RP-HPLC Method for Simultaneous Estimation of Cilnidipine and Chlorthalidone

 

Vijaykumar T. Pawar¹, Shubhangi V. Pawar², Harinath N. More¹, Anita S. Kulkarni³,

Dinanath T. Gaikwad⁴

¹Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy, Near Chitranagari, Kolhapur-416013, M. S., India

²Department of Pharmaceutical Chemistry, Shri. Santkrupa College of Pharmacy, Ghogaon

³Department of Pharmaceutical Chemistry, Government College of Pharmacy, Karad

⁴Department of Pharmaceutics, Bharati Vidyapeeth College of Pharmacy, Near Chitranagari, Kolhapur-416013, M. S., India

 

ABSTRACT:

Aim of present study was development of RP-HPLC method for simultaneous estimation of cilnidipine and chlorthalidone. RP-HPLC method for simultaneous estimation of Cilnidipine and Chlorthalidone was carried out by using methanol: water (80:20 v/v)   at a flow rate of 1 ml min-1 with UV detection at 231.6 nm. Both drugs showed linearity in the concentration range of 10-70 µg ml-1.for CIL and 10-70 µg ml-1.for CHLT. When marketed pharmaceutical preparations were analyzed, the result obtained by the proposed method was found in good agreement with labeled claims. Recoveries in all these method were in the range of 99.60-100.18%. Results obtained were statistically validated and were found to be reproducible. These methods can be applied for routine analysis of formulations containing these drugs. For analytical methods validation parameters such as linearity, precision, accuracy, specificity, limit of detection, limit of quantization and robustness were determined according to ICH Q2B guidelines.

 

 

 

1. INTRODUCTION:

Clinidipine is 2-methoxyethyl (E)-3-phenyl-2-propen-1-yl-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)- pyrimidine-3,5-dicarboxylate¹ which is calcium channel blocker. It is soluble in chloroform, acetone and slightly    soluble in methanol, ethanol and water. The drug is a kind of dihydropyridines calcium antagonist. It can be combined with the Fluzifop sites in L-type calcium channel on vascular smooth muscles cellular membrane, and inhibit the across membrane inflow of Ca2+ through L-type calcium channel, accordingly relax and expand vascular smooth muscles to reduce blood pressure.

The drug can also inhibit the arterenol releasing of sympathetic nerve activity by restraining the across membrane inflow of Ca2+ through N type calcium channel on cellular membrane of Sympathetoblast.² Chlorthalidone is 2-chloro-5-(1-hydroxy-3oxo-2,3-dihydro-1H-isoindol-1yl)benzene-1-sulphonamide which is diuretic. It is practically insoluble in water, ether and in chloroform. It is soluble in methanol, slightly soluble in ethanol. Chlorthalidone inhibits sodium ion transport across the renal tubular  epithelium in the cortical diluting segment of the ascending limb of the loop of henle. By increasing the delivery of sodium to the distal renal tubule, Chlorthalidone indirectly increases potassium excretion via the sodium –potassium exchange mechanism.^3-8

 

Figure 1 Structure of a) Clinidipine  b) Chlorthalidone

 

Cilnidipine (CIL) and Chlorthalidone (CHLT) are recently introduced in the market as combined tablet dosage form which is widely used in the treatment of Hypertension. Combination of other drugs like Cilnidipine with Olmesartan Medoxomil (Nexovas-O) is also available in market. Literature survey has revealed that there are methods reported for analysis and estimation of CIL and CHLT individually, but very few methods are reported for RP-HPLC estimation of CIL and CHLT from tablet formulations.^{9, 10} Hence it becomes necessary to develop simple, cost effective, accurate, sensitive and selective method for quantification of CIL and CHLT.

 

2. MATERIALS AND METHODS:

2.1 .MATERIALS:

2.1.1.   Reagents and Chemicals:

All chemicals and reagents used were of HPLC grade (Methanol, Distilled Water) purchased from LOBA Chemie Pvt. Ltd. Mumbai India. Pure CIL was kindly gifted from Macleoids Phrmaceuticals Ltd. Mumbai, India and CHLT gifted from IPCA Phrmaceuticals Ltd. Mumbai, India.

 

2.1.2. Instrumentation:

HPLC System (Jasco 2080 Compony) consisting of injection having capacity of 20 ul. The detector consisting of UV-Visible spectrophotometer. The software used was BORWIN, Column Gracesmart C18, 4.6 x 250mm and 5um Particle size was used for separation. AND digital balance was used for weighing, and all the solvents used in preparartion for mobile phase were degassed by using Ultrasonicator of pharma Instruments.

 

2.2.  METHODS:

2.2.1.Determination of wavelength for detection of CIL  and CHLT:

The suitable wavelength for detection of CIL and CHLT was selected from overlain spectrum of CIL and CHLT, Wavelength selected was 231.6 nm

 

2.2.2.Optimization of Detection Wavelength:

The sensitivity of HPLC method that uses UV detection depends upon proper selection of detection wavelength. An ideal wavelength is one that gives good response for the drugs that are to be detected. For good response, optimization of wavelength was done at different wavelengths by UV detector. In the present study, drug solutions of 10 μg ml -1 of CIL and 10 μg ml -1 of CHLT were prepared in Methanol. After observing UV spectra of both the drugs, wavelength of 231.6 nm was selected for further study.

 

2.2.3.Selection of Mobile Phase:

Based on sample solubility, stability and suitability various mobile phases and compositions were tried to get a good resolution and sharp peak. The standard solution containing mixture of CIL and CHLT as well as individual drugs were run in different mobile phases.

The following mobile phases were tried:

1.       ACN: Methanol [70:30 % v/v].

2.       ACN: Methanol [50:50 % v/v].

3.       Water: Methanol [50:50 % v/v].

4.       Water: Methanol [60:40 % v/v].

5.       Water: Methanol [20:80 % v/v]

 

Each mobile phase was filtered through 0.22µ membrane filter and degassed by sonication for 20 minutes. From the various mobile phases tried, mobile phase containing Methanol and Water in 80:20 % v/v proportion was selected, since it gave sharp completely resolved peaks with symmetry within limits and significant retention times for both the drugs.

 

2.2.4.Optimization of Mobile Phase Strength:

For selection of mobile phase, various mobile phase compositions containing phosphate buffer and Methanol in different ratios was tried but the resolution was not found to be satisfactory. Finally, mobile phase containing Methanol and water in 80:20 proportions was found to give best resolution for both the drugs.

 

2.2.5.Selection of Chromatographic Conditions:

The selection of HPLC method depends upon the nature of the sample, its molecular weight and solubility. RP-HPLC method was selected for the initial separations because of its simplicity and suitability. The chromatographic variables such as mobile phase, flow rate and solvent ratio were studied. The resulting chromatograms were recorded and the chromatographic parameters such as selectivity, asymmetric factor, and peak resolution were calculated. The condition that gave the best resolution, symmetry and selectivity was selected for estimation.

 

2.2.6. Separation Conditions:

Separation conditions used were as shown in Table 1.

 

Table 1: Separation conditions

Chromatographic Mode	Chromatographic Conditions
Standard Solution	100 μg ml -1 of Cilnidipine and 100 μg ml -1 of Chlorthalidone.
Stationary Phase	HIQ SII C18 column-10 (4.5 mm x 250 mm).
Mobile phase	Methanol : Water (80:20).
Detection Wavelength	231.6 nm.
Flow Rate	1 ml min-1.
Sample Size	20 μl.

 

2.2.7.   Optimization of Chromatographic Parameters:

Optimization in HPLC is the process of finding a set of conditions that adequately analyze the quantification of the analyte with acceptable accuracy, precision, sensitivity, specificity, cost, ease, and speed of analysis.

 

2.2.8. Method Validation:^11-13

Standard stock solution containing CIL and CHLT was prepared by dissolving 10 mg and 10 mg of CIL and CHLT respectively in 100 ml of Methanol. It was then sonicated for 20 minutes and then final volume of the solution was made up to 100 ml with methanol to get stock solution containing 100 μg ml -1 CIL  and 100 μg ml -1 CHLT in 100 ml volumetric flask.

 

2.2.8.1      Linearity Study of Drug at Selected Wavelength:

In to a series of 10 ml volumetric flasks, 1 to 7 ml of CIL solution (100 μg ml -1) and CHLT solution (100 μg ml -1) respectively were pipetted and to each flask 2 ml of (100 μg/ml) HCTZ was added and then final volume of the solutions was made up to 10 ml with methanol. A 20 μl of sample solution was injected into the injection port of chromatographic system having fixed volume loop injector. Chromatograms were noted and response area was plotted against concentration to get calibration curve. The data for calibration curve is given in Figure 3 for CIL and CHLT, while chromatogram of physical mixture is shown in Figure 4.

 

2.2.8.2      Analysis of Tablet Formulation:^14-18

From the triturate of 20 tablets, an amount equivalent to 10 mg of CIL and 12.5 mg of CHLT was weighed and dissolved in 50 ml of Methanol  by sonicating for 20 minutes. The solution was filtered through 0.22 μ membrane filter and then final volume of the solution was made up to 100 ml with methanol toget stock solution containing 100 μg ml -1 of CIL and 125 μg ml -1 of CHLT in 100 ml volumetric flask. After appropriate dilutions, the solutions were run on HPLC system and the concentration of each analyte was determined with the equations generated. The statistical data obtained after replicate determinations (n = 6) is shown in Table 2 and chromatogram is shown in Figure 5.

 

2.2.8.3      Accuracy (Recovery Studies):

Accuracy of analysis was determined by performing recovery studies by spiking different concentrations of pure drug that is 80, 100 and 120% of the label claim (standard addition method). These were added in the preanalyzed tablet sample. Results of recovery studies indicated that the method is rapid, accurate and reproducible and is shown in Table 3 and chromatogram is shown in Figure 6.

 

2.2.8.4      Precision/Ruggedness Studies:

The precision studies were carried out by performing repetability and intermediate precision. The repeatability (intraday) was performed by taking six concentration on the same day (n=6). Intermediate precision (interday) of the method was carried by repeating the analysis of given concentration for two different days. The results for precesion studies are given in Table 4.

 

2.2.8.5      Limit of Detection and Limit of Quantitation:

The limit of detection (LOD) and limit of quantization (LOQ) for the developed method were calculated by using following formulae.

 

LOD=3.3σ/S

LOQ=10 σ /S

 

Where

σ = Standard deviation of response.

S = Slope of Calibration Curve.

The results of LOD and LOQ were given in Table 5.

 

2.2.8.6      Robustness:

Robustness parameter were analysed by changing Lab, Analyst, Mobile phase Composition. The results are shown in Table 6.

 

2.2.8.7      System Suitability Parameters:

System suitability parameters were analyzed on freshly prepared standard stock solutions of CIL and CHLT. Both the drugs were injected into the chromatographic system under the optimized chromatographic conditions. Parameters that were studied to evaluate the suitability of the system were

a)    Number of theoretical plates.

b)    Tailing/ asymmetric factor.

c)     Resolution.

d)    Retention time.

e)     Calibration Curve.

f)     Limit of Detection and Limit of quantitation. The results are shown in Table 7.

 

3.RESULT AND DISCUSSION:

3.1.  Determination of wavelength for detection of CIL and CHLT:

 

Figure 2: Overlain spectrum of CIL and CHLT

 

3.2.  Linearity Study of Drug at Selected Wavelength:

 

Figure 3: Calibration curve for (A) CIL and (B) CHLT at 231.6 nm

 

 

Figure 4: Chromatogram of Physical Mixture

3.3.  Analysis of Tablet Formulation:

Table 2: Results of tablet analysis

*Average of six determinations; S.D., standard deviation; R.S.D., relative standard deviation

 

 

Figure 5: Chromatogram of Tablet Solution

3.4.  Accuracy (Recovery Studies):

Table 3: Results of recovery study

Level	Volume of Sample solution in ml	Volume of standard drug in ml	Drug Conc.	Drug	Label Claim	concentration recovered* (mean ± S.D.)	RSD
80	1 ml	0.8 ml	18ug/ml	CIL	10 mg	99.60 ±0.58	0.56
			22.5ug/ml	CHLT	12.5mg	99.96 ±0.58	0.55
100	1 ml	1 ml	20ug/ml	CIL	10 mg	99.61 ±0.84	0.82
			25ug/ml	CHLT	12.5mg	99.96 ± 0.99	0.96
120	1 ml	1.2 ml	22ug/ml	CIL	10 mg	99.96 ± 0.57	0.56
			27.5ug/ml	CHLT	12.5mg	100.18 ± 0.86	0.87

*Average of six determinations; S.D., standard deviation; R.S.D., relative standard deviation

 

3.5.  Precision/Ruggedness Studies:

Table 4: Results of Ruggedness

Analyst		Analyst-I		Analyst-II
Location		Lab-I		Lab-II
Date		19th May 2014		22nd May 2014
Analyte	Labelclaim (mg/tab)	% Recovery estimated* (Mean ± S. D.)	R.S.D.	% Recovery estimated* (Mean ± S. D.)	R.S.D.
CIL	10	99.62 ± 1.1690	1.1688	99.91 ± 1.8708	1.8708
CHLT	12.5	99.90 ± 1.0327	1.0322	100.37 ± 1.4719	1.4719

*Average of six determinations; S.D., standard deviation; R.S.D., relative standard deviation

 

3.6.     Limit of Detection and Limit of Quantitation:

Table 5: Sensitivity- Limit of Detection and Limit of Quantitation

 

3.7.  Robustness:

Table 6: Results of Robustness

Analyst		Analyst-I		Analyst-II
Location		Lab-I		Lab-II
Mobile Phase		Methanol: Water (80:20)		Methanol: Water (75:25)
Analyte	Label claim(mg/tab)	% Recovery estimated* (Mean ± S. D.)	R.S.D.	% Recovery estimated* (Mean ± S. D.)	R.S.D.
CIL	10	99.62 ± 1.5819	1.5820	99.57 ± 1.6718	1.6716
CHLT	12.5	99.91 ± 1.1312	1.1313	99.42 ± 1.5219	1.5217

*Average of six determinations; S.D., standard deviation; R.S.D., relative standard deviation

 

 

Figure 6: Chromatogram of Recovery Solution

 

3.8.  System Suitability Parameters:

Table 7: System Suitability Parameters

Parameters	CIL	CHLT
Theoretical Plates	2137.91	2521.93
Tailing Factor	1.36	1.52
Resolution	4.27	00
Retention Time in minutes	4.23	1.84
Selectivity	1.35	0.00
Calibration Range (µg ml -1)	10-70	10-70
Limit of Detection (µg ml -1)	1.5611	5.1516
Limit of Quantization(µg ml-1)	1.7442	5.7559

 

The proposed method describes a RP-HPLC procedure employing a C18 column and a mobile phase containing methanol and water in 80:20 proportions. To develop the method with good resolution the change in proportion of organic solvents were studied. Acetonitrile, methanol, phosphate buffer and water in various ratios were tested to get an appropriate mobile phase composition. The mixtures of acetonitrile, methanol and water at various ratios were examined, which resulted in very close retention times of the two drugs. Also in case of phosphate buffer results in broadening of peaks were observed. Best resolution of two drugs was achieved with the mobile phase having composition of methanol and water in the ratio 80:20. The linearity response of the HPLC system for both CIL and CHLT was obtained over the range of 10-70 µg ml-1.

 

Various drugs were checked for use as an internal standard to obtain well resolved peaks along with the two drugs in the formulations. Retention time of both the drugs were studied with flow rate of mobile phase at 0.5, 1, 1.2 ml min-1. Optimum retention time with greater resolution of the two drugs and internal standard eluting within six minutes was achieved with a flow rate of 1 ml min-1. The two drug solutions having concentration of 10 µg ml-1 were scanned in the UV-Vis. Range of 200 nm to 400 nm on a UV-Visible spectrophotometer for selection of sampling wavelength. After recording the spectra of the two drugs and internal standard, 231.6 nm was selected as suitable wavelength for estimation. Accuracy of the method was checked by adding known amounts of pure drug to each known concentration of the tablet formulations. The resulting mixtures were run on HPLC. The result of analysis showed excellent recoveries for both the drugs ranging from 99.75 % to 100.62 % for CIL and 98.91 % to 102.05 % for CHLT. The recovery study data by the standard addition method suggested the good accuracy of the proposed method. The results of analysis of tablet indicated that no interference due to common tablet excipients was observed with the developed method.

 

4.CONCLUSION:

The developed method is the first report for simultaneous estimation of CIL and CHLT. The developed HPLC method was found to be more accurate, precise and reproducible. The analysis of tablets containing two drugs gave the satisfactory results. The statistical parameter of this method showed good results. The recovery studies revealed excellent accuracy and high precision of the method. The statistical parameters and recovery studies of HPLC method were compared with the developed spectrophotometric method of analysis of the same dosage form. The HPLC method was found to give better results. Therefore the proposed method could be applied for routine analysis in quality control laboratories.

 

5. ACKNOWLEDGEMENT:

Authors are very thankful to Macleoids Phrmaceuticals Ltd. Mumbai and IPCA Phrmaceuticals Ltd. Mumbai, India for providing gift sample of Clinidipine and Chlorthalidone respectively.

 

6. CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

7. REFERENCES:

1.     “Drug profile for Cilnidipine”, Sep 2015, www.drugbank.ca/drugs/DB03614

2.     Rang and Dale’s Pharmacology,” antihypertensive drugs”, 6th Edition, Chap.17, 19, page no. 672-694.

3.     Goyal R.K. et al., “Derasari and Gandhi’s Elements Of Pharmacology”, 18th Edition, B. S. Shah Prakashan, 2008-2009, Page no. 214-220.

4.     “Drug profile for Chlorthalidone”, Sep 2015, http://en.wikipedia.org/wiki/ Chlorthalidone

5.     Niraimathi V, Jerad SA, Kumar SI. Uv Spectrophotometric Methods For The Estimation OF Chlorthalidone In Bulk and Oral Dosage Form. Indo American J. Pharm, 2013; 3(9): 7160-7167.

6.     Salvador SC, Luz e V-A. On- line solid –phase-extraction and high performance liquid chromatographic determination of chlorthalidone in urine. J. Chroma.1997; 36: 102-195.

7.     Mhaske RA, Sahasrabudhe S., Mhaske AA. RP-HPLC Method For Simultataneous Determination of Irbesartan, Losartan, Hydro-Chlorothiazide And Chlorthalidone–Application To Commercially Available Drug Products. Int. J. Phar. Sci. and Research, 2012; 3(4): 1116-1123.

8.     Singh B, Patel DK, and Ghosh SK. A reversed-phase high performance liquid chromatographic method for determination of chlorthalidone in pharmaceutical formulation. Int. J. Pharm. and Pharma sci, 2009; 1(2): 24-29.

9.     Kyeong RL, Yoon JC, Jong HL, Dae DK, Saeho Chong, Chang KS, Suk JC, Quantification of Cilnidipine in human plasma by liquid Chromatography-mass spectrometry.J of Liq Chromato and Related Tech 2012; 35: 308–20.

10.  Zhang X, Zhai S, Zhao R, Ouyang J, Li X, Baeyens WR. Determination of cilnidipine, a new calcium antagonist, in human plasma using high performance liquid chromatography with tandem mass spectrometric detection. Anal Chim Acta, 2007; 1: 142-146.

11.  International Conference on Harmonization (ICH). Stability Testing of New Drug Substances and Products (Q1AR2); Geneva: IFPMA, <http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q1A_R2/Step4/Q1A_R2__Guideline.pdf>  2003

12.  Center for Drug Evaluation and Research (CDER). Reviewer Guidance: Validation of Chromatographic Methods, November 1994, Division of Communications Management CDER, FDA, Rockville, MD, <http://www.fda.gov/downloads/drugs/guidances/ucm134409.pdf

13.  ICH Harmonized Tripartite Guideline. Validation of Analytical Procedure Methodology, Q2B, 1.

14.  Brijesh Singh et al, A reversed-phase high performance liquid chromatographic method for determination of chlorthalidone in pharmaceutical formulation, International Journal of Pharmacy and Pharmaceutical Sciences,  1(2), Oct-Dec. 2009.

15.  H. Padmalatha et al, Spectrophotometric estimation of chlorthalidone in Pharmaceutical formulation using mbth and fc reagent, World Journal Of Pharmacy And Pharmaceutical Sciences, 2(3), 1433-1440.

16.  Nivedita. G et al, Simultaneous Estimation of Atenolol and Chlorthalidone as Bulk and In Tablet Dosage Form Using Uv- Spectrophotometry, Journal of Pharmacy and Biological Sciences, 4(1) (July-August 2012), 2278-3008.

17.  Kreny E. Parmar, Stability indicating RP-HPLC Method for Simultaneous Determination of Telmisartan and Chlorthalidone in Bulk and Pharmaceutical Dosage Form, International Journal of PharmTech Research, 4(5), (Oct-Dec 2013),1728-1735.

18.  Santosh V. Gandhi et al, Simultaneous RP-HPLC estimation of cilnidipine and telmisartan in combined tablet dosage form, Pelagia Research Library, Der Chemica Sinica, 4(2), 2013, 6-10.

 

 

 

Accepted on 28.08.2017           © RJPT All right reserved

Research J. Pharm. and Tech 2017; 10(11): 3990-3996.