Development of Advanced Capillary Electrophoresis Techniques with UV and Mass Spectrometry Detection for Forensic, Pharmaceutical and Environmental Applications

Capillary electrophoresis (CE) is a modern analytical technique, which is electrokinetic separation generated by high voltage and taken place inside the small capillaries. In this dissertation, several advanced capillary electrophoresis methods are presented using different approaches of CE and UV and mass spectrometry are utilized as the detection methods. Capillary electrochromatography (CEC), as one of the CE modes, is a recent developed technique which is a hybrid of capillary electrophoresis and high performance liquid chromatography (HPLC). Capillary electrochromatography exhibits advantages of both techniques. In Chapter 2, monolithic capillary column are fabricated using in situ photoinitiation polymerization method. The column was then applied for the separation of six antidepressant compounds. Meanwhile, a simple chiral separation method is developed and presented in Chapter 3. Beta cycodextrin was utilized to achieve the goal of chiral separation. Not only twelve cathinone analytes were separated, but also isomers of several analytes were enantiomerically separated. To better understand the molecular information on the analytes, the TOF-MS system was coupled with the CE. A sheath liquid and a partial filling technique (PFT) were employed to reduce the contamination of MS ionization source. Accurate molecular information was obtained. It is necessary to propose, develop, and optimize new techniques that are suitable for trace-level analysis of samples in forensic, pharmaceutical, and environmental applications. Capillary electrophoresis (CE) was selected for this task, as it requires lower amounts of samples, it simplifies sample preparation, and it has the flexibility to perform separations of neutral and charged molecules as well as enantiomers. Overall, the study demonstrates the versatility of capillary electrophoresis methods in forensic, pharmaceutical, and environmental applications.

Otimização e validação de métodos empregando DLLME, SPE, HPLC-DAD e LC-ESI-MS/MS para determinação de agrotóxicos em água subterrânea

A preocupação com a poluição das águas por agrotóxicos tem aumentado, visto que aumentou o número de detecções de agrotóxicos em águas. A falta de avaliação da qualidade da água consumida pela população de áreas rurais onde não existe o abastecimento público de água potável, deve ser considerada, pois essas águas se encontram próximo a áreas de cultivo, onde há intensa aplicação de agrotóxicos. Nessas regiões, o abastecimento de água para as residências e para a irrigação é feito geralmente através das águas de poços. Neste trabalho, um método para determinação dos agrotóxicos carbofurano, clomazona, 2,4-D e tebuconazol em água subterrânea foi desenvolvido e validado. O método utilizou a Extração em Fase Sólida (SPE) e determinação por Cromatografia Líquida de Alta eficiência com Detecção por Arranjo de Diodos (HPLC-DAD) e confirmação por Cromatografia Líquida tandem Espectrometria de Massas (LC-MS/MS). Para a SPE utilizou-se cartuchos C18 de 200 mg, e eluição com 1 mL de metanol. Após a otimização dos parâmetros de extração e separação dos compostos, o método foi validado avaliando-se curva analítica, linearidade, limites de detecção e quantificação, precisão (repetitividade e precisão intermediária) e exatidão (recuperação). Todas as curvas analíticas apresentaram valores de r maiores que 0,99. Os LOQs para o método, considerando a etapa de pré-concentração de 250 vezes, foram de 0,2 µg L -1 para todos os agrotóxicos por HPLC-DAD e, por LC-MS/MS, 4,0 ng L -1 para clomazona, carbofurano e tebuconazol e de 40,0 ng L -1 para 2,4-D. As recuperações foram entre 60,3 e 107,7% para a repetitividade e entre 67,5 e 115,3% para a precisão intermediária, com RSD de 0,8 a 20,7% para todos os compostos por HPLC-DAD. Para o LC-MS/MS a precisão em termos de repetitividade, variou entre 0,97 e 20,7%, e as recuperações entre 67,0 e 108,9%. O método foi aplicado na determinação de agrotóxicos em amostras de águas subterrâneas durante um ano. Nas amostras foram detectados agrotóxicos em níveis de µg L -1 . Dentro do contexto atual da Química Analítica, de desenvolver métodos mais rápidos, que utilizem menor quantidade de solvente, de amostra e com altos fatores de enriquecimento, foi otimizado um método de extração para os agrotóxicos carbofurano, clomazona e tebuconazol utilizando a Microextração Líquido-Líquido Dispersiva (DLLME) e determinação por LC-MS/MS. Foram otimizados alguns parâmetros que influenciam no processo de extração, como: tipo e volume dos solventes dispersores e extratores, tempo de extração, força iônica e velocidade de centrifugação. Nas condições otimizadas, as recuperações para os níveis de concentração entre 0,02 e 2,0 g L -1 variaram entre 62,7 e 120,0%, com valores de RSD entre 1,9 e 9,1%. O LOQ do método foi de 0,02 µg L -1 para todos os compostos. Quando comparado com a SPE se demonstrou rápido, simples, de baixo custo, além de necessitar de menores volumes de amostra para determinação de agrotóxicos em águas. O método mostrou-se adequado à análise dos agrotóxicos em água subterrânea e todos os parâmetros de validação obtidos estão dentro dos limites sugeridos para validação de métodos cromatográficos

Otimização e validação de métodos empregando DLLME e DLLME-SFO para a extração simultânea de agrotóxicos multiclasse em água mineral

O Rio Grande Sul destaca-se no cenário nacional como grande produtor de diversas culturas, as quais demandam grande quantidade de agrotóxicos das mais diversas classes químicas e toxicidades. No entanto a intensa utilização destes compostos torna-se uma preocupação devido a possíveis contaminações das águas superficiais e subterrâneas. Em virtude da degradação dos mananciais a água mineral passou a ser uma das fontes mais utilizadas para o consumo humano, pois tem-se a percepção de que a mesma possui melhor qualidade que a água tratada, além disso acredita-se que a mesma esta isenta de substâncias orgânicas prejudiciais à saúde humana. Neste trabalho, foi realizada a determinação dos agrotóxicos atrazina, simazina, imazapique, imazetapir, imidacloprido, ciproconazol, tebuconazol e epoxiconazol em água mineral empregando a Microextração Líquido-Líquido Dispersiva (DLLME), Microextração Líquido-Líquido Dispersiva com Solidificação da Gota Orgânica Flutuante (DLLME-SFO) e Cromatografia Líquida acoplada à Espectrometria de Massas em série com fonte de ionização por Eletronebulização (LC-ESI-MS/MS). Para o método empregando DLLME e LC-ESI-MS/MS foram otimizados alguns fatores como o tipo e volume de solvente extrator e dispersor e pH. Após a otimização dos parâmetros de extração, fragmentação dos compostos e separação cromatográfica, o método foi validado avaliando-se curva analítica, linearidade, limites de detecção e quantificação, precisão (repetitividade e precisão intermediária) e exatidão (recuperação). Todas as curvas analíticas apresentaram valores de r maiores que 0,999. Os Limites de Quantificação (LOQs) para o método estiveram na faixa de 5 a 500 ng L-1. Foram obtidas recuperações entre 102 - 120% para a repetibilidade e entre 92 e 110% para a precisão intermediária, com RSD de 2 a 10% para todos os compostos. Para o método empregando DLLME-SFO e LC-ESI-MS/MS foram avaliados alguns parâmetros que afetam a eficiência da extração como, tipo e volume de solvente extrator e dispersor, força iônica e pH. Nas condições ótimas de extração todas as curvas analíticas apresentaram valores de r maiores que 0,997. Os LOQs para o método variaram entre 12,5 - 125 ng L-1. As recuperações foram entre 70 e 118% para a repetitividade e entre 76 e 95% para a precisão intermediária, com RSD de 2 a 18% para todos os compostos. Com relação ao Efeito Matriz (EM) avaliado para todos os compostos pelos dois métodos, foi observado baixo EM. Isso indicou que não é necessário utilizar a curva analítica preparada no extrato branco da matriz para a quantificação destes analitos. Ambos os métodos foram aplicados para a determinação de resíduos de agrotóxicos em amostras de água mineral provenientes de diferentes regiões do estado do Rio Grande do Sul e não foram encontrados resíduos de agrotóxicos nas amostras analisadas. Os métodos validados apresentaram como principais vantagens baixo consumo de solventes orgânicos e amostra, rapidez, altos fatores de concentração e recuperações dentro da faixa aceitável. Os limites de quantificação dos métodos ficaram abaixo dos limites máximos de resíduos permitidos pela legislação brasileira para agrotóxicos em água mineral.

Automated surface sampling of lipids from worn contact lenses coupled with tandem mass spectrometry

The deposition of biological material (biofouling) onto polymeric contact lenses is thought to be a major contributor to lens discomfort and hence discontinuation of wear. We describe a method to characterize lipid deposits directly from worn contact lenses utilizing liquid extraction surface analysis coupled to tandem mass spectrometry (LESA-MS/MS). This technique effected facile and reproducible extraction of lipids from the contact lens surfaces and identified lipid molecular species representing all major classes present in human tear film. Our data show that LESA-MS/MS is a rapid and comprehensive technique for the characterization of lipid-related biofouling on polymer surfaces.

Using ambient ozone for assignment of double bond position in unsaturated lipids

Unsaturated lipids deposited onto a range of materials are observed to react with the low concentrations of ozone present in normal laboratory air. Parent lipids and ozonolysis cleavage products are both detected directly from surfaces by desorption electrospray ionisation mass spectrometry (DESI-MS) with the resulting mass spectra providing clear evidence of the double bond position within these molecules. This serendipitous process has been coupled with thin-layer chromatography (TLC) to provide a simple but powerful approach for the detailed structural elucidation of lipids present in complex biological extracts. Lipid extracts from human lens were deposited onto normal phase TLC plates and then developed to separate components according to lipid class. Exposure of the developed plates to laboratory air for ca. 1 h prior to DESI-MS analysis gave rise to ozonolysis products allowing for the unambiguous identification of double bond positions in even low abundant, unsaturated lipids. In particular, the co-localization of intact unsaturated lactosylceramides (LacCer) with products from their oxidative cleavage provide the first evidence for the presence of three isomeric LacCer (d18:0/24:1) species in the ocular lens lipidome, i.e., variants with double bonds at the n-9, n-7 and n-5 positions.

Novel fluorinated surfactants tentatively identified in firefighters using liquid chromatography quadrupole time-of flight tandem mass spectrometry and a case-control approach

Fluorinated surfactant-based aqueous film-forming foams (AFFFs) are made up of per- and polyfluorinated alkyl substances (PFAS) and are used to extinguish fires involving highly flammable liquids. The use of perfluorooctanesulfonic acid (PFOS) and other perfluoroalkyl acids (PFAAs) in some AFFF formulations has been linked to substantial environmental contamination. Recent studies have identified a large number of novel and infrequently reported fluorinated surfactants in different AFFF formulations. In this study, a strategy based on a case-control approach using quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) and advanced statistical methods has been used to extract and identify known and unknown PFAS in human serum associated with AFFF-exposed firefighters. Two target sulfonic acids [PFOS and perfluorohexanesulfonic acid (PFHxS)], three non-target acids [perfluoropentanesulfonic acid (PFPeS), perfluoroheptanesulfonic acid (PFHpS), and perfluorononanesulfonic acid (PFNS)], and four unknown sulfonic acids (Cl-PFOS, ketone-PFOS, ether-PFHxS, and Cl-PFHxS) were exclusively or significantly more frequently detected at higher levels in firefighters compared to controls. The application of this strategy has allowed for identification of previously unreported fluorinated chemicals in a timely and cost-efficient way.

Detection of alpha(2u)-globulin and its bound putative pheromones in the preputial gland of the Indian commensal rat (Rattus rattus) using mass spectrometry

The role of pheromones and pheromone-binding proteins in the laboratory rat has been extensively investigated. However, we have previously reported that the preputial gland of the Indian commensal rat produces a variety of pheromonal molecules and preputial glands would seem to be the predominant source for pheromonal communication. The presence of pheromone-binding proteins has not yet been identified in the preputial gland of the Indian commensal rat; therefore, the experiments were designed to unravel the alpha(2u)-globulin (alpha 2u) and its bound volatiles in the commensal rat. Total preputial glandular proteins were first fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently analyzed by mass spectrometry. Further, we purified alpha 2u and screened for the presence of bound pheromonal molecules with the aid of gas chromatography/mass spectrometry (GC/MS). A novel alpha 2u was identified with a high score and this protein has not been previously described as present in the preputial gland of Indian commensal rats.This novel alpha 2u was then characterized by tandem mass spectrometry (MS/MS). Peptides with m/z values of 969, 1192, 1303 and 1876 were further fragmented with the aid of MS/MS and generated de novo sequences which provided additional evidence for the presence of alpha 2u in the preputial gland. Finally, we identified the presence of farnesol 1 and 2 bound to alpha 2u. The present investigation confirms the presence of alpha 2u (18.54 kDa) in the preputial gland of the Indian commensal rat and identifies farnesol 1 and 2 as probably involved in chemo-communication by the Indian commensal rat.Copyright (C) 2010 John Wiley & Sons, Ltd.

Identification and characterization of a library of microheterogeneous cyclohexadepsipeptides from the fungus Isaria

Ten new cyclic hexadepsipeptides, six isariins and four isaridins, from the fungus Isaria have been identified and characterized by high-performance liquid chromatography, coupled to tandem electrospray ionization mass spectrometry (LC-ESIMS/MS). The isariins possess a beta-hydroxy acid residue and five alpha-amino acids, while isaridins contain a beta-amino acid, an alpha-hydroxy acid, and four alpha-amino acids. One- and two-dimensional NMR spectroscopy confirmed the chemical identity of some of the isariin fractions. Mass spectral fragmentation patterns of [M + H](+) ions reveal clear diagnostic fragment ions for the isariins and isaridins. Previously described cyclic depsipeptides, isarfelins from Isaria felina (Guo, Y. X.; Liu, Q. H.; Ng, T. B.; Wang H. X. Peptides 2005, 26, 2384), are now reassigned as members of the isaridin family. Examination of isaridin sequences revealed significant similarities with cyclic hexadepsipeptides such as destruxins and roseotoxins. The structure of an isariin (isariin A) investigated by NMR spectroscopy indicated the presence of a hybrid alpha beta C-11 turn, formed by the beta-hydroxy acid and glycine residues and a (D)Leu-(L)Ala type II' beta-turn. Additionally, the inhibitory effect of isariins and an isaridin on the intra-erythrocytic growth of Plasmodium falciparum is presented.

Disjunct eddy covariance measurements of volatile organic compound fluxes using proton transfer reaction mass spectrometry

Volatile organic compounds (VOCs) are emitted into the atmosphere from natural and anthropogenic sources, vegetation being the dominant source on a global scale. Some of these reactive compounds are deemed major contributors or inhibitors to aerosol particle formation and growth, thus making VOC measurements essential for current climate change research. This thesis discusses ecosystem scale VOC fluxes measured above a boreal Scots pine dominated forest in southern Finland. The flux measurements were performed using the micrometeorological disjunct eddy covariance (DEC) method combined with proton transfer reaction mass spectrometry (PTR-MS), which is an online technique for measuring VOC concentrations. The measurement, calibration, and calculation procedures developed in this work proved to be well suited to long-term VOC concentration and flux measurements with PTR-MS. A new averaging approach based on running averaged covariance functions improved the determination of the lag time between wind and concentration measurements, which is a common challenge in DEC when measuring fluxes near the detection limit. The ecosystem scale emissions of methanol, acetaldehyde, and acetone were substantial. These three oxygenated VOCs made up about half of the total emissions, with the rest comprised of monoterpenes. Contrary to the traditional assumption that monoterpene emissions from Scots pine originate mainly as evaporation from specialized storage pools, the DEC measurements indicated a significant contribution from de novo biosynthesis to the ecosystem scale monoterpene emissions. This thesis offers practical guidelines for long-term DEC measurements with PTR-MS. In particular, the new averaging approach to the lag time determination seems useful in the automation of DEC flux calculations. Seasonal variation in the monoterpene biosynthesis and the detailed structure of a revised hybrid algorithm, describing both de novo and pool emissions, should be determined in further studies to improve biological realism in the modelling of monoterpene emissions from Scots pine forests. The increasing number of DEC measurements of oxygenated VOCs will probably enable better estimates of the role of these compounds in plant physiology and tropospheric chemistry. Keywords: disjunct eddy covariance, lag time determination, long-term flux measurements, proton transfer reaction mass spectrometry, Scots pine forests, volatile organic compounds

Identification and characterization of a library of microheterogeneous cyclohexa-depsipeptides from the fungus

Ten new cyclic hexadepsipeptides, six isariins and four isaridins, from the fungus Isaria have been identified and characterized by high-performance liquid chromatography, coupled to tandem electrospray ionization mass spectrometry (LC-ESIMS/MS). The isariins possess a beta-hydroxy acid residue and five alpha-amino acids, while isaridins contain a beta-amino acid, an alpha-hydroxy acid, and four alpha-amino acids. One- and two-dimensional NMR spectroscopy confirmed the chemical identity of some of the isariin fractions. Mass spectral fragmentation patterns of [M + H](+) ions reveal clear diagnostic fragment ions for the isariins and isaridins. Previously described cyclic depsipeptides, isarfelins from Isaria felina (Guo, Y. X.; Liu, Q. H.; Ng, T. B.; Wang H. X. Peptides 2005, 26, 2384), are now reassigned as members of the isaridin family. Examination of isaridin sequences revealed significant similarities with cyclic hexadepsipeptides such as destruxins and roseotoxins. The structure of an isariin (isariin A) investigated by NMR spectroscopy indicated the presence of a hybrid alpha beta C-11 turn, formed by the beta-hydroxy acid and glycine residues and a (D)Leu-(L)Ala type II' beta-turn. Additionally, the inhibitory effect of isariins and an isaridin on the intra-erythrocytic growth of Plasmodium falciparum is presented.

Headspace solid-phase microextraction with novel sol-gel permethylated-beta-cyclodextrin/hydroxyl-termination silicone oil fiber for determination of polybrominated diphenyl ethers by gas chromatography-mass spectrometry in soil

A simple, sensitive, and accurate method for determination of polybrominated diphenyl ethers (PBDEs) in soil has been developed based on headspace solid-phase microextraction (HS-SPME) followed by gas chromatography-mass spectrometry (GC-MS). Permethylated-beta-cyclodextrin/hydroxyl-termination silicone oil (PM-beta-CD/OH-TSO) fiber was first prepared by sol-gel technology and employed in SPME procedure. By exploiting the superiorities of sot-gel coating technique and the advantages of the high hydrophobic doughnut-shaped cavity of PM-beta-CD, the novel fiber showed desirable operational stability and extraction ability. After optimization on extraction conditions like water addition, extraction temperature, extraction time, salts effect, and solvents addition, the method was validated in soil samples, achieving good linearity (r>0.999), precision (R.S.D. < 10%), accuracy (recovery>78%), and detection limits (S/N =3) raging from 13.0 to 78.3 pg/g. (c) 2007 Published by Elsevier B.V.

Determination of odorous compounds in water by headspace solid-phase microextraction-gas chromatography-mass spectrometry

Three major odorous compounds are 2-methylisoborneol ( 2-MIB), geosmin and beta-cyclocitral, which in water were determined by coupling headspace solid-phase microextraction ( HS-SPME) with gas chromatography-mass spectrometry (GC-MS). The operating conditions of HS-SPME, such as fibre type, salt concentration, water temperature, stirring, absorption time and desorption time were studied and discussed.The highest absorption of the odorous compounds were obtained under the following operating conditions as the addition of 30% ( m/V) NaCl, stirring at 60 degrees C for 40 min, using 65 mu m polydimethyl siloxane/divinylbenzene coated fibre. After the odorous compounds had been absorbed in the fibre under the optimal conditions of HSSPME, they were desorbed at 250 degrees C and determined by GC-MS. The limits of detection for geosmin, beta-cyclocitral and 2-MIB in water were 1. 0, 1. 3, 1. 7 ng/L, and the relative standard deviations for them were 4. 9%, 8. 4%, 6. 2%,respectively. There were good linear correlation (the calibration coefficients were all above 0. 997) for the three odorous compounds in the range of 5 similar to 1000 ng/ L. Therefore, trace levels of the odorous compounds at ng/L in water could be quantified by the simple method with satisfactory result.

虎眼万年青化学成分及抗癌药与DNA相互作用的电喷雾质谱研究

首先，对虎眼万年青中化学成分进行了分离提取研究，采用柱层析和薄层层析等方法，并利用电喷雾质谱技术跟踪洗脱流分，首次从虎眼万年青氯仿提取物中分离得到两个新化合物。其一为单菇内酷loliolide，通过FT－ICRMS高分辨质谱、IR和NMR等手段对其结构进行了确定，并通过IHNMR和旋光法确定了其绝对构型。此外，还利用电喷雾多级串联质谱（ESI一MSn）技术对其质谱裂解规律进行了系统研究，其分析结果与NMR解析结果完全一致，建立了该类化合物结构解析的简便、快速的质谱新方法；另一化合物为生物碱类化合物，采用电喷雾多级串联质谱技术对其质谱裂解规律进行了详细研究，并对其结构进行了初步推断，进一步的NMR结构确定正在进行中。其次，对虎眼万年青多糖成分进行了分离提取研究，并结合体外抑瘤活性评价实验，确定了两种具有抑瘤生物活性的多糖成分OC一2一1一c和OC一2一卜d。并通过SDS凝胶电泳和糖基组成分析，初步证明了虎眼万年青中的活性多糖成分为阿拉伯半乳糖蛋白（AGPs），为进一步研究虎眼万年青抗肿瘤活性提供了基础数据和理论指导。此外，还对虎眼万年青中的微量元素及其溶出率进行了初步的研究，研究结果表明，其水煎液中Cu/Zn比值明显低于癌症患者血清中的Cu/Zn比值，因此，虎眼万年青可能有助于调节癌症患者体内的Cu、Zn平衡，从而达到抗癌目的；而虎眼万年青中Se、Ge含量很低，不能作为两种微量元素的药用来源。为了进一步研究抗癌药物的作用机理，我们采用电喷雾质谱技术，对三种临床疗效较好的抗癌药物与DNA相互作用进行了系统的研究，确定了复合物的化学计量比及其结合的特异性，并利用竞争实验方法，研究了抗癌药物的相对结合强度。质谱实验结果与液相行为完全一致，其中抗癌药物米托葱醒为首次利用质谱进行研究。最后，采用电喷雾质谱技术，对硝酸盐离子簇合物进行了详细研究，发现了双电荷簇合物离子，并且在离子簇合物中还发现了魔数规律的存在。利用电喷雾多级串联质谱技术，研究了双电荷簇合物离子的质谱裂解规律，并对其稳定性进行了分析。此外，还讨论了溶液浓度和毛细管温度对离子簇合物形成的影响规律。

Determination of ultra-trace concentrations of elements in high purity tellurium by inductively coupled plasma mass spectrometry after Fe(OH)(3) coprecipitation

In this work, a method was established for the determination of impurities in high purity tellurium by inductively coupled plasma mass spectrometry (ICP-MS) after Fe(OH)(3) coprecipitation. After comparison of coprecipitation ability and separation efficiency between Fe(OH), and Al(OH)(3), Fe(OH)(3) was chosen as the precipitate. A separation factor of 160 for 200 mg tellurium was obtained under conditions of pH 9 and 2 mg of Fe3(+). The 13 elements, such as Bi, Sn, Pb, In, Tl, Cd, Cu, Co, Ni, Zn, Ti, Be and Zr, could be almost completely coprecipitated under these conditions. In addition, Te memory effect imposed on the ICP-MS instrument was assessed, as well as Te matrix effect that caused the low recovery of Ga, As, Sb and V in real sample was discussed. Finally, the method was evaluated through recovery test and was applied to practical sample analysis, with detection limits of most of the elements being below 0.15 mug g(-1) and R.S.D. below or at approximately 10%, which indicated that this method could fully satisfy the requirements for analysis of 99.999% similar to 99.9999% high purity Te.