<p>LC-MS of protein drugs requires new ideas in bonded phase design rather than adapting bonded phases from the realm of small-molecule drugs. The polymer-shell bonded phase is designed to interact with larger molecules and to shield proteins from the silica substrate. The particles consist of a core of solid silica and a shell of dense polymer brush. The polymer layer is thick enough to protect the protein from interactions with silanols to reduce peak tailing. The polymer contains multiple functional groups that introduce more selectivity. This design gives unprecedented LC resolution and MS sensitivity. Our group has developed polymer shell bonded phases for hydrophobic interaction chromatography (HIC-MS) of antibody-drug conjugates (ADCs), hydrophilic interaction liquid chromatography (HILIC-MS) of glycoproteins, and reversed-phase liquid chromatography (RPLC-MS) of monoclonal antibodies. Since HIC is not in-line compatible with MS due to the high salt levels, it is laborious to identify the constituents of HIC peaks. An MS-compatible alternative to HIC is reported here: native reversed phase liquid chromatography (nRPLC). This employs a mobile phase 50 mM ammonium acetate for high sensitivity in MS, and elution with a gradient of water/isopropanol. The nRPLC-MS data show that all ADC species, ranging from drug-to-antibody ratios of 1 to 8, remained intact and native on the column. As we adapt this concept to intact proteins, we find that lysozyme and α-chymotrypsinogen A are both eluted in their native conformations. We also use the polymer-shell concept to resolve IgG1 free thiol variants by RPLC-MS with 0.5% formic acid. Since there are always other variants besides the intended ones, the need for high MS sensitivity is desired to distinguish subtle mass change between disulfide bond and free thiols. Overall, MS sensitivity increases 10X relative while all of the thiol variants are well resolved by the polymethylmethacrylate bonded phase.</p>

<p>U okviru doktorske disertacije su razvijeni i optimizovani postupci pripreme uzoraka hrane (žitarica, sirovina, poluproizvoda i prehrambenih proizvoda na bazi žitarica, voća i povrća) i stočne hrane, kao i simultano određivanje tropanskih i glikoalkaloida primenom gasne i tečne hromatografije uz maseno-spektrometrijsku detekciju. Razdvajanje alkaloida primenom gasne hromatografije je izvedeno na kapilarnoj koloni (HP-5MS UI), uz detekciju sa kvadrupolnom masenom spektrometrijom SIM tehnikom, u intervalu m/z 50&ndash;550 a.m.u. Pri optimizovanim uslovima postignuto je razdvajanje homatropina, atropina i skopolamina u vremenu od 12.75 min, pri čemu<br />tropin i anisodamin nisu mogli biti detektovani. Glikoalkaloidi nisu direktno mogli biti određivani gasnom hromatografijom usled njihove visoke molekulske mase i male isparljivosti. Za simultano određivanje tropanskih alkaloida (tropin, l-hiosciamin, atropin, skopolamin, homatropin i anisodamin) i glikoalkaloida (&alpha;-solanin i &alpha;-kakonin) validirana je brza i osetljiva multirezidualna LC&ndash;MS/MS metoda. Kori&scaron;ćena je disperzivna čvrsto-fazna ekstrakcija sa 0.5% mravlje kiseline u acotonitrilu/vodi (75:25, v/v), solima magnezijum sulfat, natrijum hlorid i natrijum citrat kao i C₁₈ sorbent. Analiti su razdvojeni na Chirobiotic V koloni, sa mobilnom fazom sastavljenom od 10 mM amonijum formata u voda/acetonitril (90:10, v/v) i metanol/acetonitril (50:50, v/v), me&scaron;anim u odnosu 20:80 (v/v). Detekcija komponenti je izvedena u pozitivnom elektrosprej modu (ESI+), kori&scaron;ćenjem reakcionog vi&scaron;estrukog monitoring akvizacionog moda (MRM). Optimizovana tečno hromatografska metoda je podrazumevala brz i jednostavan proces ekstrakcije, sa prinosom od 61-111% za pojedine alkaloide, dobru linearnost u &scaron;irokom opsegu (5-80 &mu;g/kg, r²=0.998), dobru selektivnost, robusnost i preciznost (CV &lt; 5%). Granica detekcije (LOD) za sve alkaloide je bila u opsegu od 0.74 do 0.79 ng/g dok se granica kvantifikacije (LOQ) kretala od 2.17 do 2.46 ng/g u svim žitaricama, izuzev za tropin u soji i lanenom semenu (LOD = 1.55 i LOQ = 4.91 ng/g). Pri analizi uzoraka sa trži&scaron;ta alkaloidi nisu detektovani u analiziranim uzorcima hrane, kao &scaron;to su: p&scaron;enica, raž, kukuruz, bra&scaron;no od me&scaron;anih žitarica, kukuruzno bra&scaron;no, biskvit i krekeri. Stočna hrana namenjena ishrani svinja i peradi je sadržala atropin, skopolamin, &alpha;-solanin i &alpha;-kakonin u koncentracionom opsegu od 3.6 do 21.7 ng/g. Atropin i skopolamin su detektovani u koncentracionom opsegu od 2.8 do 9.8 ng/g u hrani namenjenoj ishrani krava i zečeva, dok u hrani nemenjenoj ishrani konja i divljih životinja alkaloidi nisu detektovani. U analiziranim uzorcima voća i povrća (jabuka, kru&scaron;ka, avokado, mrkva, krastvac i paprika) po prvi put u ovom radu su detektovani glikoalkaloidi, &alpha;-solanin i &alpha;-kakonin. Koncentracija oba glikoalkaloida se kretala u opsegu od 0.3 do 15 ng/g. Sadržaj glikoalkaloida u krompiru i čipsu (8708.9-18794.7 ng/g) se nalazio u okviru preporučenog ukupnog sadržaja glikoalkaloida od 200 mg/kg (sirova odvaga) (FAO/WHO, 1999). Apsorpcija i translokacija alkaloida od strane p&scaron;enice u koren, listove i stabljiku je ustanovljena upotrebom atropina ¹⁴C. Utvrđeno je da biljka nakon 15 dana apsorbuje 4.30% i 2.28% atropina ¹⁴C za niži (13676 dpm/g soil) i vi&scaron;i (37203 dpm/g soil) sadržaj standarda sa kojim je zemlji&scaron;te spajkovano, redom. Količina apsorbovanog atropina se smanjivala tokom rasta biljke. Biljka starosti 90 dana je apsorbovala 0.38% i 0.21% atropina ¹⁴C za niži i vi&scaron;i sadržaj standarda, redom. Povećanje koncentracije supstance u zemlji&scaron;tu nije uticalo na dalje povećanje apsorpcije. Do nagomilavanja supstance je do&scaron;lo u listovima (83%) dok su niže koncentracije zabilježene u korenu (8.6%), stabljici (4.2%) i semenu (4.1%). U analiziranim uzorcima vode, koja je poticala od ispiranja zemlji&scaron;ta obogaćenog atropinom ¹⁴C, nakon 30 dana supstanca je detektovana u koncentraciji od 0.5%. Tokom vremena je ustanovljeno smanjenje ispiranja ove supstance (0.01% nakon 90 dana). Analiza uzoraka zemlji&scaron;ta nakon 30 i 60 dana je pokazala značajne adsorpcione osobine atropina, tako da je količina čvrsto adsorbovanog (neekstrahovanog) atropina iznosila 60% i 70%, redom. Zaostali atropin je bio stabilan i nakon 90 dana.</p> / <p>The methods of sample preparation have been developed and optimized for food (grains, raw materials, food products based on grains and fruits and vegetables) and feed analyses for the purpose of simultaneous determination of tropane and glycoalkaloids by gas and liquid chromatography hyphenated to mass spectrometry. Separation of alkaloids by gas chromatography was achieved by using capillary column HP-5MS UI. The alkaloids have been detected by using a quadrupole mass spectrometer inSIM mode for the range of m/z 50&ndash;550 a.m.u. Under optimised conditions, good separation of homatropine, atropine and scopolamine was achieved after 12.75 min, while tropine and anisodamine couldn`t be detected. Glycoalkaloids could not be analysed directly by gas chromatography due to their high molecular weight and low volatility. For simultaneous determination of tropane alkaloids (tropine, atropine, scopolamine, homatropine, anisodamine) and glycoalkaloids (&alpha;-solanine, &alpha;-chaconine) fast and sensitive multiresidue LC&ndash;MS/MS method was validated. In sample preparation dispersive solid phase extraction (DSPE) was performed with 0.5% formic acid in acetonitrile/water (75:25, v/v) and a mixture of magnesium sulphate, sodium chloride, sodium citrate and C18 sorbent. The analytes were separated by isocratic HPLC on a Chirobiotic V column with the mobile phase that consisted of 10 mM ammonium formate in water/acetonitrile (90:10, v/v) and methanol/acetonitrile (50:50, v/v), mixed in the ratio of 20:80 (v/v).<br />Compounds were detected in positive electrospray ionization mode (ESI+), using multi reaction monitoring (MRM). Optimised liquid-chromatographic method exhibited good linearity in the range 5-80 ng/g (r²=0.998). The method has shown to be specific, selective, accurate (recoveries from 61- 111%), precise (CV &lt; 5%) and rugged. Calculated limits of detection (LOD) for all alkaloids were in the range 0.74-0.79 ng/g, while the limits of quantitation (LOQ) were in the range 2.2-4.9 ng/g in all grains, except for tropine in soybean and linseed (LOD = 1.55 and LOQ = 4.91 ng/g). The proposed method was applied in the analyses of samples obtained from local supermarkets. The alkaloids were not detected in following food samples:<br />wheat, rye, maize, mixed grain flour, corn flour, biscuits and crackers. The feeds for pigs and chicken were the most contaminated with atropine, scopolamine, &alpha;-solanine and &alpha;-chaconine with the contents of alkaloids in the range of 3.6 to 21.7 ng/g. Atropine and scopolamine were detected in cattle and rabbit feed in concentrations ranging from 2.8 to 9.8 ng/g, while in feed for horses and wild animals none of the target alkaloids were detected. Conducted research reports for the first time the presence of glycoalkaloids, &alpha;-solanine and &alpha;-chaconine in fruit and vegetable samples (apple, pear, avocado, carrot, cucumber and paprika). The concentrations of these alkaloids were in the range of 0.3 to 15 ng/g. The content of glycoalkaloids in potato and chips complied with the recommended content of total glycoalkaloids concentration of 200 mg/kg (raw weight) (FAO/WHO, 1999). Absorption and translocation of alkaloids in wheat (roots, leaves, stem) was determined by using atropine ¹⁴C. It was found that young wheat after 15 days absorbed approximately 4.30% and 2.28% of atropine ¹⁴C for low (13676 dpm/g soil) and high (37203 dpm/g soil) spiked levels in soil, respectively. Increase in the spiked concentration in the soil did not affect higher absorption of atropine ¹⁴C in wheat. The highest accumulation of atropine ¹⁴C was observed in the leaves (83%) while lower<br />concentrations were detected in the roots (8.6%), stems (4.2%) and seeds (4.1%). In collected water samples during the soil leaching, atropine ¹⁴C was detected in the concentration of 0.5%. Leaching decreased with the time (0.01% after 90 days). Analyses of soil samples after 30 and 60 days showed strong adsorption of atropine to the soil and quantity of adsorbed atropine was 60% and 70%, respectively. Adsorbed atropine in the soil was stable after 90 days.</p>

Os métodos convencionais para a determinação de fármacos em fluidos biológicos baseiam-se em técnicas cromatográficas e imunoquímicas. O tratamento prévio de amostras biológicas, o qual abrange as etapas de extração, pré-concentração e “clean-up", tem sido requerido nas análises de fármacos, para aumentar a sensibilidade e seletividade analítica. No entanto, nos últimos anos, com o avanço das técnicas instrumentais, diversas técnicas têm sido avaliados para a análise de diferentes fármacos em fluidos biológicos, destacando-se entre elas a Microextração em Fase Sólida (SPME) e a Cromatografia Líquida acoplada a Espectrometria de Massas (LC/MS). A SPME apresenta uma série de vantagens em relação às técnicas de extração tradicionais, ou seja: não requer instrumentação analítica sofisticada, não utiliza solvente orgânico, permite automação das análises, a reutilização das fibras extratoras e integra em um único sistema, a extração, concentração e introdução da amostra no sistema cromatográfico. Neste trabalho, foi desenvolvida uma interface versátil e de baixo custo, que permite o acoplamento das técnicas SPME-LC/MS para análise dos fármacos antidepressivos tricíclicos e anticonvulsivantes. O planejamento fatorial empregado mostrou ser uma ferramenta estatística importante e simples, sendo obtido mais informações com um número menor de experimentos, avaliando não só os efeitos principais como os efeitos de interação de todas as variáveis nas respostas. As condições cromatográficas otimizadas foram adequadas para a análise por LC/MS. Os níveis de detecção alcançados ressaltam a importância e destaque da técnica de cromatografia líquida de alta eficiência acoplada à espectrometria de massas (LC/MS). O método desenvolvido, tanto para os fármacos antidepressivos tricíclicos como para os anticonvulsivantes, mostrou especificidade, precisão, linearidade e limite de quantificação adequado para a análise. / Conventional methods used for the determination of drugs in biological fluids are based on chromatographic and immunochemical techniques. The biological samples treatment - which includes extraction, pre-concentration and clean up steps – has been required in drugs analysis in order to increase both analytical sensitivity and selectivity. Nevertheless, lately, within the advancements in instrumentation, different techniques have been evaluated for the analysis of different drugs in biological fluids, such as: solid phase microextraction (SPME) and liquid chromatography coupled to mass spectrometry (LC/MS). SPME presents many advantages towards the conventional extraction techniques (soxhlet, LLE and SPE), which include: use of simple analytical instrumentation, analysis automation, reuse of extractor fibers and integration of extraction, concentration and sample introduction in the same chromatographic system. In this work, a versatile and low cost interface was developed, which allows the coupling of SPME-LC/MS techniques to tricyclic antidepressants and anticonvulsivant drugs analysis. The employed factorial design has shown to be a simple and useful statistical tool. With this device more information could be obtained with fewer experiments by evaluating not only the main interaction effects but also the interaction effects of all variables on the results. The optimized chromatographic conditions were adequate for LC/MS analysis. The obtained detection levels highlight the importance of high performance liquid chromatography coupled to mass spectrometry (LC/MS). The developed method, for both tricyclic antidepressants and anticonvulsivants drugs, has presented specificity, accuracy, linearity and adequate limit of detection for this analysis.

<p>Liquid chromatography – mass spectrometry (LC-MS) methods were developed for the analysis of positron emission tomography (PET) radiotracers in biological matrices. Additionally, radiotracer chemistry was explored as an analytical tool for supporting LC-MS method development and imaging molecular interactions in miniaturised chemical analysis systems.</p><p>Conventional radiodetection methods can offer high sensitivity in the analysis of radiotracers in biological matrices, although with the short half-life of PET tracers, this mass sensitivity decreases rapidly with time. This limits the time frame for analysis, and may compromise the precision and accuracy of the later measurements. Performing LC-MS analysis of the dominant stable isotope form of the tracer removes such time restrictions.</p><p>An LC-MS/MS method was developed for determination of the tracer flumazenil in human plasma, with high inter-assay precision (RSD < 7%) and accuracy (95 – 104%). The method was applied in a multiple scan PET study where the plasma concentration spanned from 0.07 to 0.21 nM. The method removed the time restrictions associated with radiodetection methods and thus provided the opportunity of analysing a greater number of samples than would have been possible with radioanalysis.</p><p>Furthermore, an LC-MS/MS method was developed that provided an efficient metabolic screening tool of potential PET tracers, whereby the substrates could be collected directly from 11C-labelling batches. This permitted repeated incubation experiments without the need of repeated labelling syntheses. A para-methoxy-benzamide analogue of the radiotracer WAY-100635 was thus identified as a potential tracer with improved metabolic stability. Additionally, a capillary LC-MS method was developed with rapid (0.75 min) and efficient (> 99%) on-line high flow-rate extraction for determination of metabolic stability of PET radiotracers.</p><p>Finally, the concept of radionuclide imaging of miniaturised chemical analysis systems was demonstrated with the direct study of interactions within capillary extraction columns and microchannels moulded in a plastic CD and poly(dimethylsiloxane).</p>

Liquid chromatography – mass spectrometry (LC-MS) methods were developed for the analysis of positron emission tomography (PET) radiotracers in biological matrices. Additionally, radiotracer chemistry was explored as an analytical tool for supporting LC-MS method development and imaging molecular interactions in miniaturised chemical analysis systems. Conventional radiodetection methods can offer high sensitivity in the analysis of radiotracers in biological matrices, although with the short half-life of PET tracers, this mass sensitivity decreases rapidly with time. This limits the time frame for analysis, and may compromise the precision and accuracy of the later measurements. Performing LC-MS analysis of the dominant stable isotope form of the tracer removes such time restrictions. An LC-MS/MS method was developed for determination of the tracer flumazenil in human plasma, with high inter-assay precision (RSD &lt; 7%) and accuracy (95 – 104%). The method was applied in a multiple scan PET study where the plasma concentration spanned from 0.07 to 0.21 nM. The method removed the time restrictions associated with radiodetection methods and thus provided the opportunity of analysing a greater number of samples than would have been possible with radioanalysis. Furthermore, an LC-MS/MS method was developed that provided an efficient metabolic screening tool of potential PET tracers, whereby the substrates could be collected directly from 11C-labelling batches. This permitted repeated incubation experiments without the need of repeated labelling syntheses. A para-methoxy-benzamide analogue of the radiotracer WAY-100635 was thus identified as a potential tracer with improved metabolic stability. Additionally, a capillary LC-MS method was developed with rapid (0.75 min) and efficient (&gt; 99%) on-line high flow-rate extraction for determination of metabolic stability of PET radiotracers. Finally, the concept of radionuclide imaging of miniaturised chemical analysis systems was demonstrated with the direct study of interactions within capillary extraction columns and microchannels moulded in a plastic CD and poly(dimethylsiloxane).

As agrarian society developed, the most fertile soils able to sustain the nutritional requirements needed for high crop yield were assigned to farming, while the more penurious soils were left to uphold the forest ecosystems. Some temperate forests are developed on acidic soils considered to be nutrient poor, as much of the inorganic nutrients are entrapped in poorly weatherable soil minerals and not easily accessed by plant roots. In an undisturbed ecosystem, the largest contribution of available nutrients comes from the recycling of organically bound nutrients via the decomposition of dead plant material. If biomass is removed, for instance with a more intensified exploitation of the forest ecosystems including whole tree harvesting, this source of nutrients is consequently decreased. The importance of soil mineral weathering as a source of nutrients, and especially that promoted by soil biota, is thereby emphasized. This thesis addresses biotic parameters associated with mineral weathering. Different aspects of soil solution sampling strategies and analysis of different organic ligands as well as biomarkers for the estimation of fungal biomass were investigated. These chemical parameters were also evaluated as indicators of microbial activity in relation to mineral nutrient availability in soil. With the assumption that the current nutrient status of a soil will affect the microbial interest of certain minerals as sources of inorganic nutrients, a mineral amendment trial was performed in a Swedish boreal forest soil. Overall, the amended soil presented good nutrient status, but with a possible shortage of iron. Due to this, it was hypothesized that the amended mineral with the highest iron content i.e. biotite would cause an elevation of microbial activity in its vicinity when compared to the bulk soil. The level of microbial activity in the vicinity of the amended minerals was evaluated via quantification of organic acids and siderophores, as well as estimation of fungal biomass and enzymatic activity. The highest microbial activity was measured for the O horizon of the investigated podzol, although nothing indicated an elevated association with the amended minerals. In the E horizon, however, elevation in microbial activity was observed in the vicinity of the biotite mineral when compared with bulk soil, although only a few of the investigated parameters differed significantly when evaluated separately.   To enable this study, a highly sensitive analytical method employing liquid chromatography and mass spectrometry was developed to quantify a number of hydroxamate siderophores. On-line pre-concentration enabled detection of these organic ligands in the pico-molar range – a necessity when analyzing natural samples. Furthermore, an analytical method was developed for the estimation of fungal biomass via quantification of chitin-derived glucosamine, which also employed liquid chromatography and tandem mass spectrometry. Unlike currently available methods, the one presented in this thesis did not involve analyte derivatization, which resulted in high sample throughput while simultaneously avoiding complications involved with the additional derivatization procedure. The distribution of a group of organic ligands known as aromatic low molecular mass organic acids was also studied in a boreal forest podzol soil. Different sampling and samples preparation techniques, namely tension-lysimeters, soil centrifugation and liquid-soil extraction, were compared when analyzing soil solution components. Significant differences in analyte amount and species type were found between these sampling techniques. Some of the differences could be accounted for by variation in soil composition at different depths of the investigated podzol, but others could be attributed to structural differences within the studied analyte group. This clearly illustrated the intricacy of sampling and analysis when working with a sample matrix as complex and diverse as soil. As previously, liquid chromatography and mass spectrometry was used to quantify the analytes of interest. A highly sensitive analytical method was developed that was able to detect eleven aromatic low molecular mass organic acids in the nano-molar range. High selectivity was ensured by applying multiple reaction monitoring enabled by collision induced fragmentation of the analytes. / FORE

Sulfur amino acids have critical function as intracellular redox buffers and maintain homeostasis in the external milieu by combating oxidative stress. Synthesis of glutathione (GSH) is regulated at a substrate level by cysteine, which is synthesized by homocysteine via the transsulfuration pathway. Oxidative stress and diminished glutathione pools play a sustained role in the pathogenesis of acute pancreatitis. One of the aims of this study was to experimentally address the temporal relationship between plasma sulfur amino acid levels in patients suffering from acute pancreatitis. The data indicated low concentration of cysteine initially, at levels similar to those of healthy controls. Glutathione was found reduced whilst cysteinyl-glycine and γ- glutamyl transpeptidase activity were increased in both mild and severe attacks. As the disease progressed, glutathione and cysteinyl-glycine were further increased in mild attacks and cysteine levels correlated with homocysteine and γ-glutamyl transpeptidase activity. The progress of severe attacks was associated with glutathione depletion, reduced γ-glutamyl transpeptidase activity and increased cysteinyl-glycine, that correlated with glutathione depletion. The corollary that ample supply of cysteine and cysteinly-glycine does not contribute towards glutathione synthesis in acute pancreatitis poses an important issue that merits resolution. Heightened oxidative stress and depletion of glutathione rationalized the progression of disease in severe attacks. An upsurge that reactive oxygen species can shift redox state of cells is determined by the ratio of the abundant redox couples reduced and oxidized glutathione (GSH: GSSG) in cell. The study reported a novel methodology for quantification of total oxidized glutathione (tGSSG) and total glutathione (tGSH) in whole blood using reverse phase high performance liquid chromatography. The novelty of the method is ascertained by the use of a mercaptan scavenger 1, methyl-2-vinyl-pyridinium trifluromethanesulfonate for the total oxidized glutathione determination. The results reported permit quantitation of tGSSG and tGSH and was applied to a control group. Finally, the study was also focussed in developing a liquid chromatography-mass spectrometric method to evaluate free and conjugated bile acids in patients suffering from various degrees of cholestatic-hepatobiliary disorders. The study reported low levels of ursodeoxycholic acid (UDCA) and slightly high levels of lithocholic acid (LCA). All the primary bile acids seem to be conjugated with glycine and taurine amino acid.

New solid phase microextraction (SPME) method was developed for brain tissue bioanalysis on a liquid chromatography mass spectrometry platform. To achieve set objectives, in vivo SPME desorption process was optimized for high throughput analysis through the development of a desorption device. Subsequently, new SPME coatings were developed for the extraction of polar neurotransmitters from biological matrices. In a targeted analysis, in vivo SPME was used to monitor of changes in the concentrations of endogenous compounds (multiple neurotransmitters) and exogenous drugs (carbamazepine and cimetidine) in the striatum of the rat brain extracellular fluid. For the first time, SPME was used for quantitative analysis of neurotransmitters and also study spacial distribution of other drugs in different regions of the brain extracellular fluid. A new approach was developed for improved metabolites coverage in a global non-targeted metabolomics studies. The proposed in vivo method showed how complementary results can be obtained through the combination of microdialysis and SPME for simultaneous sampling of the brain extracellular fluid. Finally, in a clinical application, SPME was used to monitor changes in the concentration of multiple neurotransmitters during deep brain stimulation of the pre-frontal cortex of the brain.

An environmentally friendly, rapid and cost effective analytical procedure based on dispersive liquid-liquid microextraction was developed for the determination of six tetracyclines (TCs) in meat destined for human consumption. Meat extracts were analyzed for TCs using a sensitive and selective analytical technique, liquid chromatography tandem mass spectrometry. Various influencing factors on the extraction, separation and determination of TCs such as pH of mobile phases, type and volume of disperser solvent, type and volume of extraction solvent and sample pH were optimized. Validation parameters such as calibration function, limit of detection (LOD), limit of quantification (LOQ), detection capability (CCα), decision limit (CCβ), accuracy and precision were established according to EU commission decision 2002/657/EC. Linearity in the range of 25-200 μg kg-1 was obtained with regression coefficients ranging from 0.9991 to 0.9998. Recoveries of spiked blank muscle samples at three levels (i.e. 50, 100 and 150 μg kg-1) ranged from 80 to 101% and reproducibility was between 2 and 7%. The LODs and LOQs ranged from 2.22 to 3.59 μg kg-1 and from 7.38 to 11.49 μg kg-1 respectively. The CCα ranged from 105 to 111 μg kg-1 while CCβ ranged from 107 to 122 μg kg-1. The proposed method compared well with the dispersive solid phase extraction method and was successfully applied to the determination of TCs in meat samples. Some of the thirty bovine muscle samples obtained from local abattoirs and butcheries were found to contain two tetracycline antibiotics residues (chlortetracycline and oxytetracycline) with oxytetracycline being the most commonly detected. The concentration levels of the TC residues detected in the eleven bovine muscle samples were between 12.4 and 68.9 μg kg-1, levels that are lower than the European Union set maximum residue level (MRL) of 100 μg kg-1 hence the meat was fit for human consumption. / Chemistry / M. Sc. (Chemistry)

Os métodos convencionais para a determinação de fármacos em fluidos biológicos baseiam-se em técnicas cromatográficas e imunoquímicas. O tratamento prévio de amostras biológicas, o qual abrange as etapas de extração, pré-concentração e “clean-up”, tem sido requerido nas análises de fármacos, para aumentar a sensibilidade e seletividade analítica. No entanto, nos últimos anos, com o avanço das técnicas instrumentais, diversas técnicas têm sido avaliados para a análise de diferentes fármacos em fluidos biológicos, destacando-se entre elas a Microextração em Fase Sólida (SPME) e a Cromatografia Líquida acoplada a Espectrometria de Massas (LC/MS). A SPME apresenta uma série de vantagens em relação às técnicas de extração tradicionais, ou seja: não requer instrumentação analítica sofisticada, não utiliza solvente orgânico, permite automação das análises, a reutilização das fibras extratoras e integra em um único sistema, a extração, concentração e introdução da amostra no sistema cromatográfico. Neste trabalho, foi desenvolvida uma interface versátil e de baixo custo, que permite o acoplamento das técnicas SPME-LC/MS para análise dos fármacos antidepressivos tricíclicos e anticonvulsivantes. O planejamento fatorial empregado mostrou ser uma ferramenta estatística importante e simples, sendo obtido mais informações com um número menor de experimentos, avaliando não só os efeitos principais como os efeitos de interação de todas as variáveis nas respostas. As condições cromatográficas otimizadas foram adequadas para a análise por LC/MS. Os níveis de detecção alcançados ressaltam a importância e destaque da técnica de cromatografia líquida de alta eficiência acoplada à espectrometria de massas (LC/MS). O método desenvolvido, tanto para os fármacos antidepressivos tricíclicos como para os anticonvulsivantes, mostrou especificidade, precisão, linearidade e limite de quantificação adequado para a análise. / Conventional methods used for the determination of drugs in biological fluids are based on chromatographic and immunochemical techniques. The biological samples treatment - which includes extraction, pre-concentration and clean up steps – has been required in drugs analysis in order to increase both analytical sensitivity and selectivity. Nevertheless, lately, within the advancements in instrumentation, different techniques have been evaluated for the analysis of different drugs in biological fluids, such as: solid phase microextraction (SPME) and liquid chromatography coupled to mass spectrometry (LC/MS). SPME presents many advantages towards the conventional extraction techniques (soxhlet, LLE and SPE), which include: use of simple analytical instrumentation, analysis automation, reuse of extractor fibers and integration of extraction, concentration and sample introduction in the same chromatographic system. In this work, a versatile and low cost interface was developed, which allows the coupling of SPME-LC/MS techniques to tricyclic antidepressants and anticonvulsivant drugs analysis. The employed factorial design has shown to be a simple and useful statistical tool. With this device more information could be obtained with fewer experiments by evaluating not only the main interaction effects but also the interaction effects of all variables on the results. The optimized chromatographic conditions were adequate for LC/MS analysis. The obtained detection levels highlight the importance of high performance liquid chromatography coupled to mass spectrometry (LC/MS). The developed method, for both tricyclic antidepressants and anticonvulsivants drugs, has presented specificity, accuracy, linearity and adequate limit of detection for this analysis.