Content Determination Of Multiple Amino Acids In Donkey-hide Glue And Lvjiao Bu Xue Granules

Objective:This project aimed to establish a content determination method for Donkey-hide Glue,by RP-HPLC using Phenylisothio-cyanate(PITC) as the pre-column derivatized reagent, then evaluate similarity of more than 10 batch materia medica. And establishing a content determination method for Lvjiao bu xue granules, evaluating similarity of more than 10 batch preparation.Methods:Put the samples in to ampoules, acid hydrolysis by 6mol/L hydrochloric acid, used 0.1mol/L hydrochloric acid solution to has a constant volume, then determined by RP-HPLC using Phenylisothio-cyanate(PITC) as the pre-column derivatized reagent. The Hypersil ODS2 C18 (4.6mm×200mm, particle size 5μm) column was used. The mobile phase A was mixing acetonitrile-0.1mol/L sodium acetate solution (3:97),and the mobile phase B was mixing acetonitrile-water (4-1), gradient elution. The wavelength for detection was 254 nm. The flow rate was 1.OmL/min.The column temperature was 30℃and the injection volume was 5μL. Confirmed the position of each peak in samples, determined the contents of L-Glutamic acid, L-Hydroxyproline, Glycine, L-Alanine, L-Arginine, L-Proline, then evaluated similarity of chromatographies of more than 10 batch materia medica or preparation, to establish compared HPLC chromatographic fingerprint, using traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2004A edition, which developed by State Pharmacopeia Committee of China. Results:1. We established a content determination method for Donkey-hide Glue and Lvjiao bu xue granules, by RP-HPLC using Phenylisothiocyanate(PITC) as the pre-column derivatized reagent. We chose 6 main amino acids to determine their contents,well reflecting the condition of protein and amino acids in Donkey-hide Glue. Donkey-hide Glue contains 17 kinds of amino acids, sum of average contents of L-Glutamic acid, L-Hydroxyproline, Glycine, L-Alanine, L-Arginine, L-Proline is 64.33% in Donkey-hide Glue, 5.900% in Lvjiao bu xue granules.2. We got HPLC chromatographic fingerprints of Donkey-hide Glue and Lvjiao bu xue granules, including various technical parameters. Similarity of 10 batch Donkey-hide Glue are above 0.998, and which of 17 batch Lvjiao bu xue granules are above 0.999.Conclusion:Relative to amino acid analyzer, experimental method of this topic is simple and convenient, stable, wide applicability, precision, accurate and reliable. It expanded the variety of amino acids which can determine simultaneously, and established a content determination method for Donkey-hide Glue and Lvjiao bu xue granules. It can be used for quality control of Donkey-hide Glue and Lvjiao bu xue granules.