HPLC法测定安立生坦片中有关物质

目的:建立高效液相色谱法测定安立生坦片中有关物质。方法:采用安捷伦ZORBAX SB-Aq(4.6 mm×250 mm,5 μm)色谱柱,流动相A为5 mmol·L^-1磷酸二氢钾溶液(pH 3.0),流动相B为乙腈,以1.0 mL·min^-1的流速进行梯度洗脱,检测波长为210 nm,柱温25 ℃,以自身对照法测定安立生坦片中有关物质。结果:安立生坦与各杂质及降解产物能够完全分离(分离度大于2.0);供试品溶液在24 h内稳定性良好;安立生坦和2-羟基-3-甲氧基-3,3-二苯基丙酸(杂质A)、(S)-1-(4-氯苯基)乙胺(杂质B)、3,3二苯基-2,3-环氧丙酸甲酯(杂质C)、2-羟基-3-甲氧基-3,3二苯基丙酸甲酯(杂质D)、4,6-二甲基-2-甲基-磺酰嘧啶(杂质E)和4,6-二甲基-2-(2,2-二苯基-乙烯氧基)嘧啶(杂质F)的定量限分别为0.66、0.64、0.65、1.31、0.65、0.63和1.32 ng,相对校正因子分别为1.00、1.08、2.20、1.09、1.10、2.54和1.07;安立生坦质量浓度在0.5~5.0 μg·mL^-1范围内线性良好(r >0.999 9),各杂质质量浓度在0.125~2.5 μg·mL^-1范围内线性良好(r >0.999),杂质加样回收率(n=9)在95.8%~103.7%之间;重复性和中间精密度符合规定。经检测,3批安立生坦片中杂质A含量均为0.01%。结论:经验证本方法准确、灵敏,精密度高,耐用性和专属性强,可用于安立生坦片中有关物质的测定。

Objective:To establish an HPLC method for the determination of related substances in ambrisentan tablets.Methods:The substances were analyzed by using an Agilent ZORBAX SB-Aq column(4.6 mm×250 mm,5 μm) with gradient elution by the mobile phase A(potassium dihydrogen phosphate buffer of pH 3.0,5 mmol·L^-1) and mobile phase B(acetonitrile) .The flow rate was 1.0 mL·min^-1,the detection wavelength was 210 nm,and the column temperature was 25 ℃.The determination of related substances in ambrisentan tablets was analyzed with self-contrast method.Results: Ambrisentan was separated completely from the impurities and degradation products(the resolution was above 2.0).The test solution was stable for at least 24 h.The LOQ of ambrisentan and 2-hydroxy-3-methoxy-3,3-diphenyl propanoic acid(impurity A),(S)-1-(4-chlorophenyl)ethylamine(impurity B),3,3-diphenyloxirane-2-carboxylic acid methyl ester(impurity C),2-hydroxy-3-methoxy-3,3-diphenyl propanoic acid methyl ester(impurity D),4,6-dimethyl-2-methylsulfonyl-pyrimidine(impurity E),2-[(2,2-diphenylvinyl)oxy]-4,6-dimethylpyrimidine(impurity F) were 0.66,0.64,0.65,1.31,0.65,0.63,and 1.32 ng and the relative correction factors were 1.00,1.08,2.20,1.09,1.10,2.54,and 1.07.The mass concentration of ambrisentan was in good linearity within the range of 0.5-5.0 μg·mL^-1(r=0.999 9),and the mass concentration of impurities obtained good linearity within the range of 0.125-2.5 μg·mL^-1(r > 0.999).The recoveries of the impurities(n=9) were between 95.80% and 103.69%.The repeatability and intermediate precision completely met the requirements.The impurity A contents in three batches of ambrisentan tablets were 0.01%,0.01%,and 0.01%,respectively.Conclusion: By method validation,the established method is accurate,sentitive with good precision,durablity and specificity,and can be used in the determination of related substances in ambrisentan tablets.