Method development and hydrolytic degradation study of doxofylline by rp-hplc and lc-ms/ms

A simple, rapid and accurate RP-HPLC method was developed for the determination of doxofylline and photolyticdegradation product. The method showed a linear response for concentrations in the range of 1-200 g/ml usingacetonitrile: formic acid (90: 10); pH-3.0 as the mobile phase with detection at 274 nm and a flow rate of 1 ml/min and retention time 2.9 min. The method was statistically validated for accuracy, precision, linearity, ruggedness,robustness, forced degradation, solution stability and selectivity. Quantitative and recovery studies of the dosage form were also carried out and analyzed; the % RSD from recovery studies was found to be less than 1. Due to simplicity, rapidity and accuracy of the method, we believe that the method will be useful for routine quality control analysis. The hydrolytic degradation product as well as pathway was characterized by LC-MS/MS.RESULT AND DISCUSSION:A sensitive, selective, precise and accurate highperformance liquid chromatographic method of analysis ofdoxofylline in both as bulk drug and in formulation wasdeveloped and validated. The mobile phase consisted ofacetonitril: 0.05M formic Acid (90: 10v/v); pH-3.0. Thedetection wavelength was 274nm. This system was found to give the sharp peak for doxofylline (RT-2.9). The methodwas validated as per ICH guideline (table 1). Stabilityindicating assay method in which hydrolytic stresscondition was used for quantitative estimation ofdoxofylline in tablet formulation and identification ofhydrolytic degradation product The separation of drugfrom its degradation product were optimized by varying theratio &amp;/or nature of organic modifier. Finally method wasdeveloped using same mobile phase composed ofacetonitrile: formic acid (90: 10); pH-3.0, in that both drugand degradation product showing good elution RT-2.9(Doxofylline) and RT-3.68 (Hydrolytic degradationproduct) and m/e-229 (fig 4). The hydrolytic degradationproduct and pure drug were identified by LC-MS/MS inorder to establish hydrolytic degradation pathway (fig 5 and6).<br>本研究建立了一种简便、快速、准确的反相高效液相色谱法（RP-HPLC），用于多索茶碱（doxofylline）及其光解降解产物的含量测定。该方法在1~200 g/ml浓度范围内呈良好线性响应，流动相为乙腈-甲酸（90:10，pH 3.0），检测波长274 nm，流速1 ml/min，目标物保留时间为2.9 min。本方法通过统计学方法验证了准确度、精密度、线性、耐用性、稳健性、强制降解、溶液稳定性及专属性。同时对制剂进行了含量测定和回收率实验，结果显示回收率实验的相对标准偏差（% RSD）小于1。鉴于本方法简便、快速、准确的特点，其可用于日常质量控制分析。本研究采用液相色谱-串联质谱法（LC-MS/MS）对水解降解产物及其降解路径进行了表征。 结果与讨论： 本研究建立了一种灵敏、专属性强、精密度良好且准确的高效液相色谱法，用于原料药及制剂中多索茶碱的含量测定，并完成了方法学验证。流动相为乙腈-0.05 mol/L甲酸（90:10，v/v，pH 3.0），检测波长为274 nm。该色谱系统下多索茶碱可获得尖锐色谱峰，保留时间（RT）为2.9 min。本方法按照人用药品注册技术要求国际协调会（ICH）指南进行了方法学验证（详见表1）。本研究采用水解强制降解条件建立了稳定性指示性含量测定方法，用于片剂中多索茶碱的定量分析及水解降解产物的鉴定。通过调整有机改性剂的比例与/或种类，优化了药物与其降解产物的色谱分离条件。最终采用乙腈-甲酸（90:10，pH 3.0）作为流动相建立分析方法，此时药物与降解产物均获得良好的色谱洗脱效果：多索茶碱保留时间为2.9 min，水解降解产物保留时间为3.68 min，其质荷比（m/e）为229（见图4）。采用液相色谱-串联质谱法（LC-MS/MS）对水解降解产物与原料药进行鉴定，以此明确多索茶碱的水解降解路径（见图5、图6）。