大鼠尿液中阿仑膦酸钠的柱前衍生化HPLC-UV测定法及药动学应用

Determination of alendronate sodium in rat urinary by pre-column derivation HPLC-UV method and its application to pharmacokinetics

目的:建立大鼠尿液中阿仑膦酸钠柱前衍生HPLC-UV测定方法,并应用于大鼠口服阿仑膦酸钠后尿液排泄动力学研究。方法:大鼠单次灌胃给予阿仑膦酸钠溶液(4.5 mg·kg^(-1)),在给药后不同时间段收集尿液,尿液固相萃取预处理后,采用异硫氰酸苯酯(PITC)对尿液中的阿仑膦酸钠衍生化。液相色谱分析采用Venusil AA分析柱(4.6 mm×250 mm,5μm),以乙酸钠溶液-乙腈为流动相,梯度洗脱,正亮氨酸为内标,流速0.8 mL·min^(-1),检测波长270 nm。结果:阿仑膦酸钠质量浓度在2.5~250.0μg·mL^(-1)范围内线性良好(r=0.999);方法的日内和日间精密度小于3.0%,提取回收率在87.5%~89.2%之间;检测限为0.2μg·mL^(-1)。尿液排泄动力学曲线显示阿仑膦酸钠溶液口服后达峰时间约为2.16 h,清除半衰期为(4.5±0.12)h,36 h尿液累积量为(42.6±1.2)μg,占给药剂量的4.7%。结论:本方法经方法学验证,可用于阿仑膦酸钠尿液排泄动力学研究。

s Objective： To develop a pre-column derivation HPLC-UV method for the determination of alendronate sodium in rat urinary and its application for pharmacokinetics. Methods： Rats were administered with a single oral dose of alendronate sodium solution （ 4.5 mg· kg^-1 ）, urine samples were collected at different time intervals. After solid phase extraction pretreatment, the urine samples were derivatized by phenyl isothiocyanate （ PITC ）. HPLC analysis was carried out on a Venusil AA column （ 4.6 mm × 250 mm, 5 μm ）, using acetate sodium and acetonitrile as mobile phase with gradient elution. The flow rate was 0.8 mL· min-1 and the detection wavelength was set at 270 nm. Norleueine was used as internal standard （ IS ）. Results： Linear calibration was established for alendronate sodium in rat urinary in the range of 2.5-250.0 μg· mL^-1 （ R=0.999 with good precision （ RSD〈3.0% ） and high accuracy （ the extraction recovery of 87.5%-89.2% ） at high, medium and low quality control levels. The limit of detection was 0.2μg· mL^-1. Urinary alendronate sodium concentration versus time curves showed the maximum excretion arrived at 2.16 h. The elimination half-life time in urine was found to be（ 4.5 ± 0.12 ）h. The cumulative excretions of alendronate sodium for 36 h were （ 42.6 ± 1.2 ） μg, which was about 4.7% of the dosage. Conclusion： The developed HPLC-UV method was validated and can be applied to alendronate sodium rat urinary excretion study.