Skip to main content

Development and validation of HPLC method for the quantification of clotrimazole in a gelatin film formulation

Abstract

Background

Clotrimazole is used to treat topical vaginal yeast infections. Vaginal gelatin film formulations of clotrimazole were prepared, and the present study aims to develop an HPLC method for quantifying clotrimazole in the film formulation.

Results

The chromatographic separation of components was achieved on a Phenomenex Luna C18(2) (150 × 4.6 mm, 5 μm) stationary phase with Phenomenex Security Guard Cartridge C18 (4 × 3 mm) using acetonitrile and water (70:30% v/v) as mobile phase. The flow rate was 1.0 ml/min and the absorbance was monitored at 210 nm. Clotrimazole was eluted at 5.6 min. The procedures and capabilities of the method were evaluated against the criteria for system suitability, specificity, linearity, precision, accuracy, and robustness as defined by the ICH guidelines. The method showed good linearity (R2 = 0.999) in the range of 10–50 μg/ml and an average recovery of 101.05%.

Conclusion

The method could be applied for the quantification of clotrimazole in the gelatin film formulation.

Background

Clotrimazole, an imidazole derivative (Fig. 1), is a broad-spectrum antifungal. It inhibits the biosynthesis of ergosterol, a component of the fungal cell membrane, and this affects the permeability of the cell membrane, which results in leakage and loss of essential intracellular compounds and finally causes cell lysis [1].

Fig. 1
figure1

Clotrimazole

Clotrimazole is used to treat vaginal and skin yeast or fungal infections, rashes, oral thrush, athlete’s foot, jock itch, ringworm, etc. This is administered orally or applied topically. It is commonly available in various dosage forms, such as a cream, vaginal tablet, or throat lozenge. Topically, clotrimazole is used for vulvovaginal candidiasis. Tablets and creams are inserted into the vagina. Clotrimazole gelatin films have been developed for vaginal application in our laboratory.

Few methods have been reported for the determination of clotrimazole individually or in combination in different formulations. These include HPLC [2,3,4,5,6,7], centrifugal partition chromatography [8], HPTLC [9], and spectrophotometry [10,11,12] for the quantification of clotrimazole. According to a survey of analytical literature, none of the reported methods describes a simple HPLC method for estimation of clotrimazole in gelatin film formulation. Furthermore, buffers were used in the mobile phase of reported HPLC methods. Thus, there was a need for a validated simple HPLC method to determine clotrimazole in gelatin film formulation without any pre-treatment to sample.

Methods

Chemicals

Clotrimazole was obtained from Glenmark Laboratories Ltd, Mumbai, India. HPLC-grade acetonitrile LiChrosolv, water LiChrosolv, and methanol were procured from Merk Pvt. Ltd., Mumbai. Gelatin and sodium lauryl sulfate were procured from S D Fine-Chem Ltd, Mumbai. Gelatin films (3% w/w of clotrimazole) were prepared in the laboratory.

Instruments

The Younglin Acme 9000 HPLC system consisting of the components, 4 Channel Vacuum Degasser and Mixer SDV50A, Gradient Pump SP930D, Rheodyne Injector, Phenomenex Luna C18(2) (150 × 4.6 mm, 5 μm) column with Phenomenex security guard cartridge C18 (4 × 3 mm), and UV–VIS Detector UV730D, was used. The equipment was controlled by PC installed with the Younglin Autochro 3000 Chromatograph data system software. Shimadzu UV-Vis Spectrophotometer 1800, Shimadzu Electronic balance AY220, Borosil All Glass Filter with Nylon 6,6 membrane 0.45 μm 47 mm filters, and Phenomenex RC membrane 0.45 μm 15 mm syringe filters were used.

Preparation of standard solution

Ten milligrams of clotrimazole was weighed and transferred into 10 ml volumetric flask. 5 ml of the methanol was added to clotrimazole and sonicated for 10 min; the volume was made up to the mark with methanol (flask A). An aliquot of 5 ml from flask A was transferred into 50 ml volumetric flask, and the volume was made up of the mark with methanol (flask B) to obtain a solution of clotrimazole (100 μg/ml).

Preparation of sample solution

Film equivalent to 10 mg of clotrimazole (332 mg) was taken in a 50 ml volumetric flask and dissolved in 40 ml of 1% sodium lauryl sulfate (SLS) solution with stirring at 50 °C. The volume was made up to the mark with SLS solution (flask C). An aliquot of 1 ml from flask C was transferred into a 10 ml volumetric flask and a mobile phase was added up to the mark to obtain a sample solution (flask D).

Selection of UV wavelength

A solution of clotrimazole in the mobile phase (10 μg/ml) was scanned in the range of 200–400 nm in a UV-VIS spectrophotometer, and λmax was selected from the UV spectrum.

Method development and optimization

To achieve satisfactory separation of clotrimazole, solvents of different proportions were tried as binary eluents on the C18 column. However, ACN achieve good satisfactory results. The optimized chromatogram and optimized conditions are decided on the basis of system suitability parameters.

Chromatographic conditions

The chromatographic separation of components was achieved on a Phenomenex Luna C18(2) (150 × 4.6 mm, 5 μm) stationary phase with Phenomenex Security Guard Cartridge C18 (4 × 3 mm) using acetonitrile and water (70:30% v/v) as mobile phase. The flow rate was 1.0 ml/min and the absorbance was monitored at 210 nm. The volume of the sample was 20 μl.

Method validation

The method has been evaluated for system suitability, specificity, linearity, precision, accuracy, robustness, and stability of solution as per ICH guideline Q2(R1) [13].

System suitability

System suitability parameters (capacity factor, tailing factor, number of theoretical plates, and resolution of peak) were assessed by injecting a blank mobile phase followed by clotrimazole solution (20 μg/ml).

Specificity

The specificity of the method has been established by comparing the chromatograms obtained by injecting blank (mobile phase), solution of clotrimazole (20 μg/ml), and sample solution of clotrimazole gelatin film. The effect of excipients used in film was checked. The parameters retention time, tailing factor, and resolution were calculated in order to prove that the method chosen was specific.

Precision

Precision was assessed by injecting six samples of clotrimazole solution (20 μg/ml) on the same day (repeatability) and a different day (intermediate precision). The precision of the method was investigated as %RSD of the response.

Linearity

Ten, 20, 30, 40, and 50 μg/ml of clotrimazole solutions were prepared by diluting the solution from flask B with mobile phase. Solutions were filtered through 0.45 μm syringe filter and injected in an HPLC system to measure the peak area. The calibration curve (peak area v/s concentration) was plotted. The correlation coefficient of the calibration curve was determined to ensure the linearity of the analytical method.

Accuracy

Recovery studies were conducted by the standard addition (spiking) technique to the matrix of gelatin film. In this method, 80, 100, and 120% of three different levels of pure drug (1.6, 2, and 2.4 ml from flask B) were added to the previously analyzed sample solution (0.5 ml from flask C) in the 10 ml volumetric flask and diluted to 10 ml with mobile phase. Solutions were filtered through 0.45 μm syringe filter and injected in an HPLC system to measure the peak area. Accuracy was evaluated as recovery values of the clotrimazole.

Robustness

Deliberate minute variations in the chromatographic conditions such as flow rate (±0.1 ml/min), acetonitrile ratio in the mobile phase (± 2%) have been made. After each change, assay results were checked by injecting the clotrimazole solution (20 μg/ml) into the chromatographic system and the results were compared with those under the original chromatographic conditions.

Solution stability

The stability of the clotrimazole solution (20 μg/ml) was assessed over 24 h at room temperature. The peak area and retention time were evaluated after injecting clotrimazole solution (20 μg/ml) into the system after every 8 h.

Analysis of clotrimazole film

Sample solution (flask D) was filtered through 0.45 μm syringe filter and injected in an HPLC system to measure the peak area.

Results

Method development

Two hundred ten nanometers was selected as the wavelength of determination from the UV spectrum of clotrimazole (Fig. 2).

Fig. 2
figure2

UV spectrum of clotrimazole

Clotrimazole was eluted at 5.6 min. Figure 3 illustrates a typical chromatogram obtained at optimum conditions.

Fig. 3
figure3

Chromatogram of clotrimazole (20 μg/ml)

Method validation

System suitability

After setting the optimum conditions, system suitability parameters for the method are determined and compared with recommended limits. The clotrimazole was repeatedly eluted at 5.6 min, tailing factor for peak less than 2 indicating good peak symmetry, and a number of theoretical plates always greater than 2000 ensure good column efficacy. Results are represented in Table 1.

Table 1 System suitability results

Specificity

The clotrimazole was eluted at 5.6 min in both a chromatogram of standard clotrimazole (Fig. 3) and clotrimazole gelatin film (Fig. 4). The clotrimazole peak in film is properly resolved from another excipient’s peak and shows no interference from the excipients of the film (Fig. 5).

Fig. 4
figure4

Chromatogram of clotrimazole gelatin film

Fig. 5
figure5

Overlay of chromatograms of std clotrimazole (blue) and clotrimazole film (red)

Linearity and range

The analytical calibration curve is linear in the range of 10–50 μg/ml and indicates the closeness of the correlation coefficient (R2) to 1 (0.999). The linear regression equation is y = 149.91x − 36.7. (Figs. 6 and 7, Table 2).

Fig. 6
figure6

Overlay of chromatograms of 10, 20, 30, 40, and 50 μg/ml of clotrimazole

Fig. 7
figure7

Calibration curve of area vs concentration in μg/ml

Table 2 Linearity of response for clotrimazole

Precision

Repeatability and intermediate precision have been expressed by the relative standard deviation of peak area response (Table 3). The results ensure the high precision of the method where data was expressed in %RSD (acceptance limit %RSD < 2).

Table 3 Precision result as the peak area of clotrimazole (20 μg/ml)

Accuracy

Accuracy was assessed by spiking of a known quantity of clotrimazole to the film sample solution of 3 concentration levels (80, 100, and 120%) and expressed as percent recovery by the assay of a known added amount of clotrimazole in the film sample. Results for recoveries are shown in Table 4.

Table 4 Result of accuracy study (recovery result)

Robustness

No significant changes were noticed on 20 μg/ml of clotrimazole assay results upon applying small variations to the chromatographic conditions, flow rate, or mobile phase ratio. Results for robustness are presented in Table 5.

Table 5 Result of robustness study

Solution stability

Clotrimazole solution was stable for 24 h at room temperature. The stability results have been assessed for the difference from zero-time injections, where no change in the peak areas was observed (Table 6).

Table 6 Result of solution stability

Analysis of clotrimazole gelatin film

The developed and validated method was applied for the determination of clotrimazole in gelatin film. The result of the assay of the film is shown in Table 7. Gelatin film contains 2.94% of clotrimazole.

Table 7 Analysis of clotrimazole gelatin

Discussion

Method development

Optimization studies are carried out to develop the HPLC method for the determination of clotrimazole. In this case, the mobile phase parameter was investigated. The optimum parameters are selected based on the retention time and peak shape.

Two hundred ten nanometers is selected as the wavelength of determination because of the maximum absorption of the analyte. Also, the baseline obtained with this wavelength was horizontal. Similarly reported by Rajameena [4], clotrimazole was detected at 254 nm in some reported methods; however, we observed no UV absorption above 250 nm (Fig. 2).

Acetonitrile is commonly used as an organic solvent for the determination of clotrimazole in the reported methods. In addition to that, it has the ability to produce a better peak shape compared to methanol. Therefore, acetonitrile is selected as an organic solvent. Different isocratic profiles were explored by increasing the acetonitrile ratio to obtain a retention time less than 10 min as well as the peak symmetry. In this study, the acetonitrile ratio was increased, because it caused a shorter retention time. In addition, acetonitrile has higher elution strength, which improves the peak shapes. The flow rate of 1.0 ml/min is chosen as the flow rate of the method. The injection volume was 20 μl, and the experiment was carried out at 25 °C. Clotrimazole was shown good retention on the C18 column. Early trials of higher organic proportion in the mobile phase retain clotrimazole on the C18 column.

Method validation

The system suitability parameters, capacity factor, tailing factor, theoretical plates, and resolution factor are within the recommended limits, so the method is suitable for the analysis.

Demonstration of the discrimination of the analyte in the presence of impurities and/or excipients is involved in the specificity. There is no interference from the excipients of the film to the clotrimazole peak. Hence, the method is specificity.

The method is linear (R2 = 0.999), precise (%RSD < 2), accurate (100–101.66%), and robust (%RSD < 2). Analysis of clotrimazole gelatin film is done by this method.

Conclusion

The developed HPLC method is simple, accurate, and precise. The results of the statistical analysis demonstrate that the values of the validation parameters are acceptable, and therefore, the method is suitable for the quantitative determination of clotrimazole in gelatin film formulation. Additionally, the method does not involve any pre-procedure such as extraction. Similar methods could be developed for the estimation of active ingredients in the gelatin film formulations. Validated HPLC methods are required in the drug development process and pharmaceutical industry.

Availability of data and materials

Available on request.

Abbreviations

HPLC:

High-performance liquid chromatography

ICH:

International Council for Harmonization

RSD:

Relative standard deviation

References

  1. 1.

    PubChem Compound Summary for CID 2812, Clotrimazole. https://pubchem.ncbi.nlm.nih.gov/compound/Clotrimazole. Accessed on 29 Mar 2021

  2. 2.

    Usman S, Zulfiqar A, Shah J, Ramesh V, Sudhir S (2017) Modification and validation of HPLC analytical method for estimation of clotrimazole in health care products. Nat Prod Chemi Res 5(1):1–7

    CAS  Google Scholar 

  3. 3.

    Beigi F, Imani M, Payehghadr M, Hosseini H (2011) SPE-HPLC method for determination of ketoconazole and clotrimazole residues in cow’s milk. J Braz Chem Soc 22(9):1679–1685. https://doi.org/10.1590/S0103-50532011000900009

    CAS  Article  Google Scholar 

  4. 4.

    Rajameena R, Rama K, Muthulakshmi C (2013) RP-HPLC method development and validation for estimation of clindamycin phosphate and clotrimazole in pharmaceutical dosage forms. Int Res J Pharm 4(7):141–146. https://doi.org/10.7897/2230-8407.04731

    CAS  Article  Google Scholar 

  5. 5.

    Dhudashia K, Patel A, Patel C (2013) Development and validation of a reversed-phase HPLC method for simultaneous estimation of clotrimazole and beclomethasone dipropionate in lotion and cream dosage form. Chronicles Young Sci 4(2):102. https://doi.org/10.4103/2229-5186.115548

    CAS  Article  Google Scholar 

  6. 6.

    Hájková R, Sklenárová H, Matysová L, Svecová P, Solich P (2007) Development and validation of HPLC method for determination of clotrimazole and its two degradation products in spray formulation. Talanta 73(3):483–489. https://doi.org/10.1016/j.talanta.2007.04.023

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Abdel-Moety E, Khattab F, Kelani K, AbouAl-Alamein A (2002) Chromatographic determination of clotrimazole, ketoconazole and fluconazole in pharmaceutical formulations. Farmaco 57(11):931–938. https://doi.org/10.1016/S0014-827X(02)01270-3

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Wang L, Li Y, Jiang B, Zhao W (2014) Determination of the active ingredients in simple-recipe ointments by centrifugal partition chromatography. Anal Methods 6(14):5245–5249. https://doi.org/10.1039/c4ay00131a

    CAS  Article  Google Scholar 

  9. 9.

    Parmar P, Mehta A (2009) Development and validation of hptlc method for the estimation of clotrimazole in bulk drug and tablet formulation. Indian J Pharm Sci 71(4):451–454. https://doi.org/10.4103/0250-474X.57299

    Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Abdelmageed O, Khashaba P (1996) Spectrophotometric determination of clotrimazole in bulk drug and dosage forms. Talanta 40:1289–1294

    Article  Google Scholar 

  11. 11.

    Bedair M, Korany M, Elsayed M, Fahmy O (1989) Derivative spectrophotometric determination of clotrimazole in single formulations and in combination with other drugs. J Assoc Off Anal Chem 72:432–435

    CAS  PubMed  Google Scholar 

  12. 12.

    Bodiwala K, Shah S, Patel Y, Prajapati P, Marolia B, Kalyankar G (2017) Simultaneous estimation of ofloxacin, clotrimazole, and lignocaine hydrochloride in their combined ear-drop formulation by two spectrophotometric methods. J AOAC Int 100(1):38–44. https://doi.org/10.5740/jaoacint.16-0229

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Validation of Analytical Procedures Q2(R1). https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf. Accessed on 29 Mar 2021

Download references

Acknowledgements

The authors are thankful to Principal, DSTS Mandal’s College of Pharmacy, Solapur for the support.

Funding

No funding was received.

Author information

Affiliations

Authors

Contributions

KP designed the study and supervised it. SM performed the experiments and was a major contributor in writing the manuscript. Both authors have read and approved the final manuscript.

Corresponding author

Correspondence to Surekha Kalshetti.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kalshetti, S., Rao, K.P. Development and validation of HPLC method for the quantification of clotrimazole in a gelatin film formulation. Futur J Pharm Sci 7, 130 (2021). https://doi.org/10.1186/s43094-021-00291-7

Download citation

Keywords

  • RP-HPLC
  • Clotrimazole
  • Gelatin film