Determination of the Content and Purity of Sublancin by Reversed-phase High Performance Liquid Chromatography

YANG Cai-xia;WU Jin-mei;HE Tao;Sinagri Yingtai Linzhou Biotechnology Park Co.,Ltd.;Beijing Sinagri Yingtai Biological Technology Institute Co.,Ltd.;Henan University of Animal Husbandry and Economy;

Sublancin;;RP-HPLC;;Content;;Purity;;Quantitative detection

[Objective] To establish a reversed-phase high performance liquid chromatography(RP-HPLC) method for detecting the content and purity of sublancin.[Method]Chromatography was performed on ZORBAX 300 SB-C_8 chormagraphic column(4.6 mm×150 mm, 5 μm),using trifluoroacetic acid-water(1∶1 000) as mobile phase A,using trifluoroacetic acid-water-acetonitrile(0.85∶200∶800)as mobile phase B;the column temperature was 25 ℃,the detection wavelength was 280 nm, the flow rate was 1.0 mL/min, the injection volume was 20 μL,linear gradient elution.[Result]At room temperature, the sublancin solution was stable within 12 hours, and the RSD was 0.61%.The RSD of the main peak retention time and peak area of subtilisin were 0.18% and 0.22%,respectively, and the RSD between different experimenters was 0.92%.The limit of quantification(LOQ) of sublancin was 120 ng, the limit of detection(LOD) was 80 ng, and the linear relationship was good between 0.031 25 and 2.000 00 mg/mL(R~2=1).The average recovery rate was 101.49%(n=9),and the RSD was 0.29%.Changed the column temperature, flow rate and chromatographic column batch number, the theoretical plate numbers of sublancin were all greater than 2 000,the degree of separation was greater than 1.5,and the absolute deviation of the relative retention time(RRT) was less than 20%.The content of three batches of sublancin were 98.8%,99.8% and 100.2%,respectively, with purity greater than 99%.[Conclusion]The detection method established in this study is sensitive, accurate and highly specific, which meets the requirements for quantitative detection of sublancin content and purity, and is suitable for quality control of sublancin.