Determining aflatoxins B1, B2, G1 and G2 in maize using florisil clean up with thin layer chromatography and visual and densitometric quantification

A method for determining aflatoxins B1 (AFB1), B2 (AFB2),G1 (AFG1) andG2 (AFG2) in maize with florisil clean up was optimised aiming at one-dimensional thin layer chromatography (TLC) analysis with visual and densitometric quantification. Aflatoxins were extracted with chloroform: water (30:1, v/v), purified through florisil cartridges, separated on TLC plate, detected and quantified by visual and densitometric analysis. The in-house method performance characteristics were determined by using spiked, naturally contaminated maize samples, and certified reference material. The mean recoveries for aflatoxins were 94.2, 81.9, 93.5 and 97.3% in the range of 1.0 to 242 µg/kg for AFB1, 0.3 to 85mg/kg for AFB2, 0.6 to 148mg/kg for AFG1 and 0.6 to 140mg/kg for AFG2, respectively. The correlation values between visual and densitometric analysis for spiked samples were higher than 0.99 for AFB1, AFB2, AFG1 and 0.98 for AFG2. The mean relative standard deviations (RSD) for spiked samples were 16.2, 20.6, 12.8 and 16.9% for AFB1, AFB2, AFG1 and AFG2, respectively. The RSD of the method for naturally contaminated sample (n = 5) was 16.8% for AFB1 and 27.2% for AFB2. The limits of detection of the method (LD) were 0.2, 0.1, 0.1 and 0.1mg/kg and the limits of quantification (LQ) were 1.0, 0.3, 0.6 and 0.6mg/kg for AFB1, AFB2, AFG1 and AFG2, respectively.

Detection and quantification of Roundup Ready® soybean residues in sausage samples by conventional and real-time PCR

The increasing presence of products derived from genetically modified (GM) plants in human and animal diets has led to the development of detection methods to distinguish biotechnology-derived foods from conventional ones. The conventional and real-time PCR have been used, respectively, to detect and quantify GM residues in highly processed foods. DNA extraction is a critical step during the analysis process. Some factors such as DNA degradation, matrix effects, and the presence of PCR inhibitors imply that a detection or quantification limit, established for a given method, is restricted to a matrix used during validation and cannot be projected to any other matrix outside the scope of the method. In Brazil, sausage samples were the main class of processed products in which Roundup Ready® (RR) soybean residues were detected. Thus, the validation of methodologies for the detection and quantification of those residues is absolutely necessary. Sausage samples were submitted to two different methods of DNA extraction: modified Wizard and the CTAB method. The yield and quality were compared for both methods. DNA samples were analyzed by conventional and real-time PCR for the detection and quantification of Roundup Ready® soybean in the samples. At least 200 ng of total sausage DNA was necessary for a reliable quantification. Reactions containing DNA amounts below this value led to large variations on the expected GM percentage value. In conventional PCR, the detection limit varied from 1.0 to 500 ng, depending on the GM soybean content in the sample. The precision, performance, and linearity were relatively high indicating that the method used for analysis was satisfactory.

Proximate composition and quantification of fatty acids in breaded chicken steak

The aim of this work was to analyze the fatty acid and proximate composition of five brands of breaded chicken steak (A, B, C, D, and E) by accurate chromatographic quantification and to compare the experimental results with food label nutrition facts. The protein values of all brands were in agreement with the Brazilian regulation values, except for that of sample E, which presented the highest lipid content. Thirteen fatty acids were identified in the studied brands and the major ones were oleic acid, linoleic acid, and palmitic acid. The polyunsaturated fatty acid/saturated fatty acid ratios were within the values considered appropriate for human health; however, the high n-6/n-3 ratios found can result in an unbalanced intake of these fat acids. Only samples D and E can be considered trans free according to the regulations. The comparison of the analyses' results and the food label nutrition facts showed little variation in protein values. Nevertheless, most brands underestimated their lipid and energy levels. Brand B and C labels declared "free of trans", but the obteined results showed levels excedding those especified by regulation to be considered trans free values.

Determination of presence and quantification of cadmium, lead and copper in Nile tilapia (Oreochromis niloticus) fillets obtained from three cold storage plants in the state of Parana, Brazil

Pisciculture is an economic activity that is steadily growing in the state of Parana, Brazil, and Nile tilapia (Oreochromis niloticus) is one of the widely cultivated species in this state. Tilapia is not only a very nutritious food, but also an important indicator of environmental contamination. This study aimed to verify contamination by cadmium, copper and lead in tilapia fillets, and to compare the found values to international legislations. Were collected 135 samples of tilapia fillets, between July 2006 and May 2007, in three fish stores located in regions west and north of Paraná State. Samples of tilapia fillet were analyzed in relation to the presence of cadmiun, lead and copper, using atomic absorption spectrophotometry. Lead has not been detected in the analyses. Cadmium has been detected in three samples, on concentrations of 0.012 µg.g-1, 0.011 µg.g-1 and 0.014 µg.g-1. Copper has been detected in all fillets, and the average concentration of each cold storage plant was of 0.122 µg.g-1, 0.106 µg.g-1 and 0.153 µg.g-1. The concentrations found in this study are within the limits allowed by both the European and the Australian legislations.

Proximate composition and quantification of fatty acids in five major Brazilian chocolate brands

The objective of the present study was to characterize the centesimal composition and quantify fatty acids in regular and diet chocolate brands with emphasis on trans fatty acids. Regular and diet dark chocolate samples from the major brands analyzed were purchased from different local supermarkets in the city of Maringá. The brands were labeled with letters and five lots of each brand with three chocolate units per lot were analyzed in triplicate. We observed that the diet chocolates from the same brands presented larger lipid contents. The main fatty acids observed were saturated fatty acids (SFA), myristic acid (14:0), palmitic acid (16:0), and estearic acid (18:0). Among the monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA) and trans fatty acids, oleic acid (18:1n-9), linoleic acid (18:2n-6), and elaidic acid (18:1-9t) predominated. The trans fatty acid contents found in the analyzed samples were lower than and/or in accordance with the limits proposed by the Brazilian regulation.

Quantification of catechins and caffeine from green tea (Camellia sinensis) infusions, extract, and ready-to-drink beverages

This study aimed to quantify the levels of catechins and caffeine in various forms of presentation of green tea: infusion tea bags, extract, and ready-to-drink beverage and, based on their content, identify the most suitable for consumption. High Performance Liquid Chromatography (HPLC) analytical method was used for the quantification of catechins and caffeine. The tea bags had the highest concentration of total catechins with 5 to 9.5% followed by the extract with 3.64 to 4.88%, and ready-to-drink green tea beverage showed low levels of catechins, from 0.14 to 0.26%. As for caffeine content, green tea extract had higher concentration (1.96 to 3.54%) compared to the tea bags (1.39 to 1.57%). Tea bags were found the most suitable for consumption because it contains higher amounts of catechins and smaller amounts of caffeine.

Development and validation of a method for detection and quantification of ochratoxin A in green coffee using liquid chromatography coupled to mass spectrometry

A method using Liquid Chromatography Tanden Mass Spectrometry (LC-MS/MS) with matrix-matched calibration curve was developed and validated for determining ochratoxin A (OTA) in green coffee. Linearity was found between 3.0 and 23.0 ng.g-1. Mean recoveries ranged between 90.45% and 108.81%; the relative standard deviation under repeatability and intermediate precision conditions ranged from 5.39% to 9.94% and from 2.20% to 14.34%, respectively. The limits of detection and quantification were 1.2 ng.g-1 and 3.0 ng.g-¹, respectively. The method developed was suitable and contributed to the field of mycotoxin analysis, and it will be used for future production of the Certified Reference Material (CRM) for OTA in coffee.

Quantification of Toxin Biosynthesis Genes In Cyanobacteria and Dinoflagellates - Genetic Factors as Predictors of Toxin Production in the Environment

Harmful algal blooms (HABs) are events caused by the massive proliferation of microscopic, often photosynthetic organisms that inhabit both fresh and marine waters. Although HABs are essentially a natural phenomenon, they now cause worldwide concern. Recent anthropogenic effects, such as climate change and eutrophication via nutrient runoff, can be seen in their increased prevalence and severity. Cyanobacteria and dinoflagellates are often the causative organisms of HABs. In addition to adverse effects caused by the sheer biomass, certain species produce highly potent toxic compounds: hepatotoxic microcystins are produced exclusively by cyanobacteria and neurotoxic saxitoxins, also known as paralytic shellfish toxins (PSTs), by both cyanobacteria and dinoflagellates. Specific biosynthetic genes in the cyanobacterial genomes direct the production of microcystin and paralytic shellfish toxins. Recently also the first paralytic shellfish toxin gene sequences from dinoflagellate genomes have been elucidated. The public health risks presented by HABs are evident, but the monitoring and prediction of toxic events is challenging. Characterization of the genetic background of toxin biosynthesis, including that of microcystins and paralytic shellfish toxins, has made it possible to develop highly sensitive molecular tools which have shown promise in the monitoring and study of potentially toxic microalgae. In this doctoral work, toxin-specific genes were targeted in the developed PCR and qPCR assays for the detection and quantification of potentially toxic cyanobacteria and dinoflagellates in the environment. The correlation between the copy numbers of the toxin biosynthesis genes and toxin production were investigated to assess whether the developed methods could be used to predict toxin concentrations. The nature of the correlation between gene copy numbers and amount of toxin produced varied depending on the targeted gene and the producing organism. The combined mcyB copy numbers of three potentially microcystin-producing cyanobacterial genera showed significant positive correlation to the observed total toxin production. However, the presence of PST-specific sxtA, sxtG, and sxtB genes of cyanobacterial origin was found to be a poor predictor of toxin production in the studied area. Conversely, the dinoflagellate sxtA4 was a good qualitative indicator of a neurotoxic bloom both in the laboratory and in the field, and population densities reflected well the observed toxin concentrations. In conclusion, although the specificity of each potential targeted toxin biosynthesis gene must be assessed individually during method development, the results obtained in this doctoral study support the use of quantitative PCR -based approaches in the monitoring of toxic cyanobacteria and dinoflagellates.

Opportunity Analysis and Category Management as a Base for Effective Indirect Sourcing

Hankintojen johtamisen kirjallisuus korostaa tehokkaan hankinnan olevan käypä keino tehostaa organisaation tulosta kokonaisvaltaisesti. Myös kasvava tietoisuus erityisesti epäsuorista hankintamenetelmistä ja työkaluista toimivat kannustimina tälle tutkimukselle. Tämän Pro Gradu -tutkimuksen päätarkoituksena on rakentaa kokonaisvaltainen ymmärrys epäsuorasta hankinnasta sekä löytää keinoja sen tehostamiseksi. Tutkimuksen tavoitteena on selvittää, miten globaali, monikansal- linen organisaatio voi parantaa kannattavuuttaan epäsuorissa hankinnoissa, sekä mitkä tekijät hankintastrategiassa vaikuttavat siihen. Tutkimus toteutettiin yksittäisenä tapaustutkimuksena suuren globaalin, monikan- sallisen yrityksen työntekijän näkökulmasta, Pääosa datasta pohjautuu vuonna 2015 toteutettuun Opportunity -analyysi projektiin, joka toteutettiin yhteistyössä ulkoisen konsulttifirman kanssa. Osa datasta pohjautuu puolistrukturoituihin haas- tatteluihin organisaation hankintajohtajan kanssa. Datan keruussa hyödynnettiin lisäksi henkilökohtaista havainnointia ja sekundääristä aineistoa organisaatiosta. Tämä Pro Gradu tutkimus on toteutettu kvalitatiivisella otteella, sisältäen joitakin kvantitatiivisia metodin piirteitä.

QUANTIFICATION AND EXTRACTION OF SURFACE FEATURES FROM DIGITAL TERRAIN MODELS

Digital Terrain Models (DTMs) are important in geology and geomorphology, since elevation data contains a lot of information pertaining to geomorphological processes that influence the topography. The first derivative of topography is attitude; the second is curvature. GIS tools were developed for derivation of strike, dip, curvature and curvature orientation from Digital Elevation Models (DEMs). A method for displaying both strike and dip simultaneously as colour-coded visualization (AVA) was implemented. A plug-in for calculating strike and dip via Least Squares Regression was created first using VB.NET. Further research produced a more computationally efficient solution, convolution filtering, which was implemented as Python scripts. These scripts were also used for calculation of curvature and curvature orientation. The application of these tools was demonstrated by performing morphometric studies on datasets from Earth and Mars. The tools show promise, however more work is needed to explore their full potential and possible uses.

Développement de techniques analytiques pour la détermination des agents anti-infectieux dans les eaux environnementales

Les agents anti-infectieux sont utilisés pour traiter ou prévenir les infections chez les humains, les animaux, les insectes et les plantes. L’apparition de traces de ces substances dans les eaux usées, les eaux naturelles et même l’eau potable dans plusieurs pays du monde soulève l’inquiétude de la communauté scientifique surtout à cause de leur activité biologique. Le but de ces travaux de recherche a été d’étudier la présence d’anti-infectieux dans les eaux environnementales contaminées (c.-à-d. eaux usées, eaux naturelles et eau potable) ainsi que de développer de nouvelles méthodes analytiques capables de quantifier et confirmer leur présence dans ces matrices. Une méta-analyse sur l’occurrence des anti-infectieux dans les eaux environnementales contaminées a démontré qu’au moins 68 composés et 10 de leurs produits de transformation ont été quantifiés à ce jour. Les concentrations environnementales varient entre 0.1 ng/L et 1 mg/L, selon le composé, la matrice et la source de contamination. D’après cette étude, les effets nuisibles des anti-infectieux sur le biote aquatique sont possibles et ces substances peuvent aussi avoir un effet indirect sur la santé humaine à cause de sa possible contribution à la dissémination de la résistance aux anti-infecteiux chez les bactéries. Les premiers tests préliminaires de développement d’une méthode de détermination des anti-infectieux dans les eaux usées ont montré les difficultés à surmonter lors de l’extraction sur phase solide (SPE) ainsi que l’importance de la sélectivité du détecteur. On a décrit une nouvelle méthode de quantification des anti-infectieux utilisant la SPE en tandem dans le mode manuel et la chromatographie liquide couplée à la spectrométrie de masse en tandem (LC-MS/MS). Les six anti-infectieux ciblés (sulfaméthoxazole, triméthoprime, ciprofloxacin, levofloxacin, clarithromycin et azithromycin) ont été quantifiés à des concentrations entre 39 et 276 ng/L dans les échantillons d’affluent et d’effluent provenant d’une station d’épuration appliquant un traitement primaire et physico- chimique. Les concentrations retrouvées dans les effluents indiquent que la masse moyenne totale de ces substances, déversées hebdomadairement dans le fleuve St. Laurent, était de ~ 2 kg. En vue de réduire le temps total d’analyse et simplifier les manipulations, on a travaillé sur une nouvelle méthode de SPE couplée-LC-MS/MS. Cette méthode a utilisé une technique de permutation de colonnes pour préconcentrer 1.00 mL d’échantillon dans une colonne de SPE couplée. La performance analytique de la méthode a permis la quantification des six anti-infectieux dans les eaux usées municipales et les limites de détection étaient du même ordre de grandeur (13-60 ng/L) que les méthodes basées sur la SPE manuelle. Ensuite, l’application des colonnes de SPE couplée de chromatographie à débit turbulent pour la préconcentration de six anti-infectieux dans les eaux usées a été explorée pour diminuer les effets de matrice. Les résultats obtenus ont indiqué que ces colonnes sont une solution de réchange intéressante aux colonnes de SPE couplée traditionnelles. Finalement, en vue de permettre l’analyse des anti-infectieux dans les eaux de surface et l’eau potable, une méthode SPE couplée-LC-MS/MS utilisant des injections de grand volume (10 mL) a été développée. Le volume de fuite de plusieurs colonnes de SPE couplée a été estimé et la colonne ayant la meilleure rétention a été choisie. Les limites de détection et de confirmation de la méthode ont été entre 1 à 6 ng/L. L’analyse des échantillons réels a démontré que la concentration des trois anti-infectieux ciblés (sulfaméthoxazole, triméthoprime et clarithromycine) était au dessous de la limite de détection de la méthode. La mesure des masses exactes par spectrométrie de masse à temps d’envol et les spectres des ions produits utilisant une pente d’énergie de collision inverse dans un spectromètre de masse à triple quadripôle ont été explorés comme des méthodes de confirmation possibles.

L’impact du polymorphisme du gène E2 sur la quantification de la charge virale du VPH-16 dans les maladies précancéreuses du col utérin

Le VPH-16 de même que certains VPH, dont le VPH-18, causent le cancer du col utérin. Son intégration dans le génome humain pourrait être un marqueur de progression de l’infection. Les charges virales totale et intégrée sont présentement mesurées en quantifiant par PCR en temps réel les gènes E6 (RT-E6) et E2 (RT-E2-1) du VPH-16. Nous avons évalué l’impact du polymorphisme du gène E2 sur la quantification de l’ADN du VPH-16 dans des spécimens cliniques. Dans un premier temps, le gène E2 de 135 isolats de VPH-16 (123 appartenaient au clade Européen et 12 à des clades non- Européens) fut séquencé. Ensuite, un test de PCR en temps réel ciblant les séquences conservées dans E2 (RT-E2-2) fut développé et optimisé. Cent trente-neuf spécimens (lavages cervicaux et vaginaux) provenant de 74 participantes (58 séropositives pour le VIH, 16 séronégatives pour le VIH) ont été étudiés avec les trois tests E2 (RT-E2-2), E6 (RT-E6) et E2 (RT-E2-1). Les ratios de la quantité d’ADN de VPH-16 mesuré avec RT-E2-2 et RT-E2-1 dans les isolats Européens (médiane, 1.02; intervalle, 0.64-1.80) et Africains 1 (médiane, 0.80; intervalle, 0.53-1.09) sont similaires (P=0.08). Par contre, les ratios mesurés avec les isolats Africains 2 (médiane, 3.23; intervalle, 1.92-3.49) ou Asiatique- Américains (médiane, 3.78; intervalle, 1.47-37) sont nettement supérieurs à ceux obtenus avec les isolats Européens (P<0.02 pour chaque comparaison). Les distributions des quantités de E2 contenues dans les 139 échantillons mesurées avec RT-E2-2 (médiane, 6150) et RT-E2-1 (médiane, 8960) étaient statistiquement différentes (P<0.0001). Nous avons observé que les charges virales totale (odds ratio (OR) OR, 2.16 95% intervalle de confiance (IC) 1.11-4.19), et épisomale du VPH-16 (OR, 2.14 95% IC 1.09-4.19), mais pas la présence de formes intégrées (OR, 3.72 95% IC 1.03-13.4), sont associées aux néoplasies intraepitheliales cervicales de haut grade (CIN-2,3), et ce, en contrôlant pour des facteurs confondants tels que l’âge, le taux de CD4 sanguin, l’infection au VIH, et le polymorphisme de VPH-16. La proportion des échantillons ayant un ratio E6/E2 > 2 pour les femmes sans lésion intraépithéliale (7 de 35) est similaire à celle des femmes avec CIN-2,3 (5 de 11, p=0.24) ou avec CIN- 1 (4 de14, P=0.65). Le polymorphisme du gène E2 est un facteur qui influence la quantification des charges intégrées de VPH-16.