A Review on Various Analytical Method Development and Validation for Simultaneous Estimation of Levo-Salbutamol and Ipratropium Bromide in Bulk and Pharmaceutical Dosage Form by RP-HPLC

Mugdivari Sangeetha*, U. Akhila, T. Rama Rao

CMR College of Pharmacy, Kandlakoya, Medchal, Hyderabad, Telangana, India-501401.

*Correspondence: sangeetha.kodiganti@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2025.4904   

INTRODUCTION

The smooth muscles of every airway, from the trachea to the terminal bronchioles, are relaxed by levosalbutamol¹. The suppression of mast cell release of mediators in the airways is similarly linked to elevated cyclic AMP concentrations. Regardless of the spasmogens involved, levosalbutamol functions as a functional agonist to relax the airway, protecting against all bronchoconstrictor difficulties. Although statistics show that there are beta receptors in the human heart, 10–50% of which are beta adrenergic receptors, beta2-adrenergic receptors are known to be the most common receptors on bronchial smooth muscle. 4-[(1R)-2-(tert-butylamino)-1-hydroxyethyl] - 2-(hydroxymethyl) phenol is its chemical name.

Figure 1: Chemical Structure of Levosalbutamol

Melting point: 228°C

Trade Name: Xopenex

By inhibiting muscarinic cholinergic receptors, Ipratropium Bromide² counteracts the effects of acetylcholine, causing bronchodilation and the drying of secretions from the respiratory tract. Without regard to subtype specificity, ipratropium inhibits muscarinic acetylcholine receptors, which encourages the breakdown of cyclic Guanosine Monophosphate (cGMP) and lowers its intracellular content. The reduced contractility of the smooth muscle in the lung, which inhibits bronchoconstriction and mucus secretion, is most likely caused by the effects of cGMP on intracellular calcium. As a nonselective muscarinic antagonist, it avoids systemic side effects by not diffusing into the blood. As a quaternary amine and a derivative of atropine, ipratropium avoids central side effects by not passing through the blood-brain barrier. In terms of chemistry, it is [8-methyl-8-(1-methylethyl)-8- 3-yl] phenyl-propanoate.
Spectrophotometric analysis of the literature indicates that 3-hydroxy-2 is found using a small number of chromatographic techniques. Levosalbutamol and ipratropium bromide estimations, both alone and in combination with other medications, were reported in
Thus, an effort has been undertaken to create and validate a straightforward, sensitive, accurate, and precise RP-HPLC technique for the simultaneous measurement of Ipratropium bromide and Levosalbutamol in a combination capsule dose form. Thus, a combination dosage form of Ipratropium bromide and Levosalbutamol may represent a new research direction.

Figure 2: Chemical Structure of Ipratropium bromide

Melting point: 230°C

Trade Name: Atrovent

LITERATURE REVIEW

HPLC METHOD:

Salma N. Ali et al., (2024) Chromatographic fingerprinting of ipratropium and fenoterol in their novel co-formulated inhaler treating major respiratory disorders; application to delivered dose uniformity testing along with greenness and whiteness assessment. Ipratropium bromide (IPR) and fenoterol hydrobromide (FEN) have recently been combined in a promising inhaler to treat two prevalent inflammatory illnesses of the airways: bronchial asthma and chronic obstructive pulmonary disease (COPD). The necessity for a single, sensitive, and trustworthy analytical approach to cover the diverse and necessary tests of in-vitro and in-vivo studies is greatly grown with the rising production of new fixed combinations. Two novel, selective and environmentally friendly LC techniques were developed in order to guarantee precise measurement of IPR and FEN in their challenging formulation. The initial technique involved high-performance thin-layer chromatography (HPTLC) in conjunction with densitometric quantification. Chromatographic separation was attained on HPTLC plates utilizing ethyl acetate - ethanol - acetic acid (5.0:5.0:0.1, by volume) as a developing system. Densitometric quantification of the separated bands was carried out at 220.0 nm over concentration ranges of 0.50–15.0 µg/band for IPR and 0.50–12.0 µg/band for FEN. High-performance liquid chromatography (HPLC) paired with diode array detection (DAD) was the core of the second technique. The optimized separation was achieved on a Zorbax SB C₁₈ (150 × 4.6 mm, 5 μm) column with a combination of 10.0 mM potassiumdihydrogen orthophosphate, pH 5.0 ± 0.1, adjusted with o-phosphoric acid and methanol (70:30, v/v) as the mobile phase and pumped at flow rate of 1.0 mL/min. The peaks were monitored at 220.0 nm using diode array detection, achieving linearity range of 5.0–200.0 µg/mL for both drugs. The ICH criteria have been verified and both methods have been confirmed to be valid, and successfully applied for assay the cited drugs in the Atrovent^® comp HFA metered dose inhaler as well as delivered dose uniformity testing of the final product. Finally, whiteness appraisal and several state-of-the-art green evaluation metrics were applied to evaluate the sustainability of the proposed methods. The suggested approaches produced promising results and are the first simple and sustainable methodologies for the simultaneous quantification of both drugs in different real samples, all of which strongly suggest their application in quality control laboratories³.

Bhaskar Musmadeet al., (2021) Development and validation of stability-indicating RP-HPLC method for the simultaneous estimation of xylometazoline hydrochloride and ipratropium bromide from nasal spray dosage form. Background: A simple, robust, precise, and an accurate HPLC method was established for simultaneous estimation of xylometazoline hydrochloride and ipratropium bromide from a nasal spray dosage form. The effective separation was obtained by injecting 10 μl of sample and standard solutions on to an Inertsil ODS column, 250 × 4.6, mm, 5 μ at 45 °C using phosphate buffer with 1-pentane sulphonic acid sodium salt at pH 4.7 as a mobile phase A and acetonitrile as the mobile phase B. The gradient was optimized with a flow rate of 1 mL/min and a wavelength of 210.0 nm. Result: The complete analytical method validation was successfully carried out as per ICH guidelines. The retrieval study was carried out at 50% to 150% level of working concentration, and results were in the range of 99 to 101% for both the analytes. The linearity was proven from 4 to 150% of working concentration with linear regression curve (R2=0.999) for both the analytes. The developed method was robust for different parameters like column temperature, flow rate, mobile phase pH, composition, and gradient. Conclusion: The developed HPLC method can be successfully used for the estimation of xylometazoline hydrochloride and ipratropium bromide from nasal spray dosage form as a release test in QC department of manufacturing units⁴.

Amol, Bansode et al., (2020)  Development and Validation of Stability Indicating Method for Ipratropium Bromide by using RP-HPLC. A simple stability indicating RP-HPLC method was validated for determination of ipratropium bromide in the bulk drug. The drug was resolved using HPLC Column (Kromasil ODS 150 x 4.6 mm C18 column) with mobile phase of HPLC grade acetonitrile: potassium di-hydrogen phosphate buffer (60:40 v/v) at a flow rate of 1 mL/min. The retention time of ipratropium bromide was 3.7 min with UV detection at 254 nm. The method was validated with respect to linearity, sensitivity, accuracy, precision and robustness as per the International Conference on Harmonization (ICH) guidelines. The method was specific and it was observed that no interference with diluents. The linearity was established over the concentration range of 20-120 μg/mL with correlation coefficients (r2) 0.9958 for ipratropium bromide. The mean recovery was found to be in the range of 99.8% for ipratropium bromide. The % R.S.D. values for intraday precision study and inter-day study were & lt; 1.0%, confirming that the method was sufficiently precise. The drug was subjected to forced degradation under different conditions. The drug was degraded more in oxidative condition (28.89%) then alkaline condition (26.39%) then acidic condition (13.42%). There was no degradation seen under the thermal conditions⁵.

Anthony J Blewett et al., (2011) Development and Validation of a Stability-Indicating High-Performance Liquid Chromatography Method for the Simultaneous Determination of Albuterol, Budesonide, and Ipratropium Bromide in Compounded Nebulizer Solutions. In recent years, there has been a large increase in the use of pharmaceutical compounding to prepare medications that are not commercially available. The treatment of asthma typically includes the use of albuterol (ALB), ipratropium bromide (IPB), and/or budesonide (BUD) nebulizer solutions. There is currently no commercially available nebulizer solution containing all three of these compounds, and patients must rely on often-unregulated compounding. There is a distinct need for methodologies that can be used to analyze compounded formulations to ensure patient safety. We report an HPLC-UV method to separate and quantitative ALB, IPB, and BUD in nebulizer solutions. The method used a gradient elution to achieve separation via an RP C₁₈ column. The method was validated, showed good selectivity, and was linear over several orders of magnitude. The method was applied to the analysis of nebulizer solutions and determination of their storage stability. Significant ALB-dependent degradation occurred within 5 h in solutions formulated with the free base of ALB, while those containing the sulphate salt of ALB produced no degradation. Alkali solutions can cause base-catalyzed hydrolysis of IPB and degradation of BUD. Compounded formulations containing ALB need to include an acid to control pH and prevent degradation⁶.

Gajanan B. Kasawar et al., (2010) Development and validation of a stability indicating RP-HPLC method for the simultaneous determination of related substances of albuterol sulfate and ipratropium bromide in nasal solution.A simple, sensitive and specific RP-HPLC method was developed for the quantification of related impurities of albuterol sulfate (AS) and ipratropium bromide (IB) in liquid pharmaceutical dosage form. The chromatographic separation employs gradient elution using an inertsil C8-3, 250 mm × 4.6 mm, 5 μm columns. Mobile phase consisting of solvent A (solution containing 2.5 g of potassium dihydrogen phosphate and 2.87 g of heptane-1-sulfonic acid sodium salt per liter of water, adjusted to pH 4 with orthophosphoric acid) and solvent B (acetonitrile) delivered at a flow rate of 1.0 ml min⁻¹. The analytes were detected and quantified at 210 nm using photodiode array (PDA) detector. The method was validated as per ICH guidelines, demonstrating to be accurate and precise (repeatability and intermediate precision level) within the corresponding linear range of known impurities of AS and IB. The specificity of the method was investigated under different stress conditions including hydrolytic, oxidative, photolytic and thermal as recommended by ICH guidelines. Relevant degradation was found to take place under hydrolytic and oxidative conditions. Robustness against small modification in pH, column oven temperature, flow rate and percentage of the mobile phase composition was ascertained. Lower limit of quantification and detection were also determined. The peak purity indices (purity angle < purity threshold) obtained with the aid of PDA detection and satisfactory resolution between related impurities established the specificity of the determination. All these results provide the stability indicating capability of the method⁷.

CONCLUSION

According to this review HPLC is a versatile, reproducible chromatographic technique for the analysis of LevoSalbutamol and Ipratropium bromide. Methanol and acetonitrile are the typical solvents used in the majority. RP-HPLC provides the highest levels of precision, repeatability, reliability, and also it is a simple, rapid, and robust quantitative analytical method.

ACKNOWLEDGEMENTS

Working under the direction of Dr. M. Sangeetha Assistant Professor, Department of Pharmaceutical Chemistry has been a privilege. I would like to extend my profound appreciation to the person who helped us to see things more clearly, who really cares about the development of my review article, and without whom our work would not have progressed to this point. I would like to take this opportunity to thank our esteemed principal, Dr. T. Rama Rao, for providing the infrastructure needed to complete the review process effectively.

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