凝胶色谱法检测特医配方婴儿乳粉中肽分子量分布

Determination of Molecular Weight Distribution of Peptides in Infant Formula for Special Medical Purposes by Gel Chromatography

摘要:

对特殊医学配方婴儿乳粉进行pH偏移处理测定其肽分子量分布。水解蛋白配方乳粉和氨基酸配方乳粉分别用水溶解，经碱偏移处理（pH值调至12静置90 min后调回至7）后收集上清液，采用TSK-Gel g2000WXL（7.8 mm×300 mm，5 μm）凝胶色谱柱，流动相为30%乙腈（含0.1%三氟乙酸，体积分数），流速为0.5 mL/min，波长为220 nm，柱温为25 ℃，进样量为10 μL，以7种蛋白标准品的分子量对数（lgMW）与保留时间做校正曲线，并使用GPC软件进行数据处理。该法用于特医乳粉中肽分子量分布测定的线性为0.994 5，精密度为0.26%~1.29%。使用本方法检测了9种品牌共29批次特医配方乳粉。结果表明，氨基酸配方乳粉中小于500 u的组分含量可达97.48 wt.%以上，乳蛋白深度水解配方乳粉中小于3 000 u的肽含量可达98.57 wt.%以上，乳蛋白部分水解配方乳粉的肽分子量分布随品牌差异结果各异。碱偏移处理可明显改善乳蛋白部分水解配方乳粉的溶解性，有利于充分展现乳粉中肽段全貌。该法前处理简单，具有良好的重复性和稳定性，可用于特殊医学配方婴儿乳粉中肽段分布的分析。

Abstract:

The peptide molecular weight distribution of infant formula for special medical purposes (FSMP) was determined by pH-shifting. The hydrolyzed protein formula and amino acid-based formula were dissolved in water and t underwent pH-shifting towards alkaline pH (pH was firstly adjusted to 12, remained at pH 12 for 90 min, then readjusted to 7) before the collection of supernatant for analysis by gel chromatography. TSK-Gel g2000WXL (7.8 mm×300 mm, 5 μm) gel chromatography column was used, with the mobile phase being 30% acetonitrile (containing 0.1% trifluoroacetic acid, volume fraction), flow rate being 0.5 mL/min, wavelength being 220 nm, column temperature was 25 ℃, and injection volume being 10 μL. Calibration curves were established between the logarithm of molecular weight (lgMW) and retention time of the 7 protein standards, and the data were processed by the GPC software. The linearity of this method for the determination of peptide molecular weight distribution in FSMP was 0.994 5 and the precision was 0.26%~1.29%. A total of 29 batches of 9 brands of FSMP products were tested by this method. The results showed that the amino acid-based formula had more than 97.48 wt.% of components smaller than 500 u, whilst the extensively hydrolyzed protein formula contained more than 98.57 wt.% of peptides smaller than 3 000 u. The peptide molecular weight distribution of partially hydrolyzed protein formula varied with different brands. The alkaline pH-shifting can significantly improve the solubility of the partially hydrolyzed protein formula, which is conducive to exhibiting fully the whole picture of peptide in the formula. The pretreatment of this method is simple. This method shows good repeatability and stability, and can be used for the analysis of peptide distribution in the FSMPs.