高效液相色法测定保加利亚乳杆菌胞内Acetyl_CoA.docVIP

高效液相色谱法（HPLC）测定保加利亚乳杆菌胞内Acetyl-CoA 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C 18（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0.011~0.359μmol/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。 关键词：高效液相色谱，保加利亚乳杆菌，Acetyl-CoA Determination of intracellular Acetyl-CoA in L.bulgaricus by HPLC Abstract：A high performance liquid chromatographic （ HPLC ） method for the determination of Acetyl -CoA in Lactobacillus bulgaricus was developed . The stationary phase used were C 18 （ 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 of 0.011 ~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. Key words：high performance liquid chromatography（HPLC）；Lactobacillus bulgaricus；Acetyl-CoA 中图分类号：TS201.3 文献标识码：A 文 章 编 号：1002-0306（2013）22-0058-05 收稿日期：2013－06－04 * 通讯联系人 作者简介：李培照（1986-），男，硕士研究生，研究方向：乳酸菌代谢。 基金项目：高等学校博士学科点专项科研基金新教师类资助