An gradient HPLC-DAD determination of phenylepherine, paracetamol, ambroxol and levocetrizine in pharmaceutical formulation

The development, validation and application of a simple and reliable gradient high-performance liquid chromatography-diode array detection (HPLC-DAD) procedure for the analysis of a complex mixture containing phenylephrine (PHE), paracetamol (PAR), ambroxol (AMB) and Levocetirizine (LEV) has been carried out . Chromatographic separation of PHE, PAR, AMB and LEV is achieved using a Phenomenex Ultracarb ODS-C18 (4.6x150 mm, 5 [mu]) column with gradient elution of the mobile phase composed of 10 mM phosphate buffer p H 3.3 and acetonitrile. A three step gradient program has been developed with step-1 elution starting with 2% (by volume) acetonitrile which ramped up linearly to 50% in 10 min, in step-2 reverting back to 20% in 5 min and in step-3 ended to achieve initial concentration of 2% in next 5 min thus contributing a total run time of 20 min. Flow rate maintained throughout the experiment is 1 mL/min. The Diode array detector (DAD) is set at 220 nm for quantification of the analytes based on measuring their peak areas. The retention times for PHE, PAR, AMB and LEV are approximately 4.4, 10.1, 14.00 and 17.90 min respectively. The proposed HPLC procedure is statistically validated with respect to linearity, ranges, precision, accuracy, selectivity and robustness. Calibration curves are found to be linear in 50 to 150% of target analyte in formulation with correlation coefficients > 0.9996. The validated HPLC method is applied successfully with good recoveries of analytes from tablet dosage; no interfering peaks were encountered from the inactive ingredients.