荧光偏振免疫分析法在小分子药物小鼠血药浓度检测中的应用

目的：建立应用荧光偏振免疫分析法（FPIA）测定小鼠血药浓度的实验方法，探讨该方法测定庆大霉素、地高辛和丙戊酸钠3种常见小分子药物小鼠血药浓度的可行性。方法：选用庆大霉素、地高辛和丙戊酸钠标准品，采用荧光偏振（FP）值非线性拟合法确定药物检出限及线性范围，在线性范围内分别设置低、中和高3个浓度组，检测质控偏差、日间精密度和小鼠血清干扰水平。选用30只KM小鼠进行药物代谢曲线测试，分为庆大霉素组（肌肉注射5 μg·g^-1）、地高辛组（灌胃20 ng·g^-1）和丙戊酸钠组（灌胃15 μg·g^-1），每组10只，给药后24 h内取5个时间点（1、3、6、12和24 h）测定小鼠血药浓度。结果：FPIA法检测3种药物的检出限均在50 μg·L^-1以下，线性范围较宽（大于10倍检出限），检测质控误差小于5%，日间精密度较高[中和高浓度组变异系数（CV）<5%]，小鼠血清对庆大霉素和地高辛的检测效果无明显影响[误差百分比（ER）<5%]，高浓度血清对丙戊酸钠的检测效果有一定的负向影响（50%血清组ER为-12.84%）。小鼠给药1 h后庆大霉素达到血药浓度峰值，半消期3 h；1 h后地高辛达到血药浓度峰值，半消期>24 h；3 h后丙戊酸钠达到血药浓度峰值，半消期6~12 h。结论：FPIA法测定小分子药物小鼠血药浓度所需样本量小，精确度高，前处理简单，具有高通量和快速检测的优势特点，可广泛应用于药理学、药剂学和临床检测研究中。

Applicationoffluorescencepolarizationimmunoassayindeterminationof small molecular drug concentrations in plasma of mice

Objective:To establish a drug concentration in plasma determination method based on fluorescence polarization immunoassay (FPIA),and to explore the feasibility of this method in monitoring the concentrations of three small molecular drugs including gentamycin,digoxin and sodium valproate in plasma of the mice. Methods:The standard samples of gentamycin,digoxin,and sodium valproate were selected. The limits and linear ranges of drugs were measured with fluorescence polarization(FP) non-linear fitting method. Three standard groups with low,medium,and high concentrations of drug were selected during liver range,and the accuracy,inter-day precisions and serum anti-interference abilities of the mice were detected.Total 30 KM mice were divided into gentamycin group (5 μg·g^-1,intramuscular injection),digoxin group(20 ng·g^-1,gavage) and sodium valproate group(15 μg·g^-1,gavage); the blood drug concentrations of the mice were measured at 1,3,6,12 and 24 h time points after administration. Results:For all three drugs detected,the detection limits detected by FPIA were lower than 50 μg·L^-1),the linear ranges were broad (more than 10 times of detection limit),the accuracy (ER) was lower than <5% and the inter-day precisions were higher (CV<5% in medium and high concentration groups).The mouse serum showed no affection to FPIA detection qualities for gentamycin and digoxin,while high concentration of serum negatively affected the detection quality for sodium valproate (ER=-12.84% in 50% serum group).After administration,gentamycin reached the peak blood concentration at 1 h with half-life period of 3 h,digoxin reached the peak blood concentration at 1 h with half-life period of >24 h,and sodium valproate reached the peak blood concentration at 3 h with half-life period of 6-12 h. Conclusion: FPIA for small molecular drug concentration in plasma of the mice has the advantages of small sample size,high accuracy,simple preprocessing,high throughput detection and rapid test,which can be applied in determination in pharmacological,pharmaceutical and clinical research.