Indiana University-Purdue University Indianapolis (IUPUI) / Solid Phase Microextraction (SPME) is a popular sampling technique that can be paired with Gas Chromatography/Mass Spectrometry (GC-MS). SPME-GC-MS is used in forensic chemistry due to its simplification of the sample preparation process. Headspace-Solid Phase Microextraction (HS-SPME) is a technique where the sample is heated to generate volatiles in the headspace of the vial. A SPME fiber is then inserted into the vial and the compounds in the headspace will bind to the fiber. Total Vaporization- Solid Phase Microextraction (TV-SPME) is a technique that is derived from the HS-SPME technique. In Chapter 1, the critical comparison of HS-SPME and TV-SPME is discussed. Samples including marijuana, essential oils, and CBD oil were utilized to compare the two techniques. The compounds of interest in marijuana are the three main cannabinoids: cannabinol (CBN), cannabidiol (CBD), and tetrahydrocannabinol (THC). The sample preparation and GC-MS parameters were kept the same for all samples to determine which SPME technique works best for these sample types and yielded the greatest sensitivity. It was found that HS-SPME shows greater sensitivity with CBN and equivalent sensitivity with essential oils, THC and CBD. In Chapter 2, the detection of synthetic cannabinoids utilizing liquid-liquid injection as well as HS-SPME and TV-SPME is discussed. The detection of these compounds is important because this type of drug has become more prevalent in the United States because they can be chemically altered slightly so they still have the effects of a drug but can evade drug legislation. The detection of synthetic cannabinoids using liquid injection was found to be successful but detection using HS-SPME and TV-SPME was found to be unsuccessful. In Chapter 3, the analyses of real and artificial saliva utilizing HS-SPME and TV-SPME is discussed. Determining the compounds present in real saliva and artificial saliva will be of importance for future research into determining if the presence of drugs in saliva can be analyzed with these techniques. The analyses of real and artificial saliva were found to be successful using HS-SPME, without derivatization, and TV-SPME, with and without derivatization. Many of the compounds present in the real saliva were detected and were confirmed to be compounds regularly found in saliva by other scientific literature.

The diploma thesis is focused on the determination of tetracyclines, sulfonamides and fluoroquinolones from soil. Antibiotics such as tetracycline, chlortetracycline, oxytetracycline. ciprofloxacin, trimethoprim, sulfamethaxazole and sulfadiazin were selected due to frequent use in veterinary medicine. Due to the fact that the soil is a complex matrix and contains many components that can interfere with the detection signal of analytes, it was necessary to use the MAX column in the SPE method, which removes fulvic aned humic acid from analytes and the HLB column Final analysis of the analytes was performed by liquid chromatography with mass detesction (LC-MS). The method was also used for real soil samples, which were delivered from ÚKZUS.

Plant signaling involves the transport of information within and between plant cells from receptors to effectors. Plants are affected by biotic and abiotic stress conditions like insect attack and extreme temperatures, respectively, which cause disease, the induction of senescence and the reduction of crop yield. To improve plant traits for feed, fiber, and energy applications, it is critical to understand the short- and long-range signaling mechanisms plants use to control growth, biomass composition, senescence and responses to environmental stresses. It is known that many plant signaling molecules have profound effects on plants, through mechanisms that remain largely obscure. A key gap in knowledge is the understanding of the mechanisms that govern the movement and fate of signaling molecules. This study seeks to synthesize signaling probes based on flagellin 22 (flg22), a 22-amino acid peptide that induces defense gene expression to trigger both local and systemic immune responses in plants. Solid-phase synthesis of fluorescently-tagged derivatives of flg22 was initiated, and studies on the uptake of labeled probes was conducted using a fiber-optic fluorescence microscope that was adapted for use in plants. Fluorescence microscopy showed uptake and internalization of TAMRA-flg22 in cells of Arabidopsis thaliana Columbia (wild-type strain), which was not observed in the fls2 strain in which FLS2, the receptor for flg22, had been knocked out.

This research study successfully completed three objectives: 1) validate liquid-liquid, supported-liquid, and solid-phase extractions for the quantitation of three barbiturates (amobarbital, butalbital, and phenobarbital) in blood and urine using liquid chromatography-tandem mass spectrometry; 2) to compare the efficiency and effectiveness among methods in accomplishing extraction of barbiturates under the laboratory setting at Boston University School of Medicine; and 3) to report all the analytical data to RTI International for interlaboratory comparison. For the validation study, a six-point linear calibration model (20-2000 ng/mL) with inversely weighted concentration (1/x) was reproducible in all three sample preparation methods for both blood and urine with r2 greater than or equal to 0.994. Bias and precision evaluated from three controls throughout the range of the curve were within ±20% and ±20%CV, respectively. Neither carryover nor interference was observed. Detection limits were evaluated down to 5 ng/mL depending on the extraction procedure. Samples were able to be diluted up to 50 times prior to instrumental analysis. Samples were stable on autosampler at room temperature up to 72 hours after their initial analysis. Recovery of barbiturates from blood and urine all ranged from 45% to 86%. The effect of ionization suppression or enhancement was found to have minimal impact on the validation. For choosing the most suitable method quantifying barbiturates, efficiency and effectiveness were studied. Efficiency evaluates the time and ease of sample preparation required to prepare a sample for analysis. Supported-liquid extraction was found to be the most efficient method for extracting barbiturates as it required the least amount of time to perform and could be easily automated with minimal training. Effectiveness is an assessment of one’s ability to selectively recover target analyte at a reasonably low concentration. By considering a method’s recovery, extract cleanliness, detection limits, and reproducibility, liquid-liquid extraction was the best at quantifying barbiturates in blood and supported-liquid extraction was the most suitable method for extracting barbiturates from urine. For interlaboratory comparison, all the data collected has been reported to RTI International. These findings can be used for examining the overall reliability and reproducibility of the validated methods. Results obtained can also be used to explore the possibility for streamlining sample preparation in the forensic laboratory, and hence reducing the case backlog.

The issue with maintaining stability of compounds during storage and transportation has been universal across many fields such as clinical, pharmaceutical, food industry and especially in forensic laboratories where case backlogs hinder immediate analysis of samples upon collection. This study compares various storage conditions and analyzes changes in compound stability over 28 days. The various storage conditions compared in this study are temperature (+4 °C and -20 °C); water sample and urine sample and solid state (loading onto Solid Phase Extraction cartridges) and storing in its liquid state (present in the urine or water sample). 51 compounds were analyzed in this study belonging to pharmaceutical and hormone classes. Two Dimensional Liquid Chromatography was used for separation allowing the analysis of varied compounds in terms of chemistry. Mass Spectrometry was used for detection of these compounds. This study conclusively helped determine that there is no significant difference in stability of 42 out of 51 of these compounds on comparing them in solid versus liquid state over a period of 28 days. This helps determine that Solid Phase Extraction (SPE) cartridges can be used as an alternative for storage and transportation of these compounds. 46 compounds showed no significant difference in +4 and -20 °C temperature storage conditions as well. Despite not including a wash step during sample preparation between loading and elution, 11 out of 51 compounds did not show suppression or enhancement due to matrix effect. This study not only highlights the importance of sample preparation prior to analysis but also shows how SPE technique could help maintain stability of compounds during storage. In future studies, stability changes using SPE for long term storage could provide beneficial results to conclude if SPE techniques can provide an advantage over liquid state for a period longer than a month.

A cannabinoid refers to any natural or synthetic compound that interacts with the CB1 and CB2 receptors. There are currently three different groups of cannabinoids: endogenous cannabinoids, phytocannabinoids and synthetic cannabinoids. The most common phytocannabinoid is delta-9-tetrahydrocannabinol (THC), which is the active component in the Cannabis sativa or marihuana plant1–3. Two examples of synthetic cannabinoids that are present in case reports from 2012 to 2018 are AB-FUBINACA and AB-PINACA4–7. THC and synthetic cannabinoids are commonly encountered drugs in forensic toxicology cases, therefore, being able to extract these compounds and their metabolites is imperative for toxicological interpretation. There are a variety of commercially available sample preparation techniques for these analytes. Companies such as UCT, Biotage, Millipore-Sigma, Tecan, and Thermo Fisher Scientific manufacture these products. The focus of this research was to evaluate these techniques for their cleanliness, efficiency and cost effectiveness. Sample preparation techniques are designed to remove the different components of the matrix and other prescription or illicit substances present in the sample that could interfere with the assay, increase the analyte recovery, extraction efficiency, decrease variability, and clean-up the sample to allow for less instrument downtime and longer column life8. This study focused on comparing a liquid-liquid extraction (LLE), solid phase extraction (SPE), and supported liquid extraction (SLE). The primary purpose of this study was to develop and validate the three above mentioned sample preparation techniques for the analysis of THC, 11-hydroxy-THC, 11-nor-9-carboxy-THC (THCCOOH), AB-FUBINACA, AB-FUBINACA metabolite 3, and AB-PINACA in blood and urine. Parameters assessed followed Academy Standards Board (ASB) Standard 036, Standard Practices for Method Validation in Forensic Toxicology, including recovery, suppression, and matrix effects. For urine and blood analysis, the calibration range was determined to be 1 ng/mL to 50 ng/mL for all three techniques. Urine recovery was highest for the LLE method, with all compounds having a recovery greater than 50%. The SLE method had the lowest LOQ results for urine, with 0.5 ng/mL for 11-hydroxy-THC and THCCOOH, 0.75 ng/mL for THC, AB-FUBINCA and AB-FUBINACA metabolite 3, and 1 ng/mL for AB-PINACA. Ion suppression was reduced using the SLE method for urine along with having the shortest sample preparation time of 1 hr for up to 48 samples. For blood analysis, the LLE method had the greatest recovery of all analytes. The LLE method also had reduced suppression and matrix effects compared to the SPE method. Sample preparation was shorter for the SPE method, consuming 2 hrs for an average sample batch, compared to 4 hrs for the LLE method, which included a 2 hr freezing step. In conclusion, for urine analysis, all three sample preparation techniques were acceptable for the analysis of THC, synthetic cannabinoids, and their metabolites, with the SLE method being the preferred method. For blood analysis a LLE and SPE method were developed and are adequate for the analysis of THC, synthetic cannabinoids, and their metabolites, with the LLE method being the preferred method.

In forensic toxicology, analysis of drugs and metabolites in biological fluids is performed to determine cause of death, suspected drug use, drug facilitated sexual assaults, or whether someone was driving under the influence. Analyte identification and concentration determination can be determined in a variety of matrices (e.g., blood, urine, or oral fluid) and can be complex. It is therefore necessary to have optimal sample preparation and instrumental conditions that work for all matrices of interests. Determining the best approach can be challenging due to the amount of time and resources to perform expansive evaluations of sample preparation, stationary/mobile phases, liquid chromatography (LC) conditions and mass spectrometry (MS) operating parameters. In this study three different sample preparation methods were validated for blood and urine. The three sample preparation methods were solid-phase extraction (SPE), supported liquid extraction (SLE), and liquid-liquid extraction (LLE). Six different drug groups were used as the analytes being tested by the methods. These drug groups were amphetamines, local anesthetics, opioids, hallucinogens, antidepressants, and novel psychoactive substances (NPS). A total of twenty-three drugs were used: amphetamine, methamphetamine, (3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), and 3,4-methylenedioxymethamphetamine (MDMA), benzoylecgonine (BZE), cocaine, lidocaine, codeine, methadone, morphine, 6-monoacetylmorphine (6-MAM), fentanyl, oxycodone, lysergic acid diethylamide (LSD), phencyclidine (PCP), amitriptyline, citalopram, fluoxetine, trazodone, ethylone, α-pyrrolidinopentiophenone (α-PVP), and 25I-NBOMe. The methods were validated according to guidelines set forth by the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation in Forensic Toxicology and the American Academy of Forensic Science (AAFS) Standards Board (ASB) draft of Standard Practices for Method Validation in Forensic Toxicology. Parameters of calibration model, bias, precision, limit of detection (LOD), limit of quantitation (LOQ), dilution integrity, ion suppression/enhancement, interference studies, and stability were evaluated. Recovery was also assessed to determine the efficiency of the extraction. Calibration models met the 0.98 R2 minimum requirement. For all sample preparations the compounds evaluated in each were found to be stable for at least 72 hours. Interferences were found to be similar across all three sample preparation methods. Parameters of bias, precision, and dilution integrity were largely comparable between all three methods. Overall for LOD, SLE resulted in lower values for blood and urine ranging for 0.1 to 5 ng/mL. Overall for LOQ, SLE resulted in lower values for blood and LLE resulted in lower values for urine in the range of 0.5-10 ng/mL. SLE resulted in the highest recovery for all twenty-three analytes, due to LLE failing to extract consistently or completely for benzoylecgonine, morphine, and 6-monoacetylmorphine. Overall, SLE resulted in the lowest percent values for ion suppression and enhancement for both blood and urine. Overall, blood resulted in high ion suppression (exceeding -20%) for SPE and LLE. Final determination overall was that SLE was the best sample preparation method for all twenty-three analytes. This was determined based on the evaluation of recovery, ion suppression/enhancement, and LOD, as well as sample preparation time. Sample preparation time for SLE was approximately 1 hour, while SPE took 2.5 hours and LLE 2 hours.

This work deals with the preparation of combinatorial libraries of peptides serving as platforms for proteolytic phenotyping. The primary objective was to prepare a solid phase fluorogenic peptide library and screen proteases by fluorescence. Further, the possibility of preparing solid phase DNA-encoded libraries was studied. Due to the non-reactivity of the specific proteases with the solid phase peptides, the solid phase was completely abandoned and DNA-encoded peptide library was prepared in the solution. Using this model of DNA-encoded dipeptide with terminal biotin, the new principle of testing proteolytic activities of proteases was verified. A combinatorial library of DNA-encoded hexapeptides was also prepared. Despite the low yield of the library, the possibility of DNA encoding, the amplifiability of the prepared molecules and the possibility of biotin-based separation were verified. The integrity of the hexapeptide sequence and the protease testing is the subject of further study.

In a forensic investigation involving oil, a comparative analysis named oil spill fingerprinting between a source and an oil spill is normally performed. It is based on detecting a certain selection of biomarkers with gas chromatography (GC) coupled to mass spectrometry (MS) which are later divided into diagnostic ratios (DRs). An international guideline formed by European Committee for Standardization (CEN) denoted CEN/TR 15522-2:2012 describes the process of conducting oil spill fingerprinting. This method is currently being inspected and adjusted for standardization into EN 15522-2:2022. One section of the upcoming standard describes sample clean-up which is important to inhibit possible matrix effect that could either enhance or reduce peak intensity in the chromatogram. There is yet no conclusive SPE (Solid Phase Extraction) protocol in the current draft version of EN 15522-2:2022. The aim with this study was to develop such a protocol. Development included comparative testing of the recommended stationary phases silica and Florisil®. Additionally, the effect of both elution solvent and elution volume were investigated. The protocol must pass validation criteria to be implemented at National Forensic Centre (NFC) and possibly be used in the final version of EN 15522-2:2022. A successful method for Heavy Fuel Oil (HFO), diesel and lubricating oil was achieved with activated Florisil® as stationary phase, eluted with 6 ml dichloromethane (DCM). If the evidence material is suspected to contain FAMEs (Fatty Acid Methyl Esters), then 2 % acetone should be added to the DCM. The final SPE column was packed with 0.5 glass wool, 4 cm Florisil® and 0.5 cm sodium sulfate. Based on successful validation result, the SPE protocol should be considered for the final version of EN 15522-2:2022 as well as for implementation at NFC.

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