HPLC梯度洗脱法测定血浆中柳氮磺吡啶及其代谢物

Quantification of sulfasalazine and its metabolites in human plasma by HPLC

目的：建立人血浆中柳氮磺吡啶（SASP）及其代谢物磺胺吡啶（SP）、乙酰磺胺吡啶（AcSP）浓度的高效液相色谱（HPLC）测定方法。方法：液相分离采用Hedera ODS-2分析柱,柱温：30℃,以甲醇0.05 mol/L乙酸铵溶液（含0.1%三乙胺）溶剂系统梯度洗脱,流速为1.0 ml/min,进样量20μl;检测波长243 nm。以苯巴比妥（PB）为内标,血浆样品用甲醇沉淀蛋白质后取上清液进样分析。结果：血浆中其他组分均不干扰样品的测定;在0.5-100mg/L范围内SP、AcSP和SASP均呈良好的线性关系,相关系数r均〉0.999 2;提取回收率（n=5）分别为97.1%-101.3%、97.7%-103.3%、94.9%-102.3%;日内相对标准偏差（RSD）分别为1.44%-3.04%、1.76%-3.85%、2.12%-3.22%,日间RSD分别为2.01%-3.69%、2.11%-3.99%、2.56%-3.56%。结论：本方法操作简便、选择性好、准确、灵敏,可用于柳氮磺吡啶的药代动力学研究。

Objective： To develop a validated, highly sensitive and selective high performance liquid chromatographic （HPLC） method for the quantitative determination of sulfasalazine （SASP） and its metabolites sulfapyridine （SP） and N-acetylsulfapyridine （AcSP） in human plasma. Methods： After protein precipitation with methanol, 20 ul of supernatant was injected onto a Hedera ODS-2 column and the effluent measured for UV absorption at 243 nm. 0.05 mol/L ammonium acetate （ containing 0. 1% triethylamine） was used as mobile phase A and methanol as mobile phase B with the flow rate of 1.0 ml/min. A gradient elution program was applied, and phenobarbital used as the internal standard. Results： The linear concentrations of SP, AcSP and SASP ranged from 0.5 to 100 mg/L （ r 〉 0.999 2） ; the rates of extraction recovery were 97.1% - 101.3% for SP, 97.7% - 103.3% for AcSP and 94.9% - 102.3% for SASP; the intra- and inter-day RSDs were 〈 4.0%. Conclusion： The HPLC method, with its high sensitivity, accuracy and repeatability, can be used in the pharmacokinetic and bioavailability studies of SASP.