HPLC法测定罗氟司特的有关物质

目的：建立罗氟司特有关物质测定的HPLC方法。方法：采用十八烷基硅烷键合硅胶为填料的色谱柱，以0.01 mol·L^-1磷酸二氢钾溶液（磷酸调节pH至3.5±0.1）为流动相A，乙腈为流动相B，梯度洗脱，流速0.5 mL·min^-1，柱温为25℃，检测波长为215 nm，对有关物质进行定性、定量分析。结果：罗氟司特和相邻杂质以及各杂质之间的分离度均大于1.5；3-环丙基甲氧基-4-二氟甲氧基苯甲酸（杂质A）、4-氨基-3，5-二氯吡啶（杂质B）、3-（环丙基甲氧基）-N-（3，5-二氯吡啶-4-基）-4-羟基苯甲酰胺（杂质C）、3-羟基-N-（3，5-二氯吡啶-4-基）-4-（二氟甲氧基）苯甲酰胺（杂质D）、3，4-二羟基-N-（3，5-二氯吡啶-4-基）苯甲酰胺（杂质E）、3-（环丙基甲氧基）-N-（吡啶-4-基）-4-（二氟甲氧基）苯甲酰胺（杂质F）、3-（环丙基甲氧基）-N-（3-氯吡啶-4-基）-4-（二氟甲氧基）苯甲酰胺（杂质G）、3-（二氟甲氧基）-N-（3，5-二氯吡啶-4-基）-4-（环丙基甲氧基）苯甲酰胺（杂质H）、3，4-二（环丙基甲氧基）-N-（3，5-二氯吡啶-4-基）苯甲酰胺（杂质I）、3，4-二（二氟甲氧基）-N-（3，5-二氯吡啶-4-基）苯甲酰胺（杂质J）和罗氟司特氮氧化物（杂质K）在各自的线性范围内线性关系良好（r>0.999 0，n=5），相对校正因子分别为1.14、1.00、1.17、0.96、0.70、1.76、1.08、1.37、1.14、1.65和1.22，检测限分别为0.1、0.03、0.1、0.1、0.1、0.03、0.1、0.1、0.1、0.1和0.1 ng；定量限分别为0.3、0.1、0.3、0.2、0.2、0.1、0.2、0.2、0.2、0.3和0.3 ng，低、中、高3种浓度的平均回收率（n=9）分别为102.9%、100.0%、107.6%、107.5%、98.1%、99.9%、98.3%、97.6%、86.9%、100.0%和98.7%。5批样品有关物质测定结果显示，各已知杂质的含量均低于0.1%，其他最大单个杂质的含量均低于0.1%，杂质总量均低于0.5%。结论：本方法可用于罗氟司特的有关物质测定。

Objective: To establish an HPLC method for the determination of related substances in roflumilast raw material.Methods: HPLC was adopted on a C₁₈ column with a gradient elution system of acetonitrile-0.01 mol·L^-1 potassium dihydrogen phosphate solution(pH was adjusted to 3.5±0.1 with phosphate acid) with a flow rate of 0.5 mL·min^-1,and the column temperature was maintained at 25℃,the detection wavelength was set at 215 nm.The related substances were qualified and quantified by the established method.Results: The resolutions between roflumilast and adjacent impurities, and between the known impurities were greater than 1.5.The calibration curves of 11 known impurities were linear in the concentration range(r>0.999 0,n=5).The relative correction factors were 1.14, 1.00, 1.17, 0.96, 0.70, 1.76, 1.08, 1.37, 1.14, 1.65 and 1.22 for 3-(cyclopropylmethoxy)-4-(difluoromethoxy)benzoic acid(impurity A),3,5-dichloropyridin-4-amine(impurity B), 3-(cyclopropylmethoxy)-N-(3, 5-dichloropyridin-4-yl)-4-hydroxybenzamide(impurity C), N-(3, 5-dichloropyridin-4-yl)-4-(difluoromethoxy)-3-hydroxybenzamide(impurity D), N-(3,5-dichloropyridin-4-yl)-3, 4-dihydroxybenzamide(impurity E), 3-(cyclopropylmethoxy)-4-(difluoromethoxy)-N-(pyridin-4-yl)benzamide(impurity F), N-(3-chloropyridin-4-yl)-3-(cyclopropylmethoxy)-4-(difluoromethoxy) benzamide(impurity G), 4-(cyclopropylmethoxy)-N-(3, 5-dichloropyridin-4-yl)-3-(difluoromethoxy) benzamide(impurity H), 3, 4-bis(cyclopropylmethoxy)-N-(3, 5-dichloropyridin-4-yl)benzamide(impurity I), N-(3, 5-dichloropyridin-4-yl)-3, 4-bis(difluoromethoxy)benzamide(impurity J)and roflumilast oxynitride(impurity K), rspectively; The detection limits were 0.1, 0.03, 0.1, 0.1, 0.1, 0.03, 0.1, 0.1, 0.1, 0.1 and 0.1 ng, and the quantitation limits were 0.3, 0.1, 0.3, 0.2, 0.2, 0.1, 0.2, 0.2, 0.2, 0.3 and 0.3 ng, respectively; And the average recovery rates(n=9)were 102.9%, 100.0%, 107.6%, 107.5%, 98.1%, 99.9%, 98.3%, 97.6%, 86.9%, 100.0% and 98.7% at the concentration level of low,medium and high concentrations, respectively.The determination results of the five batches of samples showed that the known impurities were less than 0.1%, the maximum single unknown impurity was less than 0.1%, the total impurities were less than 0.5%. Conclusion: The method is suitable for the quality control of roflumilast.

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