INTRODUCTION:

Miconazole, an antifungal drug of class imidazole, was approved by FDA in 1974. Miconazole nitrate is chemically called as 1-[(2RS)-2-[(2,4-dichlorobenzyl) oxy]-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole nitrate.^[1] It shows its action against fungi by inhibiting the ergosterol synthesis, hinders the membrane’s barrier function and enzymes bound to the membrane.^[2] It is used to treat skin infections such as Jock itch, Athelete’s foot, ringworm, etc. It is also administered orally to treat intestinal and oropharyngeal candidiasis in the form of gel.^[3]

Figure 1. Chemical structure of Miconazole nitrate

Miconazole nitrate (MCN) topical formulations are available in the market with brand name- Micogel, Micofact, Miconazole nitrate vaginal cream in 2% strength.

UV spectroscopy which also refers to as absorption or reflectance spectroscopy, uses the entire ultra-violet and visible range. The absorption of electromagnetic radiation of this region results from the transition that occurs between the electronic levels. UV spectroscopy has variety of application such as quantitative estimation of compounds which absorb UV radiations, structural elucidation (presence or absence of hetero atom and unsaturation), determination of impurities in organic molecules, etc.^[4]

As a part of the regulatory requirements for marketing a product, it is mandatory to prove that a drug substance, or developed dosage form fulfills the quality, safety, and efficacy parameters. To develop and validate a suitable analytical procedure for drug substance and its subsequent drug products is considered as one of the most important parameters among the quality requirements (ICH Q2).^[5]

Though after reviewing literature, it has been revealed that there are discrete analytical procedures exist for estimation of Miconazole nitrate, UV spectrophotometry is the simplest laboratory technique for the estimation of drugs, therefore, in the present study, an effort has been initiated to develop and validate a simple, specific, accurate and sensitive analytical UV-method to quantify MCN in bulk and topical dosage form.

METHODOLOGY:

Materials and Instrument:

MCN was taken as a gift sample from Sharon Bio-Medicine Ltd., India and commercial topical formulation (Micogel Cream; manufactured by Cipla Ltd.) containing MCN was purchased locally from a pharmacy. Ethanol used was of analytical grade. A double beam UV-visible spectrophotometer (Shimadzu corporation) was used to measure absorbance of the resulting solutions over a range of 200-400 nm.

Preparation of standard stock solution:

A standard stock solution with concentration of drug 1 mg/ml was prepared by dissolving MCN in 98% ethanol. From this solution, a solution with a strength of 100 μg/ml was prepared. Using this working solution, different concentrations ranging 2-10 μg/ml were prepared. UV spectrum of MCN solution showed absorption maximum at 232 nm after scanning between 200-400 nm.^[6]

Preparation of sample stock solution (Marketed product):

An aliquot of cream equivalent to 25 mg of drug was taken and dissolved in 25 ml of ethanol. This solution was shaken for 15 minutes and then filtered by Whatman filter paper. From the above stock solution, the solution with concentration 100μg/ml was prepared. From above solution, the concentrations of 2, 4, 6, 8, 10μg/ml were prepared using ethanol as a solvent. Absorbance of these dilutions was measured at 232nm wavelength.

VALIDATION OF DEVELOPED METHOD:

Purpose:

Analytical validation is an effective tool which establishes a documented evidence that possesses high degree of assurance and meets the required objective at each stage of drug development, giving consistent and reproducible result at its given specification and other quality characteristics. The parameters like accuracy, linearity, sensitivity, repeatability, robustness, precision, and ruggedness following ICH Q2B (R1) recommendations were used to validate the developed procedure.^[5]

Linearity:

In this study, different concentration solution of MCN drug were prepared ranging from 2-10μg/ml using the standard stock solution. The relationship between absorbance and concentration was described by Beer’s law, which states that with decrease in intensity of monochromatic light, the number of absorption molecules increases. A linearity graph obeying Beer-Lambert’s law is represented by regression equation (y= mx + c) using concentrations (2-10μg/ml). Here, ethanol was used as blank and the obtained data was used to detect the linearity in calibration curve. Correlation coefficient (r²) was also computed.^[7]

Accuracy:

Accuracy of the developed method was checked at 3 different levels by percentage recovery experiments performed at, i.e. 80, 100, and 120%. To the pre-analyzed sample solutions, known amounts of standard MCN solutions were added, re-analyzed by recording absorbances through developed method. By using given formula, the percentage recovery was calculated^[8]:

% Recovery = (A-B)/C X 100………………………(eq.1.)

Where,

A = The total amount of drug estimated;

B = The amount of drug found on pre-analyzed basis;

C = Amount of bulk drug added.

Precision:

Analysis was performed in three replicates during 3 consecutive days (inter-day precision) and same day (intra-day precision).

Intra-day precision (same day) was established by analysing standard MCN solution at three different time points with a particular concentration and inter-day precision (different days) was determined by analysing drug solution at three different time points along with %RSD (relative standard deviation) calculation.^[9]

Repeatability:

Repeatability of the method was established by evaluating 6 samples with same drug concentrations (10 μg/ml). From the resulting absorbances, the standard deviation and RSD were calculated.^[10]

Sensitivity:

Sensitivity of the method was evaluated on the basis of 2 parameters namely limit of quantitation (LOQ). and limit of detection (LOD). LOQ is the minimum quantifiable concentration while LOD is the lowest concentration of the analyte that can be detected by the method.

It is based on residual standard deviation of regression line and slope. For this purpose, the sample containing analyte in the range of detection limit and quantization limit was studied through the specific calibration curve. LOD and LOQ were calculated using following equations^[11]:

LOD (μg/ml)= 3.3 x SD/S…………………..…….(eq.2.)

LOQ (μg/ml)= 10 x SD/S……………………........(eq.3.)

where,

SD = the standard deviation of the response

S = the slope of the calibration curve (mean).

Robustness:

Robustness of the analytical method was carried out to know the effect, if small changes in wavelength for the analytical performance was made. In this parameter, the absorbance of standard solution (10μg/ml) was measured by varying detection wavelengths (±2 nm) of optimized conditions from the standard detection wavelength (232 nm). The variation at two different wavelengths (230 nm and 234 nm) form the assay value was measured.^[12]

Ruggedness:

Ruggedness of the method was determined by checking the standard solution having 10𝜇g/ml concentration of MCN repeatedly for six times using the same environmental and experimental conditions by two different analysts. Standard deviation and % RSD were determined.^[7]

Analysis of pharmaceutical formulation:

A sufficient amount of cream equivalent to 25mg of Miconazole nitrate was taken and solubilised in few ml of ethanol. The resulting solution was sonicated for 5 minutes and then after filtered by Whatman filter paper and the final volume was made up to 25 ml by adding ethanol to it. From the clear solution, further dilution was made to get 10µg/ml of MCN. The resulting solution was evaluated through the developed method. The data obtained was substituted in the regression equation and percentage purity was determined.^[13]

RESULT AND DISCUSSION:

The purpose of the study was to develop an analytical method for estimation of Miconazole nitrate under the suitable spectral conditions. Ethanolic solution of MCN showed absorbance maximum (𝜆_max) at 232 nm, thus, reported analysis was done at 232 nm.

Linearity and Range:

The linearity of MCN drug and marketed formulation was observed to be in the range of 2-10μg/ml with correlation coefficient 0.999 and 0.992 respectively. Calibration data is shown in table 1 and calibration curve is shown in Figure 2(drug) and 3(marketed formulation).

Accuracy (Recovery studies):

The recovery studies were performed by standard addition method to the pre-analyzed samples, at 80%, 100% and 120% levels of drug concentration to ensure the accuracy of the developed method, and percent recovery values were also calculated. The mean % recovery for MCN was calculated to be 98.36%. The obtained result has been presented in Table 2.

Precision:

The intra-day precision (same day) was established by evaluating the sample for 3 times on the taking time intervals of 3 hrs at 10:00 am, 01:00 pm and 04:00 pm respectively at particular concentration. The inter-day precision (3 consecutive days) was determined similarly. The results obtained from intraday and inter day precision have been presented in table 3.

Repeatability:

The repeatability of the instrument was validated by taking the absorbance of six samples of the same concentration (10 μg/ml). The mean concentration was found to be 9.009 μg/ml with %RSD 0.216.

Sensitivity:

The mean LOQ and LOD of MCN were observed to be as 2.760 μg/ml and 1.868 μg/ml respectively indicating the sensitivity of the developed method. The final results of this study are given in table 4.

Robustness:

Robustness studies assumed that the small variations in any of the variables did not significantly affect the results. The results obtained are given in the table 5.

Ruggedness:

The obtained results did not show any major statistical difference between 2 different analysts suggesting the ruggedness of the method developed. The results have been shown in table 6.

Analysis of pharmaceutical formulation:

The percentage of MCN in the marketed formulation (Micogel Cream) was determined using the prepared calibration curve of drug. The labelled claim percentage present in marketed formulation was found to be 99.74 ± 0.254.

Table 1. Calibration data of standard MCN and its marketed formulation

Sr. No.	Conc. (µg/ml)	Mean Absorbance (n=3) (standard MCN)	Mean Absorbance (n=3) (MCN formulation)
1	2	0.103	0.102
2	4	0.200	0.158
3	6	0.294	0.269
4	8	0.407	0.358
5	10	0.498	0.448

Figure. 2. Calibration curve of Miconazole nitrate at 232 nm

Figure.3. Calibration curve of Miconazole nitrate cream at 232 nm

Table 2. Accuracy studies of Miconazole Nitrate

Level of % Recovery	Initial conc. (μg/ml)	Added drug conc. (μg/ml)	Total conc. (μg/ml)	Total conc. Recovered (μg/ml)	% Recovery	Mean Recovery
80	10	8	18	17.49	88.55	98.36
100	10	10	20	19.73	99.09
120	10	12	22	21.84	99.25

Table 3. Precision studies of Miconazole nitrate

Intra-Day Precision
Concentration taken (µg/ml)	*Concentration found (µg/ml) (n=3)**			Mean	SD	% RSD
Concentration taken (µg/ml)	10:00 am	01:00 pm	04:00 pm	Mean	SD	% RSD
10	9.79	9.611	9.683	9.6947	0.090	0.929
9	8.727	8.588	8.501	8.605	0.114	1.325
8	7.424	7.628	7.815	7.622	0.196	2.566
Inter-Day Precision
Concentration taken (µg/ml)	*Concentration found (µg/ml) (n=3)**			Mean	SD	%RSD
Concentration taken (µg/ml)	Day 1	Day 2	Day 3	Mean	SD	%RSD
10	9.416	9.619	9.701	9.5787	0.147	1.532
9	8.667	8.398	8.457	8.507	0.141	1.662
8	6.395	6.626	6.716	6.579	0.166	2.517

Table 4. LOD and LOQ of Miconazole nitrate

Drug	LOD (µg/ml) (n=3)	LOQ (µg/ml) (n=3)
Miconazole nitrate	1.868	2.760

Table 5. Robustness studies of Miconazole nitrate

Sr. No.	Conc. taken (µg/ml)	Conc. (µg/ml) at 232 nm	Conc. (µg/ml) at 234 nm
1	10	10.22	8.77
2	10	10.22	8.66
3	10	10.32	8.77
4	10	10.38	9.19
5	10	10.43	9.32
Mean		10.300	10.314
Standard deviation		0.890	0.094
% RDS		0.860	0.913

Table 6. Ruggedness studies of Miconazole nitrate

Sr. No.	Conc. taken (µg/ml)	Conc. observed (µg/ml) by analyst 1	Conc. observed (µg/ml) by analyst 2
1	10	9.71	7.995
2	10	9.83	8.172
3	10	10.11	8.273
4	10	10.246	8.389
Mean		9.974	8.207
Standard deviation		0.246	0.166
% RDS		2.475	2.034

Table 7. Summary of validation parameters

Sr. No.	Parameters	Results
1	λ_max	232 nm
2	Linearity RANGE	2- 10 µg/ml
3	Regression equation (y)	0.0499x + 0.0011
4	Regression coefficient (R²)	0.999
5	Repeatability (%RDS)	0.216
6	Accuracy	98.36
7	Precision
	Intra-day	1.606
	Inter-day	2.077
8	Sensitivity (µg/ml)
	Limit of detection	1.868
	Limit of quantitation	2.760
9	Robustness (%RDS)
	232 nm	0.860
	234 nm	0.913
10	Ruggedness (%RDS)
	Analyst 1	2.475
	Analyst 2	2.034

CONCLUSION:

Analytical method by UV spectrophotometry method provides precise, simple, accurate and rapid analytical method for the estimation of Miconazole nitrate. The present analytical method was validated as per ICH Q2 (R1) guideline and it was able to meet to specific acceptance criteria. The statistical data of the developed method were reproducible. In addition, the reliability, validity and usability of the method was sound and could be replicated with ease for future research practices and endeavours. It is concluded that the developed analytical method was precise, accurate, robust, specific, linear, and possess stability indicating characteristics. The present developed analytical method can be used for its intended purpose.

CONFLICT OF INTEREST:

Authors have no conflicts to declare.

ACKNOWLEDGEMENT:

The authors are thankful to Dr. Monica Gulati, Dean, Lovely School of Applied medical Sciences, LPU, Phagwara for valuable suggestions, inspiration and providing facilities.

REFERENCES:

1. Bennett JE. Antimicrobial Agents: Antifungal Agents in The Pharmacological Basis of Therapeutics, J.G. Hardman, L.E. Limbird, and A. Goodman Gillman, McGraw-Hill, New York, 2001, 1175–1190.

2. Fothergill AW. Miconazole: a historical perspective. Expert Review of Anti-infective Therapy. 4: 2006:171-175.

3. Cuesta CG, Pérez MGS and Bagán JV. Current treatment of oral candidiasis: A literature review. Journal of Clinical and Experimental Dentistry. 6(5): 2014: e576-e582.

4. Araujo P. Key aspects of analytical method validation and linearity evaluation. Journal of chromatography B, 877(23):2009:2224-2234.

5. International Conference on Harmonization, Validation of Analytical Procedures: Text and Methodology Q2 (R1), EMEA, Netherlands, 2006.

6. Abbas N, Arshad M S, Hussain A, Irfan M, Ahsan M. Rasool MF and Rehman MH. Development and validation of a spectroscopic method for the simultaneous analysis of Miconazole nitrate and hydrocortisone acetate in pharmaceutical dosage form. Tropical Journal of Pharmaceutical Research, 16(2): 2017: 413-420.

7. Kumar R., Singh S, Kamal S.S, Kaur D, Singh M and Katual MK. Development and Validation of Spectrophotometric Method for Estimation of Levosulpiride in Bulk and Tablet Dosage Form. Eurasian Journal of Analytical Chemistry. 12(3): 2017: 265-273.

8. Bhimani S, Sanghvi G, Pethani T, Dave G, Airao V, Sharma T and Vaishnav D. Development of the UV spectrophotometric method of Azithromycin in API and stress degradation studies. Internationl Letters of Chemistry, Physics and astronomy. 68:2016: 48-53.

9. Eticha T, Kahsay G, Hailu T, Gebretsadikan T, Asefa F, Gebretsadik H and Thangabalan B. Development and Validation of an Extractive Spectrophotometric Method for Miconazole Nitrate Assay in Pharmaceutical Formulations. Journal of Analytical Methods in Chemistry. 2018: 1-5.

10. Kamal SS, Singh S, Kaur D, Sharma A, Katual MK and Kumar R. Development and Validation of UV-Spectrophotometric Method for Estimation of Ibuprofen in Bulk and Marketed Tablets. American Journal of Pharmtech Research. 6(1):2015: 197-203.

11. Kaur M, Bhardwaj P, Kaur B, Sharma A, Kaur C and Kumar R. Development and Validation of a Novel Stability Indicating UV Spectrophotometric Method for Estimation of Febuxostat in Bulk and Pharmaceutical Formulation (Tablets). Pharmaceutical Methods, 9(1):2018: 23-28.

12. Das J and Dhua M. UV-spectrophotometric assay method development and validation of metronidazole in bulk and tablet formulation. Journal of Pharmaceutical Science and Technology. 3(2):2014:106-109.

13. Sheliya K, Shah K and Kapupara P. Development and validation of analytical method for simultaneous estimation of mometasone furoate, hydroquinone and tretinoin in topical formulation by RP-HPLC. Journal of Chemical and Pharmaceutical Research. 6(4):2014: 934-940.

Received on 04.05.2019 Modified on 10.06.2019

Research J. Pharm. and Tech. 2019; 12(10):4999-5003.

DOI: 10.5958/0974-360X.2019.00866.7