高效液相色谱法测定曲伏噻吗滴眼液中曲伏前列素有关物质含量

Determination of the Related Substances of Travoprost in Travoprost and Timolol Maleate Eye Drops by HPLC

目的建立高效液相色谱法测定曲伏噻吗滴眼液中曲伏前列素有关物质的含量。方法采用Phenomenex LUNA苯基己基色谱柱(3.0 mm×150 mm,3μm)色谱柱;流动相A为20 mmol·L^(-1)庚烷磺酸钠溶液(用磷酸调pH值至2.5),流动相B为甲醇,流动相C为乙腈,洗脱梯度;检测波长220 nm;柱温40℃;流速0.5 mL·min^(-1)。结果在该色谱条件下,曲伏前列素与各杂质均可良好分离;曲伏前列素、曲伏前列素杂质A、曲伏前列素15-表非对映异构体、曲伏前列素5,6-反式杂质异构体、曲伏前列素15-酮衍生物分别在0.02~219.29μg·mL^(-1)(r=1.0000),0.02~3.10μg·mL^(-1)(r=0.9999),0.02~3.87μg·mL^(-1)(r=1.0000),0.02~4.37μg·mL^(-1)(r=1.0000),0.02~3.98μg·mL^(-1)(r=0.9999)浓度范围内与峰面积呈良好线性关系,其最低检测限分别为2,1,2,2和2 ng;曲伏前列素平均加样回收率99.7%(RSD 0.60%)。结论该方法可用于曲伏噻吗滴眼液中曲伏前列素及其有关物质的含量测定。

Objective To establish a method for determining the related substances of travoprost in travoprost and timolol maleate eye drops by HPLC.Methods The determination was performed on Phenomenex LUNA phenyl-hexyl column(3.0 mm×150 mm,3μm)with gradient elution using 20 mmol·L^(-1)sodium heptane sulfonate solution(adjusted to pH 2.5 with phosphoric acid)as mobile phase A,methanol as mobile phase B and acetonitrile as mobile phase C,the detection wavelength was 220 nm;the temperature of the column was 40℃;the flow rate was 0.5 mL·min^(-1).Results Under the optimized condition of HPLC chromatography,an excellent separation was achieved for travoprost and the impurities.The calibration curves for travoprost,travoprost related compound A,15-epi diastereomer,5,6-trans isomer and 15-keto derivative revealed good linearities over the range of 0.02-219.29μg·mL^(-1)(r=1.0000),0.02-3.10μg·mL^(-1)(r=0.9999),0.02-3.87μg·mL^(-1)(r=1.0000),0.02-4.37μg·mL^(-1)(r=1.0000),0.02-3.98μg·mL^(-1)(r=0.9999),respectively.The LODs of the above compounds were 2,1,2,2 and 2 ng,respectively.The average recoveries of travoprost was 99.7%(RSD=0.60%).Conclusion The method is specific,accurate and sensitive,which can be used for the determination of travoprost and timolol maleate eye drops and related substances of travoprost in travoprost and timolol maleate eye drops.