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高效液相色谱-蒸发光散射法测定蛇皮中胶原蛋白含量
Determination of collagen content in snake skin by HPLC-ELSD
丁瑞敏, 周华锋, 李赟高, 蔡衡, 段明星, 靳清贤
2017 (11):
659-662.
doi: 10.13218/j.cnki.csdc.2017.11.013
摘要
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建立高效液相色谱-蒸发光散射检测(HPLC-ELSD)法测定蛇皮胶原蛋白含量的方法。胶原蛋白经盐酸水解后,采用Thermo Hypersil Gold C18(250 mm×4.6 mm,5 μm)色谱柱进行分离,以乙腈(含0.2%甲酸,A相)-0.2%甲酸水溶液(B相)为流动相进行梯度洗脱,洗脱时间15 min,柱温30 ℃,流速0.4 mL/min。ELSD参数:雾化器温度85 ℃,蒸发器温度85 ℃,氮气作载气,蒸发器气流1.2 L/min。进样量10 μL。在此条件下,羟脯氨酸的线性范围为10~100 mg/L,R2=0.999 6,检测限为5.56 mg/L,定量限为10 mg/L。蛇皮胶原蛋白的平均回收率为94.53%,RSD为2.15%。蛇皮中胶原蛋白质量分数平均值为40.21%,为蛇皮的主要蛋白质。
A high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) method for determination of content of collagen in snake skin was established.After the collagen in snake skin sample was hydrolyzed with hydrochloric acid,separation was performed by a Thermo Hypersil Gold C18(250 mm×4.6 mm,5 μm) chromatographic column and acetonitrile (containing 0.2% formic acid,mobile phase A)-water (containing 0.2% formic acid,mobile phase B) as the mobile phase were used for gradient elution.The elution time is 15 min.The column temperature is 30 ℃;and the flow rate is 0.4 mL/min.The ELSD detector parameters are as follows:nebulization temperature,85 ℃;evaporation temperature,85 ℃;carrier gas,N2;air flow of the evaporator,1.2 L/min.The injection volume of the sample is 10 μL.Under these chromatographic conditions,the linearity range of hydroxyproline is 10 - 100 mg/L,R2 = 0.999 6;the lowest limit of detection is 5.56 mg/L,and the lowest limit of quantification is 10 mg/L.The average recovery rate of snake skin collagen is 94.53%;the RSD is 2.15%;and the average of the mass fraction of collagen in snake skin is 40.21%.
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