Stability-Indicating Validated Novel RP-HPLC Method for Simultaneous Estimation of Methylparaben, Ketoconazole, and Mometasone Furoate in Topical Pharmaceutical Dosage Formulation

A simple, specific, precise, and accurate RP-HPLCmethod has been developed and validated for simultaneous estimation ofMethylparaben (MP), Ketoconazole (KT), andMometasone Furoate (MF) topical pharmaceutical dosage formulation.The separation was achieved by Waters X Terra C18 column using mobile phase consisting of buffer (triethyl amine in water, pH adjusted to 6.5 with glacial acetic acid)-acetonitrile (40 : 60, v/v) at a flow rate of 1.5mL/min and detection at 250 nm. The method showed linearity with correlation coefficient <0.9999 over the range of 0.12–15.2 μg/mL, 0.67–149.4 μg/mL, and 0.42–7.6 μg/mL for MP, KT, andMF, respectively.Themean recoveries were found to be in the range of 99.9–101.1% for all the components.Themethod was validated as per the ICH guidelines for linearity, limit of detection, limit of quantification, accuracy, precision, robustness and solution stability. Stability indicating capability of the developedmethodwas established by analyzing forced degradation of samples inwhich spectral purity of MP, KT, and MF along with separation of degradation products from analytes peak was achieved. The method can be successfully applied for routine analysis of quantitative determination of MP, KT, and MF in pharmaceutical dosage form.

The preservative system is an important part of semisolid formulations in preventing the deterioration of formulations from microbial contamination.Methylparaben (MP) (Figure 1(c)) and its salts are most commonly used as preservatives for many years.To establish their effectiveness throughout the shelf life of the product, the actual concentrations of preservatives must be determined, as also required by regulatory agencies [8].
The finished product release specifications should include an identification test and a content determination test with acceptance criteria and limits for each antimicrobial preservative present in the formulation [9].The finished product self-life specification should also include an identification test and limits for the antimicrobial preservatives present [10].A detailed literature survey for Mometasone Furoate, Ketoconazole, and Methylparaben revealed that determination of individual compound or combination with other drugs has been reported by high performance liquid chromatography (HPLC) [1,2,4,9,, polarographic techniques [34], liquid chromatography-mass spectrometry (LCMS) [35][36][37][38], electrophoresis [39,40], subcritical-fluid chromatography (SFC) [41], spectrophotometric techniques [42][43][44], and high performance thin layer chromatography (HPTLC) [45].To the best of our knowledge, there is no stability-indicating liquid chromatographic method reported for the simultaneous estimation of Mometasone furoate, Ketoconazole, and Methylparabenin topical pharmaceutical dosage formulation.
The drug product stability guideline Q1A (R2) issued by the International Conference on Harmonisation (ICH) [46] suggests that stress studies should be carried out on a drug to establish its inherent stability characteristics, leading to identification of degradation products and, hence, supporting the suitability of the proposed analytical procedures.It also requires that analytical procedures for testing the stability of samples should be stability indicating and should be fully validated.

Materials and Reagents.
MF and KT sample working standards were provided by Dr. Reddy's Lab, India.HPLC grade acetonitrile, triethylamine, and glacial acetic acid were used of Rankem, India.0.22 m nylon membrane, 0.22 m PVDF syringe filter, and 0.22 m Nylon syringe filter were used of Millipore, India.Water for HPLC was generated using Milli-Q Plus water purification system (Millipore, Milford, MA, USA).

Chromatographic Parameters and Equipments.
All chromatographic experiments were performed on Waters Alliance HPLC system (Waters, Empower 2 software, USA), photo stability chamber (Sanyo, Leicestershire, UK), dry air oven (Cintex, Mumbai, India), XS205 dual range balance (Mettler Toledo), and cintex digital water bath were used for specificity study.All chromatographic experiments were performed in the isocratic mode.Separation was achieved on Waters X-Terra (250 × 4.6 mm, 5 m) column as stationary phase by using mixture of buffer (0.2% v/v triethyl amine in water, pH adjusted to 6.5 with glacial acetic acid) : acetonitrile (40 : 60, v/v) as mobile phase.Other parameters such as run time 6.0 minutes, 1.5 mL/min as flow rate, injection volume of 10 L, and column temperature of 50 ∘ C were finalized during development.MP, KT, and MF were detected at 250 nm.Mixture of acetonitrile : water, 80 : 20, v/v was used as diluent.
The stress-degraded samples and the solution stability samples were analyzed using a photo diode array (PDA) detector covering the range of 200-400 nm.

Standard Solution Preparation.
The stock solutions of MP (100 g/mL), KT (1000 g/mL), and MF (100 g/mL) were prepared by dissolving an appropriate amount of analyte in diluent separately.Working standard solution was prepared by mixing above stock solutions of MP, KT, and MF with final concentration of 10 g/mL, 100 g/mL, and 5 g/mL respectively.

Sample
Preparation.An accurately weighed 0.5 g sample (equivalent to 10 mg of KT, 0.5 mg of MF) was taken into 100 mL volumetric flask.About 70 mL mixture of acetonitrile and water (80 : 20, v/v) was added to this volumetric flask and sonicated in an ultrasonic bath for 15 min.with intermittent shaking, diluted to the volume with acetonitrile.and mixed well.A portion of solution was filtered through 0.22 m Nylon syringe filter.

Placebo
Preparation.An accurately weighed 0.5 g placebo sample was taken into 100 mL volumetric flask.About 70 mL mixture of acetonitrile and water (80 : 20, v/v) was added to this and sonicated for 15 min with intermittent shaking, diluted to the volume with acetonitrile, and mixed well.A portion of solution was filtered through 0.22 m Nylon syringe filter.

2.4.
Procedure for Method Validation.The method was validated for linearity, precision, accuracy solution stability, filter compatibility, limit of detection (LOD), limit of quantification (LOQ), specificity-forced degradation studies, and robustness as per ICH guidelines.

System Suitability.
To ensure that HPLC testing system was suitable for the intended application, the system suitability was assessed by five replicate analyses of system suitability solution (standard solution) and chromatographic parameters were evaluated.The acceptance criteria were not more than 2.0% for the RSD of the peak areas and tailing factor of the analyte peaks.The acceptance criteria were not less than 3000 for the plate count of the MP, KT and MF peaks.

Specificity-Forced Degradation Studies.
The forced degradation studies were executed to demonstrate whether the analytical method was stability indicating and could unequivocally assess the analyte in the presence of impurities and degradation products.Combined lotion of MP, MF, and KT was stressed under thermolytic, photolytic, acid hydrolytic, base hydrolytic, and oxidative stress conditions to result in partial degradation of the drugs.All stress decomposition studies were performed at an initial drug concentration of 10, 100, and 5 g/mL for MP, KT, and MF, respectively.
(1) Thermal Stressed Sample.For thermal stress testing, the lotion sample was placed in convection oven and exposed to heat at 75 ∘ C for 6 h.
(2) Photolytic Light Stressed Sample.For photo stress testing, the lotion sample was placed in photolytic chamber and expose to UV and visible light (1.2 million lux hours and 200 wh/m 2 ).
(3) Acid Degradation Sample.For acid hydrolysis, solution was prepared by dispersing and dissolving lotion sample into 15 mL mixture of acetonitrile and water (80 : 20, %v/v).Acid hydrolysis was performed by adding 1 mL of 5 N HCl and mixture was kept at 70 ∘ C on water bath for 30 minutes.
The solution was neutralized with 1 mL of 5 N NaOH and further proceeded as per sample preparation.
(4) Base Degradation Sample.For base hydrolysis, solution was prepared by dispersing and dissolving lotion sample into 15 mL mixture of acetonitrile and water (80 : 20, %v/v).Base hydrolysis was performed by adding 1 mL of 0.05 N NaOH and mixture was kept at room temperature for 15 minutes.The solution was neutralized with 1 mL of 0.05 N HCl and proceeded further as per sample preparation.
(5) Peroxide Oxidation Sample.For oxidation study, solution was prepared by dispersing and dissolving lotion sample into 15 mL mixture of acetonitrile and water (80 : 20, %v/v).Oxidative study was performed by adding 1 mL of 30% v/v hydrogen peroxide (H 2 O 2 ) and the mixture was kept at room temperature for 45 minutes.It was processed further as per sample preparation.

Precision.
The precision of the developed method was assessed by performing repeatability and intermediate precision (interday) at an initial drug concentration of 10, 100, and 5 g/mL for MP, KT, and MF, respectively, in one day.%RSD was calculated to determine repeatability of precision.These studies were also repeated on different days to determine interday precision.

Accuracy.
To confirm the method's accuracy, recovery experiments were checked by standard addition method.The recovery experiments were performed in triplicate at 50, 100, and 150% concentration levels of the amount of the analytes in in-house mixture of lotion excipients (placebo).

Limit of Detection and Limit of Quantification.
The standard solutions of MP, KT, and MF for the limit of detection (LOD) and limit of quantification (LOQ) were prepared by diluting them in mixture of acetonitrile and water (80 : 20, %v/v) sequentially.The LOD and LOQ were determined by analyzing signal-to-noise (S/N) ratio for each compound involving a series of diluted solutions until the S/N ratio yield 3 for LOD and 10 for LOQ, respectively.
2.4.6.Linearity.Six levels of calibration standard solutions were prepared from the stock solutions at concentration range 0.12-15.2g/mL for MP, 0.67-149.4g/mL for KT, and 0.42-7.6g/mL for MF to encompass the expected concentration in measured samples.Calibration curves were constructed by plotting areas versus concentrations of MP, KT, and MF and then subjected to least-square linear regression analysis.

Robustness.
To determine the robustness of the method the experimental conditions were deliberately changed.The flow rate of the mobile phase (1.5 ± 0.2 mL/min), column oven temperature (50 ± 5 ∘ C), mobile phase buffer pH (6.5 ± 0.2), and acetonitrile composition (60 ± 3.5%) were varied.In each case, the %RSD values were calculated for the obtained peak area.The number of theoretical plates and tailing factors was compared with that obtained under the optimized conditions.

Solution Stability.
In order to demonstrate the stability of sample solutions, the solution was tested at intervals of 0, 12, 24 h by its storage at ambient temperature.The stability of solutions was appreciated by comparing assay results of peak area of MP, KT and MF.
2.4.9.Filter Compatibility.Filter compatibility was performed for nylon 0.22 m syringe filter (Millipore) and PVDF 0.22 m syringe filter (Millipore).To confirm the filter compatibility in the proposed method, filtration recovery experiment was carried out by sample filtration technique.Sample was filtered through both syringe filters, and percentage assay was determined and compared against centrifuged sample.

Method Development and Optimization.
The prime objective of this study was an RP-HPLC method development for determination of Methylparaben, Ketoconazole, and Mometasone furoate in pharmaceutical dosage form and determination of assay of drug in single run which should be accurate, reproducible, robust, and stability indicating.All degradation products from stress conditions should be well separated from each other and the method should be simple to be effective in analytical research and quality control laboratory for routine use.

Mobile Phase and Chromatographic Conditions Optimization.
Optimization of column selection and mobile phase selection were done simultaneously.An isocratic method was employed using buffer (0.2% v/v triethyl amine in water, pH was adjusted to 6.5 with glacial acetic acid) : acetonitrile (60 : 40 v/v) as mobile phase.X-Terra C18 (150 × 4.6 mm, 5 ) column with flow rate 1.5 mL/min and column temperature 50 ∘ C were used in HPLC equipped with photo diode array detector.MF peak fronting was observed and peak eluted too late.To reduce run time and improve MF peak shape, an attempt was made by increasing acetonitrile composition in mobile phase, which then became buffer (0.2% v/v triethyl amine in water, pH 6.5 adjusted with glacial acetic acid) : acetonitrile (40 : 60 v/v).Due to early elution of MP peak (1.2 minute), plate count was found to be 1500.To retain the MP peak, an attempt was made by increasing column length X-Terra C18 (250 × 4.6 mm, 5 ).MP, KT, and MF peaks eluted at RT 2.3, 3.0, and 4.6 minutes, respectively.Plate count for MP peak was more than 4000.Resolution between MP, KT was 4.8 and KT, MF was 6.5.Considering solubility of all the component mixture of acetonitrile : water, 80 : 20 %v/v was used as diluent and satisfactory recovery was achieved.

Analytical Method Validation.
After satisfactory development of method, it was subjected to method validation as per ICH guideline [47].  1.

Specificity.
Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities [47].Forced degradation studies were performed to demonstrate selectivity and stability indicating capability of the proposed RP-HPLC method.Figure 2 shows that there is no interference at the RT (retention time) of MP, KT and MF in presence of blank and placebo.
Force degradation studies of drug product containing MP, KT, and MF were also performed to evaluate the stabilityindicating property and specificity of the proposed method.The solutions of drug product and placebo were exposed to acid hydrolysis, alkali, peroxide oxidation, thermal exposure, and photolytic exposure.KT was found sensitive to acid hydrolysis than MF.During acid hydrolysis process, about 54.1% of KT degraded and one major degradation peak was observed at 2.852 min, 8.1% of MP degraded while no degradation was observed in case of MF.MF was found to be more sensitive to base hydrolysis than KT.During base hydrolysis process, about 11.3% of MF degraded and main degradation peaks were observed at 4.911, 5.164 min, while no degradation was observed in case of KT and MP.During peroxide oxidation process, about 12.4% of KT degraded, but no significant degradation was observed for MP and MF.During photolytic degradation process, about 17.8% of KT degraded and main degradation peak was observed at 2.636 min, while no degradation was observed in case of MF and MP.No significant degradation was observed in case of thermal degradation of sample.Typical chromatograms obtained from assay of stressed lotion sample are shown in Figure 3, acid degradation (a), base degradation (b), peroxide oxidation (c), thermal exposed (d), and photo exposed (e) sample.MF, KT and MP were investigated for spectral purity in the chromatogram of all exposed samples and found spectrally pure.The purity and assay of MP, MF, and KT were unaffected by the presence of its degradation products and  thus confirms the stability-indicating power of the developed method.Results from forced degradation study are given in Table 2.

Method Precision.
The Precision of assay method was evaluated by carrying out six independent determinations of 10 g/mL of MP, 100 g/mL of KT, and 5 g/mL of MF lotion samples against qualified working standards.The average % assay ( = 6) of MP, KT, and MF were 100.5%, 99.7% and 100.2%, respectively, with RSD of below 0.8%.Low values of % RSD indicate that the method is precise.Results are presented in Table 3.

Conclusions
An isocratic RP-HPLC method was successfully developed for the estimation of Methylparaben, Ketoconazole, and Mometasone Furoate in topical pharmaceutical dosage form.
The method validation results have proved that the method is selective, precise, accurate, linear, robust, filter compatible and stability indicating.Forced degradation data proved that the method is specific for the analytes and free from the interference of placebo/known impurities/and degradation products.The run time (6.0 min) enables rapid determination of drug.Moreover, it may be applied for determination of Methylparaben, Ketoconazole, and Mometasone Furoate in the study of content uniformity, tube homogeneity, and invitro release test profiling of topical dosage forms, where sample load is higher and high throughput is essential for faster delivery of results.The developed method is stabilityindicating and can be used for quantifying Methylparaben, Ketoconazole, and Mometasone Furoate in topical pharmaceutical dosage forms and their combinations, that is, MP+MF, MP+KT, MF+KT, and MP+MF+KT.

Figure 3 :
Figure 3: Typical overlay chromatograms of (a) acid degradation sample and placebo, (b) base degradation sample and placebo, (c) peroxide oxidation sample and placebo, (d) thermal exposed sample and placebo, and (e) photolytic light exposed sample and placebo.
The accuracy was evaluated applying the proposed method to the analysis of the in-house mixture of cream excipients with known amount of the drug, to obtain solutions at concentrations of 5.05, 10.10, and 15.15 g/mL for MP; 49.80, 99.60, and 149.40 g/mL for KT; 2.53, 5.05

Table 2 :
Results of forced degradation study for MP, KT, and MF.

Table 3 :
Method precision, intermediate precision result, LOD, LOQ evaluation, and linearity data for MP, KT, and MF.

Table 4 :
Accuracy results for MP, KT, and MF.Table1along with system suitability parameters of precision and intermediate precision study.3.3.9.Stability of Sample Solution.Stability of sample solution was established by storage of sample solution at ambient temperature for 24 h.The assay of MP, KT, and MF was analyzed.It was found that % labeled amounts of MP at 0, 12, and 24 hrs were 99.8, 100.5, and 99.8, respectively; % labeled amounts of KT were 98.9, 99.1, and 98.7, respectively, and % labeled amount of MF were 100.1, 100.8, and 100.1, respectively.3.3.10.Filter Compatibility.Sample solution did not show any significant change in assay percentage with respect to centrifuged sample.It was found that % labeled amount of MP at centrifuged sample, 0.22 m PVDF syringe filter, and 0.22 m Nylon syringe filter were 100.5, 100.2, and 100.7, respectively; KT were 98.6, 98.4, and 98.8, respectively; and MF were 100.0, 99.5, and 100.9, respectively.In the result, difference in % assay was not more than ±1.0, which indicates that both syringe filters have a good compatibility with sample solution.
* Determined on three values.#Mean of three determinations.KT, and MF, respectively, demonstrating that the method is accurate within the desired range and also there is no interference due to excipients present in placebo cream sample.3.3.6.Limit of Detection (LOD) and Limit of Quantification (LOQ).The LOD and LOQ were determined at a signal to ∘ C), mobile phase buffer pH (±0.2), and mobile phase organic composition (±3.5% acetonitrile).No significant effect was observed on system suitability parameters such as theoretical plates, tailing factor, and % RSD of MP, KT, and MF.The results are presented in