Institute of Sciences of Food Production, National Research Council of Italy (ISPA-CNR), Bari, Italy.
Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2011 Oct;28(10):1304-14. doi: 10.1080/19440049.2011.593191. Epub 2011 Jul 21.
A method based on LC-ESI-high-resolution (HR)-MS analysis, using a single-stage Orbitrap mass spectrometer, was developed for the quantification of casein allergens potentially present in white wines as a result of fining by caseinate. The method consists of (1) extraction from the matrix by ultrafiltration, (2) digestion with trypsin and (3) detection/quantification of residual caseins, obtained by monitoring the LC-MS response of representative tryptic peptides (peak areas in extracted-ion chromatograms). Method linearity was assessed first on caseinate solutions prepared either in water or in wine matrix (the ultrafiltration residue of a protein-free white wine). Limits of detection (LOD) ranged from 0.1 to 0.3 µg ml(-1) (S/N = 3) in water, and between 0.15 and 0.7 µg ml(-1) in wine matrix, depending on the selected peptide. Method repeatability and reproducibility, measured as response variability (standard deviation) due to LC-MS analysis alone and to both enzymatic digestion and LC-MS analysis, were assessed on caseinate standard solutions in water and ranged from 5 to 12% and from 8 to 20%, respectively. A higher variability was usually observed for the peptide marker response in the case of matrix-matched samples, the only exception being peptide GPFPIIV from β-casein, the marker also providing the highest sensitivity. The method was finally applied to a casein-free white wine ('Greco di Tufo') fined with caseinate at different concentrations, after discarding the precipitate due to casein-wine components aggregation. Minimum detectable added caseinate concentrations (i.e. those corresponding to responses with S/N = 3) were estimated between 39 and 51 µg ml(-1), according to the peptide marker chosen. These limits are compatible with caseinate concentrations typically adopted for wine-fining purposes. Moreover, a cross-check with the calibration performed in wine matrix led to an estimation of the concentration of dissolved caseinate to be in the low ng ml(-1) range.
一种基于 LC-ESI-高分辨率 (HR)-MS 分析的方法，使用单级轨道阱质谱仪，被开发用于定量分析由于乳清蛋白酸盐澄清而潜在存在于白葡萄酒中的酪蛋白过敏原。该方法包括（1）通过超滤从基质中提取，（2）用胰蛋白酶消化，以及（3）通过监测代表性胰蛋白酶肽（提取离子色谱图中的峰面积）的 LC-MS 响应来检测/定量残留的酪蛋白。首先在乳清蛋白酸盐溶液中评估方法的线性度，这些溶液是在水或葡萄酒基质（不含蛋白质的白葡萄酒的超滤残留物）中制备的。在水中，检测限 (LOD) 范围为 0.1 至 0.3μg/ml（信噪比 = 3），而在葡萄酒基质中，取决于所选肽，LOD 范围为 0.15 至 0.7μg/ml。方法的重复性和再现性，以 LC-MS 分析本身以及酶解和 LC-MS 分析的响应变异性（标准偏差）来评估，在水中的乳清蛋白酸盐标准溶液中进行，范围为 5 至 12%，在水中分别为 8 至 20%。对于基质匹配样品中的肽标记物响应，通常观察到更高的变异性，唯一的例外是来自 β-酪蛋白的肽 GPFPIIV，该标记物也提供了最高的灵敏度。该方法最后应用于一种用乳清蛋白酸盐在不同浓度下澄清的无酪蛋白白葡萄酒（“Greco di Tufo”），在由于酪蛋白-葡萄酒成分聚集而丢弃沉淀后。根据所选的肽标记物，估计最低可检测添加的乳清蛋白酸盐浓度（即响应信噪比 = 3 时对应的浓度）在 39 至 51μg/ml 之间。这些限制与通常用于葡萄酒澄清目的的乳清蛋白酸盐浓度兼容。此外，与在葡萄酒基质中进行的校准进行交叉检查，导致对溶解的乳清蛋白酸盐浓度的估计处于低 ng/ml 范围。
