京都大学 / 新制・論文博士 / 博士(薬科学) / 乙第13485号 / 論薬科博第5号 / 新制||薬科||17(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 石濱 泰, 教授 松﨑 勝巳, 教授 土居 雅夫 / 学位規則第4条第2項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

<p>The comprehensive analysis of human blood plasma metabolome has been completed using derivatization gas chromatography-mass spectrometry (GC-MS) analysis, liquid chromatography-mass spectrometry (LC-MS) analysis and a comprehensive LC-GC-MS analysis approach wherein LC fractions were collected, derivatized and analyzed using GC-MS. In all cases blood plasma samples were deproteinized using solvent precipitation prior to chromatography and MS analysis.</p> <p>In GC-MS analyses, the progress of all derivatization reactions was monitored by adding 9-anthracenemethanol and 1,3-diphenylacetone to all reaction mixtures; their conversions to 9-anthracenemethanol trimethylsilyl ether and the oxime derivative of 1,3-diphenylacetone were used as measures of the completion of these derivatization reactions. Any reactions with completions less than 99% were repeated.</p> <p>GC-MS analysis of blood plasma samples detected 100 peaks; 44 were positively identified by comparing retention indices and mass spectra with those of authentic standards. LC-MS analyses were conducted on a HILIC column (aminopropyl phase) with MS detection in both negative ion and positive ion modes and resulted in the identification of 97 peaks; 47 were observed in the positive ion mode, 58 in the negative ion mode with 8 peaks observed in both modes.</p> <p>The multi-dimensional LC-GC approach was not designed as a routine analytical method; rather the purpose of this approach was to see how many compounds could be observed in the sample and to obtain better quality mass spectra and retention index values. The LC separation afforded 16 fractions which upon derivatization GC-MS analysis gave an additional 176 peaks from a total of 276 peaks. The MS data from these additional spectra can be used to develop selected ion monitoring GC-MS or tandem mass spectrometry analytical methods. This thesis has demonstrated the power of off-line comprehensive methods to identify compounds that neither the GC nor the LC methods detected.</p> / Master of Science (MSc)

An optimized method for the chiral resolution of enantiomers of amino acids in bacterial supernatants is reported. This LC-MS/MS method is performed using a chiral Teichoplanin LC column and does not require sample clean up or chemical derivitization. This method allows for the determination of the relative amounts of the D and L enantiomers of 20 proteinogenic amino acids. The detection limits and response factors for the 20 amino acids were determined. Calibrations over three orders of magnitude showed least squares coefficient values (R^2) greater than 0.996 for eighty percent of the amino acids and greater than 0.992 for the remainder. The amino acids and their enantiomers were identified based on their retention times and their unique Multiple Reaction Monitoring (MRM) transitions for each amino acid. L-Aspartic acid-2,3,3-d3 was used as the internal standard. Cultures of Sinorhizobium meliloti (a nitrogen-fixing soil bacterium) were grown on minimal media; thus, all amino acids were biosynthesized by the bacterium. After centrifugation, supernatants were freeze dried, reconstituted in a small volume of methanol/water with internal standard and injected onto the LC column. The amino acids detected in the bacterial supernatant and the concentrations of the enantiomers were reported as the L and D isomers respectively: arginine [L, 12.6 ± 3.1 μg/L; D, 10.1 ± 3.2 μg/L], serine [L, 7.2 ± 1.16 μg/L; D, n.d.], threonine [L, n.d.; D, 11.2 ± 2.7 μg/L] and valine [L, 15.5 ± 4.3 μg/L; D, 11.3 ± 3.7 μg/L], where the term n.d. means below detection limit. The limits for detection for all amino acids ranged from 1.3 μg/L - 5.1 μg/L. In media with no added phosphate, the amino acid profiles changed somewhat under these stress conditions. Arginine was no longer detected while alanine and proline were now observed; the concentrations of the amino acids were: alanine [L, 7.7 ± 1.2 μg/L; D, 13.4 ± 2.5 μg/L], proline [L, n.d.; D, 8.63 ± 1.3 μg/L], serine [L, 7.6 ± 1.2 μg/L; D, n.d.], threonine [L, n.d.; D, 10.2 ± 3.2 μg/L] and valine [L, 11.6 ± 2.3 μg/L; D, 10.1 ± 3.1 μg/L]. These data represent the mean values of three independent bacterial growth experiments conducted over a 3 month period; the data came from the analysis of five separate aliquots from each growth experiment. The percent standard deviation for these data ranged from 15% to 33% and averaged 24%. Under both the normal and stressed growth conditions of S. meliloti produced the L enantiomer of serine, the D enantiomer of threonine and racemic valine. While racemic arginine was observed under normal growth conditions, levels were below detection under stressed conditions; under stress conditions only the D enantiomer of proline was observed while alanine was found in 1:2, L:D ratio. / Thesis / Master of Science (MSc)

No description available.

Folate, also known as vitamin B9, is an essential dietary vitamin that provides the donor group for one carbon transfer reactions. Deficiency in folate is associated with neural tube birth defects (NTDs), cancer, cardiovascular disease, and anemia. In the US enriched food products including bread, pasta, and cereal are fortified with folic acid, the synthetic analog of folate. While effective in reducing NTDs, this practice is costly and not economically practical in developing countries. Folate biofortification, increasing the natural folate level in foods by metabolic engineering, has been proposed as a sustainable alternative to food fortification with folic acid. To increase folate levels in peanut seed, GTP cyclohydrolase I from Arabidopsis thaliana (AtGCHI) was introduced into peanut by biolistic transformation. Plant transformation vectors were constructed using publicly available or licensable vector components to avoid intellectual property restrictions that hinder commercialization. Thirteen peanut cultivars were evaluated for transformation efficiencies and regeneration potential. Expression levels of the AtGCHI transgene were determined by quantitative real-time PCR. The endogenous peanut GCHI (AhGCHI) was isolated and sequenced. Studies were conducted to test whether heterologous over-expression of AtGCHI altered expression of the endogenous AhGCHI. Seed-specific expression of AtGCHI does not affect AhGCHI transcript accumulation. For validation of the proposed folate biofortification strategy, vitamin quantification will be required. A liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed to identify and quantify the different forms of folate. However, additional work will be needed to determine sensitivity of the instrument, to optimize vitamin extraction, and to increase sufficient seed for vitamin extraction and analysis. Peanut products derived from folate biofortified peanut kernels will have a niche market in the United States, but there is a larger global implication as a mechanism for sustainable delivery of essential vitamins to populations that can not adopt synthetic vitamin supplementation/fortification. Successful demonstration of increased folate in peanut will result in better vitamin availability for populationssonsuming peanut based foods as a dietary staple. / Ph. D.

Liquid chromatography coupled with mass spectrometry (LC-MS) has been widely used in various omic studies for biomarker discovery. Appropriate LC-MS data preprocessing steps are needed to detect true differences between biological groups. Retention time alignment is one of the most important yet challenging preprocessing steps, in order to ensure that ion intensity measurements among multiple LC-MS runs are comparable. In this dissertation, we propose a Bayesian alignment model (BAM) for analysis of LC-MS data. BAM uses Markov chain Monte Carlo (MCMC) methods to draw inference on the model parameters and provides estimates of the retention time variability along with uncertainty measures, enabling a natural framework to integrate information of various sources. From methodology development to practical application, we investigate the alignment problem through three research topics: 1) development of single-profile Bayesian alignment model, 2) development of multi-profile Bayesian alignment model, and 3) application to biomarker discovery research. Chapter 2 introduces the profile-based Bayesian alignment using a single chromatogram, e.g., base peak chromatogram from each LC-MS run. The single-profile alignment model improves on existing MCMC-based alignment methods through 1) the implementation of an efficient MCMC sampler using a block Metropolis-Hastings algorithm, and 2) an adaptive mechanism for knot specification using stochastic search variable selection (SSVS). Chapter 3 extends the model to integrate complementary information that better captures the variability in chromatographic separation. We use Gaussian process regression on the internal standards to derive a prior distribution for the mapping functions. In addition, a clustering approach is proposed to identify multiple representative chromatograms for each LC-MS run. With the Gaussian process prior, these chromatograms are simultaneously considered in the profile-based alignment, which greatly improves the model estimation and facilitates the subsequent peak matching process. Chapter 4 demonstrates the applicability of the proposed Bayesian alignment model to biomarker discovery research. We integrate the proposed Bayesian alignment model into a rigorous preprocessing pipeline for LC-MS data analysis. Through the developed analysis pipeline, candidate biomarkers for hepatocellular carcinoma (HCC) are identified and confirmed on a complementary platform. / Ph. D.

No / Piperacillin, a potent ß-lactam antibiotic, is effective in a large variety of Gram+ and Gram¿ bacterial infections but its administration is limited to the parenteral route as it is not absorbed when given orally. In an attempt to overcome this problem, we have synthesized a novel series of charged liposaccharide complexes of piperacillin comprising a sugar moiety derived from d-glucose conjugated to a lipoamino acid residue with varying side-chain length (cationic entity) and the piperacillin anion. A complete multiple reaction monitoring LC¿MS/MS method was developed to detect and characterize the synthesized complexes. The same method was then successfully applied to assess the in vitro apparent permeability values of the charged liposaccharide complexes in Caco-2 monolayers. / BBSRC

Per- and polyfluoroalkyl substances (PFAS) are organic synthetic compounds used in several industries because of their unique properties and thermal and chemical stability. Perfluorooctanesulfonic acid (PFOS) and perfluorooctaonic acid (PFOA) are two of the most prominent PFAS that are undegradable and accumulate in nature. To study the impact of PFOS and PFOA on the liver in a controlled environment, organoids can be used. A sensitive and selective LC-MS/MS method for individual and simultaneous analysis of PFOS and PFOA in liver organoid media and equipment used in organoid analyses was developed. For detection of low concentrations, ability to analyse complex organoid samples, and limit background contamination, a solid phase extraction (SPE) column, automatic filter (AFFL) and a trap column was included. The AFFL-SPE-LC-MS/MS was optimised efficiently through Design of Experiment (DoE) regarding the loading phase in the LC and six MS parameters for PFOS and PFOA. Validation was controlled against Eurachem’s guideline showing high sensitivity, detecting LOD at 6 pg/mL. The method demonstrated high repeatability with an RSD below 8 % for most samples. Simultaneous analysis of PFOS and PFOA demonstrated high selectivity. Nevertheless, the method showed low intermediate precision and varying reliability, as well as persistent background contamination limiting detection of lower concentrations. The method was fit for purpose and allowed rapid analysis of PFOS and PFOA in organoid media and equipment used in organoid analyses. Result from studies of PFAS in liver organoids through analysis with this method can aid in understanding the connection between PFAS and metabolic diseases. / Populärvetenskaplig sammanfattning Per- och polyfluorerade alkylsubstanser (PFAS) är en grupp av människoskapta, syntetiska ämnen med unika egenskaper. Dessa egenskaper gör att de är olja- och vattenavvisande, och har många applikationsområden. De finns i textiler, livsmedelsförpackningar, brandsläckningsskum och andra industriprodukter. De är väldigt termiskt och kemisk stabila, vilket gör att de inte bryts ner och därmed ackumulerar i miljön. Flera studier har också visat koppling mellan PFAS och många kroniska sjukdomar, som hormonstörningar, cancer, immunsuppression och metabolt associerad fettlever (MAFLD, tidigare nonalkoholisk fettlever (NAFLD)). Kopplingen mellan MAFLD och PFAS har fått mycket uppmärksamhet då levern har visats sig vara ett målorgan för PFAS. Eftersom PFAS är ihärdiga, har ett komplicerat spridningsbeteende och ackumulerar i naturen är det svårt att studera kopplingen mellan MAFLD och PFAS i en kontrollerad miljö. För att studera effekten av PFAS kan man använda organoider, laboratorieodlade 3D modeller gjord från stamceller för att imitera ett äkta organ.    Någon av de mest omtalade PFAS ämnen är perfluoroktansyra (PFOA) och perfluoroktansulfonat (PFOS), vilket är fokus för detta arbete. Leverorganoiderna kan utsättas för PFOS och PFOA, och mediet de ligger i kan extraheras och studeras med konventionella analytiska metoder för att få en bild av hur PFAS påverkar levern. I detta arbete vill analysen ske via vätskekromatografi med masspektrometri som detektion (LC-MS/MS). Med LC-MS/MS separeras den studerade molekylen, analyten från lösningen baserat på dess kemiska egenskaper. Analyten detekteras baserat på dess massa, mer bestämd massa/laddning-fördelningen (m/z). För att anpassa LC-MS metoden till injektion av komplexa organoidprover inkluderades ett automatiskt filter (AFFL) samt ett extra automatiskt separationssteg med en kolonn med fastfasextraktion (SPE). I övrigt ger SPE möjligheten att små mängder PFAS kan uppkoncentreras och fokuseras på kolonnen, vilket ger en sensitiv metod som kan detektera låga koncentrationer. SPE och AFFL implementerades båda för att bättre kunna separera och detektera PFOS och PFOA från andra ämnen, samt filtrera bort föroreningar och stora molekyler som kan skada LC-MS/MS instrumentet i längden. Då PFAS hopar upp sig i vår omgivning, visade det sig att kontamination av PFAS från systemet blev en utmaning under metodutvecklingen. Därför implementerades PFAS fritt utstyr, samt en extra kolonn för att fånga PFAS från systemet och på så sätt minska bakgrundskontaminationen som detekterades.    AFFL-SPE-LC-MS/MS metoden optimerades via en maskininlärningsbaserad optimeringsmetod baserad på parametrar i LC och MS. Metoden baserar sig på att, med tre värden för varje parameter, uppger programmet ett antal experiment som måste utföras för att kunna beräkna ett optimalt värde för varje parameter. Med resultatet från experimenten kan modellen matematiskt, genom en Bayes baserat Gaussian modell, uppskatta optimala värden för metoden. På så sätt kunde metoden optimeras systematiskt och tidseffektivt.    Innan rutinanvändning måste den optimerade metoden valideras. Validering blev gjord genom at följa Eurachem’s riktlinjer. Metoden visade hög repeterbarhet, selektivitet och riktighet. Den har hög sensitivitet, och kan detektera låga mängder, men bakgrundskontaminationen kunde inte elimineras totalt, och gör att man måste korrigera för detta i rutinanalyser. Komplexiteten av AFFL-SPE-LC-MS/MS med flera kolonner och filter gjorde att metoden visade låg robusthet och behövde justeras ofta. AFFL-SPE-LC-MS/MS metoden gör det möjligt att snabbt studera PFOS och PFOA i leverorganoider och utstyr använt i organoidanalyser, och kan bidra i forskningen för att bättre förstå hur PFAS påverkar levern. / Health Effects of Persistent Organic Pollutants

Die Rückstandsanalytik tierischer Lebensmittel ist eine anspruchsvolle Aufgabe aufgrund des hohen Lipidanteils der Proben sowie des sich stetig vergrößernden Wirkstoffspektrums. Heutzutage werden für die Probenaufarbeitung die DFG S 19 Methode, mit der vorrangig unpolare Analyten nachgewiesen werden und zunehmend die QuEChERS Methode eingesetzt, die insbesondere auf die Erfassung polarer Pestizide abzielt. In dieser Arbeit wurde eine moderne Multirückstandsmethode für Hühnereier entwickelt, um sowohl gas- als auch flüssigkeitschromatographisch (GC, LC) erfassbare Wirkstoffe zu analysieren. Dazu gehören unpolare PCBs, Pyrethroide und Organochlorpestizide, aber auch polarere Organophosphate, Triazole und Carbamate. Das Verfahren basiert auf der Extraktion mittels Matrix Solid Phase Dispersion, der Reinigung auf Grundlage einer modifizierten Gelpermeationschromatographie (GPC) und zwei verschiedenen Festphasenextraktionen (SPEs) für GC- und LC-erfassbare Pestizide sowie der Quantifizierung mittels GC- und LC-MS/MS. Dünnschichtchromatographisch wurde die effektive Entfernung hochmolekularer Lipide durch die modifizierte GPC und niedrigmolekularer Fette durch die SPEs belegt. Laut der für Ei durchgeführten Validierung erfüllten 164 der 172 untersuchten Pestizide und alle sechs PCBs die Leistungskriterien für die amtliche Rückstandskontrolle - zumeist am niedrigsten validierten Level (5 µg/kg bzw. 0,5 µg/kg). Ausnahmen bildeten sehr polare LC-Pestizide (z.B. Aminopyralid, Clopyralid, MCPA, Quinmerac) und pH-Wert-abhängige GC-Analyten (Nicotin, Tolylfluanid, Dichlofluanid), die auch mit den etablierten Verfahren schwierig zu analysieren sind. Weiterhin verdeutlichte die erfolgreiche Untersuchung von verschiedenen Ringversuchsmaterialien, dass die ursprünglich für Eier entwickelte Methode auch für mageres Geflügelfleisch und Sahne genutzt werden kann. Gegenüber den etablierten Verfahren wies die neue Methode deutliche Vorzüge auf. So belegte die Dünnschichtchromatographie, dass mit der neuen Methode Cholesterin, aber auch freie Fettsäuren besser abgetrennt werden als mit den etablierten Verfahren. Die neue Methode verbrauchte im Vergleich zur DFG S 19 Methode 46 % weniger Lösungsmittel und ermöglichte eine Verdopplung des Probendurchsatzes innerhalb von 8 h. Zudem eignete sich das entwickelte Verfahren laut den Validierungsdaten für GC-Analyten deutlich besser als die QuEChERS Methode und etwas besser als die DFG S 19 Methode (v.a. für Pyrethroide). Hinsichtlich der LC-Analyten unterschieden sich die neue und die QuEChERS Methode nur bei wenigen Analyten. Mit dem neuen Verfahren konnten folglich im Gegensatz zu den etablierten Methoden sowohl unpolare GC- als auch polare LC-Analyten sicher erfasst werden.

O preparo da amostra é uma das etapas mais importantes em toda a análise química. O isolamento e a concentração dos componentes da amostra são cruciais e busca-se sempre que essas etapas sejam as mais simples e consumam o mínimo possível de tempo e reagentes. Nos últimos anos, um tipo de material tem se mostrado bastante útil para análises químicas a partir de fluidos biológicos, os polímeros molecularmente impressos (MIPs). Os MIPs são sintetizados por reações de polimerização, na presença de uma molécula molde (template). A molécula molde se liga aos monômeros funcionais do polímero durante a reação de polimerização e permanece ligada à superfície das cadeias poliméricas quando a reação se completa. Terminada a polimerização, realiza-se a completa lavagem das moléculas molde, assim, restam na superfície polimérica cavidades tridimensionais complementares à molécula empregada como molde. Essas cavidades permitem a ligação reversível e preferencial da molécula molde ou outras com estrutura química semelhante. A Cannabis sativa é a droga ilícita mais consumida em todo o mundo e nos últimos anos muita atenção tem se voltado a seus efeitos toxicológicos no corpo humano e a aplicações medicinais. Nesta dissertação, foi sintetizado um MIP com a molécula molde catequina para a extração e posterior análise por LC-MS/MS dos canabinóides &Delta;9-tetrahidrocanabinol (THC), 11-hidroxi-&Delta;9-tetrahidrocannabinol (THC-OH) e 11-nor-&Delta;9-tetrahidrocannabinol-9-ácido carboxílico (THC-COOH) em amostras de urina. O MIP produzido foi empacotado em microdispositivo e empregado no preparo das amostras de urina por microextração por sorvente empacotado (MEPS). O método desenvolvido apresentou boa linearidade (valores de r de 0,977 para o THC e 0,994 para THC-OH e THC-COOH). Os limites de detecção e de quantificação foram respectivamente de 5 ng mL-1 e 20 ng mL-1, para os compostos THC e THC-OH, na faixa linear de 25 a 250 ng mL-1. Para o composto THC-COOH os limites de detecção e quantificação alcançados foram de 1 ng mL-1 e 5 ng mL-1, respectivamente, na faixa linear de 5 a 170 ng mL-1. O método apresentou valores razoáveis de precisão entre 3,2% (THC-COOH) e 25,1% (THC) e de exatidão, que variou entre -18,4 e 17,4 (ambos para o THC). O MIP empregado no preparo da amostra mostrou-se mais seletivo e específico do que materiais normalmente empregados para a extração dos canabinoides das amostras de urina, além de a técnica de extração por MEPS apresentar baixo consumo de solventes e amostra para a extração dos analitos e posterior análise por LC-MS/MS. / The sample preparation is one of the most important steps in every chemical analysis. The isolation and concentration of the sample components are crucial and it is always sought that these steps are simple and consume the lowest amount of time and reagents. In the recent years, a type of material has proved to be very useful for chemical analyzes of biological fluids, the molecularly imprinted polymers (MIPs). MIPs are synthesized by polymerization reactions in the presence of a template molecule. The template molecule binds to the functional monomers of the polymer during the polymerization reaction and remains bonded to the surface of the polymeric chains after the reaction is complete. After the polymerization is finished, the complete washing of the template molecules is carried out, thus, three-dimensional cavities, complementary to the molecule used as a template, remain on the polymer surface. These cavities allow the reversible and preferential bonding of the template molecule or others with similar chemical structure. Cannabis sativa is the most commonly consumed illicit drug in the world and in recent years much attention has focused on its toxicological effects on human body and for medical applications. In this master dissertation, a MIP was synthesized with the catechin molecule as template, for extraction and subsequent analysis by LC-MS/MS of the cannabinoids &Delta;9-tetrahydrocannabinol (THC), 11-hydroxy-&Delta;9-tetrahydrocannabinol (THC-OH), and 11-nor-&Delta;9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine samples. The MIP produced was packed in a microdevice and used in the preparation of the urine samples by microextraction by packed sorbent (MEPS). The developed method showed good linearity (r values of 0.977 for THC and 0.994 for THC-OH and THC-COOH). The detection and quantification limits were respectively 5 ng mL-1 and 20 ng mL-1 for THC and THC-OH in the linear range from 25 to 250 ng mL-1. For the compound THC-COOH the limits of detection and quantification achieved were 1 ng mL-1 and 5 ng mL-1, respectively, in the linear range from 5 to 170 ng mL-1. The method presented reasonable values of precision, between 3.2% (for THC-COOH) and 25.1% (for THC) and displayed accuracy ranging from -18.4 to 17.4 (both for THC). The MIP used in the sample preparation was more selective and specific than other materials usually employed for the extraction of the cannabinoids from the urine samples. The MEPS technique also showed low consumption of solvents and sample for sample preparation, extraction of analytes and subsequent analysis by LC-MS/MS.