柱前衍生化HPLC-FD法测定大鼠体内当归多糖组分ASP1的含量

Determination of Angelica Sinensis Polysaccharide(ASP1) in Rats by HPLC-FD with Pre-column Derivatization

目的:建立高效液相色谱-荧光检测法(HPLC-FD)测定大鼠体内当归多糖ASP1含量的方法,为其药代动力学研究奠定基础。方法:采用Belder和Granath的方法将异硫氰酸荧光素(FITC)标记到精制当归多糖组分ASP1上,得到标记产物ASP1-FITC;组织样品经30%三氯乙酸溶液和11%氢氧化钠溶液处理后进样。采用HPLC-FD法,以PL aquagel-OH MIXED(300 mm×7.5 mm,8μm)为色谱柱,以pH 7.0的磷酸盐缓冲液(取磷酸二氢钠2.34 g、磷酸氢二钠4.33 g与氯化钠11.70 g,加水使溶解成1000 ml)为流动相,流速0.5 ml·min^(-1),荧光检测器λex 494 nm、λem 520 nm。结果:ASP1-FITC在组织样品中的定量下限为0.20μg·ml^(-1),在0.25~40.00μg·ml^(-1)范围浓度内与峰面积呈良好的线性关系(r﹥0.999 6),回收率在91.98%~114.20%之间,日内精密度RSD和日间精密度RSD分别低于8.31%和2.94%,均符合要求。结论:本试验建立了当归多糖ASP1柱前荧光标记衍生、HPLC-FD法测定大鼠组织样品中当归多糖组分ASP1含量的方法,该方法样品用量少、灵敏度高、特异性强、精密度和重复性较好,适用于当归多糖组分ASP1药代动力学和组织分布试验的检测。

Objective ： To establish an HPLC-FD method for determining the content of Angelica sinensis polysaccharide （ ASP1 ） to lay foundation for its pharmacokinetic study in rats. Methods： Purified ASP1 was labeled with FITC by the method of Belder and Granath to obtain ASP1-FITC. The tissue samples were treated with 30% trichloroacetie acid and 11% NaOH before injection. The samples were determined by HPLC-FD. A PL aquagel-OH MIXED column was used,and the mobile phase was phosphate buffer（ dis- solve NaH2PO3 2.34g , N%HPO34.33 g and NaC1 11.70 g into 1 000 ml water） with pH of 7.0. The flow rate was 0.5 ml min-1. The excitation and emission wavelengths was set at 495 nm and 520 nm, respectively. Results： The linear calibration curve was within the concentration range of 0.25-40.00 μg ml-1（ r = 0. 999 6）with the lower limit of quantification of 0.20μg m1-1 in tissue sam- ples. The extraction recovery of ASP1 was determined at low, medium and high concentration with the recovery of 91.98%-114.20%. The intra and inter-day RSDs were lower than 8.31% and 2.94%, respectively. Conclusion： The method to determine the content of ASP1 in rats by HPLC-FD with pre-column derivatization has been esablished. It is simple, accurate and reliable, which can be suc- cessfully applied in the study of pharmacokinetics and tissue distribution of ASP1 in rats.