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Vol 1 | Issue 3 | Oct - Apr 2022                                                                                    Indian J Pharm Drug Studies | 102  

Original Article 

RP-HPLC Method Development and Validation for Simultaneous Estimation 

of Benidipine Hydrochloride and Chlorthalidone in Pharmaceutical Dosage 

Form  

Hasan I Shaikhmulani, Ashpak M Tamboli, Naziya A Tamboli,   Rohan T Kshirsagar, Harshvardhan T 

Suryawanshi, Omkar Khandare  

From, Department of Pharmaceutical Chemistry, Sahyadri College of Pharmacy, Sangola, Solapur, Maharashtra, India  

Correspondence to: Hasan I Shaikhmulani, Department of Pharmaceutical Chemistry, Sahyadri College of Pharmacy, Methwade, 

Sangola, Solapur, Maharashtra, India - 413307 Email: shaikhmulani9881@gmail.com   

ABSTRACT 

A simple, precise, rapid and accurate reverse phase High Performance Liquid Chromatography (HPLC) method was developed for the 

estimation of Benidipine Hydrochloride and Chlorthalidone in bulk and tablet dosage form. C18 C-18 Agilent Zorbax Bonus – RP (250 

x 4.6 mm, Particle size -5 Micron) was used in this method. The mobile phase comprises of Methanol and 0.1% Orthophosphoric acid 

(45:55v/v) with flow rate 1 mL/min (Photodiode array Detector). The retention time for Benidipine Hydrochloride was 1.09 min and 

for Chlorthalidone was 3.52 min. The detection concentration was linear over 32-48 µg/mL for Benidipine Hydrochloride and 100-150 

µg/mL for Chlorthalidone. The regression equation of Benidipine Hydrochloride and Chlorthalidone were found to be y = 50184x-

30070 and y = 37102x-74642 respectively with regression coefficient of Benidipine Hydrochloride and Chlorthalidone were 0.999 and 

0.999. So, the present work is aimed for development of simple, reproducible chromatographic RP-HPLC method for simultaneous 

estimation of Benidipine Hydrochloride and Chlorthalidone. The developed method was successfully validated in accordance to ICH 

guideline. Hence the method can be conveniently adopted for routine analysis in quality control laboratories.  

Key words: Benidipine Hydrochloride, Chlorthalidone, RP-HPLC, Method Development, Validation 

enidipine  Hydrochloride  is  a  5-O-[(3R)-

1benzylpiperidin-3-yl]  3-O-methyl  (4R)-2, 

6dimethyl-4-(3-nitrophenyl)-1, 4-dihydropyridine3, 5-

dicarboxylate;  hydrochloride  [1-2]. Benidipine 

Hydrochloride is a long acting anti-hypertensive drug class of 

Ca2+channel blocker (Dihydropyridine Derivative) its longer 

duration of action to slow dissociation from the DHP receptor 

on smooth muscle cell [3].It is a Triple L, T and N channel 

blocker for treatment of Hypertension and Angina Pectoris 

mostly used in Japan and India [1-3]. Chlorthalidone is a 2-

chloro-5-(1-hydroxy-3-oxo-2Hisoindol-1-yl) 

benzenesulfonamide [4]. It is a long-acting thiazide, diuretic 

drug used in treatment of hypertension and certain kidney 

disorder [5].   

 
Fig. 1- Structure of Benidipine Hydrochloride  

Molecular formula: - C28H32ClN3O6  

Molecular Weight: - 542 g/mol   

Chlorthalidone has longest duration of action but similar 

diuretic effect at maximum therapeutic doses. It reduces 

reabsorption of sodium and chloride primarily through 

inhibition of the Na+ / Cl- symporter in the apical membrane of 

distal convoluted tubule cells in kidney [6-7]. Chlorthalidone 

was first introduced in Switzerland in1959 it is also available as 

generic medicine [4].  

 

Fig. 2 – Structure of Chlorthalidone 

Molecular Formula: - C14H11ClN2O4S  

Molecular Weight: - 338.8g/mol   

B 

  



Shaikhmulani et al.                                             RP-HPLC method development and validation of BEN.HCL & CTD 

Vol 1 | Issue 3 | Oct - Apr 2022                                                                                    Indian J Pharm Drug Studies | 103  

MATERIALS AND METHODS  

Chemicals used: A pure drug in powder form Benidipine 

Hydrochloride and Chlorthalidone received from Alkumentis 

Pharmaceuticals, Mumbai and Ipaca Pharmaceutical, Pvt. Ltd 

Mumbai, Methanol (AR grade) and Orthophosphoric acid 

utilized as diluent and received from Fine Chem laboratories, 

Mumbai and Loba Chemie Pvt. Ltd. and a commercial product 

manufactured by Alkumenties Pharmaceuticals “Benitowa-CH” 

which contain both the Benidipine Hydrochloride and 

Chlorthalidone received from market.  

Instrument used: Agilent 1260 Infinity Quaternary HPLC 

device equipped with Photodiode array detector and the output 

signal was checked and processed using openLab EZChrome 

software.  

Chromatographic conditions were as follows:  

Oven Temperature: - 300 C,  

Flow Rate was:-1mL/min, 

Run Time was: - 10 min,  

Injection Volume: - 10µL,  

Wavelength: -238 nm,  

Column: - C-18Agilent Zorbax Bonus –RP (250 x 4.6 mm, 

Particle size -5 Micron) and diluent: - Methanol: 0.1% 

Orthophosphoric acid (50:50).  

Preparation of Mobile Phase: The mobile phase was prepared 

by mixing Methanol and 0.1% orthophosphoric acid in the ratio 

of 55:45 (v/v). The resultant solution filtered through Micron 

filter 0.45µm pore size by means of vacuum pump and then 

degassed by using Ultasonicator to remove gases in solvent.  

Preparation of Standard solution: Standard solution of both 

the drug were prepared by weighing 5mg and 12.5mg of  

Benidipine Hydrochloride & Chlorthalidone and transfer in to 

10mL of volumetric flask and make volume up to 10mL using 

mobile phase and prepared solutions concentration were 

500µg/mL and 1250 µg/mL respectively.  

Preparation of Working Standard solution: The Working 

Standard solution of both the drug were prepared by weighing 

5mg and 12.5mg of Benidipine Hydrochloride & 

Chlorthalidone and transfer in to 10mL of volumetric flask and 

make volume up to 10mL using mobile phase then pipette out 

0.8mL and 1.0mL respectively and add into another 10mL 

Volumetric flask and make up volume up to 10mL using mobile 

phase. The resultant solution was 40µg/mL and 125µg/mL, 

respectively.  

Selection of Wavelength: The sample was scanned from 200-

400 nm Photodiode array detectors. The wavelength selected for 

analysis chosen was 238 nm on basis of appropriate intensity of 

both the drug.  

Analysis of Marketed Formulation  

Take 5 tablets each tablet contains 8 mg of Benidipine 

Hydrochloride and 12.5 mg of Chlorthalidone and weighed and 

powdered from that transfer 5mg of Benidipine Hydrochloride 

and 12.5 mg of Chlorthalidone in 10 mL of volumetric flask and 

make up volume up to 10 mL with help of diluent and further 

dilution would make final concentration of solution were 

40µg/mL and 125 µg/mL respectively. Then solution was 

filtered by 0.45 µm nylon membrane filter by using vacuum 

filter. Tablet formulation analysis was carried out as mentioned 

under section tablet formulation analysis. Procedure was 

repeated for 5 times. Sample solution was injected and area was 

recorded for each drug concentration and percentage purity was 

determined as shown in Table 1.  

Analysis is the most important aspect of any drug 

development; a suitable method must be developed so as to 

ensure that any drug in dosage from. With help of method 

development ensure that the amount of particular drug can easily 

determine. The validation parameter confirms that the 

developed method is precise, accurate and reproducible and can 

be used for used for routine evaluation of Benidipine 

Hydrochloride and Chlorthalidone in combined dosage form [8-

12]. In the present study suitable for RP-HPLC method was 

developed with the aim of making detection of Benidipine 

Hydrochloride and Chlorthalidone more accurate and precise 

with addition of validation parameters like Specificity, 

Linearity, Accuracy, and LOD & LOQ [8]. 

Table 1 - Assay result with Benidipine Hydrochloride & Chlorthalidone 

S.no  Benidipine Hydrochloride  Chlorthalidone  

Peak area Amt. recovered in 

µg/mL 

% Recovery Peak area Amt. recovered in 

µg/mL 

% Recovery 

1  1612593 32 98.54 3800032 100 100.41 

2  1612895 31.97 98.56 3765482 99.81 99.67 

3  1623583 31.82 99.22 3768221 99.98 99.57 

4  1624896 32.03 99.31 3798523 100 97.67 

5  1628369 32 99.52 3799213 100 100.39 

Mean  1620467 31.96 99.03 3766294 99.95 99.54 

% RSD  0.44 0.23 0.45 1.09 0.08 1.12 
                    



Shaikhmulani et al                                               RP-HPLC method development and validation of BEN.HCL & CTD 

Vol 1 | Issue 3 | Oct – Dec 2022                                                                             Indian J Pharm Drug Studies | 104  

Table 2 - Concentration and Area of Benidipine 

Hydrochloride  

% Level Conc (µg/ml) Area 

80 32 1584314 

90 36 1762564 

100 40 1979466 

110 44 2181734 

120 48 2378413 

Table 3 - Concentration and Area of Chlorthalidone  

Chlorthalidone 

% Level Conc (µg/ml) Area 

80 100 3800057 

90 112.5 4214430 

100 125 4721514 

110 137.5 5199512 

120 150 5626385 
 

Table 4- Linearity values of Benidipine Hydrochloride and 

Chlorthalidone  

Parameter  Benidipine 

Hydrochloride  

Chlorthalidone  

  

Range 32-48 µg/mL 100-150µg/mL 

Slope 50184 37102 

Intercept 30070 74642 

Correlation 

Coefficient 

0.999 0.999 

Method Validation  

1. Linearity: To get desired analyte concentrations the standard 

solution was created by adding 5 mg of Benidipine 

Hydrochloride and 12.5mg of Chlorthalidone in 10 mL of 

volumetric flask and make up volume up to 10 mL using diluent 

then further dilution was made to get 32-48µg/mL of 

Benidipine Hydrochloride and 100-150µg/mL respectively. 

The correlation coefficient for calibration curve Benidipine 

Hydrochloride and Chlorthalidone was found to be 0.999 and 

0.999 respectively (Table 4).  

 
Fig. 3 - Chromatogram of Standard mixture of Benidipine 

Hydrochloride & Chlorthalidone 

2. Precision: Precision of an analytical procedure express the 

closeness of agreement between a series of measurement 

obtained from multiple sampling of same homologous sample 

under précised condition. Intraday precision for Benidipine 

hydrochloride and Chlorthalidone are shown in Table no. 

(5&6). The %RSD for Benidipine Hydrochloride and 

Chlorthalidone was found to be 0.161and 0.390, respectively. 

Interday precision for Benidipine hydrochloride and 

Chlorthalidone are shown in Table no. (7&8). The %RSD or 

Benidipine Hydrochloride and Chlorthalidone was found to be 

0.27and 0.41, respectively. 

 

Fig. 4 - Linearity graph of Benidipine Hydrochloride 

3. LOD & LOQ:  Limit of Detection (LOD) means the 

detection of an individual analytical procedure express the 

closeness of agreement between a series of measurement 

obtained from multiple sampling of same homologous sample 

under précised condition and Limit of Quantitation (LOQ) 

means the quantitation of an analytical procedure is the lowest 

amount of an analyte in a sample which can be quantitatively 

determine with suitable precision and accuracy. It can be 

calculated using formula 3.3 σ/S and 10 σ/S, respectively. 

Where σ is SD of y- intercepts of regression line and S is slope 

of calibration curve (Table 13).  

 

4. Robustness: This was done by minute changes in the 

Chromatographic condition and found to be unchanged by 

minute changes like ±2% changes in volume of organic solution 

of mobile phase.  

Fig. 5 - Linearity graph of Chlorthalidone 

5. Accuracy: Accuracy is the closeness of agreement between 

the values found. The value accepted as the conventional true 

value or the accepted reference value. The accuracy of the 

  
      

y = 37102x + 74642  
R² = 0.999  

3400000  
3900000  
4400000  
4900000  
5400000  
5900000  

80 130 180  
Concentration (µ 

g/ml) 

Linearity of Chlorthalidone 

  
    

y = 50184x  - 30070 
R² = 0.9993 

1200000  

1400000 

1600000  

1800000 

2000000 

2200000 

2400000 

2600000 

28 38 48 58  

Concentration (ug/ml)  

Linearity of Benedipine 



Shaikhmulani et al                                               RP-HPLC method development and validation of BEN.HCL & CTD 

Vol 1 | Issue 3 | Oct – Dec 2022                                                                             Indian J Pharm Drug Studies | 105  

method was confirmed by recovery study from the marketed 

formulation at three level of standard addition (Table 9 & 10).  

6. System suitability parameters:   

7. Specificity:  Specificity is ability to assess the analyte for the 

presence of various components that may be present. 

Specificity of the RP_HPLC method was determined by entire 

separation of Benidipine Hydrochloride and Chlorthalidone 

with parameter like Retention Time (RT), resolution (Rs) and 

Tailing factor (T). The peak obtained for Benidipine 

Hydrochloride and Chlorthalidone were sharp and have a clear 

baseline separation. The results are shown in Table 12.  

 Table 5 - Intraday Precision study of Benidipine Hydrochloride 

Concentration in µg/mL  Area  % Recovery  Mean % Recovery ± SD  Mean % Recovery ±RSD  

  1612694  98.55      

32 µg/mL  1613005  98.57  98.59±0.064    

  1614658  98.67      

  1825799  99.39    99.21±0.161  

36 µg/mL  1836154  99.97  99.79±0.34    

  1837001  100.01      

  2023149  99.28      

40 µg/mL  2024153  99.33  99.25±0.08    

  2020746  99.16      

 

Table 6 - Intraday Precision study of Chlorthalidone 

Concentration in µg/mL  Area  % Recovery  Mean % Recovery ± SD  Mean % Recovery ±RSD  

  3794817  100.26    

99.53±0.66  

  

  

100 µg/mL  3745628  98.94    

  3767898  99.41    

  4256897  100.19    

99.75±0.49  

  

  

122.5 µg/mL  4215783  99.21  99.75±0.39  

  4243162  99.86    

  4712539  100    

99.98±0.02  

  

  

125 µg/mL  4710986  99.96    

  4711656  99.98    

Table 7 - Interday Precision study of Benidipine Hydrochloride 

Concentration in µg/mL  Area  % Recovery  Mean % Recovery ± SD  Mean % Recovery ±RSD  

32 µg/mL  

1611936  98.50  

98.45±0.04  

97.93±0.270  

1611127  98.45  

1610456  98.41  

36 µg/mL  

1796321  97.76  

97.48±0.44  1795854  97.73  

1789500  96.97  

40 µg/mL  

1999120  98.04  

97.88±0.33  1887664  97.49  

1998664  98.06  

Table 8 - Interday Precision study of Chlorthalidone  

Concentration in µg/mL  Area  % Recovery  Mean % Recovery ± SD  Mean % Recovery ±RSD  

  3689541  97.48    

97.79±0.354  

  

  

100 µg/mL  3698627  97.73    

  3715496  98.18    

  4158946  97.89    

98.62±0.637  

  

  

122.5 µg/mL  42011993  98.93  98.67±0.421  

  4207234  99.05    

  4699137  99.75    

99.62±0.273  

  

  

125 µg/mL  

  

4678789  99.31    

  4701856  99.81  



Shaikhmulani et al                                               RP-HPLC method development and validation of BEN.HCL & CTD 

Vol 1 | Issue 3 | Oct – Dec 2022                                                                             Indian J Pharm Drug Studies | 106  

Table 9 - Recovery study of Benidipine Hydrochloride 

Std Area  1968846      

%Level Reps Spiked Conc. (µg/ml) Area Amount  Recovered µg/ml)  %Recovery AVG STDEV  RSD  

80%  

Rep 1  32  1584314 32.19  100.59  

99.86  0.702637  0.70  Rep 2  32  1572215 31.94  99.82  

Rep 3  32  1562213 31.74  99.18  

100%  

Rep 1  40  1979466 40.22  100.54  

99.72  0.726323  0.73  Rep 2  40  1957783 39.78  99.44  

Rep 3  40  1952473 39.67  99.17  

120%  

Rep 1  48  2378413 48.32  100.67  

99.62  1.149973  1.15  Rep 2  48  2324579 47.23  98.39  

Rep 3  48  2357896 47.90  99.80  

Table 10 - Recovery study of Chlorthalidone  

Table 11 - System suitability parameters 

 

RESULTS AND DISCUSSION 

In this method development 100 mL of mobile phase was 

prepared by mixing 550 Ml of 1% orthophosphoric acid and 450 

mL of Methanol. The mobile phase filtered through 0.45µm 

filters to avoid the column clogging due to smaller particle size. 

The flow rate was found to be 1 mL/min resulting in short 

retention time, good baseline stability with low noise level. In 

this present developed RP-HPLC method, the standard and 

sample preparation required less me. By the use of proposed 

method the retention time of Benidipine Hydrochloride and 

Chlorthalidone was found to be 1.09 and 3.52 minutes respectively.    

Table 12 - Specificity Parameter  

Parameter  Benidipine 

Hydrochloride  

Chlorthalidone  

Tailing Factor  1.09  1.05  

Resolution  0.00  9.62  

Retention 

Time  

1.09  3.52  

 The assay of Benidipine Hydrochloride and Chlorthalidone in 

bulk drug and combined dosage form was found to be 100.99% 

and 100.15% respectively. A good linear relationship, r2 = 0.999 

for Benidipine Hydrochloride and r2 = 0.999 for chlorthalidone 

was observed between the concentration range of 32-48µg/mL 

and 100-150µg/mL, respectively. The LOD and LOQ values for 

Benidipine Hydrochloride was found to be 2.05µg/mL and 6.22 

µg/mL and for Chlorthalidone 7.71 µg/mL and 23.38 µg/mL, 

respectively.  

The low values of standard deviation of retention time and peak 

area indicates high precision of method from recovery study 

data it has found to be mean % recovery was within a limit, 

indicates high accuracy of proposed method. There are no 

additional peaks are observed in the chromatogram which 

indicates no interference of common ingredients used in 

formulation. No marked changes were observed in % assay of 

the optimized condition with that of alternate condition in the 

robustness study indicating that the method is robust.  

Std Area   4735952      

% Level Reps  Spiked Conc. µg/ml)   Area  Amount  Recovered (µg/ml) % Recovery AVG  STDEV  RSD  

80%  

Rep 1  100  3800057  100.30  100.30  

100.58  0.282482  0.28  Rep 2  100  3821454  100.86  100.86  

Rep 3  100  3810244  100.57  100.57  

100%  

Rep 1  125  4721514  124.62  99.70  

100.06  0.444878  0.44  Rep 2  125  4732156  124.90  99.92  

Rep 3  125  4762145  125.69  100.55  

120%  

Rep 1  150  5626385  148.50  99.00  

98.96  0.047238  0.05  Rep 2  150  5621020  148.36  98.91  

Rep 3  150  5623887  148.44  98.96  

Sr.no  Parameters  Benidipine Hydrochloride  Chlorthalidone  

1  Retention time (RT)  1.09  3.52  

2  Area  1979466  4731515  

3  Theoretical plates  5344  8346  

4  Resolution  0.0  9.62  



Shaikhmulani et al                                               RP-HPLC method development and validation of BEN.HCL & CTD 

Vol 1 | Issue 3 | Oct – Dec 2022                                                                             Indian J Pharm Drug Studies | 107  

Table 13 - LOD & LOQ Parameter  

Name of Drug  LOD (µg/mL)  LOQ (µg/mL)  

Benidipine 

Hydrochloride  

2.05  6.22  

Chlorthalidone  7.71  23.38  

 CONCLUSION  

A simple competent method was developed for the simultaneous 

equation of Benidipine Hydrochloride and Chlorthalidone 

within a short analysis time with no interference of common 

additives present in tablet formulation. 

 

 

The proposed method can be applied for routine quality control 

and applied for bulk and in tablet dosage form.  

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Jaypee brother’s medical publishers, New Delhi p.n-549-551.  

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How to cite this article: Shaikhmulani HI, Tamboli AM, 

Tambili NA, Kshirsagar RT, Suryawanshi HT, Khandare 

O.  RP-HPLC Method Development and Validation for 

Simultaneous Estimation of Benidipine Hydrochloride and 

Chlorthalidone in Pharmaceutical Dosage Form. Indian J 

Pharm Drug Studies. 2022: 1(3) 102-107. 

Funding: None                  Conflict of Interest: None Stated 

  

  
  Fig. 6 - Chromatogram of 80% Accuracy   

Fig.  7   -   Chromatogram of 100% Accuracy.   

  
Fig .   8 - Chromatogram of 120% Accuracy.   

https://engo.drugbank.com/drug/DB09231
https://en.m.wikipedia.org/wiki/chlorthalidone
http://dx.doi.org/10.20959/wjpr201710-9417
http://dx.doi.org/10.20959/wjpr201710-9417

