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

Hypertension is one of the most common worldwide disease affecting humans. Because of the associated morbidity and mortality and the cost to society, hypertension is an important public health challenge. Over the past several decades, extensive research, widespread patient education, and a concerted effort on the part of health care professionals have led to decreased mortality and morbidity rates from the multiple organ damage arising from years of untreated hypertension. Hypertension is defined as systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg. Hypertension is divided into two stages: Stage 1: includes patients with systolic blood pressure 140 – 159 mmHg or diastolic blood pressure 90 – 99 mmHg. Stage 2: includes patients with systolic blood pressure ≥ 160 mmHg or diastolic blood pressure ≥ 100 mmHg. _(1-3) In August 2017, CDSCO has approved a Efonidipine HCl Ethanolate for the treatment of hypertension. Efonidipine hydrochloride ethanolate is chemically 2-(N-benzyl anilino) ethyl 5-(5, 5-dimethyl-2-oxo-1, 3,2λ5-dioxaphosphinan-2-yl)-2, 6-dimethy l-4-(3-nitrophenyl) -1, 4-dihydropyridine-3-carboxylate, hydrochloride. (Figure 1) Efonidipine is a dihydropyridine, calcium channel blocker with anti-hypertensive activity. Efonidipine HCl Ethanolate works as a Calcium channel Blocker, Specifically Inhibiting both L-type & T-type calcium channels, leading to Vasodilation & decrease in heart rate by primarily acting on the (SA node) Sinoatrial node, thereby lowering blood pressure. (Figure 2) _(4-10) A review of the literature indicates that several UV spectrophotometric methods have been reported for the estimation of Efonidipine HCl Ethanolate in bulk and pharmaceutical dosage forms.₍₁₁₎ 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 Efonidipine HCl Ethanolate in bulk and pharmaceutical dosage forms using a Class 3 solvent, ensuring compliance with Q3C guidelines and principles of green chemistry.

Figure 1: Mechanism of action of efonidipine HCl ethanoate

Figure 2: Structure of efonidipine HCl ethanoate

1. MATERIALS AND METHODS
   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

Efonidipine HCl ethanoate was gifted sample from reputed company, India. Iso- propyl alcohol AR was purchased from Chemdyes Corporation Rasayan Ghar, Rajkot. The marketed formulation EFNOCAR -40 (Zuventus Healthcare Limited, India) tablet was purchased form local pharmacy.

1. 3. Preparation Of Solution

Accurately weighted quantity of Efonidipine HCl Ethanolate 10 mg was transferred to 10 mL volumetric flask, dissolved and diluted to the mark with Isopropyl Alcohol (1000 μg/mL). Aliquot 1 ml was withdrawn from standard stock solution of Efonidipine HCl Ethanolate and was transferred to 10 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 Efonidipine HCl Ethanolate 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 Efonidipine HCl Ethanolate (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 Efonidipine HCl Ethanolate.

1. 5. Prepartion Of Calibration Curve

From working standard solution A of Efonidipine HCl Ethanolate (100 μg/mL) aliquot of 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL were taken and transferred into 10 mL volumetric flasks and diluted up to mark with IPA which resulted in the concentration range of 5 – 25 μg/mL respectively. The absorbance of solutions was measured at 251 nm. Graph of absorbance vs concentration was plotted at wavelengths and regression line equation was found.

1. 6. Validation
      1. Linearity

Linearity was determined by analysing 5 independent levels of calibration curves in range of 5 - 25 μg/mL for Efonidipine HCl Ethanolate 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 15 μg/mL of Efonidipine HCl Ethanolate 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 15 μg/mL Efonidipine HCl Ethanolate 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 5- 25 μg/mL for of Efonidipine HCl. 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 5 - 25 μg/mL for of Efonidipine HCl. 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 Efonidipine HCl Ethanolate 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 Efonidipine HCl ethanolate was added to the pre-analysed sample at three levels (i.e. 80%, 100%, 120%) and analysed.

Procedure:

Tablet powder equivalent to 40 mg of Efonidipine HCl ethanolate was accurately weighed and transferred to four individual 100 mL volumetric flasks. Standard Efonidipine HCl ethanolate 32 mg, 40 mg and 48 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 0.25 mL aliquot was withdrawn and diluted up to mark with 10 ml with IPA. (80% level: 18 μg/mL EFO; 100% level: 20 μg/mL EFO; 120% level: 22 μg/mL EFO) The absorbance was measured at 251 nm. From the calibration curve of Efonidipine HCl ethanloate, the amount of Efonidipine HCl ethanolate was calculated and % recovery was determined.

1. 7. ASSAY

Twenty tablets were accurately weighed and powdered. Tablet powder equivalent to 40 mg of Efonidipine HCl ethanolate 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. (400 μg/mL EFO) Make final solution 15 μg/mL from above solution. The measured absorbance of resulting solution at 251 nm. The amount of Efonidipine HCl ethanolate in marketed formulation was determined.

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

To determine wavelength for measurement, 15 μg/mL solution of Efonidipine HCl Ethanolate was scanned in range of 200 – 400 nm keeping IPA as blank. The absorbance maxima (λ max) of Efonidipine HCl Ethanolate were obtained 251 nm respectively. (Figure 3)

Figure 3: UV Spectrum of Efonidipine HCl ethanolate in IPA

1. 2. VALIDATION
      1. LINERITY

Representative calibration curve of Efonidipine HCl Ethanolate was obtained by plotting absorbance of Efonidipine HCl Ethanolate at 251 nm against concentration over the range of 5 – 25 μg/mL (n=5). It was found to be linear in the above-mentioned range with regression coefficient 0.9976 at 251 nm. The average linear regressed equation for the calibration curve was ????=0.0268????+0.3213 at 251 nm. Linearity data is depicted in figure 4 and 5.

Table 1: Linearity data

Figure 4: Calibration curve of Efonidipine HCl Ethanolate

Figure 5: Calibration curve of linearity of Efonidipine HCl ethanolate

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

The %RSD for measurement of absorbance for of Efonidipine HCl Ethanolate was found to be 0.693%. The data for 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 Efonidipine HCl Ethanolate was found to be 0.636%. The data for repeatability of application is depicted in table 2.

Table 2: Repeatability data

1. 1. 3. Intermediate Precision

The %RSD for Intra-day and inter-day precision of Efonidipine HCl Ethanolate were found to be 0.200-0.987% and 0.213-1.192% respectively, as shown in table 3. The result indicate that method is precise for measurement of drugs.

Table 3: Intra day and Inter day precision data

1. 1. 4. LOD AND LOQ

The LOD and LOQ for Efonidipine HCl Ethanolate were found to be 0.612 μg/mL and 1.856 μg/mL respectively. The data for Efonidipine HCl Ethanolate are 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 Efonidipine HCl ethanolate was found to be 98.89-100.43%. The data for accuracy of method for Efonidipine HCl ethanolate 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 Efonidipine HCl ethanolate. The amount of Efonidipine HCl ethanolate was found to be 99.82 %. The result is within the range of acceptance limit.

1. 4. Summary Of Validation Parameters

Table 6: Summary of validation parameters