Determination of Bovine Type¢ñ Collagen by High Performance Liquid Chromatography-Mass Spectrometry

KeyWord:high performance liquid chromatography-mass spectrometry(HPLC-MS)  marker peptide  bovine type¢ñ collagen  quantitation

Abstract:

      A high performance liquid chromatography-mass spectrometric(HPLC-MS) method was developed for the determination of bovine type¢ñ collagen.Collagen sponge samples were digested with trypsin,then the digesting mixture was separated with a C18 column.The detection was performed with electrospray ionization in positive mode and using single ion monitoring(SIM) mode for data collection,and the quantitation was achieved by external standard method.A marker peptide GFSGLDGAK was obtained in the tryptic digestion of bovine type¢ñ collagen.There was a good linearity for the marker peptide in the concentration range of 0.645-21.5 ¦Ìg/mL(r2=0.999 4).The quantitation limit of the method was 6.45¡Á10-4 ¦Ìg/mL.Recoveries for the target at three spiked levels of 3.10,2.48 and 1.55 ¦Ìg/mL ranged from 98.2% to 102%,with relative standard deviations(n=6) of 0.80%.The collagen sponge samples were tested for uniformity,stability and constant value to ensure the effectiveness of the method and the quality of the product,and the content of bovine type¢ñ collagen was determined to be £¨0.926¡À0.014£© mg/mg.The method is simple,rapid,replicable and high stable,and could meet the requirements for determination of bovine type¢ñ collagen.