Center for Metabolomics and Bioanalysis, Facultad de Farmacia, Universidad San Pablo-CEU, Urbanización Montepríncipe, Boadilla del Monte, 28668 Madrid, Spain.
J Pharm Biomed Anal. 2012 Mar 5;61:230-6. doi: 10.1016/j.jpba.2011.12.014. Epub 2011 Dec 19.
The classification of an impurity of a drug substance as genotoxic means that the "threshold of toxicological concern" (TTC) value of 1.5 μg/day intake, considered to be associated with an acceptable risk, should be the admissible limit in the raw material and that leads to new analytical challenges. In this study, reliable chromatographic methods were developed and applied as limit tests for the control of three genotoxic impurities (GTIs) in cloperastine fendizoate, drug widely used as an antitussive active pharmaceutical ingredient (API). In particular, GC-MS was applied to the determination of one alkyl halide (2-chloroethanol, 2-CE), while HPLC-DAD was selected for the analysis of two sulfonate esters (methyl p-toluenesulfonate, MPTS, and 2-chloroethyl p-toluenesulfonate, CEPTS). Regarding GC-MS, strong anion-exchange (SAX)-SPE was applied to remove fendizoate from the sample solutions, due its low volatility and its high amount in the raw material. The GC-MS analysis was performed on a Factor Four VF-23 ms capillary column (30 m × 0.25 mm I.D., film thickness 0.25 μm, Varian). Single ion-monitoring (SIM) detection mode was set at m/z 80. In the case of HPLC-DAD, a suitable optimization of the chromatographic conditions was carried out in order to obtain a good separation of the impurity peaks from the drug substance peaks. The optimized method utilizes a SymmetryShield RP(8) column (250 mm × 4.6 mm, 5 μm, Waters) kept at 50°C, with phosphate buffer (pH 3.0; 10 mM)-methanol (containing 10% ACN) (45:55, v/v) as the mobile phase, at the flow-rate of 1.7 mL/min and UV detection at 227 nm. The required sensitivity level was achieved by injecting 80 μL of sample solution, purified from fendizoate by SAX-SPE, followed by a 1:1 (v/v) dilution of the SPE eluate with water. For both GC-MS and HPLC-DAD, the method validation was performed in relation to specificity and limit of detection (LOD), as required by ICH guidelines in relation to limit assays. The developed methods were successfully applied for the determination of GTIs in five different batches of cloperastine fendizoate. In all the analyzed batches, the three target GTIs were below the concentration limit.
作为一种药物杂质，其被分类为遗传毒性物质意味着，每日摄入 1.5μg 被认为是可接受风险相关的“毒理学关注阈值”（TTC）值，应成为原料药的可接受限度，这也带来了新的分析挑战。在本研究中，开发并应用了可靠的色谱方法作为限度试验，以控制氯苯那敏非索酯中的三种遗传毒性杂质（GTIs）。氯苯那敏非索酯是一种广泛用作镇咳药物活性药物成分（API）的药物。具体而言，GC-MS 用于测定一种卤代烷（2-氯乙醇，2-CE），而 HPLC-DAD 则用于分析两种磺酸酯（甲基对甲苯磺酸酯，MPTS 和 2-氯乙基对甲苯磺酸酯，CEPTS）。对于 GC-MS，由于非索酯的挥发性低且在原料药中的含量高，因此应用强阴离子交换（SAX）-SPE 从样品溶液中去除非索酯。GC-MS 分析在 Factor Four VF-23 ms 毛细管柱（30 m×0.25mm ID，膜厚 0.25μm，Varian）上进行。在单离子监测（SIM）检测模式下，设定 m/z 80。对于 HPLC-DAD，为了获得药物杂质峰与药物主峰的良好分离，对色谱条件进行了适当的优化。优化后的方法利用 SymmetryShield RP(8)柱（250mm×4.6mm，5μm，Waters）在 50°C 下运行，以磷酸盐缓冲液（pH 3.0；10mM）-甲醇（含 10% ACN）（45:55，v/v）作为流动相，流速为 1.7mL/min，在 227nm 处进行紫外检测。通过 SAX-SPE 从非索酯中纯化出 80μL 的样品溶液，然后用 1:1（v/v）的水稀释 SPE 洗脱液，以达到所需的灵敏度水平。对于 GC-MS 和 HPLC-DAD，均按照 ICH 指南中关于限度分析的要求，针对专属性和检测限（LOD）进行了方法验证。所开发的方法成功地应用于五种不同批次氯苯那敏非索酯中 GTIs 的测定。在所有分析的批次中，三种目标 GTIs 的浓度均低于浓度限度。
