Stability Indicating RP-HPLC Method For Simultaneous Estimation of Ranolazine and Atenolol

Ranolazine is used to treat a specific type of chronic stable angina and lowers the frequency of chest pain. Angina symptoms can be alleviated, allowing you to exercise and complete strenuous chores more comfortably. Atenolol is a beta blocker. It is recommended for the management of hypertension and arrhythmia. It can also be used to treat chest pain caused by angina pectoris.
Taking Atenolol for high blood pressure lowers the risk of future heart disease, heart attacks, and strokes. Ranolazine, an antianginal drug known to partly inhibit fatty acid oxidation, enhanced treadmill workout performance; however, its long-term efficacy and safety were investigated when used with calcium antagonists or beta-blockers in a significant number of patients with acute chronic angina ^[1]. Twice-daily Ranolazine dosage enhances the ability to exercise as well as offer supplementary antianginal relief to patients suffering from symptoms with acute chronic angina who are taking routine Atenolol doses, with no obvious negative long-term survival consequences even after 1 to 2 years of treatment ^{[1, 2]} . Combination medicine therapy, which involves prescribing two or more drugs to a patient, is quite common these days. Only a few medications can be combined; the rest must be administered separately. When both medications are present in plasma, we must evaluate the sample concurrently rather than separating them.

As a result, the use of simultaneous analytical procedures to estimate drugs in multi-component pharmaceutical formulations is becoming more common due to their inherent advantage of avoiding time-consuming extraction and separation procedures while also being economical in terms of depreciation of expensive reagents, and these methods are equally accurate and precise.
The analysis of combined dose formulations is crucial in analytical chemistry and aids in the routine quality monitoring of pharmaceutical dosage forms in industries. Many pharmacological formulations combining Ranolazine (RAN) and Atenolol (ATN) are available in the market, both individually and in conjuction with other drugs.

Forced degradation of both the drugs was carried out to develop stability indicating RP-HPLC method.

MATERIAL AND METHODS:

Instrumentation:

SHIMADZU Prominence HPLC system including a pump (LC-20 Ai), system controller, auto injector, and UV detector was used in this study. The data was analyzed and processed using LabSolutions software. Radwag analytical balance and a Grant sonicator. Purified water was collected with a Millipore Milli-Q (Nanopure Diamond, Barnstead thermolyne, USA) water purification system.

Standards and chemicals:

Ranolazine (RAN) and Atenolol (ATN) were gift samples obtained from the Poornayu research facility in Bangalore, India. Methanol of HPLC quality was obtained from Merck Ltd, o-phosphoric acid of A.R. grade from S.D. Fine Chemicals Ltd, and potassium dihydrogen phosphate of A.R. grade from Loba Chemie Pvt. Ltd.

Preparation of standard stock solutions:

30.16 mg of RAN and 10.23 mg of ATN were weighed into a clean, dry 100 ml volumetric flask and mixed with the appropriate diluent (water : Methanol 50:50 v/v). To achieve the desired concentrations of 300.16 μg/ml RAN and 100.23 μg/ml ATN (Standard Stock-I), the final volume was filled with diluent.

To prepare a solution with 20.46 μg/ml of RAN and 75.40 μg/ml of ATN, 1.0 ml of standard stock I of ATN into a 50 ml volumetric flask. Fill the flask up to the mark with mobile phase.

Sample preparation for pharmaceutical formulation:

Twenty pills with 750.0 mg of RAN and 100.4 mg of ATN each were weighed and powdered. The powder containing 350.45 mg of RAN and 25.21 mg of ATN was carefully weighed and transported to 100 ml volumetric flasks, where it was dissolved in a small amount of diluent and sonicated for 20 minutes. The resulting solution was filtered via a 0.45 μ membrane filter. The final volume was increased to 50 ml with diluent to achieve a concentration of 350.45 μg/ml of RAN and 250.21 μg/ml of ATN. Pipetted 1.0 ml into a 50 ml and 1 ml into a 10 ml volumetric flask, and made up to the mark with diluent, to obtain the concentration of 35.34 μg/ml of RAN and 25.04 μg/ml of ATN, respectively.

Method Development

During method development, the main focus was on totally separating RAN and ATN from solvent peaks. RAN is soluble in methanol but very marginally so in water. ATN is readily soluble in water. Several parameters were changed to optimize the chromatographic process, including flow rate, mobile phase buffer, and mobile phase pH. Several mobile phases were tested until a satisfactory resolution was found between two drugs. The Shimadzu C18 column (Phenomenex Gemini NX 100×4.6µm, 5µm) was tested using a mobile phase of 0.05 M potassium dihydrogen phosphate buffer and methanol using the gradient method. Phosphate buffer and methanol were used as mobile phases, and the resolution and retention time were satisfactory. After several studies, the process was optimized as a gradient approach using 0.05 M potassium dihydrogen phosphate buffer and methanol, with a flow rate of 1.2 mL/min at 240 nm and a run time of 13 minutes. These chromatographic parameters achieved adequate resolution, retention time, and tailing factor for RAN and ATN. The results are presented in Table 1.                         

Table 1: Developed RP-HPLC method chromatographic parameters.

Method validation ^{[6, 7, 8]}

The method was validated based on the ICH Q2 R1 recommendations for system applicability, linearity, precision, accuracy, limit of detection, limit of quantification, robustness, specificity, and assay. System suitability Six replicated standard sample solutions were injected to test system appropriateness and characteristics such as area%, tailing factor (T), theoretical plates, and retention time (Rt). The results are shown in Table 2 and Figure 1.

Linearity

The linearity of this approach was assessed using linear regression analysis using the Least Squares method. The drug was linear at concentrations ranging from 30.16-120.64 µg/ml for RAN and 8.18-32.74 µg/ml for ATN. Calibration standards were prepared by spiking required volume of working standard solution 0.4, 0.8, 1.0, 1.20, and 1.60 ml of RAN and 0.5, 1.25, 1.50,1.75, 2.00 ml into different 50 ml volumetric flasks and volume made with diluent to yield concentrations of 30.16, 60.32, 75.40, 90.48, and 120.64 µg/ml for RAN and 8.18, 16.37, 20.46, 24.55, and 32.74 µg/ml for ATN. A 20 µl sample was put into the analytical column. The resulting peak areas of the medicines were measured.

The calibration curve was plotted between the drug's peak regions and its concentration. These findings reveal a strong link between peak area and analyte concentration. The linearity findings are shown in Table 2 and Figure 2.

Intra-day and Inter-day precision

Precision was tested using quality control sampling of standard solutions with medium, lower, and high linearity range concentrations. Peak areas for three replicated injections of each concentration were measured.

Six replica measurements at three different concentrations were taken on the same day to evaluate intra-day precision. Inter-day precision was tested over three days using the system's usual technique. The method's accuracy was assessed by calculating recovery analyses. Statistical study revealed that the drug's relative standard deviation at different concentration levels for six injections was less than two. The results are displayed in Table 2.

Limit of Detection and Quantification:

The detection limits for RAN and ATN were 0.069 µg/ml and 0.102 µg/ml, respectively, while the quantification limits were 0.210 µg/ml and 0.310 µg/ml, respectively. The LOD and LOQ were determined using the response's standard deviation and slope.

Robustness:

The established method's robustness was enhanced by modest changes to parameters such as mobile phase composition, flow rate, and wavelength. When the acute parameters were changed, the current approach showed no significant difference. The tailing factor for both medications was consistently less than 2.0, and the components remained well separated throughout all of the permutations. Given the changes in the system suitability parameters and the technique's specificity, as well as the fact that the experiment was conducted at room temperature, one can conclude that the method conditions were resilient.

Accuracy:

Preparation of standard and sample mixture of RAN and ATN:

Level I (80%): Transfer 0.5 ml of sample stock solutions and 0.5 ml of RAN and 0.3 of ATN standard stock solutions of RAN and ATN into separate 10 ml volumetric flasks, and fill to the mark with methanol (3 repetitions).

Level II (100%): Transfer 0.5 ml of sample stock solutions and 0.75 ml of RAN and 0.5 ml of ATN from standard stock solutions of RAN and ATN into separate 10 ml volumetric flasks, and fill to the mark with methanol (3 repetitions).

Level III (120%): Transfer 0.5 ml of sample stock solutions and 1.0 ml of RAN and 0.7 ml of ATN standard stock solutions into separate 10 ml volumetric flasks, and fill to the mark with methanol(3 repetitions).

Level III (150%): Transfer 0.5 ml of sample stock solutions and 1.25 ml of RAN and 0.9 ml of ATN standard stock solutions into separate 10 ml volumetric flasks, and fill to the mark with methanol (3 repetitions).

Assay:

Twenty tablets containing 750 mg of RAN and 100.4 mg of ATN each were weighed and then powdered. Powder equivalent to 35.45 mg of RAN and 25 mg of ATN were carefully weighed and transferred to 100 ml volumetric flasks. The powder was then dissolved in a tiny amount of diluent and sonicated for 20 minutes. The final solution was filtered using a 0.45 μ membrane filter. The final volume was increased to 50 ml with diluent to accomplish concentration of 350.45 μg/ml of RAN and 250 μg/ml of ATN (stock solution). To obtain concentrations of 35.45 μg/ml of RAN and 5.0 μg/ml of ATN, 1 ml of the RAN stock solution was transferred to a 50 ml volumetric flask and 1 ml of the ATN stock solution was transferred to a 50 ml volumetric flask. Volume was adjusted with diluent to reach the mark.

Forced Degradation Study:

To achieve a concentration of 1000 µg/ml, 25 mg of RAN and ATN were weighed separately into a 25 ml volumetric flask and diluent was added to the volume. Degradation investigations were conducted by pipetting out certain ml of working stock solution based on the standard concentration required for analysis.

Acid Degradation Studies:

In a 25 ml quantity volumetric flask, transfer 5ml of normal working solution and add 5ml of 0.5 N HCl. This acidic solution was refluxed on a water bath at 70°C for 2 hours before being neutralized with 5.0 ml of 0.5 N NaOH and diluted to the desired concentration. The chromatogram for acid degradation tests for each medication is obtained and analyzed. The results were provided in Table 3.

Base Degradation Studies:

In a 25 ml volumetric flask, transfer 5ml of normal working solution and add 5ml of 0.5 N NaOH. This acidic solution was refluxed on a water bath at 70°C for 2 hours before being neutralized with 5.0 ml of 0.5 N HCl and diluent to the desired concentration. The refluxed solutions for certain medications are obtained and examined. Table 3 shows the collected results.

Oxidative Degradation Studies:

In a 25 mL volumetric flask, add 5 mL of normal working solution and 5 mL of 3% hydrogen peroxide. This solution was stored at room temperature for 24 hours before being diluted to the required volume. The chromatogram for oxidative degradation tests on particular medicines is obtained. The collected findings are shown in Table 3.

Thermal Degradation Studies:

5 mg of RAN and ATN were added to a 25 mL volumetric flask. The flask was placed in a hot air oven at 70°C for 24 hours, and diluent was added to make up the capacity. The chromatogram for heat degradation tests on individual medicines was obtained and analyzed. The results were provided in Table 3.

Photolytic Degradation Studies:

5 mg of RAN and ATN were added to a 25 mL volumetric flask. The flask was placed under UV light at 254 nm for 6 hours before diluent was added to make up the capacity. The chromatogram for photolytic degradation tests on various medicines was obtained and examined. The results were provided in Table 3.

RESULTS

Fig 1: Chromatogram for simultaneous estimation of RAN and ATN

(a)

(b)

Table 2: Result summary for validation parameters.

When accuracy was investigated using tablet formulation, the mean% recovery for RAN and ATN at four different levels was found to be 101.8-104.2% and 100.3%-104.3%, respectively.
The percent assay for RAN and ATN in tablet dosage form was determined to be 100.8% and 100.9%, respectively. The results reveal that no excipient interference or contaminants were detected in the sample using the devised RP-HPLC method.

Table 3: Degradation study data of RAN and ATN: