以腺苷为对照的高效液相色谱法测定三磷酸腺苷二钠注射液含量

目的: 建立以腺苷为对照的高效液相色谱法测定三磷酸腺苷二钠注射液含量。方法: 采用依利特Hypersil C₈(4.6 mm×250 mm,5 μm),柱温35 ℃,以0.05 mol·L^-1磷酸盐缓冲液(含0.161%四丁基溴化铵,pH 7.0)为流动相,流速为1.0 mL·min^-1,检测波长259 nm,以磷酸腺苷二钠和腺苷相对分子质量的理论比值2.062为系数、腺苷为对照外标法计算三磷酸腺苷二钠的含量。结果: 腺苷质量浓度(C)在5.056~404.4 ng·mL^-1范围时与峰面积(A)呈良好的线性关系,线性方程为A=3.219×10⁴C+31.70,r=0.999 9;三磷酸腺苷二钠质量浓度(C)在9.939~795.1 ng·mL^-1范围时与峰面积(A)呈良好的线性,线性方程为A=1.567×10⁴C+22.16,r=1.000。腺苷和三磷酸腺苷二钠的定量限分别为7.6×10^-5 mg·mL^-1和2.6×10^-4 mg·mL^-1,重复性和中间精密度的RSD均为1.0%,三磷酸腺苷二钠回收率为101.1%(RSD=0.9%,n=9)。含量测定结果范围为94.2%~109.2%。结论: 本实验建立的分析方法解决了未能获得含量确定且稳定的三磷酸腺苷对照的问题,提高了方法的专属性和定量准确性,可用于测定三磷酸腺苷二钠注射液的含量。

Objective: To establish an HPLC method for determination of adenosine triphosphate disodium injection using adenosine as the reference. Methods: The method was established using the Elite Hypersil C₈ column(4.6 mm×250 mm,5 μm)at the determination wavelength of 259 nm.The mobile phase was 0.05 mol·L^-1 phosphate buffer(with 0.161% tetrabutylammonium bromide,pH 7.0).The flow rate was 1.0 mL·min^-1 and the column temperature was 35 ℃.The content was calculated by HPLC-external standard method using the relative molecular mass ratio(2.062)of adenosine triphosphate disodium(ATP-2Na)to adenosine as the coefficient and adenosine as the reference. Results: The adenosine calibration curve was linear over the concentration range of 5.056-404.4 ng·mL^-1(linear regression equation was A=3.219×10⁴C+31.70,r=0.999 9)and the ATP-2Na calibration curve was linear over the concentration range of 9.939-795.1 ng·mL^-1(linear regression equation was A=1.567×10⁴C+22.16,r=1.000).The limit of quantification was 7.6×10^-5 mg·mL^-1 for adenosine and was 2.6×10^-4 mg·mL^-1 for ATP-2Na.RSDs of both repeatability and intermediate precision were 1.0%.The recovery of ATP-2Na was 101.1%(RSD=0.9%,n=9).The contents were 94.2%-109.2%. Conclusion: The proposed HPLC method solved the issue of obtaining the ATP-2Na reference with accurate and stable content,improving the specificity and accuracy of the method,which can be successfully applied in determination of the content of adenosine triphosphate disodium injection.