Browsing by Subject "analyysimenetelmä"

3-Chloro-1,2-propanediol (3-MCPD), 2-chloro-1,3-propanediol (2-MCPD) and 2,3-epoxy-1-propanol (glycidol) and their fatty acid esters are contaminants formed during processing fat containing foodstuffs at high temperatures. Mainly MCPD and glycidyl esters have been found to be formed in the deodorization process of oils, and in vegetable oils, such as palm oil, they have been measured at high concentrations. In accordance with the restrictions imposed by the European Commission, the levels of glycidyl esters must be especially monitored, as they have been identified as potentially carcinogenic compounds. The aim of the study was to introduce and validate a gas chromatographic analysis method for glycidyl esters and MCPD esters for the Customs Laboratory. The method was validated for two matrices: first for oils and then for powdered infant formulas. In addition, the success of the validation was examined by analyzing various oil samples previously received by the Customs Laboratory. The Customs Laboratory is also involved in the activities of the European Union Reference Laboratory, for which it was intended to participate in the reference measurement organized by the EU Reference Laboratory. The method for the determination of 3-MCPD, 2-MCPD and glycidyl esters in oils and infant formulas was carried out according to the guidelines of the European Union Reference Laboratory for Contaminants (EURL-PC). Determination of MCPD and glycidyl ester concentrations in oils and infant formulas included the following steps: fat extraction by liquid-liquid extraction (for infant formulas), addition of standards, solid-phase extraction, conversion of glycidyl esters to 3-MBPD esters, transesterification, neutralization, salting out, derivatization and analysis with gas chromatography-mass spectrometry system. Concentrations were determined using internal standard method. The method was validated for the following parameters: specificity, selectivity, limit of detection and quantitation, reproducibility, repeatability, trueness, linearity and working range, stability and measurement uncertainty. The analytical method developed for the determination of MCPD and glycidyl esters was successfully validated for oils and powdered infant formulas. The developed method proved to be specific and selective. The limit of determination was found to be 6.3 µg/kg, 1.3 µg/kg and 0.8 µg/kg for the oil matrix 3-MCPD, 2-MCPD and glycidyl esters. The limits of determination for the infant formula were 5.4 µg/kg, 3.0 µg/kg and 1.6 µg/kg for 3-MCPD, 2-MCPD and glycidyl esters. Recoveries for MCPD and glycidyl esters in the oil and powdered infant formulas were 83-105%. R2 for calibration lines were greater than 0.99, and the lines were linear over the entire measurement range of 2-1000 µg/kg. The relative standard deviation of repeatability and reproducibility was less than 20% for both matrices. The expanded measurement uncertainty for the MCPD and glycidyl esters of the oil and powdered infant formula was less than 50%. For all parameters, the requirements set by the Customs Laboratory and the performance requirements of Regulation (EU) 1881/2006 were met. A method validated for two matrices can then be accredited. The customs laboratory may use the developed method in the future to control 3-MCPD, 2-MCPD and glycidyl esters levels of oils and powdered infant formulas. In the future, the method could also be validated for new matrices, such as liquid infant formulas.

Avenanthramides are hydroxycinnamic acids unique to oat (Avena sativa L., Poaceae). They consist of an anthranilic acid part (anthranilic acid or hydroxylated and/or methoxylated derivative of anthranilic acid) that is conjugated to a cinnamic acid part via an amide bond. More than 25 different avenanthramides are found and identified in oat. However, the most common forms are esters of 5-hydroxyanthranilic acid with caffeic (2c), ferulic (2f) and p-coumaric (2p) acids. Avenanthramides have been shown to possess antioxidant and anti-inflammatory properties. Currently there is a great interest towards oat and bioactive compounds of oat, like avenanthramides. In the previous studies there has been a lot of diversity concerning the extraction method used for analysis of avenanthramides and usually quantitation methods were based on high-performance liquid chromatography (HPLC). The aim of this research was to develop a method based on ultra-high-performance liquid chromatography (UHPLC) for the analysis of the most common forms of avenanthramides (2c, 2f and 2p) in oat and oat products. In addition, also other forms of avenanthramides, like 2fd and 2pd, were identified and quantified using liquid chromatography-mass spectrometry (UHPLC-QTOF-MS). The extraction method of avenanthramides was optimized and the UHPLC-PDA method used for quantitation was validated. Finally, the study measured the differences in concentrations of avenanthramides in different oat products. In this study it was recognized that the concentrations and the extent of variation of the concentrations of avenanthramides were affected by the sample amount, the homogeneity of the samples and the extraction time used. Especially bigger sample amount of oat flour (0.5 g) led to larger and more reproducible results than smaller amount (0.1 g). The ratio of sample and solvent 1:10 worked excellently and as an extraction solvent ethanol:water (80:20) was more efficient than tested ethanol:water (80:20) with a phosphate buffer at pH 2.8. Smaller particle size of oat flour and extraction overnight led to better extractability of avenanthramides than a short extraction of sample with larger particle size. The UHPLC method was optimized using chromatographic parameters. The avenanthramides separated from each other acceptably and were eluted as follows during the 16-minute run: 2c, 2p, 2f. The response of the UHPLC instrument was linear in the tested concentration range for all three avenanthramides. The results were reproducible, and the accuracy of the UHPLC instrument was acceptable. The recovery % for avenanthramides were 99–117%. Also, other forms of avenanthramides, like 2fd, 2pd, 5p, 5f ja 3f, were identified in different oat samples using UHPLC-QTOF-MS technique. The total amount of avenanthramides in analyzed oat samples varied between 3–41.3 µg/g per fresh weight. Oat bran included them the most and oat snack the least. Avenanthramide 2c was dominant in oat flour which included 2p the least. In oat bran, in oat drink, in oat snack, in Nyhtökaura and in Muru the avenanthramide 2f was dominant over the two other forms. The UHPLC method developed in this study can be applied to the analysis of the most common forms of avenanthramides 2c, 2f and 2p in oat and in oat products and can be used in oat research in the future. The method can also be used to identify and quantitate more widely other forms of avenanthramides in different oat products. The optimized extraction was shown to be functional and reproducible to already homogeneous and processed samples, but the extraction of non-homogeneous samples could be optimized further in the future.