Gabriel Rübensam

A liquid-liquid extraction procedure followed by a low temperature purification step for the analysis of macrocyclic lactones in milk by liquid chromatography-tandem mass spectrometry and fluorescence detection.

In this work a method is proposed and demonstrated for the analysis of the macrocyclic lactones abamectin, doramectin, eprinomectin, ivermectin and moxidectin in bovine milk by liquid chromatography coupled to mass spectrometry (LC-MS/MS) and liquid chromatography with fluorescence detection (LC-FL). The method is based on liquid-liquid extraction followed by a low temperature purification (LLE-LTP) step. Moreover, the proposed method was validated according to the Commission Decision 2002/657/EC, using LC-MS/MS and LC-FL for confirmatory and quantitative analysis, respectively. For LC-MS/MS the recovery rates observed ranged from 101.2 to 141.6% with coefficient of variation from 2.6 to 19.8%. For LC-FL the recovery rates observed ranged from 100.2 to 105% and coefficient of variations from 2.9 to 8.8%. Matrix effects were negligible due to the low temperature purification step. The quantification limits were far below the maximum limits established by regulations of all countries consulted. The proposed method proved to be simple, easy, and adequate for high-throughput analysis of a large number of samples per day at low cost.

Cashew (Anacardium occidentale) apple juice lowers mutagenicity of aflatoxin B1 in S. typhimurium TA102

Cashew (Anacardium occidentale) is a medicinal plant native to Brazil and also yields a nutritious fruit juice. Its large pulpy pseudo-fruit, referred to as the cashew apple, contains high concentrations of vitamin C, carotenoids, phenolic compounds and minerals. Natural and processed cashew apple juice (CAJ/cajuina) are amongst the most popular juices in Brazil, especially in the north-east. Both juices have antioxidant potential and suppress mutagenicity of hydrogen peroxide. In the present study we evaluated the inhibitory effects of CAJ/cajuina on Aflatoxin B1(AFB1)-induced mutation, using the Salmonella/microsome assay with the experimental approaches of pre-, co- and post-treatments. Both CAJ/cajuina suppress AFB1-induced mutagenesis in strain TA102 when applied in co- and in post-treatment. Possible mechanisms for anti-mutagenicity in co-treatment are (a) interaction with S9 enzymes, (b) metabolization to non-mutagenic compounds of AFB1 or (c) inactivation of S9 potential. Total suppression of AFB1 mutagenicity was observed in co-treatment with both CAJ and cajuina. Post-treatment anti-mutagenicity of both juices suggests a modulation of activity of error-prone DNA repair. CAJ/cajuina may be considered promising candidates for control of genotoxicity of AFB1 and may thus be considered as health foods with anti-carcinogenic potential. This promising characteristic warrants further evaluation with in vivo studies.

Antimutagenic activity of cashew apple (Anacardium occidentale Sapindales, Anacardiaceae) fresh juice and processed juice (cajuína) against methyl methanesulfonate, 4-nitroquinoline N-oxide and benzo[a]pyrene

Cashew apple juice (CAJ), produced from the native Brazilian cashew tree (Anacardium occidentale), and has been reported to have antibacterial, antifungal, antitumor, antioxidant and antimutagenic properties. Both the fresh unprocessed juice and the processed juice (cajuina in Portuguese) has been shown to consist of a complex mixture containing high concentrations of anacardic and ascorbic acids plus several carotenoids, phenolic compounds and metals. We assessed both types of juice for their antimutagenic properties against the direct mutagens methyl methanesulfonate (MMS) and 4-nitroquinoline-N-oxide (4-NQO) and the indirect mutagen benzo[a]pyrene (BaP) using pre-treatment, co-treatment and post-treatment assays with Salmonella typhimurium strains TA100, TA102, and TA97a. In pre-treatment experiments with strains TA100 and TA102 the fresh juice showed high antimutagenic activity against MMS but, conversely, co-treatment with both juices enhanced MMS mutagenicity and there was an indication of toxicity in the post-treatment regime. In pre-, co-, and post-treatments with TA97a as test strain, antimutagenic effects were also observed against 4-NQO and BaP. These results suggest that both fresh and processed CAJ can protect the cells against mutagenesis induced by direct and indirect mutagens.

Sample stacking in CZE using dynamic thermal junctions I. Analytes with low dpKa/dT crossing a single thermally induced pH junction in a BGE with high dpH/dT

The possibility to compress analyte bands at the beginning of CE runs has many advantages. Analytes at low concentration can be analyzed with high signal‐to‐noise ratios by using the so‐called sample stacking methods. Moreover, sample injections with very narrow initial band widths (small initial standard deviations) are sometimes useful, especially if high resolutions among the bands are required in the shortest run time. In the present work, a method of sample stacking is proposed and demonstrated. It is based on BGEs with high thermal sensitive pHs (high dpH/dT) and analytes with low dpKa/dT. High thermal sensitivity means that the working pKa of the BGE has a high dpKa/dT in modulus. For instance, Tris and Ethanolamine have dpH/dT=−0.028/°C and −0.029/°C, respectively, whereas carboxylic acids have low dpKa/dT values, i.e. in the −0.002/°C to+0.002/°C range. The action of cooling and heating sections along the capillary during the runs affects also the local viscosity, conductivity, and electric field strength. The effect of these variables on electrophoretic velocity and band compression is theoretically calculated using a simple model. Finally, this stacking method was demonstrated for amino acids derivatized with naphthalene‐2,3‐dicarboxaldehyde and fluorescamine using a temperature difference of 70°C between two neighbor sections and Tris as separation buffer. In this case, the BGE has a high pH thermal coefficient whereas the carboxylic groups of the analytes have low pKa thermal coefficients. The application of these dynamic thermal gradients increased peak height by a factor of two (and decreased the standard deviations of peaks by a factor of two) of aspartic acid and glutamic acid derivatized with naphthalene‐2,3‐dicarboxaldehyde and serine derivatized with fluorescamine. The effect of thermal compression of bands was not observed when runs were accomplished using phosphate buffer at pH 7 (negative control). Phosphate has a low dpH/dT in this pH range, similar to the dKa/dT of analytes. It is shown that ∣dKa/dT−dpH/dT∣≫0 is one determinant factor to have significant stacking produced by dynamic thermal junctions.

Multivariate Optimization for Extraction of Pyrethroids in Milk and Validation for GC-ECD and CG-MS/MS Analysis

A simple and inexpensive method based on solvent extraction followed by low temperature clean-up was applied for determination of seven pyrethroids residues in bovine raw milk using gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) and gas chromatography with electron-capture detector (GC-ECD). Sample extraction procedure was established through the evaluation of seven different extraction protocols, evaluated in terms of analyte recovery and cleanup efficiency. Sample preparation optimization was based on Doehlert design using fifteen runs with three different variables. Response surface methodologies and polynomial analysis were used to define the best extraction conditions. Method validation was carried out based on SANCO guide parameters and assessed by multivariate analysis. Method performance was considered satisfactory since mean recoveries were between 87% and 101% for three distinct concentrations. Accuracy and precision were lower than ±20%, and led to no significant differences (p < 0.05) between results obtained by GC-ECD and GC-MS/MS techniques. The method has been applied to routine analysis for determination of pyrethroid residues in bovine raw milk in the Brazilian National Residue Control Plan since 2013, in which a total of 50 samples were analyzed.

Sample stacking in CZE using dynamic thermal junctions II: Analytes with high dpKa/dT crossing a single thermal junction in a BGE with low dpH/dT

In a previous work [M. Mandaji, et al., this issue] a sample stacking method was theoretically modeled and experimentally demonstrated for analytes with low dpKa/dT (analytes carrying carboxylic groups) and BGEs with high dpH/dT (high pH–temperature‐coefficients). In that work, buffer pH was modulated with temperature, inducing electrophoretic mobility changes in the analytes. In the present work, the opposite conditions are studied and tested, i.e. analytes with high dpKa/dT and BGEs that exhibit low dpH/dT. It is well known that organic bases such as amines, imidazoles, and benzimidazoles exhibit high dpKa/dT. Temperature variations induce instantaneous changes on the basicity of these and other basic groups. Therefore, the electrophoretic velocity of some analytes changes abruptly when temperature variations are applied along the capillary. This is true only if BGE pH remains constant or if it changes in the opposite direction of pKa of the analyte. The presence of hot and cold sections along the capillary also affects local viscosity, conductivity, and electric field strength. The effect of these variables on electrophoretic velocity and band stacking efficacy was also taken into account in the theoretical model presented. Finally, this stacking method is demonstrated for lysine partially derivatized with naphthalene‐2,3‐dicarboxaldehyde. In this case, the amino group of the lateral chain was left underivatized and only the alpha amino group was derivatized. Therefore, the basicity of the lateral amino group, and consequently the electrophoretic mobility, was modulated with temperature while the pH of the buffer used remained unchanged.