As micotoxinas são compostos produzidos por fungos, sendo potencialmente perigosos à saúde humana e animal. A Ocratoxina A (OTA) é uma das micotoxinas mais amplamente estudadas, sendo encontrada em várias matrizes alimentícias. A concentração dessa micotoxina nos alimentos é geralmente muito baixa (da ordem de ng g-1), sendo, portanto, necessário o emprego de técnicas de preparo de amostras que realizem a purificação e pré-concentração no analito. Os métodos de separação e detecção empregados na análise de OTA também devem oferecer sensibilidade adequada para a quantificação do analito. Logo, os objetivos do trabalho foram desenvolver uma metodologia utilizando a técnica in-tube SPME no modo de extração única (flow through extraction) com partículas de C18 como fase extratora e separação e detecção por HPLC-MS/MS. Além disso, otimizar e validar a metodologia proposta e detectar e quantificar a OTA em amostras de vinho. Para isso, a técnica in-tube SPME foi configurada e um tubo de PEEK foi empacotado com partículas de C18, o qual foi utilizado na extração. A otimização do método foi realizada utilizando-se um planejamento experimental composto central 22 + 3 pontos centrais, tendo como fatores a porcentagem de ACN e o tempo durante o carregamento da amostra. A validação da metodologia empregada foi realizada conforme o guia de validação da ANVISA e, posteriormente, amostras de vinho tinto seco e vinho branco seco foram analisadas. O método proposto foi desenvolvido, tendo sua funcionalidade atestada e suas condições de análise melhoradas, sendo utilizado 22 % de ACN e 6 minutos no carregamento da amostra. O método foi validado, mostrando sensibilidade adequada, com limites de detecção e quantificação iguais à 0,02 e 0,05 µg L-1, respectivamente. A linearidade e precisão da metodologia foram avaliadas, apresentando coeficiente de correlação igual a 0,996 e DPR menor que 6%, respectivamente. O método mostrou-se exato nos níveis de concentração médio e alto e a recuperação máxima obtida para o nível alto foi próximo 73%. Amostras brasileiras e estrangeiras de vinho tinto seco e vinho tinto seco branco foram analisadas e a OTA não foi detectada em nenhuma delas. Entretanto, a OTA pode essa estar presente nas amostras analisadas em concentrações mais baixas que as determinadas pelos limites de detecção e quantificação, não sendo potencialmente perigosos à saúde. / Mycotoxins are compounds produced by fungus, being a potential danger to human and animal health. Ochratoxin A (OTA) is a mycotoxin widely studied, being found in a varied of food matrices. OTA concentration in food is generally low (the order of ng g-1), being, therefore, necessary the use of sample preparation techniques to perform the analyte purification and preconcentration in the sample. The separation and detection methods used in OTA analysis also should offer proper sensibility in order to quantify the analyte adequately. In short, the goals of the present work were to develop a methodology using in-tube SPME in flow through extraction mode with C18 particles as extraction phase and separation and detection by HPLC-MS/MS. Further, optimize and validate the proposed methodology and detect and quantify OTA in wine samples. For this, in-tube SPME was configured and a PEEK tube was packed with C18 particles, which was used in the extraction step. The method optimization was achieved using a central composite 22 + 3 central points experimental design, having as factors the percentage of ACN and time during sample loading step. Validation method was done following ANVISA validation guide after which red dry wine and white dry wine samples were analyzed. The proposed method was developed, having its functionalities attested and its analysis conditions enhanced, using 22% of ACN and 6 minutes in sample loading. The method was validated, demonstrating proper sensitivity, with detection and quantification limits equal to 0.02 and 0.05 µg L-1, respectively. Linearity and precision were evaluated, exhibiting correlation coefficient equal to 0,996 e RSD under 6%, respectively. The method was accurate in medium and high concentration levels and maximum recovery was 73% in high concentration level. Brazilian and foreign red dry wine and white dry wine samples were analyzed and OTA was not detected in any of them. However, OTA may be present in samples analyzed in lower concentrations than that ones determined by detection and quantification limits, not being a potential danger to human health.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2018-08-20T12:37:22Z No. of bitstreams: 1 2010 - Raquel Duarte da Costa Cunha Bandeira.pdf: 1478855 bytes, checksum: f7637b4df6dd802e4e0003f4fb48d8cd (MD5) / Made available in DSpace on 2018-08-20T12:37:22Z (GMT). No. of bitstreams: 1 2010 - Raquel Duarte da Costa Cunha Bandeira.pdf: 1478855 bytes, checksum: f7637b4df6dd802e4e0003f4fb48d8cd (MD5) Previous issue date: 2010-09-24 / Coffee is an extremely complex food matrix and has an important role in the world?s economy, especially in producing and exporting countries like Brazil. However this product may suffer from technical barriers imposed for exportation because of the possible presence of ochratoxin A, which is nefrotoxic and carcinogenic mycotoxin found in many foods including coffee. The aim of this study was to implement chemical metrology concepts in the development and validation of Liquid Chromatography with Mass Spectrometry in tandem (CLAE-EM/EM) method for identification and quantification of ochratoxin A in green and roasted coffee estimating uncertainty of measurement according to directive 2002/657/EC and Inmetro guidelines (DOC-CGCRE-2010). The extraction method was based on Pittet (1998) and chromatographic parameters were: flow rate of 0.3 mL/min, mobile phase 80:20 water trifluoracetic acid 0.05 %: methanol trifluoracetic acid 0.05 %, injection volume of 50 PL, injection mode Full loop, isocratic mode. The column was Synergi Hydro C18. The mass spectrometry parameters were optimized and transitions selected based on the colision energies monitored were m/z 404 >358 (-10.5 V) and m/z 404 >239 (-20.5 V). From the validation procedure, methods were considered seletive. The evaluation and verification of matrix effect was performed by comparing variances and averages using F and t test. Value of Fcalculated for green coffee (25.2152) and roasted coffee (104.0353), were higher than Ftable (4.0426). Value of t calculated for green (5.0214) and roasted coffee (10.1997) were higher than ttable (2.0106). Both methods were considered linear in the working range of calibration curve with linear correlation coefficients (r) of 0.98188 and 0.91754 for green and roasted coffee, respectively.The quantification and detection limits were 1.2 Pg/kg and 3.0 Pg/kg; 0.36 Pg/kg and 1.0 Pg/kg, for green and roasted coffee respectively. The average recoveries, RSDr and RSDR were in range of 90.45 ? 108.81 %, 5.39 ? 9.94 % and 2.20 ? 14.34 % for green coffee and 89.02 ? 108.85 %, 2.43 ? 13.73 % and 12.57 ? 17.84 % for roasted coffee. All results obtained were considered within acceptable levels according to literature. Measurement value and expanded uncertainties (U) for ochratoxin A were mass fraction w = (11.50 ? 1.11) and w = (4.63 ? 0.63) for green coffee and roasted coffee. Both methods developed and validated using a high sensitivity technique, that allowed detection, confirmation and quantification of ochratoxin A in green and roasted coffee with a estimated uncertainty of measurement, and in the future these methods can be used to help overcome possible technical barriers imposed for exportation of Brazilian coffee. / O caf? constitui uma matriz extremamente complexa e tem importante papel na economia mundial, especialmente nos pa?ses produtores e exportadores como o Brasil. No entanto tem sido alvo de barreiras t?cnicas devido a uma subst?ncia denominada ocratoxina A, micotoxina potencialmente nefrot?xica e nefrocarcinog?nica encontrada em muitos alimentos inclusive o caf?. O presente trabalho tem como objetivo implantar os conceitos da metrologia qu?mica no desenvolvimento, e valida??o do m?todo para identifica??o e quantifica??o de ocratoxina A no caf? verde e caf? torrado estimando a incerteza da medi??o e utilizando a t?cnica de Cromatografia L?quida acoplada a Espectrometria de Massas em s?rie (CLAE-EM/EM) seguindo os crit?rios da diretiva EC-657/2002 e o documento orientativo do Inmetro (DOCCGCRE- 2010). A metodologia de extra??o baseou-se em Pittet (1998) e os par?metros cromatogr?ficos foram: fluxo de 0,3 mL/min, fase m?vel 80:20 ?gua ?cido trifluoroac?tico 0,05%: metanol ?cido trifluoroac?tico 0,05 %, volume de inje??o de 50 PL, com o modo de inje??o Full loop e sistema de elui??o isocr?tico. A coluna utilizada foi Synergi Hydro C18. As condi??es do espectr?metro de massas foram otimizadas e a transi??o selecionada de acordo com suas energias de colis?o foram m/z 404 >358 (-10,5 V) e m/z 404 >239 (-20,5 V). A partir da valida??o os m?todos propostos foram considerados seletivos, a avalia??o e comprova??o do efeito matriz foi realizada atrav?s da compara??o das vari?ncias e das m?dias atrav?s do teste F e teste t. O Fcalculado para o m?todo caf? verde (25,2152) e caf? torrado (104,0353), apresentaram valores maiores que o Ftabelado (4,0426). O tcalculado para o caf? verde (5,0214) e torrado (10,1997) apresentaram valores superiores ao ttabelado (2,0106). Os m?todos foram considerados lineares em toda a faixa de trabalho da curva de calibra??o com os coeficientes de determina??o linear (r) de 0,98188 e 0,91754 para matriz caf? verde e caf? torrado, respectivamente. O limite de quantifica??o e detec??o para os m?todos propostos foram de 1,2 Pg/kg e 3,0 Pg/kg para caf? verde e 0,36 Pg/kg e 1,0 Pg/kg para caf? torrado. Os valores das recupera??es m?dias, DPRr e DPRR variaram na faixa de 90,45 - 108,81 %, 5,39 - 9,94 % e 2,20 - 14,34 % para caf? verde; e de 89,02 - 108,85 %, de 2,43 - 13,73 % e 12,57 - 17,84 %, para caf? torrado. Todos os resultados obtidos encontram-se dentro dos limites comumente aceit?veis na literatura. Todos os resultados de medi??o e as incertezas expandidas (U) para ocratoxina A foram as fra??es m?ssicas W = (11,50 ? 1,11) Pg/kg e W = (4,63 ? 0,63) Pg/kg para caf? verde e caf? torrado, respectivamente. Os m?todos desenvolvidos e validados utilizaram t?cnica de elevada sensibilidade, permitindo a detec??o, confirma??o e a quantifica??o de ocratoxina A no caf? verde e caf? torrado com c?lculo da incerteza, podendo auxiliar futuramente na supera??o das barreiras t?cnicas para exporta??o do caf? brasileiro.

As micotoxinas são compostos produzidos por fungos, sendo potencialmente perigosos à saúde humana e animal. A Ocratoxina A (OTA) é uma das micotoxinas mais amplamente estudadas, sendo encontrada em várias matrizes alimentícias. A concentração dessa micotoxina nos alimentos é geralmente muito baixa (da ordem de ng g-1), sendo, portanto, necessário o emprego de técnicas de preparo de amostras que realizem a purificação e pré-concentração no analito. Os métodos de separação e detecção empregados na análise de OTA também devem oferecer sensibilidade adequada para a quantificação do analito. Logo, os objetivos do trabalho foram desenvolver uma metodologia utilizando a técnica in-tube SPME no modo de extração única (flow through extraction) com partículas de C18 como fase extratora e separação e detecção por HPLC-MS/MS. Além disso, otimizar e validar a metodologia proposta e detectar e quantificar a OTA em amostras de vinho. Para isso, a técnica in-tube SPME foi configurada e um tubo de PEEK foi empacotado com partículas de C18, o qual foi utilizado na extração. A otimização do método foi realizada utilizando-se um planejamento experimental composto central 22 + 3 pontos centrais, tendo como fatores a porcentagem de ACN e o tempo durante o carregamento da amostra. A validação da metodologia empregada foi realizada conforme o guia de validação da ANVISA e, posteriormente, amostras de vinho tinto seco e vinho branco seco foram analisadas. O método proposto foi desenvolvido, tendo sua funcionalidade atestada e suas condições de análise melhoradas, sendo utilizado 22 % de ACN e 6 minutos no carregamento da amostra. O método foi validado, mostrando sensibilidade adequada, com limites de detecção e quantificação iguais à 0,02 e 0,05 µg L-1, respectivamente. A linearidade e precisão da metodologia foram avaliadas, apresentando coeficiente de correlação igual a 0,996 e DPR menor que 6%, respectivamente. O método mostrou-se exato nos níveis de concentração médio e alto e a recuperação máxima obtida para o nível alto foi próximo 73%. Amostras brasileiras e estrangeiras de vinho tinto seco e vinho tinto seco branco foram analisadas e a OTA não foi detectada em nenhuma delas. Entretanto, a OTA pode essa estar presente nas amostras analisadas em concentrações mais baixas que as determinadas pelos limites de detecção e quantificação, não sendo potencialmente perigosos à saúde. / Mycotoxins are compounds produced by fungus, being a potential danger to human and animal health. Ochratoxin A (OTA) is a mycotoxin widely studied, being found in a varied of food matrices. OTA concentration in food is generally low (the order of ng g-1), being, therefore, necessary the use of sample preparation techniques to perform the analyte purification and preconcentration in the sample. The separation and detection methods used in OTA analysis also should offer proper sensibility in order to quantify the analyte adequately. In short, the goals of the present work were to develop a methodology using in-tube SPME in flow through extraction mode with C18 particles as extraction phase and separation and detection by HPLC-MS/MS. Further, optimize and validate the proposed methodology and detect and quantify OTA in wine samples. For this, in-tube SPME was configured and a PEEK tube was packed with C18 particles, which was used in the extraction step. The method optimization was achieved using a central composite 22 + 3 central points experimental design, having as factors the percentage of ACN and time during sample loading step. Validation method was done following ANVISA validation guide after which red dry wine and white dry wine samples were analyzed. The proposed method was developed, having its functionalities attested and its analysis conditions enhanced, using 22% of ACN and 6 minutes in sample loading. The method was validated, demonstrating proper sensitivity, with detection and quantification limits equal to 0.02 and 0.05 µg L-1, respectively. Linearity and precision were evaluated, exhibiting correlation coefficient equal to 0,996 e RSD under 6%, respectively. The method was accurate in medium and high concentration levels and maximum recovery was 73% in high concentration level. Brazilian and foreign red dry wine and white dry wine samples were analyzed and OTA was not detected in any of them. However, OTA may be present in samples analyzed in lower concentrations than that ones determined by detection and quantification limits, not being a potential danger to human health.

No description available.

No description available.

L’utilisation de lentilles cornéennes peut servir à améliorer le profil d’administration d’un principe actif dans les yeux. Avec une efficacité d’administration de 5% par l’utilisation de gouttes, on comprend rapidement que l’administration oculaire doit être améliorée. Cette faible administration a donné naissance à plusieurs tentatives visant à fabriquer des lentilles cornéennes médicamentées. Cependant, à cause de multiples raisons, aucune de ces tentatives n’a actuellement été mise sur le marché. Nous proposons dans cette étude, une possible amélioration des systèmes établis par le développement d’une lentille cornéenne à base de 2-(hydroxyéthyle)méthacrylate (HEMA), dans laquelle des microgels, à base de poly N-isopropylacrylamide (pNIPAM) thermosensible encapsulant un principe actif, seront incorporé. Nous avons donc débuté par développer une méthode analytique sensible par HPCL-MS/MS capable de quantifier plusieurs molécules à la fois. La méthode résultante a été validée selon les différents critères de la FDA et l’ICH en démontrant des limites de quantifications et de détections suffisamment basses, autant dans des fluides simulés que dans les tissus d’yeux de lapins. La méthode a été validée pour sept médicaments ophtalmiques : Pilocarpine, lidocaïne, proparacaïne, atropine, acétonide de triamcinolone, timolol et prednisolone. Nous avons ensuite fait la synthèse des microgels chargés négativement à base de NIPAM et d’acide méthacrylique (MAA). Nous avons encapsulé une molécule modèle dans des particules ayant une taille entre 200 et 600 nm dépendant de la composition ainsi qu’un potentiel zêta variant en fonction de la température. L’encapsulation de la rhodamine 6G (R6G) dans les microgels a été possible jusqu’à un chargement (DL%) de 38%. L’utilisation des isothermes de Langmuir a permis de montrer que l’encapsulation était principalement le résultat d’interactions électrostatiques entre les MAA et la R6G. Des cinétiques de libérations ont été effectuées à partir d’hydrogels d’acrylamide chargés en microgels encapsulant la R6G. Il a été trouvé que la libération des hydrogels chargés en microgels s’effectuait majoritairement selon l’affinité au microgel et sur une période d’environ 4-24 heures. La libération à partir de ces systèmes a été comparée à des formules d’hydrogels contenant des liposomes ou des nanogels de chitosan. Ces trois derniers (liposomes, microgels et nanogels) ont présenté des résultats prometteurs pour différentes applications avec différents profils de libérations. Enfin, nous avons transposé le modèle développé avec les gels d’acrylamide pour fabriquer des lentilles de contact de 260 à 340 µm d’épaisseur à base de pHEMA contenant les microgels avec une molécule encapsulée devant être administrée dans les yeux. Nous avons modifié la composition de l’hydrogel en incorporant un polymère linéaire, la polyvinylpyrrolidone (PVP). L’obtention d’hydrogels partiellement interpénétrés améliore la rétention d’eau dans les lentilles cornéennes. L’encapsulation dans les microgels chargés négativement a donné de meilleurs rendements avec la lidocaïne et cette dernière a été libérée de la lentille de pHEMA en totalité en approximativement 2 heures qu’elle soit ou non encapsulée dans des microgels. Ainsi dans cette étude pilote, l’impact des microgels n’a pas pu être déterminé et, de ce fait, nécessitera des études approfondies sur la structure et les propriétés de la lentille qui a été développée. En utilisant des modèles de libération plus représentatifs de la physiologie de l’œil, nous pourrions conclure avec plus de certitude concernant l’efficacité d’un tel système d’administration et s’il est possible de l’optimiser. / The development of corneal contact lenses initially aimed to correct vision troubles but more recently targets to improve administration of ophthalmic drugs. Eye drops from ophthalmic solutions has a poor administration efficiency of 5% or less and is currently the most used method to deliver drugs to the eye. Such administration technique needs to be improved and contact lenses could be the solution according to many opticians. However, no marketed therapeutic contact lenses has been marketed up to date. In this project we have developed a model of a contact lens made of 2-(hydroxyethyl)methacrylate embedding microgels of poly N-isopropylacrylamide (pNIPAM), encapsulating a model drug. We first developed an analytical method capable to quantify simultaneously seven ophthalmic drugs: Pilocarpine, lidocaine, proparacaine, atropine, triamcinolone acetonide, timolol and prednisolone. This method was developed on a HPLC-MS/MS device and was validated according to FDA and ICH criteria. Using this method, we achieved very low detection and quantitation limits with high precision and accuracy in both simulated lachrymal fluids and in rabbit ocular tissues. Each seven drugs was validated using this method. We proceeded with the synthesis of negatively charged microgels of NIPAM using methacrylic acid (MAA) as comonomer. Resulting size were ranging between 200-600 nm and zeta potential was found to increase (absolute value) with temperature. The microgels were used to encapsulate a model molecule, rhodamine 6G (R6G), in different medium and were loaded in the microgel up to 38% (drug loading, DL%). Using Langmuir isotherms to measure affinity and adsorption of R6G, it was found well correlated to MAA content in microgels, suggesting electrostatic interaction was the main parameter for drug loading. Release kinetics was performed using a model hydrogel of acrylamide embedding the R6G-loaded microgels. The measured release was found to follow an affinity-based mechanism for over 4-24 hours. The release kinetics were then compared to a formulation of liposomes and nanogels of chitosan embedded in hydrogel. All formulations exhibited interesting release profiles making them promising systems for different therapeutic applications. Finally, we changed the acrylamide gels for pHEMA designed to reproduce contact lenses containing drug-loaded microgels. The hydrogel composition, in terms of monomer / cross-linker ratio, was first optimized to fit contact lenses properties of 260-340 µm thick contact lenses. We also made use of semi-interpenetrated polyvinylpirrolidone (PVP) in the pHEMA hydrogel matrix to increase its water content. The highest DL% of negatively charged microgels were obtained using lidocaine and were used for release studies, where the total content of lidocaine was released in approximately 2 hours with and without microgels. In the end, this was a pilot study aiming to evaluate the potential of microgel usability in contact lenses. However, the impact of microgels on release was not fully conclusive. Additional studies should be undertaken to achieve a better comprehension and characterization of the release mechanism such as using more eye relevant physiological models. Such studies would provide further insights on the use of such materials for eye drug delivery and its applicability.

Honey has been used to heal wounds since ancient times. There are many references in ancient literature that cite honey for its medicinal uses. It is used as an alternative agent to cure infections of wounds, burns, ulcers etc. Researchers have shown some of the antimicrobial properties of honey when used as an ointment. When applied to an affected area, it helps to promote the growth of healthy tissue. One of the factors on which the quality of the honey depends, is its geographical origin. Based on the location, honey types can vary as much as 100-fold from each other in color, aroma, viscosity, and antimicrobial properties. The important components in honey that play an essential part in healing wounds and contributing to the antimicrobial properties are enzymes. Their presence allows honey to kill various types of pathogenic bacteria, viruses, fungi etc. A higher antimicrobial effect is seen in monofloral honey (when a single plant species is the source of nectar), which is often more potent than other types of honey in terms of antibacterial activity. Resistance of pathogens to these antimicrobial actions has never been shown, which makes honey a more promising source of antimicrobial research. Presently, infections of burns and wounds are very challenging to treat, especially when they are caused by antibiotic-resistant bacteria. The purpose of this study was to examine the antimicrobial properties of honey from Utah and other locales, and to identify promising antimicrobial activities that could be useful in treating infections caused by resistant bacteria.

One of the greatest threats to human health is increasing antimicrobial resistance among pathogens, and finding alternatives for treatment of bacterial infections is of highest importance together with a more controlled use of antibiotics. Porphyrins and heme have both been shown to be a promising class of compounds for inactivation of bacteria; porphyrins by their excellent properties to act as a photosensitizer, and heme by its importance as an iron source during a bacterial infection in vertebrates. This thesis describes the development of analytical methods for the identification and determination of porphyrins and heme using liquid chromatography coupled to tandem mass spectrometry. Subsequently, these developed methods were applied to bacterial samples to investigate different culture conditions and additives effect to the intracellular porphyrin and heme composition. Singlet oxygen production of three naturally occurring porphyrins have been determined together with the photosensitivity for blue light and the porphyrin content in E. coli. Toothbrushes equipped with a LED, emitting light with a wavelength of 450 nm, were used in an eight week randomized clinical trial to investigate any positive periodontal effect of blue light. Porphyrin and heme content in Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were highly affected by the different cultivation conditions. The culture age of A. actinomycetemcomitans affected the porphyrin profile, while only small changes were observed for P. gingivalis during growth. A large change of the porphyrin profile could be observed when the bacteria were passaged onto a new growth medium. Additional porphyrins were detected and the total porphyrin content increased up to 28 times. These findings highlight the need for more standardized cultivation procedures when performing in vitro experiments. Heme content in Escherichia coli was affected when different additives related to biosynthesis of heme were added to the growth medium. The uptake of heme could be reduced with 52% when a compound that chemically looks similar to heme was added to the growth medium. Since heme acquisition is important for many pathogens, this could be a promising target for antimicrobial drugs. E. coli showed no sensitivity for 405 nm light using light doses up to 172.8 J/cm2 and only low concentrations of porphyrins could be quantified. By adding a porphyrin precursor to E. coli the intracellular concentration of porphyrins increased remarkably and a light dose of 57.6 J/cm2 reduced the bacterial number with &gt; 5 log10 steps. This shows that E. coli can be killed due to their endogenous porphyrins. In the clinical study we could see a weak trend that the 450 nm LED toothbrush possessed a phototherapeutic effect for three clinical indices. All indices were decreased in the intervention group, but there were no statistically significant difference compared to the control group. However, four inflammation markers were significantly decreased in the intervention group while only one decreased significantly in the control group. In conclusion, this thesis has shown that porphyrins and heme are produced endogenously in microorganisms and that the porphyrin profiles vary depending on culture conditions and different additives. Furthermore, porphyrins may be used as endogenous photosensitizers to inactivate bacteria, but more research is necessary to determine if there is a specific porphyrin that contributes more to the photosensitivity. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.</p>

During the industrialization of the 19th century the negative health effects of vitamin D was discovered as children in the cities developed osteomalacia or more commonly known as rickets caused by vitamin D deficiency. Vitamin D is produced in the skin from 7-dehydrocholesterol during sun-exposure and enhances intestinal phosphor and calcium absorption thus enhancing the bone remodeling process. Now, in the 21st century, Vitamin D is still relevant as positive health effects have been recognized and with it an increased number of samples and a demand for accurate analyzing. Vitamin D is commonly believed to be sensitive to ultraviolet radiation in serum and blood samples and therefore have traditionally been kept protected from light exposure from the time of sampling until the finished analyze. However recent studies have proven 25- hydroxyvitamin D (25[OH]D) to be stable in both whole blood and serum. As previous studies have been primarily conducted in research laboratories with the aim to study vitamin D under specific research-laboratory conditions the aim of this study was to study the stability of 25[OH]D in serum and whole blood within both primary care- and hospital laboratories under normal and exaggerated conditions with the purpose to evaluate possible pre-analytical issues with everyday handling processes. The assay used was high pressure liquid chromatography-tandem mass spectrometry, HPLCMS/MS, and the sought analytes 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, 25[OH]D2 and 25[OH]D3. The results showed that 25-hydroxyvitamin D is stable in serum for 24 hours at room temperature whilst exposed to light both ultraviolet and fluorescent. The analyte is also stable for up to four freeze-thaw cycles rendering the process of light-protection and samples frozen immediately after centrifugation superfluous. The results also ensure reliable results even if samples are accidently left on benchtops or saved refrozen to be reanalyzed at a later date. / Under den industriella revolutionen på 1800 talet upptäcktes de negativa hälsoeffekterna av vitamin D-brist då barnen i städerna utvecklade rakit (osteomalaci) eller engelska sjukan som sjukdomen också kallas på grund av brist på sol och D-vitamin. Vitamin D produceras i huden från 7-dehydrokolesterol vid solexponering och ökar upptaget av fosfor och kalcium i tarmen som i sin tur förbättrar återuppbyggnaden av skelettet. Vitamin D är fortfarande aktuell även nu i vår tid men då för dess nyupptäckta hälsofrämjande egenskaper som till exempel förebyggandet av coloncancer. Detta medför även en ökning av antalet analyser och kräver därmed en adekvat analysmetod. Traditionellt har det antagits att vitamin D är ljuskänsligt i alla former därför har blod och serum ljusskyddats, från provtagningstillfället fram tills dess att analysen är utförd. Dock har nya studier visat att 25-hydroxyvitamin D (25[OH]D) är mycket stabilt bundet till vitamindbindande protein i både serum och helblod. Syftet med studien var att utvärdera om 25[OH]D i serum och helblod behöver ljusskddas genom att studera stabiliteten hos 25[OH]D i både serum och helblod under normala primärvårdslaboratorie- och sjukhuslaboratorieförhållanden samt under extrema förhållanden för att utvärdera eventuella preanalytiska problem eller fel relaterade till den vardagliga hanteringen av vitamin D prover. Proverna analyserades med högupplösande vätskekromatografi-tandem masspektrometri, HPLC-MS/MS, och de sökta analyterna var 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, 25[OH]D2 och 25[OH]D3. Resultat från studien visade att 25-hydroxyvitamin D är stabilt i serum i 24 timmar i rumstemperatur med ljusexponering från både ultraviolett och fluorescerande ljus. 25-hydroxyvitamin D är även stabil i serum upp till fyra frys- och tiningscykler. Detta gör att provhanteringen kan förenklas genom att dessa prover inte behöver ljusskyddas samt att serumet ej behöver frysas in direkt efter centrifugering. Resultatet säkerställer även tillförlitliga resultat om prover lämnas framme på bänken av misstag eller om prover behöver sparas och frysas om för att analyseras vid senare tillfälle.

Acrylamide is an undesirable carcinogenic component of thermally processed foods being formed from reducing saccharides and asparagine. In this work, the effect of ammonium and sodium raising agents themselves or in their combination with L-asparaginase enzyme catalyzing the conversion of asparagine into aspartic acid resulting in the reduction of acrylamide in gingerbreads was studied. Also, the influence of selected inorganic salts on the content of acrylamide in a model matrix simulating a composition of cereal products was observed. Simultaneously, the impact of these salts on activity of L-asparaginase was examined to find optimal conditions for its application in cereal technology. Based on experiments it was found, that addition of L-asparaginase reduces acrylamide content by 40 % while inorganic salts addition decreases acrylamide content in the range of 30 - 99 % when the most effective compounds were NH4Cl and CaCl2.