[关键词]
[摘要]
目的 建立一种快速测定高尿酸模型小鼠血浆中尿酸浓度的高效液相色谱(HPLC)法,并将其用于典型药物Lesinurad的降尿酸作用评价。方法 采用Laballiance Series III HPLC系统,色谱柱为Kromasil C18柱(100 mm×4.6 mm,5 μm);流动相为甲醇/0.5%乙酸(10:90),等度洗脱,体积流量0.4 mL/min,检测波长为283 nm;应用建立的HPLC法测定小鼠ip给予250、500 mg/kg尿酸0.5、1.0、2.0 h后血浆中尿酸浓度;ig给予小鼠50、150 mg/kg Lesinurad 0.5 h后,ip 500 mg/kg尿酸制备模型,1 h后HPLC法测定血浆中尿酸浓度。结果 在建立的HPLC法中,血浆尿酸浓度在7.5~150 μg/mL与峰面积呈良好线性关系(r=0.997),方法专属性、重复性、精密度、样品稳定性、回收率均符合生物样品检测指导原则规定。与对照组小鼠内源性血尿酸浓度比较,250 mg/kg剂量组在0.5 h血浆尿酸浓度显著升高(P<0.01),500 mg/kg组在0.5、1.0、2.0 h血浆尿酸浓度均显著升高(P<0.01)。与模型组比较,50、150 mg/kg Lesinurad组血尿酸浓度均显著下降(P<0.05、0.01),且呈剂量相关性。结论 本研究建立的HPLC法简便、快速、准确,可用于小鼠血浆尿酸浓度测定及相关药物的药效评价,为小鼠高尿酸模型建立及后续降尿酸药物的筛选提供了快速可靠的分析手段。
[Key word]
[Abstract]
Objective To establish an efficient HPLC method for the determination of uric acid in plasma of hyperuricemia model mice, and the evaluation of uric acid lowering effect of Lesinurad. Methods The Laballiance Series III HPLC system was adopted with Kromasil C18 column (100 mm×4.6 mm, 5 μm). The mobile phase consisted of methanol-0.5% acetic acid (10:90) for isocratic elution with a flow rate of 0.4 mL/min. The detection wavelength was set at 283 nm. The established HPLC method was used to detect the plasma uric acid level of mice at 0.5, 1.0, and 2.0 h time points after which being ip injected with 250 and 500 mg/kg uric acid. Lesinurad of 250 and 500 mg/kg was ig given to mice, 0.5 h later, mice were ip injected with 500 mg/kg uric acid to establish hyperuricemia model, and 1 h later, the established HPLC method was used to detect the plasma uric acid level of mice. Results There was a good linear relationship between peak area and the concentration of plasma uric acid in the range of 7.5-150 μg/mL (r=0.997). The specificity, repeatability, precision, stability, and recovery of the established HPLC method was in accordance with the guiding rules of biological sample determination. Compared with the endogenous serum uric acid concentration of control group mice, serum uric acid concentration of 250 mg/kg dose group was significantly increased 0.5 h after ip administration with uric acid (P<0.01), and serum uric acid concentration of 500 mg/kg dose group was significantly increased 0.5, 1.0, and 2.0 h after ip administration with uric acid. Compared with model group, the concentration of uric acid in plasma decreased significantly in low dosage group administered with Lesinurad (P<0.05), while decreased more significantly in high dosage group (P<0.01). Conclusion This convenient, rapid, and accurate method can be applied to the determination of uric acid in mouse plasma and the evaluation of relative drugs, which provide an efficient analysis way for establishing hyperuricemia model and screening relative drugs.
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[基金项目]
“精准医学研究”国家重点研发计划(2016YFC0904903),天津市科技计划项目(16YFZCSY00910)