Versapharm Incorporated, 1035 Louis Drive, Warminister, PA 18974, USA.
J Pharm Biomed Anal. 2010 May 1;52(1):142-8. doi: 10.1016/j.jpba.2009.12.011. Epub 2009 Dec 14.
The objective of the current study was to develop a validated, specific and stability-indicating reverse phase liquid chromatographic method for the quantitative determination of acetazolamide and its related substances. The determination was done for an active pharmaceutical ingredient, its pharmaceutical dosage form in the presence of degradation products, and its process-related impurities. The drug was subjected to stress conditions of hydrolysis (acid and base), oxidation, photolysis and thermal degradation as per International Conference on Harmonization (ICH) prescribed stress conditions to show the stability-indicating power of the method. Significant degradation was observed during acid and base hydrolysis, and the major degradant was identified by LC-MS, FTIR and (1)H/(13)C NMR spectral analysis. The chromatographic conditions were optimized using an impurity-spiked solution and the generated samples were used for forced degradation studies. In the developed HPLC method, the resolution between acetazolamide and, its process-related impurities (namely imp-1, imp-2, imp-3, imp-4 and its degradation products) was found to be greater than 2. The chromatographic separation was achieved on a C18, 250mmx4.6mm, 5microm column. The LC method employed a linear gradient elution, and the detection wavelength was set at 254nm. The stress samples were assayed against a qualified reference standard and the mass balance was found to be close to 99.6%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.
本研究的目的是开发一种经过验证的、专属性强且能够显示稳定性的反相高效液相色谱法，用于定量测定乙酞唑胺及其有关物质。该测定方法适用于原料药、制剂（在降解产物存在的情况下）以及其工艺相关杂质。根据国际人用药品注册技术协调会（ICH）规定的降解条件，对药物进行了水解（酸、碱）、氧化、光解和热降解等强制条件降解，以证明该方法的稳定性指示能力。在酸、碱水解过程中观察到明显的降解，通过 LC-MS、FTIR 和（1）H/（13）C NMR 光谱分析确定了主要降解产物。通过杂质加标溶液优化了色谱条件，并使用生成的样品进行了强制降解研究。在开发的 HPLC 方法中，乙酞唑胺与其工艺相关杂质（即 imp-1、imp-2、imp-3、imp-4 及其降解产物）之间的分离度大于 2。色谱分离在 C18、250mmx4.6mm、5μm 柱上实现。LC 方法采用线性梯度洗脱，检测波长设定为 254nm。对强制降解样品进行测定时，使用的是合格的参比标准品，且质量平衡接近 99.6%。该反相高效液相色谱法已针对线性、准确性、精密度和耐用性进行了验证。
