New, precise and selective HPLC and UHPLC methods have been upgraded and verified for simultaneous estimation of a quaternary mixture of cephalexin (CPH), sodium benzoate (SB), methylparaben (MP) and propylparaben (PP) in their powder for oral suspension (POS) dosage form and applied to holding time study. The bulk holding time study plays an important role in determination of the maximum allowable periods for holding the bulk material starting from preparation till primary packaging and evaluating the quality of the product during the hold time. The analytes are determined by isocratic chromatography condition using 0.01 M ammonium dihydrogen phosphate (pH 4.5 ± 0.1) : acetonitrile : methanol (60:30:10, v/v) as mobile phase and separated on a reverse-phase C8 column (25 cm × 0.46 cm) with injection volume 20 μL and a flow rate of 1.5 mL/min for RP-HPLC and stationary phase C18 column (5 cm × 0.46 cm, 2.7 μm particle size) with injection volume 0.2 μL and a flow rate of 0.3 mL/min for RP-UHPLC with a detection wavelength at 230 nm. Two ecofriendly spectrophotometric methods are successfully deveoped for resolving spectral over-lap of drugs. Method A: first derivative of ratio spectra (1stDD) spectrophotometric method in which CPH is selected at 322.2 nm, while paraben group and SB determined at 285.1 nm, 313.9 nm and 219 nm, 231 nm respectively. Method B: mean centering of ratio (MCR) spectra method at 247 nm, 279 nm and 241 nm for CPH, paraben group and SB, respectively. The validated methods are efficiently accomplished for the pure, pharmaceutical formulation and mixture with a high correlation coefficient ≥0.9995 and recovery 100 ± 1.0%. The reported methods are described for simultaneous determination of quaternary mixture in one injection with shorter run time at high sensitivity and selectivity.
