminant, estimation of approximate timing of the release and its distribution in the environment, appropriation of the liability for the damage among the sources, and finally prosecution of the responsible person or parties. As such, it encompasses all aspects of environmental pollution and contamination within air, water, and soil. The major US regulation known as “The Comprehensive Environmental Response, Compensation and Liability Act (CERCLA)”119 is the principal safeguard for environmental forensics which is funded through a tax on the chemical and oil industries in the USA and the generated revenue is used to remediate abandoned site(s) where responsibility of the pollution cannot be determined. Another important legislation known as the Clean Water Act120 authorizes each state to establish its own water-quality criteria and to limit maximum limit of disposal for a particular contaminant. US EPA has classified 126 compounds as the priority pollutants and monitors closely their disposal, distribution, and fate in the environment. EPA priority pollutants include polycyclic aromatic hydrocarbons (PAHs), asbestos, pesticides, heavy metals, and polychlorinated biphenyls (PCBs). In addition to these 126 priority pollutants, US EPA has also identified 116 compounds, which are listed in the Contaminant Candidate List 3 (CCL 3). Compounds of this class include pesticides, disinfection byproducts,

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Sampling Theory and Methodology

commercial chemicals, waterborne pathogens, pharmaceuticals, and biological toxins. CCLs are not regulated by national primary drinking water regulations, however, may be regulated by Safe Drinking Water Act (SDWA). R.D. Morrison extensively reviewed all major techniques generally used in environmental forensics.121 Also, a number of books were published in recent years covering the whole spectrum of environmental forensics.119,122,123 Although the jurisdiction of environmental forensics encompasses all current or potential man-made pollutants, only a few of them have drawn attention to environmental forensic scientists which include asbestos, sewage, heavy metals, radioactive compounds, pesticides, perchlorate, polychlorinated biphenyls (PCBs), chlorinated solvents, dioxin and furans, polycyclic aromatic hydrocarbons (PAHs), and petroleum hydrocarbons.123 However, among all the pollutants, petroleum hydrocarbons have been investigated the most. In addition, environmental pollution originating from illicit drugs and pharmaceuticals has drawn attention in recent years.124,125 Sampling strategy in environmental forensics plays a very important role as the target contaminant(s) is often not homogeneously distributed across the affected area. Therefore, it is critical to pay utmost attention in selecting the decision unit (region of the affected area to be investigated), establishing realistic level of confidence in data collection, collecting the sample from the decision unit, preserving the integrity of the sample prior to analysis, maintaining chain of custody of the samples beginning from the sample collection through the analysis, and collecting the analytical subsample from the field sample. Another important factor in the sampling strategy is to establish a viable quality control measure that ensures and safeguards the quality of the whole analytical process from any systematic error and often includes trip blanks, field blanks, decontamination check blanks, splits, and replicates.119 In the US, often regulatory standards specify the proper sampling containers to be used for water, air, and soil samples. Samples with low concentration of the target pollutant require refrigeration or addition of appropriate preservatives to minimize any potential physico-chemical or biological changes. Saxton and Engel126 surveyed the soil-sample handling procedures of different state pesticide regulatory agencies and found that 10 states use bags, 30 states use glass jars, 2 states use bags/jars, and 4 states use plastic tubes/aluminum foil/ boxes. As such, it is important to follow the regulation of the particular state in which the jurisdiction of the environmental site falls. US EPA has developed a number of standard methods119 in order to measure volatile and semi-volatile hydrocarbon compounds in water, soil, tissue, oil, air, and other matrices. Among these methods, the following are most frequently used: 1. Compendium Method TO-15, Determination of volatile organic compounds (VOCs) in air collected in specially prepared canisters and analyzed by gas chromatography/mass spectrometry (EPA, 1999). 2. Method 8015B, Nonhalogenated organics using GC/FID (EPA, 1997; SW-846). 3. Method 8260B, Volatile organic compounds by gas chromatography/mass spectrometry (EPA, 1997a; SW-846). 4. Method 8270C, Semi-volatile organic compounds by gas chromatography/mass spectrometry (EPA, 1997a; SW-846). 5. Method 8082, Polychlorinated biphenyls (PCBs) by gas chromatography (EPA, 1997a; SW-846). 6. Method 680, Determination of pesticides and PCBs in water and soil/sediment by gas chromatography/mass spectrometry (EPA, 1985). 7. Method 1668A, Chlorinated biphenyl congeners in water, soil, sediment, and tissue by HRGC/HRMS (EPA, 1999b). In addition to these chromatographic methods, EPA Method 1664, N-Hexane extractable material (HEM; oil, and grease) and silica gel treated N-Hexane extractable material (SGT-HEM; nonpolar material) by extraction and gravimetry (EPA, 1999a) is a frequently used wet chemical method that aims at analyzing total petroleum hydrocarbons (TPHs).

1.23.8.1

Volatile Hydrocarbon Fingerprinting

Volatile hydrocarbons include light refined petroleum products and tar distillates with hydrocarbons range from C4 to C12. Regulated compounds, e.g., benzene, toluene, ethylbenzene, and xylene isomers (BTEX), belong to this class. Refined petroleum often contains different toxic additives and/or bleeding agents, e.g., MTBE, ETBE, TBA, which also offer invaluable information in regard to their source or origin. Volatile hydrocarbon data are often used to identify the type and source of the pollutant, degree of weathering, and approximate timing of the disposal in the environment.119

1.23.8.1.1

Air

Petroleum products and tar distillate emanate many volatile hydrocarbons to the atmosphere and their efficient capture and analysis may provide valuable information for the fingerprinting. EPA Method TO-15 is generally used for potential indoor air pollutants. Suspected air sample is drawn through a sampling loop comprised of regulators to control the rate of flow as well as the duration of flow into the empty canister. Before analyzing the air containing the suspected pollutant(s), a known volume of air is transferred through a multisorbent concentrator. The multisorbent concentrator is then thermally desorbed into the GC/MS system for chromatographic analysis of the contaminants. This method has been proven to be extremely useful in generating qualitative and quantitatve data for VOCs in air and subsurface vapors.

1.23.8.1.2

Soil

The collection soil samples for VOC analysis is generally governed by EPA Method 5035A. This method illustrates a closed purgeand-trap system and extraction for volatile organic compounds in soil and waste samples. Soil samples are collected using EnCoreÔ

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sampler/Purge-and-Trap soil sampler or similar samplers. Care must be taken in order to minimize the potential loss of VOCs during sampling. The sampler is then placed in a prelabeled foil container and shipped back to the analytical laboratory. If the sample can be processed on-site, the soil samples are collected in small quantity using EasyDrawÔ syringe and PowerStop HandleÔ. The 5-gram sample is transferred into a 40-ml septum-capped VOA vial containing 5 ml of deionized water and a Teflon stir bar. If the sample is not analyzed immediately, then it must be kept in the refrigerator. VOCs are purged through Tenax GC/Methyl silicone packing OV-1 (3%) on Chromosorb-W/Cocoanut charcoal/Carbopack/ Carbosieve trap to preconcentrate the VOCs on the trap. Finally, the trapped VOCs are thermally desorbed and introduced into the gas chromatographic system for separation and analysis. Chromatographic analysis of the VOCs can be carried out following EPA Method 8015/8021/8260 or any other suitable GC methods. When the VOC concentration in soil sample is relatively high, direct headspace analysis can be used. Efficiency in headspace analysis greatly depends on the optimization of several factors, e.g., temperature, matrix agitation, headspace volume, and injection volume to the GC. Sewage samples and sediments have also been analyzed using this method.

1.23.8.1.3

Water

Water samples for VOC analysis are collected with pre-cleaned Teflon bailers. Subsamples are prepared by draining water into a 40 ml septum-capped VOA vial containing HCl to adjust the pH of water to 2. Samples are then stored at 4
C. Long storage of water samples containing VOCs are not recommended as Bravo-Linares127 demonstrated that even storing for 2–4 h may potentially decrease VOCs at 5–30% per hour. During analysis, VOCs are stripped from water samples by a continuous stream of an inert gas (He/N2). The purged volatiles are trapped on a sorbent cartridge or a cryotrap. The trapped analytes are then transferred to GC system by thermal desorption. Although time consuming and complicated, the dynamic headspace or purgeand-trap system has been proven to be rugged and reliable and has been used to analyze VOCs in water,128 sea water,129 and drinking water.130 Although purge-and-trap is the most predominantly used technique for VOC analysis from water, solid-phase microextraction has been used with favorable results. Bravo-Linares et al.127 has demonstrated the advantage of using SPME over purge-and-trap in sea water. A modified version of SPME designed to take advantage of purging the VOCs through the SPME fiber has successfully identified and quantified a wide range of VOCs from sea water including sulfur-containing compounds, halogenated compounds, nonmethane hydrocarbons, BTEX, aldehydes, and terpenes in a single analysis.

1.23.8.1.4

Nonaqueous Phase Liquid (NAPL) Samples

Nonaqueous samples are collected from the field using pre-cleaned Teflon bailers or other suitable collection systems capable of transferring easily in subsampling VOA vials. The collected samples are transferred into a 25 ml or 40 ml VOA vials with minimal headspace. In case the available NAPL samples are low, the VOA vials can be filled up with water from the bailer to reduce the headspace. The NAPL samples are then stored at 4
C and shipped to the analytical laboratory. The VOCs present in NAPL samples are also analyzed in a similar fashion as mentioned for water samples.

1.23.8.2

Semi-Volatile Hydrocarbon Fingerprinting

Although US EPA does not define semi-volatile hydrocarbons in order to explicitly differentiate them from volatile hydrocarbons, sample preparation for semi-volatile hydrocarbons differs from that of volatiles to some extent and deserves careful consideration. Semi-volatile hydrocarbons include crude oil, refinery intermediate, and petroleum products like kerosene, diesel, and residual fuel oils.119 In addition, coal tars, oil tars, wood tars, and their derivatives are also considered to be semi-volatile. Oftentimes, when the environmental samples (generally, soils, and sediments) are highly contaminated and contain a plethora of unwanted materials that may be coextracted with the target analyte and inflict serious matrix interferences, these samples are required to cleanup prior to extraction. Some of the cleanup processes include alumina solid-phase adsorbent, gel permeation chromatography, and silica gel solid-phase adsorbent.

1.23.8.2.1

Water

Environmental water samples containing semi-volatile hydrocarbons are typically extracted using solvent extraction techniques illustrated in EPA Method 3510, Separatory Funnel Liquid–Liquid Extraction. As per the protocol, 1 l of water is added to a 2 l separatory funnel. The water sample is then extracted three times with 60 ml of dichloromethane (DCM). The combined dichloromethane extracts are then passed through anhydrous sodium sulfate to remove trace water present in dichloromethane. Prior to instrumental analysis, the extracts need to be concentrated by evaporating much of the dichloromethane.

1.23.8.2.2

Soil and Sediment

Semi-volatile hydrocarbons from soil and sediment samples are generally extracted using solvent extraction process. If the sample is bulky, it might require physical disintegration by crushing/drilling/cutting. Biological debris should be removed prior to extraction. The homogenized solid samples are then transferred to the extraction vessel. Dichloromethane is the most commonly used solvent for solvent extraction. In order to achieve near complete extraction, al least 3 extractions should be carried out for each sample. Finally, all extracts are combined and the solvent is evaporated prior to the introduction into the analytical instrument.

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1.23.8.2.3

Sampling Theory and Methodology

Nonaqueous Phase Liquid (NAPL) Samples

Petroleum and tar products, the major constituents of NAPL samples, are generally processed using EPA Method 3580, Waste Dilution. Upon receiving such a sample, an aliquot is quantitatively transferred into the dilution vessel. A known volume of dichloromethane is added to the dilution vessel and rigorously mixed until it becomes apparently homogeneous. The mixture is then filtered through a glass filter to remove any particulates material.

1.23.9

Analysis of Human Odor Profile

Human scent is the ultimate result of the complex combination of the body’s metabolism, gland secretions, hormonal control, and the interaction with the bacterial populations residing on the skin surface. Human scent is a complex mixture of hundreds of compounds with different functional groups, e.g., alcohols, aldehydes, aliphatic/aromatic hydrocarbons, carboxylic acids, carboxylic acid methyl esters, ketones.131 It is believed that every human being has his/her own distinct odor profile, analogous to a signature fingerprint.132 Individual body odor of a human being is dependent upon several factors (e.g., genetic makeup, environmental and internal physiological conditions) and can be classified as: the primary odor if the odor constituents are stable over time regardless of the diet or environmental factors, as the secondary odor if the odor constituents are contributed from diet and environmental factors, and as the tertiary odor if the odor constituents are originated from an outside source (e.g., personal hygene/ cosmetic products).133 The individual odor hypothesis has influenced the interest of many researchers including our research group to investigate the human scent profile and its implications to canine training. As the primary odor of an individual attributes to the ‘uniqueness’ of the individual, forensic scientists generally focus in understanding VOCs that constitute the primary odor. A comprehensive account on human scent collection and identification can be found in recently published review articles.134,135 For both qualitative and quantitative identification of human scent components, it is often recommended to use a suitable sampling and/or preconcentration step so that each individual compound of the complex mixture (human scent) reaches the limit of detection for the particular analytical instrument (GC/MS, in general). Being a simple, rapid, selective, and efficient sample preparation technique, SPME has gained enormous popularity among forensic chemists who are engaged in human scent research. As such, most of the articles published in the last decade on human scent profiling used SPME in conjugation with other scent collection media. The complexity and the wide range of polarity of VOCs in human scent have made the selection of the right SPME fiber type quite challenging. Divinylbenzene/Carboxen/polydimethylsiloxane (DVB/CAR/PDMS) 50/30 mm has been found to be the most efficient in human scent profiling136 although Z-M. Zhang137 reported polydimethylsiloxane/divinylbenzene (PDMS/DVB) 65 mm to have performed the best. When extraction is carried out directly from the body part, it needs to be enclosed within a defined headspace where the SPME fiber can be exposed for a predetermined time. Another approach uses a flow-sampling chamber (Figure 6) where human hand emanation can be carried to the SPME fiber via inert gas, and after the predetermined extraction time, SPME fiber can be introduced into GC or GC/MS.135,137 In aligning with the common practice followed by the law enforcement community to capture the volatiles on a sorbent media so that it can be presented to a trained canine for identification purpose, our (K.F) research group utilized different sorbent media (cotton, polyester, etc.) to capture emanating VOCs from the human skin, equilibrated in an SPME vial for 24 h and subsequently extracted on the SPME fiber and finally introduced into the injection port of the GC/MS for analysis and identification.136 A study in our lab designed to identify suitable sorbent media (cotton, cotton blend) for human scent collection revealed that pure cotton seems to have strong affinity toward polar compounds (e.g., alcohol) due to the exposed hydroxyl groups on the surface and tends to release such compounds rather slowly so that it can be extracted from the headspace by the SPME fiber. As such, cotton blend was found to be more efficient in human scent collection and release for instrumental analysis.138 The sorbent materials frequently used for human scent collection are often found to contain a variety of compounds which are reported as human scent component. Therefore, the sorbent materials need to be analytically cleaned prior to use in human scent collection. Our research group compared different extraction techniques in order to remove all the VOCs from the sorbent media that can be potentially found in human scent. Supercritical fluid extraction was found to be the most efficient to clean the sorbent media from human scent VOCs.131 Recent trend in human scent collection favors the scent collection on the sorbent media with the aid of a scent transfer unit (STU-100), a hand-held portable device that uses a vacuum to force VOCs to pass through the sorbent media for trapping onto its surface.The major advantage of this procedure is its dynamic noncontact sampling mode that allows collection of VOCs without making any physical contact to it, thus the evidential integrity of the object remains intact.139

1.23.10 Analysis of Human Decomposition Products The last couple of years have witnessed an increased interest in the study of human remains among the forensic scientist community. Although the sample matrix is the same, different research groups have different research goals. Some research groups aim at understanding the chemical processes of decomposition, others at estimating postmortem interval, whereas some focus at developing advanced methodologies or techniques to uncover buried dead bodies from clandestine burial sites. The matrix, human remains, is extremely complex with a varying degree of decomposition containing protein, lipid, and carbohydrate macromolecules with microbial reaction byproducts, free fatty acids, amines, other VOCs compounded with environmental factors as well as VOCs already present in the soil matrix.

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Figure 6 Systems for sampling volatile compounds emitted from the skin. (a) Direct SPME in sealed glass globes, (b) direct SPME in flow-sampling chambers and (c) liquid sampling in glass cup. Reproduced with permission from Kataoka, H.; Saito, K. Recent advances in SPME techniques in biomedical analysis. J. Pharm. Biomed. Anal. 2011, 54, 926–950, Copyright 2011, Elsevier Science.

As such, it poses a serious challenge to concerned forensic scientists as far as sample preparation is concerned. Sample preparation pertaining to human remains mostly dependent upon the objective of the research outcome: (1) the estimation of postmortem interval, (2) understanding the complex decompositional pathway including the impact of different environmental and geochemical factors on the decomposition process, and (3) developing victim recovery (VR) canine training aid to locate clandestine gravesites.140 One of the major implications of estimating postmortem interval (PMI) of the dead body is to make a connection or exonerate a suspect based on the whereabouts of the suspect at the time of death. A wide array of techniques are used to determine PMI, all having their own merits and shortcomings. Adipocere is defined as a late-stage postmortem decomposition product consisting of a mixture of free fatty acids (FFAs) formed under favorable conditions due to the hydrolysis of triglycerides in adipose tissue. The major constituents of adipocere are myristic acid, palmitic acid, and stearic acid. In addition, triacylglycerides (TAGs), hydroxy fatty acids, and sodium, potassium, calcium, and magnesium salts of fatty acids have been identified as minor adipocere constituents. The identification and quantitation of adipocere is of considerable interest as it provides valuable information regarding the state of decomposition of the cadaver and its surrounding environment.141 As in many cases, suspected grave soils are analyzed to determine the presence of adipocere; a suitable sample preparation technique is required to isolate adipocere from the complex soil matrix as well as from other potential interfering components, e.g., undecomposed adipose tissue. Common sample preparation techniques for adipocere include thin-layer chromatography (TLC), liquid–liquid extraction, and column chromatography.142 However, low recovery of adipocere, high volume of organic solvent usage, and the potential oxidation of polyunsaturated fatty acids caused by prolonged exposure to air have made these sample preparation techniques less attractive. As a result, faster, less solvent consuming, and environment friendly sample preparation techniques are of continuous demand. S. L. Forbes et al.141 developed a rapid method where adipocere was extracted with chloroform and derivatized with hexamethylenedisilazane (HMDS). The derivatization process converts adipocere into fatty acid trimethylsilyl esters and allows the identification of individual esters in ppm range in GC/MS analysis. Since extraction of adipocere using chloroform does not fractionate lipid classes (triacylglycerides and free fatty acids), a new solid-phase extraction (SPE) method has been developed using aminopropyl disposable cartridge columns that can isolate free fatty acids from neutral lipid components in adipocere samples. After extracting adipocere samples onto the SPE cartridge, neutral lipid fraction was eluted from the column using a mixture of chloroform and 2-propanol (2:1 v/v). The free fatty acids (FFA) were eluted using diethyl ether containing 2% acetic acid. Both the fractions were then subjected to derivatization using bis(trimethylsilyl) trifluoroacetamide (BSTFA). The TMS fatty acid derivatives were then analyzed in GC/MS.142,143

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Due to the fact that both human and pig adipocere possess similar fatty acid distribution, oftentimes pigs are used to mimic human remains. S. J. Notter et al.143 conducted a study to verify the suitability of pigs as models for human decomposition in aqueous environment. To make a fair comparison, both pig and human adipocere tissue were submerged in warm, anaerobic, and aquatic environment. Over a period of six months, samples were collected and total lipids were extracted from homogenized adipose tissue using chloroform–methanol mixed solvent. The extract was evaporated to dryness and redissolved in hexane followed by solid-phase extraction using disposable aminopropyl cartridge columns. Finally, the extract was derivatized using BSTFA before injecting aliquot into the GC/MS system for analysis. In addition to the chromatographic methods that offer both qualitative and quantitative profiles of the decomposition fluid, non-chromatographic methods are also used primarily to identify chemical markers that can be used to estimate postmortem interval (PMI). As such, vitreous humor samples of dead bodies were analyzed to quantify potassium ions (Kþ), hypoxanthine (Hx), and amino acids, and different models have been developed to plug in these analytical data in order to estimate the postmortem interval. Another important facet of human remains study is to identify and use the volatile organic compounds (VOCs) associated with human remains decomposition for developing canine training aids. Such training aids have been proven to be very effective to train canines and later deploy them for detecting clandestine graves.144,145 E. M. Hoffman et al.145 performed a SPME analysis on 14 different tissue types, which were previously used as victim recovery (VR) canine training aids. The samples included blood clot, blood clot from placenta, blood, muscle, testicle, skin, body fat attached to skin and teeth, adipocere, fat tissue, bone, etc. The headspace above the human tissues was sampled at room temperature using polydimethylsiloxane/divinylbenzene (PDMS/DVB) 65 mm SPME fiber for 40 min. GC/MS analysis of SPME extracted VOCs yielded 33 compounds that included acids, esters, alcohols, aldehydes, halogens, ketones, aromatic hydrocarbons, and sulfides. Arpad A. Vass et al.144,146 performed human remains decompositional odor analysis by employing triple sorbent traps (TSTs) into 4 artificially created gravesites to long-term study of VOC emanation from human remains during the decomposition process. The TSTs was comprised of Carbotrap, Carbotrap C, and Carbosieve S-III sorbents. The sorbents were housed in a 76-mm-long stainless steel tube with 6 mm O.D. and 4 mm I.D. The TSTs were connected to a specially designed sampling manifold with controllable flow rates. After the extraction of VOCs into the TSTs, analytes were transferred into the cryocooled GC inlet by heating TSTs to 350
C for 5 min. These studies have identified 478 specific volatile organic compounds liberated from the human body during the decomposition process. In a similar fashion, M. Statheropoulos et al.147 studied VOCs released during the decomposition process. He used three layers of sorbents comprised of 300 mg carbograph 2, 200 mg carbograpg 1, and 125 mg carbosieve S-III packed in a glass tube. The tubes were conditioned for 2 h at 300
C for background removal. In a recent study by our group (K.F),148 human remains volatiles were collected by a noncantact, dynamic airflow sampling device (Scent Transfer Unit, STU-100). Figure 7 represents the diagram of the STU-100. The major advantage of such sampling method is its noncontact nature that maintains the integrity of the sample (very critical for forensic samples) and also, minimizes the possibility of contamination. VOCs from human remains were first collected on a pre-cleaned Dukal Gauze using STU-100. The captured analytes on the Dukal gauge were then extracted on a SPME fiber (DVB/Carboxen/PDMS) and introduced into the GC/MS for separation and identification.

Figure 7 Diagram of the Scent Transfer Unit. Reproduced with permission from DeGreeff, L. E.; Furton, K. G. Collection and identification of human remains volatiles by non-contact, dynamic airflow sampling and SPME-GC/MS using various sorbent materials. Anal. Bioanal. Chem. 2011, 401, 1295–1307, Copyright 2011, Elsevier Science.

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1.23.11 Nuclear Forensics Since the break down of the Soviet Union in the early 1990s, there have been more than 1000 cases of illicit trafficking involving radioactive or nuclear materials recorded in the International Atomic Energy Agency illicit trafficking database. During that period, there have been numerous efforts to enhance the international safeguard systems to discourage illicit trafficking of nuclear materials as well as interdict and analyze such materials. In 2010, DeMuth published a detailed chapter on proliferation resistance and safeguards in the Handbook of Nuclear Engineering.149 The field of nuclear forensics involves the detection, collection, analysis, and case development of illicit nuclear materials. A model action plan (shown below) has been developed by the International Technical Working Group on combating nuclear smuggling to provide guidance on incident response Figure 8.149 A recent paper summarizes the model action plan to deter illicit nuclear trafficking based on reports prepared for the International Atomic Energy Agency as a framework for international nuclear forensic support.150 The generally accepted sequence of analysis has been prioritized into techniques that should be performed within 24 h, one week, or months from arrival at the nuclear forensics laboratory. The table below details the sequence of analysis categorized by time and laboratory techniques and methods employed (Table 2).150 The first primary reference source for nuclear forensic science was published in 2005151 which details the nuclear forensic protocols and techniques as well as case studies of actual investigations. This book contains detailed chapters including principles, extraordinary sampling issues, field detection kits and laboratory analysis methods and attribution. Most of the techniques utilized in nuclear forensics are the same as those used in traditional forensic science (primarily criminalistics) as well as nuclear chemistry. Nuclear forensics poses the unusual problem due to the possible admixture of radioactive contamination with evidentiary signatures of conventional forensic analysis.151 The list of techniques which have actually been used for nuclear forensic investigations is extensive and divided into five major categories as follows: (1) Isotopes (alpha spectrometry, beta spectrometry, gamma spectrometry, secondary-ion mass spectrometry, thermal-ionization mass spectrometry, multicollector plasma-source mass spectrometry, noble gas mass spectrometry, stable-isotope mass spectrometry, accelerator mass spectrometry); (2) Elemental composition/ major and trace elements (inductively-coupled-plasma mass spectrometry, inductively-coupled-plasma, optical-emission spectroscopy, X-ray fluorescence spectroscopy, atomic absorption spectroscopy, scanning electron microscopy, scanning electron microscopy with X-ray analysis, electron microprobe microanalysis, secondary-ion mass spectrometry, particle-induced X-ray emission, high-performance liquid chromatography, ion chromatography, thin-layer chromatography, capillary electrophoresis); (3) Organic species (gas chromatography-mass spectrometry, ion-trap mass spectrometry with MS-MS, high-performance liquid chromatography/electrospray ionization/triple-quadrupole mass spectrometry; (4) DNA (polymerase chain reaction, DNA sequencing, electrophoresis, immunoassay); (5) Physical and structural characteristics (optical microscopy, scanning electron microscopy, transmission electron microscopy, X-ray diffraction, neutron and X-ray radiography, metallurgical techniques, FTIR, NMR). Depending on the materials to be analyzed, forensic tools can be prioritized as essential, important, specialized, or not relevant. Moody et al. have published a prioritization table of forensic tools categorizing the major materials analyzed (biological,

Figure 8 The model action plan developed by the Nuclear Smuggling International Technical Working Group. Adopted with modification from Cacuci, D. G., Ed.; In Handbook of Nuclear Engineering; Springer, 2010; Vol. 1–5, pp 3600, Copyright 2010, Springer.

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Table 2

Suggested sequence for laboratory techniques and methods

Techniques/Methods

24-h

Radiological

Estimated total activity Dose rateða; b; g; nÞ Surface contamination Visual inspection Radiography Photography Weight Dimensions Optical microscopy Density Fingerprints, fibers Gamma-spectroscopy Alpha-spectroscopy

Physical characterization

Traditional forensic analysis Isotope analysis Elemental/chemical

One week

Two months

SEM/EDS, XRD

TEM

Mass spectrometry (SIMS, TIMS, ICP-MS) ICP-MS, XRF Assay (titration, IDMS)

Radiochemical separations GC/MS

SEM/EDS ¼ Scanning electron microanalysis with energy dispersive sensor. TEM ¼ Transmission electron microscopy. SIMS ¼ Secondary-ion mass spectrometry. TIMS ¼ Thermal-ionization mass spectrometry. ICP-MS ¼ Inductively-coupled-plasma mass spectrometry. XRF ¼ X-ray fluorescence analysis. IDMS ¼ Isotope dilution mass spectrometry. GC/MS ¼ Gas chromatography/mass spectrometry. Reproduced with permission from Smith, D. K.; Kristo, M. J.; Niemeyer, S.; Dudder, G. B. Documentation of a model action plan to deter illicit nuclear trafficking. J. Radioanal. Nucl. 2008, 276, 415–419 (Copyright 2008, Springer).

geological, industrial, and packaging material) and the priorities of essential, important, specialized, or not relevant for the measurements of isotope abundances, major element composition, trace element composition, organic constituents, DNA, and physical structural characteristics.151 The major sample preparation techniques for alpha, beta, and gamma counting include stippling, vacuum volatilization, and electrodeposition. For inorganic analysis the sample preparation methods are those commonly used in the radioanalytical laboratory with samples adsorbed onto filter or as a dry deposit in a Teflon or other suitable container which must be determined by the radioanalytical chemist and the mass spectrometrist working together to minimize cross-contamination and adsorption losses. For organic analysis, extraction methods include classical liquid–liquid extraction, ultrasonic extraction, microwave-assisted extraction, accelerated solvent extraction, and solid-phase microextraction.151 A recent review of the use of atom counting in forensic investigations highlighted the application of accelerator mass spectrometry (AMS) to extend the application of the radiocarbon method and to determine the isotopic ratios of nuclear materials such as traces of plutonium and uranium as well as other important isotopes such as I.152 In a recent tutorial review of nuclear forensics, the authors stress the need for nuclear forensic scientists (or teams) to have knowledge in areas including radiochemistry, nuclear physics, reactor physics, materials science, and the nuclear fuel cycle.153 These authors categorize analysis methods in the categories of radiological, physical characterization, traditional forensic analysis, isotope analysis, and elemental/chemical analysis and recommend a group of first analysis methods followed by another group of methods for more detailed analysis. Another recent review focuses on the characteristic parameters found in nuclear materials including microscopic and elemental compositions, geolocation methods, and the interface with classical forensic methods and identifies the main challenges of nuclear forensics in identifying additional parameters characteristic for the origin of materials as well as expanded databases for comparison.154 Numerous sample preparation and analysis methods have been published with some recent examples highlighted here. A recent dedicated nuclear forensic methodology focused on plutonium has been developed and validated with a six-laboratory round-robin exercise.155 Schwantes et al. utilized state-of-the-art analytical method combined with simulations and historical records to identify a sample from a waste burial site as the oldest located reactor-produced sample of Pu in the world.156 A variety of techniques have been developed focusing on the sampling and analysis of microparticles. One recent article demonstrated the importance of applying a suite of methods to extract the maximum amount of useful information. In this study, particles from cotton swipe samples were examined by SEM-EDX as well as secondary-ion mass spectrometry with particle manipulation and relocation techniques capable of sequential examination of a single particle yielding morphological, elemental, isotopic, and depth-profiling information.157 A method developed that utilizes intrinsic dosimetry via thermoluminescence and electron paramagnetic resonance dosimetry has been demonstrated to be useful in the identification of glass containers used to transport nuclear material.156 A laboratory method and analysis via neutron interrogation has been developed for the analysis of unknown U-containing material without opening its holder.158 Many techniques have been published recently that utilize mass spectrometry. One recent ICP-MS method that makes use of nano-second laser ablation as a sampling source which was demonstrated to provide a screening protocol capable of producing results within an hour allowing for multi-isotope screening of nuclear and non-nuclear solid samples for a number of natural and non-natural isotopic compositions.159 A simple and low-background sample preparation method has been published for the simultaneous separation of analytes followed by multicollector ICP-MS.160 Analytical protocols have been developed for isotopic ratio analysis of actinides, fission products, and geolocators utilizing multicollector thermal-ionization mass spectrometry.159

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Finally, one of the greatest sampling challenges in nuclear forensics is where to locate sensors or sampling sites. A recent article focuses on locating a limited set of fixed and mobile radiation sensors in a transportation network in order to maximize the information gain and minimize the estimation error.161

1.23.12 Field Sampling Methods for Analytes of Forensic Interests The majority of forensic tests are conducted within a laboratory under presumably sanitary and controlled conditions. There has been considerable effort in the last decade to improve the sensitivity and specificity of analytical techniques used for these tests. However, similar efforts are still needed in the field where samples are initially collected to be analyzed in the lab. Ribaux et al.162 in 2010 stated it best by reiterating the phrase ‘rubbish in, rubbish out’ to emphasize the importance that field-sampling methods have on laboratory results. This section will discuss some of the advances that have occurred in field sampling over the last decade, focusing on field sampling strategies that have improved identification of latent evidence and storage of evidence. This section does not attempt to provide guidelines for the seemingly limitless types of samples encountered during a forensic case. Rather, it aims to highlight some of the areas that should be considered when conducting field sampling of analytes and introduces some new techniques used to analyze samples commonly encountered in the field. Forensic sampling entails identifying anomalies in a particular area or crime scene that may be used as evidence during the course of a criminal investigation. The discovery of these anomalies comes from a targeted search as opposed to a statistically random approach. As highlighted by Budowic et al.,163 procedures should be used that have the ‘highest diagnostic yield’. A targeted approach conserves resources of time and manpower in order to gather evidence essential to an investigation particularly when the search area is large or the number of items numerous. Knowledge of the type of evidence to be collected (whether biological or inorganic), the amount of sample expected to be recovered (whether trace or large amounts), and the technology available for analysis are also crucial in this targeted approach. The Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) has stated that either a statistical or a nonstatistical sampling approach can be used.164 Jamieson proposed a hypotheticodeductive method that may also be used as a generic guideline for examining a crime scene for forensic evidence.165 Also crucial to forensic sampling is the maintenance of a chain of custody. The chain of custody document simply authenticates the origin of a sample between the crime scene and the laboratory, no mention of field sampling would be complete without also making mention of this essential document since any inconsistency with the document renders the sample inadmissible and useless for criminal proceedings.

1.23.12.1 Field-Sampling Strategies The task of searching, sampling, packaging, and transporting evidence back to the laboratory to be analyzed by the forensic scientist falls on specialized individuals called crime scene investigators/technicians. It is important for these investigators to have a previously implemented sampling strategy to ensure consistency in sample collection irrespective of the person doing the actual collection. The crime scene investigator starts the chain of custody by identifying where the sample was collected and a brief description of the sample. There are three approaches that can be taken to collect evidence from a crime scene: 1. Removal of the entire item for transportation back to the laboratory. 2. Removal of a portion of the item. 3. Swabbing of the item. Depending on the size and capabilities of the laboratory, the appropriate field-sampling strategy should be employed. If items are small then the entire item may be transported to the laboratory, larger items may need to be disassembled for a portion to be transported to the laboratory. Swabbing is normally done using cotton-tipped applicators to collect trace evidence from items. Care should be taken when swabs are taken of trace samples. A 2008 study revealed that though swabs are useful, special training of crime scene investigators is needed in order for the collected swabs to yield reliable results in the laboratory.166 Collected samples are typically stored in paper bags or envelopes as these do not gather moisture or condensation. Liquids are collected in nonbreakable leak-proof containers, bodily fluids stored in plastic containers, while arson samples are collected in airtight metal cans; however, one study reported that compounds from the storage material were found to leach into the evidence being stored.167 When collecting trace evidence an additional consideration must be made, the amount of sample to collect. Consider, glass fragments, fiber, pills, powders, or any other matrix that when analyzed represents a much smaller portion of the original batch. Key statistical considerations for sample size determination in these instances should be adhered to in order for collected samples to be representative of the population. Sample heterogeneity will determine the sample size, as more heterogeneous samples will require large sample sizes. Methods for selecting an appropriate sample size may be found elsewhere in literature.168 There are also published guidelines by ASTM International ASTM E141-10169 and ASTM E 105-10170 which are good starting points from which to create sampling plans to determine sample size. Colon et al.171 reported a simplified approach commonly used by many forensic laboratories in determining sample size when more than 20 exhibits are to be seized. n ¼ 20 þ 0:10ðN  20Þ;

where N > 20

The above equation implies that if there are fewer than 20 samples, then all should be collected for analysis.

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One disadvantage to the crime scene investigator is often the inability to identify evidence in the field. To aid in this area some new developments have emerged in recent years. A latent fingerprint is one such example, and is still one of the most important items of physical evidence used in criminal proceedings. New dyes such as Oil Red O were introduced within the last decade allowing for the development of fingerprints on wet and porous surfaces.172 For the identification of latent bodily fluids, ultraviolet light from an alternate light source (ALS) and luminol tests have been traditionally used and are still in use today due to their low level false positive and false negative results. A review of this and other newly developed field sampling tools may be found in the literature.173 For the collection of bodily fluids, there has been a proliferation of immunoassay-based field-sampling kits that have proved to be robust and cost effective in quickly identifying both the presence and type of bodily fluid. Care should always be taken while conducting these particular field tests, as the tests themselves degrade the sample, resulting in limited amounts available for laboratory analyses when only trace quantities are discovered.173 To resolve this issue of sample degradation, some nondestructive tests have been developed that utilize laser excitation sources, these portable instrumental-based methods preserve the sample while providing confirmatory results.173 Other field-sampling techniques recently developed include Ferrotrace, a reagent that is used to reveal the recent contact of a suspect with a weapon (Figure 9).174 These techniques reveal latent evidence and allow for timely retrieval of said evidence. Though there are instances when in-field testing is beneficial such as acquiring evidence before it deteriorates under poor environmental conditions or if the test will eliminate a large number of suspects in a short time,174 every effort should be made by the crime scene investigator to collect samples to be analyzed in the laboratory.

1.23.12.2 Storage Once samples have been taken from the field, storage is important prior to and after analysis. Storage conditions prior to analysis ensure that the integrity of the sample is maintained by avoiding degradation that will reduce or alter the makeup of the sample. Storage conditions after analysis is also important in the event that repeated analyses are required during the criminal proceedings. The time between sample collection and actual analysis may be significant in some instances, which makes the storage conditions of these collected samples even more crucial. There have been numerous reports on storage of samples in the last decade, particularly for biological specimens. As smaller and smaller amounts of analyte are able to be detected; it becomes more difficult to maintain low levels of analytes during sample storage. Human scent samples, which are collected on cotton sorbent pads, were studied and revealed that light and extremes in temperature degrade the sample.175 In general, most forensic samples showed higher preservation rates when stored in cool temperatures and away from direct light. Samples submitted for ignitable liquid residue (ILR) analysis were also found to be prone to degradation. A 2009 study by Turner et al. highlighted the propensity of microbes to attack even number n-alkanes of ILR samples while in storage resulting in changes in the chromatographic profiles.176 Despite efforts to improve sample storage over the past decade, excessively long backlog times between sample collection and sample analysis have overshadowed any progress made. Time is still the most significant factor in determining the integrity of samples during storage, the shorter the storage time, the less degradation a sample experiences. A review of the literature has indicated much emphasis on laboratory or analytical techniques while emphasis on the sampling procedures for the collection of the evidence used for these techniques has lapsed.

Figure 9 Ferrotrace used to show recent contact of a gun on a suspect. Reproduced with permission from Almog, J. Forensic science does not start in the lab: The concept of diagnostic field tests. J. Forensic Sci. 2006, 51, 1228–1234, Copyright 2011, Elsevier Science.

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1.23.13 Conclusion The nature of forensic evidence is such that it can be found as gases (volatiles), liquids, and solids. There are natural and biological sources as well as man-made materials that range from fairly homogeneous matrices (blood components) to very heterogeneous matrices (soil samples) and everything in between. The nature of the sample is such that it is often dirty and requires cleanup steps that are appropriate to the analyte–matrix combination. Finally, every forensic case is different and so the standardization of sampling and sample preparation that is often commonplace in other disciplines may not readily apply to forensic samples. Finally, a common question that is often asked of forensic scientists, in addition to analyte identification and analyte quantification is, ‘could this evidence sample have originated from the known source’ in a comparison. While the analyte identification and quantification may be straightforward, it is usually very difficult to make statements regarding the source attribution with certainty and we are often satisfied with general statements about attribution that do not overstate the value of the evidence but also, hopefully, do not understate the value of the evidence.

See also: Recent Advances in Solid-Phase Microextraction for Environmental Applications

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Relevant Websites www.epa.gov (US Environmental Protection Agency) www.astm.org (ASTM International) www.aafs.org (American Academy of Forensic Science) www.soft-tox.org (Society of Forensic Toxicologist) www.abft.org (American Board of Forensic Toxicology)