生物转化L-脯氨酸生成反式-4-羟基-L-脯氨酸的定量分析方法

Quantitative Analysis of trans-4-Hydroxy-L-proline by Microbial Conversion of L-Proline

以芴甲氧羰酰氯（FMOC-Cl）为柱前衍生剂，四硼酸钠为缓冲溶液，建立了一种柱前衍生反相高效液相色谱（RP-HPLC）测定微生物转化L-脯氨酸生成反式-4-羟基-L-脯氨酸的定量分析方法。优化了衍生反应条件，当衍生反应体系中水和乙腈的比例为66∶34，pH值为9.9时衍生效果最佳。采用Agilent Extend C-18柱进行分离，以0.1%三氟乙酸水溶液和乙腈作为流动相，梯度洗脱，检测波长263 nm。L-脯氨酸和反式-4-羟基-L-脯氨酸均在0.01~500 mg/mL范围内线性关系良好，相关系数均大于0.999，检出限为5.00~7.00 ng/L，不同浓度下的平均回收率分别为98.9%~102%和97.9%~100%。该方法重现性好，精密度高，为定量分析微生物发酵液中的L-脯氨酸和反式-4-羟基-L-脯氨酸提供了有效方法。

trans-4-Hydroxy-L-proline is one of the valuable chiral building block in the production of many pharmaceuticals.A quantification method of trans-4-hydroxy-L-proline and L-proline from microbial transformation by using reverse phase high performance liquid chromatography （RP-HPLC） was developed.9-Fluorenylmethyl chloroformate（FMOC-Cl） was used as pre-column derivation agent,and the derivatization was proceeded in sodium tetraborate buffer solution（pH 9.9） containing acetonitrile-water（66∶34）.The solution then was separated on an Agilent Extend C-18 column by using 0.1% trifluoroacetic acid and acetonitrile as mobile phase,and detected with UV detector at 263 nm.The linear ranges of two amino acid were in the range of 0.01-5.00 mg/mL,with correlation coefficients greater than 0.999.The limits of detection（LOD） were in the range of 5.00-7.00 ng/L,and the average recoveries of trans-4-hydroxy-L-proline and L-proline were 98.9%-102% and 97.9%-100%,respectively.This method provides an effective way to quantify L-proline and trans-4-hydroxy-L-proline in microorganism fermented liquid system with good reproducibility,and high precision.