OBJECTIVE： To isolate and purify three principal degraded impurities of mitiglinide calcium （impurity A， B， C） and identify their structures， establish HPLC method for content determination of impurity A，B，C. METHODS： Mitiglinide calcium was used as raw material and reacted with acid； 3 impurities were then separated by HPLC and their structures were elucidated by IR， MS， 1H NMR， 13C NMR， LC-ESI-MS and ORD. 3 impurities of 3 batches of mitiglinide calcium were determined， and the determination was performed on Agilent Extend-C18 column with mobile phase consisted of 0.01 mol/L sodium acetate solution-acetonitrile-triethylamine （60 ∶ 40 ∶ 0.1， pH＝3.0） at the flow rate of 1.0 ml/min. The detection wavelength was set at 210 nm and sample size was 20 μl. The response tests of 3 impurities and mitiglinide calcium were conducted. RESULTS： After treated with acid， impurity A， B， C had been obtained， and their purity were 99.05％， 98.87％， 99.98％， respectively after isolation and purification； after identifying the structure， 3 impurities were S-2-bezylsuccinic acid， S-2-bezylsuccinic acid-4-methyl ester， methyl （2S）-2-benzyl-3-（cis-hexahydroisoindolin-2-ylcarbonyl） propionate； methodological study of content determination of impurities were all up to the requirement. The linear range of impurity A， B， C were 0.387 5-3.875， 0.395-3.95 and 0.392 5-3.925 μg/ml （all r were 1.000 0）. The response value of impurity A， B， C and mitiglinide calcium were 2.316 1， 2.636 1， 2.617 8 and 2.620 4， respectively. CONCLUSIONS： The structures of 3 principal degraded impurities of mitiglinide calcium have been identified and confirmed； the content of them can be determined by HPLC main component self-comparison method.