Comparison of medical and forensic profiling potential of volatile biomarkers from different biological specimens from individuals and across populations

There is limited scientific knowledge on the composition of human odor from different biological specimens and the effect that physiological and psychological health conditions could have on them. There is currently no direct comparison of the volatile organic compounds (VOCs) emanating from different biological specimens collected from healthy individuals as well as individuals with certain diagnosed medical conditions. Therefore the question of matching VOCs present in human odor across various biological samples and across health statuses remains unanswered. The main purpose of this study was to use analytical instrumental methods to compare the VOCs from different biological specimens from the same individual and to compare the populations evaluated in this project. The goals of this study were to utilize headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC/MS) to evaluate its potential for profiling VOCs from specimens collected using standard forensic and medical methods over three different populations: healthy group with no diagnosed medical or psychological condition, one group with diagnosed type 2 diabetes, and one group with diagnosed major depressive disorder. The pre-treatment methods of collection materials developed for the study allowed for the removal of targeted VOCs from the sampling kits prior to sampling, extraction and analysis. Optimized SPME-GC/MS conditions has been demonstrated to be capable of sampling, identifying and differentiating the VOCs present in the five biological specimens collected from different subjects and yielded excellent detection limits for the VOCs from buccal swab, breath, blood, and urine with average limits of detection of 8.3 ng. Visual, Spearman rank correlation, and PCA comparisons of the most abundant and frequent VOCs from each specimen demonstrated that each specimen has characteristic VOCs that allow them to be differentiated for both healthy and diseased individuals. Preliminary comparisons of VOC profiles of healthy individuals, patients with type 2 diabetes, and patients with major depressive disorder revealed compounds that could be used as potential biomarkers to differentiate between healthy and diseased individuals. Finally, a human biological specimen compound database has been created compiling the volatile compounds present in the emanations of human hand odor, oral fluids, breath, blood, and urine.

The discrimination and association of float glass and the quantitative analysis of liquids from aerosols and microdrops using laser induced breakdown spectroscopy

Glass is a common form of trace evidence found at many scenes of crimes in the form of small fragments. These glass fragments can transfer to surrounding objects and/or persons and may provide forensic investigators valuable information to link a suspect to the scene of a crime. Since the elemental composition of different glass sources can be very similar, a highly discriminating technique is required to distinguish between fragments that have originated from different sources. ^ The research presented here demonstrates that Laser Induced Breakdown Spectroscopy (LIBS) is a viable analytical technique for the association and discrimination of glass fragments. The first part of this research describes the optimization of the LIBS experiments including the use of different laser wavelengths to investigate laser-material interaction. The use of a 266 nm excitation laser provided the best analytical figures of merit with minimal damage to the sample. The resulting analytical figures of merit are presented. The second part of this research evaluated the sensitivity of LIBS to associate or discriminate float glass samples originating from the same manufacturing plants and produced at approximately the same time period. Two different sample sets were analyzed ranging in manufacturing dates from days to years apart. Eighteen (18) atomic emission lines corresponding to the elements Sr, K, Fe, Ca, Al, Ba, Na, Mg and Ti, were chosen because of their detection above the method detection limits and for presenting differences between the samples. Ten elemental ratios producing the most discrimination were selected for each set. When all the ratios are combined in a comparison, 99% of the possible pairs were discriminated using the optimized LIBS method generating typical analytical precisions of ∼5% RSD. ^ The final study consisted of the development of a new approach for the use of LIBS as a quantitative analysis of ultra-low volume solution analysis using aerosols and microdrops. Laser induced breakdown spectroscopy demonstrated to be an effective technique for the analysis of as low as 90 pL for microdrop LIBS with 1 pg absolute LOD and 20 µL for aerosol LIBS with an absolute LOD of ∼100 fg.^

Determination of signature volatile odor chemicals emanating from novel biological specimens by non-invasive analytical techniques for the potential use in forensic identifications

Human scent, or the volatile organic compounds (VOCs) produced by an individual, has been recognized as a biometric measurement because of the distinct variations in both the presence and abundance of these VOCs between individuals. In forensic science, human scent has been used as a form of associative evidence by linking a suspect to a scene/object through the use of human scent discriminating canines. The scent most often collected and used with these specially trained canines is from the hands because a majority of the evidence collected is likely to have been handled by the suspect. However, the scents from other biological specimens, especially those that are likely to be present at scenes of violent crimes, have yet to be explored. Hair, fingernails and saliva are examples of these types of specimens. ^ In this work, a headspace solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) technique was used for the identification of VOCs from hand odor, hair, fingernails and saliva. Sixty individuals were sampled and the profiles of the extracted VOCs were evaluated to assess whether they could be used for distinguishing individuals. Preliminary analysis of the biological specimens collected from an individual (intra-subject) showed that, though these materials have some VOCs in common, their overall chemical profile is different for each specimen type. Pair-wise comparisons, using Spearman Rank correlations, were made between the chemical profiles obtained from each subject, per a specimen type. Greater than 98.8% of the collected samples were distinguished from the subjects for all of the specimen types, demonstrating that these specimens can be used for distinguishing individuals. ^ Additionally, field trials were performed to determine the utility of these specimens as scent sources for human scent discriminating canines. Three trials were conducted to evaluate hair, fingernails and saliva in comparison to hand odor, which was considered the standard source of human odor. It was revealed that canines perform similarly to these alternative human scent sources as they do to hand odor implying that, though there are differences in the chemical profiles released by these specimens, they can still be used for the discrimination of individuals by trained canines.^

Elemental analysis of biological matrices by laser ablation high resolution inductively coupled plasma mass spectrometry (LA-HR-ICP-MS) and high resolution inductively coupled plasma mass spectrometry (HR -ICP -MS)

The need for elemental analysis of biological matrices such as bone, teeth, and plant matter for sourcing purposes has emerged within the forensic and geochemical laboratories. Trace elemental analyses for the comparison of materials such as glass by inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS has been shown to offer a high degree of discrimination between different manufacturing sources. Unit resolution ICP-MS instruments may suffer from some polyatomic interferences including 40Ar16O+, 40Ar 16O1H+, and 40Ca 16O+ that affect iron measurement at trace levels. Iron is an important element in the analysis of glass and also of interest for the analysis of several biological matrices. A comparison of the analytical performance of two different ICP-MS systems for iron analysis in glass for determining the method detection limits (MDLs), accuracy, and precision of the measurement is presented. Acid digestion and laser ablation methods are also compared. Iron polyatomic interferences were reduced or resolved by using dynamic reaction cell and high resolution ICP-MS. MDLs as low as 0.03 μg g-1 and 0.14 μg g-1 for laser ablation and solution based analyses respectively were achieved. The use of helium as a carrier gas demonstrated improvement in the detection limits of both iron isotopes (56Fe and 57Fe) in medium resolution for the HR-ICP-MS and with a dynamic reaction cell (DRC) coupled to a quadrupole ICP-MS system. ^ The development and application of robust analytical methods for the quantification of trace elements in biological matrices has lead to a better understanding of the potential utility of these measurements in forensic chemical analyses. Standard reference materials (SRMs) were used in the development of an analytical method using HR-ICP-MS and LA-HR-ICP-MS that was subsequently applied on the analysis of real samples. Bone, teeth and ashed marijuana samples were analyzed with the developed method. ^ Elemental analysis of bone samples from 12 different individuals provided discrimination between individuals, when femur and humerus bones were considered separately. Discrimination of 14 teeth samples based on elemental composition was achieved with the exception of one case where samples from the same individual were not associated with each other. The discrimination of 49 different ashed plant (cannabis) samples was achieved using the developed method. ^

Development of advanced capillary electrophoresis techniques with UV and mass spectrometry detection for forensic, pharmaceutical and environmental applications

Capillary electrophoresis (CE) is a modern analytical technique, which is electrokinetic separation generated by high voltage and taken place inside the small capillaries. In this dissertation, several advanced capillary electrophoresis methods are presented using different approaches of CE and UV and mass spectrometry are utilized as the detection methods. ^ Capillary electrochromatography (CEC), as one of the CE modes, is a recent developed technique which is a hybrid of capillary electrophoresis and high performance liquid chromatography (HPLC). Capillary electrochromatography exhibits advantages of both techniques. In Chapter 2, monolithic capillary column are fabricated using in situ photoinitiation polymerization method. The column was then applied for the separation of six antidepressant compounds. ^ Meanwhile, a simple chiral separation method is developed and presented in Chapter 3. Beta cycodextrin was utilized to achieve the goal of chiral separation. Not only twelve cathinone analytes were separated, but also isomers of several analytes were enantiomerically separated. To better understand the molecular information on the analytes, the TOF-MS system was coupled with the CE. A sheath liquid and a partial filling technique (PFT) were employed to reduce the contamination of MS ionization source. Accurate molecular information was obtained. ^ It is necessary to propose, develop, and optimize new techniques that are suitable for trace-level analysis of samples in forensic, pharmaceutical, and environmental applications. Capillary electrophoresis (CE) was selected for this task, as it requires lower amounts of samples, it simplifies sample preparation, and it has the flexibility to perform separations of neutral and charged molecules as well as enantiomers. ^ Overall, the study demonstrates the versatility of capillary electrophoresis methods in forensic, pharmaceutical, and environmental applications.^

The Effect of Sample and Sample Matrix on DNA Processing: Mechanisms for the Detection and Management of Inhibition in Forensic Samples

The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments were conducted to establish the inhibition tolerance of quantification and amplification kits using common inhibitory substances in order to determine if current laboratory practices are optimal for identifying potential problems associated with inhibition. DART mass spectrometry was used to determine the amount of inhibitor carryover after sample purification, its correlation to the initial inhibitor input in the sample and the overall effect in the results. Finally, a novel alternative at gathering investigative leads from samples that would otherwise be ineffective for DNA typing due to the large amounts of inhibitory substances and/or environmental degradation was tested. This included generating data associated with microbial peak signatures to identify locations of clandestine human graves. Results demonstrate that the current methods for assessing inhibition are not necessarily accurate, as samples that appear inhibited in the quantification process can yield full DNA profiles, while those that do not indicate inhibition may suffer from lowered amplification efficiency or PCR artifacts. The extraction methods tested were able to remove >90% of the inhibitors from all samples with the exception of phenol, which was present in variable amounts whenever the organic extraction approach was utilized. Although the results attained suggested that most inhibitors produce minimal effect on downstream applications, analysts should practice caution when selecting the best extraction method for particular samples, as casework DNA samples are often present in small quantities and can contain an overwhelming amount of inhibitory substances.

The Construction and Optimization on an Ion Mobility Spectrometer for the Analysis of Explosives and Drugs

Today, over 15,000 Ion Mobility Spectrometry (IMS) analyzers are employed at worldwide security checkpoints to detect explosives and illicit drugs. Current portal IMS instruments and other electronic nose technologies detect explosives and drugs by analyzing samples containing the headspace air and loose particles residing on a surface. Canines can outperform these systems at sampling and detecting the low vapor pressure explosives and drugs, such as RDX, PETN, cocaine, and MDMA, because these biological detectors target the volatile signature compounds available in the headspace rather than the non-volatile parent compounds of explosives and drugs. In this dissertation research volatile signature compounds available in the headspace over explosive and drug samples were detected using SPME as a headspace sampling tool coupled to an IMS analyzer. A Genetic Algorithm (GA) technique was developed to optimize the operating conditions of a commercial IMS (GE Itemizer 2), leading to the successful detection of plastic explosives (Detasheet, Semtex H, and C-4) and illicit drugs (cocaine, MDMA, and marijuana). Short sampling times (between 10 sec to 5 min) were adequate to extract and preconcentrate sufficient analytes (> 20 ng) representing the volatile signatures in the headspace of a 15 mL glass vial or a quart-sized can containing ≤ 1 g of the bulk explosive or drug. Furthermore, a research grade IMS with flexibility for changing operating conditions and physical configurations was designed and fabricated to accommodate future research into different analytes or physical configurations. The design and construction of the FIU-IMS were facilitated by computer modeling and simulation of ion’s behavior within an IMS. The simulation method developed uses SIMION/SDS and was evaluated with experimental data collected using a commercial IMS (PCP Phemto Chem 110). The FIU-IMS instrument has comparable performance to the GE Itemizer 2 (average resolving power of 14, resolution of 3 between two drugs and two explosives, and LODs range from 0.7 to 9 ng). The results from this dissertation further advance the concept of targeting volatile components to presumptively detect the presence of concealed bulk explosives and drugs by SPME-IMS, and the new FIU-IMS provides a flexible platform for future IMS research projects.

Environmental analysis of polar herbicides in complex organic-rich matrices by high performance liquid chromatography atmospheric pressure ionization mass spectrometry (HPLC-API-MS)

A comprehensive forensic investigation of sensitive ecosystems in the Everglades Area is presented. Assessing the background levels of contamination in these ecosystems represents a vital resource to build up forensic evidence required to enforce future environmental crimes within the studied areas. This investigation presents the development and validation of a fractionation and isolation method for two families of herbicides commonly applied in the vicinity of the study area, including phenoxy acids like 2,4-D, MCPA, and silvex; as well as the most common triazine-based herbicides like atrazine, prometyne, simazine and related metabolites like DIA and DEA. Accelerated solvent extraction (ASE) and solid phase extraction (SPE) were used to isolate the analytes from abiotic matrices containing large amounts of organic material. Atmospheric-pressure ionization (API) with electrospray ionization in negative mode (ESP-), and Chemical Ionization in the positive mode (APCI+) were used to perform the characterization of the herbicides of interest.

The Use of Hyphenated Spectrometric Techniques for the Environmental Forensic Assessment of Non-Traditional Pollutants and Degradates in the Greater Florida Everglades

A comprehensive investigation of sensitive ecosystems in South Florida with the main goal of determining the identity, spatial distribution, and sources of both organic biocides and trace elements in different environmental compartments is reported. This study presents the development and validation of a fractionation and isolation method of twelve polar acidic herbicides commonly applied in the vicinity of the study areas, including e.g. 2,4-D, MCPA, dichlorprop, mecroprop, picloram in surface water. Solid phase extraction (SPE) was used to isolate the analytes from abiotic matrices containing large amounts of dissolved organic material. Atmospheric-pressure ionization (API) with electrospray ionization in negative mode (ESP-) in a Quadrupole Ion Trap mass spectrometer was used to perform the characterization of the herbicides of interest. The application of Laser Ablation-ICP-MS methodology in the analysis of soils and sediments is reported in this study. The analytical performance of the method was evaluated on certified standards and real soil and sediment samples. Residential soils were analyzed to evaluate feasibility of using the powerful technique as a routine and rapid method to monitor potential contaminated sites. Forty eight sediments were also collected from semi pristine areas in South Florida to conduct screening of baseline levels of bioavailable elements in support of risk evaluation. The LA-ICP-MS data were used to perform a statistical evaluation of the elemental composition as a tool for environmental forensics. A LA-ICP-MS protocol was also developed and optimized for the elemental analysis of a wide range of elements in polymeric filters containing atmospheric dust. A quantitative strategy based on internal and external standards allowed for a rapid determination of airborne trace elements in filters containing both contemporary African dust and local dust emissions. These distributions were used to qualitative and quantitative assess differences of composition and to establish provenance and fluxes to protected regional ecosystems such as coral reefs and national parks.

Validation of an Analytical Method for the Identification of SmartWater CSI Forensic Marking Technology

SmartWater is a chemical taggant used as a crime deterrent. The chemical taggant is a colorless liquid that fluoresces yellow under ultra-violet (UV) light and contains distinctive, identifiable and traceable elemental composition. For instance, upon a break and entry scenario, the burglar is sprayed with a solution that has an elemental signature custom-made to a specific location. The residues of this taggant persist on skin and other objects and can be easily recovered for further analysis. The product has been effectively used in Europe as a crime deterrent and has been recently introduced in South Florida. In 2014, Fourt Lauderdale Police Department reported the use of SmartWater products with a reduction in burglaries of 14% [1]. The International Forensic Research Institute (IFRI) at FIU validated the scientific foundation of the methods of recovery and analysis of these chemical tagging systems using LA-ICP-MS. Analytical figures of merit of the method such as precision, accuracy, limits of detection, linearity and selectivity are reported in this study. Moreover, blind samples were analyzed by LA-ICP-MS to compare the chemical signatures to the company’s database and evaluate error rates and the accuracy of the method. This study demonstrated that LA-ICP-MS could be used to effectively detect these traceable taggants to assist law enforcement agencies in the United States with cases involving transfer of these forensic coding systems.

Elemental Analysis of Biological Matrices by Laser Ablation High Resolution Inductively Coupled Plasma Mass Spectrometry (LA-HR-ICP-MS) and High Resolution Inductively Coupled Plasma Mass Spectrometry (HR-ICP-MS)

The need for elemental analysis of biological matrices such as bone, teeth, and plant matter for sourcing purposes has emerged within the forensic and geochemical laboratories. Trace elemental analyses for the comparison of aterials such as glass by inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS has been shown to offer a high degree of discrimination between different manufacturing sources. Unit resolution ICP-MS instruments may suffer from some polyatomic interferences including 40Ar16O+, 40Ar16O1H+, and 40Ca16O+ that affect iron measurement at trace levels. Iron is an important element in the analysis of glass and also of interest for the analysis of several biological matrices. A comparison of the nalytical performance of two different ICP-MS systems for iron analysis in glass for determining the method detection limits (MDLs), accuracy, and precision of the measurement is presented. Acid digestion and laser ablation methods are also compared. Iron polyatomic interferences were reduced or resolved by using dynamic reaction cell and high resolution ICP-MS. MDLs as low as 0.03 ìg g-1 and 0.14 ìg g-1 for laser ablation and solution based analyses respectively were achieved. The use of helium as a carrier gas demonstrated improvement in the detection limits of both iron isotopes (56Fe and 57Fe) in medium resolution for the HR-ICP-MS and with a dynamic reaction cell (DRC) coupled to a quadrupole ICP-MS system. The development and application of robust analytical methods for the quantification of trace elements in biological matrices has lead to a better understanding of the potential utility of these measurements in forensic chemical analyses. Standard reference materials (SRMs) were used in the development of an analytical method using HR-ICP-MS and LA-HR-ICP-MS that was subsequently applied on the analysis of real samples. Bone, teeth and ashed marijuana samples were analyzed with the developed method. Elemental analysis of bone samples from 12 different individuals provided discrimination between individuals, when femur and humerus bones were considered separately. Discrimination of 14 teeth samples based on elemental composition was achieved with the exception of one case where samples from the same individual were not associated with each other. The discrimination of 49 different ashed plant (cannabis)samples was achieved using the developed method.

Development of Advanced Capillary Electrophoresis Techniques with UV and Mass Spectrometry Detection for Forensic, Pharmaceutical and Environmental Applications

Capillary electrophoresis (CE) is a modern analytical technique, which is electrokinetic separation generated by high voltage and taken place inside the small capillaries. In this dissertation, several advanced capillary electrophoresis methods are presented using different approaches of CE and UV and mass spectrometry are utilized as the detection methods. Capillary electrochromatography (CEC), as one of the CE modes, is a recent developed technique which is a hybrid of capillary electrophoresis and high performance liquid chromatography (HPLC). Capillary electrochromatography exhibits advantages of both techniques. In Chapter 2, monolithic capillary column are fabricated using in situ photoinitiation polymerization method. The column was then applied for the separation of six antidepressant compounds. Meanwhile, a simple chiral separation method is developed and presented in Chapter 3. Beta cycodextrin was utilized to achieve the goal of chiral separation. Not only twelve cathinone analytes were separated, but also isomers of several analytes were enantiomerically separated. To better understand the molecular information on the analytes, the TOF-MS system was coupled with the CE. A sheath liquid and a partial filling technique (PFT) were employed to reduce the contamination of MS ionization source. Accurate molecular information was obtained. It is necessary to propose, develop, and optimize new techniques that are suitable for trace-level analysis of samples in forensic, pharmaceutical, and environmental applications. Capillary electrophoresis (CE) was selected for this task, as it requires lower amounts of samples, it simplifies sample preparation, and it has the flexibility to perform separations of neutral and charged molecules as well as enantiomers. Overall, the study demonstrates the versatility of capillary electrophoresis methods in forensic, pharmaceutical, and environmental applications.

Determination of Signature Volatile Odor Chemicals Emanating from Novel Biological Specimens by Non-invasive Analytical Techniques for the Potential use in Forensic Identifications

Human scent, or the volatile organic compounds (VOCs) produced by an individual, has been recognized as a biometric measurement because of the distinct variations in both the presence and abundance of these VOCs between individuals. In forensic science, human scent has been used as a form of associative evidence by linking a suspect to a scene/object through the use of human scent discriminating canines. The scent most often collected and used with these specially trained canines is from the hands because a majority of the evidence collected is likely to have been handled by the suspect. However, the scents from other biological specimens, especially those that are likely to be present at scenes of violent crimes, have yet to be explored. Hair, fingernails and saliva are examples of these types of specimens. In this work, a headspace solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) technique was used for the identification of VOCs from hand odor, hair, fingernails and saliva. Sixty individuals were sampled and the profiles of the extracted VOCs were evaluated to assess whether they could be used for distinguishing individuals. Preliminary analysis of the biological specimens collected from an individual (intra-subject) showed that, though these materials have some VOCs in common, their overall chemical profile is different for each specimen type. Pair-wise comparisons, using Spearman Rank correlations, were made between the chemical profiles obtained from each subject, per a specimen type. Greater than 98.8% of the collected samples were distinguished from the subjects for all of the specimen types, demonstrating that these specimens can be used for distinguishing individuals. Additionally, field trials were performed to determine the utility of these specimens as scent sources for human scent discriminating canines. Three trials were conducted to evaluate hair, fingernails and saliva in comparison to hand odor, which was considered the standard source of human odor. It was revealed that canines perform similarly to these alternative human scent sources as they do to hand odor implying that, though there are differences in the chemical profiles released by these specimens, they can still be used for the discrimination of individuals by trained canines.

High-performance liquid chromatography (HPLC) and high-performance liquid chromatography mass spectrometry (HPLC/MS) for the analysis of date rape drugs

The drugs studied in this work have been reportedly used to commit drug-facilitated sexual assault (DFSA), commonly known as "date rape". Detection of the drugs was performed using high-performance liquid chromatography with ultraviolet detection (HPLC/UV) and identified with high performance-liquid chromatography mass spectrometry (HPLC/MS) using selected ion monitoring (SIM). The objective of this study was to develop a single HPLC method for the simultaneous detection, identification and quantitation of these drugs. The following drugs were simultaneously analyzed: Gamma-hydroxybutyrate (GHB), scopolamine, lysergic acid diethylamide, ketamine, flunitrazepam, and diphenhydramine. The results showed increased sensitivity with electrospray (ES) ionization versus atmospheric pressure chemical ionization (APCI) using HPLC/MS. HPLC/ES/MS was approximately six times more sensitive than HPLC/APCI/MS and about fifty times more sensitive than HPLC/UV. A limit of detection (LOD) of 100 ppb was achieved for drug analysis using this method. The average linear regression coefficient of correlation squared (r2) was 0.933 for HPLC/UV and 0.998 for HPLC/ES/MS. The detection limits achieved by this method allowed for the detection of drug dosages used in beverage tampering. This method can be used to screen beverages suspected of drug tampering. The results of this study demonstrated that solid phase microextraction (SPME) did not improve sensitivity as an extraction technique when compared to direct injections of the drug standards.