INTRODUCTION:

Azelnidipine (AZL) is a dicarboxylate of ()-3(1-diphenylmethylazetidin3yl)5isopropy12amino1,4dihydro-6 methyl4(3-nitrophenyl)3,5pyridine^1-2. It is a calcium channel blocker (CCB) of the dihydropyridine (DHP) type used to treat hypertension. AZL has two enantiomers due to an asymmetric carbon at the 4 position of the DHP ring^3-4. The pharmacological action of AZL resides in the (R) enantiomer. In contrast to other CCBs, where the (S) enantiomer is responsible for biological activity. The peculiar three-dimensional structure of the active enantiomer of AZL may be related to its unique pharmacological features that are not shared by other DHPs, such as a long-lasting reduction in blood pressure, decreased heart rate, and anti-atherosclerosis effect. AZL also shows a diuretic effect by increasing urine volume and thus reducing the retention of ions^5-7.

Figure-1 Chemical Structure of Azelnidipine

Smartphone-based colorimetry has been gaining relevance because of the widespread advancement of devices with increasing computational power, their relatively low cost and portable designs with user-friendly interfaces, and their compatibility with data acquisition^8-9. Various methods has been reported for the estimation of drugs by using smart phone application in which mostly the RGB (Red, Blue and Green) principle has been used. In this study, the mobile phone application, called PhotoMetrix, which employs the techniques of simple linear correlation for univariate analysis and principal components analysis (PCA) for multivariate exploratory analysis was used. This Photo Metrix application is available free in Google Play Store. The method was based on the detection of color intensities and the evaluation of relationship between measured color and concentration of sample¹⁰.

MATERIALS AND METHODS:

Apparatus and Software:

A Shimadzu UV-1700 double-beam spectrophotometer connected to a computer with Shimadzu UV-Probe 2.10 software installed was used for all the spectrophotometric measurements. The absorbance spectra of the reference and test solutions were carried out in 1 cm² quartz cells over the range of 400–800nm. The samples were weighed on an electronic balance (A120) by Shimadzu. A smart phone having the application Photo Metrix was used to take images.

Chemicals and Reagents:

Purechem LTD., Ankleshwar, supplied the Azelnidipine (API).

Preparation of Ethanolic Ninhydrin Solution (3% W/V):

Two grammes of ninhydrin were dissolved in 95 volumes of methanol and five volumes of glacial acetic acid.

Preparation of ammonium acetate buffer (pH 4.9):

10mM of ammonium acetate buffer was used, and for pH adjustment, acetic acid was used. (All chemicals used in the present study were of analytical grade.)

Preparation of standard stock solution:

10mg of azelnidipine was weighed accurately and transferred into a separate 100ml volumetric flask. This results in an azelnidipine concentration of 100μg/mL.

Preparation of working solutions for calibration graph:

0.2ml, 0.4ml, 0.6ml, 0.8ml, and 1ml of standard stock solution were transferred to a 10ml volumetric flask to achieve a concentration range of 2-10g/ml.Add 2ml of freshly prepared Ninhydrin reagent and 1ml of buffer solution to the flask. All solutions were heated in a water bath for 25minutes. After heating time, solutions were cooled down, and volume was made up to the mark using methanol.

Preparation of sample solution for assay:

Twenty tablets containing 8 mg of azelnidipine (Azedax-8) were precisely weighed. The average tablet weight was determined, and the tablets were powdered. The tablet powder equivalent of 8mg of Azelnidipine was weighed and transferred into a 100ml volumetric flask, and the volume was made up to the mark using distilled water to get an 800μg/mL solution. The content was filtered through the Whatman filter paper to get a clear solution. From the above solution, 0.5ml was withdrawn into the 10 ml volumetric flask to get an 8μg/ml concentration. Add 2ml of reagent and 1ml of buffer, and heat the solution in a water bath for 25min. Distilled water was used to make up the remaining volume.

METHOD DEVELOPMENT:

UV-Vis Spectroscopy Prepared working standard solutions in the range of 2–10μg/ml were scanned between 400–800nm in the UV-Spectrophotometer by using Ninhydrin reagent as a blank. At 574nm, the maximum absorbance was observed, and it was selected as the detection wavelength. A calibration graph was plotted for the concentration range of 2–10μg/ml, and overlay spectra are mentioned below.

Figure- 2 Overlay Spectra of AZL at 574nm

Experimental optimization:

1. Optimization for Concentration of Ninhydrin reagent :

By holding all other parameters constant, the effect of a 1% to 5% change in reagent concentration was achieved.Maximum absorbance was observed when the concentration of the Ninhydrin reagent was set at 3%, so that concentration was chosen.

Figure –3 Optimization of Concentration

2. Optimization of Reagent volume :

The effect of reagent volume on the drug reagent complex was carried out in the range of 1-3ml. For the colour intensity and absorbance in the UV spectrophotometer, the optimum concentration was found to be 2ml of reagent for the method.

Figure -4 Optimization of Reagent volume

3. Optimization of heating time:

The effect of heating time on complex formation was examined. The drug-reagent reaction occurred between 5 and 30 minutes of heating time. A slight increase in colour intensity was observed after heating for 25 min.

Figure -5 Optimization of heating time

4. Optimization of Temperature:

To 1ml of the stock solution of azelnidipine, 2ml of ninhydrin solution (3% w/v) were added. The reaction mixtures were heated for 10minutes at 50–100°C. The coloured product was diluted up to 10ml with methanol, and the absorbance was measured against a reagent blank at 574nm. The results showed that the highest absorbance was obtained at 702°C. The developed colour remained stable for 48hrs.

Figure-6 Optimization of temperature

Optimized conditions for the colorimetric estimation of Azelnidipine

Parameters	Optimized value
℅Ninhydrin	3℅
Volume of Ninhydrin	2ml
Heating time	25min
Temperature	70⁰C

Reaction Mechanism:

Reaction of Ninhydrin with amines, alpha amino acids, peptides, and proteins yields an aldehyde with one carbon atom less than the alpha-amino acid; carbon dioxide in stoichiometric amounts; and varying amounts of ammonia, hydronation, and a chromophoric compound known as Ruhlmann’s Purple (2-(3-hydroxy-1-oxo-1H-inden-2-ylimino)—2Hindene-1,3-dione; and a chromo)—asThis pigment serves as the basis for detection and quantitative estimation of alpha-amino acids. The proposed mechanism (Figure 7) for the reaction involves the removal of a water molecule from ninhydrin hydrate 1 in the first step to generate 1,2,3-indantrione 2, which then forms a Schiff's base with the amino group of pregabalin to produce ketamine 3. Removal of the aldehyde RCHO generates an intermediate amine (4, 2-aminoethane, 3-indandione). The condensation of this intermediate amine with another molecule of ninhydrin follows to form the expected chromophore 5 (Ruhlmann’s purple).

ESTIMATION OF AZELNIDIPINE USING SMARTPHONE APPLICATION:

Experimental Setup:

A self-designed box was built in the lab to improve the accuracy and precision of the measurements. On the upper side of the box, an LED bulb was fitted to provide a consistent incident light source. All the inner side walls of the box were covered with white paper to provide full reflection of the light. The front side is made in such a manner that it can be opened to insert a cuvette inside the box (Figure 8A). On the front side of the box, a small square-shaped hole was made exactly in the middle to allow the camera to take photos of the object placed inside the box. Also, a cuvette holder was made from thermocol and fixed in the middle of the box. The entire experimental setup is illustrated in Figure 8.

Figure : 8 A. Cuvette placed in the arranged setup B. Image captured by mobile phone camera in the arranged setup

Preparation of calibration graph by smart phone application:

An aliquot of the standard solution of Azelnidipine, corresponding to 2–10µg/mL, was taken into a 10mL volumetric flask. 70°C for each flask, 2ml of 3% Ninhydrin solution and 1ml of buffer were added, followed by sand, and the solution was heated at 70°C for 25minutes. The solution was allowed to cool at room temperature, and then the volume was made up to 10ml with methanol. Once the standard solutions were prepared, the images were captured one by one in the Photo Metrix Pro application. The interface of the application as well as the options to be chosen in a stepwise manner are shown in Figure 9. In the application, a univariate analysis was performed first, and then vector RGB was selected in the univariate analysis. Then, once you click on calibration, the app will ask about the number of samples. In this case, 6 samples (1 blank and 5 standards) were written. Then, first, the blank solution was filled in the cuvette and was inserted in the box, and after writing 0 in the concentration section, an image was captured by putting the camera at the middle hole of the box. Similarly, the image of each standard solution was captured one by one in increasing order of concentration. Then the save button was clicked, and the calibration graph as well as the regression equation were shown by the application itself.

Figure : 9 Graphic interface of the Photo Metrix Application and steps to generate calibration graph in application

Figure-10 Colour intensities of captured images in Photo Metrix

Once the regression equation was obtained, the concentration of the sample solution from the formulation was estimated. Here, instead of calibration, the sampling button was clicked, and the image of sample solution prepared for assay was captured in a manner similar to standard solution. Then the save button was clicked, and the concentration of the sample was given by application of the generated calibration graph.

Method Validation:

Linearity :

Azelnidipine was linear with a concentration range of 2–10µg/ml at 574nm, obeying Beer’s law (Figure 11). A calibration curve was plotted between concentration and absorbance. The plot was found to be linear, as shown in the figure below.

Figure -11 Calibration curve of Azelnidipine (2-10ug/ml)

Assay of formulation:

The assay was carried out using both methods on the marketed formulation azedex-8, which has a label claim of 8 mg of Azelnidipine. Sample solutions were analyzed, and concentration was estimated as a percentage recovery from a linear regression equation. Assay results were found to be in an acceptable range and significant for both methods. Results of assays are shown in the table below.

Table-1 Assay results obtained from both methods

Method

Amount Lebeled 2 (mg)

Amount Estimated (mg)

%Recovery ±

SD (n=6)

%RSD

UV method

7.94

99.65 ± 0.321

0.80

Photometric

7.89

99.2 ± 0.131

0.83

Result table: Statistical data for the regression equation of the proposed method.

Parameters	Azelnidipine(uv)	Photo matrix
Analytical wavelength	574nm	-
Linearity	2-10ug/ml	2-10ug/ml
Regression equation	0.0894x+0.0232	14.825x+7.738
Slope	0.0894	14.825
Intercept	0.0232	7.738
Correlation coefficient	0.9993	0.993
Assay	99.65	99.2

CONCLUSION:

A novel and rapid colorimetric detection method for azelnidipine is developed using a smartphone-based PhotoMetrix application. The method used a simple colouring agent in a simple and less time-consuming procedure. The main aim of this study was to make the colorimetric estimation of drug content easier with the help of such smartphone-based applications. The method was also compared with an UV method developed with the same reagent and procedure, and it was found that there was no significant difference in the assay results. This novel method can be used as an alternative to analytical science in quantitative drug estimation in pharmaceutical dosage forms.

REFERENCES:

1. https://pubchem.ncbi.nlm.nih.gov/compound/Azelnidipine

2. Rele RV, Patil SP. Ultra-Violet Spectrophotometric Method for Estimation of Azelnidipine from Bulk Drug and Pharmaceutical Formulation. Asian Journal of Research in Chemistry. 2010; 3(4): 1077-9

3. Mandale D, Mistry R, Chauhan N. An analytical approach of azelnidipine: a review. Muralidharan S, S. Parasuraman and V. Venugopal, Simple Validation of Azelnidipine By RP-HPLC Method. Rapports De Pharmacie. 2015; 1(1): 43-45.

4. Raskapur KD, Patel MM, Captain AD. UV-Spectrophotometric method development and validation for determination of Azelnidipine in pharmaceutical dosage form. Toxicology. 2010; 106: 135-43.

5. Shewale VU, et al. Azelnidipine: A Review on Therapeutic Role in Hypertension. Journal of Drug Delivery and Therapeutics. 2019; 9: 1002-1005.

6. Mellon MG. The Role of Spectrophotometry in Colorimetry. Industrial and Engineering Chemistry Analytical Edition. 1937; 9(2): 51-6. https://doi.org/10.1021/ac50106a001

7. D. Prabhakar, J. Sreekanth, K.N. Jayaveera. Method Development and Validation of Azelnidipine By RP-HPLC. International Journal of ChemTech Research. 2018; 11(1): 7-12.

8. S. Yuvasri, S. Murugan and T. Vetrichelvan. FIRST- Order Derivative and UV-Spectrophotometric Methods For Simultaneous Determination Of Telmisartan And Azelnidipine In Bulk And Tablet Dosage Form. European Journal of Biomedical and Pharmaceutical Science. 2021; 8(5): 290-294.

9. Bhati KN, Mashru R. Development and Validation of Extractive Spectrophotometric Method for Estimation of Hydroxychloroquine Sulphate by using Smartphone Application. Journal of Drug Delivery and Therapeutics. 2022; 12(3): 49-56 DOI: http://dx.doi.org/10.22270/jddt.v12i3.5460

10. Saiyed SA, Jadeja P, Mashru R. Development and Validation of Extractive Spectrophotometric Methods for the Estimation of Telmisartan by Using Smartphone Application. Journal of Drug Delivery and Therapeutics. 2022; 12(3-S): 178-190 DOI: http://dx.doi.org/10.22270/jddt.v12i3-s.5527

Received on 07.12.2022 Modified on 14.07.2023

Asian J. Pharm. Ana. 2024; 14(1):1-5.

DOI: 10.52711/2231-5675.2024.00001