Determination of hyaluronic acid by high-performance liquid chromatography of the oligosaccharides derived therefrom as 1-(4-methoxy)phenyl-3-methyl-5-pyrazolone derivatives.
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.
Kakehi
K
K
Faculty of Pharmaceutical Sciences, Kinki University, Higashi-Osaka, Japan.
Ueda
M
M
Suzuki
S
S
Honda
S
S
D000818:Animals; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D006820:Hyaluronic Acid / Q000032:analysis*; D008297:Male; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D011720:Pyrazoles / Q000737:chemistry*; D047069:Pyrazolones; D012756:Sheep; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013552:Swine
D009844:Oligosaccharides; D011720:Pyrazoles; D047069:Pyrazolones; C074355:1-(4-methoxy)phenyl-3-methyl-5-pyrazolone; D006820:Hyaluronic Acid