OPTIMIZATION OF THE HPLC METHOD FOR THE DETERMINATION OF AMLODIPINE IN TABLETS USING THE APPROACH “QUALITY BY DESIGN”

Abstract

Introduction. To ensure the safety and effectiveness of medicinal products, reliable, express and simple methods for quantitative determination of active pharmaceutical ingredients in drugs are required. Calcium channel blockers (CCBs) are often used in the treatment protocols of arterial hypertension, both in monodrugs and in combination drugs. Amlodipine (AML) is a representative of CCBs. The methods for quantitative determination of amlodipine given in pharmacopoeias have some drawbacks, therefore they need to be optimized using modern approaches. The aim of the work was to develop a rapid, simple and "green" HPLC method for the determination of amlodipine besylate in medicinal products using Quality by Design (QbD) approaches. Research Methods. The study was performed using a Shimadzu LC-2050 C liquid chromatograph with a diode array detector, LabSolutions software was used to obtain chromatograms and integrate the results. Chromatographic columns: Luna C8 (100 x 4.6 mm 3 μm), Luna C18 (100 x 4.6 mm 3 μm), InertClone ODS (150 x 4.6 mm 3.5 μm), Zorbax SB C8 (150 x 4.6 mm 3.5 μm) and Zorbax SB Phenyl (150 x 4.6 mm 3.5 μm). Amlodipine besylate (AML) CRS (purity ≥98%) was purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA), amlodipine besylate tablets "Amlodipine" 10 mg "Farmak" was purchased from a local pharmacy. Results and Discussion. The method was optimized using QbD, using a three-level factorial design, taking into account the main factors of the method (ratio of methanol (MeOH), triethylamine (TEA) and pH value) that affect the chromatographic response (number of theoretical plates (NTP), peak area (A), retention time (Rt) and asymmetry coefficient (Tf)). The proposed chromatographic conditions were based on isocratic elution of amlodipine besylate with a mobile phase composed of: acetonitrile (AСN) – MeOH – 0.7% TEA with adjustment to pH 2.81 using 88% phosphoric acid in the ratio 30-35-35. Optimal chromatographic conditions: temperature – 30°C, flow rate – 1 ml/min, detection wavelength – 237 nm, retention time up to 3 min. The described conditions with satisfactory parameters of the chromatographic system were tested on various columns: Luna C8 (100 x 4.6 mm 3 μm), Luna C18 (100 x 4.6 mm 3 μm), InertClone ODS (150 x 4.6 mm 3.5 μm), Zorbax SB C8 (150 x 4.6 mm 3.5 μm). For the optimized HPLC method for the determination of amlodipine besylate in drugs, various alkyl columns can be used as a stationary phase. The developed method for the determination of amlodipine besylate is universal, because when testing different types of columns, it remains express and with satisfactory parameters of the chromatographic system. The obtained results expand the possibilities of using the developed method for laboratories with a small arsenal of chromatographic columns, since the work also shows columns with different pricing policies (Phenomenex – cheaper, Agilent – expensive). The validation of the HPLC method was carried out according to the following parameters: linearity, robustness, accuracy and precision in accordance with the requirements of the International conference on harmonization (ICH). The limit of detection (LOD) and the limit of quantification (LOQ) of the method are 1.39 μg/ml and 4.21 μg/ml, respectively. The range of application was within 10 to 50 μg/ml. The results of the study of the accuracy (99.88-101.35%) and precision (RSD < 2.0%) of the developed HPLC method meet the acceptance criteria. The principles of "green" chemistry were successfully implemented in the developed HPLC method, which was confirmed by the results of the study of environmental friendliness using the AGREE (0.78) and MOGAPI (79) tools. Conclusions. A simple, express and "green" HPLC method for the determination of amlodipine has been developed for quality control of finished dosage forms, studying the stability of finished dosage forms, studying the impact of changes on finished dosage forms when introducing new manufacturers of excipients, when changing manufacturing technology, changing the composition of drugs and other processes of the life cycle of finished dosage forms of amlodipine.