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 | 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. REFERENCES 1. Wikipedia contributors. Benidipine [Internet]. Wikipedia, The Free Encyclopedia. Available from: https://en.m.wikipedia.org/wiki/Benidipine 2. Drugbank.com. [cited 2022 Jul 28]. Available from: https://engo.drugbank.com/drug/DB09231 3. Tripathi K.D, Essential of Medical Pharmacology 7th edition Jaypee brother’s medical publishers, New Delhi p.n-549-551. 4. Wikipedia contributors. Chlortalidone [Internet]. Wikipedia, The Free Encyclopedia. Available from: https://en.m.wikipedia.org/wiki/chlorthalidone 5. Tripathi K.D, Essential of Medical Pharmacology 7th edition Jaypee brothers medical publishers, New Delhi p.n- .559-560 6. Chaudhary BR. Development and validation of stability indicating gradient RP-hplc method for simultaneous estimation of telmisartan and chlorthalidone in bulk api and fixed dose combination. World J Pharm Res [Internet]. 2017; 1015 - 29. 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ICH Q2B Text on validation of analytical procedure, International Conference on Harmonisation. Tripartite guideline. 1994; 1 - 10. 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