Green Solvent Assisted UV–Spectrophotometric Method for Estimation of Ranolazine in Bulk and Pharmaceutical Dosage Form

Angina pectoris is a clinical syndrome characterized by ischemic chest pain due to transient myocardial ischemia. The anginal pain occurs due to an imbalance between the oxygen demand and oxygen supply in the ischemic area of the myocardium. It is periodic episodes of substernal chest pain due to coronary artery atherosclerosis or vasospasm leading to compromised blood flow and oxygen supply to a particular region of the heart. This chest pain may radiate to the neck, jaw, back, and arms. In angina pectoris, due to myocardial ischemia, the myocardial tissues are deprived of oxygen and nutrients for aerobic metabolism. As a result, there is the inclusion of anaerobic metabolism which leads to the accumulation of lactic acid. Due to an increase in lactic acid, myocardial nerve Fibers are irritated and this transit a pain message to cardiac nerves, and all this led to cardiac pain which is angina pectoris. The occurrence of angina pectoris depends upon two factors (a) coronary blood flow and (b) oxygen consumption by the myocardium. Cardiac oxygen consumption increases with the increase in heart size, heart rate, systemic blood pressure, and cardiac contractability.

Various pharmaceutical dosage forms are available in the market for the treatment of angina. Ranolazine is an anti-anginal medication used alone or in combination with other drugs to manage chronic angina—persistent chest pain or pressure resulting from inadequate oxygen supply to the heart. In January 2006, FDA has approved a ranolazine for the treatment of angina pectoris. Structurally, it is n-(2,6-dimethylphenyl)-2-[4-[2-hydroxy-3-(2-methoxyphenoxy) propyl] piperazine-1yl] acetamide. ranolazine is believed to have its effects via altering the trans-cellular late sodium current.it is by altering the intracellular sodium level that ranolazine affects the sodium-dependent calcium channels during myocardial ischemia. thus, ranolazine indirectly prevents the calcium overload that causes cardiac ischemia. Ranolazine is indicated for the treatment of chronic angina.  A review of the literature indicates that several UV spectrophotometric methods have been reported in methanol for the estimation of ranolazine in bulk and pharmaceutical dosage forms. However, no UV-based method has been developed using a class 3 solvent, as classified by the ich Q3C guideline. Therefore, this study aims to develop a UV-spectrophotometric method for the estimation of ranolazine in bulk and pharmaceutical dosage forms using a class 3 solvent, ensuring compliance with Q3C guidelines and principles of green chemistry. _(5-11)
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1. MATERIALS AND METHODS

2.1 Instruments And Software

The study was performed using UV- Spectrophotometer (SHIMADZU UV 1900i; Software: Lab solution UV-Vis), which features a double beam with a 1 cm matched cell. The Electronic analytical balance (Scaletac; Model: MH-0095) was used for the weighing.

1. 2. Materials And Reagents

Ranolazine was gifted sample from reputed company, India. Iso- propyl alcohol AR was purchased from Chemdyes Corporation Rasayan Ghar, Rajkot. The marketed formulation RANCAD (Hetero labs Limited, India) tablet was purchased form local pharmacy.

1. 3. Preparation Of Solution

Accurately weighted quantity of Ranolazine 10 mg was transferred to 10 mL volumetric flask, dissolved and diluted to the mark with Isopropyl Alcohol (1000 μg/mL). Aliquot 2.5 ml was withdrawn from standard stock solution of Ranolazine and was transferred to 25 mL volumetric flask and was diluted to the mark with IPA resulting 100 μg/mL concentration of solution. (Working standard solution A) Aliquot 1 ml was withdrawn from working standard solution A of Ranolazine and was transferred to 10 mL volumetric flask and was diluted to the mark with IPA resulting 10 μg/mL concentration of solution. (Working standard solution B)

2.4 Determination Of Wavelength For Measurement

The working standard solution of Ranolazine (10 μg/mL) was scanned in the range of 200 – 400 nm keeping IPA as blank. Each solution was scanned between 200 – 400 nm. Wavelength was selected from the spectra of Ranolazine.

2. Prepartion Of Calibration Curve

From working standard solution A of Ranolazine (100 μg/mL) aliquot of 1 mL, 2 mL, 3 mL, 4 mL, 5 mL were taken and transferred into 10 mL volumetric flasks and diluted up to mark with IPA which resulted in the concentration range of 10 – 50 μg/mL respectively. The absorbance of solutions was measured at 274 nm. Graph of absorbance vs concentration was plotted at wavelengths and regression line equation was found.

1. 5. Validation
      1. Linearity

Linearity was determined by analysing 5 independent levels of calibration curves in range of 10 - 50 μg/mL for Ranolazine 5 times. Plot the curve of absorbance vs respective concentration and regression coefficient & regression line equation was determined.

1. 1. 2. Repeatability
         1. Repeatability Of Sample Application

Repeatability of the sample application was determined by preparing solution of 30 μg/mL of Ranolazine respectively for seven times and measure the absorbance at selected wavelengths. The results of repeatability sample preparation were reported in terms of %RSD.

1. 1. 1. 2. Repeatability Of Sample Measurement

Repeatability of the sample measurement was determined by measuring the absorbance of 30 μg/mL Ranolazine solution respectively for seven times at selected wavelengths. The results of repeatability sample measurement were calculated in terms of %RSD

1. 1. 3. Intermediate Precision
         1. Intraday Precision

Intraday precision was determined by measuring the corresponding response for 3 times on same day for each level of calibration curve in a range of 10 - 50 μg/mL for of Ranolazine. The results of intraday precision were calculated in terms of %RSD.

1. 1. 1. 2. Interday Precision

Interday precision was determined by measuring the corresponding response for on 3 different days for each level of calibration curve in a range of 10 - 50 μg/mL for of Ranolazine. The results of Interday precision were calculated in terms of %RSD.

1. 1. 4. LOD AND LOQ

The limit of detection and limit of quantitation were calculated from the standard deviations of the intercepts and mean slope of the calibration curves of Ranolazine using the equation given below

                            ????????????=3.3????/???? and ????????????=10????/????

Where σ = the standard deviation of the Y-intercept of the five calibration curves; S = mean slope of the five calibration curves

1. 1. 5. Accuracy

The accuracy in terms of the extraction efficiency of the method was determined by the standard addition method. The known amount of Ranolazine was added to the pre-analysed sample at three levels (i.e. 80%, 100%, 120%) and analysed.

Procedure:

Tablet powder equivalent to 500 mg of ranolazine was accurately weighed and transferred to four individual 100 mL volumetric flasks. Standard ranolazine 400 mg, 500 mg and 600 mg was spiked in the first, second and third volumetric flask respectively. Fourth flask was kept as control. The flasks were filled to about 80% with IPA, sonicated for 20 minutes and diluted up to mark with IPA. These solutions were filtered through Whatman filter paper (paper no. 42) individually; the first few mL was discarded. From the resulting solutions 1 mL aliquot was withdrawn and diluted up to mark with 10 ml with IPA. (80% level: 900 μg/mL ranolazine; 100% level: 1000 μg/mL ranolazine; 120% level: 1100 μg/mL ranolazine) 0.2 mL aliquot was withdrawn and diluted up to mark with 10 ml with IPA. (80% level: 18 μg/mL ranolazine; 100% level: 20 μg/mL ranolazine; 120% level: 22 μg/mL ranolazine). The absorbance was measured at 274 nm. From the calibration curve of ranolazine, the amount of ranolazine was calculated and % recovery was determined.

1. 6. ASSAY

Twenty tablets were accurately weighed and powdered. Tablet powder equivalent to 500 mg of ranolazine was accurately weighed and transferred to four individual 100 mL volumetric flasks. The flasks were filled to about 80% with IPA, sonicated for 20 minutes and diluted up to mark with IPA. (5000 μg/mL Ranolazine) By applying further dilution make final concentration solution 30 μg/mL Ranolazine. The absorbance of resulting solution was measured at 274 nm. The amount of ranolazine in marketed formulation was determined.

2. RESULTS AND DISCUSSION
   1. Determination Of Wavelength For Measurement

To determine wavelength for measurement, 10 μg/mL solution of Ranolazine was scanned in range of 200 – 400 nm keeping IPA as blank. The absorbance maxima (λ max) of Ranolazine were obtained 274 nm respectively. (Figure 3)

Figure 3: Wavelength selection from Ranolazine in IPA

1. 2. Validation
      1. Linerity

Representative calibration curve of Ranolazine was obtained by plotting absorbance of Ranolazine at 274 nm against concentration over the range of 10 – 50 μg/mL (n=5). It was found to be linear in the above-mentioned range with regression coefficient 0.9955 at 274 nm. The average linear regressed equation for the calibration curve was ????=0.0085x – 0.073 at 274 nm. Linearity data is depicted in figure 4 and 5.

Table 1: Linearity data

Figure 4: Overlain UV spectrum of Ranolazine in IPA

Figure 5: Calibration curve of Ranolazine

1. 1. 2. Repeatability
         1. Repeatability Of Sample Measurement

The % RSD for repeatability of sample measurement for absorbance of Ranolazine was found to 1.2001. The repeatability of measurement is depicted in table 2

1. 1. 1. 2. Repeatability Of Sample Application

The % RSD for repeatability of sample application for absorbance of Ranolazine was found to be 0.9967. The repeatability of application is depicted in table 2.

Table 2: Repeatability data

1. 1. 3. Intermediate Precision

The % RSD For Intraday and Interday Precision of Ranolazine were found to be 0.380-1.364 and 0.328-1.219 respectively as Shown in Table 3. The Results Indicate That the method is Precise for measurement of the drug

Table 3: Intraday and Inter day precision data

1. 1. 4. LOD AND LOQ

The LOD and LOQ of Ranolazine were found to be 0.902 and 2.733 respectively. The data for Ranolazine is depicted in table 4.

Table 4: LOD and LOQ data

1. 1. 5. ACCURACY

Accuracy was determined in terms of recovery study and the recoveries were done at three levels i.e., 80 %, 100 % and 120%. The recovery of Ranolazine was found to be 99.56 -100.01%. The data for accuracy of method for Ranolazine was depicted in table 5.

Table 5: Recovery data

3.3 ASSAY

Applicability of the proposed method was tested by analysis pharmaceutical dosage form of Ranolazine. The amount of Ranolazine was found to be 99.36 %. The result is within the range of acceptance limit.

1. 4. Summary Of Validation Parameters

Table 6: Summary of validation parameters