Novel method for metalloproteins determination in human breast milk by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry.

Levels of essential metals in human breast milk (HBM) have been determined by different analytical techniques, but there is few woks about human whey milk fractions. However, the current trend lies in metalloproteomic and identification of different metalloproteins. In this sense, native separative techniques (N-PAGE and SEC) coupled to ICP-MS provide us with valuable information. Besides it is necessary the development of new methodologies in order to determine with accuracy and precision the profile of such metals and metalloproteins in the different whey protein fractions of HBM. Thus, the aim of this work was to develop a new method for metals and metalloproteins determination by SEC-ICP-MS in whey protein fractions of HBM. Human whey fractions were obtained of HBM samples by ultracentrifugation. Then, protein fractions of whey milk were separated by SEC coupled to ICP-MS for metalloproteins and Mn, Co, Cu and Se quantification. Besides, protein profile of whey milk was determined by N-PAGE and computer assisted image analysis. SEC-ICP-MS results indicated that first and second protein fractions showed detectable levels of the Mn, Co, Cu, and Se. Protein profile determined by N-PAGE and image analysis showed that molecular weight of protein fractions ranged between 68,878-1,228.277 Da. In this work, metalloproteins were analyzed by SEC coupled to ICP-MS, with adequate sensitivity and accuracy. Our study has shown the presence of Mn, Co, Cu and Se bound to two protein fractions in whey milk of HBM. Metals levels analyzed were within the ranges reported in the literature.

Liquid chromatography coupled to molecular fluorescence with postcolumn UV sensitization for thimerosal and derivative compounds monitoring in environmental samples.

A HPLC coupled with molecular fluorescence (MF) spectrometry method for determination of thimerosal (THM, sodium ethylmercurythiosalicylate, C9 H9 HgNaO2 S), and derivatives is proposed. A sensitization of MF was provoked by UV irradiation of analytes in a home-made photoreactor that served as interface between the LC column and MF spectrometer. This method is applied to determination of THM, ethyl mercury, and thiosalicylic acid in samples of pharmaceutical industry effluents, and waters of La Carolina and Jáchal rivers situated in the center-west side of San Luis city and in the east of San Juan city (Middle West, Argentine) where the effluents are dumped. The LODs calculated on basis of 3σ criterion were 1.8, 5, and 0.05 µmol/L for THM, ethyl mercury, and for thiosalicylic acid, respectively.

Determination of seleno-amino acids bound to proteins in extra virgin olive oils.

An analytical method has been developed to determine seleno-amino acids in proteins extracted from extra virgin olive oils (EVOOs). Different aqueous/organic solvents were tested to isolate proteins, an acetone:n-hexane combination being the best protein precipitant. In a first dimension chromatography, extracted proteins were analysed by size exclusion chromatography (SEC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) to identify S and Se associations as proteins marker. Two fractions of 66 kDa (A) and 443 kDa (B) were identified. These fractions were submitted to microwave-assisted acid hydrolysis (MAAH) to release seleno-amino acids. In a second dimension chromatography seleno-amino acids were determined by reversed-phase chromatography (RPC) coupled to ICP-MS. Seleno-methylselenocysteine was determined with values ranging from 1.03-2.03±0.2 µg kg(-1) and selenocysteine at a concentration of 1.47±0.1 µg kg(-1). Variations of protein and seleno-amino acid concentrations were observed between EVOO varieties, contributing to EVOO cultivar differentiation.

Preconcentration of seleno-amino acids on a XAD resin and determination in regional olive oils by SPE UPLC-ESI-MS/MS.

This study describes a method for seleno-amino acids determination in Argentinean olive oils. Preliminary total selenium determination in olive oils probed low concentrations (62.8±1.6 to 117.4±3.0 µg/kg) and the necessity of implementing a preconcentration method. To this end a XAD® resin was employed as sorbent for selenomethionine (Se-Met), selenomethylselenocysteine (Se-MetSeCys), and selenocysteine (Se-Cys) preconcentration. Determinations were performed by UPLC-ESI-MS/MS. Recoveries were between 84% and 97% for the seleno-amino acids studied, reaching a detection limit of 0.09 µg/kg, a precision of 10% (RSD, n=6), and an enhancement factor of 60-fold (6 for the extraction system and 10 for the preconcentration approach). The only detected Se species in the olive oils was Se-MetSeCys in concentrations ranging from 2.0 to 8.3 µg/kg.