Method Development and Validation of Azelnidipine Drug by Ultraviolet Visible Double Beam Spectrophotometer

Introduction

Azelnidipine (AZEL) is chemically (±)-3- (1-diphenyl methylazetidin3- yl) 5-isopropy12-amino-1, 4-dihydro6-methyl-4-(3-nitrophenyl) 3,5- pyridinedi carboxylate (Figure 1) and is a ca2+ channel blocker used to treat HT blood pressure.¹ Due to an unbalanced carbon at the 4-position of 1,4 dihydropyridine ring, it appears as two enantiomers. The drug has renoprotective effects, as well as anti-atherosclerotic effects. Azelnidipine inhibits trans membrane Ca+2 influx into cell and its membrane, lowers PVR and arterial pressure through the voltage dependent medium of smooth muscle in vascular walls.^2-4

Materials and Method Instrumentation:

A Thermo scientific GENESYS 10S UV-Visible spectrophotometer with 1.0-centimetre matching quartz cells were used for absorbance measurements. The UV spectrum was captured over the wavelength 200‐400 nm.^5-6

Chemicals and reagents:

16 mg of AZEL (Azovas) was obtained from Apollo pharmaceuticals. The sample of azelnidipine was moderately dissolved in methanol, freely dissolved in acetone, soluble in ethyl acetate and sparingly dissolved in water.⁷

Preparation of standard stock solution:

A 100 milligram of Azel standard was weighed exactly and shifted to a 100 mL volumetric flask and solubilized in diluents to give a solution containing 1000 microgram/ millilitre standard stock solution of Azel.^8-9

Preparation of working standard stock solution:

10 millilitre of standard stock solution was retrieved and transferred to 100 millilitre volumetric flask. Capacity was made up to the mark with diluents to get the working standard solution of 100 microgram/millilitre.¹⁰

Preparation of calibration curve of AZEL:

Suitable volumes of liquids 2, 4, 6, 8, 10, 12 and 14 microgram/ millilitre for linearity from Azel stock solutions were tranferred to distinct volumetric flasks of 10 millilitre capacity.^11-12 The magnitude was adjusted to the mark with diluents to obtain concentration of 2-14 µg/mL. The absorbance of the solutions was measured at 257 nm. The calibration curve was constructed by plotting absorbance versus concentration of the drug and regression equations were determined.¹³

Method Validation

The process was validated to assess the reliability of outcome of the analysis for different parameters like Linearity, Range, Accuracy, Precision, Limit of detection (LOD), Limit of quantification (LOQ), and system suitability parameters of Specificity and Robustness.^14-15

Linearity

Linearity was estimated by analysing the absorbance of acetone as blank standard concentration (2-14 µg/mL) at a wavelength of 257 nm.¹⁶ The calibration curve was plotted using concentration against absorbance.^17-18 Ar2 equation and correlation coefficient were estimated for Azel standard concentrations (2-14 μg/mL) which is shown in Table 1.

Range

The data retrieved from the linearity and accuracy studies was used to assess the range of the method.

Accuracy

Accuracy was established by producing three samples of the solution 50, 100 and 150% of working standard and adding known concentrations of Azel to each sample solution and solubilized in 10 mL of volumetric flask with analytical grade Acetone. Accuracy was evaluated using a minimum of nine determinations over a minimum of three concentration levels for each sample.¹⁹ Absorbance values at a wavelength of 257 nm and the calculated % RSD is shown in Table 2

Precision

The instrument precision was evaluated by determining the absorbance of the standard solution of AZEL six times repeatedly.^20-21 The results are reported in terms of relative standard deviation. The intra- and inter-day difference for the estimation of AZEL was carried out in triplicate for the standard solution and is shown in Table 3 and 4. 

Limit of Detection (LOD)

The identification limit of an independent analytical procedure is the lowest amount of analyte in a sample which can be identified but not definitely quantified as an exact value. The LOD was determined from the set of five calibration curves used to estimate method linearity. LOD was calculated as follows from the formula^24-

LOD= (3.3*SD)/slope

Where, SD = the standard deviation of y-intercept of five calibration curves

Slope = the mean slope of five calibration curves as shown in Table 5

Limit of Quantification (LOQ)

The Quantitation limit is a parameter of quantitative assays for low levels of compounds in sample. The LOQ was evaluated from the set of five calibration curves used to estimate method linearity as shown in Table 5.

The LOQ may be calculated as LOQ = 10 × (SD/S)

Where, σ = Standard deviation of the Y- intercepts of the five calibration curves. 

S = Mean slope of the five calibration curves.

Specificity

In Ultraviolet-Visible spectroscopy, specificity was established by examining of each Azel standard solution (1-14 μg/mL) in range of 200-800 nm against acetone as blank (Figure 2).

Robustness

The results remain unaffected by slight, but deliberate differences in method parameters, change in wavelength, analysis by person to person and changing room temperature. 

Absorbance of any one concentration was measured at  three different wavelengths i.e. 254, 257, 260 nanometres and mean and % RSD were calculated.

Consent and ethical approval

It is not applicable Competing interest Writers have announced that no competing interests exists.