Validated uv-spectrophotometric method for quantitative estimation of ciclopirox olamine in API and ethosomal gel

Abstract

Ultra-visible Spectroscopy is one of the most widely used analytical techniques in pharmaceutical research, university's, industries and quality control laboratories. This technique offers high sensitivity, accuracy, precision, and reproducibility, providing huge platform for drug discovery, development, and routine quality assurance. This short communication presents a comprehensive overview of Ultra-visible Spectroscopy, method development strategies, validation parameters as per International Council for Harmonisation (ICH) guidelinesQ2(R2) for the determination of Ciclopirox olamine in API as well as Ethosomal gel. Ultra Violet-Spectroscopy process developed for Ciclopirox Olamine shows the maximum absorbance at wavelength 302 nm. Ciclopirox Olamine showed the linearity, range 2.5–25 µg per ml for this procedure with Correlation Coeff. (R²) found to be 0.9996. The technique was found definite as no intervention was detected with excipients. The precision studies include Repeatability, Inter-day Precision, Intra-day Precision, Specificity and Accuracy studies and their R.S.D value found to be 0.98,1.55,1.448,0.032 and 0.27. (Acceptable criteria: RSD should be less than 2%). The proposed analytical process is suggested for analysis of Ciclopirox olamine as API and ethosomal dosage forms. This article aims to serve as a useful reference for researchers, academicians, and quality control analysts involved in pharmaceutical analysis. UV–Visible spectroscopy is widely used in pharmaceutical analysis due to rapid analysis, cost effective, minimal solvent consumption and non-destructive technique.

Keywords

spectroscopy validation repeatability inter-day precision intra-day precision specificity and accuracy

Introduction

The development of new antifungal medicines has not kept pace with the increasing number of invasive fungal infections. Simultaneously, serious skin-related disorders are becoming more common, creating a strong need for effective treatments which can work against serious fungal conditions. Ciclopirox olamine (CPO) belong to the hydroxypyridone class of compounds class II and show strong anti-fungal activity. These drugs act through a complex mechanism, affecting several metabolic processes in fungal cells, which makes them effective antifungal agents.¹ These synthetic, broad-spectrum antifungal drugs have been used since 1998 for the topical treatment of superficial fungal infections of the skin.² Diseases treated with ciclopirox have not shown the development of drug resistance, even after many years of continuous use. This suggests that the drug acts on molecular targets that are essential for the survival of fungal cells.³ Ciclopirox olamine is widely used for the treatment of various superficial fungal infections.⁴

From a pharmaceutical perspective, the presence of main group elements such as carbon, oxygen, and nitrogen in Ciclopirox Olamine plays a critical role in determining its physicochemical properties, including solubility, stability, and interaction with biological systems. These elements also contribute to the chromophoric behaviour essential for UV absorption, enabling accurate quantitative analysis.

The proposed analytical method is therefore suitable for the determination of Ciclopirox Olamine in both API and ethosomal dosage forms. This work highlights not only the analytical efficiency of UV–Visible spectroscopy but also the fundamental importance of main group chemistry in governing drug behaviour and analytical response. The method is rapid, cost-effective, requires minimal solvent consumption, and is non-destructive, making it highly valuable for routine pharmaceutical analysis.

This article aims to serve as a useful reference for researchers, academicians, and quality control analysts, while also emphasizing the critical role of main group elements in modern pharmaceutical analytical science.

Chemical structures

Ciclopirox, chemically known as 6-cyclohexyl-1-hydroxy-4-methylpyridin-2(1H)-one (Figure 1(a)), is a cyclic hydroxamic acid. Its olamine (ethanolamine) salt form is known as ciclopirox olamine (6-cyclohexyl-1-hydroxy-4-methylpyridin-2-one and 2-aminoethanol) (Figure 1(b)).

Figure 1.

Chemical structure of ciclopirox(a) and ciclopirox olamine (b).

Chemical formula

The formula of ciclopirox olamine is C₁₄H₂₄N₂O₃, and it has a molecular mass of 268.4 g/mol.

Physical and chemical properties

Ciclopirox olamine is a white to off-white crystalline powder that is freely soluble in water. It is stable under normal temperature and pressure conditions and does not readily react with other chemical groups or undergo decomposition. Additionally, it is non-volatile, meaning it does not easily evaporate into the air. The pyridin-2(1H)-one core of ciclopirox, which is a six-membered aromatic ring, is important for its chemical stability. The hydroxamic acid group at position 1 is able to form hydrogen bonds because of the lone pair of electrons on the nitrogen atom, and it also shows resonance delocalization within the ring structure.^1,5

Therapeutic activity

Ciclopirox Olamine shows a broad range of antifungal activity. It is effective against dermatophytes such as Trichophyton species, Microsporum species, and Epidermophyton floccosum; yeasts including Candida species, Malassezia furfur, Cryptococcus neoformans, and Saccharomyces cerevisiae; and molds such as Aspergillus species and Fusarium solani.

Depending on the concentration and duration of treatment, ciclopirox olamine can act as either a fungicidal or fungistatic agent. Overall, ciclopirox olaminehas been found to be more effective against many tested fungal species than itraconazole and ketoconazole, with particularly strong activity against yeasts compared to dermatophytes.^6–8

Influence of structure and physicochemical properties on therapeutic activity

The pyridinone ring of ciclopirox shows a unique combination of physicochemical properties, such as good metabolic stability, high water solubility, and balanced lipophilicity. These properties allow it to behave like a non-peptide structure and interact effectively with biological targets. Both the free hydroxyl group and the nitrogen atom in the pyridinone ring can act as hydrogen bond donors or acceptors. This enables ciclopirox to form multiple hydrogen bonds with biological targets, including enzymes and receptors. Such interactions are essential for its antifungal and antibacterial activity, as they improve binding strength and specificity.^2,9

Mechanism of action

Ciclopirox Olamineis a hydroxypyridone antifungal agent that forms stable complexes with trivalent metal ions, particularly iron (Fe³⁺). This chelation inhibits metal-dependent enzymes responsible for mitochondrial energy production, electron transport, and detoxification of reactive oxygen species. Furthermore, ciclopirox alters fungal cell membrane permeability and inhibits DNA repair processes, contributing to its fungistatic and fungicidal effects depending on concentration and exposure duration.¹⁰ It inhibit squalene epoxidase and thus interferes with ergosterol biosynthesis.¹¹

Materials and methods

Materials

Reference Standard of Ciclopirox Olamine Active Pharmaceutical Ingredient was provided by Kumar Organics Products Limited, Bangalore, and Ethanol from Central Drug House Pvt. Ltd

Instruments

The Ultra Violet-Spectroscopy double Beam manufacturer by Agilent Tech, Digital Weighing Balance TX323L by Shimadzu was used.

Preparation of standard stock solution of ciclopirox olamine [solvent: distilled water and ethanol (50:50)]

Weigh Accurately about 100 mg of the drug and transferred to 100 ml volumetric flask. Add a few ml of solvent to dissolve the drug completely and volume make up was done up to the mark.

Transferred 2.5 ml of stock solution to 50 ml volumetric flask and dilute up to the mark with solvent. This solution contained 50μg of drug per ml of the solution.

Selection of wavelength maxima (λ max)

0.5,1, 2, 3, 4 and 5 ml of the standard stock solution were pipette out and transferred into a series of 10 ml volumetric flask. The volumes were made up to the mark with solvent and mixed to obtain solutions in the concentration rang 2.5, 5, 10, 15,20, 25μg/ml.

The absorbance of this solution was scanned in the UV range of 180–400 nm against solvent as blank and was found to be 302 nm. The result was shown (Figure 2 and Figure 3).

Figure 2.

U.V scan of ciclopirox olamine in the range of 180–400 nm and found to be at 302 nm.

Figure 3.

Overlay Spectra of ciclopirox olamine.

Preparation of calibration curve for ciclopirox olamine at 302 nm

The absorbance of these resultant solutions was measured at 302 nm against solvent as blank and graph were plotted between absorbance obtained versus concentrations. The linearity of drug was found 2.5 to 25 ug/ml at 302 nm with co-relation coefficient 0. 9996.The results were shown in (Figure 4) & (Table 1).

Figure 4.

Calibration curve of ciclopirox olamine.

Table 1.

Linearity, range, E^1%_1CM, absorptivity (L gm-¹ cm-¹), & molar absorptivity (L mol-¹ cm-¹).

S. no.	Concentration (microgram/Ml)	Absorbance	Absorptivity	E1%1cm	Molar absorptivity (Mwt-268.35)
1	2.5	0.112	44.80	448.00	12022.08
2	5	0.217	43.40	434.00	11646.39
3	10	0.435	43.50	435.00	11673.23
4	15	0.658	43.87	438.67	11771.62
5	20	0.853	42.65	426.50	11445.13
6	25	1.092	43.68	436.80	11721.53
Mean			43.65	436.49	11713.33

Λ max determination

Maximum absorbance observed at 302 nm (Table 2)

Table 2.

Validation parameters (ICH Q2(R2)).

Parameter	Result	Acceptance criteria
Linearity	2.5–25 µg/mL	R² ≥ 0.999
Repeatability (%RSD)	0.98	≤ 2%
Intra-day Precision	1.448	≤ 2%
Inter-day Precision	1.55	≤ 2%
Accuracy (%RSD)	0.27	≤ 2%
Specificity	No interference	No interference
Parameter	Result	Acceptance Criteria

Spectrum recorded in the range of 200–400 nm

Sample preparation details

Stock solution: Prepared by dissolving accurately weighed Ciclopirox Olamine in suitable solvent (Ethanol: water)

Working solutions: Prepared by serial dilution to obtain required concentrations

Analysis of ethosomal gel

Sample extracted using suitable solvent

Filtered and diluted appropriately

Analysed at λmax = 302 nm

Statistical analysis

Mean, standard deviation (SD), and %RSD calculated for all validation parameters

All results were within acceptable ICH limits.

Ultraviolet–Visible (UV–Visible) spectroscopy is one of the most widely used analytical techniques in pharmaceutical research, universities, industries, and quality control laboratories. This technique offers high sensitivity, accuracy, precision, and reproducibility, providing a robust platform for drug discovery, development, and routine quality assurance.

This short communication presents a comprehensive overview of UV–Visible spectroscopy, method development strategies, and validation parameters in accordance with International Council for Harmonisation (ICH) guidelines Q2(R2) for the determination of Ciclopirox Olamine in both active pharmaceutical ingredient (API) and ethosomal gel formulations.

The UV spectroscopic method developed for Ciclopirox Olamine showed maximum absorbance (λ max) at a wavelength of 302 nm. The drug exhibited good linearity in the concentration range of 2.5–25 µg/mL, with a correlation coefficient (R²) of 0.9996. The method was found to be specific, as no interference from excipients was observed.

Precision studies, including repeatability, inter-day precision, intra-day precision, specificity, and accuracy, showed %RSD values of 0.98, 1.55, 1.448, 0.032, and 0.27, respectively. All values were within the acceptable limit (%RSD < 2%).

The proposed analytical method is suitable for the analysis of Ciclopirox Olamine in both API and ethosomal dosage forms. This article aims to serve as a useful reference for researchers, academicians, and quality control analysts involved in pharmaceutical analysis.

UV–Visible spectroscopy is widely used in pharmaceutical analysis due to its rapid analysis, cost-effectiveness, minimal solvent consumption, and non-destructive nature (Table 3).

Table 3.

Comparative structural features and mechanisms of antifungal compounds.

Compound	Core structure	Key functional group	Mechanism
Ciclopirox	Pyridinone	Hydroxamic group	Metal chelation
Ciclopirox Olamine	Salt form	Amine + hydroxamic	Enhanced solubility
Hydroxamic Acid	Simple chain	–CONHOH	Metal binding
Azoles	Imidazole/Triazole	N-heterocycles	CYP450 inhibition

All experimental data were expressed as mean ± standard deviation (SD). The % relative standard deviation (%RSD) was calculated to assess precision. The acceptance criterion for validation parameters was set as %RSD < 2%, in accordance with ICH guidelines.

Validation of the method for estimation of ciclopirox olamine according to I.C.H. guideline (Q2R2)

Repeatability

0.5 ml of the standard stock solution were pipette out and transferred into a series of six 10 ml volumetric flask. The volumes were made up to the mark with solvent (ethanol and water) and mixed to obtain solutions concentration 2.5 μg/ml. The absorbance of this solution was measured six times and recorded at 302 nm. The results are shows in table (Acceptance criteria: RSD should be less than 2%)

NOTE: Keeping the concentration of the drug same, procedure was repeated for 6times. The calculated %RSD for repeatability study is 0.98 which is acceptable; this shows good repeatability of method in below table 4.

Table 4.

Results of repeatability studies for ciclopirox olamine.

Nominal conc.(µg/ml)	Absorbance	Observed conc.(µg/ml)	Mean conc.(µg/ml)	SD	%RSD
2.5	0.1125 0.1132 0.1154 0.1143 0.1154 0.1132	2.58 2.59 2.64 2.62 2.64 2.59	2.61	0.03	0.98

Inter-Day precision

3 ml of the standard stock solution was taken in 10 ml volumetric flasks and volume was made up to the mark with solvent system to obtain solution of concentration 15 µg/ml. The absorbance of nine solutions individually was measured thrice different days and recorded. The result are shows in the table 5. (Acceptable criteria: RSD should be less than 2%)

Table 5.

Results of inter-day precision.

Nominal conc. (µg/ml)	Absorbance			Observed conc.(µg/ml)			Mean conc. (µg/ml)	SD	%RSD
Nominal conc. (µg/ml)	0 h	24 h	48 h	0 h	24 h	48 h	Mean conc. (µg/ml)	SD	%RSD
15	0.658	0.638	0.662	15.07	14.98	15.16	14.99	0.23	1.55
15	0.656	0.661	0.674	15.02	15.14	15.44
15	0.623	0.681	0.641	14.89	15.6	14.99

The reading was taken in triplicate for each concentration at 0hr, 24hr,48 h in three different days. The calculated mean RSD was 1.55.

Intra-Day precision

3 ml of the standard stock solution was taken in 10 ml volumetric flasks and volume was made up to the mark with solvent system to obtain solution of concentration 15 µg/ml. The absorbance of these nine solutions was measured thrice within a day and recorded. The result are shows in the table 6. (Acceptable criteria: RSD should be less than 2%)

Table 6.

Results of intra-day precision.

Nominal conc.(µg/ml)	Absorbance			Observed conc.(µg/ml)			Mean conc.(µg/ml)	S. D	%RSD
Nominal conc.(µg/ml)	0 h.	3 h.	6 h.	0 h.	3 h.	6 h.	Mean conc.(µg/ml)	S. D	%RSD
15	0.64	0.628	0.654	14.99	14.98	14.98	15.03	0.218	1.448
15	0.693	0.631	0.673	15.12	14.99	15.41
15	0.659	0.652	0.679	15.09	14.93	15.55

The reading was taken in triplicate for each concentration at 0hr, 3hr,6hr. within a day. The calculated mean RSD was 1.448.

Specificity

A volume of 3 mL of the standard stock solution was transferred into 10 mL volumetric flasks, and the volume was made up to the mark with the ethanol–water solvent system to obtain solutions of 15 μg/mL concentration, prepared both with and without excipients. In total, twelve solutions were prepared under these conditions. The absorbance of each solution was measured at 302 nm using a UV–Visible spectrophotometer, and the results are presented in Table 7.

Table 7.

Results of specificity study.

Nominal	Without excipients		With excipients		%Interference
Conc.(µg/ml)	Absorbance	Observed Conc.(µg/ml)	Absorbance	Observed Conc.(µg/ml)	%Interference
15	0.658	15.07	0.653	14.95	0.12
15	0.656	15.02	0.654	14.98	0.04
15	0.655	15	0.658	15.07	−0.07
15	0.657	15.05	0.656	15.02	0.03
15	0.654	14.98	0.653	14.95	0.03
15	0.659	15.09	0.657	15.05	0.04
Mean					0.032

It can be concluded from the result that developed method is specific and there is no interference of excipients, since the %interference was negligible (0.032).

Accuracy studies

Pipette out 2 ml standard solution of API and 1.6 ml of standard solution of formulation and transfer into three 10 ml volumetric flasks respectively. Again 2 ml of standard solution of API and 2 ml of standard solution of formulation and transfer into three 10 ml volumetric flasks respectively. Again 2 ml of standard solution of API and 2.2 ml of standard solution of formulation and transfer into three 10 ml volumetric flasks respectively.

Absorbance of the resultant solutions was measured at 302 nm using ethanol and water as blank. The result obtained is summarized in the table 8 below.

Table 8.

Results of accuracy study.

S.NO	Recovery at 80%, 100% & 120%	Absorbance	Conc.(µg/ml)	Observed con(µg/ml)	%Recovery	S. D	R.S. D
	80%	0.782	18	17.92	99.53	0.60	0.27
1.	80%	0.77890	18	17.84	99.13
	80%1	0.7785	18	17.84	99.08
	100%	0.863	20	19.75	98.77
2.	100%	0.8717	20	19.97	99.85
	100%	0.865	20	19.85	99.26
	120%	0.9621	22	22.04	100.19
3.	120%	0.9655	22	22.12	100.54
	120%	0.9621	22	22.04	100.19

Percentage assay of ciclopirox olamine ethosomal gel (0.77/100gm)

An accurately weighed quantity of ethosomal gel equivalent to 7.7 mg of Ciclopirox Olamine was transferred into a 100 mL volumetric flask, and the volume was made up to the mark with the ethanol–water solvent system. The solution was shaken thoroughly and sonicated for 5 min to ensure complete dispersion, followed by filtration. From the filtrate, 2 mL was transferred into each of three separate 10 mL volumetric flasks, and the volume in each flask was made up to the mark with the same solvent system. This procedure was repeated for five formulations, and the absorbance of the resulting solutions was measured at 302 nm using a UV–Visible spectrophotometer. The result was shown in table 9 and Overlay Spectra Graph of Validation Methods Figure 5)

Figure 5.

Overlay Spectra graph of validation methods.

Table 9.

Percentage assay of ciclopirox olamine in pharmaceutical ethosomal dosage form.

S.NO	FORMULATIONS	Conc.(µg/ml)	Absorbance	Observed conc.(µg/ml)	%ASSAY	Mean	MEAN	SD	RSD
1	F1	10	0.422	9.7	97.01	98.62	98.19	1.6	1.612
		10	0.434	9.98	99.77
		10	0.431	9.91	99.08
2	F2	10	0.438	10.07	100.69	98.77
		10	0.427	9.82	98.16
		10	0.424	9.75	97.47
3	F3	10	0.415	9.54	95.4	97.47
		10	0.428	9.84	98.39
		10	0.429	9.86	98.62
4	F4	10	0.423	9.72	97.24	97.16
		10	0.425	9.77	97.7
		10	0.42	9.66	96.55
5	F5	10	0.436	10.02	100.23	98.93
		10	0.419	9.63	96.32
		10	0.436	10.02	100.23

Result

The wavelength maximum of Ciclopirox Olamine was determined to be 302 nm in the ethanol–water solvent system. The drug exhibited excellent linearity across the investigational concentration range of 2.5–25 μg/mL, with a correlation coefficient of 0.9996. The calibration equation was obtained as y = 0.044x – 0.0008. Compliance with Beer–Lambert's law was confirmed in the range of 5–25 μg/mL.

The absorptivity and molar absorptivity of Ciclopirox Olamine were calculated as 43.65 L·mol⁻¹·cm⁻¹ and 11,713.33 μg/cm²/0.001, respectively. The relative standard deviation (RSD) for the repeatability study was 0.98, which is within acceptable limits, indicating good repeatability of the method. The mean RSD values for inter-day precision and intra-day precision were 1.55 and 1.448, respectively, both meeting the acceptable criterion of RSD < 2%.

The method demonstrated high specificity, with negligible interference from excipients (% interference = 0.032). The accuracy was confirmed with an RSD of 0.27%, well below the 2% threshold. The assay of Ciclopirox Olamine in pharmaceutical dosage form yielded a mean value of 98.19%, with an RSD of 1.612, confirming the reliability and robustness of the developed spectroscopic method.

Statistical analysis

Discussion

A novel, modest, and delicate spectroscopic method was developed for the quantitative estimation of Ciclopirox Olamine in both pure form and ethosomal gel dosage, using for the first time a solvent system composed of ethanol and water. Ciclopirox Olamine exhibited a wavelength maximum at 302 nm in this solvent system. The developed method demonstrated excellent linearity across the concentration range of 2.5–25 μg/mL, with a correlation coefficient of 0.9996. The regression equation was determined as Y = 0.044x + 0.0008.

This technique, established for the first time in the ethanol–water solvent system, provided precise results. The relative standard deviation (RSD) was consistently below 2% for repeatability, inter-day precision, intra-day precision, specificity, and accuracy. No significant interference was observed in the absorbance of Ciclopirox Olamine in the presence of common excipients. The method was statistically validated in accordance with ICH Guidelines (Q2R2).

Furthermore, the technique was successfully applied for the quantitative determination of Ciclopirox Olamine in ethosomal gel dosage forms. In conclusion, the developed spectroscopic method is simple, specific, reproducible, and reliable, making it suitable for routine analysis of Ciclopirox Olamine in bulk drug and novel ethosomal gel formulations using the newly established ethanol–water solvent system.^12–15

Footnotes

ORCID iD

Saloni Jaswal

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

References

Singh

Kaur

Chintamaneni

, et al. Exploring the versatility of ciclopirox—from antifungal to anticancer agent and beyond. European Journal of Clinical and Experimental Medicine 2023; 21: 896–908.

Lin

Liu

Zhao

, et al. Recent advances of pyridinone in medicinal chemistry. Front Chem 2022; 10: 869860.

Zangi

Donald

Casals

, et al. Synthetic derivatives of the antifungal drug ciclopirox are active against herpes simplex virus-2. Eur J Med Chem 2022; 238: 114443–114462.

Tang

Pudney

PDA

Lane

. Investigation of piroctone olamine delivery to the skin from single, binary and ternary solvent systems. Int J Cosmet Sci 2024; 46: 357–367.

Shen

Huang

. Repositioning the old fungicide ciclopirox for new medical uses. Curr Pharm Des 2016; 22: 4443–4450.

Zhang

Pike

. Pyridones in drug discovery: recent advances. Bioorg Med Chem Lett 2021; 38: 127849.

Lin J, Zangi M, Kumar TVNH, et al. Synthetic derivatives of ciclopirox are effective inhibitors of Cryptococcus neoformans. ACS Omega 2021; 6: 8477–8487.

Seidl HP, Jackel A, Müller J, et al. Sporicidal effect of amorolfine and other antimycotics used in the therapy of fungal nail infections. Mycoses 2015; 58: 610–619.

Mucha

Borkowski

Erkiert-Polguj

, et al. Ciclopirox and ciclopirox olamine: antifungal agents in dermatology with expanding therapeutic potential. Appl Sci 2024; 14: 11859.

10.

Pant

, et al. Ciclopirox suppresses poxvirus replication by targeting iron. Science Direct 2025. Antiviral Research 2025; 244: 106290.

11.

Kumar

Chaudhary

Rathi

, et al. Validated analytical method development for drug residue (clonazepam) in pharmaceutical industries by RP-HPLC. In: AIP Conference Proceedings, 2800(1), 2017.

12.

Rawat

, et al. Validated method development and validation for estimation of telmisartan as API and in pharmaceutical dosage form by UV-spectroscopy. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2014; 5: 679–685.

13.

Chaudhary

, et al. Validated method development for estimation of ketamine HCl as API and in pharmaceutical dosage forms by UV-spectroscopy. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2014; 5: 686–692.

14.

Dahiya

Saini

Singh

, et al. A novel quality by design compliant approach for the determination of albendazole in bulk and pharmaceutical dosage form using RP-HPLC. AIP Conf Proc 2020; 2800: 020030.

15.

Chaudhary

Kumar

Chaudhary

, et al. Validated method development for estimation of Ketamine HCL as API and in pharmaceutical dosage forms by UV-spectroscopy. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2014; 5: 686–692.