同时测定血浆 INH、AcINH、RFP 和 PZA 的高效液相色谱法建立及其在肺结核患者中的应用

摘要

Objective To establish the high-performance liquid chromatography ( HPLC) in order to determine the concentration of isoniazid (INH), rifampicin (RFP), pyrazinamide (PZA) and acetylisoniazid (AcINH) simultaneously in the plasma of patients with tuberculosis , and to verify its effect .Methods The HPLC method was carried out on a Wa-ters Xselect-HSS T3 (250 mm ×4.6 mm, 5 μm) column with 0.05 mol/L KH2 PO4 ( adjusting PH to 5.6 with triethyl-amine)-acetonitrile as mobile phase .The column temperature was 40 ℃, the flow rate was 0.8-1.0 mL/min, and the in-ject volume was 20μL.Meanwhile, the plasma containing reference substances of INH , RFP, PZA and AcINH were injec-ted.Then we tested the specificity , linear relationship, precision, accuracy, stabilization and extraction recovery .We de-tected the concentrations of INH , AcINH, RFP and PZA in the plasma of 10 tuberculosis patients who took INH , RFP and PZA as treatment program by HPLC method .Results This method had a good separation effect on INH , AcINH, RFP, and PZA without interference of each other , and all calibration curves had good linearity ( r2 >0.99) within the test ran-ges.The intra-and inter-day precision was less than 10%, the extraction recovery was between 80%-120%.Except 1 case of AcINH and 1 case of RFP below the linear range , during the 10 tuberculosis patients , the rest were within the linear range.Conclusions HPLC that can determine the concentrations of INH , PZA, RFP and AcINH simultaneously in plas-ma of tuberculosis patients is successfully established .The method is sensitive , simple and rapid .It is suitable for analyti-cal require of biological samples .%目的：建立同时测定血浆异烟肼（ INH）、乙酰异烟肼（ AcINH）、利福平（ RFP）、吡嗪酰胺（ PZA）的高效液相色谱法（ HPLC），并验证其在肺结核患者中的应用效果。方法 HPLC色谱柱为Waters Xselect-HSS T3（250 mm ×4．6 mm，5μm），以乙腈和磷酸二氢钾为流动相，柱温40℃，流速0．8～1．0 mL／min，进样体积20μL。同时进样含INH、AcINH、RFP、PZA对照品的血浆，检验方法的专属性、线性关系、精密度和准确度、稳定性及回收率。取联合服用INH、乙胺丁醇、RFP和PZA的10例肺结核患者的血浆样本，采用上述HPLC测定INH、AcINH、RFP和PZA的血药浓度。结果血浆样本中INH、AcINH、RFP和PZA各组分有较好的分离，互不干扰；在线性范围内与峰面积线性关系良好（r2＞0．99）；日内、日间精密度均＜10％；稳定性较好；回收率为80％～120％。肺结核患者中除1例AcINH和1例RFP血药浓度低于线性范围外，其余数据均在线性范围内。结论成功建立了同时测定血浆INH、AcINH、RFP、PZA的HPLC方法，该方法专属性好，样品处理简单，符合生物样品分析要求。