Solid-phase combinatorial chemistry and scintillation proximity assays

The Scintillation Proximity Assay (SPA) is a method that is frequently used to detect and quantify the strength of intermolecular interactions between a biological receptor and ligand molecule in aqueous media. This thesis describes the synthesis of scintillant-tagged-compounds for application in a novel cell-based SPA. A series of 4-functianlised-2,5-diphenyloxazole molecules were synthesised. These 4-functionalised-2,5-diphenyloxazoles were evaluated by Sense Proteomic Ltd. Accordingly, the molecules were evaluated for the ability to scintillate in the presence of ionising radiation. In addition, the molecules were incorporated into liposomal preparations which were subsequently evaluated for the ability to scintillate in the presence of ionising radiation. The optimal liposomal preparation was introduced into the membrane of HeLa cells that were used successfully in a cell-based SPA to detect and quantify the uptake of [14C]methionine. This thesis also describes the synthesis and subsequent polymerisation of novel poly(oxyethylene glycol)-based monomers to form a series of new polymer supports. These Poly(oxyethylene glycol)-polymer (POP) supports were evaluated for the ability to swell and mass-uptake in a variety of solvents, demonstrating that POP-supports exhibit enhanced solvent compatibilities over several commercial resins. The utility of POP-supports in solid-phase synthesis was also demonstrated successfully. The incorporation of (4’-vinyl)-4-benzyl-2,5-diphenyloxazole in varying mole percentage into the monomer composition resulted in the production of chemically functionalised scintillant-containing poly(oxyethylene glycol) polymer (POP-Sc) supports. These materials are compatible with both aqueous and organic solvents and scintillate efficiently in the presence of ionising radiation. The utility of POP-Sc supports in solid-phase synthesis and subsequent in-situ SPA to detect and quantify, in real-time, the kinetic progress of a solid-phase reaction was exemplified successfully.In addition, POP-Sc supports were used successfully both in solid-phase combinatorial synthesis of a peptide nucleic acid (PNA)-library and subsequent screening of this library for the ability to hybridise with DNA, which was labelled with a suitable radio-isotape. This data was used to identify the dependence of the number and position of complimentary codon pairs upon the extent of hybridisation. Finally, a further SPA was used to demonstrate the excellent compatibility of POP-Sc supports for use in the detection and quantification of enzyme assays conducted within the matrix of the POP-Sc support.

Solid phase synthesis of a metronidazole oligonucleotide conjugate

Direct, solid phase synthesis of an oligonucleotide conjugate of the antibiotic drug metronidazole was accomplished by the phosphoramidite method. Removal of protecting groups and cleavage from the controlled pore glass (CPG) solid support was successful using mild conditions (20% EtN in pyridine, then conc. NH (aq) at rt for 30 min) whereas standard conditions (conc. NH (aq) at 55°C for 16 h) cleaved the drug. © 2006 by MDPI.

Evaluation of new processes for the preparation of iron by gas/solid phase systems

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The development of an optimized system of narcotic and explosive contraband mimics for calibration and training of biological detectors

Current commercially available mimics contain varying amounts of either the actual explosive/drug or the chemical compound of suspected interest by biological detectors. As a result, there is significant interest in determining the dominant chemical odor signatures of the mimics, often referred to as pseudos, particularly when compared to the genuine contraband material. This dissertation discusses results obtained from the analysis of drug and explosive headspace related to the odor profiles as recognized by trained detection canines. Analysis was performed through the use of headspace solid phase microextraction in conjunction with gas chromatography mass spectrometry (HS-SPME-GC-MS). Upon determination of specific odors, field trials were held using a combination of the target odors with COMPS. Piperonal was shown to be a dominant odor compound in the headspace of some ecstasy samples and a recognizable odor mimic by trained detection canines. It was also shown that detection canines could be imprinted on piperonal COMPS and correctly identify ecstasy samples at a threshold level of approximately 100ng/s. Isosafrole and/or MDP-2-POH show potential as training aid mimics for non-piperonal based MDMA. Acetic acid was shown to be dominant in the headspace of heroin samples and verified as a dominant odor in commercial vinegar samples; however, no common, secondary compound was detected in the headspace of either. Because of the similarities detected within respective explosive classes, several compounds were chosen for explosive mimics. A single based smokeless powder with a detectable level of 2,4-dinitrotoluene, a double based smokeless powder with a detectable level of nitroglycerine, 2-ethyl-1-hexanol, DMNB, ethyl centralite and diphenylamine were shown to be accurate mimics for TNT-based explosives, NG-based explosives, plastic explosives, tagged explosives, and smokeless powders, respectively. The combination of these six odors represents a comprehensive explosive odor kit with positive results for imprint on detection canines. As a proof of concept, the chemical compound PFTBA showed promise as a possible universal, non-target odor compound for comparison and calibration of detection canines and instrumentation. In a comparison study of shape versus vibration odor theory, the detection of d-methyl benzoate and methyl benzoate was explored using canine detectors. While results did not overwhelmingly substantiate either theory, shape odor theory provides a better explanation of the canine and human subject responses.

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.

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.^

Online solid phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) method for the determination of sucralose in reclaimed and drinking waters and its photo degradation in natural waters from South Florida

Background Sucralose has gained popularity as a low calorie artificial sweetener worldwide. Due to its high stability and persistence, sucralose has shown widespread occurrence in environmental waters, at concentrations that could reach up to several μg/L. Previous studies have used time consuming sample preparation methods (offline solid phase extraction/derivatization) or methods with rather high detection limits (direct injection) for sucralose analysis. This study described a faster and sensitive analytical method for the determination of sucralose in environmental samples. Results An online SPE-LC–MS/MS method was developed, being capable to quantify sucralose in 12 minutes using only 10 mL of sample, with method detection limits (MDLs) of 4.5 ng/L, 8.5 ng/L and 45 ng/L for deionized water, drinking and reclaimed waters (1:10 diluted with deionized water), respectively. Sucralose was detected in 82% of the reclaimed water samples at concentrations reaching up to 18 μg/L. The monthly average for a period of one year was 9.1 ± 2.9 μg/L. The calculated mass loads per capita of sucralose discharged through WWTP effluents based on the concentrations detected in wastewaters in the U. S. is 5.0 mg/day/person. As expected, the concentrations observed in drinking water were much lower but still relevant reaching as high as 465 ng/L. In order to evaluate the stability of sucralose, photodegradation experiments were performed in natural waters. Significant photodegradation of sucralose was observed only in freshwater at 254 nm. Minimal degradation (<20%) was observed for all matrices under more natural conditions (350 nm or solar simulator). The only photolysis product of sucralose identified by high resolution mass spectrometry was a de-chlorinated molecule at m/z 362.0535, with molecular formula C12H20Cl2O8. Conclusions Online SPE LC-APCI/MS/MS developed in the study was applied to more than 100 environmental samples. Sucralose was frequently detected (>80%) indicating that the conventional treatment process employed in the sewage treatment plants is not efficient for its removal. Detection of sucralose in drinking waters suggests potential contamination of surface and ground waters sources with anthropogenic wastewater streams. Its high resistance to photodegradation, minimal sorption and high solubility indicate that sucralose could be a good tracer of anthropogenic wastewater intrusion into the environment.

Evaluation of non-contact sampling and detection of explosives using receiver operating characteristic curves

The growing need for fast sampling of explosives in high throughput areas has increased the demand for improved technology for the trace detection of illicit compounds. Detection of the volatiles associated with the presence of the illicit compounds offer a different approach for sensitive trace detection of these compounds without increasing the false positive alarm rate. This study evaluated the performance of non-contact sampling and detection systems using statistical analysis through the construction of Receiver Operating Characteristic (ROC) curves in real-world scenarios for the detection of volatiles in the headspace of smokeless powder, used as the model system for generalizing explosives detection. A novel sorbent coated disk coined planar solid phase microextraction (PSPME) was previously used for rapid, non-contact sampling of the headspace containers. The limits of detection for the PSPME coupled to IMS detection was determined to be 0.5-24 ng for vapor sampling of volatile chemical compounds associated with illicit compounds and demonstrated an extraction efficiency of three times greater than other commercially available substrates, retaining >50% of the analyte after 30 minutes sampling of an analyte spike in comparison to a non-detect for the unmodified filters. Both static and dynamic PSPME sampling was used coupled with two ion mobility spectrometer (IMS) detection systems in which 10-500 mg quantities of smokeless powders were detected within 5-10 minutes of static sampling and 1 minute of dynamic sampling time in 1-45 L closed systems, resulting in faster sampling and analysis times in comparison to conventional solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) analysis. Similar real-world scenarios were sampled in low and high clutter environments with zero false positive rates. Excellent PSPME-IMS detection of the volatile analytes were visualized from the ROC curves, resulting with areas under the curves (AUC) of 0.85-1.0 and 0.81-1.0 for portable and bench-top IMS systems, respectively. Construction of ROC curves were also developed for SPME-GC-MS resulting with AUC of 0.95-1.0, comparable with PSPME-IMS detection. The PSPME-IMS technique provides less false positive results for non-contact vapor sampling, cutting the cost and providing an effective sampling and detection needed in high-throughput scenarios, resulting in similar performance in comparison to well-established techniques with the added advantage of fast detection in the field.

The Development of an Optimized System of Narcotic and Explosive Contraband Mimics for Calibration and Training of Biological Detectors

Current commercially available mimics contain varying amounts of either the actual explosive/drug or the chemical compound of suspected interest by biological detectors. As a result, there is significant interest in determining the dominant chemical odor signatures of the mimics, often referred to as pseudos, particularly when compared to the genuine contraband material. This dissertation discusses results obtained from the analysis of drug and explosive headspace related to the odor profiles as recognized by trained detection canines. Analysis was performed through the use of headspace solid phase microextraction in conjunction with gas chromatography mass spectrometry (HS-SPME-GC-MS). Upon determination of specific odors, field trials were held using a combination of the target odors with COMPS. Piperonal was shown to be a dominant odor compound in the headspace of some ecstasy samples and a recognizable odor mimic by trained detection canines. It was also shown that detection canines could be imprinted on piperonal COMPS and correctly identify ecstasy samples at a threshold level of approximately 100ng/s. Isosafrole and/or MDP-2-POH show potential as training aid mimics for non-piperonal based MDMA. Acetic acid was shown to be dominant in the headspace of heroin samples and verified as a dominant odor in commercial vinegar samples; however, no common, secondary compound was detected in the headspace of either. Because of the similarities detected within respective explosive classes, several compounds were chosen for explosive mimics. A single based smokeless powder with a detectable level of 2,4-dinitrotoluene, a double based smokeless powder with a detectable level of nitroglycerine, 2-ethyl-1-hexanol, DMNB, ethyl centralite and diphenylamine were shown to be accurate mimics for TNT-based explosives, NG-based explosives, plastic explosives, tagged explosives, and smokeless powders, respectively. The combination of these six odors represents a comprehensive explosive odor kit with positive results for imprint on detection canines. As a proof of concept, the chemical compound PFTBA showed promise as a possible universal, non-target odor compound for comparison and calibration of detection canines and instrumentation. In a comparison study of shape versus vibration odor theory, the detection of d-methyl benzoate and methyl benzoate was explored using canine detectors. While results did not overwhelmingly substantiate either theory, shape odor theory provides a better explanation of the canine and human subject responses.

Pore water and solid-phase data from site Scharendijke (Lake Grevelingen, NL)

Will be submitted by the author