A STUDY OF ANALYTICAL METHOD DEVELOPMENT, VALIDATION AND FORCED DEGRADATION FOR SIMULTANEOUS ESTIMATION OF GLYCOPYRROLATE AND FORMOTEROL FUMARATE IN BULK DRUG BY UHPLC METHOD

A STUDY OF ANALYTICAL METHOD DEVELOPMENT, VALIDATION AND FORCED DEGRADATION FOR SIMULTANEOUS ESTIMATION OF GLYCOPYRROLATE AND FORMOTEROL FUMARATE IN BULK DRUG BY UHPLC METHOD

Ramya Kuber Banoth ^* and Rama Lakshmi Nethikoppula

Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam (Women’s University) Tirupati - 517502, Andhra Pradesh, India.

ABSTRACT: The present study was proposed to develop a simple, rapid, and economical UHPLC method was described for the method development, validation and forced degradation for the simultaneous estimation of glycopyrrolate and formoterol fumarate in bulk drug. The chromatographic conditions were observed in the column using Zorbax RXC18 (150 × 4.6 mm) 5 µm using a mobile phase composition of Water: Acetonitrile: Methanol (20:30:50 v/v) and pH was adjusted to 3.0 with dilute orthophosphoric acid. The flow rate was 1.0 ml/min, and the analytes were recorded at 279 nm. The Retention time (RT) was observed in 1.208 for Glycopyrrolate and 5.897 for Formoterol fumarate. A calibration curve was linear with a coefficient correlation between 0.999 to 1.0 over a concentration range of 20-60µg/ml for glycopyrrolate and formoterol fumarate. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.003,0.011µg/ml for Glycopyrrolate and 0.108,0.32µg/ml for formoterol fumarate. All the validation parameters were within the acceptance range according to ICH norms. The validation and forced degradation study was performed for the proposed method and applied successfully to simultaneously estimate glycopyrrolate and formoterol fumarate. This method was precise, accurate, robust and economical.

Glycopyrrolate, Formoterol fumarate, UHPLC, HPLC, Forced degradation

INTRODUCTION:

Ultra High-Performance Liquid Chromato-graphy: Totally porous, spherical silica dominated the HPLC market from the 1980s to the 2000s. The average particle size continually decreased from 10 µm to 5 µm to 3 µm ^{1, 2}. The trend towards smaller particles is driven by improved performance in terms of speed, sensitivity, and resolution.

Smaller particles, however, require higher operating pressures, and in 2004 instruments were commercialized that could utilize columns prepared with sub-2 µm particles. Fig. 1 illustrates the chromatographic performance of four typical particle sizes used in HPLC.

FIG. 1: CHROMATOGRAPHIC PERFORMANCE FOR FOUR COMMON PARTICLE SIZES

Principle: The UHPLC is based on the principle of use of stationary phase consisting of particles less than 2.5 μm (while HPLC columns are typically filled with particles of 3 to 5 μm). The underlying principles of this evolution are governed by the “Van Deemeter equation” which is an empirical formula that describes the relationship between linear velocity (flow rate) and plate height (HETP or column efficiency) ³.

H=A+ B/v+ Cv

Where; A, B , and C are constants; V is the velocity of the sample.

Analytical Method Development of Glyco-pyrrolate and Formoterol Fumarate: Glyco-pyrolate is a quaternary ammonium salt. Chemically, Glycopyrrolate is (RS) - [3 (SR) – Hydroxy - 1, 1 - dimethylpyrrolidinium bromide] α-cyclopentylmandelate. The chemical formula is C₁₉H₂₈BrNO₃. The molecular weight is 398.33 g / mol. Glycopyrolate is a crystalline white powder. It is completely dissolvable in water and alcohol and much insoluble in chloroform and ether. Glycopyrrolate, as other anticholinergic (anti muscarinic) drugs, the action of acetylcholine on structures innervated by postganglionic cholinergic nerves and on smooth muscles that respond to acetylcholine it require cholinergic innervations ⁴. Thus, it diminishes the volume and free acidity of gastric secretions and controls excessive pharyngeal, tracheal and bronchial secretions ⁵. Formoterol acts as a bronchodilator. It extends the airways of the lungs, so that it helps to inhale all the more effortlessly.

FIG. 2: STRUCTURE OF FORMOTEROL FUMARATE

It may even be utilized to forestall respiratory issues caused by exercise. It can also be utilized for the long-term treatment of chronic obstructive pulmonary disease (COPD) ⁶. Chemically, Formoterol is N-[2-Hydroxy5-[(1RS)-1 - hydroxyl - 2 - [(1RS) - 2(4 - methoxyphenyl) - 1 - methylethyl] - amino] ethyl] phenyl] formamide (E) - 2 - butenedioatedihydrate. The chemical formula is C₁₉H₂₄N₂ O. C₄H₄O₂H₂ O. The molecular weight is 840.91 g / mol.

FIG. 3: STRUCTURE OF GLYCOPYRROLATE

The main aim of the work is to develop a simple, accurate, precise, and economical new RP-UHPLC method development, validation, and forced degradation for the simultaneous estimation of Glycopyrrolate and Formoterol fumarate in bulk drug. It includes the objective of the experimental work has been planned as to following the methods i.e Solubility determination of Glycopyrrolate and Formoterol fumarate in various solvents and buffers and to determine the absorption maxima of both the drugs in UV–Visible region in different solvents/buffers and selecting the solvents for UHPLC method development, Optimize the mobile phase and flow rates for proper resolution and retention times and Validate the developed method and forced degradation as per ICH guidelines.

Literature survey revealed that it has very few analytical methods had been reported for the simultaneous estimation of glycopyrrolate and formoterol fumarate by using UV-spectroscopy ⁷, RP-HPLC ^8-15, gas chromatography by individually or simultaneously with other drugs. Form this survey; it confirms that there is no method has been reported for the simultaneous estimation of glycopyrrolate and formoterol fumarate in bulk drug by using UHPLC.

The present method has so many advantages like decrease the runtime and increase the sensitivity and reduce the time consuming and easy to handle, and simple preparation of the mobile phase and standard solutions with low cost of the solvents. All the parameters are satisfied with the ICH guidelines for validation of the simultaneous estimation of glycopyrrolate and formoterol fumarate in bulk drugs.

MATERIALS AND METHODS:

Instruments: The method has made up of Agilent infinity 1290 with having Zorbax RX C18 (150 × 4.5 mmID) 3 µm column with UV-Detector.

Chemical Solutions and Reagents: Glycopyrrolate and formoterol fumarate obtained from madras pharmaceuticals, Chennai, India and potassium hydrogen phosphate buffer analytical grade from Rankem and water, Acetonitrile, methanol from Merck chemicals private limited.

Preparation of Standard Solution: About 10 mg of Glycopyrrolate and 10 mg of Formoterol fumarate were weighed into a 50 mL volumetric flask, to this 50 mL of mobile phase was added, sonicated, and the volume was made up to mark with the mobile phase. 1ml was Pipetted out from the above stock solution and transferred into 10 ml volumetric flask and made up to 10 ml with the mobile phase.

Preparation of Orthophosphoric Acid Buffer pH: Buffer solution was made with dissolving 1ml of orthophosphoric acid in 1000 ml of water. Buffer was filtered through 0.45 µm filters to remove all fine particles and gases.

Mobile Phase Pomposition: Simultaneous estimation of glycopyrrolate and formoterol fumarate was carried out with different combinations of solvents like water: Acetonitrile: methanol.

Sample Solution: Crush 20 tablets, then weigh the quantity of powder equivalent to 50 mg of glycopyrolate and 100 mg of formoterol fumaratein 100 mL volumetric flask and add 70 mL of mobile phase then sonicated for 30 min intermittent shaking after 30 min make up the volume with the mobile phase. Pipetted 5 mL of the clear solution in to 50 mL volumetric flask and make up the volume with mobile phase. Filter the solution through 0.45 µm filter paper.

Chromatographic Conditions: The method development for separation of glycopyrrolate and formoterol fumarate using different solutions finally optimized method was obtained with mobile phase water: Acetonitrile: methanol (20:30:50 v/v) pumped at flow rate is 1 ml / min. The separation of the peaks was scanned at 279 nm by observing of UV-Detector. Mobile phase is filtered with vacuum filtration by using 0.45 µm membrane filter.

Method Validation: The developed method was validated according to ICH guidelines it followed by ICHQ2 (R1) include system suitability, linearity, precision, LOD, LOQ, accuracy, robustness ¹⁶.

RESULTS:

Optimization of Chromatographic Conditions: A number of the eluting system was examined for optimization of the mobile phase for the separation of the drugs. Mixtures containing phosphate buffer and Acetonitrile and methanol were used as an eluting system based on drug polarity, the mixture of water, Acetonitrile, and methanol in the ratio of 20:30:50 v/v proved an efficient separation of the drugs with good peak shapes and retention time. The flow rate 1.0 ml/min, and the injection volume were 10 µl; the obtained peaks were scanned at 279 nm using UV-detector.

TABLE 1: OPTIMIZED CHROMATOGRAPHIC CONDITION FOR THE SIMULTANEOUS ESTIMATION OF GLYCOPYRROLATE AND FORMOTEROL FUMARATE

 FIG. 4: CHROMATOGRAM FOR OPTIMIZED METHOD

This gives retention times of 1.208 for glycol-pyrolate and 5.897 for formoterol fumarate with good efficiency, peak shape and good resolution. So, this method was considered and validated according to ICH guidelines respectively. Hence this method was finalized for the simultaneous estimation of glycopyrolate and formoterol fumarate shown in the Table 1 and Fig. 4.

Method Validation:

System suitability:

FIG. 5: CHROMATOGRAM OF SYSTEM SUITABILITY INJECTION

The developed method has been produced at theoretical plates above 2000 for glycopyrrolate and formoterol fumarate with a tailing factor less than 2, which ensures the suitability of the developed method. The results of the system suitability results were shown in Fig. 5 and Table 2.

TABLE 2: SYSTEM SUITABILITY OF THE DEVELOPED METHOD [N=6]

Linearity: Linearity of the calibration curve was obtained in the concentration ranges from 20-60µg/ml for glycopyrrolate and formoterol fumarate. The linear response of the drug was found to be in the selected concentration range. The correlation coefficients for glycopyrrolate and formoterol fumarate were found to be 0.999 and 0.999, respectively. The results of the linearity summarized in Table 3, 4, 5, and Fig. 6.

TABLE 3: LINEARITY DATA OF GLYCOPYRROLATE

TABLE 4: LINEARITY DATA OF FORMOTEROL FUMARATE

TABLE 5: REGRESSION EQUATION OF THE LIN-EARITY PLOTS AND REGRESSION COEFFICIENT

FIG. 6:  GRAPH FOR LINEARITY DATA FOR GLYCOPYRROLATE [A] AND FORMOTEROL FUMARATE [B]

Accuracy: The accuracy of the method was determined by recovery studies. The recovery studies were carried out three times, and the percentage recovery and percentage mean recovery were calculated for drug and shown in Fig.7 and Table 6, 7.

FIG. 7: CHROMATOGRAM FOR ACCURACY -100%

The % mean recovery of the glycopyrrolate and formoterol fumarate should be not less than 98.0% and not more than 102%. The results of the recovery studies were described in Table 6, 7.

TABLE 6: ACCURACY DATA FOR GLYCOPYROLATE

TABLE 7: ACCURACY DATA FOR FORMOTEROL FUMARATE

Precision: The developed method has shown in % RSD less than 2. It indicates that this method was precise is shown in Fig. 8 and Table 8.

LOD and LOQ: Limit of detection and limit of quantification were quantified from the standard deviation of the y-intercepts and slope of the calibration curve of glycopyrrolate and formoterol fumarate. The LOD and LOQ were found to be 0.003 µg/ml, 0.011 µg/ml for glycopyrrolate and 0.108 µg/ml, 0.32 µg/ml for formoterol fumarate respectively. This data showed that the developed method could detect and quantify at lower concentration was highly sensitive.

Specificity: Specificity was carried out by evaluation of standard solution injections. The chromatogram of standard and spiking sample solution was compared, the correlation was good, there is no interference of excipients with drug was observed and shown in Fig. 9, 10, 11.

FIG. 8: CHROMATOGRAM FOR SYSTEM PRECISION

TABLE 8: PRECISION OF THE DEVELOPED METHOD OF GLYCOPYROLATE AND FORMOTEROL FUMARATE [N=6]

N is number of determinations, SD is standard deviation, RSD is the relative standard deviation

FIG. 9: SPECIFICITY CHROMATOGRAM OF STANDARD INJECTION

FIG. 10: SPECIFICITY CHROMATOGRAM OF SPIKED SAMPLE INJECTION

FIG. 11: SPECIFICITY OF PLACEBO INJECTION

Robustness: The Robustness of the proposed method was described. The results were obtained by the effect of variation in method parameters are summarized below.

These results are satisfied with acceptance criteria (i.e. plate count is˃2000 and tailing factor ˂2) and are mentioned in Table 9 and Fig. 14.

TABLE 9: RESULTS FOR ROBUSTNESS OF GLYCOPYRROLATE AND FORMOTEROL FUMARATE

Ruggedness: The ruggedness of the method was studied by determining the analyst to analyst variation by performing the method by two different analysts.

From this observation, the % RSD between two analysts Assay values not greater than 2.0%, hence the method was rugged Table 10.

FIG. 12: CHROMATOGRAM OF ANALYST 01 STANDARD PREPARATION

TABLE 10: RESULTS FOR RUGGEDNESS

Assay: The assay was estimated by injecting the prepared concentration of tablet formulation into UHPLC system.

Assay results were calculated by comparing the peak area of tablet formulation with peak area of the standard solution.

TABLE 11: ASSAY DATA FOR THE FORMULATION FOR GLYCOPYROLATE AND FORMOTEROL FUMARATE (N=5)

The % assay of glycopyrrolate and formoterol fumarate was found to be 99.81% and 99.60%, respectively. The percentage assay of both drugs was found to be more than 99.5%. Hence, the method was successfully applied for estimation of glycopyrrolate and formoterol fumarate in the bulk and pharmaceutical dosage form. The results of assay were described in Table 11.

Forced Degradation: Degradation studies were carried out with acid, base, peroxide, thermal, UV and water. It was observed that the response of peak area and retention time of glycopyrrolate and formoterol fumarate nearly same as obtained results. Degradation was found in peroxide and thermal conditions because extra peaks were observed and no degradation was found in UV, acid, base, water. Because they were no extra peaks. The observed data showed in Table 12, 13, and Fig. 15-19.

FIG. 13: CHROMATOGRAM FOR PEROXIDE DEGRADATION

FIG. 14: CHROMATOGRAM FOR PHOTOLYTIC DEGRADATION

FIG. 15: CHROMATOGRAM FOR ACIDIC DEGRADATION

FIG. 16: CHROMATOGRAM FOR ALKALINE DEGRADATION

FIG. 17: CHROMATOGRAM FOR THERMAL DEGRADATION

TABLE 12: RESULTS OF GLYCOPYRROLATE

TABLE 13: RESULTS FOR FORMOTEROL FUMARATE

DISCUSSION: Stability indicating UHPLC method is a simple, rapid, precise, accurate method for analyzing each component in a mixture. The previous study had reported in the literature survey. In this UHPLC method, we used UV-detector to prove the selectivity of the method. The method was validated according to the ICH guidelines on validation of analytical procedures and stability testing of new drug substance and products.

In order to develop a UHPLC method for estimation of glycopyrrolate and formoterol fumarate, different buffer ratios and flow rates were applied. Water: Acetonitrile: Methanol (20:30:50) v/v as mobile phase and discovery C18 column was selected. Separation of glycopyrrolate 1.208 min and formoterol fumarate is 5.897 min was detected by the wavelength of 279 nm. In this method UV- detector is able to identify the glycopyrrolate and formoterol fumarate and degradation products. Method was validated. The results of validation parameters had shown in compliance of ICH guidelines. The range of linearity had good correlation with concentration and peak area. The correlation coefficients for glycopyrrolate and formoterol fumarate were 0.999 and 0.999, respectively. Which indicates that the concentration range was highly linear. In the assay the amount of both drugs recovered was found to be 99.81% and 99.60%, respectively.

Hence, the stability-indicating assay method was found to be appropriate for the analysis of the drug. The separation of degradation peak for degradants products was observed under peroxide and thermal hydrolysis.

CONCLUSION: A simple and sensitive stability-indicating UHPLC method was developed for simultaneous estimation of glycopyrrolate and formoterol fumarate. It concludes that all the parameters are within limits and meet the acceptance criteria of ICH guidelines for method validation. The proposed method was simple, accurate, specific precise, robust, and economical. Hence this method is validated and can be used for routine and stable sample analysis.

ACKNOWLEDGEMENT: The authors are thankful to the Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam, Tirupati, and Chandra Labs Pvt. Ltd, Hyderabad, for provided pure drugs and to carry out this research work.

CONFLICTS OF INTEREST: Authors have no conflicts of interest

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    How to cite this article:

    Banoth RK and Nethikoppula RL: A study of analytical method development, validation and forced degradation for simultaneous estimation of glycopyrolate and formoterol fumarate in bulk drug by uhplc method. Int J Pharm Sci & Res 2021; 12(7): 3781-90. doi: 10.13040/IJPSR.0975-8232.12(7).3781-90.

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