Contributo para a determinação simultânea, por cromatografia líquida de alta resolução, de carotenoides, vitamina A e vitamina E em amostras compostas por diferentes matrizes alimentares

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia Química e Biológica

QuEChERS: a sample preparation for extraction of carbaryl from rat feces

Carbamate compounds are an important group of cholinesterase inhibitors. There is a need for creating awareness regarding the risks of the inadequate carbamate use in the residential areas due to potential adverse human effects. Carbaryl is a commonly used pesticide worldwide. A simple, fast, and high throughput method was developed employing liquid chromatography with fluorescence detector to determine carbaryl residues in rat feces. The extraction was performed by using a rapid, easy, cheap, effective, reliable, and safe (QuEChERS) method, using acetonitrile as the extracting solvent. The parameters for the performance of the extraction method were optimized, such as ratio of mass of sample per volume of extraction solvent, QuEChERS content, and cleanup columns. Linear response was obtained for all calibration curves (solven and matrix-matched) over the established concentration range (5 500 mg/L) with a correlation coefficients higher than 0.999. The achieved recovery was 97.9% with relative standard deviation values of 1.1% (n D 4) at 167 mg/kg fortified concentration level and the limits of detection and quantification were 27.7 and 92.3 mg/kg respectively.

Relationship between plasma and red blood cell concentrations of quinine in Brazilian children with uncomplicated Plasmodium falciparummalaria on oral therapy

We determined the relationship between plasma and red blood cell concentrations of quinine in children with uncomplicated falciparum malaria from an endemic area of Amazonian region. Quinine was determined by high performance liquid chromatography with ultraviolet detection. In the steady state the ratio between plasma and red blood cell quinine concentration was 1.89 ± 1.25 ranging from 1.05 to 2.34. This result demonstrated that quinine do not concentrate in red blood cell of Brazilian children and characterize the absence of interracial difference in this relationship.

Homogneous time resolved fluorescence: a new technique for detection and quantifications of infliximab in autoimmune patients

RESUMO:As terapias biológicas revolucionaram o tratamento das doenças autoimunes nos últimos anos. Tipicamente têm como alvos mediadores importantes no mecanismo das doenças. Os antagonistas do fator de necrose tumoral-α (TNF-α) são um grupo de agentes biológicos muito prescrito, pois estão indicados no tratamento de doenças imuno-mediadas comuns, tais como artrite reumatoide, artrite idiopática juvenil, artrite psoriática, espondilite anquilosante, doença de Crohn e colite ulcerosa. Com o uso frequente de inibidores do TNF-α, tem-se tornado evidente que estes agentes têm um potencial imunogénico importante, que pode comprometer o prognóstico a longo prazo dos doentes cronicamente tratados. A produção de anticorpos anti-fármaco parece causar falência terapêutica secundária em muitos doentes. Um dos efeitos dos anticorpos anti-fármaco é o aumento da eliminação do fármaco. A eliminação do fármaco, por sua vez, varia entre indivíduos, refletindo diferentes perfis farmacocinéticos. A determinação dos níveis séricos mínimos do agente anti-TNF-α é assim muito informativa e pode auxiliar nas decisões terapêuticas. Contudo, os testes imunológicos para determinar as concentrações séricas do fármaco não estão facilmente disponíveis na prática clínica. De forma a investigar uma nova técnica potencialmente fidedigna e prática para a deteção e quantificação dos agentes biológicos anti-TNF-α, foi testada a técnica por HTRF (homogeneous time-resolved fluorescence resonance energy transfer) para a determinação de concentrações séricas de infliximab. Apesar de apresentar algumas limitações relacionadas com as condições de leitura da fluorescência, esta técnica provou obter resultados próximos das concentrações obtidas por ELISA (enzyme-linked immunosorbent assay) bridging. Adicionalmente, tem a vantagem de ser de execução muito mais fácil e rápida. Deste modo, a técnica por HTRF poderá ser otimizada e tornar-se uma valiosa ferramenta laboratorial para orientar as decisões terapêuticas em doentes autoimunes com falência da terapêutica anti-TNF-α.--------- ABSTRACT: Biologic therapies revolutionized the treatment of autoimmune diseases in the last years. Typically, they target important disease mediators. Tumor necrosis factor-alpha (TNF-α) antagonists constitute a very prescribed group of biologic agents as they are indicated for the treatment of common immune-mediated diseases, such as rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn’s disease and ulcerative colitis. With the increasing use of TNF-α inhibitors it has been noticed that they have an important immunogenic potential that can compromise long-term outcomes in chronically treated patients. The production of anti-drug antibodies seems to cause secondary therapeutic failure in many patients. One of the effects of anti-drug antibodies is the enhancement of drug clearance. Drug clearance, in turn, varies among individuals, reflecting different pharmacokinetic profiles. Determination of serum anti-TNF-α drug trough levels is though very informative and could support treatment decisions. However, immunologic assays to determine drug serum concentrations are not readily available in clinical practice. In order to investigate a potentially reliable and practical new technique for detection and quantification of anti-TNF-α biologic agents, homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technique was tested for determination of serum infliximab concentrations. Although presenting some limitations related with fluorescence reading conditions, this technique proved to give results close to the concentrations obtained by the widely used bridging enzyme-linked immunosorbent assay (ELISA). In addition, it has the advantage of being much easier and faster to perform. Thus, HTRF technique can be optimized and become a valuable laboratorial tool to guide treatment decisions in autoimmune patients with anti-TNF-α therapy failure.

MSight: an image analysis software for liquid chromatography-mass spectrometry.

Images obtained from high-throughput mass spectrometry (MS) contain information that remains hidden when looking at a single spectrum at a time. Image processing of liquid chromatography-MS datasets can be extremely useful for quality control, experimental monitoring and knowledge extraction. The importance of imaging in differential analysis of proteomic experiments has already been established through two-dimensional gels and can now be foreseen with MS images. We present MSight, a new software designed to construct and manipulate MS images, as well as to facilitate their analysis and comparison.

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.

A validated assay for measuring doxorubicin in biological fluids and tissues in an isolated lung perfusion model: matrix effect and heparin interference strongly influence doxorubicin measurements.

Doxorubicin is an antineoplasic agent active against sarcoma pulmonary metastasis, but its clinical use is hampered by its myelotoxicity and its cumulative cardiotoxicity, when administered systemically. This limitation may be circumvented using the isolated lung perfusion (ILP) approach, wherein a therapeutic agent is infused locoregionally after vascular isolation of the lung. The influence of the mode of infusion (anterograde (AG): through the pulmonary artery (PA); retrograde (RG): through the pulmonary vein (PV)) on doxorubicin pharmacokinetics and lung distribution was unknown. Therefore, a simple, rapid and sensitive high-performance liquid chromatography method has been developed to quantify doxorubicin in four different biological matrices (infusion effluent, serum, tissues with low or high levels of doxorubicin). The related compound daunorubicin was used as internal standard (I.S.). Following a single-step protein precipitation of 500 microl samples with 250 microl acetone and 50 microl zinc sulfate 70% aqueous solution, the obtained supernatant was evaporated to dryness at 60 degrees C for exactly 45 min under a stream of nitrogen and the solid residue was solubilized in 200 microl of purified water. A 100 microl-volume was subjected to HPLC analysis onto a Nucleosil 100-5 microm C18 AB column equipped with a guard column (Nucleosil 100-5 microm C(6)H(5) (phenyl) end-capped) using a gradient elution of acetonitrile and 1-heptanesulfonic acid 0.2% pH 4: 15/85 at 0 min-->50/50 at 20 min-->100/0 at 22 min-->15/85 at 24 min-->15/85 at 26 min, delivered at 1 ml/min. The analytes were detected by fluorescence detection with excitation and emission wavelength set at 480 and 550 nm, respectively. The calibration curves were linear over the range of 2-1000 ng/ml for effluent and plasma matrices, and 0.1 microg/g-750 microg/g for tissues matrices. The method is precise with inter-day and intra-day relative standard deviation within 0.5 and 6.7% and accurate with inter-day and intra-day deviations between -5.4 and +7.7%. The in vitro stability in all matrices and in processed samples has been studied at -80 degrees C for 1 month, and at 4 degrees C for 48 h, respectively. During initial studies, heparin used as anticoagulant was found to profoundly influence the measurements of doxorubicin in effluents collected from animals under ILP. Moreover, the strong matrix effect observed with tissues samples indicate that it is mandatory to prepare doxorubicin calibration standard samples in biological matrices which would reflect at best the composition of samples to be analyzed. This method was successfully applied in animal studies for the analysis of effluent, serum and tissue samples collected from pigs and rats undergoing ILP.

A phase I pharmacokinetic study of hypoxic abdominal stop-flow perfusion with gemcitabine in patients with advanced pancreatic cancer and refractory malignant ascites

PURPOSE: As no curative treatment for advanced pancreatic and biliary cancer with malignant ascites exists, new modalities possibly improving the response to available chemotherapies must be explored. This phase I study assesses the feasibility, tolerability and pharmacokinetics of a regional treatment of gemcitabine administered in escalating doses by the stop-flow approach to patients with advanced abdominal malignancies (adenocarcinoma of the pancreas, n = 8, and cholangiocarcinoma of the liver, n = 1). EXPERIMENTAL DESIGN: Gemcitabine at 500, 750 and 1,125 mg/m(2) was administered to three patients at each dose level by loco-regional chemotherapy, using hypoxic abdominal stop-flow perfusion. This was achieved by an aorto-caval occlusion by balloon catheters connected to an extracorporeal circuit. Gemcitabine and its main metabolite 2',2'-difluorodeoxyuridine (dFdU) concentrations were measured by high performance liquid chromatography with UV detection in the extracorporeal circuit during the 20 min of stop-flow perfusion, and in peripheral plasma for 420 min. Blood gases were monitored during the stop-flow perfusion and hypoxia was considered stringent if two of the following endpoints were met: pH </= 7.2, pO(2) nadir ratio </=0.70 or pCO(2) peak ratio >/=1.35. The tolerability of this procedure was also assessed. RESULTS: Stringent hypoxia was achieved in four patients. Very high levels of gemcitabine were rapidly reached in the extracorporeal circuit during the 20 min of stop-flow perfusion, with C (max) levels in the abdominal circuit of 246 (+/-37%), 2,039 (+/-77%) and 4,780 (+/-7.3%) mug/ml for the three dose levels 500, 750 and 1,125 mg/m(2), respectively. These C (max) were between 13 (+/-51%) and 290 (+/-12%) times higher than those measured in the peripheral plasma. Similarly, the abdominal exposure to gemcitabine, calculated as AUC(t0-20), was between 5.5 (+/-43%) and 200 (+/-66%)-fold higher than the systemic exposure. Loco-regional exposure to gemcitabine was statistically higher in presence of stringent hypoxia (P < 0.01 for C (max) and AUC(t0-20), both normalised to the gemcitabine dose). Toxicities were acceptable considering the complexity of the procedure and were mostly hepatic; it was not possible to differentiate the respective contributions of systemic and regional exposures. A significant correlation (P < 0.05) was found between systemic C (max) of gemcitabine and the nadir of both leucocytes and neutrophils. CONCLUSIONS: Regional exposure to gemcitabine-the current standard drug for advanced adenocarcinoma of the pancreas-can be markedly enhanced using an optimised hypoxic stop-flow perfusion technique, with acceptable toxicities up to a dose of 1,125 mg/m(2). However, the activity of gemcitabine under hypoxic conditions is not as firmly established as that of other drugs such as mitomycin C, melphalan or tirapazamine. Further studies of this investigational modality, but with bioreductive drugs, are therefore warranted first to evaluate the tolerance in a phase I study and later on to assess whether it does improve the response to chemotherapy.

Fate of the herbicide glyphosate and its metabolite AMPA in soils and their transfer to surface waters: A multi-scale approach in the Lavaux vineyards, western Switzerland

The use of herbicides in agriculture may lead to environmental problems, such as surface water pollution, with a potential risk for aquatic organisms. The herbicide glyphosate is the most used active ingredient in the world and in Switzerland. In the Lavaux vineyards it is nearly the only molecule applied. This work aimed at studying its fate in soils and its transfer to surface waters, using a multi-scale approach: from molecular (10-9 m) and microscopic scales (10-6 m), to macroscopic (m) and landscape ones (103 m). First of all, an analytical method was developed for the trace level quantification of this widely used herbicide and its main by-product, aminomethylphosphonic acid (AMPA). Due to their polar nature, their derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) was done prior to their concentration and purification by solid phase extraction. They were then analyzed by ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The method was tested in different aqueous matrices with spiking tests and validated for the matrix effect correction in relevant environmental samples. Calibration curves established between 10 and 1000ng/l showed r2 values above 0.989, mean recoveries varied between 86 and 133% and limits of detection and quantification of the method were as low as 5 and 10ng/l respectively. At the parcel scale, two parcels of the Lavaux vineyard area, located near the Lutrive River at 6km to the east of Lausanne, were monitored to assess to which extent glyphosate and AMPA were retained in the soil or exported to surface waters. They were equipped at their bottom with porous ceramic cups and runoff collectors, which allowed retrieving water samples for the growing seasons 2010 and 2011. Results revealed that the mobility of glyphosate and AMPA in the unsaturated zone was likely driven by the precipitation regime and the soil characteristics, such as slope, porosity structure and layer permeability discrepancy. Elevated glyphosate and AMPA concentrations were measured at 60 and 80 cm depth at parcel bottoms, suggesting their infiltration in the upper parts of the parcels and the presence of preferential flow in the studied parcels. Indeed, the succession of rainy days induced the gradual saturation of the soil porosity, leading to rapid infiltration through macropores, as well as surface runoff formation. Furthermore, the presence of more impervious weathered marls at 100 cm depth induced throughflows, the importance of which for the lateral transport of the herbicide molecules was determined by the slope steepness. Important rainfall events (>10 mm/day) were clearly exporting molecules from the soil top layer, as indicated by important concentrations in runoff samples. A mass balance showed that total loss (10-20%) mainly occurred through surface runoff (96%) and, to a minor extent, by throughflows in soils (4%), with subsequent exfiltration to surface waters. Observations made in the Lutrive River revealed interesting details of glyphosate and AMPA dynamics in urbanized landscapes, such as the Lavaux vineyards. Indeed, besides their physical and chemical properties, herbicide dynamics at the catchment level strongly depend on application rates, precipitation regime, land use and also on the presence of drains or constructed channels. Elevated concentrations, up to 4970 ng/l, observed just after the application, confirmed the diffuse export of these compounds from the vineyard area by surface runoff during main rain events. From April to September 2011, a total load of 7.1 kg was calculated, with 85% coming from vineyards and minor urban sources and 15% from arable crops. Small vineyard surfaces could generate high concentrations of herbicides and contribute considerably to the total load calculated at the outlet, due to their steep slopes (~10%). The extrapolated total amount transferred yearly from the Lavaux vineyards to the Lake of Geneva was of 190kg. At the molecular scale, the possible involvement of dissolved organic matter (DOM) in glyphosate and copper transport was studied using UV/Vis fluorescence spectroscopy. Combined with parallel factor (PARAFAC) analysis, this technique allowed characterizing DOM of soil and surface water samples from the studied vineyard area. Glyphosate concentrations were linked to the fulvic-like spectroscopic signature of DOM in soil water samples, as well as to copper, suggesting the formation of ternary complexes. In surface water samples, its concentrations were also correlated to copper ones, but not in a significant way to the fulvic-like signature. Quenching experiments with standards confirmed field tendencies in the laboratory, with a stronger decrease in fluorescence intensity for fulvic-like fluorophore than for more aromatic ones. Lastly, based on maximum concentrations measured in the river, an environmental risk for these compounds was assessed, using laboratory tests and ecotoxicity data from the literature. In our case and with the methodology applied, the risk towards aquatic species was found negligible (RF<1).

Determinants of the essential one-carbon metabolism metabolites, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine and folate, in cerebrospinal fluid.

BACKGROUND: Disturbances in the levels of one-carbon (1C) metabolism metabolites have been associated with a wide variety of neuropsychiatric diseases. Cerebrospinal fluid (CSF) levels of homocysteine (Hcy) and the other 1C metabolites, nor their interrelatedness and putative determinants, have been studied extensively in a healthy population. METHODS: Plasma and CSF samples from 100 individuals free from neuropsychiatric diseases were analyzed (55 male, 45 female; age 50±17 years). In blood, we measured plasma Hcy, serum folate and serum vitamin B12. In CSF, we measured total Hcy, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and 5-methyltetrahydrofolate (5-methylTHF). Highly selective analytical methods like liquid chromatography combined with either mass spectrometry or fluorescence detection were used. RESULTS: CSF Hcy was inversely correlated with CSF 5-methylTHF and positively with plasma Hcy, independent of serum folate status. CSF SAH correlated with age, lower CSF 5-methylTHF and higher CSF Hcy. CSF 5-methylTHF showed independent negative correlations with age and positive correlations with serum folate. CSF SAM did not correlate with any of the 1C metabolites. CONCLUSIONS: Aging is characterized by a reduction in CSF 5-methylTHF levels and increased CSF levels of the potentially neurotoxic transmethylation inhibitor SAH. CSF 5-methylTHF, which is itself determined in part by systemic folate status, is a powerful independent determinant of CSF levels of Hcy and SAH.

Fast and sensitive supercritical fluid chromatography - tandem mass spectrometry multi-class screening method for the determination of doping agents in urine.

This study shows the possibility offered by modern ultra-high performance supercritical fluid chromatography combined with tandem mass spectrometry in doping control analysis. A high throughput screening method was developed for 100 substances belonging to the challenging classes of anabolic agents, hormones and metabolic modulators, synthetic cannabinoids and glucocorticoids, which should be detected at low concentrations in urine. To selectively extract these doping agents from urine, a supported liquid extraction procedure was implemented in a 48-well plate format. At the tested concentration levels ranging from 0.5 to 5 ng/mL, the recoveries were better than 70% for 48-68% of the compounds and higher than 50% for 83-87% of the tested substances. Due to the numerous interferences related to isomers of steroids and ions produced by the loss of water in the electrospray source, the choice of SFC separation conditions was very challenging. After careful optimization, a Diol stationary phase was employed. The total analysis time for the screening assay was only 8 min, and interferences as well as susceptibility to matrix effect (ME) were minimized. With the developed method, about 70% of the compounds had relative ME within the range ±20%, at a concentration of 1 and 5 ng/mL. Finally, limits of detection achieved with the above-described strategy including 5-fold preconcentration were below 0.1 ng/mL for the majority of the tested compounds. Therefore, LODs were systematically better than the minimum required performance levels established by the World anti-doping agency, except for very few metabolites.

Alendronato de sódio: metodologias para análise quantitativa

This paper presents a review of some published proposals for the analysis of sodium alendronate. The drug is an aminobisphosphonate compound used to inhibit the osteoclastic resorption of bone, and different methods were developed for its quantitative determination. These methodologies employed reversed-phase or ion-exchange HPLC analysis, both associated with different detectors: UV and fluorescence detection after derivatization of the drug, conductivity and refractive index detectors, as well as the indirect UV detection. Titrimetry and spectrophotometry (with previous complexation of the drug), which are simpler procedures, were also described, but they showed poor specificity when compared to liquid chromatography.

Avaliação de solventes de extração por ultrassom usando-se cromatografia líquida de alta eficiência para a determinação de hidrocarbonetos policíclicos aromáticos em solos contaminados

A method using ultrasonication extraction for the determination of 17 polycyclic aromatic hydrocarbons (PAHs), selected by the USEPA and NIOSH as "consent decree" priority pollutants, in soil by High Performance Liquid Chromatography (HPLC) was studied. Separation and detection were completed in 20 min with a C18 columm, acetonitrile-water gradient elution and ultraviolet absorption and fluorescence detections. The detection limits, for a 10 µL of solution injection, were less than 9,917 ng/g in UV detection and less than 1,866 ng/g in fluorescence detection. Several organic solvents were tested for extraction of the 17 PAHs from soils. Acetone was the best solvent among the three solvents tested, and the order of the extraction efficiencies was: acetone>methanol>acetonitrile. Ultrasonication using acetone as solvent extraction was used to evaluate the biodegradation of those compounds in contaminated soil during a vermicomposting process.

Super/subcritical fluid chromatography with packed columns: state of the art and applications

Separations using supercritical fluid chromatography (SFC) with packed columns have been re-discovered and explored in recent years. SFC enables fast and efficient separations and, in some cases, gives better results than high performance liquid chromatography (HPLC). This paper provides an overview of recent advances in SFC separations using packed columns for both achiral and chiral separations. The most important types of stationary phases used in SFC are discussed as well as the most critical parameters involved in the separations and some recent applications.

A simple HPLC-fluorescence method for the measurement of R,S-sotalol in the plasma of patients with life-threatening cardiac arrhythmias

R,S-sotalol, a ß-blocker drug with class III antiarrhythmic properties, is prescribed to patients with ventricular, atrial and supraventricular arrhythmias. A simple and sensitive method based on HPLC-fluorescence is described for the quantification of R,S-sotalol racemate in 500 µl of plasma. R,S-sotalol and its internal standard (atenolol) were eluted after 5.9 and 8.5 min, respectively, from a 4-micron C18 reverse-phase column using a mobile phase consisting of 80 mM KH2PO4, pH 4.6, and acetonitrile (95:5, v/v) at a flow rate of 0.5 ml/min with detection at lex = 235 nm and lem = 310 nm, respectively. This method, validated on the basis of R,S-sotalol measurements in spiked blank plasma, presented 20 ng/ml sensitivity, 20-10,000 ng/ml linearity, and 2.9 and 4.8% intra- and interassay precision, respectively. Plasma sotalol concentrations were determined by applying this method to investigate five high-risk patients with atrial fibrillation admitted to the Emergency Service of the Medical School Hospital, who received sotalol, 160 mg po, as loading dose. Blood samples were collected from a peripheral vein at zero, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0 and 24.0 h after drug administration. A two-compartment open model was applied. Data obtained, expressed as mean, were: CMAX = 1230 ng/ml, TMAX = 1.8 h, AUCT = 10645 ng h-1 ml-1, Kab = 1.23 h-1, a = 0.95 h-1, ß = 0.09 h-1, t(1/2)ß = 7.8 h, ClT/F = 3.94 ml min-1 kg-1, and Vd/F = 2.53 l/kg. A good systemic availability and a fast absorption were obtained. Drug distribution was reduced to the same extent in terms of total body clearance when patients and healthy volunteers were compared, and consequently elimination half-life remained unchanged. Thus, the method described in the present study is useful for therapeutic drug monitoring purposes, pharmacokinetic investigation and pharmacokinetic-pharmacodynamic sotalol studies in patients with tachyarrhythmias.