Development and validation of a Rapid Stability Indicating chromatographic determination of Cilnidipine in Bulk and Dosage form.

 

Mohammed M Safhi, Manohara Yagaina Nagaraj*

College of Pharmacy, Jazan University, Jazan, Kingdom of Saudi Arabia

*Corresponding Author E-mail: manohara_yn@yahoo.com.

 

A rapid, simple and specific reversed-phase HPLC method has been developed for estimation of Cilnidipine in Pharmaceutical Formulation. HPLC analysis was performed on a C₁₈ column using a 80:20 (v/v) mixture of Methanol and 0.05 M Phosphate Buffer at pH 3.0 as isocratic mobile phase at a flow rate of 1 ml min^-1. UV detection was carried at 254 nm. The method was validated for accuracy, precision, linearity, specificity. Validation revealed the method is specific, accurate, precise, reliable and reproducible. Good linear correlation coefficients (r² > 0.9999) were obtained for calibration plots in the ranges tested. Limit of detection was 0.179 μg/ml and limit of quantification was 0.544 μg/ml. Intra and inter-day RSD of retention times and peak areas were less than 1.729% and recovery was between 99.95% and 101.09%. The established HPLC method is appropriate enabling efficient quantitative analysis of Cilnidipine in Pharmaceutical Formulation.

 

 

 

1. INTRODUCTION:

Cilnidipine [2-methoxyethyl-(E)-3-phenyl-2-propen-1-yl(_)-1,4-dihydro- 2,6-dimethyl-4-(3-nitrophenyl) pyridine-3,5-dicarboxylate]. The structural formula is given in Fig.1

 

Fig-1. Structure of Cilnidipine

 

It is a dihydropyridine calcium channel antagonist and antihypertensive agent, and has been reported to have a long-lasting anti-hypertensive effect and unique inhibitory actions on sympathetic neurotransmission^1-3 Because of these pharmacokinetic properties, this hypertensive agent appears to have potential for clinical use^4,5.

 

A single dose of 10mg cilnidipine is routinely prescribed for patients with hypertension. However, the plasma concentration required for achieving a blood pressure lowering effect would be expected to be low² due to the low dosage used and the relatively large volume of distribution⁶ of the drug in humans. Furthermore, a number of dihydropyridine calcium channel antagonists, including cilnidipine, are known to inhibit the human CYP3A4 isozyme^7,8. Since this isozyme of cytochrome P-450s primarily governs the biotransformation of many other xenobiotics⁹   drug-drug interactions are a real possibility when cilnidipine is prescribed along with other drugs.  Therefore, various assay methods are developed to quantify the cilnidipine for its application in the various studies.

 

A number of analytical procedures have been reported for the quantification of cilnidipine in various matrices. For example, an assay involving high performance liquid chromatography (HPLC), in conjunction with UV detection, was reported for this anti-hypertensive agent¹⁰.  In addition, the derivatization of cilnidipine by reaction with 1-naphthaleneboronic acid was reported to be useful in the quantification of the drug, by HPLC with fluorescence detection¹¹.  Although the HPLC method coupled with tandem mass spectrometry (HPLC-MS/MS) assay that reported sufficient sensitivity for the drug was available¹².  The drug is not official in Pharmacopeia. This paper describes a simple, accurate and validated HPLC method for the quantification of Cilnidipine as bulk drug and in tablet dosage form. The proposed method is optimized and validated as per the International Conference on Harmonization (ICH) guidelines¹³.

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2. MATERIALS AND METHODS:

2.1 Chemicals and Reagents:

Cilnidipine was provided by the J.B. Chemicals and Pharmaceutical Ltd. (Mumbai, India). Methanol, and Sodium Phosphate were purchased from Fischer Scientific (NJ, USA). Ultra-pure water was produced by purification system (Millipore Co., Bedford, MA, USA). All solvents were HPLC grade and used without further purification.

 

2.2 Instrument:

The experiment was performed with Shimadzu HPLC Model LC-10 AT (VP).

 

2.3 Preparation of Stock and Standard working solutions:

The stock solution of Cilnidipine (1mg/ml) was prepared by dissolving 100 mgm of Cilnidipine in 100 ml methanol then the solution was left in an ultrasonic bath for 15 min. The standard solution was further diluted to obtain 5- 30 µg/ml. The solution was filtered through 0.22 μm membrane filter and filtrate was used for analysis.

 

2.3.1 Preparation of Sample solutions:

Twenty tablets were weighed and finely powdered. The powder equivalent to 100 mgm of cilnidipine was weighed and suspended in 50 ml. Methanol, and then the solution was left in an ultrasonic bath for 15 min and then centrifuged for 10 min. Then the solution was filtered through whatman filter paper No. 41 volume of the filtrate made up to 100 ml with methanol. The sample solution was further diluted to obtain 5- 30 µg/ml. The solution was filtered through 0.22 μm membrane filter and filtrate was used for analysis.

 

2.3.2 Stability Test.

Forced degradation studies were performed to ensure the stability-indicating properties and specificity of the method. Intentional degradation was attempted using acid, base, hydrogen peroxide and UV-radiation. A degradation sample was prepared by dissolving 10 mg of Cilnidipine in 50 mL methyl alcohol through shaking and sonication. Then 10 mL of this solution was taken in each of three 50 mL round bottomed flasks to perform the first three degradation tests. To the first flask, 10 mL of 1 N HCl was added for acidic degradation. To the second flask, 10 mL of 1 N NaOH was added for basic degradation. To the third flask, 10 mL of 30% H₂O₂ was added for oxidative degradation. All the three flasks were refluxed for about 1 h. After completing degradation treatments, samples were allowed to cool to room temperature and treated as follows: The pH values of the first and second flasks were neutralized with 1 N NaOH and 1 N HCl, respectively. To the third flask 1 N sodium bisulfite solution was added to destroy H₂O₂. The volume of all the three flasks was adjusted to 50 mL with methyl alcohol. For degradation through UV-radiation 2 mL of the sample solution was left in UV radiation for 1 h then the radiated solution was diluted with methyl alcohol to 10 mL, then finally injected into chromatograph and compared with control sample.

 

2.3.3 Selection of Detection Wavelength:

The solution of drug was scanned over the range of 200-400 nm. It was observed that the drug showed considerable absorbance at 254 nm. So 254 nm was selected for the estimation.

 

2.3.4 Chromatographic conditions:

The following Chromatographic conditions were maintained for analysis of the drug throughout the experimental work.

 

System : Shimadzu HPLC Model LC-10AT(VP)

Column : Grace (4.6 mm I.D x 250 mm) C-18

Detector : Variable wavelength programmable UV/VIS detector SPD-10A (VP series)

Mobile phase : Methanol : 0.05 M phosphate buffer at pH 3 :: (80:20)

Detection wavelength : 254 nm

Mode : Isocratic

Syringe : Hamilton syringe. (705 NR, 50 μL )

Flow rate : 1.0 ml/min.

Injector : Rheodyne-7725i

Type of Injector: Manual

Temperature : Room temperature

 

2.3.5 Selection of mobile phase:

The pK_b value of cilnidipine is 11.39 and its partition coefficient is 5.36. For the selection of mobile phase, various solvents individually and in combinations were tried. Finally the mobile phase consisting of Methanol: 0.05 Phosphate buffer at pH 3 (80:20) was selected for study. The drug was eluted within a time period of 10 minutes with sharp peak. The mobile phase was degassed before use.

 

2.3.6 System suitability parameters

According to USP, system suitability tests were carried out on standard stock solution of Cilnidipine 20 μL of the solution was injected into the chromatographic column. Parameters studied to evaluate the suitability of system were: retention time, area under curve, asymmetry, capacity factor and number of theoretical plates. (Table-1). Acceptance criteria for system suitability, Asymmetry not more than 2.0, theoretical plate not less than 4000 and   % RSD of peak area not more than 2.0, were fulfilled during all validation parameter.

 

 

 

Table 1.  System Suitability Parameters

3. VALIDATION:

3.1 Linearity and Range

Linearity of the method was studied by injecting six concentrations of the drug prepared in the Methanol in the range  of 05- 30 μg/ml. into the HPLC system by injector by keeping the injection volume constant (20 μL). The peak areas were plotted against the corresponding concentrations to obtain the standard curve. Figure. 2.

 

Figure. 2.Standard Graph

 

The results are given in Table- 2.

 

Table-2 Linearity and Range of Study

 

Table - 3. Intraday and Interday precision data of Cilnidipine in tablet.

Sl. No	Std.Conc. (µg/ml)	Average concentration found.(µg/ml)	S.D	R.S.D
1	5	4.886	0.0166	0.3397
2	10	9.985	0.0304	0.3044
3	15	15.210	0.2631	1.7297
4	20	19.950	0.0664	0.3331
5	25	24.875	0.0444	0.1784
6	30	30.36	0.3753	1.2361

The results obtained by One-way ANOVA, found that P value is < 0.0001. Hence the method is considered extremely significant.

 

3.2 Precision:

Precision is determined by using the same method used to assay the sample and repeated for a sufficient number of times to obtain statistically valid result. The precision is then expressed as the relative standard deviation. (Table No-3).The retention time for all the samples was determined and found that mean retention time was 8.184, Standard deviation is 0.0470 and % RSD is 0.5745.

 

3.3 Accuracy (Recovery Studies)

To check the accuracy of the method, recovery studies were carried out by the addition of standard drug solution to pre-analyzed sample solution at three different levels 80, 100 and 120 %. At each level of the amount, three determinations were carried out. The results of recovery studies were expressed as percent recovery and in the range of 99.95 – 101.09.

 

Statistical comparison of the results obtained from the analysis is given in Table.4

 

3.4 Limit of detection (LOD) and Limit of quantitation (LOQ):

LOD and LOQ for both the drug were calculated by using the values of slopes and intercepts of the calibration curve. The values are given below Table- 5

 

3.5 Robustness Studies:

In the robustness study, the influence of small, deliberate variations of the analytical parameters on peak area of the drugs were examined. Factors varied were pH of the mobile phase, Temperature, flow rate, Content of Methyl alcohol in mobile phase, Wavelength of detection, buffer pH-value, injection volume. One factor was changed at a time to estimate the effect. Robustness of the method was checked at

a)      pH 2.8 and pH 3.2.

b)      Temperature of 35^oCand 36^oC.

c)      Flow rate 0.95 ml/min/1.05 ml/min.

d)      Percentage of Methyl alcohol 79% and 81%.

e)      Injecting volume 19µL and 21 µL.

f)       Wave length of detection 252 nm and 256 nm.

g)      Buffer concentration 79:21 and 81:19

 

The retention time for the standard was between 8.130-8.278 mins. The retention time was observed for all the effect was within the retention time observed for the pure drug. The results show that the proposed method is robust.

 

3.6. Stability of the analytical solution and stability tests:

Stability of the standard solution was studied by injection of the prepared solution at periodic intervals into the chromatograph up to five days. The results indicate that the RSD of the peak area was within 1.25%. The results of stress degradation indicate that Cilnidipine was not affected with reflux with HCl or NaOH or H₂O₂. Exposure to UV radiation leads to degradation of Cilinidipine. Although there are several degradants, there is no interference with the peak of the drug, indicating that the method is stability-indicating.

 

Table: 4 – Accuracy study Data

Sl. no	Drug in pre analysed Sample (µg/ml)	Standard Drug added (µg/ml)	Total amount found. (µg/ml) Mean	%Recovery*	S.D*	RSD*
1	10	8	18.016	100.33	0.046	0.029
2	10	10	20.253	101.28	0.083	0.048
3	10	12	21.990	99.950	0.283	0.163

Where * indicates mean of six determinations.

The results obtained by One-way ANOVA, found that P value is < 0.0001. Hence the method is considered extremely significant.

 

Table: 5 - LOD and LOQ Values

 

3.6 Selectivity and Specificity:

The comparison between the chromatogram of the raw Cilnidipine and that of extractd Cilnidipine from dosage form indicates that the excipients in the formulation did not interfere with the determination of Cilnidipine. No interfering endogenous compound peak was observed at the retention time of analyte, under chromatographic conditions. The results obtained during the course of work clearly show that the proposed method is more selective and specific for drug. No interference was observed of Tolbutamide, Chlorpropamide, Glibenclamide, Glimperide, Metformin, Phenformin when they were injected simultaneously with Cilnidipine.

 

4. RESULTS AND DISCUSSION:

The mobile phase Methanol: 0.05M Phosphate buffer at pH 3.0 (80:20) was selected, because it was found to give sharp peaks of Cilnidipine with retention time of 8.189 ± 0.032 min. Wavelength was selected by scanning standard solution of drugs over 200 nm to 400 nm. The compound showed good response at 254 nm. The low value of standard deviation indicates system suitability parameters as stable over the given chromatographic conditions. The value of coefficient of correlation reflects the method is linear over the concentration range of 05-30 μg/ml. The percentage recovery of drug from the formulation is close to 99.95–101.09%, and its low relative standard deviation values, indicates a high accuracy of the method.

 

5. CONCLUSION:

The proposed RP-HPLC method is simple, sensitive, precise and accurate. Since the analysis is completed within 10 minutes, it clearly indicates that the method is rapid and thus it could be used for routine analysis of Cilnidipine in bulk drug and tablet dosage form.

 

6. REFERENCES:

1.       Ikeda, K.; Hosono, M.; Iida, H.; Ohnishi, H. Antihypertensive and Cardiovascular Profiles of a Newly Sythesized Dihydropyridine Derivative 2-methoxyethyl (E)-3-phenyl-2-propen-l-yl (_)-1, 4-dihydro-2, 6-dimethyl-4-(3-nitrophenyl) pyridine-3, 5-dicarboxylate (FRC-8653). Pharmacometrics 1992, 44,433–442.

2.       Zhang, X.; Zhai, S.; Zhao, R.; Ouyang, J.; Li, X.; Baeyens, W. Determination of Cilnidipine, a New Calcium Antagonist, in Human Plasma Using High Performance Liquid Chromatography with Tandem Mass Spectrometric Detection. Anal. Chim. Acta 2007, 600, 142–146.

3.       Hosono, M.; Fujii, S.; Hiruma, T.; Watanabe, K.; Hayashi, Y.; Ohnishi, H.; Takata, Y.; Kato, H. Inhibitory Effect of Cilnidipine on Vascular Sympathetic Neurotransmission and Subsequent Vasoconstriction in Spontaneously Hypertensive Rats. Jpn. J. Pharmacol. 1995, 69, 127–134.

4.       Hiruma, T.; Hosono, M.; Watanabe, K.; Hayashi, Y.; Ohnishi, H. Changes in Heart Rate and Plasma Catecholamine Levels Accompanied with Hypotensive Action of Calcium Channel Blockers in Conscious SHR. Jpn. J. Pharmacol. 1995, 67, 267.

5.       Tominaga, M.; Ohya, Y.; Tsukashima, A.; Kobayashi, K.; Takata, Y.; Koga, T.; Yamashita, Y.;Fujishima, Y.; Abe, I.; Fujishima, M. Ambulatory Blood Pressure Monitoring in Patients with Essential Hypertension Treated with a New Calcium Antagonist, Cilnidipine. Cardiovasc. Drugs Ther. 1997, 11, 43–48.

6.       Henrich, W. Principles and Practice of Dialysis, Lippincott Williams and Wilkins:Philadelphia,  USA,2009, 307.

7.       Katoh, M.; Nakajima, M.; Yamazaki, H.; Yokoi, T. Inhibitory Potencies of 1,4-dihydropyridine Calcium Antagonists to P-glycoprotein-mediated Transport: Comparison with the Effects on CYP3A4. Pharm. Res. 2000, 17, 1189–1197.

8.       Katoh, M.; Nakajima, M.; Shimada, N.; Yamazaki, H.; Yokoi, T. Inhibition of Human Cytochrome P450 Enzymes by 1,4-dihydropyridine Calcium Antagonists: Prediction of In Vivo Drug-Drug Interactions. Eur. J. Clin. Pharmacol. 2000, 55, 843–852.

9.       Pelkonen, O.; Maeenpaea, J.; Taavitsainen, P.; Rautio, A.; Raunio, H. Inhibition and Induction of Human Cytochrome P450 (CYP) Enzymes. Xenobiotica 1998, 28, 1203–1253.

10.     Shen, W.; Du, Y.; Huang, C.; Xu, Y. Determination of Cilnidipine and Its Tablets by RP-HPLC. J. Chin. Pharm. U. 2002, 33, 544–547.

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13.     FDA, Guidance for Industry: Bioanalytical Method Validation, CDER: Rockville, MD, 2001.

 

 

Received on 05.02.2013       Modified on 01.03.2013

Accepted on 10.03.2013      © RJPT All right reserved