A validated rp-HPLC method for simultaneous determination of olopatadine hydrochloride and its related substances in eye drop

Olopatadine Hydrochloride, a H1-receptor antagonist, widely used for Allergic Conjunctivitis. A HPLC method was created and validated for the quantification of olopatadine hydrochloride and its related substances in eye formulation in accordance with ICH standards. The separation of olopatadine hydrochloride was done by using a 4.6-mm 15-cm; 5 µm packing USP L7 column with a mobile phase made up of a mixer of phosphate buffer, pH 3.0: Acetonitrile (72:28) ratio at a flow rate of 1.0 mL/min and injection volume 30 µL. A photodiode array detector was used at a wavelength of 299 nm. The validation parameters including specificity, LOD/LOQ, linearity, accuracy, precision, robustness, and solution stability, were verified for performance of the method. The linearity ranges were 0.02–0.40, 0.0004–0.008 and 0.0001–0.002 mg/mL for olopatadine hydrochloride, olopatadine-related compound B and olopatadine carbaldehyde, respectively. The % recovery was within the 80–120%. When the column temperature and flow rate were changed, the system suitability resolution was still within the acceptable range. The solutions were stable after 24 hr at both room temperature and 5°C temperature. For the analysis of olopatadine hydrochloride and its impurity in pharmaceutical finished eye dosage form, the confirmed HPLC method can be a workable analytical approach.

盐酸奥洛他定（Olopatadine Hydrochloride）是一种H1受体拮抗剂（H1-receptor antagonist），被广泛应用于过敏性结膜炎（Allergic Conjunctivitis）的治疗。本研究依据ICH（International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use）指南标准，建立并验证了一种可用于眼用制剂中盐酸奥洛他定及其有关物质定量分析的高效液相色谱法（High Performance Liquid Chromatography, HPLC）。该方法采用规格为4.6 mm×15 cm、填充粒径5 μm的USP L7色谱柱进行盐酸奥洛他定的分离；流动相为pH 3.0的磷酸盐缓冲液（phosphate buffer）与乙腈（Acetonitrile）的混合溶液，体积比为72:28，流速设定为1.0 mL/min，进样体积为30 μL。检测采用光电二极管阵列检测器（Photodiode Array Detector），检测波长为299 nm。本研究对该方法的专属性、检出限/定量限（Limit of Detection / Limit of Quantitation, LOD/LOQ）、线性范围、准确度、精密度、耐用性以及溶液稳定性等验证指标进行了考察。其中，盐酸奥洛他定、奥洛他定相关杂质B（olopatadine-related compound B）以及奥洛他定缩醛（olopatadine carbaldehyde）的线性范围分别为0.02–0.40 mg/mL、0.0004–0.008 mg/mL与0.0001–0.002 mg/mL；平均回收率处于80%–120%范围内。当柱温与流速发生变动时，系统适用性分离度仍处于可接受范围内。供试溶液在室温与5℃条件下放置24小时后仍保持稳定。综上，该经验证的高效液相色谱法可作为药物成品眼用制剂中盐酸奥洛他定及其杂质定量分析的可行分析手段。