高效液相色谱法（HPLC）测定保加利亚乳杆菌胞内Acetyl-CoA

李培照; 李春; 梁婉婷; 刘丽波

doi:10.13386/j.issn1002-0306.2013.22.035

高效液相色谱法（HPLC）测定保加利亚乳杆菌胞内Acetyl-CoA

Determination of intracellular Acetyl-CoA in L.bulgaricus by HPLC

摘要

摘要: 建立了一种快速测定保加利亚乳杆菌胞内Acetyl-CoA含量的HPLC分析方法。以C18（Hypersil ODS2 5μm）色谱柱为固定相,以缓冲液A（0.2mol/L磷酸钠,pH=5）和缓冲液B（800mL 0.25mol/L磷酸钠,pH=5和200mL乙腈的混合物）为流动相,梯度洗脱,流速为1mL/min,柱温25℃,采用紫外检测器（254nm）进行检测。结果表明:该方法可以在17min内实现Acetyl-CoA与其他物质完全分离和定量,Acetyl-CoA在0.011⁰.359μmol/mL范围内的线性关系良好,回归方程线性相关系数R2=0.9995,检出限（以信噪比（S/N）为3计）为0.28mg/L,定量限（以S/N为10计）为0.32mg/L。该方法用于保加利亚乳杆菌胞内Acetyl-CoA含量的测定,相对标准偏差（n=6）为0.75%,重现性良好,三个水平的加标回收率为94.8%¹03.3%,该方法适用于保加利亚乳杆菌胞内Acetyl-CoA快速、高效分离和定量。

Abstract: A high performance liquid chromatographic ( HPLC) method for the determination of Acetyl-CoA in Lactobacillus bulgaricus was developed. The stationary phase used were C18 ( Hypersil ODS2 5μm) chromatographic column and the two mobile-phase solvents used were buffer A (0.2mol/L sodium phosphate, pH5) and buffer B (800mL of 0.25mol/L sodium phosphate mixed with 200mL pH5 of acetonitrile) , gradient elution and at a flow rate of 1mL/min, temperature of column at 25℃, the UV detector was employed to monitor column effluent at 254nm. Results indicated that : Acetyl- CoA and other substances were completely separated and determined in 17min, the linear correlation coefficients were above 0.9995 in the range of0.011 ⁰.359μmol/mL. The limit of detection of acetyl coenzyme A was 0.28mg/L, the limit of quantitation of acetyl coenzyme A was 0.32mg/L. Under these optimized conditions, the recovery of the Acetyl-CoA in Lactobacillus bulgaricus at three spiked levels were in the range of 94.8%¹03.3% with the relative standard deviations (n=6) of 0.75%. This method was fast and accurate for the quantitative analysis of the Acetyl-CoA in Lactobacillus bulgaricus.