Simultaneous Quantification of Mometasone Furoate and Formoterol Fumarate in Bulk and Formulations by RP-HPLC Method

 

Sivagami B¹*, Bhaskar M¹, Pavan Kumar V¹, Meena D¹, Satheesh Kumar G², Chandrasekar R²

¹Associate Professor, Faculty of Pharmacy, Pharmaceutical Sciences, Department of Pharmaceutical Analysis, Seven Hills College of Pharmacy, Venkatramapuram, Tirupati, Chittoor, Andhra Pradesh, India.

¹PGStudent, Pharmaceutical Sciences, Department of Pharmaceutical Analysis,

Seven Hills College of Pharmacy, Venkatramapuram, Tirupati, Chittoor, Andhrapradesh, India.

¹Associate Professor, Faculty of Pharmacy, Pharmaceutical Sciences, Department of Pharmaceutical Analysis, Seven Hills College of Pharmacy, Venkatramapuram, Tirupati, Chittoor, Andhra Pradesh, India.

¹Assistant Professor, Faculty of Pharmacy, Pharmaceutical Sciences, Department of Pharmaceutical Analysis, Seven Hills College of Pharmacy, Venkatramapuram, Tirupati, Chittoor, Andhra Pradesh, India.

²Professor, Faculty of Pharmacy, Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, Seven Hills College of Pharmacy, Venkatramapuram, Tirupati, Chittoor, Andhra Pradesh, India.

²Associate Professor, Faculty of Pharmacy, Pharmaceutical Sciences, Department of Pharmacognosy,

Seven Hills College of Pharmacy, Venkatramapuram, Tirupati, Chittoor, Andhra Pradesh, India.

 

ABSTRACT:

A simple and robust reversed-phase high pressure liquid chromatographic method was developed and validated for the quantification of Mometasone and Formoterol in bulk and fixed dosage forms. The column utilized for study was Inertsil C18, ODS was chosen for good peak shape. Ambient temperature was found to be suitable for the nature of drug solution. The mobile phase was streamlined with a ratio of 35:65 Methanol: Water was optimized for symmetrical peaks and good resolution. The flow rate was streamlined at 1.0 ml/min because of good peak area, satisfactory retention time and good resolution.  Common λ-max was found to be at 278 nm and Injection volume was 20 µl which gave a good peak area. The percentage recovery was found to be 98.0-101.50 was linear and precise over the same range. Both system and method precision was found to be accurate and well within the acceptable limits.  Detection limit was found to be 0.25 Mometasone and 0.34 for Formoterol. The analytical method was found linearity over the range of 20-80 ppm of the target concentration for both the drugs. The LOD and LOQ values for Mometasone, was observed to be 0.25 and 0.77 and for Formoterol was in the range of 0.34 and 1.05 consecutively. The analytical method passed both intermediate precision and robustness tests. On both cases, relative standard deviation was well satisfactory. The validation experiments met ICH standards. It inferred the method found to be simple, accurate, precise and linear. The method was found to be having suitable application in routine laboratory analysis with high degree of accuracy and precision.

 

 

 

INTRODUCTION:

Mometasone furoate is a topical corticosteroid (MF), 9α, 21-dichloro-11β, 17-dihydroxy-16α- methylpregna-1, 4-diene-3, 20-dione 17-(2-furoate), (Fig. 1). It has various therapeutic properties such as anti-inflammatory, anti-pruritic, and vasoconstrictive. It is used to treat various ailments, including allergic reactions, eczema, and psoriasis. Corticosteroids work by inducing phospholipase A2 inhibitory proteins known as lipocortins. It is presumed that these proteins regulate the biosynthesis of potent inflammatory mediators like prostaglandins and leukotrienes by inhibiting the release of their common precursor arachidonic acid. ¹

 

Figure 1: Chemical Structure of Mometasone

 

Formoterol, ((RR)-(±)-N-[2-hydroxy-5-[1-hydroxy-2-[[2-(4- methoxyphenyl)-1 methylethyl]amino] ethyl] phenyl]formamide), (Fig. 2) is an adrenergic agent with high selectivity for the β2 receptor (a β2-agonist) that has proven to be an extremely effective bronchodilator. As a result, it is frequently used therapeutically in the form of metered dose inhalers for the treatment of asthma, bronchospasm, and the prevention of exercise-induced bronchospasm.²

Z

Figure 2: Chemical Structure of Formoterol Fumarate

 

Literature from various studies revealed that few UV, HPLC, UPLC, HPTLC methods for the concurrent estimation and single and multi-component analysis of Mometasone furoate and Formoterol fumarate in combination with other drugs were reported. Few methods were reported by UV spectroscopy for the quantification of Mometasone and Formoterol in single and multi-component analysis,^2,3 few methods were reported by RP-HPLC for the quantification of Mometasone and Formoterol and in combination with other drugs, ^4-13one method by UPLC was reported for the quantification of Mometasone and Formoterol, ¹⁴and two methods by HPTLC was investigated for the determination of Mometasone.^{15, 16}

 

MATERIALS AND METHODS:

Instruments:

The instrument used for the study was HPLC –Waters Model NO.2690/5 series Compact System Consisting of Inertsil-C₁₈ ODS column.

 

Chemicals:

Methanol HPLC Grade(Merck), Buffer (KH₂PO₄) HPLC Grade Sigma Aldrich, all other chemicals were procured from standard suppliers.

 

Optimized Method:

Preparation of stock solution:

The solution was prepared by dissolving 20.0 mg of accurately weighed Mometasone and 220.0 mg Formoterol fumarate in Mobile phase, in two 100.0 mL volumetric flasks separately and sonicated for 20min. From the above solutions 10.0 mL was taken from each solution into a 50.0 mL volumetric flask and then makeup with mobile phase and sonicated for 10min.

 

 

Figure 1: Chromatogram of sample

 

Figure 2: Chromatogram of Standard

Preparation of working standard solution:

The stock solutions equivalent to 20ppm to 80ppm with respect to both drugs were prepared in combination of Mometasone and Formoterol fumarate above, sonicated and filtered through 0.45µ membrane. The optimized chromatogram for Mometasone and Formoterol fumarate is depicted in Figure no 1 and 2

 

Method Validation:

System Suitability:

A standard solution was prepared using the test method's Mometasone and Formoterol fumarate working standards and injected five times into the HPLC system. The system suitability parameters were determined by calculating the % RSD from five replicate injections of Mometasone and Formoterol fumarate, retention times, and peak areas from standard chromatograms.¹⁷

 

Specificity:

Mometasone and Formoterol fumarate:

Solutions of standard and sample were prepared as per the test method and injected into chromatographic system.

 

Repeatability:

System precision:

Standard solution were prepared as per test method and injected five times into the HPLC system.

 

Method precision:

Six sampleswere prepared individually as per test method and each solution was injected into the instrument.¹⁸

 

Accuracy (Recovery):

A study of Accuracy was conducted, drug assay was performed in triplicate as per test method with equivalent amount of Mometasone and Formoterol fumarate into each volumetric flask for each spike level to get the concentration of Mometasone and Formoterol fumarate equivalent to 50%, 100%, and 150% of the labeled amount as per the test method. The average % recovery of Mometasone and Formoterol fumarate was calculated.¹⁹

 

Linearity of Test Method:

A Series of solutions were prepared using Mometasone and Formoterol fumarate working standards at concentration levels from 20ppm to 80 ppm of target concentration. Measure the peak area response of solution at Level 1 and Level 6 six times and Level 2 to Level 5 two times.²⁰

 

 

Ruggedness of Test Method:

System to system variability:

Various HPLC systems were used to study system to system variability under similar conditions at different times. Six samples were prepared and analyzed according to the test method. The comparison of the results obtained on two different HPLC instruments demonstrates that the assay test method is resistant to system-to-system variability.²¹

 

Robustness:

Effect of variation of flow rate:

A study was carried out to determine the effect of flow rate variation. The test method's standard solution was injected into the HPLC system at flow rates of 1.0 ml/min and 1.2 ml/min. The system suitability parameters were evaluated and found to be within the limits for 1.0 and 1.2 ml/min flow rates. Mometasone and Formoterol fumarate were separated from all other peaks, and the retention times were comparable to those obtained for mobile phase flow rates of 1.0 ml/min.²²

 

Limit of Detection and Quantitation (LOD and LOQ):

From the linearity data the limit of detection and quantitation was calculated, using the following formula. From the linearity plot the LOD and LOQ are calculated, the LOD for Mometasone is 0.25 and LOQ for Mometasone was 0.77, LOD for Formoterol is 0.34 and LOQ for Formoterol is 1.05.²³

 

RESULTS AND DISCUSSION:

Precision:

Repeatability:

System precision:

Standard solution were prepared as per test method and injected five times injected into the HPLC system. The Data of Repeatability (System precision) for Mometasone and Formoterol Fumarate are shown in Table 1 and 2

 

Table 1: Data of Repeatability (System precision) for Mometasone and Formoterol

 

Table2: Data of Repeatability (Method precision) for Mometasone and Formoterol

 

Method precision:

Six samples were prepared individually as per test method and each solution was injected into the HPLC system. Data of Repeatability (Method precision) for Mometasone and Formoterol are shown in Table 3 and 4

 

Table 3: Data of Linearity (Mometasone)

 

Figure 3: Linearity Plot (Concentration Vs Response) of Mometasone

 

Linearity:

A Series of solutions were prepared using Mometasone and Formoterol fumarate working standards at concentration levels from 20 ppm to 80 ppm of target concentration. Measure the peak area response of solution at Level 1 and Level 6 six times and Level 2 to Level 5 two times. The linearity data for Mometasone and Formoterol is shown in Table 5 and 6 and Linearity Plot of Mometasone and Linearity Plot of Formoterol are depicted in Figures 3 and 4

 

Figure 4: Linearity Plot (Concentration Vs Response) of Formoterol Fumarate

 

Accuracy:

A study of Accuracy was conducted. Drug Assay was performed in triplicate as per test method with equivalent amount of Mometasone and Formoterol fumarate into each volumetric flask for each spike level to get the concentration of Mometasone and Formoterol fumarate equivalent to 50%, 100%, and 150% of the labeled amount as per the test method. The average % recovery of Mometasone and Formoterol fumarate was calculated.²⁴ The Data of Accuracy for Mometasone and Formoterol Fumarate are shown in Table 7 and 8.

 

 

Table4: Data of Accuracy for Mometasone

 

 

Table 5: Data for Effect of variation in flow rate (Mometasone)

 

Robustness:

A study was carried out to determine the effect of flow rate variation. The test method's standard solution was injected into the HPLC system at flow rates of 1.0 ml/min and 1.2ml/min. The system suitability parameters were evaluated and found to be within the limits for 1.0 and 1.2ml/min flow rates. Mometasone and Formoterol fumarate were separated from all other peaks, and the retention times were comparable to those obtained for mobile phase flow rates of 1.0ml/min.²⁵ The Data for Effect of variation in flow rate Mometasone and Formoterol Fumarate are shown in Table 9 and 10.

 

DISCUSSION:

This method has been validated in accordance with the requirements for new methods established by the United States Pharmacopoeia, which include accuracy, precision, selectivity, robustness, linearity, and range. This method exhibits sufficient selectivity, accuracy, precision, linearity, and range to satisfy, λmax for Mometasone was observed to be 276nm and λmax for Formoterol is 237nm. The common maximum was observed to be at 278nm, and the injection volume was 20l, resulting in a good peak area. The column used for the study was Inertsil C18, with ODS selecting a good peak shape. The ambient temperature was found to be appropriate for the nature of the drug solution. Because of the good peak area, satisfactory retention time, and good resolution, the flow rate was optimized at 1.0 ml/min. The mobile phase with ratio of 35:65 Methanol: Water was optimized for symmetrical peaks and good resolution. The percentage recovery was found to be 98.0-101.50 was linear and precise over the same range. Both system and method precision was found to be accurate and well within range. Detection limit was found to be 0.25 Mometasone and 0.34 for Formoterol. The analytical method was found linearity over the range of 20-80ppm of the target concentration for both the drugs. The analytical method passed both robustness and ruggedness tests. On both cases, relative standard deviation was well satisfactory.²⁶

 

CONCLUSION:

The proposed chromatographic method provides an accurate, reproducible, simple and sensitive method for quantification of Mometasone and Formoterol in bulk and combined dosage forms. The newly developed RP-HPLC method is reproducible and sensitive. It has the advantages of having a short run time, a large sample capacity, and using a small amount of solvent. The HPLC method provides good resolution between the proposed components in a reasonable amount of time; it is highly specific but less expensive. The proposed methods have the advantage than other published methods of being more sensitive, simple, less time consuming, and can be applicableto different dosage forms.

 

ACKNOWLEDGEMENTS:

Authors express their sincere gratitude to Seven Hills College of Pharmacy, Tirupati, for continuous motivation, support, and guidance for research activity and for providing all required facilities to accomplish the entitled work.

 

COMPETING INTEREST:

Authors have declared that no competing interests exist.

 

AUTHOR CONTRIBUTION:

All the authors have equally contributed.

 

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Received on 07.11.2022       Modified on 09.03.2023

Accepted on 16.06.2023   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2023; 13(4):267-272.