FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / AtravÃs da cromatografia lÃquida de alta eficiÃncia com detecÃÃo por espectrometria de massa no modo MS/MS, desenvolveu-se um mÃtodo sensÃvel e altamente seletivo para determinar os nÃveis de acetato de megestrol no plasma humano, usando a betametasona como padrÃo interno. O analito e o padrÃo interno foram extraÃdos das amostras do plasma atravÃs da soluÃÃo de extraÃÃo hexano-acetato de etila (1:1 v/v), e cromatografados em uma coluna C18. A fase mÃvel consistiu de acetonitrila-Ãgua (80:20 v/v), contendo 0,1% de Ãcido fÃrmico. A detecÃÃo foi realizada em um triplo-quadrupolo, atravÃs de um espectrÃmetro de massa no modo de monitoramento de reaÃÃo mÃltipla via eletrospray. O mÃtodo tem um tempo de corrida de 3 minutos e limite de quantificaÃÃo de 2 ng/mL. As curvas de calibraÃÃo foram obtidas utilizando uma faixa de concentraÃÃo de 2 a 150 ng/mL. As precisÃes intralote foram 3,16%, 4,65% e 2,68%, e a acurÃcia intralote foi de 6,77%, 6,23% e 5,73% para 6, 60 e 120 ng/mL, respectivamente. As precisÃes interlote foram 7,76%, 6,23% e 6,37%, e a acurÃcia interlote foi de 0,08%, 1,55% e 2,11% para as mesmas concentraÃÃes. Este mÃtodo validado foi aplicado com sucesso para a determinaÃÃo dos parÃmetros farmacocinÃticos dos comprimidos de acetato de megestrol administrados a 30 voluntÃrios sadios. / A sensitive and highly selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed to determine megestrol acetate in human plasma using betamethazone as the internal standard. The analyte and internal standard were extracted from plasma samples by hexane/ethyl acetate (1:1 v/v), and chromatographed on a C18 column. The mobile phase consisted of acetonitrila-water (80:20 v/v) including formic acid 0.1%. Detection was performed on a triple quadropole tandem mass spectrometer by multiple reaction mode via electrospray source. The method has a chromatographic run time of 3 min and a limit of quantification of 2 ng/mL. The linear calibration curves were obtained in the concentration range 2 â 150 ng/mL. The intra-batch precisions were 3.16%, 4.65%, and 2.68%; and the intra-batch accuracy was 6.77%, 6.23%, and 5.73% for 6, 60 and 120 ng/mL, respectively. The inter-batch precision was 7.76%, 6.23%, and 6.37% and the inter-batch accuracy was 0.08%, 1.55%, and 2.11% for the same concentrations. This validated method was successfully applied for the determination of pharmacokinetic profiles of megestrol acetate tablets administered to 30 healthy volunteers.

O quadro evolutivo da agricultura brasileira resulta em benefícios à população exigindo crescentes avanços tecnológicos no setor. Constantemente, novos agrotóxicos são introduzidos estimulando estudos científicos com a finalidade de determinar e avaliar os impactos na população e no meio ambiente. No presente trabalho, a matriz avaliada foi o lodo gerado no processo de tratamento de água para consumo humano, coletado na região do Vale do Ribeira, SP. A técnica empregada foi a cromatografia líquida de fase reversa acoplada à espectrometria de massas triploquadrupolar em tandem com ionização por electrospray. Os compostos foram extraídos previamente da matriz. O desenvolvimento da metodologia exigiu tratamento dos dados para que esses pudessem ser utilizados e transformados em informações confiáveis. Os processos envolvidos foram avaliados usando o conceito da validação de ensaios químicos. Os indicadores avaliados foram seletividade, linearidade, intervalo de trabalho, sensibilidade, exatidão, precisão, limite de detecção, limite de quantificação e robustez. Esses indicadores produziram valores quantitativos e qualitativos que foram estatisticamente evidenciados de forma objetiva. A metodologia desenvolvida e validade é simples. Como resultado, mesmo explorando a sensibilidade da técnica, os compostos estudados não foram encontrados no lodo da ETA de Registro. Isso leva a crer que esses compostos podem estar presentes em concentrações muito baixas, podem sofrer degradação durante o tratamento da água ou não são retidos completamente pela ETA. 7 / The evolving scenario of Brazilian agriculture brings benefits to the population and demands technological advances to this field. Constantly, new pesticides are introduced encouraging scientific studies with the aim of determine and evaluate impacts on the population and on environment. In this work, the evaluated sample was the sludge resulted from water treatment plant located in the Vale do Ribeira, São Paulo, Brazil. The technique used was the reversed phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Compounds were previously liquid extracted from the matrix. The development of the methodology demanded data processing in order to be transformed into reliable information. The processes involved concepts of validation of chemical analysis. The evaluated parameters were selectivity, linearity, range, sensitivity, accuracy, precision, limit of detection, limit of quantification and robustness. The obtained qualitative and quantitative results were statistically treated and presented. The developed and validated methodology is simple. As results, even exploring the sensitivity of the analytical technique, the work compounds were not detected in the sludge of the WTP. One can explain that these compounds can be present in a very low concentration, can be degraded under the conditions of the water treatment process or are not completely retained by the WTP.

Apesar da rápida expansão das aplicações da cromatografia líquida acoplada à espectrometria de massas em química clínica, a análise de metabólitos de baixo peso molecular e alta polaridade em matrizes biológicas ainda permanece como um grande desafio analítico. Dentre os compostos de grande importância no diagnóstico de doenças metabólicas que ainda carecem de melhores alternativas bioanalíticas destacam-se os aminoácidos. O presente estudo descreve o desenvolvimento e a validação de um novo método para a quantificação de 24 aminoácidos em plasma explorando a cromatografia líquida acoplada a espectrômetros de massas em tandem. Foi construído um método de detecção baseado em SRM (múltiplas reações selecionadas) com duas transições de massas para cada um dos 24 aminoácidos e os 19 padrões internos marcados com isótopos estáveis. Foram avaliadas três estratégias de separação cromatográfica e o melhor desempenho foi obtido com fase reversa com octadecilsilano (C18) com pareamento iônico com o ácido perfluoropentanoico. O método cromatográfico final permitiu a separação dos 24 aminoácidos, com resolução completa dos isômeros: leucina, isoleucina e allo-isoleucina, em 11 minutos incluindo o tempo de re-estabilização da coluna cromatográfica. Os limites de quantificação variaram em 113 fmol a 6 pmol injetados na coluna cromatográfica. A imprecisão obtida nos níveis testados para todos os aminoácidos foi inferior a 14%. O método apresentou linearidade para os intervalos testados chegando a 1,5 mmol.L-1 para vários compostos. Os ensaios de arraste mostraram que os limites máximos obtidos na linearidade não geram nenhuma interferência subsequente. A exatidão do método foi avaliada com amostras provenientes do programa de referência interlaboratorial European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism (ERNDIM) e com o material de referência certificado do National Institute of Standards and Technology (NIST). Todos os analitos mostraram equivalência estatística com o método desenvolvido. / Despite the widespread use of liquid chromatography coupled to mass spectrometry applications in clinical chemistry, the analysis of low molecular weight and high polar metabolites in biological matrices remains as a major analytical challenge. Notwithstanding the key role played by amino acids in the diagnosis of metabolic diseases, there is still need for improvements in bioanalytical process of these analytes. The present study describes the development and validation of a new method for quantification of 24 amino acids in plasma based on liquid chromatography coupled to tandem mass spectrometry. Detection and quantification were achieved building a selected reaction monitoring method two mass transitions for each 24 amino acids and 19 stable isotope internal standards. Three chromatographic strategies for separation were evaluated, and best performance was achieved using reversed-phase octadecylsilane with perfluropentanoic acid as ion pairing agent. The separation method allowed separation of 24 amino acids with full resolution for isomers leucine, isoleucine and alloisoleucine in 11 minutes, including column equilibration time. The limits of quantification ranged from 113 fmol to 6 pmol (on column injection). Imprecisions for all evaluated levels and amino acids were less than 14%. The method is linear in all clinical intervals and extending up to 1.5 mmol.L-1. Carryover evaluation demonstrated absence of interference in the following injection throughout the analytical interval. Method accuracy was evaluated analyzing reference samples from European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism (ERNDIM) and National Institute of Standards and Technology (NIST). Statistical equivalence was demonstrated for all analytes using the present method.

Patients that have undergone organ transplantation are life-long treated with immunosuppressant drugs and these have to be monitored regularly to get the desired effect of suppressing the immune system. To monitor the drug concentration normally a venous blood sample is collected at a clinic but the use of dried blood spots (DBS) as a matrix for drug monitoring for immunosuppressant drugs will make home sampling possible for this patient group. The aim of this study was to develop and validate a bioanalytical method for quantifying cyclosporine A and tacrolimus in dried blood spots. The method consist of punching out a 5 mm disc from a blood spot , followed by extracting the spot in a 96-well hydrophobic filter plate with 150 µL extraction solution containing internal standard (ascomycin and cyclosporine A d12) in a methanol water solution (80:20v/v%). The extract is then centrifuged through the filter plate down in a 96-deep well plate and injected on the LC-MS/MS, with an analysis time of 2.5min. The method will be validated in accordance with the guidelines set by the European Medicines Agency with additions specific to DBS. The method is not fully validated but will be in due time. The validated parameters show a robust and fast analysing method that has the prospects of being used for analysing DBS samples for patients and in the future can possibly be used by patients in home environment.

Synthetic piperazines are chemically-produced compounds that contain a six-member ring with two opposing nitrogen atoms. Several piperazine derivatives, namely 1- benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)-piperazine (TFMPP), and 1-(3- chlorophenyl)-piperazine (mCPP), have fallen into the “designer drugs” category due to their increasing recreational use as a “legal” alternative to ecstasy (3,4-methylenedioxymethamphetamine). These compounds share similar stimulant and physiological effects with amphetamines which make them desirable to young adults in party-type atmospheres. BZP, a Schedule I drug for its high abuse potential and no accepted medical use, is the only recreationally-abused synthetic piperazine currently federally controlled in the United States. The purpose of this research was to develop and validate a reliable method to identify and quantify eight forensically significant synthetic piperazines in blood and urine using ultra-fast liquid chromatography-electrospray ionization-tandem mass spectrometry (UFLC-ESI-MS/MS). The method was validated according to the Scientific Working Group for Forensic Toxicologists (SWGTOX) guidelines for quantitative analysis for both matrices and includes the following analytes: 1-benzylpiperazine (BZP), 1-(4-fluorobenzyl)-piperazine (FBZP), 4-methyl-1-benzylpiperazine (MBZP), 1-(4-methoxyphenyl)-piperazine (MeOPP), 1-(para-fluorophenyl)-piperazine (pFPP), 1-(3-chlorophenyl)-piperazine (mCPP), 2,3-dichlorophenylpiperazine (DCPP), and 1-(3-trifluoromethylphenyl)-piperazine (TFMPP). All samples were prepared by fortifying 100 µL of certified drug-free whole blood and urine (UTAK Laboratories, Inc., Valencia, CA, U.S.A.) with certified reference standards (Cayman Chemical, Ann Arbor, MI, U.S.A.) of each analyte at desired concentrations and standard additions of 1-benzylpiperazine-d7, 1-(3-chlorophenyl)-piperazine-d8, and 1(-3-trifluoromethylphenyl)-piperazine-d4 internal standards (Cerilliant, Round Rock, TX, U.S.A). After pretreatment with 1 mL phosphate buffer, samples underwent solid phase extraction (SPE) on mixed-mode copolymeric columns (Clean Screen®, UCT Inc., Levittown, PA, U.S.A.). Eluents were evaporated to dryness with low heat (65°C) and nitrogen gas. Samples were reconstituted with a 50:50 mixture of methanol and 2mM ammonium formate buffer with 0.2% formic acid before being analyzed by a UFLC (Shimadzu Corporation, Kyoto, Japan) and 4000 QTRAP ESIMS/MS (SCIEX, Framingham, MA, U.S.A.) system. Analyses were performed with multiple reaction monitoring scans in positive ionization mode using ions and voltages obtained from a manual compound optimization. Analytes were separated on a reversed phase column (Kinetex® F5, Phenomenex®, Torrance, CA, U.S.A.) with a binary gradient consisting of a 2mM ammonium formate buffer with 0.2% formic acid and methanol with 0.1% formic acid. The flow rate was 0.400 mL/min. Analyst™ (SCIEX) software was used for data collection and MultiQuant™ (SCIEX) software was used for quantitation. The total run time was 11.5 minutes with equilibrations. All calibration curves in both matrices exhibited R2 values > 0.99 using a weighting factor of 1/x. A linear dynamic range of 20-2000 ng/mL was used for all analytes in both matrices, except for BZP in urine which ranged from 50-2000 ng/mL. In blood, the limit of quantitation was 10 ng/mL for mCPP and TFMPP and 20 ng/mL for BZP, FBZP, MBZP, MeOPP, pFPP and DCPP. In urine, the limit of quantitation was 10 ng/mL for MeOPP, mCPP, TFMPP and DCPP, 20 ng/mL for FBZP, MBZP and pFPP and 50 ng/mL for BZP. When a 200 ng/mL concentration was evaluated, the SPE procedure showed percent recoveries ranging from 80-95% for blood; except for BZP, FBZP, and MeOPP which had recoveries of 60%, 60%, and 105%, respectively. Percent recoveries ranged from 82-94% for urine; except for BZP and FBZP which had recoveries of 66% and 68%, respectively. Bias and precision were assessed at concentrations of 50, 200, and 700 ng/mL. All samples were calculated within ±20% bias and within ±20% coefficient of variation. The highest concentration evaluated that did not produce carryover in subsequent matrix blanks was 5000 ng/mL. Ionization was suppressed for all analytes in both matrices by 45-95%. Matrix effects were present but were determined to be insignificant. Of the drugs evaluated, caffeine, dibenzylpiperazine, and 1-(4-chlorophenyl)-piperazine (pCPP) produced chromatographic peaks in the method; however, pCPP was the only substance that affected quantitation of an analyte. It increased the peak area of mCPP by almost 50% when present at the same concentration which suggests this method is unable to differentiate between isomeric pairs. This is a sensitive, reliable, and robust method with a wide linear dynamic range to account for the presence of these analytes in both blood and urine. This research will provide for the identification and quantitation of these substances in forensic casework.

L’utilisation massive des antibiotiques contribue à leur accumulation dans les boues des stations d’épurations. L’application directe des boues est parmi les sources de dissémination des antibiotiques et des gènes de résistance aux antibiotiques. Le compostage et la méthanisation sont parmi les bioprocédés de traitement des boues qui permettent d’éliminer ou réduire les teneurs de certains antibiotiques. Dans ce travail, une boue primaire de la STEP de Marrakech a été contaminée par trois familles d’antibiotiques (macrolides, tétracyclines, fluoroquinolones) pour conduire 4 essais de compostage à différentes doses (dont un essai témoin) et un essai deméthanisation en mode semi-continu. Les résultats du compostage ont montré que l’augmentation des concentrations d’antibiotiques retarde la dégradation de la matière organique et affecte le ratioC/N. De même, la phase thermophile est perturbée, retardée et réduite dans le temps. Pour la méthanisation, une concentration unique et réaliste a été testée. Dans ces conditions, aucun effet sur la production du biogaz ou sur la dégradation de la matière organique n’a été observé. Afin de suivre la dissipation des trois familles d’antibiotiques utilisées au cours du compostage et de la méthanisation, une approche analytique basée sur l’extraction accélérée par solvant (ASE) suivie par l’application d’une méthode des ajouts dosés avant quantification par chromatographie liquide couplée à de la spectrométrie de masse en tandem (UPLC-MS/MS) a du être mise en point. Le compostage et la méthanisation permettent de réduire significativement les concentrations des molécules parents appartenant à la famille des macrolides et des tétracyclines. Par contre,l’élimination des fluoroquinolones est non-significative et ne dépasse pas 30%. Au cours du compostage, la dissipation des macrolides se fait en phase de stabilisation tandis que la phase de maturation est impliquée dans la dissipation des tétracyclines. Les concentrations encirprofloxacine (fluoroquinolone) semblent légèrement évoluer au cours du procédé probablement en raison d’une adsorption/désorption sur le co-substrat lignocellulosique utilisé. Concernant la méthanisation, l’élimination des macrolides et des tétracyclines est significative durant la stabilisation du procédé mais n’atteinds pas les rendements observés lors du compostage. Ladiminution des concentrations des molécules parents est probablement accompagnée par une biotransformation des antibiotiques sous forme de métabolites qui à ce stade ne sont pas connus.La question de la rémanence de certaines molécules comme les fluoroquinolones, interpelle quand au risque d’antibiorésistance. Ainsi, la valorisation des composts/digestats comme amendements organiques des sols dois à terme conduire à une réflexion concernant la réglementation qui inclus la présence de molécule de la classe des antibiotiques.

In the field of forensic toxicology, several challenges exist with quantification analysis of cocaine and metabolites in post mortem samples. Cocaine can prove difficult to detect and quantify in blood, urine, and soft tissues following extensive decomposition. Alternative matrices, such as hair, nails, and bone could prove useful in detecting chronic drug use in post-mortem toxicology cases. Detection and quantification of drugs in complex matrices is difficult to accomplish due to time-consuming extraction processes, and inability to detect an analyte at trace levels. Further, analysis of drugs in hard tissues, such as hair and bone, has only been attempted in recent years. Even fewer studies have investigated detection of drugs following decomposition of remains, specifically outdoor decomposition. The objective of this study was to develop a robust extraction and clean up methodology, in which a homogenization step precedes, to efficiently extract drugs from complex matrices, reach a target limit of detection (LOD) and to maintain instrument performance using multidimensional chromatography. Multi-dimension chromatography platform such as two dimensional liquid chromatography tandem mass spectrometry ( 2D-LC/MS/MS,) offers options not compatible with single dimension v units. With large volume injection capabilities of aqueous and organic extracts, the analytical process be reduced from multiple hours to minutes. All rat specimens used for this study fell under an Institutional Animal Care and Use Committee (IACUC) protocol. The rodents underwent a 10-12 weeks chronic intravenous self-administration of cocaine. This was followed by a six-week period of abstinence, followed again by a three-week period of cocaine self-administration before being euthanized. Average daily dosages for each rat fell within a range of 13-19 mg/kg. A total of 14 cocaine positive rats were placed outside and above ground in the Boston University Forensic Anthropology Outdoor Research Facility (Holliston, MA, U.S.A) for a period of 12 months. All recoverable skeletal samples were collected for testing. Drug free control rat bones were also acquired by placing drug-free rats outdoors, above ground, until full decomposition occurred. In this study, a method analyzing cocaine and its major metabolites benzoylecgonine and ecgonine methyl ester was developed. After homogenization of whole bones, the extraction process was performed using a mixed mode reversed-phase/ion exchange sorbent. The use of a 2D LC/MS/MS technology eliminates the need for a lengthy evaporation step in the extraction method. The chosen 2D LC/MS/MS used in this application was identified using a 6x6 automated method development protocol. The manual extraction of the bone samples was completed in less than an hour. The analysis was performed using 100μL of the final organic solvent (MeOH) extracts. vi The limit of quantitation (LOQ) for cocaine and benzoylecgonine was measured at 0.05ng/g (0.05ng/mL or 50pg/g) of sample material and the LOQ for ecgonine methyl ester was measured at 0.1ng/g (0.1 ng/mL or 100pg/g). The extraction method for cocaine proved to give a linear dynamic range of 2.5 orders of magnitude (0.05 ng/g to 10ng/g with an R2 = 0.998. The micro extraction protocol combined with a multi-dimension chromatography used in this study decreased sample preparation time without sacrificing the quality seen with current single dimension chromatography techniques. The procedure developed in this study can be utilized on bone and completed in less than an hour before injection into the 2D-LC/MS/MS system.

Antibiotics are used as prophylactic agents to promote growth and for treating infections in animals. However, the irrational use of antibiotics in livestock management is a significant cause of the development of antibioticresistant genes in the environment. Each year 2 million people suffer from the infections caused by bacteria which are resistant to antibiotics and 23,000 of these people are estimated to die because of antibiotic resistance. New drugs are continually coming into the market but are at the risk of developing resistance. Thus, there is a need for the development of analytical methods which can be used to monitor these antibiotic concentrations in environmental samples. This research is focused on developing and validating a Solid Phase Extraction (SPE) procedure and liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying tylosin antibiotic in cattle waste. Tylosin was extracted from cattle waste samples using Strata polymeric weak cation cartridges by adding a sodium-EDTA buffer solution and methanol. Chemical analysis of the extracted tylosin was performed using a Varian 212-LC HPLC and Agilent 500 Ion Trap mass spectrometric detector. The concentrations of tylosin in study group animals were compared with respect to the date of sampling and cattle body weight with a control group and results are presented.

The purpose of the studies included in this thesis is to develop an effective an efficient method to study the proteome using separation and detection of peptides, when only a limited amount of sample, 10 micrograms of total protein or less, is available. The analysis will be applied to multiple myeloma cancer cells using ultra high-performance liquid chromatography-mass spectrometry for expression proteomics to illustrate utility. To detect low abundance peptides in a complex proteome, we use different strategies, including basic pH reversed-phase liquid chromatography (bRPLC), mass-to-charge fractionation in the mass spectrometer, and various liquid chromatography gradients to increase peptide separation to improve opportunities for detection and quantification. The different methods are optimized and compared by the number of peptides detected. Step-wise elution of bRP spin columns proved to yield more than 36,000 peptides using only 10 μg of protein. Mass-to-charge (m/z) fractionation was tested in mass analyzer Q-Exactive Plus (Thermo Scientific). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of an unfractionated sample was analyzed 4 times at different mass ranges, each mass range width of 150 m/z, resulting from 4 spectra combined, 31,732 peptides representing 3,967 proteins. Showingcomparable results to those form high pH reversed phase fractionation spin columns 5 fractions. Establishing a benchmark where the LC-MS/MS analysis of 600 μg of 10plex TMT-labeled peptides fractionated with bRPLC into 24 fractions yielded over 74,000 peptides from 7,700 proteins, we compared those results with analysis of 10 μg of total TMT-labeled peptides fractionated by bRP spin columns into 5 fractions, which produced 14,019 peptides from 3,538 proteins. These experiments were used to relatively quantify protein expression in naïve and drug resistant multiple myeloma cells lines as an example application in cancer research.

The field of proteomics encompasses the study of identities, interactions, and dynamics of all proteins expressed by a living system. Research in this dissertation blends biochemical and quantitative proteomics techniques to increase the latitude of biological applications for the bottom-up mass spectrometry proteomics approach. Together, isolation of selected protein “targets,” such as multiprotein complexes, and quantitative characterization yields information essential for more detailed understanding of microbial cell function. Often, a challenging aspect of characterizing a variety of biochemically enriched samples is limited protein yield. This dissertation describes an enzymatic proteolysis protocol employing an organic/aqueous solvent that alleviates excessive handling steps to reduce losses during sample preparation for small quantities of protein samples. Presence of artifactual, non-specific proteins in enriched protein complex isolates complicates biological interpretation of specific protein interactions. Heterologous expression of affinity-tagged bait proteins may also cause unintended collateral effects. A series of local and global protein isotope ratio measurements were performed to differentiate authentic interactions from artifactual interactions among affinity-isolated complexes and assess collateral effects, respectively. Protein localization provides clues regarding protein function. To infer protein localization, quantitative proteomics techniques were used to estimate protein enrichment of cold osmotic shock periplasmic isolates. Protein isotope ratios indicating enrichment, combined with identification of amino-terminal signal peptide cleavages, increase confidence of periplasmic localization. Collectively, this dissertation provides a framework for tailoring biochemical and quantitative techniques for targeted characterization of microbial protein isolates.