基于HPLC的标准加入法同时测定小鼠血清中犬尿氨酸和色氨酸

Simultaneous determination of kynurenine and tryptophan in serum of mice by standard addition method based on HPLC

目的 建立一种同时测定小鼠血清中犬尿氨酸和色氨酸浓度的高效液相色谱（HPLC）法，用于评价小鼠给予IDO抑制剂后体内IDO活性。方法 血清用6%高氯酸沉淀蛋白法处理,离心后取上清进样，采用标准加入法定量。选用ThermoHypersil GOLD色谱柱（250 mm×4.6 mm，5 μm）分离，流动相为乙腈-醋酸钠（15 mmol/L，冰醋酸调pH至 4.0），流速1.1 mL/min，柱温30 ℃，犬尿氨酸和色氨酸的紫外检测波长分别为360 nm和280 nm。结果 犬尿氨酸保留时间为6.10 min，线性范围为0.25～10 μmol/L，检测限为0.09 μmol/L；色氨酸保留时间10.12 min，线性范围为25～1 000 μmol/L，检测限为0.25 μmol/L。精密度、回收率及稳定性等均符合生物样品定量分析方法验证指导原则的要求，并成功应用于62只小鼠血清犬尿氨酸和色氨酸浓度的测定。结论 建立了一种简单、快速、准确的HPLC分析方法，采用标准加入定量方式，可同时测定小鼠血清中犬尿氨酸和色氨酸血药浓度，并成功应用于实际生物样本的测定。

Objective A high performance liquid chromatography （HPLC） method has been developed for the simultaneous determination of kynurenine and tryptophan in the serum of mice, which was used to evaluate IDO activity in vivo in mice treated with IDO inhibitors. Methods Sample was precipitated with 6% perchloric acid to remove protein before centrifugation, then the clear layer was injected.Standard addition method was used for quantitative analysis. Separation was achieved by an Thermo Hypersil GOLD column （250 mm×4.6 mm, 5 μm）. The mobile phase composed of 15 mmol/L sodium acetate （acetic acid adjust pH to 4.0） and acetonitrile, with a constant flow rate of 1.1 mL/min at 30 ℃. Kynurenineand tryptophan were measured at UV detection wavelength of 360 nm and 280 nm, respectively. Results The retention time of kynurenine was 6.10 min,the linear range was from 0.25 to 10 μmol/L, and the detection limit was 0.09 μmol/L. The retention time of tryptophan was 10.12 min, the linear range was from 25 to 1 000 μmol/L, and the detection limit was 0.25 μmol/L. Precision, accuracy and stability all were accorded with guiding principles for quantitative analysis of biological samples. This method was applied to determinate kynurenine and tryptophan in serum of 62 mice successfully. Conclusions The HPLC method with standard addition quantitative analysis was a simple, rapid and accurate tool for simultaneous determination of kynurenine and tryptophan in serum of mice, which was applied to biological samples successfully.