Table of Contents  
ORIGINAL ARTICLE
Year : 2013  |  Volume : 4  |  Issue : 2  |  Page : 102-107  

Development and validation of a reversed-phase HPLC method for simultaneous estimation of clotrimazole and beclomethasone dipropionate in lotion and cream dosage form


Department of Quality Assurance, Shri Sarvajanik Pharmacy College, Mehsana, Gujarat, India

Date of Web Publication24-Jul-2013

Correspondence Address:
Komal R Dhudashia
Department of Quality Assurance, Shri Sarvajanik Pharmacy College, Mehsana - 384 001, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2229-5186.115548

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   Abstract 

Background: The combination of Clotrimazole and Beclomethasone dipropionate is used as anti-fungal and anti-inflammatory for external use in the form of cream and lotion. Aim: A simple, specific, economic, precise, and accurate reversed-phase high performance liquid chromatographic method development for the simultaneous estimation of clotrimazole (CT) and beclomethasone dipropionate (BD) in lotion and cream formulations. Materials and Methods: The chromatographic separation was achieved on a Kromasil C18 (150 mm × 4.6 mm, 5 μm) analytical column. A mixture of acetonitrile-water (70:30, v/v) was used as the mobile phase, at a flow rate of 1 ml/min and detector wavelength at 254 nm. The validation of the proposed method was carried out for specificity, linearity, accuracy, precision, limit of detection, limit of quantitation, and system suitability test as per ICH guideline. Results: The retention time of CT and BD was found to be 5.4 and 4 min, respectively. The linear dynamic ranges were from 2-16 μg/ml and 80-640 μg/ml for BD and CT, respectively. Limit of detection and quantification for BD were 0.039 and 0.12 μg/ml, for CT 1.24 and 3.77 μg/ml, respectively. Conclusions: The developed method was validated and found to be simple, specific, accurate and precise and can be used for routine quality control analysis of titled drugs in combination in lotion and cream formulation.

Keywords: Beclomethasone dipropionate, clotrimazole, reversed-phase high performance liquid chromatographic


How to cite this article:
Dhudashia KR, Patel AV, Patel CN. Development and validation of a reversed-phase HPLC method for simultaneous estimation of clotrimazole and beclomethasone dipropionate in lotion and cream dosage form. Chron Young Sci 2013;4:102-7

How to cite this URL:
Dhudashia KR, Patel AV, Patel CN. Development and validation of a reversed-phase HPLC method for simultaneous estimation of clotrimazole and beclomethasone dipropionate in lotion and cream dosage form. Chron Young Sci [serial online] 2013 [cited 2019 Dec 7];4:102-7. Available from: http://www.cysonline.org/text.asp?2013/4/2/102/115548


   Introduction Top


Clotrimazole is 1-[(2-chlorophenyl)(diphenyl) methyl]-1h- imidazole. [1] It is a prescription drug indicated for the treatment and prophylaxis of fungal infections. Clotrimazole interacts with yeast 14-α demethylase, a cytochrome p-450 enzyme that converts lanosterol to ergosterol, an essential component of the membrane. In this way, clotrimazole inhibits ergosterol synthesis, resulting in increased cellular permeability. [2],[3],[4] Beclomethasone dipropionate is 9a-chloro-11b-hydroxy-16b-methyl-3,20-dioxopregna 1,4-diene-17,21-diyldipropionate. [5] It is a synthetic halogenated glucocorticoid with anti-inflammatory and vasoconstrictive effects, is used for treating steroid-dependent asthma, allergic or non-allergic rhinitis. The anti-inflammatory actions of corticosteroids are thought to involve phospholipase A2 inhibitory proteins, lipocortins, which control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes. [2],[3],[4]

The chemical structures of CT and BD are shown in [Figure 1]a and b.
Figure 1: Chemical structure of (A) Clotrimazole and (B) Beclomethasone dipropionate[4,5]

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A detailed survey of analytical literature for CT revealed several methods based on varied techniques, viz, HPLC, [6],[7],[8],[9] Spectrophotometry, [10],[11],[12] Spectrofluorimetry, [13] High-Performance Thin‐Layer Chromatography (HPTLC), [14],[15] and stability-indicating HPLC method. [16] Similarly, a survey of the analytical literature for BD revealed methods based on HPLC for determination in pharmaceuticals. [6],[7],[8],[17] According to detailed survey of analytical literature, none of the reported analytical methods available for simultaneous estimation of CT and BD in their combined dosage form. None of the reported analytical procedures describes a simple and satisfactory RP-HPLC method for simultaneous determination of CT and BD in their combined dosage forms. So, the objective of this work was to develop simple, precise, and rapid RP-HPLC methods for combination drug products containing CT and BD.


   Materials and Methods Top


Apparatus and instruments

  • A Shimadzu (Kyoto, Japan) HPLC system (LC-2010CHT) equipped with autosampler, UV and photodiode array (PDA) detector, Rheodyne injector with 20 μl loop volume
  • Balance, Model ALC 210.4 (Acculab)
  • Ultra Sonicator (Fast Clean Ultrasonic Cleaner)
  • pH analyzer (Chemiline CL 180 μc based pH meter)
  • Volumetric flasks - 10 ml, 100ml (Borosil)
  • Pipettes - 1 ml, 2 ml, 5 ml, 10 ml (Durasil)
  • Separating funnel
  • Beaker - 250 ml, 100 ml (Borosil).


Reagents and materials

  • Clotrimazole (Glenmark pharmaceuticals Ltd.)
  • Beclomethasone dipropionate (Glenmark pharmaceuticals Ltd.)
  • HPLC grade acetonitrile, water (Finar Chemicals Pvt. Ltd, Ahmedabad, India)
  • Cyclohexane (SD Fine Chemicals Pvt. Ltd., Ahmedabad, India)
  • Whatman filter paper no. 41.


Formulation

  • CANDID-B lotion (Glenmark pharmaceuticals Ltd.

    Label claim: Clotrimazole IP (1% w/v) and beclomethasone dipropionate IP (0.025% w/v) in 15 ml.
  • CANESTEN-S cream (Bayer pharmaceuticals Ltd.)

    Label claim: Clotrimazole IP (10 mg) and beclomethasone dipropionate IP (0.25 mg)


Chromatographic condition

  • Column: Kromasil C18 (150 mm × 4.6 mm, 5 μm)
  • Mobile phase: Acetonitrile:Water (70:30)
  • Detection wavelength: 254 nm
  • Flow rate: 1 ml/min
  • Injection volume: 10 μl
  • Temperature: 40°C


Preparation of combined standard stock solution of CT and BD

Accurately weighed CT (40 mg) and BD (1 mg) were transferred into 10 ml volumetric flask and diluted up to the mark with ACN (Acetonitril) to give a stock solution having strength of 4000 μg/ml:100 μg/ml of CT:BD.

Preparation of test solution

Lotion


1 ml of lotion (10 mg:0.25 mg of CT:BD) was taken in 10 ml volumetric flask, and volume was made up with methanol. Then, extraction of prepared solution was done with cyclohexane using separating funnel. cyclohexane layer was discarded, and methanol layer was collected. 3 ml was pipetted out from methanol layer into 10 ml volumetric flask and diluted up to mark with mobile phase to get the concentration of 300 μg/ml:7.5 μg/ml of CT:BD.

Cream

1 gm of cream (10 mg:0.25 mg of CT:BD) was taken in beaker containing 10 ml of methanol, mixed well, and filtered. The solution was warmed for 5 min at 50 o C, then cooled in ice-bath for 15 min and promptly centrifuged. The supernatant layer was taken and extracted with cyclohexane using separating funnel. cyclohexane layer was discarded, and methanol layer was collected. 3 ml was pipetted out from methanol layer into 10ml volumetric flask and diluted up to mark with mobile phase to get the concentration of 300 μg/ml:7.5 μg/ml of CT:BD.

Method development and optimization

The wavelength for the analysis of was selected from the UV spectrum of CT and BD by scanning in the range of 200-400 nm. From this, the wavelength of 254 nm was selected for the final method as these drugs has shown good absorbances. For HPLC analysis, initially various mobile phases and stationary phase were tried in attempts to obtain the best separation and resolution between CT and BD. The mobile phase consisting a combination of acetonitrile and water in the ratio of 70:30 v/v was found to be an appropriate mobile phase allowing adequate separation of two drugs using a Kromasil C18 (150 mm × 4.6 mm, 5 μm) with flow rate of 1 ml/min at 254 nm and 40 o C temperature. 40 o C Temperature was used for good resolution and faster elution of compounds. A HPLC chromatogram of separation of CT and BD were shown in [Figure 2].
Figure 2: HPLC chromatogram of CT (400 μg/ml) and BD (10 μg/ml)

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   Method Validation Top


As per the ICH guidelines Q2R1, the method validation parameters studied were specificity, linearity, accuracy, precision, limit of detection, limit of quantitation, and system suitability test. [18]

Specificity

Specificity of an analytical method is its ability to measure the analyte accurately and specifically in the presence of component that may be expected to be present in the sample matrix. Chromatograms of standard and sample solutions of CT and BD were compared, and peak purity spectra obtained from using photo diode array detector (PDA) were recorded in order to provide an indication of specificity of the method.

Linearity

Different volume (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 ml) were withdrawn from combined standard stock solution containing 4000:100 μg/ml of CT:BD and diluted upto 10 ml with mobile phase to get the linear concentrations of CT (80-640 μg/ml) and BD (2-16 μg/ml). Each solution was injected under the operating chromatographic conditions. Calibration curves were constructed by plotting peak areas versus concentrations, and the regression equation was calculated. Each response was average of three determinations.

Precision

The repeatability was checked by repeatedly (n = 6) injecting CT (400 μg/ml) and BD (10 μg/ml) standard solutions and recording the responses. The intra-day and inter-day precisions of the proposed method was determined by measuring the corresponding responses 3 times on the same day and on 3 different days over a period of 1 week for test mixture of 3 different concentration of CT (320,400,480 μg/ml) and BD (8, 10, 12 μg/ml). The results were reported in terms of relative standard deviation.

Accuracy (% Recovery)

The accuracy of the method was determined by calculating recoveries of CT and BD by the standard addition method. Known amount of standard solutions of CT (0,192,240,288 μg/ml) and BD (0,4.8,6.0,7.2 μg/ml) were added to a pre-quantified sample solution of CT (240 μg/ml) and BD (6 μg/ml). Each solution was injected in triplicate, and the percentage recovery was calculated by measuring the peak areas and fitting these values into the regression equation of the respective calibration curves.

Limit of detection and limit of quantification

The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using the standard deviation of y-intercept of calibration curve (N) and slope (S) of the calibration curve.

LOD = 3.3 × N/S, LOQ = 10 × N/S

System-suitability test

System suitability tests are used to verify that the resolution and repeatability of the system are adequate for the analysis intended. The parameters used in this test were retention time, capacity factor, theoretical plate, tailing factor, resolution.


   Result and Discussion Top


Method validation

Specificity


The analytical method was found to be specific as no interference of excipients was found in separation. Study has shown that the CT and BD peaks were free from excipients and co-eluting impurities, as the peak purity index was >0.99 [Table 1].
Table 1: Peak purity table of CT and BD

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(The peak mentioned as excipient in chromatogram was not observed in case of standard and blank, and it was only observed in test sample so we can confirm it as excipient. As we had used marketed sample, a detail about placebo preparation was not available; so, we had not prepared it and also not injected it.) Comparative HPLC chromatogram of std, test and blank of CT and BD were shown in [Figure 3].
Figure 3: Comparative HPLC chromatogram of std and test of CT and BD

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Linearity

Linear correlation was obtained between peak area and concentration of CT and BD in the range of 80-640 μg/ml and 2-16 μg/ml, respectively. The linearity of the calibration curves was validated by the value of correlation coefficients of the regression [Table 2] and [Table 3].
Table 2: Linearity of CT and BD

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Table 3: Linearity data of developed method

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Overlay chromatogram of CT (80-640 μg/ml) and BD (2-16 μg/ml) were shown in [Figure 4].
Figure 4: Overlay chromatogram of CT (80-640 μg/ml) and BD (2-16 μg/ml)

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Precision

The % RSD for repeatability of CT and BD were found to be 0.45 and 0.18, respectively. For lotion, the % RSD for intra-day precision was found to be in the range of 0.22-0.56 and 0.11-0.71; while inter-day precision was found to be in the range of 0.99-1.12 and 0.98-1.22 for CT and BD, respectively. While for cream, the % RSD for intra-day precision was found to be in the range of 0.16-0.62 and 0.28-0.73; while inter-day precision was found to be in the range of 1.07-1.25 and 1.05-1.19 for CT and BD, respectively, which indicates the method is precise [Table 4] and [Table 5].
Table 4: Results of repeatability (n=6)*

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Table 5: Results of intra‑day and inter‑day precision (n=3)*

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Accuracy (% Recovery)

The accuracy study was carried out by the standard addition method. For lotion, the percent recovery was found in the range of 99.97-100.46% and 100-100.62% for CT and BD, respectively. For cream, the percent recovery was found in the range of 98.95-99.62% and 99.37-99.58% for CT and BD, respectively, which indicates accuracy of the method [Table 6] and [Table 7].
Table 6: Results of recovery study (lotion) (n=3)*

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Table 7: Results of recovery study (cream) (n=3)*

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Limit of detection and limit of quantification

LOD and LOQ value of CT and BD were listed in following table [Table 8].
Table 8: LOD and LOQ of CT and BD

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System suitability test

The % RSD of system-suitability test parameters was found satisfactory [Table 9].
Table 9: System suitability test parameter (n =3)*

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Analysis of formulation

The proposed RP-HPLC method was successfully applied for determination of CT and BD from lotion and cream. The percentage of CT and BD was found to be satisfactory, which is comparable with the corresponding claim amount [Table 10].
Table 10: Result of analysis of formulation (n=3)*

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HPLC chromatogram of test (lotion) containing CT (400 μg/ml) and BD (10 μg/ml) were shown in [Figure 5].
Figure 5: HPLC chromatogram of test (lotion) containing CT (400 μg/ml) and BD (10 μg/ml)

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HPLC chromatogram of test (cream) containing CT (400 μg/ml) and BD (10 μg/ml) were shown in [Figure 6].
Figure 6: HPLC chromatogram of test (cream) containing CT (400 μg/ml) and BD (10 μg/ml)

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   Conclusion Top


A simple, specific, economic, linear, precise, and accurate RP-HPLC method has been developed and validated for quantitative determination of clotrimazole and beclomethasone dipropionate in lotion and cream formulation using simple mobile phase. The method is very simple and specific as both peaks are well separated from excipient peaks, which makes it especially suitable for routine quality control analysis work.


   Acknowledgments Top


The authors are thankful to the Management, Shri Sarvajanik Pharmacy College, Mehsana for providing laboratory facilities, and authors greatly acknowledge Glenmark Pharmaceuticals Ltd. for providing the gift sample of clotrimazole and beclomethasone dipropionate.

 
   References Top

1."Clotrimazole". Available from: http://en.wikipedia.org/wiki/clotrimazole [Last accessed on October 2011].  Back to cited text no. 1
    
2.Rang HP, Dale MM, Pitter JM, Moore PK. Pharmacology; 15 th Ed, New Delhi: Elesvier; 2005.  Back to cited text no. 2
    
3.Joel GH, Lee EL. Goodman and Gilman's The Pharmacological basis of Therapeutics. 9 th Ed, New York: McGraw Hill Publishers; 1996.  Back to cited text no. 3
    
4.Tripathi KD. Essentials of Medical Pharmacology, 6 th Ed; New Delhi: Jaypee Brothers Medical Publishers; 2008.  Back to cited text no. 4
    
5."Beclomethasone", available from: http://en.wikipedia.org/wiki/beclomethasone [Last accessed on October 2011].  Back to cited text no. 5
    
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9.WANG LK, LAI YJ. Develop a HPLC method to determine the Clotrimazole in Clotrimazole Ointment. Strait Pharm J 2005;23:56.  Back to cited text no. 9
    
10.Abdelmageed OH, Khashaba PY. Spectrophotometric determination of clotrimazole in bulk drug and dosage form. Talanta 1993;40:1289-94.  Back to cited text no. 10
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15.Parmar P, Mehta A. Development and validation of HPTLC method for the estimation of clotrimazole in bulk drug and tablet formulation. Indian J Pharm Sci 2009;71:451-4.  Back to cited text no. 15
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16.Prodduturi S, Shah A, Stodghill S, Avery B, Repka M. Stability-indicating HPLC method development and validation for determination of clotrimazole in hot-melt extruded polymeric matrices. Planar chromatogr 2003;13:128.  Back to cited text no. 16
    
17.Darwisa Y, KokKhiangaand P, Tan YT, high performance liquid chromatography (HPLC) method was developed and validated for the determination of aerodynamic characteristics of the emitted dose of beclomethasone dipropionate from Beclate-50 Inhaler®. J Liq Chromatogr Relat Technol 2011;34:446-8.  Back to cited text no. 17
    
18.ICH: Proceedings of the International Conference on Harmonisation of Technical Requirement of Registration of Pharmaceuticals for Human Use (ICH Harmonised Tripartite Guidelines). Validation of Analytical Procedures: Methodology, Q2B, Geneva, Switzerland: 1996.  Back to cited text no. 18
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]



 

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