CONICET | Buscador de Institutos y Recursos Humanos

Introduction Lipid oxidation in foods constitutes a complex chain of reactions that yields to primary products (peroxides), which once exposed to extended oxidation conditions, give rise to secondary oxidation products, including aldehydes, ketones, epoxides and hydroxy compounds. Most of these secondary oxidation products produce undesirable sensorial and biological effects. To avoid rancidification process oils should be stored in a cold and dark environment. The oxidation process becomes more pronounced with the higher number of double bonds in lipids. Despite being intermediate compounds of lipid oxidation process, hydroperoxides are relatively stable (depending on the lipid structure) and can be used to assess lipid oxidation status in food samples, as long as the sample is not in an advanced oxidative status. Because of this feature, temperature conditions during analysis must be controlled to avoid hydroperoxide decomposition and addition of an antioxidant is often required. The probe electrospray ionization (PESI) is an ESI-based ionization technique that generates electrospray from the tip of a solid metal needle. This technique is versatile in the analysis of biological samples as it requires only a small amount of the material picked up without any special sample preparation. Methanolic extract of several nuts were chosen for the investigation of oxidation process, as it contains highly insaturated lipids. The sample was exposed to air and sunlight for several days. PESI mass spectra were recorded before and after treatment. Moreover, statistical analysis was used to compare PESI mass spectra of nut, soybean, macademia nut and peanut. Materials and Methods All reagents used in this study were analytical grade or higher and were used without further purification. Water was purified by a Milli-Q system (Millipore, Bedford, MA, USA). Ammonium acetate, glacial acetic acid, chloroform and methanol were provided by Kanto Chemicals (Tokyo, Japan). Soybeans and nuts were obtained from local market (Kofu, Japan). Seeds were mashed in a mortar. Lipids were extracted and washed with small portions of methanol (total volume of methanol used was 2 mL). The liquid extract was collected and then centrifugated. The PESI experimental procedures were similar to those described in our previous papers.2 Brieﬂy, the needle was moved up and down along a vertical axis using a custom made linear actuator system. When the needle was at the bottom position, the tip of the needle was adjusted to touch the surface of the sample. When the needle or wire was moved up to the highest position, a high voltage of about 2?3 kV was applied to it. The distance of the needle stroke was 10 mm. As electrospray emitters, disposable acupuncture needles (Seirin, Shizuoka, Japan: 0.3 x 50 mm and 0.14 x 30 mm) with sub-micrometer tip diameter and a tangentially cut titanium (Ti) wire (Nilaco Corporation, Tokyo, Japan) with 0.5 mm diameter, were used throughout the PESI-MS experiments. After the Ti wire surface was oxidized under a burner flame, the wire was incubated in 100% pentafluorophenyl triethoxysilane for 1 h at room temperature. After incubation, the wire was rinsed with methanol and pure water. The seeds were cut in two pieces and kept on the sample holder using double fixing tape or a metallic hook. The sample was stitched directly by the probe needle. The stitching depth was not precisely determined, but was estimated to be less than 0.5 mm. Since PESI is based on electrospray, it can only be applicable to liquid samples. In some cases, however, the surface of biological samples was not juicy enough; only a small amount of biofluid could be attached to the needle tip. In general, some drops of methanol or methanol/water (1:1) were deposited over the tissue to solve that problem. The fluid picked up by the needle was electrosprayed and analysed. Two methods were used to acquire the mass spectra: the droplet was left to be electrosprayed until the electrospray current decreased to zero (single-shot) or the droplet was periodically renewed (continuous-shot). In the latter, the PESI mass spectra were obtained with an acquisition time of 1 s with 3 Hz of the probe motion. For PESI, the sample was sprayed by electrostatic means, and was not assisted by any sheath flow or nebulising gas. The ions generated from the electrospray were sampled through the ion-sampling oriﬁce with a diameter of 0.4 mm into the vacuum chamber and mass analyzed by an orthogonal time-of-ﬂight mass spectrometer (AccuTOF; JEOL, Akishima, Japan). Spectra were obtained and analyzed with the program Mass Center (JEOL). Results and Discussion The triglyceride spectra of various seeds were compared by principal component analysis (PCA). The data set consist of m/z values and the corresponding signal intensities. For example, the positive ion PESI mass spectra of soybean, macademia nut, nut and peanut were recorded using acupuncture needle and continuous-shot mode in order to detect possible differences in triglyceride composition. The triglyceride distribution is very similar for all the samples. However, PCA shows differences in spectra with very similar signals. This shows that the variation in the intensity distribution contain enough information to obtain a clear differentiation between the four seeds studied. On the other hand, methanolic extracts of nuts were exposed to air and sunlight for several days. PESI-MS spectra were recorded. Because seeds are rich in phospholipids, strong signals of phosphatidylcholine, phosphatidylethanolamine and triacylglycerols of different fatty acid residues were detected as major ions in the positive ion mode. Lipids were observed mostly as adducts with Na+ or K+. With the aging of the oil, the formation of hydroperoxides with various numbers of oxygen atoms was observed in the PESI spectra. The relative intensity of oxidized triglycerides increased with days of exposition. The great advantage of PESI-MS, compare with other techniques used for the study of lipid oxidation, is that minimal sample preparation is needed.

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