Elemental analysis and forensic comparison of soils by laser-induced breakdown spectroscopy (LIBS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)

The elemental analysis of soil is useful in forensic and environmental sciences. Methods were developed and optimized for two laser-based multi-element analysis techniques: laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and laser-induced breakdown spectroscopy (LIBS). This work represents the first use of a 266 nm laser for forensic soil analysis by LIBS. Sample preparation methods were developed and optimized for a variety of sample types, including pellets for large bulk soil specimens (470 mg) and sediment-laden filters (47 mg), and tape-mounting for small transfer evidence specimens (10 mg). Analytical performance for sediment filter pellets and tape-mounted soils was similar to that achieved with bulk pellets. An inter-laboratory comparison exercise was designed to evaluate the performance of the LA-ICP-MS and LIBS methods, as well as for micro X-ray fluorescence (μXRF), across multiple laboratories. Limits of detection (LODs) were 0.01-23 ppm for LA-ICP-MS, 0.25-574 ppm for LIBS, 16-4400 ppm for μXRF, and well below the levels normally seen in soils. Good intra-laboratory precision (≤ 6 % relative standard deviation (RSD) for LA-ICP-MS; ≤ 8 % for μXRF; ≤ 17 % for LIBS) and inter-laboratory precision (≤ 19 % for LA-ICP-MS; ≤ 25 % for μXRF) were achieved for most elements, which is encouraging for a first inter-laboratory exercise. While LIBS generally has higher LODs and RSDs than LA-ICP-MS, both were capable of generating good quality multi-element data sufficient for discrimination purposes. Multivariate methods using principal components analysis (PCA) and linear discriminant analysis (LDA) were developed for discriminations of soils from different sources. Specimens from different sites that were indistinguishable by color alone were discriminated by elemental analysis. Correct classification rates of 94.5 % or better were achieved in a simulated forensic discrimination of three similar sites for both LIBS and LA-ICP-MS. Results for tape-mounted specimens were nearly identical to those achieved with pellets. Methods were tested on soils from USA, Canada and Tanzania. Within-site heterogeneity was site-specific. Elemental differences were greatest for specimens separated by large distances, even within the same lithology. Elemental profiles can be used to discriminate soils from different locations and narrow down locations even when mineralogy is similar.

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.^

Elemental Analysis of Glass and Ink by Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) and Laser Induced Breakdown Spectroscopy (LIBS)

The necessity of elemental analysis techniques to solve forensic problems continues to expand as the samples collected from crime scenes grow in complexity. Laser ablation ICP-MS (LA-ICP-MS) has been shown to provide a high degree of discrimination between samples that originate from different sources. In the first part of this research, two laser ablation ICP-MS systems were compared, one using a nanosecond laser and another a femtosecond laser source for the forensic analysis of glass. The results showed that femtosecond LA-ICP-MS did not provide significant improvements in terms of accuracy, precision and discrimination, however femtosecond LA-ICP-MS did provide lower detection limits. In addition, it was determined that even for femtosecond LA-ICP-MS an internal standard should be utilized to obtain accurate analytical results for glass analyses. In the second part, a method using laser induced breakdown spectroscopy (LIBS) for the forensic analysis of glass was shown to provide excellent discrimination for a glass set consisting of 41 automotive fragments. The discrimination power was compared to two of the leading elemental analysis techniques, µXRF and LA-ICP-MS, and the results were similar; all methods generated >99% discrimination and the pairs found indistinguishable were similar. An extensive data analysis approach for LIBS glass analyses was developed to minimize Type I and II errors en route to a recommendation of 10 ratios to be used for glass comparisons. Finally, a LA-ICP-MS method for the qualitative analysis and discrimination of gel ink sources was developed and tested for a set of ink samples. In the first discrimination study, qualitative analysis was used to obtain 95.6% discrimination for a blind study consisting of 45 black gel ink samples provided by the United States Secret Service. A 0.4% false exclusion (Type I) error rate and a 3.9% false inclusion (Type II) error rate was obtained for this discrimination study. In the second discrimination study, 99% discrimination power was achieved for a black gel ink pen set consisting of 24 self collected samples. The two pairs found to be indistinguishable came from the same source of origin (the same manufacturer and type of pen purchased in different locations). It was also found that gel ink from the same pen, regardless of the age, was indistinguishable as were gel ink pens (four pens) originating from the same pack.

Elemental Analysis and Forensic Comparison of Soils by Laser-Induced Breakdown Spectroscopy (LIBS) and Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS)

The elemental analysis of soil is useful in forensic and environmental sciences. Methods were developed and optimized for two laser-based multi-element analysis techniques: laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and laser-induced breakdown spectroscopy (LIBS). This work represents the first use of a 266 nm laser for forensic soil analysis by LIBS. Sample preparation methods were developed and optimized for a variety of sample types, including pellets for large bulk soil specimens (470 mg) and sediment-laden filters (47 mg), and tape-mounting for small transfer evidence specimens (10 mg). Analytical performance for sediment filter pellets and tape-mounted soils was similar to that achieved with bulk pellets. An inter-laboratory comparison exercise was designed to evaluate the performance of the LA-ICP-MS and LIBS methods, as well as for micro X-ray fluorescence (μXRF), across multiple laboratories. Limits of detection (LODs) were 0.01-23 ppm for LA-ICP-MS, 0.25-574 ppm for LIBS, 16-4400 ppm for µXRF, and well below the levels normally seen in soils. Good intra-laboratory precision (≤ 6 % relative standard deviation (RSD) for LA-ICP-MS; ≤ 8 % for µXRF; ≤ 17 % for LIBS) and inter-laboratory precision (≤ 19 % for LA-ICP-MS; ≤ 25 % for µXRF) were achieved for most elements, which is encouraging for a first inter-laboratory exercise. While LIBS generally has higher LODs and RSDs than LA-ICP-MS, both were capable of generating good quality multi-element data sufficient for discrimination purposes. Multivariate methods using principal components analysis (PCA) and linear discriminant analysis (LDA) were developed for discriminations of soils from different sources. Specimens from different sites that were indistinguishable by color alone were discriminated by elemental analysis. Correct classification rates of 94.5 % or better were achieved in a simulated forensic discrimination of three similar sites for both LIBS and LA-ICP-MS. Results for tape-mounted specimens were nearly identical to those achieved with pellets. Methods were tested on soils from USA, Canada and Tanzania. Within-site heterogeneity was site-specific. Elemental differences were greatest for specimens separated by large distances, even within the same lithology. Elemental profiles can be used to discriminate soils from different locations and narrow down locations even when mineralogy is similar.

Assessment of the occurrence and potential risks of antibiotics and their metabolites in South Florida waters using liquid chromatography tandem mass spectrometry

An automated on-line SPE-LC-MS/MS method was developed for the quantitation of multiple classes of antibiotics in environmental waters. High sensitivity in the low ng/L range was accomplished by using large volume injections with 10-mL of sample. Positive confirmation of analytes was achieved using two selected reaction monitoring (SRM) transitions per antibiotic and quantitation was performed using an internal standard approach. Samples were extracted using online solid phase extraction, then using column switching technique; extracted samples were immediately passed through liquid chromatography and analyzed by tandem mass spectrometry. The total run time per each sample was 20 min. The statistically calculated method detection limits for various environmental samples were between 1.2 and 63 ng/L. Furthermore, the method was validated in terms of precision, accuracy and linearity. ^ The developed analytical methodology was used to measure the occurrence of antibiotics in reclaimed waters (n=56), surface waters (n=53), ground waters (n=8) and drinking waters (n=54) collected from different parts of South Florida. In reclaimed waters, the most frequently detected antibiotics were nalidixic acid, erythromycin, clarithromycin, azithromycin trimethoprim, sulfamethoxazole and ofloxacin (19.3-604.9 ng/L). Detection of antibiotics in reclaimed waters indicates that they can't be completely removed by conventional wastewater treatment process. Furthermore, the average mass loads of antibiotics released into the local environment through reclaimed water were estimated as 0.248 Kg/day. Among the surface waters samples, Miami River (reaching up to 580 ng/L) and Black Creek canal (up to 124 ng/L) showed highest concentrations of antibiotics. No traces of antibiotics were found in ground waters. On the other hand, erythromycin (monitored as anhydro erythromycin) was detected in 82% of the drinking water samples (n.d-66 ng/L). The developed approach is suitable for both research and monitoring applications.^ Major metabolites of antibiotics in reclaimed wates were identified and quantified using high resolution benchtop Q-Exactive orbitrap mass spectrometer. A phase I metabolite of erythromycin was tentatively identified in full scan based on accurate mass measurement. Using extracted ion chromatogram (XIC), high resolution data-dependent MS/MS spectra and metabolic profiling software the metabolite was identified as desmethyl anhydro erythromycin with molecular formula C36H63NO12 and m/z 702.4423. The molar concentration of the metabolite to erythromycin was in the order of 13 %. To my knowledge, this is the first known report on this metabolite in reclaimed water. Another compound acetyl-sulfamethoxazole, a phase II metabolite of sulfamethoxazole was also identified in reclaimed water and mole fraction of the metabolite represent 36 %, of the cumulative sulfamethoxazole concentration. The results were illustrating the importance to include metabolites also in the routine analysis to obtain a mass balance for better understanding of the occurrence, fate and distribution of antibiotics in the environment. ^ Finally, all the antibiotics detected in reclaimed and surface waters were investigated to assess the potential risk to the aquatic organisms. The surface water antibiotic concentrations that represented the real time exposure conditions revealed that the macrolide antibiotics, erythromycin, clarithromycin and tylosin along with quinolone antibiotic, ciprofloxacin were suspected to induce high toxicity to aquatic biota. Preliminary results showing that, among the antibiotic groups tested, macrolides posed the highest ecological threat, and therefore, they may need to be further evaluated with, long-term exposure studies considering bioaccumulation factors and more number of species selected. Overall, the occurrence of antibiotics in aquatic environment is posing an ecological health concern.^

Improved dynamic headspace sampling and detection using capillary microextraction of volatiles coupled to gas chromatography mass spectrometry

Sampling and preconcentration techniques play a critical role in headspace analysis in analytical chemistry. My dissertation presents a novel sampling design, capillary microextraction of volatiles (CMV), that improves the preconcentration of volatiles and semivolatiles in a headspace with high throughput, near quantitative analysis, high recovery and unambiguous identification of compounds when coupled to mass spectrometry. The CMV devices use sol-gel polydimethylsiloxane (PDMS) coated microglass fibers as the sampling/preconcentration sorbent when these fibers are stacked into open-ended capillary tubes. The design allows for dynamic headspace sampling by connecting the device to a hand-held vacuum pump. The inexpensive device can be fitted into a thermal desorption probe for thermal desorption of the extracted volatile compounds into a gas chromatography-mass spectrometer (GC-MS). The performance of the CMV devices was compared with two other existing preconcentration techniques, solid phase microextraction (SPME) and planar solid phase microextraction (PSPME). Compared to SPME fibers, the CMV devices have an improved surface area and phase volume of 5000 times and 80 times, respectively. One (1) minute dynamic CMV air sampling resulted in similar performance as a 30 min static extraction using a SPME fiber. The PSPME devices have been fashioned to easily interface with ion mobility spectrometers (IMS) for explosives or drugs detection. The CMV devices are shown to offer dynamic sampling and can now be coupled to COTS GC-MS instruments. Several compound classes representing explosives have been analyzed with minimum breakthrough even after a 60 min. sampling time. The extracted volatile compounds were retained in the CMV devices when preserved in aluminum foils after sampling. Finally, the CMV sampling device were used for several different headspace profiling applications which involved sampling a shipping facility, six illicit drugs, seven military explosives and eighteen different bacteria strains. Successful detection of the target analytes at ng levels of the target signature volatile compounds in these applications suggests that the CMV devices can provide high throughput qualitative and quantitative analysis with high recovery and unambiguous identification of analytes.

Determination of Extracellular Molecules Produced by Vibrio Harveyi Using MS/MS

Quorum sensing (QS) is a process that allows bacteria to sense the population density of cells around them by communicating with each other via autoinducer molecules. This cross-communication is crucial in the regulation of bacterial processes such as bioluminescence, virulence, and biofilm formation. Previous research by Milburn and Makemson on Vibrio harveyi suggested that in addition of the known biosynthesis of three well-characterized autoinducers, dozens of unknown molecules are also produced and released to the environment by V. harveyi. This study was performed using electrospray tandem mass spectrometry with the purpose of detection and characterization of the extracellular molecules produced by V. harveyi, and assessment of their relationship to QS. A total of 11 molecules were characterized, from which three could be related to QS. These findings provide a glimpse of the nature of novel secondary metabolites produced by V. harveyi and provide the groundwork for further research.

Assessment of the Occurrence and Potential Risks of Antibiotics and their Metabolites in South Florida Waters Using Liquid Chromatography Tandem Mass Spectrometry

An automated on-line SPE-LC-MS/MS method was developed for the quantitation of multiple classes of antibiotics in environmental waters. High sensitivity in the low ng/L range was accomplished by using large volume injections with 10-mL of sample. Positive confirmation of analytes was achieved using two selected reaction monitoring (SRM) transitions per antibiotic and quantitation was performed using an internal standard approach. Samples were extracted using online solid phase extraction, then using column switching technique; extracted samples were immediately passed through liquid chromatography and analyzed by tandem mass spectrometry. The total run time per each sample was 20 min. The statistically calculated method detection limits for various environmental samples were between 1.2 and 63 ng/L. Furthermore, the method was validated in terms of precision, accuracy and linearity. The developed analytical methodology was used to measure the occurrence of antibiotics in reclaimed waters (n=56), surface waters (n=53), ground waters (n=8) and drinking waters (n=54) collected from different parts of South Florida. In reclaimed waters, the most frequently detected antibiotics were nalidixic acid, erythromycin, clarithromycin, azithromycin trimethoprim, sulfamethoxazole and ofloxacin (19.3-604.9 ng/L). Detection of antibiotics in reclaimed waters indicates that they can’t be completely removed by conventional wastewater treatment process. Furthermore, the average mass loads of antibiotics released into the local environment through reclaimed water were estimated as 0.248 Kg/day. Among the surface waters samples, Miami River (reaching up to 580 ng/L) and Black Creek canal (up to 124 ng/L) showed highest concentrations of antibiotics. No traces of antibiotics were found in ground waters. On the other hand, erythromycin (monitored as anhydro erythromycin) was detected in 82% of the drinking water samples (n.d-66 ng/L). The developed approach is suitable for both research and monitoring applications. Major metabolites of antibiotics in reclaimed wates were identified and quantified using high resolution benchtop Q-Exactive orbitrap mass spectrometer. A phase I metabolite of erythromycin was tentatively identified in full scan based on accurate mass measurement. Using extracted ion chromatogram (XIC), high resolution data-dependent MS/MS spectra and metabolic profiling software the metabolite was identified as desmethyl anhydro erythromycin with molecular formula C36H63NO12 and m/z 702.4423. The molar concentration of the metabolite to erythromycin was in the order of 13 %. To my knowledge, this is the first known report on this metabolite in reclaimed water. Another compound acetyl-sulfamethoxazole, a phase II metabolite of sulfamethoxazole was also identified in reclaimed water and mole fraction of the metabolite represent 36 %, of the cumulative sulfamethoxazole concentration. The results were illustrating the importance to include metabolites also in the routine analysis to obtain a mass balance for better understanding of the occurrence, fate and distribution of antibiotics in the environment. Finally, all the antibiotics detected in reclaimed and surface waters were investigated to assess the potential risk to the aquatic organisms. The surface water antibiotic concentrations that represented the real time exposure conditions revealed that the macrolide antibiotics, erythromycin, clarithromycin and tylosin along with quinolone antibiotic, ciprofloxacin were suspected to induce high toxicity to aquatic biota. Preliminary results showing that, among the antibiotic groups tested, macrolides posed the highest ecological threat, and therefore, they may need to be further evaluated with, long-term exposure studies considering bioaccumulation factors and more number of species selected. Overall, the occurrence of antibiotics in aquatic environment is posing an ecological health concern.

Improved Dynamic Headspace Sampling and Detection using Capillary Microextraction of Volatiles Coupled to Gas Chromatography Mass Spectrometry

Sampling and preconcentration techniques play a critical role in headspace analysis in analytical chemistry. My dissertation presents a novel sampling design, capillary microextraction of volatiles (CMV), that improves the preconcentration of volatiles and semivolatiles in a headspace with high throughput, near quantitative analysis, high recovery and unambiguous identification of compounds when coupled to mass spectrometry. The CMV devices use sol-gel polydimethylsiloxane (PDMS) coated microglass fibers as the sampling/preconcentration sorbent when these fibers are stacked into open-ended capillary tubes. The design allows for dynamic headspace sampling by connecting the device to a hand-held vacuum pump. The inexpensive device can be fitted into a thermal desorption probe for thermal desorption of the extracted volatile compounds into a gas chromatography-mass spectrometer (GC-MS). The performance of the CMV devices was compared with two other existing preconcentration techniques, solid phase microextraction (SPME) and planar solid phase microextraction (PSPME). Compared to SPME fibers, the CMV devices have an improved surface area and phase volume of 5000 times and 80 times, respectively. One (1) minute dynamic CMV air sampling resulted in similar performance as a 30 min static extraction using a SPME fiber. The PSPME devices have been fashioned to easily interface with ion mobility spectrometers (IMS) for explosives or drugs detection. The CMV devices are shown to offer dynamic sampling and can now be coupled to COTS GC-MS instruments. Several compound classes representing explosives have been analyzed with minimum breakthrough even after a 60 min. sampling time. The extracted volatile compounds were retained in the CMV devices when preserved in aluminum foils after sampling. Finally, the CMV sampling device were used for several different headspace profiling applications which involved sampling a shipping facility, six illicit drugs, seven military explosives and eighteen different bacteria strains. Successful detection of the target analytes at ng levels of the target signature volatile compounds in these applications suggests that the CMV devices can provide high throughput qualitative and quantitative analysis with high recovery and unambiguous identification of analytes.