Orientador: Profa. Dra. Ivanise Gaubeur / Dissertação (mestrado) - Universidade Federal do ABC. Programa de Pós-Graduação em Ciência e Tecnologia/Química, 2015. / O cromo e utilizado em varias atividades industriais e com o aumento da sua presenca no ambiente se tem uma preocupacao em relacao a contaminacao ambiental e considerando que as duas principais especies do elemento quimico apresentam diferentes efeitos na saude humana e importante a realizacao da especiacao quimica. Este trabalho propoe o desenvolvimento de metodo analitico para especiacao de cromo combinada a microextracao liquido-liquido dispersiva com gota organica flutuante (DLLME-FO) e deteccao por espectrometria de absorcao atomica com chama de fonte continua com alta resolucao (HR-CS F AAS). As variaveis que afetam a complexacao (valor de pH, tipo e concentracao dos complexantes: dietilditiocarbamato de sodio (DDTC) e pirrolidinaditiocarbamato de amonio (APDC), microextracao (volume dos solventes dispersor e extrator e concentracao salina) e deteccao como a composicao da chama foram otimizadas de forma univariada e multivariada. Apos as otimizacoes foram escolhidas como melhores condicoes para complexacao, o complexante APDC (6,0x10-3 mol L-1) e valor de pH 2,0 e 7,0, para Cr(VI) e cromo total, respectivamente. E as condicoes da microextracao foram: 5%(m/v) de NaCl, 50 ¿ÊL de 1-undecanol (solvente extrator) e 300 ¿ÊL e 275 ¿ÊL de etanol (solvente dispersor). As caracteristicas analiticas obtidas foram: faixa linear de 20-100 ¿Êg L-1 para Cr(VI) e cromo total. Para o Cr(VI) foram obtidos limite de deteccao 0,35 ¿Êg L-1, coeficiente de variacao igual a 7% (n=10) e fator de enriquecimento igual a 14. Para o cromo total foram obtidos limite de deteccao 6,7 ¿Êg L-1, coeficiente de variacao igual a 18% (n=10) e fator de enriquecimento igual a 10. A exatidao foi avaliada em material de referencia certificado de agua e as concentracoes determinada e certificada foram concordantes em um nivel de confianca de 95%. O metodo foi aplicado em amostras de agua e as concentracoes de cromo encontradas estao dentro dos limites estabelecidos pelas legislacoes vigentes. / Chromium is used in various industrial activities and increasing its presence in the environment has been a concern in relation to environmental contamination and considering that the two main species of the chemical element have different effects on human health is important the realization of chemical speciation.This work proposes the development of analytical method for chromium speciation combined dispersive liquid-liquid microextraction based on floating organic drop (DLLME-FO) and detection by high-resolution continuum source flame atomic absorption spectrometric (HR-CS F AAS) . The variables that affect the complexation (pH, type and concentration of complexant: sodium diethyldithiocarbamate (DDTC) and ammonium pyrrolidinedithiocarbamate (APDC), microextraction (volume of disperser and extractant solvents and salt concentration) and detection as the composition of the flame were optimized using univariate and multivariate analysis. After the optimizations were chosen as the best conditions for complexing the complexant APDC (6.0x10-3 mol L-1) and pH value 2.0 to 7.0 for Cr (VI) and total chromium, respectively. And the microextraction conditions were: 5% (w/v) NaCl, 50 ìL of 1- undecanol (extractant solvent) and 300 ìL of ethanol and 275 ìL (disperser solvent). The analytical characteristics were obtained: linear range of 20-100 ìg L-1 to Cr (VI) and total chromium. For the Cr (VI) was obtained limit of detection 0.35 ìg L-1, coefficient of variation 7% (n=10) and enrichment factor of 14. For the total chromium was obtained limit of detection 6.7 ìg L-1, coefficient of variation 18% (n=10) and enrichment factor of 10. The accuracy was evaluated in water certificate reference material and determined and certified concentrations were agreed at the 95% confidence level. The method was applied to water samples and chromium concentrations found are within the limits established by current legislation.

The widespread use of pesticides in agriculture has brought many benefits such as, increasing quantity and quality of crops grown. However, there is a concern regarding to the presence of pesticide residues in the consumed manufactured food products. Due to this, it is necessary to monitor pesticides in these products in order to verify compliance with maximum residue limits. Wine is a beverage obtained from grape must, but the grapes grown for this purpose are susceptible to pest attack and the occurrence of fungal diseases. Since the wine quality is related to the grapes quality, its production is dependent on the use of pesticides. Currently, the most useful way to determine the pesticides residues in food is the application of multiresidue methods that allow the determination of several compounds from different classes in a single analytical process. Based on that, the aim of this work was to evaluate two multiresidue methods for the extraction of pesticides in wine, one of them being based on the QuEChERS method and the dispersive liquid-liquid microextraction with the use of demulsifying solvent (SD-DLLME). Parameters that affect the extraction efficiency of both methods were studied and after established the best extraction conditions the methods were validated. The use of ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) allowed to perform the determination with high selectivity and sensitivity. For the QuEChERS method, 97 compounds were validated and the limit of quantification of the method (LOQ) ranged from 10 to 20 μg L-1. For SD-DLLME, 74 compounds were validated and LOQ were between 0.1 and 0.2 μg L-1. Among the compounds not validated by SD-DLLME are compounds with polar characteristics, not suitable for this technique. Pesticides residues of different classes were found in the samples the concentrations of 0.8 to 55.3 μg L-1. In view of the results obtained, the use of SD-DLLME proved to be a viable alternative to the QuEChERS method for the multiresidue determination of pesticides in wine. / O uso generalizado de agrotóxicos na agricultura trouxe muitos benefícios em relação ao aumento da quantidade e qualidade das culturas produzidas. Entretanto, existe uma preocupação com relação à presença de resíduos de agrotóxicos nos alimentos e produtos comerciais consumidos. Devido a isso, torna-se necessário o monitoramento de agrotóxicos nesses produtos a fim de verificar a observância dos limites máximos de resíduos. O vinho é uma bebida obtida a partir do mosto da uva, porém as uvas cultivadas para esse fim são suscetíveis ao ataque de pragas e a ocorrência de doenças fúngicas. Uma vez que a qualidade do vinho depende fortemente da qualidade das uvas, a sua produção é dependente do uso de agrotóxicos. Atualmente, a maneira mais útil para determinação de resíduos de agrotóxicos em alimentos é a aplicação de métodos multirresíduo que permitam a determinação de inúmeros compostos, de diversas classes, em um único processo analítico. Pensando nisso, esse trabalho teve como objetivo a avalição de dois métodos de extração multirresíduo de agrotóxicos em vinho, sendo um deles baseado no método QuEChERS e outro na técnica de extração denominada microextração líquido-líquido dispersiva com uso de solvente demulsificante (SD-DLLME). Os parâmetros que afetam a eficiência de extração foram estudados para ambos e depois de encontradas as melhores condições de extração os mesmos foram validados. A determinação por cromatografia líquida de ultra-alta eficiência acoplada à espectrometria de massas em série (UHPLC-MS/MS) permitiu a realização das análises com alta seletividade e sensibilidade. Para o método QuEChERS foram validados 97 compostos sendo que o limite de quantificação do método (LOQm) variou de 10 a 20 μg L-1. Para a SD-DLLME, 74 compostos foram validados e o LOQm entre 0,1 e 0,2 μg L-1. Dentre os compostos que não foram validados pela SD-DLLME estão compostos com características polares, não adequados para essa técnica. Os métodos validados foram utilizados para a determinação de agrotóxicos em amostras de vinho. Foram encontrados resíduos de agrotóxicos de diferentes classes nas concentrações de 0,8 a 55,3 μg L-1. Tendo em vista os resultados obtidos, a utilização da SD-DLLME demonstrou ser uma alternativa viável ao método QuEChERS para determinação multirresíduo de agrotóxicos em vinho.

MSc (Chemistry) / Department of Chemistry / In this work, modern pre-concentration techniques, namely dispersive liquid-liquid micro-extraction (DLLME) and QuEChERS, were used to analyse pesticides and heavy metals in complex matrices. The work is divided into six papers. In Papers 1, the recent developments and applications of DLLME during analysis of pesticides in food matrices were reviewed. The DLLME technique has captured the interests of many researchers in recent years. The major advantage, among others, of DLLME is miniaturisation in which the acceptor-to-donor ratio is reduced tremendously leading to high enrichment compared to other sample preparation techniques. In the present work, the different complex matrices where the DLLME technique has been employed for the analysis of pesticides are reviewed as well as the challenges associated with this technique. Papers II reviewed the recent applications and developments of the QuEChERS technique during the analysis of pesticides in food matrices. QuEChERS is a versatile pre-concentration method whose application spans the whole breath of organic compounds. There are three common standard methods used during QuEChERS and these are the original QuEChERS, AOAC and the EN methods. In this paper, recent developments and applications of QuEChERS techniques in the analysis of pesticides in food samples were reviewed. In Paper III, green pre-concentration techniques employed during analysis of pesticides were reviewed. Recently, the parameter of “greenness” during sample pre-concentration of pesticides in food matrices is as important as selectivity in order to avoid using large amounts of harmful organic solvents during sample preparation. Developing new green pre-concentration techniques is one of the key subjects in green chemistry in order to minimize the release of large volume of toxic organic solvents into the environment. Thus, to reduce the impact on the environment during trace analysis of pesticides in food matrices, new developments in pesticide pre-concentration have gone in three separate directions (which are reviewed in this paper): one is the search for more environmentally friendly solvents, the second one is miniaturization and the third one is the development of solvent-free pre-concentration techniques. Eco-friendly solvents such as supercritical fluids, ionic liquids and natural deep eutectic solvents have been developed for use as extraction solvents during pre-concentration of pesticides in food matrices. Also miniaturized pre-concentration techniques such as QuEChERS, dispersive liquid-liquid micro-extraction and hollow-fibre liquid phase micro-extraction have been used during trace analysis of pesticides in food samples as well as solvent-free techniques such as solid phase micro-extraction and stir bar sorptive extraction. All these developments are geared to ensure that pesticide pre-concentration in food matrices is green and were reviewed in this paper. The effect of vehicular emissions on the concentrations of selected heavy metals was investigated in Paper IV. The samples were pre-concentrated using DLLME prior to analysis with flame atomic absorption spectroscopy. Dithizone, chloroform and methanol were used as chelating agent, extraction solvent and dispersion solvent respectively during the DLLME technique. The pH of the sample was adjusted to around 8 using sulphuric acid or sodium hydroxide solution. The influential DLLME parameters, such as pH volume and type of extraction solvent, and voume of disperser solvent, were optimized prior to the application of the developed method to real samples (roadside dust, fruits and vegetables). In Paper V, chromium speciation in fruits and vegetables was studied. The chromium in fruit and vegetable sample juices was pre-concentrated using DLLME prior to analysis with flame atomic absorption spectroscopy. Diphenylcarbazide (DPC) was used as a chelating agent in this study, and salting out of the complex from the aqueous medium into the organic phase was effected using sodium acetate. Chloroform and methanol were used as extraction and dispersion solvents respectively in the DLLME method for the determination of chromium (VI). For total chromium, the trivalent chromium was oxidised using acidified KMnO4 to hexavalent chromium before performing the DLLME technique. The concentration of chromium (III) was determined by finding the difference between total chromium and concentration of chromium (VI). The important parameters that influence the efficiency of the DLLME technique were also optimized using the univariate approach. After optimization, the developed method was applied to real samples. In Paper VI, the concentration of malathion pesticide in fruits was determined using QuECHERS for pre-concentration and UV-Vis spectrophotometry for instrumental analysis. Acetonitrile was used as the extraction solvent and Z-sep+/PSA sorbent combination was used for sample clean-up. The acetonitrile extract from QuEChERS was then hydrolysed using KOH followed by reaction with acidified potassium bromate for colour development. The coloured product formed was then analysed using UV-Vis spectrophotometry. Among the fruits analysed, Oranges had no malathion residue in them. However, trace amounts of malathion, below WHO maximum allowable limits, were found in pears and apples. / NRF

Submitted by Ana Hilda Fonseca (anahilda@ufba.br) on 2014-09-22T12:14:30Z No. of bitstreams: 1 DISSERTAÇÃO MESTRADO 2013 - Versão Final.pdf: 1294177 bytes, checksum: fcc79b2fe855be99bc86d8a653bb2da8 (MD5) / Approved for entry into archive by Fatima Cleômenis Botelho Maria (botelho@ufba.br) on 2014-09-23T13:02:35Z (GMT) No. of bitstreams: 1 DISSERTAÇÃO MESTRADO 2013 - Versão Final.pdf: 1294177 bytes, checksum: fcc79b2fe855be99bc86d8a653bb2da8 (MD5) / Made available in DSpace on 2014-09-23T13:02:35Z (GMT). No. of bitstreams: 1 DISSERTAÇÃO MESTRADO 2013 - Versão Final.pdf: 1294177 bytes, checksum: fcc79b2fe855be99bc86d8a653bb2da8 (MD5) / O consumo de drogas de abuso é uma prática crescente no mundo moderno. Dessa forma, as drogas sintéticas e mais particularmente os derivados da anfetamina, ocupam um lugar de destaque. O consumo de estimulantes anfetamínicos tais como fenproporex (FEN), dietilpropiona (DIE) e sibutramina (SIB) se tornou uma prática comum entre motoristas profissionais nas estradas brasileiras nos últimos anos e entre pessoas que desejam perder peso, mas fazem o uso dessas substâncias de forma indiscriminada. O presente trabalho constitui o desenvolvimento de um método analítico, empregando microextração líquido-líquido dispersiva (DLLME), capaz de detectar e quantificar esses medicamentos em baixas concentrações em amostras de urina. As análises foram realizadas utilizando cromatografia à gás acoplada a espectrometria de massas (GC-MS). O método apresentou boa performance analítica, com excelente linearidade na faixa dinâmica de 1 a 1000 ng/mL para DIE, FEN e SIB, com valores de R2 iguais a 0,9926 , 0,9994 e 0,9951, respectivamente. Os limites de detecção (LD), foram de 0,1 ng/mL para DIE e FEN e de 0,05 ng/mL para SIB. O coeficiente de variação intradia variou de 6,63% a 7,95% enquanto o interdia variou de 3,89% a 5,47%. A recuperação relativa encontrada foi superior a 82,88% para DIE, 88,58% para FEN e 95,60% para SIB. O método desenvolvido mostrou-se bastante útil na análise dos compostos estudados em amostras de urina postmortem ou in vivo. / The drug abuse consumption is a growing practice in the modern world. Thus, synthetic drugs and more particularly amphetamine derivatives, occupy a prominent place. The consumption of stimulants-type amphetamines such as fenproporex (FEN), diethylpropion (DIE) and sibutramine (SIB) has become a common practice among professional drivers on Brazilian roads in recent years and among people who wish to lose weight, but make use of these substances indiscriminately. The present work is the development of an analytical method using dispersive liquid-liquid microextraction (DLLME) capable of detecting and quantifying these drugs at low concentrations in urine samples. Analyses were performed using gas chromatography coupled to mass spectrometry (GC-MS). The method showed good analytical performance with excellent linearity in a dynamic range from 1 to 1000 ng/mL for DIE, FEN and SIB, with R2 values equal to 0.9926, 0.9994 and 0.9951, respectively. The limits of detection (LOD) were 0.1 ng/ml for DIE and FEN and 0.05 ng/mL to SIB. The intraday variation coefficient ranging from 6.63% to 7.95% while the interday ranged from 3.89% to 5.47%. The relative recovery founded was 82.88% for DIE, 88.58% for FEN and 95.60% for SIB. The method proved to be very useful in the analysis of these compounds in postmortem or in vivo urine samples.

Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Cocaine is a stimulant that features a strong ability to cause dependence. Often adulterants are added to this drug in order to mimic its action or minimize its adverse effects. When there are other pharmacologically active components in the drug composition, severe problems can occur to users’ health, such as intoxication symptoms. Thus, the aim of this study was to develop a method for the determination of the main adulterants of cocaine (caffeine, levamisole, lidocaine, phenacetin, diltiazem, and hydroxyzine) in human urine. The high-performance liquid chromatography with a photodiode array detector and the dispersive liquid-liquid microextraction based on solidification of floating organic drop were used as analysis technique and as sample preparation technique, respectively. The reversed-phase chromatographic separation was obtained with a C18 column (250 x 4.6 mm; 5 μm; 80 Å) in gradient elution mode using acetonitrile-trifluoroacetic acid 0.026% (v/v) at 1 mL min-1 as mobile phase, at 25°C, and detection at 235 nm. The analysis time was 25 min. Under optimum conditions, human urine samples were alkalized with 0.5 mol.L-1 sodium phosphate buffer (pH 10) and added sodium chloride (20% m/v). Acetonitrile (150 μL) and 1-dodecanol (30 μL) were used as dispersive and extraction solvent, respectively. The method presented linear range of 312.5 – 3125 ng.mL−1 for caffeine and levamisole and 187.5 – 1875 ng.mL−1 for lidocaine, phenacetin, diltiazem, and hydroxyzine, with limit of quantification of 187.5 ng.mL-1 to lidocaine, phenacetin, diltiazem, and hydroxyzine and 312.5 ng.mL-1 for caffeine and levamisole. Recovery mean values were between 6.0 and 42.6%. The method showed good precision and accuracy, with within- and between-run relative standard deviation and relative error less than 15%. The samples were stable after freeze-thaw cycle and short-term room temperature stability tests. Additionally, this method was applied in samples of urine of five cocaine users and at least one adulterant was identified in all samples. It is expected that this method will contribute to the precision in the diagnosis of cocaine adulterants’ intoxication and to the proper planning of therapeutic measures. / A cocaína é uma droga estimulante que apresenta capacidade de causar dependência. Frequentemente são adicionados a esta droga adulterantes com o intuito de mimetizar sua ação ou minimizar seus efeitos adversos. Quando há nessa droga outros componentes farmacologicamente ativos, agravos à saúde dos usuários podem ocorrer, como quadros de intoxicação. Assim, o objetivo deste trabalho foi desenvolver um método de determinação dos principais adulterantes da cocaína (cafeína, levamisol, lidocaína, fenacetina, diltiazem e hidroxizina) em urina humana. A cromatografia líquida de alta eficiência com detector de arranjo de fotodiodos foi utilizada como técnica de análise e a microextração líquido-líquido dispersiva com solidificação da gota orgânica flutuante, como técnica de preparo das amostras. A separação cromatográfica dos analitos em fase reversa foi obtida em uma coluna C18 (250 x 4,6 mm; 5 μm; 80 Å) em modo gradiente e usando acetonitrila-ácido trifluoroacético 0,026% (v/v) a 1 mL.min-1 como fase móvel (25°C e detecção a 235 nm). O tempo de análise foi de 25 min. Para o preparo da amostra, a urina foi alcalinizada com tampão fosfato de sódio 0,5 mol.L-1 (pH 10) e adicionada de cloreto de sódio (20% m/v). Acetonitrila (150 μL) e 1-dodecanol (30 μL) foram utilizados como solvente dispersor e extrator, respectivamente. O método apresentou intervalos lineares de concentração de 312,5 – 3125 ng.mL−1 para cafeína e levamisol e de 187,5 – 1875 ng.mL−1 para lidocaína, fenacetina, diltiazem e hidroxizina, com limite de quantificação de 187,5 ng.mL-1 para lidocaína, fenacetina, diltiazem e hidroxizina e 312,5 ng.mL-1 para cafeína e levamisol. Os valores médios de recuperação variaram de 6,0 a 42,6%. O método mostrou boa precisão e exatidão intra e intercorrida com coeficientes de variação e erros relativos menores que 15%. As amostras apresentaram-se estáveis após ciclos de congelamento-descongelamento e após serem mantidas por 24h em temperatura ambiente. Ainda, o método foi aplicado em cinco amostras de urina de usuários de cocaína e pelo menos um adulterante foi identificado em todas as amostras. Espera-se que este método possa contribuir para a precisão no diagnóstico das intoxicações por adulterantes da cocaína e para o adequado planejamento das medidas terapêuticas.

<p>Insekticidi novije generacije, neonikotinoidi, odlikuju se specifičnim načinom &nbsp;delovanja na&nbsp;nervni sistem insekata. Radi dobijanja &scaron;to brže i kvalitetnije informacije o izloženosti životne sredine&nbsp;ovim insekticidima i količinama njihovih ostataka u hrani potrebno je raspolagati odgovarajućim&nbsp;instrumentalnim metodama za njihovo određivanje. Razvijene su i optimizovane analitičke metode&nbsp;zasnovane na tečnoj hromatografiji za određivanje sedam odabranih neonikotinoida (dinotefurana,&nbsp;nitenpirama, tiametoksama, klotianidina, imidakloprida, acetamiprida &nbsp;i tiakloprida) u medu i likeru od&nbsp;meda. Ispitivana je mogućnost određivanja klotianidina pomoću tečne hromatografije visoke efikasnosti&nbsp;sa detektrorom od niza dioda (HPLC-DAD) primenom kombinacije tečno-tečne i ekstrakcije na čvrstoj&nbsp;fazi iz uzoraka meda. Na osnovu preliminarnih rezultata može se zaključiti da kori&scaron;ćenje &nbsp;faznih-čvrsto&nbsp;kolona u kombinaciji sa tečno-tečnom ekstrakcijom dihlormetanom rezultira prihvatljivim prinosom&nbsp;klotianidina u uzorcima meda pri koncentraciji od oko 0,5 &micro;g g^-1&nbsp;klotianidina. Radi dobijanja većih&nbsp;prinosa odabrana je disperzna tečno-tečna mikroekstrakcija (DLLME) kao tehnika pripreme uzoraka&nbsp;meda. Testirana je upotreba acetonitrila kao disperznog sredstva. Pored hloroforma, kori&scaron;ćen je i&nbsp;dihlormetan kao drugo ekstrakciono sredstvo, kako bi se &nbsp;uporedila efikasnost ekstrakcije. Zabeleženi su&nbsp;prinosi klotianidina od 69,7 i 68,3% &nbsp;u zavisnosti da li je kori&scaron;ćen hloroform, odnosno DHM kao rastvor&nbsp;za ekstrakciju. Može se zaključiti da je prinos ekstrakcije bio povoljniji pri odnosu 0,5 mL ACN i 2,0&nbsp;mL DHM. Prinosi su se kretali od 68,4% do 92,1%, &scaron;to je ukazalo da su parametri DLLME ekstrakcije optimalni. Kako bi se detaljnije ispitali ključni parametri DLLME tehnike, kori&scaron;ćena je metodologija povr&scaron;ine odziva (RSM), kao i detekcija na osetljivijem kuplovanom masenom detektoru (MS/MS). Optimizovani su HPLC-MS/MS &nbsp;parametri kako bi se obezbedilo zadovoljavajuće hromatografsko &nbsp;razdvajanje i niske granice detekcije (GD, 0,5&ndash;1,0 &mu;g kg^-1) i određivanja (GO, 1,0&ndash;2,5 &mu;g kg^-1) ispitivanih neonikotinoida u medu. Upotrebom centralno kompozitnog dizajna konstruisani su kvadratni modeli ispitivanih faktora: zapremine ekstrakcionog (DHM, 1,0&ndash;3,0 mL) i disperznog (ACN, 0,0&ndash;1,0 mL) sredstva, izračunati statistički parametri i optimizovan proces DLLME upotrebom <em>Derringer</em>-ove funkcije poželjnih odgovora. Upotrebom MMC i SC krivih u opsegu GO&ndash;100,0 &mu;g kg^-1&nbsp;ispitan je uticaj matriksa pri čemu zaključeno je da je najveći uticaj matriksa bio na odziv analitičkog signala nitenpirama, dinotefurana i klotianidina. Ispitani su prinosi odabranih neonikotinoida (R, 74,3&ndash;113,9%), kao i preciznost metode u uslovima ponovljivosti (RSD, 2,74&ndash; 11,8%) i intermedijerne reproduktivnosti (RSD, 6,64&ndash;16,2%). Brza (retenciona vremena 1,5&ndash;9,9 min) i osetljiva metoda, koja tro&scaron;i malu količinu rastvarača, primenjena je za ispitivanje 15 realnih uzoraka meda različitog cvetnog porekla. Rezultati su pokazali da ispitivani med nije sadržao ostatke ispitivanih neonikotinoida u koncentracijama iznad GD. Dalje istraživanje je bilo usmereno ka&nbsp; razvijanju i optimizaciji HPLC-DAD analitičke metode upotrebom DLLME i QuEChERS tehnika za &nbsp;pripremu uzoraka za određivanje 7 neonikotinoida u uzorcima meda. U ovom delu istraživanja optimizovani su i hromatografski parametri, upotrebom RSM sa Box-Behnken-ovim dizajnom i Derringer-ovom funkcijom poželjnih odgovora. Od &nbsp;ispitivanih neonikotinoida dinotefuran i imidakloprid su bili u najvećoj meri izloženi uticaju matriksa, bez obzira na proceduru pripreme uzoraka. Može se istaći da je uticaj matriksa na analitički signal dinotefurana bio izraženiji u slučaju MS/MS, apostrofirajući manju robusnost ove metode određivanja. Prinosi neonikotinoida su &nbsp;bili (R, 73,1&ndash;118,3%), preciznost u uslovima ponovljivosti (RSD, 3,28&ndash;10,40%) i intermedijerne reproduktivnosti (RSD, 6,45&ndash;17,70%), a granice detekcije (GD, 1,5&ndash;2,5 &micro;g kg^-1) i određivanja (GO, 5,0&ndash;10,0 &micro;g kg^-1). Metoda je primenjena za ispitivanje 7 neonikotinoida u 104 uzorkameda različitog cvetnog porekla sa &nbsp;teritorije Autonomne Pokrajine Vojvodine. Detektovano je prisustvo tiakloprida, imidakloprida i tiametoksama u količinama koje su bile ispod MDK RS i EU. Analizirani su uzorci likera od meda - medice. Upoređivane su dve tehnike pripreme uzoraka, DLLME i QuEChERS i primenjeni optimizovani hromatografski &nbsp;uslovi i MS/MS parametri. U slučaju nitenpirama, dinotefurana i tiametoksama uticaj matriksa bio je najizraženiji. Metoda je validovana određivanjem prinosa neonikotinoida (R, 69,2&ndash;113,4%), preciznosti u uslovima ponovljivosti (RSD, 3,21&ndash;12,81%) i intermedijerne reproduktivnosti (RSD, 9,11&ndash;16,63%), kao i granice detekcije (GD, 0,5&ndash;2,5 &micro;g kg^-1) i određivanja (GO, 1,0&ndash;10,0 &micro;g kg^-1). Analizom 10 komercijalno dostupnih likera od meda otkriveno je&nbsp;prisustvo klotianidina i tiakloprida, evčzokinot&scaron; z&nbsp; na neophodnost daljeg kontrolisanja ovog proizvoda na&nbsp;prisustvo neonikotinoida. Ispitana je mogućnost uklanjanja odabranih neonikotinoida (dinotefurana,&nbsp;klotianidina i tiakloprida) iz vodene sredine (reke Dunav). Ispitivanje efikasnosti 6 različitih vrsta&nbsp;uklanjanja odabranih neonikotinoida (u prisustvu prirodne insolacije u laboratorijskim uslovima, sa&nbsp;dodatkom H2O2, sa dodatkom MWCNT, sa dodatkom MWCN+H&nbsp;₂O₂, sa dodatkom Fe-MWCNT, sa dodatkom Fe-MWCNT+H₂O₂) vr&scaron;eno je upotrebom prethodno razvijene HPLC-MS/MS metode. Krive uklanjanja odabranih neonikotinoida, pokazale su da tokom 60 minuta pri prirodnoj insolaciji&nbsp; u laboratorijskim uslovima koncentracija smanjenje oko 25%. Analitički signal dinotefurana dobijen u prisustvu H₂O_2&nbsp;pod istim uslovima ukazuje na uklanjanje ciljnog analita od oko 40%, tiakloprida od oko 70%, a klotianidina u potpunosti. Testirana je adsorpcija ciljnog analita na vi&scaron;ezidnim ugljeničnim nanocevima (MWCNT). Ovim postupkom može da se ukloni oko 30% dinotefurana, oko 50% klotianidina i 60% tiakloprida. U kombinaciji sa H₂O_2&nbsp;, MWCNT pokazuju bolju sposobnost uklanjanja za 15&ndash;50% u zavisnosti od ispitivanog neonikotinoida. Upotreba Fe-MWCNT i njihova kombinacija sa H₂O₂ otvorila je mogućnost za dalja &nbsp;ispitivanja mehanizma uklanjanja. Ustanovljeno je nastajanje intermedijera kojima odgovaraju m/z od 117,5 i 140,6 u slučaju razgradnje dinotefurana u sistemima sa H₂O₂, MWCNT+H₂O₂, Fe-MWCNT+H₂O_2&nbsp;i klotianidina u sistemu Fe-MWCNT+H₂O₂.</p> / <p>Neonicotinoid insecticides, as one of the fastest growing new generation of insecticides, have&nbsp;contributed to a significant reduction of toxicity for the environment; therefore, monitoring and&nbsp;determination of trace levels of the neonicotinoids in honey are necessary and demands highly efficient,&nbsp;selective and sensitive analytical techniques. The objective of the present work was to develop a rapid,&nbsp;sensitive, optimized and accurate analytical method based on liquid chromatography for determining&nbsp;seven neonicotinoid insecticides, dinotefuran, nitenpyram, thiamethoxam, clothianidin, imidacloprid,&nbsp;acetamiprid and thiacloprid in honey and honey liqueur samples. The possibility for determination of&nbsp;clothianidin in honey samples was investigated by HPLC with a diode array detector (HPLC-DAD).&nbsp;Based on preliminary results, it can be concluded that the use of a solid-phase column in combination&nbsp;with a liquid-liquid extraction with dichloromethane results in an acceptable recovery of clothianidin in&nbsp;the samples with a clothianidin concentration of about 0.5 &micro;g g^-1. After obtaining low recovery of&nbsp;clothianidin, dispersed liquid-liquid microextraction (DLLME) was selected as a technique for the&nbsp;preparation of honey samples.. The adequacy of acetonitrile as a dispersing agent was investigated.&nbsp;Besides the chloroform, a dichloromethane was used as a second extracting agent , in order to compare&nbsp;the relative efficiency of the extraction solvents. It can be concluded that the extraction recovery (68.4&ndash;92.1%) was more favorable with the use of 0.5 mL ACN and 2.0 mL DHM. Furthermore, LC-MS/MS&nbsp;parameters were optimized to unequivocally provide good chromatographic separation, low detection&nbsp;(LOD, 0.5&ndash;1.0 &mu;g L^&minus;1) and quantification (LOQ, 1.0&ndash;2.5 &mu;g L^&minus;1) limits for acetamiprid, clothianidin,&nbsp;thiamethoxam, imidacloprid, dinotefuran, thiacloprid and nitenpyram in honey samples. Using different&nbsp;<br />types (chloroform, dichloromethane) and volumes of extraction (1.0&ndash;3.0 mL) and dispersive&nbsp;(acetonitrile; 0.0&ndash;1.0 mL) solvent and by mathematical modeling it was possible to establish the optimal&nbsp;sample preparation procedure. Matrix-matched calibration and blank honey sample spiked in the&nbsp;<span style="font-size: 12px;">concentration range of LOQ&ndash;100.0 &mu;g kg</span>^{<span style="font-size: 12px;">&minus;1&nbsp;</span>}<span style="font-size: 12px;">were used to compensate the matrix effect and to fulfill the&nbsp;</span><span style="font-size: 12px;">requirements of SANCO/12495/2011 for the accuracy (R 74.3&ndash;113.9%) and precision (expressed in&nbsp;</span><span style="font-size: 12px;">terms of repeatability (RSD 2.74&ndash;11.8%) and within-laboratory reproducibility (RSDs &nbsp;6.64&ndash;16.2%)) of&nbsp;</span><span style="font-size: 12px;">the proposed method. The rapid (retention times 1.5&ndash;9.9 min), sensitive and low solvent consumption&nbsp;</span><span style="font-size: 12px;">procedure described in this work provides reliable, simultaneous, and quantitative method applicable for&nbsp;</span><span style="font-size: 12px;">the routine laboratory analysis of seven neonicotinoid residues in 15 real honey samples. Neonicotinoid &nbsp;</span><span style="font-size: 12px;">residues were not detected in any of the investigated samples. The objective of next study was to&nbsp;</span><span style="font-size: 12px;">develop and optimize HPLC-DAD analytical method with dispersive liquid-liquid microextraction&nbsp;</span><span style="font-size: 12px;">(DLLME) and QuEChERS sample preparation procedures for the simultaneously analysis of seven&nbsp;</span><span style="font-size: 12px;">neonicotinoids in honey samples. The liquid chromatographic conditions were optimized by response&nbsp;</span><span style="font-size: 12px;">surface methodology with <em>Box-Behnken</em> design and the global <em>Derringer</em>&acute;s desirability. The optimized&nbsp;</span><span style="font-size: 12px;">method was &nbsp;validated to fulfill the requirements of SANCO/12495/2011 standard for both sample&nbsp;</span><span style="font-size: 12px;">pretreatment procedures providing results for accuracy (R, 73.1&ndash;118.3%), repeatability (RSD, 3.28&ndash;</span><span style="font-size: 12px;">10.40%) and within-laboratory reproducibility (RSD, 6.45&ndash;17.70%), limits of detection (LOD, 1.5&ndash;2.5&nbsp;</span><span style="font-size: 12px;">g&micro; kg</span>^{<span style="font-size: 12px;">-1</span>}<span style="font-size: 12px;">) and quantification (LOQ, 5.0&ndash;10.0 &micro;g kg</span>^{<span style="font-size: 12px;">-1</span>}<span style="font-size: 12px;">). For the first time, more than 100 honey samples&nbsp;</span><span style="font-size: 12px;">collected from all 7 counties of Autonomous Province of Vojvodina were analyzed. The presence of&nbsp;</span><span style="font-size: 12px;">thiacloprid, imidacloprid and thiametoxam was discovered in a small number of samples. The objective&nbsp;</span><span style="font-size: 12px;">of next study was to develop an optimized LC-MS/MS analytical method with DLLME and QuEChERS&nbsp;</span><span style="font-size: 12px;">procedures for analysis of 7 neonicotinoids in honey liqueur. The method was validated to fulfill the&nbsp;</span><span style="font-size: 12px;">requirements of SANCO/12495/2011 for both sample pretreatment procedures providing results for&nbsp;</span><span style="font-size: 12px;">accuracy (R, 69.2&ndash;113.4% for DLLME; 71.8&ndash;94.9% for QuEChERS), precision (RSD expressed in&nbsp;</span><span style="font-size: 12px;">terms of repeatability (3.21&ndash;10.20% for DLLME; 4.19&ndash;12.81% for QuEChERS) and within-laboratory&nbsp;</span><span style="font-size: 12px;">reproducibility (9.11&ndash;16.63% for DLLME; 11.32&ndash;16.40% for QuEChERS)), limits of detection (LOD,&nbsp;</span><span style="font-size: 12px;">0.5&ndash;1.5 g&micro; L</span>^{<span style="font-size: 12px;">-1&nbsp;</span>}<span style="font-size: 12px;">for DLLME; 1.0&ndash;2.5 g&micro; L</span>^{<span style="font-size: 12px;">-1&nbsp;</span>}<span style="font-size: 12px;">for QuEChERS) and quantification (LOQ, 1.0&ndash;5.0 g&micro; L</span>^{<span style="font-size: 12px;">-1&nbsp;</span>}<span style="font-size: 12px;">for&nbsp;</span><span style="font-size: 12px;">DLLME; 2.5&ndash;10.0 &micro;g L</span>^{<span style="font-size: 12px;">-1&nbsp;</span>}<span style="font-size: 12px;">for QuEChERS). Analysis of real honey liqueur samples obtained from local&nbsp;</span><span style="font-size: 12px;">markets showed the presence of clothianidin or thiacloprid in four of the analyzed samples, therefore&nbsp;</span><span style="font-size: 12px;">implicating the necessity of ongoing control of this type of traditional product. &nbsp;Removal of selected&nbsp;</span><span style="font-size: 12px;">neonicitinoid insecticides - dinotefuran, clothianidin and thiacloprid using MWCNT and H</span><span style="font-size: 12px;">₂O_2&nbsp;</span><span style="font-size: 12px;">from&nbsp;</span><span style="font-size: 12px;">Danube water matrix was investigated. &nbsp;Efficiency of different systems for neonicotinoids removal&nbsp;</span><span style="font-size: 12px;">(under natural insolation in laboratory, with H</span><span style="font-size: 12px;">₂O₂, with MWCNT, with MWCNT+ H</span><span style="font-size: 12px;">₂O₂, with Fe-MWCNT, with Fe-MWCNT+H₂O₂) was evaluated with developed LC-MS/MS method. Analysis of&nbsp;</span><span style="font-size: 12px;">degradation rates revealed loss of 25% of the initial neonicotinoid concentration under natural insolation in&nbsp;</span><span style="font-size: 12px;">the laboratory conditions during 60 min. Addition of chemical agent H₂O_2&nbsp;promoted loss of 40% of the&nbsp;</span><span style="font-size: 12px;">initial dinotefuran, 70% of thiacloprid concentration and total removal of clothianidin under same&nbsp;</span><span style="font-size: 12px;">conditions. With the addition &nbsp;of MWCNT concentration of dinotefuran, clothianidin and thiacloprid&nbsp;</span><span style="font-size: 12px;">decayed for 30, 50 and 60%, respectively. Iron modification of MWCNT in combination with H</span><span style="font-size: 12px;">₂O_2&nbsp;</span><span style="font-size: 12px;">increased the removal rate of selected neonicotinoid for 15&ndash;50%. Presence of intermediates was&nbsp;</span><span style="font-size: 12px;">discovered in systems of dinotefuran with H₂O₂, MWCNT+H</span><span style="font-size: 12px;">₂O₂, e-MWCNT+H₂O_2&nbsp;</span><span style="font-size: 12px;">and of&nbsp;</span><span style="font-size: 12px;">clothianidin in systems with Fe-MWCNT+H₂O_2&nbsp;</span><span style="font-size: 12px;">with m/z of 117,5 and 140,6.&nbsp;</span></p>

Submitted by MICHAEL PÉREZ RODRÍGUEZ null (michaelpr@iq.unesp.br) on 2016-09-16T20:43:06Z No. of bitstreams: 1 TESE MICHAEL VERSÃO FINAL.pdf: 4495815 bytes, checksum: 5d4a4f8ffcd143bb08ad336b289fe7ab (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-09-22T14:44:30Z (GMT) No. of bitstreams: 1 perezrodriguez_m_dr_araiq_par.pdf: 1340231 bytes, checksum: 9e639833f42ff1e6fc87e7f9f965f8fc (MD5) / Made available in DSpace on 2016-09-22T14:44:30Z (GMT). No. of bitstreams: 1 perezrodriguez_m_dr_araiq_par.pdf: 1340231 bytes, checksum: 9e639833f42ff1e6fc87e7f9f965f8fc (MD5) Previous issue date: 2016-08-26 / Pró-Reitoria de Pós-Graduação (PROPG UNESP) / Este trabalho descreve o desenvolvimento de métodos analíticos ambientalmente mais amigáveis utilizando sistemas de screening e procedimentos de análise por injeção em fluxo para a detecção e quantificação de antibióticos, da classe das tetraciclinas, em amostras de medicamentos veterinários e alimentos de origem animal como leite bovino e suplementos de ovos. Os métodos desenvolvidos foram baseados na reação de acoplamento diazo obtida entre as tetraciclinas e o ácido p-sulfanílico diazotado em meio básico, resultando na formação de azo compostos com máximos de absorção em torno de 435 nm, permitindo determinar esses antibióticos por espectrofotometria. As condições analíticas foram otimizadas através de análise multivariada utilizando planejamentos experimentais fatoriais e o desempenho dos métodos propostos foi avaliado através de parâmetros como linearidade, efeito matriz, precisão, exatidão, limite de detecção, limite de quantificação, recuperação e estudo de interferências. O primeiro trabalho desenvolvido fez uso de um procedimento de injeção em fluxo contínuo para a rápida determinação de tetraciclinas em formulações veterinárias comerciais, visando maior eficiência no controle de qualidade na indústria farmacêutica. Na sequência, foi desenvolvido um método de screening mediante análise por injeção em fluxo empregando uma cela capilar de longo caminho óptico (LWCC) para a detecção de resíduos de tetraciclinas em amostras de leite bovino, sem a necessidade de passos de extração dispendiosos e demorados ou complicados tratamentos de amostras. Finalmente, um novo método de microextração líquido-líquido dispersiva assistida por ultrassom (US-DLLME) foi desenvolvido para a simples, rápida e eficiente extração de resíduos de tetraciclinas a partir de amostras de suplemento de ovos e, posterior análise por injeção em fluxo baseada em uma cela capilar de longo caminho óptico (LWCC), empregando um banho de aquecimento com controle de temperatura. Os métodos descritos apresentaram importantes características como simplicidade operacional, possibilidade de automação, rapidez e baixo custo das análises. Além disso, estes métodos seguiram os princípios da química verde, de modo que permitiram realizar determinações ambientalmente mais limpas, através da redução considerável do consumo de reagentes e amostras, evitando ou minimizando o emprego de solventes tóxicos. Também, os métodos desenvolvidos para a análise de resíduos mostraram excelente desempenho para aplicação no controle de qualidade dos produtos alimentares estudados na indústria alimentícia, a fim de proteger os consumidores. / This work describes the development of environmentally friendly analytical methods using screening systems and flow injection procedures for detection and quantification of tetracycline antibiotics in veterinary pharmaceuticals and animal-derived foods such as bovine milk and egg-based protein supplements. The developed methods were based on the diazo coupling reaction between the tetracyclines and diazotized p-sulfanilic acid in basic medium, resulting in the formation of azo compounds that present maximum absorption around 435 nm, which allows determining these antibiotics by spectrophotometry. The analytical conditions were optimized by means of multivariate analysis using factorial experimental designs. Performance of the proposed methods was assessed through parameters such as linearity, selectivity, precision, accuracy, detection limit, quantification limit, matrix effect and recovery. The first developed work used a continuous flow injection procedure for the rapid determination of tetracyclines in commercial veterinary formulations, aiming greater efficiency in quality control in the pharmaceutical industry. Thereafter, it was developed a screening method by flow injection analysis using a liquid waveguide capillary cell (LWCC) for detecting tetracyclines residues in bovine milk samples, without the need for expensive extraction steps that are time-consuming or complicated sample treatments. Finally, a new ultrasound-assisted dispersive liquid-liquid microextraction (US-DLLME) method was developed for simple, rapid, and efficient extraction of tetracyclines residues from egg supplement samples, followed by flow injection analysis based on a liquid waveguide capillary cell (LWCC), using a controlled temperature heating bath. The described methods presented important features such as operational simplicity, possibility of automation, quickness and low cost of the analysis. Furthermore, these methods followed the principles of green chemistry, making possible to perform environmentally cleaner determinations, due to the considerable reduction of reagents and samples consumption, avoiding or minimizing the use of toxic solvents. Besides, the methods developed for residues analysis showed excellent performance for application in the food industry for quality control of the studied food products in order to protect consumers.

Steroid hormones belong to a group of compounds known as endocrine disruptors. They are hydrophobic compounds and are categorized as natural and synthetic estrogens. Some common household products have been implicated as estrogen mimics. Exposure effects of these compounds are felt by human and wildlife, such reproductive alterations in fish and frogs. They mainly introduced into the environment through veterinary medicines administration to animals and the discharges from wastewater treatment plants (WWTPs). In this study, a new alternative analytical procedure that is simple, rapid and fast for the determination and quantification of five steroidal hormones: estriol (E3), beta estradiol (β-E2), alpha estradiol (α-E2), testosterone (T), progesterone (P) and bisphenol A (BPA) using the High pressure liquid chromatography coupled to a charged aerosol detector (HPLC-CAD). These compounds were studied because of their strong endocrine-disrupting effects in the environment. Under optimum conditions, a linear graph was obtained with correlation coefficient (R2) ranging from 0.9952 - 0.9996. The proposed method was applied to the analysis of water samples from a wastewater plant and the results obtained were satisfactory. The limits of detection (LOD) for the target analytes in wastewater influent was between 0.0002 – 0.0004 μg/L and the limit of quantification (LOQ) was 0.001 μg/L respectively for each of the analytes. Enrichment factors of 148- 258, and extraction efficiency 84- 102% were obtained for the target analytes; relative standard deviations (% RSD) for m = 6 were between 2.8 and 7.6%. The concentration of the EDCs in environment sample was between 0.2 - 2.3 μg/L. / Chemistry / M. Sc. (Chemistry)

Steroid hormones belong to a group of compounds known as endocrine disruptors. They are hydrophobic compounds and are categorized as natural and synthetic estrogens. Some common household products have been implicated as estrogen mimics. Exposure effects of these compounds are felt by human and wildlife, such reproductive alterations in fish and frogs. They mainly introduced into the environment through veterinary medicines administration to animals and the discharges from wastewater treatment plants (WWTPs). In this study, a new alternative analytical procedure that is simple, rapid and fast for the determination and quantification of five steroidal hormones: estriol (E3), beta estradiol (β-E2), alpha estradiol (α-E2), testosterone (T), progesterone (P) and bisphenol A (BPA) using the High pressure liquid chromatography coupled to a charged aerosol detector (HPLC-CAD). These compounds were studied because of their strong endocrine-disrupting effects in the environment. Under optimum conditions, a linear graph was obtained with correlation coefficient (R2) ranging from 0.9952 - 0.9996. The proposed method was applied to the analysis of water samples from a wastewater plant and the results obtained were satisfactory. The limits of detection (LOD) for the target analytes in wastewater influent was between 0.0002 – 0.0004 μg/L and the limit of quantification (LOQ) was 0.001 μg/L respectively for each of the analytes. Enrichment factors of 148- 258, and extraction efficiency 84- 102% were obtained for the target analytes; relative standard deviations (% RSD) for m = 6 were between 2.8 and 7.6%. The concentration of the EDCs in environment sample was between 0.2 - 2.3 μg/L. / Chemistry / M. Sc. (Chemistry)

Koffein (1,3,7-Trimethylxanthin) und Coprostanol (5beta-cholestan-3beta-ol) wurden im Berliner Oberflächenwasser nachgewiesen. Ihre Konzentrationen korrelierten mit dem Verunreinigungsgrad der Proben, was nahelegt, dass sie sich als Marker für menschliche Aktivität eignen. Bemerkenswerterweise wurde Koffein in jeder einzelnen Oberflächenwasserprobe oberhalb der Bestimmungsgrenze von 0,025 µg/L gefunden. Um Oberflächenwasserproben in größeren Serien zu untersuchen, war die Entwicklung zweier neuer Methoden erforderlich: ein Immunoassay, basierend auf einem monoklonalen Antikörper für Koffein und eine dispersive flüssig-flüssig Mikroextraktionsmethode (DLLME), gefolgt von Flüssigkeitschromatographie gekoppelt mit Tandem-Massenspektrometrie (LC-MS/MS) für Coprostanol. Der entwickelte Koffein-Immunoassay zeigt die beste je erhaltene Nachweisgrenze für Koffein (0,001 µg/L), erlaubt Hochdurchsatz-Analysen und erfordert keine Probenvorbereitung. Der Assay wurde auch erfolgreich für die Messung von Koffein in Getränken, Haarwaschmitteln, Koffeintabletten und menschlichem Speichel angewendet. Antikörper gegen Coprostanol sind nicht kommerziell erhältlich. Eine neue Strategie Anti-Coprostanol-Antikörper zu generieren wurde erarbeitet, die eine analoge Verbindung – Isolithocholsäure (ILA) – als Hapten verwendet, mit der eine Gruppe von Mäusen immunisiert wurde. Ein polyklonales Anti-ILA-Serum wurde produziert, welches Coprostanol bindet, aber die niedrige Affinität erlaubte nicht den Aufbau eines Immunoassays, der die Messung von Umweltkonzentrationen des Anayten (im Bereich ng/L) zulässt. Spezifische Anti-ILA-Immunglobuline G wurden auch in den Faeces der Mäuse gefunden. Coprostanol wurde in den Wasserproben durch die Verwendung einer neuentwickelten LC-MS/MS-Methode unter APCI-Ionisation (atmospheric pressure chemical ionisation) gemessen. Konzentrationen oberhalb von 0,1 µg/L wurden nach Voranreicherung der Probe mittels DLLME bestimmt. / Caffeine (1,3,7-trimethylxanthine) and coprostanol (5beta-cholestan-3beta-ol) were detected in samples of Berlin’s surface water. Their concentrations correlated with the contamination status of the samples, suggesting their usefulness as markers of human activity. Remarkably, caffeine concentrations were always well above the limit of quantitation of 0.025 µg/L. In order to screen surface water samples in larger series, the development of two novel methods was required: a monoclonal antibody-based immunoassay for caffeine and a dispersive liquid-liquid microextraction (DLLME) method, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for coprostanol. The caffeine immunoassay developed shows the best analytical limit of detection (LOD) obtained so far for caffeine (0.001 µg/L), allows high-throughput analysis, and does not require sample pre-treatment. The assay was also successfully employed to measure caffeine in beverages, shampoos, caffeine tab-lets, and human saliva. Antibodies to coprostanol are not commercially available. A new strategy to generate anti-coprostanol antibodies was elaborated using an analogous com-pound as hapten – isolithocholic acid (ILA) – and immunizing a group of mice. A polyclonal anti-ILA serum was produced, which binds coprostanol but the low affinity did not permit setting up an immunoassay to measure environmental concentrations of the analyte (in the range of ng/L). Specific anti-ILA immunoglobulin G were also found in the faeces of the immunized mice. Coprostanol was quantified in the water samples using a newly developed LC-MS/MS method using atmospheric pressure chemical ionisation (APCI). Concentrations above 0.1 µg/L were determined after sample preconcentration using DLLME. This extraction method also proved to be successful for enrichment of coprostanol-related compounds such as cholesterol, cholestanol, cholestanone, ergosterol, and stigmasterol.