HPLC法同时测定人血浆中异烟肼及乙酰异烟肼的浓度

Simultaneous determination of isoniazid and acetylisoniazid in human plasma by HPLC

目的建立HPLC法同时检测人血浆中异烟肼及其代谢产物乙酰异烟肼的浓度。方法采用HPLC法,血浆用20%三氯乙酸沉淀蛋白后,旋涡混匀1 min,离心10 min,取上清液进行分析,以外标法定量。色谱柱为SinoChrom ODS-BP(250 mm×4.6 mmm,5μm),保护柱为Hypersil ODS(C18),流动相为乙腈-0.05 mol·mL^(-1)磷酸二氢钾溶液(含0.04%三乙胺)(3:97),流速为1.0mL·min^(-1),柱温为30.0℃,检测波长为265 nm。结果异烟肼和乙酰异烟肼质量浓度在0.25~20.00 mg·L^(-1)内线性关系良好,回归方程分别为y=1 113.0 x-150.9(r=0.999 8)和y=529.8x-131.9(r=0.999 7),定量下限均为0.25 mg·L^(-1);异烟肼的回收率为100%~104%,乙酰异烟肼为90%~106%;低、中、高浓度的日内、日间精密度良好(RSD均<15%)。结论本方法专属性好,样品处理简单,适用于人血浆中异烟肼及乙酰异烟肼的含量测定。

AIM To establish an HPLC method for the determination of isoniazid（INH） and acetylisoniazid （Ac- INH） in human plasma. METHODS By HPLC method, serum protein was precipitated with 20% trichloroacetic acid, mixed vortically for 1 min, then centrifuged for 10 min, and the supernatant was taken for analysis. Separation was performed on a SinoChrom ODS-BP column with a Hypersil ODS（C18） guard column. The mobile phase was acetontrile - 0.05 mol· mL^- 1 KH2PO4（0.04% triethylamine） with the ratio of 3：97. The column temperature was 30.0℃. The flow rate was 1.0 mL· min^- 1 and the detection wavelength was 265 nm. RESULTS Both calibration curves had good linearity in the range of 0.25- 20.00 mg· L^-1. The calibration curves of isoniazid and acetylisoniazid were y = 1 113.0 x- 150.9（ r = 0.999 8）and y = 529.8x - 131.9（ r = 0.999 7） respectively. The limit of quantification was 0.25 mg·L^- 1 for both isoniazid and acetylisoniazid. The recovery of isoniazid was in the range of 100% - 104%, and that of acetylisoniazid was in the range of 90% - 106%. The intra-day and inter-day RSD for assaying the sample containing three concentrations of isoniazid and acetylisoniazid were all lower than 15%. CONCLUSION The method is sensitive, simple and quick. It is suitable for the analysis of isoniazid and acetylisoniazid in human plasma.