复合固相萃取净化-液相色谱法同时测定奶粉中的5种核苷酸

Simultaneous determination of 5 kinds of nucleotides in milk powder by liquid chromatography with composite solid phase extraction

目的建立复合固相萃取净化-液相色谱法同时测定婴幼儿配方奶粉中的5种核苷酸含量的分析方法。方法样品用纯水涡旋提取, 10%乙酸水溶液调pH到4.1,离心将上清液全部转移到25 mL容量瓶中,用纯水定容,混匀。提取液用复合固相萃取小柱(弱阴离子交换和强阳离子交换固相萃取小柱)净化,以磷酸盐缓冲液和甲醇为流动相,等度洗脱, Spursil C18柱(250 mm×4.6 mm, 5μm)色谱分离,液相色谱-紫外检测器进行测定,外标法定量。结果 5种核苷酸在一定范围内(0.1-10 mg/L、0.2-20 mg/L)线性关系良好,相关系数均大于0.999,定量限为1.0、2.0 mg/kg,检出限为0.3、0.6 mg/kg。3水平加标浓度(1.0、5.0、10.0 mg/kg, 2.0、10.0、20.0 mg/kg)的回收率在91.0%-103%之间,精密度在8%以内。结论该方法具有操作简单、灵敏度高、重现性好等特点,为奶粉中核苷酸含量测定提供一个简单准确的方法。

Objective To establish a method for the simultaneous determination of 5 kinds of nucleotides in infant formula by liquid chromatography(LC) with composite solid phase extraction. Methods The sample was extracted with ultrapure water in vortex. The pH was adjusted to 4.1 with 10% acetic acid water. The supernatant was centrifugally transferred to 25 mL volumetric flask, constanted to scale mark with ultrapure water, and mixed well. The extract was purified by composite solid-phase extraction column(weak anion exchange and strong cation exchange solid-phase extraction column). Phosphate buffer and methanol were used as mobile phase on isocratic elution. spursil C18 column(250 mm×4.6 mm, 5 μm) was used for chromatographic separation and quantification by external standard method. Results Under the optimized conditions, good linearity(0.1-10 mg/L, 0.2-20 mg/L) was observed for nucleotides. The quantitative limits were 1.0 and 2.0 mg/kg, and the detection limits of the method were 0.3 and 0.6 mg/kg. The method recoveris at the three standard levels(1.0, 5.0 and 10.0 mg/kg;2.0, 10.0 and 20.0 mg/kg) were between 91.0%-103% and the precision was within 8%. Conclusion The method has the characteristics of simple operation, high sensitivity and good reproducibility, which provides a simple and accurate method for the determination of nucleotide content in infant formula.