对甲氧基苯磺酰氯柱前衍生测定尿中游离羟脯氨酸与脯氨酸

以对甲氧基苯磺酰氯为衍生试剂,建立了分离测定人尿中游离羟脯氨酸和脯氨酸的高效液相色谱方法。考察了衍生温度、衍生缓冲液pH值、衍生时间、衍生剂用量对衍生反应的影响以及流动相组成、流动相缓冲液浓度、pH值和柱温对分离的影响。在优化条件下,羟脯氨酸和脯氨酸分别在5～100μmol/L和5～250μmol/L范围内呈良好线性,相关系数分别为0.999 4、0.999 5,检出限(S/N=3)分别为0.50 nmol/L和0.20 nmol/L,回收率(n=5)分别为95%～99%和96%～102%,相对标准偏差分别为2.1%～4.3%和2.0%～4.8%。该方法可用于人尿中游离羟脯氨酸和脯氨酸的定性定量分析。

Determination of Free Hydroxyproline and Proline in Human Urine by Precolumn Derivation of MOBS-Cl

A highly sensitive precolumn high performance liquid chromatographic(HPLC) method for the simultaneous determination of free hydroxyproline(Hyp) and proline(Pro) in human urine was developed.Effects of reaction temperature,pH value,time,concentration of labeling reagent,composition of moblie phase,concentration and pH value of buffer,and column temperature were investigated in detail.Under the optimal conditions,the calibration curves showed good linear relationship in the concentration ranges of 5-100 μmol/L for hydroxyproline and 5-250 μmol/L for proline.The correlation coefficients were 0.999 4 for hydroxyproline and 0.999 5 for proline.The detection limits(S/N=3) for hydroxyproline and proline were 0.50 nmol/L and 0.20 nmol/L,respectively.The recoveries(n=5) for hydroxyproline and proline were in the ranges of 95%-99% and 96%-102% with RSDs of 2.1%-4.3%and 2.0%-4.8%,respectively.This method can be used for the qualitative analysis of free hydroxyproline and proline in human urine sample.