Development And Validation of a Simple Stability Indicating High Performance Liquid Chromatographic Method for The Determination of Miconazole Nitrate in Bulk and Cream Formulations

Miconazole is an antifungal imidazole derivative with a broad spectrum of activity, effective against dermatophytes, Candida albicans, and other Gram-positive bacteria. It is utilized in diverse pharmaceutical formulations, encompassing injections, pills, oral gels, creams, ointments, topical powders, and vaginal suppositories. The predominant dosage forms comprise creams, ointments, gels and additional ointments for dermatitis, diaper dermatitis, mucoadhesive buccal patches and extended-release miconazole bio adhesive buccal tablets for oropharyngeal candidiasis. Pessaries including metronidazole and miconazole nitrate are utilized for vulvovaginal infections. The methodologies outlined by the USP and British Pharmacopoeia for the assay of miconazole nitrate and its related substances or degradation products are varied. The assay of bulk material is conducted via potentiometric altitrimetry, while related compounds are assessed using an isocratic high- performance liquide chromatography (HPLC) method. No analytical chromatographic techniques have been documented for the routine quantitative determination of miconazole nitrate in bulk, and only a few procedures for its assessment in pharmaceutical formulations have been introduced. Cream analysis generally represents a difficult task for analysts, essentially due to the complexity of the base cream or matrix in which the analyte of interest is immersed. It usually contains vase line, paraffine, cetotheriid alcohol or other lipophilic components which are poorly soluble in the solvents commonly used in HPLC. For this reason, the assay method requires an extractive procedure prior to the chromatographic step in order to ensure the elimination of interfering components, avoiding the precipitation in the chromatographic system.In the present report, we present a strategy which allows for the simultaneous quantification and stability evaluation of miconazole nitrate in bulk and cream formulations through a simple, rapid, precise, accurate and specific HPLC-UV method with an efficient and extremely easy extractive procedure. It is interesting to note thata reversed-phase HPLC method for determination of econazole nitrate in cream formulations has been recently developed using miconazole nitrate as an internal standard. However, in the presently proposed method, the separation was achieved in a shorter retention time and consuming a lower amount of sol vent. On the other hand, the results obtained in the validation step suggest that it is not necessary to employ an internal standard, simplifying the work. Moreover, in comparison with the methods described in the

Littérature, Our chromatographic system needs a simpler mobile phase composition and no buffer, reaching excellent resolution and peak symmetry.

Fig. 1. Structural Formulae for Miconazole Nitrate

Drug profile:

Experimentation

Chemicals and Reagents

A miconazole nitrate working standard was prepared in-house from raw material, which was characterized and assayed in accordance with the specifications of the 30th edition of the United States Pharmacopeia (USP). Miconazole nitrate bulk material, creams containing (i) 2.0% w/w miconazole nitrate, or (ii) 2.0% miconazole nitrate, 0.1% betamethasone 17-valerate, and 0.1% gentamicin sulfate, as well as the base cream used in the formulations, were all obtained from

Laformedsa (Formosa, Argentina).

The base cream formulation included liquid and solid paraffin, ceto-stearyl alcohol, benzyl alcohol, ceto macrogol 1000, monobasic sodium phosphate, phosphoric acid, propylene glycol, methylparaben, propylparaben, and triethanolamine. All raw materials for these components were also supplied by LAFORMEDSA. Methanol (HPLC grade) was purchased from Aberson Química, Argentina. Glacial acetic acid (analytical grade) was obtained from Laboratorios Cicarelli, Argentina. Purified water (HPLC grade) was produced using a Milli-Q® system (Millipore, Milford, MA, USA). Acidic methanol was prepared by diluting glacial acetic acid in methanol to achieve a final concentration of 0.1% (v/ v).

HPLC Instrumentation and Conditions

The HPLC system used was a Shimadzu LC-10AT, equipped with a pump, membrane degasser, thermostated column compartment, autosampler, and diode array detector (DAD). Data acquisition and processing were performed using Che Station software, version B 0103. Chromatographic separation was carried out on a ZORBAX Eclipse C18 column (4.6 mm × 150 mm, 5 µm particle size) from Agilent Technologies. The mobile phase consisted of methanol and water, delivered using a gradient elution program over 15 minutes (see Table 1 for gradient details). All solvents were filtered through a 0.45 µm pore size Nylon membrane filter prior to use. The column was maintained at 25 °C, with an injection volume of 10 µL. The DAD was set to monitor at 232 nm (bandwidth: 4 nm) with a reference wavelength of 360 nm (bandwidth: 100 nm), and operated in scan mode over a wavelength range of 200–500 nm.

Table No 1 Gradient Program Used For The Separation Of Miconazole

Method

Solutions of Standard and Stock

A stock standard solution of miconazole nitrate, at the strength of eleven milligrams per milliliter (11.0 mg/mL), was prepared by carefully weighing and dissolving a portion of the working standard in pure methanol of the highest grade. This solution, which proved stable for a span of two months, was kept in a cold chamber at four degrees Celsius (4°C), shielded from the light as if preserved in a sealed vessel. Before use, this solution was allowed to return to the warmth of the room. From this stock, daily working standard solutions were prepared by dilution in methanol, adjusting to a final concentration of 0.53 mg/mL.

Préparation of Cream for Analysis

Approximately two and two-tenths grams (2.2 g) of the thoroughly mixed cream samples were weighed with precision into vessels holding 250 milliliters (mL). To these, fifteen milliliters (15 mL) of acidic methanol were added. The vessels were covered with a glass watch and placed into a heated bath set at ninety degrees Celsius (90°C) with agitation, until the components of the cream melted. Thereafter, the vessels were removed, shaken vigorously on a magnetic stirrer for five minutes, and left to cool until the sample resolidified. This cycle of heating, shaking, and cooling was repeated twice to ensure the complete dissolution and extraction of miconazole nitrate from the matrix. The resulting suspensions were transferred to volumetric flasks of twenty-five milliliters (25.0 mL), rinsing the vessels with further acidic methanol to collect all contents. The volume was then adjusted to the mark with the same solvent. Ten milliliters (10.0 mL) of these suspensions were placed in centrifuge tubes, capped, frozen at minus twenty degrees Celsius (-20°C) for twenty minutes, and then centrifuged at 2000 times gravity (2000×g) for fifteen minutes to precipitate lipophilic cream components. The miconazole nitrate remained dissolved in the acidic methanol. Three milliliters (3.0 mL) of the clear supernatant, warmed to room temperature, were transferred to a ten milliliter (10.0 mL) flask and diluted with the mobile phase to match the initial solvent composition of the mobile phase. These solutions were placed again in fresh centrifuge tubes, frozen for twenty minutes at -20°C, and centrifuged once more under the same conditions to remove remaining lipophilic components. Finally, suitable amounts of these filtered solutions, containing approximately 0.53 mg/mL of miconazole nitrate, were passed through a 0.45  micron nylon filter into injection vials for analysis.

Validation Samples

Matrix and Excipients Solutions

A blank portion of base cream (without miconazole nitrate or active ingredients) weighing about 2.2 grams was processed identically as above. Solutions of common excipients — cetostearyl alcohol, benzyl alcohol, propylene glycol, methylparaben, propylparaben, and triethanolamine — were prepared at concentrations of 0.5 mg/mL in methanol.

Linearity Standard Solutions

Portions of stock standard solution were diluted to obtain concentrations of 0.26, 0.40, 0.53, 0.66, and 0.79 mg/mL, covering 50% to 150% of the expected analyte concentration in assay solutions.

Linearity Cream Solutions

Base cream portions (~2.2 g) were treated as before and spiked with miconazole nitrate stock solution to prepare cream solutions matching the concentrations of the linearity standard solutions.

Recovery Solutions

Samples of laboratory-prepared cream were spiked with known amounts of miconazole nitrate powder to reach 80%, 100%, and 120% of the expected analyte amount in actual samples and processed as described.

Method Development Extraction

Ancient methods described in pharmacopoeias for cream samples used simple suspensions in methanol or mixtures, by agitation or sonication at room temperature or mild heating (40–45°C), followed by filtration. However, such methods yield foamy emulsions, complicating filtration, reducing recovery, and lowering precision. Further, late precipitation of excipients threatened column and instrument integrity. The method here described, while more laborious and lengthier, is straightforward and requires no costly apparatus like solid-phase extraction or supercritical fluids.

Chromatography

The column chosen, densely packed and double end-capped with ultra-pure silica, allowed use of pure solvent mobile phases, yielding sharp and symmetric peaks. This choice offered advantages: no buffers needed, shorter stabilization times, and longer column life. Optimized gradients of methanol and flow rates ensured separation of miconazole from excipients such as parabens and degradation products within a short time. The retention time for miconazole was 8.3 minutes, stable across injections. The analysis time was set at 15 minutes to ensure all degradation products eluted without extra stabilization time. Detection was performed at 232 nm, the absorption maximum of miconazole in the eluent.

Validation According to ICH Recommendations

Limits of detection and quantitation were not evaluated here, as the method targets quantification of a major component in bulk or pharmaceutical products.

RESULT

Selectivity The selectivity of the method was evaluated injecting by the following solutions: (a) injection solvent (and: methanol), (b) a pure standard solution of miconazole nitrate 0.53mg mL−1, (c) solutions of excipients, (d) solution of matrix, and (e) a cream sample solution. No peaks were observed in solvent chromatograms, whereas two peaks were obtained with the standard solution, corresponding to nitration with retention time(tr)equal to G32 min and miconazole (tr=8.38min). Only methyl paraben and propylparaben solutions showed peak sat the selected wavelength. Two peaks were observed in chromatograms for matrix solutions. They were identified by comparing both the retention times and the UV spectra to those for the reference excipient solutions, and corresponded to methyl paraben (tr=1.80min) and propyl paraben (tr=2.60min). Interestingly, no peaks were observed at tr values near the miconazole retention time. Finally, when analyzing a cream sample solution, there solution between the analyte and propyl paraben peaks was highly satisfactory. Typical chromatograms for standard and sample solutions are shown in Fig.2.

Fig. 2. Typical chromatograms obtained at 232 nm for (A) standard solution of miconazolenitrate0.53mgmL−1and(B)miconazolenitratecreamsampleshowing (1) nitration, (2) miconazole,(3) methylparaben, (4) propylparaben

On the other hand, the peak purity of the analyte was evaluated by the following procedure: (a) recording all spectra in the peak by using a diode array detector, (b) computing the average spectrum, and (c) comparing the difference values between each spectrum and the average with an estimation of the noise threshold. A peak purity factor was calculated as the mean value of all spectra in the peak that are within the threshold [21]. Peak purity factors were 99.95%for miconazole in sample solutions and 99.93% in standard solutions, computed on135 spectra for each peak. Moreover, spectral matching between analyte peak in standard and sample solutions was of 99.96%. On the basis of these results, it can be concluded that the reared interfering components in the analyte peak.

Table 2 System Suitability Parameters

Degradation Study

To investigate the degradation of the active ingredient and assess the stability-indicating capability of the analytical method, the International Council for Harmonisation (ICH) guidelines were followed. Three different batches of miconazole nitrate cream, naturally aged for 18 months, were analyzed using the procedure described above. A minor secondary peak corresponding to isovalerate was observed, which was well separated from the principal peaks. The peak purity of the analyte was maintained throughout the study, with no interference detected in the assay.

System Suitability

System suitability testing was conducted by injecting six replicates of the standard solution, according to ICH recommendations. The evaluation of analyte peak parameters yielded high-quality results, as summarized in Table 2. These results complied with the specifications outlined in the relevant pharmacopoeias and ICH guidelines, confirming that the chromatographic system was both adequate and reliable.

Solution Stability

To ensure the stability of miconazole standard and sample solutions during typical routine analysis periods, aliquots of each solution were divided into four injector vials and placed in the injector tray. Each vial was injected every 2 hours over a total period of VI hours. The percentage of peak area relative to the initial area at time zero (t = 0) was calculated. The validity period for each solution was defined as the duration during which the peak area remained within 98.0% to 102.0% of the initial value. The study established solution stability for VI hours for the standard solution and IV hours for the sample solution.

Linearity

Linearity was assessed for both the pure standard and analyte in the sample matrix using solutions prepared as described in Section 2.5. Peak areas of miconazole were plotted against concentration, and a least-squares linear regression analysis was performed. To verify homoscedasticity of the data, an F-test was conducted to compare the extreme variances; the difference between the observed and critical F-values was not significant. Linearity between response and concentration over the studied range was evaluated using the statistical test recommended by the International Union of Pure and Applied Chemistry (IUPAC). Calculated F- values did not exceed the tabulated critical values. Confidence intervals were calculated to compare the regression intercepts with zero. A t-test was also applied to compare both the intercept and slope of the regression lines, revealing no significant differences. The results of these analyses are summarized in fig 3.

Precision

Precision was evaluated at the repeatability and intermediate precision levels. For repeatability analysis, six independent portions of a cream sample were processed through the full analytical method. The results were evaluated, obtaining an associated R.S.D. (%) value of 0.58. Intermediate precision was assessed using a new series of six portions of the same sample employed in the repeatability assay, processed on a different day (one week later) and by a different analyst. The corresponding R.S.D. (%) was 0.54. A statistical F-test was applied to compare the variance with that obtained in the repeatability analysis. The computed F value was 1.29, while the critical value (F_crit(5,5, α = 0.05)) was 5.05. Therefore, it can be concluded that no significant differences exist between the variances obtained.

Both variances are lower than 2%, indicating extremely good repeatability of the proposed methodology. In addition, an R.S.D. (%) value was computed using the mean values of the analysed series, resulting in an R.S.D. of 0.90. As this value is lower than 2.8%, the maximum accepted value according to AOAC recommendations for active ingredients the intermediate precision can be considered excellent.

Accuracy

Solutions prepared as described in Section 2.5 were injected, and the recovery of the known amount of added analyte was computed for each sample. The results obtained indicate good accuracy of the proposed methodology, since the mean recovery was within the interval 100 ± 2% at each level over the range of 80–120% of the target concentration. Detailed results are shown in Table 4. In addition, the global recovery, computed from nine determinations (of the total analytical procedure), was 100.5% (R.S.D. = 1.70%). A statistical Student’s t-test was applied, confirming that no significant difference exists between the recovery obtained and the ideal value of 100% at a 95% confidence level.

Assay of Commercial Cream Préparations

Once validated, the developed method was applied to the assay of miconazole nitrate and the evaluation of degradation products in commercial cream formulations. The results, corresponding to three batches of miconazole nitrate cream, are presented in Table 5. The percentage of drug recovery, relative to the label claim by the manufacturer, indicates that the active ingredient in the samples was present within the USP-specified range of 90.0–110.0% of the labeled amount of miconazole nitrate. To further assess the capability of the developed methodology, three additional batches of aged creams—containing miconazole nitrate, gentamicin sulphate, and betamethasone 17-valerate— were analyzed using the complete analytical procedure. As shown in Figure 4, several additional chromatographic peaks appeared. These corresponded to betamethasone 17-valerate and its degradation and isomerization products. Min Li and colleagues previously demonstrated, using a combined LC–MS strategy with forced degradation studies, that betamethasone 17-valerate undergoes at least two degradation pathways. The isomerization pathway produces betamethasone 21-valerate, dexamethasone 17-valerate, and dexamethasone 21-valerate, while the hydrolytic pathway leads to the formation of betamethasone and desamethasone as degradants Using DAD (diode array detection), we confirmed that all degradant peaks observed in the chromatograms of these aged samples showed high spectral correlation with betamethasone 17- valerate. Notably, miconazole was well-resolved from the other components. Furthermore, the peak purity matched that observed in the standard solution, indicating the specificity and robustness of the method. Fig. 4, gentamicin does not interfere in the analysis, owing to the fact that the absorbance in the UV region for this compound is negligi.