Despite efforts to control synthetic cannabinoids, clandestine manufacturers continue to modify their structures to avoid legal consequences, creating an ever-changing analytical target for forensic laboratories (1). Forensic toxicology laboratories often lack the needed resources or do not have the capabilities to test for these compounds and metabolites, requiring specimens to be submitted to reference laboratories (2). Drug stability can be affected by long storage times, temperature and preservatives (3). Although these factors can be controlled, systematic research is necessary to identify their impacts on the stability of these new synthetic cannabinoids that are continually emerging. The purpose of this research is to assess the stability of 17 synthetic cannabinoids in human whole blood and 10 synthetic cannabinoid metabolites in human urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) over thirty-five weeks. The analysis methods were validated in accordance to the Academy Standards Board (ASB) method validation guidelines for quantitative analysis and stability evaluation of the following analytes in blood: 4-cyano CUMYL-BUTINACA, ADB-PINACA, EMB-FUBINACA, JWH-250, MO-CHMINACA, 5-fluoro-3,5-ABPFUPPYCA, 5-fluoro ADB-PINACA, APP-PICA, CUMYL-THPINACA, PB-22, XLR11, 5-fluoro PY-PINACA, MDMB-FUBICA, MEP-CHMICA, NM2201, RCS-8, and UR144. The stability analysis in urine includes the following metabolites: 5-fluoro MDMB-PICA metabolite 7, 5-fluoro PB-22 3-carboxyindole, AB-FUBINACA metabolite 3, ADB-PINACA N-(4-hydroxypentyl), ADB-PINACA pentanoic acid, UR-144 Degradant N-pentanoic acid, PB-22 N-(5-hydroxypentyl), MDMB-FUBICA metabolite 3, UR-144 N-(5-hydroxypentyl), and JWH-250 N-pentanoic acid. Research samples were prepared by spiking with certified reference standards (Cayman Chemical, Ann Arbor, MI, USA) of each select synthetic cannabinoid in certified drug-free human whole blood (Boston Medical Center, Boston, MA, USA; Biological Specialty Corporation, Colmar, PA) and drug-free urine that was received as donations following the approved Institutional Review Board guidelines (Boston University School of Medicine, Boston, MA, USA). Blood samples were aliquoted into 6 mL BD Vacutainer Plastic Collection Tubes (Fisher Scientific, Waltham, MA, USA) and urine samples were stored in 15 mL Falcon Conical Centrifuge Tubes (Fisher Scientific, Waltham, MA, USA). Stability under room temperature (20ºC), refrigerator (4ºC), and freezer (-20ºC) at low and high concentrations were evaluated at select time points. A 5% solution of potassium oxalate and sodium fluoride or ethylenediaminetetraacetic acid (EDTA) was added to the preserved blood samples by the manufacturer prior to storage. The anticoagulant, potassium oxalate, was only added in solution to the preserved samples whereas none was added to the nonpreserved samples. Short-term urine samples were preserved with 1% of sodium fluoride prior to storage. Extraction of analytes was conducted using supported-liquid extraction (SLE) ISOLUTE 1 mL cartridges (Biotage, Charlotte, NC, USA) and reconstituted in 100 μL of 50:50 mixture of 0.1% formic acid in millipore deionized water and 0.1% formic acid in acetonitrile (Fisher Scientific, Waltham, MA, USA). Analysis was performed in triplicate using a reverse-phase C18 column (Waters XBridge C18 3.5 μM, 2.1 x 50 mm, Milford, MA, USA) on the Shimadzu Prominence Ultra-Fast Liquid Chromatography (UFLC, Kyoto, Japan) with SCIEX 4000 Q-Trap Electrospray Ionization Tandem Mass Spectrometry (ESI/MS/MS, Waltham, MA, USA) in positive ionization mode. The total run time was 8 minutes with a flow rate of 0.6 mL/min and injection volume of 10 μL. Linear calibration curves for each analyte with the exception of a quadratic regression for PB-22, all had acceptable R2 values > 0.99 using a weighting factor of 1/x. A linear dynamic range of 0.5 – 25 ng/mL was used for all analytes in blood except for NM2201 and APP-PICA with a limit of quantitation (LOQ) of 0.1 ng/mL and MO-CHMINACA with a working range of 0.5 – 15 ng/mL. A linear working range of 5 – 40 ng/mL was utilized for all metabolites in urine. No signs of carryover were observed. In general, analytes were considered stable if the average area ratio between the analyte and internal standard at the time point was within ± 20% of the average area ratio response at time point zero. In some cases, it was necessary to evaluate the complete picture of the stability data by reviewing analyte area, concentration, and overall stability data trend between timepoints at the low and high concentrations. In certain situations, an analyte was considered stable even if specific timepoints for a concentration were outside the ±20% range. For example, in cases where one concentration at a timepoint was within the ±20% range and the other concentration fell within ±30% range the analyte was considered stable overall. Long-term stability results revealed that all synthetic cannabinoids were stable at 21 to 35 weeks in frozen blood preserved with sodium fluoride except for APP-PICA. The preservatives are recommended to be added to blood to reduce the possibility of matrix inferences and minimize detrimental impacts on the stability of synthetic cannabinoids. Analytes experienced lower degradation in the order of samples that were kept frozen, refrigerated, and then at room temperature. Blood analytes that were stable up to 35 weeks in freezer generally had a core structure of a carbonyl substituent on a pyrazole or pyrrole with surrounding nonpolar groups; whereas compounds with two polar carbonyl functional groups present were found to experience degradation much earlier at 1 week or less in room temperature and refrigerator storage conditions. 5-fluoropentyl analogs, like XLR11 and 5-fluoro ADB-PINACA, in comparison to their counterpart analyte, UR144 and ADB-PINACA, were unstable at earlier time points under all storage conditions. Instability in the majority of the urine metabolites was not observed until after 9 weeks and was generally consistent across all storage conditions. The validated methods demonstrate a sensitive and reliable way to positively identify 17 different synthetic cannabinoids in human whole blood and 10 synthetic cannabinoid metabolites in urine for rapid time stability analysis at various storage conditions. The use of SLE improved sample preparation efficiency by decreasing the extraction time from 1 hour to 30 minutes compared to traditional extraction methods, such as solid-phase extraction (SPE) and liquid-liquid extraction (LLE). Further studies into additional matrices, such as oral fluid, longer storage times, and other emerging synthetic cannabinoid analytes would expand the scope of this research.

Liquid chromatography, coupled with tandem mass spectrometry, presents a powerful tool for the quantification of the sex steroid hormones 17-β estradiol, progesterone and testosterone from biological matrices. The importance of accurate quantification with these hormones, even at endogenous levels, has evolved with our understanding of the role these regulators play in human development, fertility and disease risk and manifestation. Routine monitoring of these analytes can be accomplished by immunoassay techniques, which face limitations on specificity and sensitivity, or using gas chromatography–mass spectrometry. LC–MS/MS is growing in capability and acceptance for clinically relevant quantification of sex steroid hormones in biological matrices and is able to overcome many of the limitations of immunoassays. Analyte specificity has improved through the use of novel derivatizing agents, and sensitivity has been refined through the use of high-resolution chromatography and mass spectrometric technology. This review highlights these innovations, among others, in LC–MS/MS steroid hormone analysis captured in the literature over the last decade.

学位授与年月日: 2007-07-23 ; 学位の種類: 新制・課程博士 ; 学位記番号: 工博第2837号 / Perfluorochemicals (PFCs) were produced by industries and consumed “safely” as surfactants, repellents, additives, fire-fighting foams, polymer emulsifiers and insecticides for almost fifty years. However they are now considered as persistent, bioaccumulated and toxic (PBT) chemicals, and ubiquitously distributed in waster, air, human body and biota. Although some efforts were contributed to reduce PFCs in environment, such as development of alternatives and recycling processes, huge amount of persisted PFCs have already been discharged in environment and accumulated in biota including humans. In some industrialized areas, such as Yodo river basin in Japan, water environment and human blood were polluted by some PFCs, and thus reduction and control of PFCs were urgently required for the purpose of environmental safety and human health in these areas. Unfortunately, some studies implied that current water and wastewater treatment processes seemed ineffective to remove PFCs in trace levels. Therefore, this study will try to develop some proper technologies to treat trace level of PFCs in wastewater. In order to achieve this main objective, several works have been accomplished as follows. 　Current available literature has been reviewed to obtain a solid background for this study. Basic information of PFCs was summarized in physiochemical properties, PBT properties, productions and applications, regulations and etc.. Analytical methods for PFCs, especially of LC-ESI-MS/MS, were reviewed including pretreatment processes in diverse matrices, which derived objectives of chapter III. Distributions and behavior of PFCs were briefly discussed in water environments, biota sphere and human bloods. Available control strategies were shown in detail about alternatives, industrial recycling processes, and newly developed treatment processes. Current wastewater treatment processes showed inefficient removal for some PFCs, deriving objectives of chapter IV on the PFC behavior in treatment process. Newly developed treatment technologies seemed able to decompose PFCs completely but unsuitable for application in WWTP. Therefore, granular activated carbon (GAC) adsorption and ultra violet (UV) photolysis were developed in chapter V and VI as removal and degradation processes respectively. 　Fifteen kinds of PFCs were included in this study, consisting of twelve kinds of perfluorocarboxylic acids (PFCAs) with 4~18 carbons and three kinds of perfluoroalkyl sulfonates (PFASs) with 4~8 carbons. An integral procedure was developed in chapter III to pretreat wastewater samples. LC-ESI-MS/MS was applied to quantify all PFCs in trace level. Pretreatment methods were optimized between C18 and WAX-SPE processes for aqueous samples, and between IPE, AD-WAX and ASE-WAX processes for particulate samples. Standard spiking experiments were regularly conducted for each wastewater sample to calculate recovery rate and control analytical quality. As the result, WAX-SPE showed better performance on samples with very high organics concentrations, and C18-SPE performed better for long-chained PFCs. ASE-WAX was proposed as the optimum method to pretreat particulate samples because of the simple and time saving operations. 9H-PFNA was used as internal standard to estimate matrix effect in wastewater. 　Behavior of PFCs in a municipal WWTP has been studied in chapter IV by periodical surveys for six times in half a year. All PFCs used in this study were detected in WWTP influent and effluent. According to their carbon chain lengths, all PFCs can be classified into “Medium”, “Long” and “Short” patterns to simplify behavior analysis. PFCs in same pattern showed similar properties and behavior in wastewater treatment facilities. Very high concentrations of PFCs existed in WWTP influent, indicating some point sources of industrial discharge in this area. “Medium” PFCs, such as PFOA(8), PFNA(9) and PFOS(8), were primary contaminants in the WWTP and poorly removed by overall process. Performances of individual facilities were estimated for removal of each PFC. Primary clarification and secondary clarification were helpful to remove all PFCs in both aqueous phase and particulate phase. “Medium” PFCs in aqueous phase were increased after activated sludge process, but other PFCs can be effectively removed. Ozone seemed ineffective to decompose PFCs because of the strong stability of PFC molecules. Sand filtration and biological activated carbon (BAC) filtration in this WWTP can not remove PFCs effectively too, which required further studies. Performances of combined processes were estimated by integrating individual facilities along the wastewater flow. Activated sludge process coupled with clarifiers showed satisfied removal of most PFCs in the investigated WWTP except “Medium” PFCs. 　Adsorption characteristics of PFCs onto GAC have been studied by batch experiments in chapter V. Freundlich equation and homogenous surface diffusion model (HSDM) were applied to interpret experimental data. Isothermal and kinetics experiments implied that PFC adsorption on GAC was directly related with their carbon chain lengths. By ascendant carbon chain length, adsorption capacity for specific PFC was increased, and diffusion coefficient (Ds) was decreased. Ds of GAC adsorption was also decreased gradually in smaller GAC diameters. Coexisted natural organic matters (NOMs) reduced adsorption capacities by mechanism of competition and carbon fouling. Carbon fouling was found reducing adsorption capacity much more intensively than competition by organics. Acidic bulk solution was slightly helpful for adsorption of PFCs. However adsorption velocity or kinetics was not affected by NOM and pH significantly. GAC from Wako Company showed the best performance among four kinds of GACs, and Filtra 400 from Calgon Company was considered more suitable to removal all PFCs among the commercial GACs. Preliminary RSSCT and SBA results implied that background organics broke through fixed GAC bed much earlier than trace level of PFCs. Medium-chained PFCs can be effectively removed by fixed bed filtration without concerning biological processes. 　Direct photolysis process has been developed in chapter VI to decompose PFCAs in river water. Irradiation at UV254 nm and UV254+185 nm can both degrade PFCAs. Stepwise decomposition mechanism of PFCAs was confirmed by mass spectra analysis, and consecutive kinetics was proposed to simulate experimental data. PFASs can also be degraded by UV254+185 photolysis, although the products have not been identified yet. Coexisted NOMs reduced performance of UV photolysis for PFCAs by competition for UV photons. Sample volume or irradiation intensity showed significant influence on degradation of PFCAs. Local river water polluted by PFOA can be cleaned up by UV254+185 photolysis effectively. Ozone-related processes were also studied but ineffective to degrade PFC molecules. However, PFCs could be removed in aeration flow by another mechanism. / 京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第13340号 / 工博第2837号 / 新制||工||1417(附属図書館) / UT51-2007-M963 / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 田中 宏明, 教授 藤井 滋穂, 教授 伊藤 禎彦 / 学位規則第4条第1項該当 / Doctor of Engineering / Kyoto University / DFAM

Aripiprazol är den aktiva substansen i läkemedel för bipolaritet och schizofreni och metaboliseras av två enzym till den aktiva metaboliten dehydroaripiprazol. Till följd av interindividuella skillnader i aktiviteten hos enzymen samt att koncentrationen in vivo kan påverkas av andra läkemedel rekommenderas terapeutisk läkemedelsövervakning (TDM). Därmed har en selektiv och känslig vätskekromatografi- tandem masspektrometri (LC-MS/MS) validerats för kvantifiering av aripiprazol och dess aktiva metabolit dehydroaripiprazol i humant serum vid Specialkemi, Klinisk kemi och farmakologi vid Lunds universitetssjukhus utifrån interna kriterier byggda på riktlinjer från European Medicine Agency (EMA). LC-MS/MS analys utfördes på Tripple Quad 6500+ från AB Sciex med joniserande elektrospray (ESI) och multi reaction monitoring (MRM). Valideringen fastslog metodens mätområde till 4 – 2 500 nmol/L och kvantifieringsgräns till 4 nmol/L för respektive analyt. Metodens inomdags- och mellandags noggrannhet (variationskoefficient (CV%)) och riktighet (nominell differens) för kontrollprover (10 och 1 000 nmol/L) var mellan 3,5 – 9,1 % och mellan -6,8 – -13,0 för respektive analyt vilket var inom godkända kriterier. Innan metoden implementeras på kliniska prover bör framtida utvärdering undersöka om minimering av provsmittan är möjlig samt utvärdera långtidsstabiliteten av analyterna.

This project focused on the development and validation of an ultra-performance liquid chromatography tandem mass spectrometer (UPLC-MS/MS) method for the determination of Patulin in apple-based products. Patulin is one of the many mycotoxins that are secondary metabolites from about 60 filamentous fungi. The mold often appears as black or blue on fruit, vegetables or crops. To determine the concentration of Patulin in consumer products is important since it may affect consumer health. The symptoms are often flu-like and can lead to kidney-failure and neurotic damage. The Swedish Food Agency is tasked to analyze consumer products to determine if they are safe to ingest. The European Commission has set maximum residue limits for several toxins that can potentially appear in groceries on the market. Using an UPLC-MS/MS allows for the accurate qualification and quantification of Patulin in apple juice and purees. The method was validated by analyzing several lots of apple juices and a proficiency test from Fapas®. The recovery rate ranged between 70.5-103.8% and were accepted because they met the recovery criteria in Regulation (EC) No. 401/2006 for Patulin.

Introduction Cytochromes P450 (CYPs) are the major family of enzymes responsible for detoxification and metabolism of a wide range of both endogenous and xenobiotics chemicals in living organisms. The use of CYPs to activate prodrugs to cytotoxins selectively in tumours has been explored including AQ4N, Phortress and Aminoflavone. CYP1A1, CYP1B1, CYP2W1, and CYP4F11 have been identified as expressed in tumour tissue and surrounding stroma at high frequency compared to most normal tissues. Aim is to investigate the differential metabolism of novel chloromethylindoline by high frequency expressed CYPs in tumours. This differential may be exploited to elicit a selective chemotherapeutic effect by metabolising inert small molecules to potent cytotoxins within the tumour environment. Materials and Methods Sensitive and specific LC/MS/MS techniques have been developed to investigate the metabolism of chloromethylindolines. Recombinant enzymes and transfected cell lines were used to investigate the metabolic profiles with a focus on production of the cytotoxic derivatives of chloromethylindolines. Results Detailed metabolic studies show that (1-(Chloromethyl)-1,2-dihydropyrrolo [3,2-e]indol-3(6H)-yl)(5-methoxy-1H-indol-2-yl) methanone (ICT2700) and other chloromethylindolines are converted by CYP1A1 mediated hydroxylation at the C-5 position leading to highly potent metabolites. In vitro cytotoxicity studies showed differentials of up to 1000-fold was achieved between CYP1A1 activated compared to the non-metabolised parent molecules. The reactivity of metabolites of ICT2700 was also explored using glutathione as a nucleophile. The metabolites were identified by a combination of LC/MS and LC MS/MS techniques. Investigations using mouse and human liver microsomes show that a large number of metabolites are created though none were shown to be associated with a potential anticancer effect. Studies focused on CYP2W1 show that this isoform metabolised ICT2706 to a cytotoxic species and a pharmacokinetic study showed a good distribution of ICT2706 into mouse tissues including tumour. However metabolism of ICT2726 by CYP2W1 resulted only in a non-toxic metabolite profile and may have potential as a biomarker for functional CYP2W1 in tissues. Preliminary studies show that palmitic acid hydroxylation is a useful marker of functional CYP4F11. Summary and conclusion The in vitro results show that the chloromethylindolines are a novel class of agent with potential as prodrugs that following specific hydroxylation by CYP1A1 and CYP2W1 are converted to ultra-potent cytotoxins. Other metabolites are also evident which are not cytotoxic. Studies in vivo show that selected chloromethylindolines possess a good pharmacokinetic profile and show potential as prodrug anticancer agents that require activation by CYP1A1 or CYP2W1. The methods, results, progress and suggestions for future work are presented in this thesis.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24678号 / 農博第2561号 / 新制||農||1100(附属図書館) / 学位論文||R5||N5459(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 菅原 達也, 教授 舟場 正幸 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

We investigated the fungus Aspergillus fumigatus PD-18 responses when subjected to the multimetal combination (Total Cr, Cd2C, Cu2C, Ni2C, Pb2C, and Zn2C) in synthetic composite media. To understand how multimetal stress impacts fungal cells at the molecular level, the cellular response of A. fumigatus PD-18 to 30 mg/L multimetal stress (5 mg/L of each heavy metal) was determined by proteomics. The comparative fungal proteomics displayed the remarkable inherent intracellular and extracellular mechanism of metal resistance and tolerance potential of A. fumigatus PD-18. This study reported 2,238 proteins of which 434 proteins were exclusively expressed in multimetal extracts. The most predominant functional class expressed was for cellular processing and signaling. The type of proteins and the number of proteins that were upregulated due to various stress tolerance mechanisms were post-translational modification, protein turnover, and chaperones (42); translation, ribosomal structure, and biogenesis (60); and intracellular trafficking, secretion, and vesicular transport (18). In addition, free radical scavenging antioxidant proteins, such as superoxide dismutase, were upregulated upto 3.45-fold and transporter systems, such as protein transport (SEC31), upto 3.31-fold to combat the oxidative stress caused by the multiple metals. Also, protein–protein interaction network analysis revealed that cytochrome c oxidase and 60S ribosomal protein played key roles to detoxify the multimetal. To the best of our knowledge, this study of A. fumigatus PD-18 provides valuable insights toward the growing research in comprehending the metal microbe interactions in the presence of multimetal. This will facilitate in development of novel molecular markers for contaminant bioremediation.

In postmortem toxicology, certain cases require the examination of embalmed biological specimens to investigate the presence and potential role drugs may have played in a person’s death. Key factors, like postmortem distribution, which can be greatly affected by temperature, may alter drug concentrations in different areas of the body. In the United States, the involvement of cocaine in overdose deaths has significantly increased between 2012 and 2019 (1). The purpose of this project was to examine the stability of cocaine and its primary metabolite, benzoylecgonine, in perfused postmortem rat tissues stored at different temperatures over a one month. Twelve frozen cocaine positive rat specimens, intracardially perfused with a saline and formaldehyde solution, were received from a chronic cocaine rat study at Boston University Department of Psychological and Brain Sciences (Dr. Kathleen Kantak, Boston, MA, USA). The specimens were dissected, and the spleen, one kidney, and one lung were removed from each specimen. A fine-needle aspiration biopsy was performed on each organ to collect a time zero (T0) sample. One set of four rat specimens were stored at room temperature (20-22°C), another four were stored at refrigerator temperature (4°C), and another four were stored at freezer temperature (-20°C). A section of each organ was collected for analysis at two weeks (T1) and one month (T2). Samples underwent solid-phase extraction before liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using a QSight 220 CR Laminar Flow Triple Quadrupole Mass Spectrometer with electrospray ionization, operated in positive ion mode (PerkinElmer, Shelton, CT, USA). Simplicity 3QTM software (PerkinElmer) was used for all data collection, analysis, and quantification. All calibration curves generated for each analyte had acceptable R2 values greater than 0.98 using a weighted linear regression model (1/x). Between T1 and T2, eight samples demonstrated a 15-873% increase in cocaine concentration and four samples had a 13-45% decrease in cocaine concentration. For benzoylecgonine, nine samples demonstrated an 18-289% increase in concentration between T1 and T2 and six samples had a 3-57% decrease in concentration. In samples collected at one month, concentration values for cocaine were highest in samples stored at freezer temperature (-20°C) and lowest in samples stored at refrigerator temperature (4°C). The highest benzoylecgonine values were found in samples stored at freezer temperature (-20°C) as well, and the lowest concentrations were in samples stored at room temperature (20-22°C). Due to the variability in analyte concentration in the organs of the intracardially perfused specimens, the impact storage conditions had on analyte stability could not be determined.

Reports of declining insect populations suggest that more research focusing on this phenomenon is needed, especially in pollinator insects. Climate change, habitat destruction, and usage of certain pesticides have all been implicated in insect decline. Neonicotinoid pesticides are highly toxic to bees, can have drastic sub-lethal effects on behavior, and are persistent in the environment; likewise, they have been implicated as a major factor affecting bee populations. However, there are limited studies on native bees regarding their interactions with neonicotinoids, even regarding simple questions such as exposure levels. In this study, we aimed to assess concentrations of common neonicotinoids in native bees and soils from a threatened habitat in our region, the Black Belt Prairie, by using a modified QuEChERS LC/MS-MS protocol. Our results showed that specific taxa of native bees- Bombus spp., Xylocopa spp., and Mellissodes spp. (Family: Apidae)- were exposed to neonicotinoids. In contrast, no concentration of neonicotinoids was detected in our soil samples.