Kakehi K, Ueda M, Suzuki S, Honda S
Faculty of Pharmaceutical Sciences, Kinki University, Higashi-Osaka, Japan.
J Chromatogr. 1993 Feb 5;630(1-2):141-6. doi: 10.1016/0021-9673(93)80449-i.
Hyaluronic acid (HA) was digested with various kinds of depolymerizing enzymes and the products were analysed by high-performance liquid chromatography (HPLC) after derivatization with 1-(4-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). As hyaluronate 4-glycanohydrolase (EC 3.2.1.35) from sheep testis showed a high efficiency for depolymerization, giving the tetra- and hexasaccharides abundantly, and is inexpensive, a method for the specific determination of HA was established, based on digestion by this enzyme followed by determination of the tetra- or hexasaccharide derived therefrom as the PMPMP derivatives by HPLC with UV detection. This method allowed the determination of HA in the range 0.5-50 micrograms with high reproducibility.
用各种解聚酶消化透明质酸（HA），并用1-（4-甲氧基）苯基-3-甲基-5-吡唑啉酮（PMPMP）衍生化后，通过高效液相色谱（HPLC）分析产物。由于来自绵羊睾丸的透明质酸4-聚糖水解酶（EC 3.2.1.35）显示出高效的解聚效率，能大量产生四糖和六糖，且价格低廉，因此建立了一种特异性测定HA的方法，该方法基于用这种酶消化，然后通过带紫外检测的HPLC测定由此衍生的四糖或六糖的PMPMP衍生物。该方法能够在0.5 - 50微克范围内以高重现性测定HA。
