酶解高效液相色谱法测定硫酸软骨素的含量

Determination of chondroitin sulfate content by enzymatic HPLC

目的建立酶解高效液相色谱法测定硫酸软骨素含量的方法。方法采用硫酸软骨素ABC酶制备样品,高效液相色谱法测定含量。以Ultimate XB-NH2柱(4.6 mm×25 cm,5μm)分离样品,醋酸钠缓冲液(pH 5.6)-乙腈(950∶50)为流动相,流速1.0 mL/min,检测波长232 nm,进样量10μL,柱温30℃。结果在此色谱条件下,硫酸软骨素A、B、C三组分分离良好;硫酸软骨素浓度在300～3 000μg/mL之间与峰面积呈良好线性关系;高、中、低三个浓度平均加样回收率为102.1%,100.4%和97.5%。结论本法简便、准确、可靠,可用于硫酸软骨素的含量测定。

Purpose To establish an enzymatic HPLC method for determination of chondroitin sulfate content. Methods The sample was prepared by chondroitinase ABC and the content was determined by HPLC. The sample was separated and analyzed on a Ultimate XB-NH2 column （25 cm× 4.6 mm ,5 μm） , and a salt acetate buffer solution（pH 5.6）- acetonitrile（950：50）was used as mobile phase. The flow rate was 1.0 mL/min. The detective wavelength was 232 nm. The injection volume was 10μL. The column temperature was 30 ℃. Results The ehondroitin sulfate A, B,C three ingredient peaks could be separated each other completely. The calibration curve was linear at a range of 300-3 000 μg/mL. The average re- coveries（ n = 3 ） of high, middle, low concentrations were 102. 1% , 100.4% and 97.5% respectively. Conclusion The method was simple,accurate,reliable. It can be used for the determination of content in chondroitin sulfate.