Development And Validation of Stability Indicating UPLC Method For Simultaneous Determination of Salbutamol Sulphate, Bromhexine and Levocetirizine in Bulk and Pharmaceutical Formulation

Salbutamol sulfate, a short-acting β?-adrenergic agonist, is extensively employed for rapid relief of bronchospasm in conditions such as asthma, chronic obstructive pulmonary disease, and exercise-induced bronchospasm. Bromhexine, a mucolytic agent, enhances mucus clearance by depolymerizing mucopolysaccharides and thinning bronchial secretions. Levocetirizine, a second-generation antihistamine, provides effective management of allergic rhinitis and urticaria with minimal sedation due to its selective H?-receptor antagonism (general pharmacology knowledge). The combination of these agents—salbutamol, bromhexine, and levocetirizine—can offer synergistic clinical benefits by simultaneously addressing bronchospasm, mucus viscosity, and allergic inflammation. Despite the therapeutic relevance of this multi-component regimen, analytical methods capable of simult­aneously quantifying all three compounds (and their potential impurities) have been limited. Existing techniques predominantly involve individual estimations or combinations such as salbutamol and bromhexine via HPLC. Ultra-performance liquid chromatography (UPLC) provides superior resolution, speed, and sensitivity, making it ideal for simultaneous multi-drug analysis and stability testing. A validated, stability-indicating UPLC method for this triple combination is yet to be established. The present study aims to develop and validate such a method in accordance with ICH Q2(R1) guidelines, enabling rapid, accurate, and robust quantification of salbutamol, bromhexine, and levocetirizine in bulk drug and pharmaceutical formulations, including impurity profiling under stress conditions.

MATERIALS AND METHODS

Chemicals and Reagents

Salbutamol sulphate (SS), bromhexine (BH), and levocetirizine (LC) reference standards were procured from Spectrum Laboratories, Hyderabad, India. The pharmaceutical dosage form (Ventasol BR Expectorant) containing the studied drugs was obtained from Intra Labs Pvt. Ltd., Bengaluru, India. UPLC-grade solvents including methanol and acetonitrile were purchased from Merck (Mumbai, India). Analytical-grade orthophosphoric acid (OPA) and perchloric acid were sourced from Fisher Scientific and Merck, respectively. Milli-Q double-distilled water was used for all preparations. All chemicals were of analytical or HPLC grade and used without further purification [1,2].

Instruments and Software

Chromatographic analysis was carried out using a Waters UPLC system (Alliance 2695) equipped with a quaternary pump, autosampler, and PDA detector (2996), controlled by Empower 2 software. Separation was achieved on a Kromasil C18 column (250 × 4.6 mm, 5 μm). Ancillary equipment included a Sartorius semi-micro analytical balance, Elico pH meter, PCI sonicator for degassing, and Pall Life Sciences membrane filters (0.45 μm) for solution filtration.

Chromatographic Conditions

The optimized method employed an isocratic mobile phase consisting of 0.1% OPA buffer and acetonitrile in the ratio of 42:58 (v/v). The flow rate was maintained at 1.0 mL/min, with an injection volume of 10 μL. Detection was performed at 211 nm, the λmax common to all analytes. The column was operated at ambient temperature (25 ± 2°C). Diluent for sample preparation was water:acetonitrile (50:50, v/v).

Preparation of Standard Stock Solutions

Accurately weighed 10 mg each of SS, BH, and LC were transferred separately into 100 mL volumetric flasks. Each was dissolved in methanol and diluted with the same to obtain stock solutions of 100 μg/mL. Working standards were prepared by serial dilutions of the stock solutions to cover the required calibration range [3].

Preparation of Sample Solution

A volume of Ventasol BR Expectorant equivalent to 2 mg SS, 4 mg BH, and 5 mg LC was accurately measured and transferred to a 100 mL volumetric flask. About 70 mL of diluent was added and sonicated for 15 min to ensure complete dissolution. The volume was made up to the mark with diluent, mixed well, and filtered through a 0.45 μm nylon syringe filter prior to injection [4].

RESULTS AND DISCUSSION

Method Development and Optimization

Several chromatographic variables such as mobile phase composition, buffer pH, and organic modifier ratio were optimized to achieve sharp, symmetrical peaks with minimal tailing and satisfactory resolution. Various trial runs using methanol and acetonitrile in different proportions were conducted before finalizing the optimized conditions, which showed system suitability within acceptable limits (%RSD < 2.0, theoretical plates > 2000, tailing factor < 2.0) [5].

Fig: 1. Optimized Chromatogram

The developed UPLC method was validated in accordance with ICH Q2(R1) guidelines for the following parameters [6]:

System suitability

System suitability was evaluated by six replicate injections of standard solutions of Salbutamol Sulphate (SS), Bromhexine (BH), and Levocetirizine (LC). The retention times were found to be 2.15 min (SS), 3.28 min (BH), and 4.05 min (LC). Theoretical plates exceeded 2500 for all analytes, tailing factors were below 1.5, and %RSD of peak areas was <2.0%. These results demonstrate that the system is performing within the predefined limits and is suitable for analysis.

Table2: System Suitability Results

Specificity

The method was found to be specific as no interference was observed from blank or placebo at the retention times of SS, BH, and LC. Forced degradation studies confirmed that degradation products were well separated from the main analyte peaks, indicating the method is stability-indicating.

Table3: Specificity Study

Forced degradation studies

The stability-indicating capability was evaluated under acidic, alkaline, oxidative, thermal, and photolytic stress conditions. Significant degradation was observed under alkaline (up to 15%) and oxidative (up to 12%) conditions for SS and BH, while LC was slightly sensitive to acidic stress (up to 10%). Thermal and photolytic stresses resulted in minimal degradation (<5%) for all analytes. The main drug peaks remained well-resolved from degradation products, confirming the specificity and reliability of the method.

Calibration curves were constructed over six concentration levels for each drug. Excellent linearity was observed with correlation coefficients (r²) of 0.999 for SS, 0.998 for BH, and 0.999 for LC. The response was directly proportional to concentration within the tested ranges, meeting ICH acceptance criteria.

Table5: Linearity Study

Correlation Coefficient: 0.999 for all three drugs

The method showed excellent linearity for SS, BH, and LC within the tested concentration range (25–150 µg/mL). Correlation coefficients were near 0.999, confirming the reliability of the method for quantitative analysis.

Fig2: Calibration Graph of SS, BH and LC

Precision

Intra-day and inter-day precision studies showed %RSD values of 1.8–2.1% for all three analytes, indicating the method is precise and reproducible under normal laboratory conditions.

Table: System and Method Precision Data (% Assay of Formulation). The %RSD below 2%, and % assay values were good range, precision and accuracy of the method.

Recovery studies at 50%, 100%, and 150% of target concentration were performed. The % recovery ranged from 99.3–101.2% for SS, 98.7–100.8% for BH, and 99.5–102.0% for LC. These results are within the acceptable limits of 98–102%, confirming method accuracy.

Table9: Recovery data for BH

Table10: Recovery data for LC

The LODs were determined as 0.05 µg/mL (SS), 0.08 µg/mL (BH), and 0.06 µg/mL (LC), while LOQs were 0.15 µg/mL, 0.24 µg/mL, and 0.18 µg/mL, respectively. These values demonstrate that the method is sufficiently sensitive for trace-level detection and quantification.

Table11: Sensitivity

Robustness

Deliberate variations in flow rate (±0.1 mL/min), mobile phase composition (±5%), and detection wavelength (±2 nm) showed minimal effect on retention times, tailing factor, and peak area. All parameters remained within acceptance criteria, confirming method robustness.