柱前衍生-反相高效液相色谱法测定扶正补血食疗方胶原蛋白含量

Precolumn derivatization-reversed-phase high-performance liquid chromatography for the determination of collagen in Fuzheng Buxue dietary supplement

目的基于RP-HPLC法对扶正补血食疗方中胶原蛋白含量进行测定,并通过正交试验优选出扶正补血食疗方中胶原蛋白的最佳提取方法。方法于2021年12月至2022年1月将样品以6 mol/L盐酸于110℃水解24 h,利用AQC试剂进行衍生,采用Diamonsil-C_(18)色谱柱(250 mm×4.6 mm,5μm),以流动相A:无水乙酸钠11.48 g,三乙胺1.72 g,EDTA 1 mg,溶于1 L水中,用磷酸调pH为4.95;流动相B:乙腈水溶液(体积分数60%),梯度洗脱;柱温37℃;检测波长254 nm;流速1 mL/min;进样量10μL。以羟脯氨酸含量为评价指标,考察煎煮时间、料水比、煎煮温度对胶原蛋白含量的影响,优化其制备工艺。结果羟脯氨酸在10~500 mg/L浓度范围内线性关系良好(Y=0.0365X+0.5173,R^(2)=0.9991),平均回收率95.11%,相对标准偏差(RSD)为1.12%(n=6)。猪蹄胶原蛋白的最佳提取工艺为:A_(3)B_(1)C_(3),即加热温度为100℃,料水比为1∶8,提取时间为3 h。结论该方法快速准确,稳定可行,可作为扶正补血食疗方中胶原蛋白含量的测定方法,为该产品质量控制及临床应用提供科学依据。

Objective To determine the collagen content in Fuzheng Buxue dietary supplement based on the RP-HPLC method and to optimize the best extraction method of collagen in Fuzheng Buxue dietary supplement by orthogonal testing.Methods Samples were hydrolyzed with 6 mol/L hydrochloric acid at 110℃for 24 h from December 2021 to January 2022 and derived using AQC reagent on a Diamonsil-C_(18) column(250 mm×4.6 mm,5μm)with mobile phase A:anhydrous sodium acetate 11.48 g,triethylamine 1.72 g,EDTA 1 mg,dissolved in 1 L of water.Mobile phase B:aqueous acetonitrile(60%v/v),gradient elution;column temperature 37℃;detection wavelength 254 nm;flow rate 1 mL/min;injection volume 10μL.The effects of decoction time,ratio of material to water,and decoction temperature on the collagen content were investigated to optimize the preparation process using the hydroxyproline content as the evaluation index.Results Hydroxyproline showed good linearity in the concentration range of 10-500 mg/L(Y=0.0365X+0.5173,R^(2)=0.9991)with an average recovery of 95.11%and RSD value of 1.12%(n=6).The best extraction process for pig trotter collagen was A_(3)B_(1)C_(3),i.e.,the heating temperature was 100℃,the ratio of material to water was 1∶8,and the extraction time was 3 h.Conclusion The method is rapid,accurate,stable and feasible and can be used as a method for the determination of collagen content in Fuzheng Buxue dietary supplement and provides a scientific basis for product quality control and clinical application.