Zielińska Anna, Wicherkiewicz Sławek, Łuniewski Wojciech, Sidoryk Katarzyna, Pesta Edyta, Kutner Andrzej
Acta Pol Pharm. 2016 Jul;73(4):865-874.
Due to the raising requirements of drug quality, there is an increasing need for fast liquid separations of pharmaceutical substances with high efficiency and good resolution. The ultra-high pressure liquid chromatography (UHPLC) has been considered to meet this challenge. However, it was found that this fast method has also serious disadvantages. The range of applications of the UHPLC in the analysis of pharmaceutical substances and dosage forms is currently extensively discussed. In this study we investigated the consequences of the shortening of the analysis time of the liquid chromatographic method. Bosentan, a non-peptide antagonist of human endothelin receptors, was chosen as an example in this study, due to its therapeutic importance and lack of the reported analytical methods of the drug product. Two high-performance, reversed phase liquid chromatography methods with UV detection at 220 nm were developed for this purpose. Both methods were validated and the resulting performance characteristics were compared. The first separation (method A) was achieved on Kinetex column, (2.6 μ C18 1OOA, 150 x 4.6 mm), the second-fast (method B) employed Kinetex column, (1.7 μ XB-C18 100A 50 x 3.0 mm). Both methods were performed with a buffered mobile phase containing 0.1% of triethylamine in water brought to the pH 2.5 with phosphoric acid and methanol as the solvent A and acetonitrile as the solvent B. Gradient program was used and flow rate of 0.8 mL/min and 0.4 mL/min, for the methods A and B, respectively. The methods were validated according to the ICH guidelines for specificity, precision on the specified and LOQ limits, intermediate precision, accuracy, linearity (correlation co-efficient =0.999) and robustness. The robustness was confirned using four factors: the mobile phase pH, the flow rate of the mobile phase, column temperature and the second column of the same kind. The limits of detection and quantification were established as 0.0132 and 0.1505 μg/mL for methods A and B, respectively. Both validated methods complied with the acceptance criteria. The method B was 3.5 times faster than the method A, but the method A showed much better sensitivity. The resolution between compound B and bosentan was 3.39 and 1.75 for methods A and B, respectively. The lower sensitivity limits the use of Method B, especially in the analyses at low levels of active substances (e.g., bioanalysis, validation of the cleaning procedures) and makes method A more suitable for this purpose.
由于对药品质量要求的提高，对药物高效快速液相分离的需求日益增加。超高压液相色谱法（UHPLC）被认为能够应对这一挑战。然而，人们发现这种快速方法也存在严重缺点。目前，UHPLC在药物分析和剂型分析中的应用范围正在广泛讨论。在本研究中，我们考察了液相色谱法分析时间缩短所带来的影响。波生坦作为一种人内皮素受体的非肽拮抗剂，因其治疗重要性以及缺乏该药物产品已报道的分析方法，在本研究中被选作示例。为此开发了两种在220nm波长下进行紫外检测的高效反相液相色谱法。两种方法均经过验证，并对所得性能特征进行了比较。第一次分离（方法A）使用Kinetex柱（2.6μm C18 100A，150×4.6mm）完成，第二种快速方法（方法B）采用Kinetex柱（1.7μm XB-C18 100A 50×3.0mm）。两种方法均采用缓冲流动相，流动相为含0.1%三乙胺的水溶液，用磷酸调至pH 2.5，以甲醇作为溶剂A，乙腈作为溶剂B。采用梯度洗脱程序，方法A和方法B的流速分别为0.8mL/min和0.4mL/min。按照国际人用药品注册技术协调会（ICH）指南对方法进行特异性、规定限度和定量限的精密度、中间精密度、准确度、线性（相关系数=0.999）和稳健性验证。使用流动相pH、流动相流速、柱温以及同一类型的第二根色谱柱这四个因素来确认稳健性。方法A和方法B的检测限和定量限分别确定为0.0132μg/mL和0.1505μg/mL。两种经验证的方法均符合验收标准。方法B比方法A快3.5倍，但方法A的灵敏度更高。方法A和方法B中化合物B与波生坦之间的分离度分别为3.39和1.75。较低的灵敏度限制了方法B的使用，尤其是在低水平活性物质分析（如生物分析、清洁程序验证）中，这使得方法A更适合此用途。
