一种新的高效液相色谱法选择性测定尿羟脯氨酸

Selective determination of hydroxyproline in urine by high performance liquid chromatography

目的 建立一种新的高效液相色谱法，选择性测定尿中羟脯氨酸的含量。方法 采用邻苯二甲醛和芴甲基氯甲酸酯两个衍生剂，先后处理尿标本，以国产 Ｃ８ 柱为固定相，在醋酸钠缓冲液中加入２３ ％ 的乙腈作为洗脱液，３ ，４脱氢脯氨酸作为内标。结果 测定结果的批内、批间变异系数分别为１ ．９２ ％ 和３ ．５８ ％ ，回收率为（９７ ．０ ±３ ．０） ％ ，在３１ ．２５ ～５００ ．００ μｍｏｌ／ Ｌ 范围内线性关系良好（ｒ 为０ ．９９７） 。结论 该法排除了尿液中具有一级胺结构的氨基酸在羟脯氨酸测定中的干扰，特异性好，无需梯度洗脱，适合临床检测需要。

Objective To establish a new method for selective determination of urinary hydroxyproline by HPLC. Method After acid hydrolysis of urine, primary amino acids were derivatized with ortho phthaldialdehyde and removed from the sample by use of silica gel, followed by devivatized of hydroxyproline with 9 fluorenylmethyl chloroformate, and then injected into a C8 reversed phase column. Using sodium acetate buffer (50 mmol/L, pH4.2) /acetomitrile (77:23) as mobile phase, 3,4 dehydroproline was used as internal standard.Results Within and between run were 1.92% and 3.58%. Analytical recovery of hydroxyproline added to urine samples was (97.6±3.0)%. The linear range of the detection was 31.25～500.00 μmol/L ( r =0.997). Conclusions The method was ensured to eliminate the interference from primary amino acid completely it is specific, accurate and suitable for clinical laboratory diagnosis.