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Vol 2 | Issue 1 | Jan – Mar 2023                                                                                     Indian J Pharm Drug Studies | 27  

Research Article 

Advanced UV spectrophotometric method development and validation for 

simultaneous estimation of Azelnidipine and Telmisartan in Pharmaceutical 

Dosage Form 

Pallavi Suthar1, Rajashree Mashru2  

From, 1 Assistant Professor, Department of Chemistry, Shree Swaminarayan Sanskar Pharmacy College, Gujarat, 2 Professor, HOD 

of Pharmaceutical Quality Assurance Department, Maharaja Sayajirao University of Baroda, India.   

Correspondence to: Pallavi Suthar, Assistant Professor, Department of Chemistry, Shree Swaminarayan Sanskar Pharmacy college, 

Near Zundal Circle, S. P. Ring Road, Between Chandkheda-Adalaj, Zundal, Gandhinagar- 382421, Gujarat, India. Email: 

pallaveemali12@gmail.com   

ABSTRACT 

The presented research work aims to develop and validate three advanced UV spectrophotometric methods for the simultaneous 

estimation of Azelnidipine (AZL) and Telmisartan (TEL). These methods offer a higher degree of sensitivity than already-existing 

methods of analysis. By implementing advanced spectroscopic techniques such as the simultaneous method, Q-ratio method, and first 

derivative spectroscopy method. These methods offer a higher degree of sensitivity than already-existing methods of analysis. By 

implementing advanced spectroscopic techniques such as the simultaneous method, Q-ratio method, and first derivative spectroscopy 

method. The linearity of the three methods was in the range of 2 µg/ml to 12 µg/ml for AZL and 10 µg/ml to 50 µg/ml for TEL. The 

correlation coefficients for simultaneous estimation were 0.999 and 0.998 for AZL and TEL, respectively, and 0.9992 and 0.9989 for 

AZL and TEL, respectively, for the first derivative method, whereas the correlation coefficients for the Q-ratio method were 0.999 and 

0.9988, respectively. The LOD values obtained by the simultaneo us estimation method were found to be lower as compared to those 

obtained by the first derivative method and the Q-ratio method, proving that the sensitivity of the simultaneous estimation method is high. 

Key words: Method development, Validation, Azelnidipine, Telmisartan, Concentration. 

he chemical formula for Azelnidipine (AZL) is 

3(1diphenylmethylazetidin3-yl)-5-isopropyl-12amino-

1,4-dihydro-6-methyl-4-(3-nitrophenyl)-3,5pyridine 

dicarboxylate. It is a dihydropyridine (DHP) type 

calcium channel blocker (CCB) used for the treatment of 

hypertension [1, 2]. AZL has two enantiomers due to an 

asymmetric carbon at the 4-position of the DHP ring. The 

pharmacological action of AZL resides in the (R) enantiomer. 

This is in marked contrast to other CCBs in which the (S) 

enantiomer is responsible for the biological activity (Figure 1) 

[3, 4]. 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 [6, 8]. AZL also shows 

a diuretic effect by increasing urine volume and thus reducing 

the retention of ions [9, 10].  

Telmisartan [TEL] is a chemical compound that is 2-(4[4-

methyl-6-(1-methyl-1H-1, 3-benzodiazol-2-yl)-2-propyl1H-1, 

3-benzodiazol-1-yl] methyl phenyl) benzoic acid [1113]. It is an 

angiotensin II receptor antagonist used in the management of 

hypertension. Generally, angiotensin II receptor blockers 

(ARBs) such as telmisartan bind to the angiotensin II type 1 

(AT1) receptors with high affinity, causing inhibition of the 

action of angiotensin II on vascular smooth muscle and 

ultimately leading to a reduction in arterial blood pressure [14–

17]. Recent studies suggest that telmisartan may also have 

PPAR-gamma agonistic properties that could potentially confer 

beneficial metabolic effects [17–18].  After a literature review, 

it was known that numerous methods had already been 

described for the estimation of Telmisartan, such as visible 

spectrophotometric methods, stability-indicating UV 

spectrophotometric methods, and RP-HPLC methods. For the 

determination of telmisartan in human plasma, advanced 

techniques such as LC-MS were also found [19-20].  

  
Figure 1 – Chemical structure of Azelnidipine 

T 



Suthar and Mashru                                       Advanced UV Spectrophotometric Method Development and Validation 

Vol 2 | Issue 1 | Jan – Mar 2023                                                                                     Indian J Pharm Drug Studies | 28  

 
Figure 2 – Chemical Structure of Telmisartan  

MATERIAL AND METHODS   

Apparatus and Software: A Shimadzu UV-1700 doublebeam 

spectrophotometer connected to a computer with Shimadzu 

UV-Probe 2.10 software installed was used for all the 

spectrophotometric measurements. The samples were weighed 

on an electronic balance (A120) by Shimadzu.  

Chemicals and Reagents: All the dilutions were made using 

Methanol as the diluent.  

Preparation of Standard Stock Solution: The standard stock 

solutions of both AZL and TEL are prepared by taking 10 mg 

of the standard drug in a 10 ml volumetric flask and making up 

the volume using methanol as the diluent to achieve a 

concentration of 1000 µg/ml.  

Preparation of Working Standard Solution: The working 

stock solution of AZL is prepared by taking 1 ml of the standard 

stock solution and transferring it to a 10 ml volumetric flask. 

The volume is made up to the mark using methanol to get a 

concentration of 100 µg/ml. Similarly, the standard working 

solution of TEL is prepared by taking 1 ml of the standard stock 

solution and transferring it to a 10 ml volumetric flask. The 

volume is made up to the mark using methanol to get a 

concentration of 100 µg/ml.  

Preparation of Series for Calibration Curves for  

Simultaneous Method, First Derivative Method, and QRatio 

Method: For preparing the solutions used in obtaining the 

calibration curve, a series of previously calibrated volumetric 

flasks were used. To prepare the linearity of Azelnidipine, 0.2 

ml, 0.4 ml, 0.6 ml, 0.8 ml, 1.0 ml, and 1.2 ml were withdrawn 

from the working standard solution of AZL and taken into 

separate volumetric flasks, and the volume was made up with 

methanol to prepare a series of solutions having concentrations 

in the range of 2 µg/ml to 12µg/ml. Similarly, to prepare the 

linearity of Telmisartan, 1 ml, 2 ml, 3 ml, 4 ml, and 5 ml were 

withdrawn from the working standard solution of TEL and 

taken into separate volumetric flasks, and the volume was made 

up with methanol to prepare a series of solutions having 

concentrations in the range of 10 µg/ml to 50 µg/ml.  

Preparation of Sample Solutions (Test Solutions): Various 

pharmaceutical dosage forms are available for this particular 

drug combination, including the azova-T40 tablet dosage 

form. The dosage of each of these forms would vary according 

to the conditions of the patient. For the estimation of the 

azova-t40 formulation, one just needs to empty the content of 

azova-t40 into a 100ml volumetric flask and make up the 

volume using methanol. Then 2.5 ml of this solution is taken 

into another volumetric flask, and the volume is made up using 

methanol again. This solution is then placed in the UV 

spectrophotometer for quantitative analysis against a methanol 

blank.   

Simultaneous Equation Method: This method uses the 

absorbance at two selected wavelengths, one at λ max of one 

drug where other drug also shows considerab le absorbance (λ2) 

and other being the wavelength at which the first drug has 

practically nil absorbance (λ1). Absorptivity of Azelnidipine 

and Telmisartan were calculated at both the wavelengths. The 

concentration of Azelnidipine and Telmisartan can be calculated 

from following equations:   

Cy = A1ax2-A2ax1/ax2ay1 –ax1 ay2 … (1)  

Cx = A2 ay1-A1ay2/ax2ay1- ax1ay2 … (2)  

Where, A1 and A2 are the absorbance of mixture at λ1 and λ2 

respectively, ay1 and ay2 are absorptivity of y at λ1 and λ2 

respectively, ax1 is absorptivity of X at λ2, Cx is concentration 

of X, Cy is concentration of Y.  

  
Figure 3 – Overlay Spectra of Azelnidipine and  

Telmisartan  

Calculation for Azelnidipine  

Cx = A2 ay1-A1ay2/ax2ay1- ax1ay2  

= 1.477*0.0282-1.466*0.356/0.084*0.282-0.366*0.356  

= 7.92PPM  

CALCULATION FOR TELMISARTAN  

Cy = A1ax2-A2ax1/ax2ay1 –ax1 ay2  

= 1.466*0.084-1.477*0.366/0.282*0.084-0.366*0.356  

= 39.54PPM  

Q-absorbance ratio method: This method, also called the 

"Absorption ratio method," is a modification of the 

simultaneous equation’s method. According to this method, the 

ratio of absorbance at any two wavelengths for a substance, 

which obeys Beer's law, is a constant value independent of the 

concentration and path length. This constant is termed 

"Hufner's Quotient" or Qvalue.  

 The method involves the measurement of absorbance at two 

wavelengths, one being the λ max of one of the components (λ 



Suthar and Mashru                                       Advanced UV Spectrophotometric Method Development and Validation 

Vol 2 | Issue 1 | Jan – Mar 2023                                                                                     Indian J Pharm Drug Studies | 29  

2) and the other being a wavelength of equal absorptivity of the 

two components (λ 1), called the iso- absorptive point.  

Cx = (Qm - Qy/Qx - Qy) *A/a1….(3)  

Cy=(Qm-Qy/Qy-Qx) *A/a2….(4)  

Where, Cx and Cy are the concentrations of x and y 

respectively, A is absorbance of sample at iso-absorpitive 

wavelength and a1 and a2 are the absorptivity of x and y 

respectively at iso-absorpitive wavelength.  

 
Figure 4 – Overlay Spectrum of Azelnidipine and  

Telmisartan   

First Derivative Zero Crossing Point Method: Derivative 

spectroscopy involves the conversion of a normal spectrum (a 

fundamental or zero-order spectrum) to its first, second, or 

higher derivative spectra by differentiating the absorbance of the 

sample with respect to the wavelength. The advantages of using 

derivative spectroscopy are that it leads to the separation of 

overlapped signals, the elimination of background caused by the 

presence of other compounds in a sample, and an improvement 

in the resolution of mixtures.  

Sensitivity and Specificity: If the measured height of 

derivative peak of analyte is performed at those wavelengths at 

which the spectra of other components are undergoing zeroing 

(cross through the zero line), the measured amplitude is 

proportional only to concentration of the analyte in 

consideration – ZERO CROSSING TECHNIQUE.   

 

Figure 5 – Zero Crossing Point of Azelnidipine  

 
Figure 6 – Zero Crossing Point of Telmisartan  

Validation Parameters  

Linearity: In three of the developed methods, three sets of 

calibration curves were plotted between the absorbance and 

concentration. The calibration curve that showed the best values 

is represented below in Figures 7–8. The linearity of the 

simultaneous equation method was determined by the zero-

order spectra of both drugs individually in the range of 200–400 

nm. Thus, at 255 nm (the λ max of AZL), whereas at 297 nm 

(the λ max of TEL), the linearity of the simultaneous equation 

method was found in the range of 2 µg/ml to 12 µg/ml for AZL 

(r2 = 0.9996) and 10 µg/ml to 50µ g/ml for TEL (r2 = 0.9989).  

 
Figure 7 – Calibration Curve of Azelnidipine  

 
Figure 8 – Calibration Curve of Telmisartan  

 
Figure 9 – Calibration Curve of Azelnidipine  

 

Figure 10 – Calibration Curve of Telmisartan  



Suthar and Mashru                                       Advanced UV Spectrophotometric Method Development and Validation 

Vol 2 | Issue 1 | Jan – Mar 2023                                                                                     Indian J Pharm Drug Studies | 30  

 

Figure 11 – Calibration Curve of Azelnidipine 

 
Figure 12 – Calibration Curve of Telmisartan  

The absorbance ratio method compares absorbances at two 

different wavelengths, one being an isoabsorptive point and the 

other being the λ max of one of the two components. From the 

overlay spectra of two drugs, it is evident that AZL and TEL 

show an isoabsorptive point at 274.4 nm. The second 

wavelength employed is 297 nm, which corresponds to the λ 

max of TEL. The linearity shown in Figure 9 & 10. The Q-

absorbance ratio method was found in the range of 10 µg/ml to 

50 µg/ml for TEL (r2 = 0.999) and 2 µg/ml to 12 µg/ml for AZL 

(r2 = 0.998).  

Then these zero-order spectra were converted into their 

respective first derivative spectra using the UV Probe software 

itself. A ∆ = 5 nm and a scaling factor of 10 were used to convert 

zero-order spectra to first derivative spectra. Thus, here at 234 

nm (the zero crossing point of TEL), the estimation of AZL is 

possible, whereas at 274 nm (the zero crossing point of AZL), 

the estimation of TEL is done as shown in Figures 11 and 12. 

The linearity of the First Order Derivative Method was found in 

the range of 10 µg/ml to 50 µg/ml for TEL (r2 = 0.9989) and 2 

µg/ml to 12 µg/ml for AZL (r2 = 0.9989).  

Limit of Detection and Limit of Quantification 

The Limit of Detection and Limit of Quantification was 

calculated using the series of calibration curves plotted. The 

LOD and LOQ values were determined using the following 

equations and the data is represented in below Table 1. 

Table 1 – Limit of Detection and Limit of Quantification for Azelnidipine and Telmisartan  

Parameter Drag Simultaneous Method Q-Absorbtion Method First Derivative 

LOD AZL 0.35 0.16 1.8 

TEL 0.73 0.77 1.71 

LOQ AZL 0.927 0.048 5.4 

TEL 2.16 2.34 5.16 

 

Precision  

The precision of an analytical method expresses the closeness 

of agreement between a series of measurements which are 

obtained by performing multiple samplings of the same 

homogenous sample under the given conditions of the method. 

In this section of the article the two developed methods have 

been analyzed for precision at two levels:  

1. Repeatability (precision under the same operating conditions 

over a short interval of time)  

2. Intermediate precision (variations in the results obtained at 

different intervals)  

From the results of precision, it may be concluded that both 

the methods developed are precise as the %RSD values are less 

than 2. It also may be concluded here that the Dual Wavelength 

Method is more precise than the 1st Derivative Method. In the 

data below represents the repeatability data, whereas Table- 2 

(A) and Table- 2 (B) present the data for intra-day and inter-day 

precision respectively. Precision data are represented in terms 

of %RSD and the nominal concentration of Azelnidipine (TEL) 

was kept 8µg/ml and 10µ (AZL) and Telmisartan g/ml 

respectively.  

Accuracy: Recovery studies for the UV-Spectrophotometric 

methods were conducted using the Standard Addition Method 

by taking a nominal concentration of 10µg/ml for Azelnidipine 

and 20µg/ml for Telmisartan from the formulation (test sample) 

and then spiking this solution by 80%, 100% and 120% of 

standard drug (API) (Table 3).  

Assay of Marketed Formulation; 20 tablets of formulation 

(Azova-T40) containing 8 mg of Azelnidipine and 40 mg of 

Telmisartan were weighed accurately. The average weight of 

tablets was found and tablets were powdered. The tablet powder 

equivalents to 40 mg of Telmisartan was weighed and 

transferred into 100ml volumetric flask and volume is made 

upto the mark using methanol to get 400 μg/ml solution.  

The content was filtered through the Whatman filter paper 

to get clear solution. From the clear sample stock solution 

dilutions 1 ml was withdrawn and taken into 10 ml volumetric 



Suthar and Mashru                                       Advanced UV Spectrophotometric Method Development and Validation 

Vol 2 | Issue 1 | Jan – Mar 2023                                                                                     Indian J Pharm Drug Studies | 31  

flask and volume is made upto the mark using methanol to 

obtain 40 μg/ml of Telmisartan and 8μg/ml of Azelnidipine. 

The resulting solutions were analyzed for drug content by 

spectrophotometric method at 255 nm and 297 nm for AZL and 

TEL, respectively. Assay was repeated 6 times and standard 

deviation was calculated. The drug content in AZL and TEL 

was found by three methods and results are mention in below 

tables (Table 4).  

RESULTS  

The two methods discussed above may be compared with each 

other by comparing their Limits of Detections and Limits of 

Quantifications as shown in the Table 5. ANOVA and the t-

test were used to obtain the assay results from the simultaneous 

equation method and the QAbsorbance Ratio method, and the 

results are shown in Table 6.   

Table 2 – Precision (A) Repeatability (B) Interday Precision  

Repeatability 

Azelnidipine   Telmisartan   

Concentration Parameter Simultaneous 

method 

Q-Ratio First 

derivative 

Concentration Parameter Simultaneous 

method 

Q- 

Ratio 

First 

derivative 

8 µg/ml Mean 0.0285 0.2803 0.0135 10 µg/ml Mean 0.0155 0.1816 0.0155 

SD 0.00164 0.00136 0.0016 SD 0.00055 0.0013 0.0005 

%RDS 0.001 0.4872 0.0010 %RSD 0.001 0.7519 0.001 

Table 3 – Accuracy of Azelnidipine and Telmisartan  

Simultaneous Q-ratio First derivative 

Interday – Azelnidipine 

Conc. µg/ml Mean SD (n=3) %RSD Mean SD (n=3) %RSD Mean SD (n=3) %RSD 

6 0.217333 0.00152 0.702 0.142 0.0017 1.219 0.0106 0.0011 0.0010 

8 0.284667 0.00115 0.405 0.181 0.0015 0.840 0.049 0.0615 0.010 

10 0.344 0.002 0.581 0.22 0.001 0.454 0.016 0.0017 0.001 

Interday –Tel misartan 

Conc. µg/ml Mean  SD (n=3) %RSD Mean SD (n=3) %RSD Mean SD (n=3) %RSD 

10 0.354  0.0015 0.283 0.280 0.0015 0.544 0.016 0 0 

20 0.714  0.0020 0.280 0.537 0.0025 0.468 0.0303 0.0005 1.903 

30 1.075  0.002 0.186 0.802 0.0011 0.143 0.044 0.0005 1.29 

Table 4 – Assay Results  

 Drug   Concentration taken (μg/ml)   Concentration of μg/ml   %Recovery  %RSD 

Assay Result-Simultaneous method 

 AZL   8   7.93   99.12   0.0144 

 TEL   40   39.93   99.98   0.0724 

Assay Result –Q Absorption ratio method 

 AZL  8   7.98  99.75   0.0147  

 TEL   40   39.24  98.1   0.0723  

Assa y Result –First derivative method 

 AZL  8  7.8   97.5   0.147  

 TEL   40  39.65   99.12   0.0145  

Table 5 – Comparison of Methods  

 Para-meter   Drug   Simultaneous method   Q-absorption ratio method   First derivative method  

 LOD   AZL   0.35   0.16   1.8  

 TEL   0.73   0.77   1.71  

 LOQ  

 

 AZL   0.927   0.048   5.4  

 TEL   2.16   2.34   5.16  

Table 6 – Two way ANOVA  

ANOVA 

Source of Variation SS DF MS F P-Value F Crit 

Sample 0.59535 3 0.19845 0.710972 0.55952 3.238872 



Suthar and Mashru                                       Advanced UV Spectrophotometric Method Development and Validation 

Vol 2 | Issue 1 | Jan – Mar 2023                                                                                     Indian J Pharm Drug Studies | 32  

Columns 0.464816667 1 0.464816669 1.665263 0.215232 4.493998 

Interaction 1.100816667 3 0.366938889 1.314604 0.304208 3.238872 

Within 4.466 16 0.279125    

Total 6.626983333 23     
  
CONCLUSION   

The simple, rapid, accurate, and precise simultaneous methods, 

Q absorbance ratio and first derivative methods of UV 

spectroscopy have been developed and validated for the routine 

analysis of AZL and TEL in API and pharmaceutical dosage 

form. This method was validated as per the ICH guidelines. The 

values of the standard deviation and coefficient of variation 

calculated were satisfactory, which indicates the suitability of 

the proposed methods for routine estimation of AZL and TEL. 

According to LOD, LOQ, and assay results, the simultaneous 

method is more accurate and precise than the Q-ratio method 

and the first derivative method. There was a significant 

difference between the three methods for AZL and TEL.  

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How to cite this article: Pallavi Suthar, Rajashree Mashru. 

Advanced UV spectrophotometric method development 

and validation for simultaneous estimation of Azelnidipine 

and Telmisartan in Pharmaceutical Dosage Form. Indian J 

Pharm Drug Studies. 2023; 2(1) 27-32. 

Funding: None                  Conflict of Interest: None Stated 

 


