RP-HPLC Method Development and Validation for the Simultaneous Estimation of Anti-Hypertensive Drugs in Bulk and its Tablet Dosage Form

Hypertension is a major global health concern and a leading risk factor for cardiovascular diseases such as stroke, myocardial infarction, and renal failure. Effective management of hypertension often requires combination therapy to achieve better therapeutic outcomes. Olmesartan Medoxomil (OLMT), an angiotensin II receptor blocker (ARB), and Azelnidipine (AZLD), a calcium channel blocker (CCB), are commonly used together in the treatment of hypertension due to their complementary mechanisms of action.[1]

Olmesartan Medoxomil works by blocking the angiotensin II receptors, leading to vasodilation and reduced blood pressure. Azelnidipine, on the other hand, inhibits calcium ion influx into vascular smooth muscle, resulting in relaxation of blood vessels and improved blood flow. The combination of these two drugs provides enhanced antihypertensive effects and improved patient compliance.[2]

For ensuring the quality, safety, and efficacy of pharmaceutical formulations containing these drugs, it is essential to develop reliable analytical methods for their simultaneous estimation. Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) is one of the most widely used techniques in pharmaceutical analysis due to its accuracy, precision, and sensitivity.[3]

The present study focuses on the development and validation of a simple, precise, and robust RP-HPLC method for the simultaneous estimation of Olmesartan Medoxomil and Azelnidipine in bulk and combined tablet dosage forms. The method was validated according to standard guidelines to ensure its suitability for routine quality control analysis.[4]

Table: Chemical Structure and Pharmacological Characteristics of Azelnidipine and Olmesartan Medoxomil

MATERIALS & METHODS

Materials

The accuracy of an analytical method depends on the proper selection of materials and experimental conditions. In this study, all reagents and solvents used were of analytical or HPLC grade to ensure reliable results.The drug samples, Olmesartan Medoxomil (OLMT) and Azelnidipine (AZLD), were obtained as gift samples from MG Lab, Hyderabad, and used for method development.

Reagents and Apparatus

All reagents and chemicals used in the study were of analytical (AR) grade and HPLC grade to ensure accuracy and reliability of results. The chemicals included acetonitrile (HPLC grade), methanol (HPLC grade), ortho-phosphoric acid, and HPLC grade water, all procured from Merck Ltd., India.The instruments used for the analysis included a UV-Visible spectrophotometer (Shimadzu, double beam UV-1900i), an HPLC system (Agilent 1220 series Infinity LC with UV detector), and an FTIR spectrophotometer (Bruker-Alpha) for spectral analysis. A pH meter (Equip-tronich EQ-610) was used for pH adjustments. Chromatographic separation was carried out using an Intersil ODS C18 analytical column (4.6 × 150 mm). Additionally, a Phoenix Gold analytical balance (300 P) was used for accurate weighing, and a Labman ultrasonicator (LMUC2) was used for degassing and sample preparation.

 Chromatographic Conditions

Chromatographic separation of Azelnidipine (AZD) and Olmesartan (OLS) was carried out using a Kromstar™ RP-Vertex RP-Purosphere Star C18 column (5 µm, 4.6 × 250 mm). The mobile phase consisting of methanol: acetonitrile: water (50:30:20 v/v) was prepared, filtered through a 0.2 µm nylon filter, and degassed using an ultrasonicator for 5 minutes. The mobile phase was pumped at a flow rate of 1.0 mL/min. The analysis was performed at ambient temperature with detection at 255 nm. The injection volume was 20 µL, and the total run time was approximately 14 minutes.[5]

Preparation of Standard Solutions[13,14]

Olmesartan Standard Stock Solution (1000 µg/mL)

An accurately weighed 10 mg of Olmesartan was transferred into a 10 mL volumetric flask and dissolved in 70% v/v methanol using ultrasonication for 10 minutes. The volume was made up to the mark with methanol and filtered through a 0.45 µm membrane filter.[6]

Olmesartan Working Solution (100 µg/mL)

1 mL of stock solution was diluted to 10 mL with 70% methanol.

Azelnidipine Standard Stock Solution (1000 µg/mL)

10 mg of Azelnidipine was dissolved in 10 mL volumetric flask using 70% methanol with ultrasonication for 10 minutes. The volume was adjusted to mark and filtered.

Azelnidipine Working Solution (100 µg/mL)

1 mL of stock solution was diluted to 10 mL with 70% methanol.

 Preparation of Sample Solution[8]

Twenty tablets were weighed, powdered, and an amount equivalent to 10 mg of Olmesartan and 10 mg of Azelnidipine was transferred into a 100 mL volumetric flask. The mixture was dissolved in mobile phase (methanol: acetonitrile: water, 50:30:20 v/v) and ultrasonicated for 30 minutes. The volume was made up to the mark and filtered through Whatman filter paper followed by a 0.45 µm membrane filter.

The solution was further diluted to obtain final concentrations of:

• Olmesartan → 20 µg/mL
• Azelnidipine → 8 µg/mL

Selection of Detection Wavelength

All standard solutions were scanned in the UV region, and the overlain spectra indicated that 255 nm was the optimum wavelength, showing maximum absorbance for both drugs

Method Validation[9,12,15,16,17,8]

Table: Method Validation Parameters for AZD and OLS

RESULT

Results and Discussion Summary of AZD and OLS

Fig: Overlay Calibration Curve of Olmesartan Medoxomil (OLMT) and Azelnidipine (AZLD)

Table Observation of standard curves of OLMT and AZLD

Table Result of System Suitability Study

Fig. Chromatogram obtained by formulation of OLMT and AZLD

Table: Validation Parameters for OLMT and AZLD