VALIDATED STABILITY-INDICATING HPTLC AND HPLC METHODS FOR DETERMINATION OF PIPAZETHATE AND ITS DEGRADANT

Abstract

This paper describes validated high-performance thin-layer chromatography and high-performance liquid chromatography methods for the simultaneous determination of pipazethate hydrochloride (PZ) and its degradant. The HPTLC separation was carried out on Merck HPTLC aluminum sheets of silica gel 60 F254 as stationary phase using chloroform:diethylamine:methanol (9.4:0.1:0.5, v/v/v) as a mobile phase. Quantification was achieved using densitometric measurements at 225 nm. The isocratic reversed phase HPLC separation was performed on 5 µm CN column (Luna, Phenomenex®, USA.). Good resolution between PZ and its degradants was achieved using a mixture of acetonitrile:12 mM ammonium acetate:diethylamine (35:65:0.1, v/v/v, pH 4.0) as a mobile phase. Quantitation was achieved with UV detection at 225 nm based on peak area. Forced degradation studies were performed on bulk sample of PZ using acid (1N hydrochloric acid), alkaline (0.1N sodium hydroxide), oxidation (0.33% hydrogen peroxide), heat (75°C), and photolytic degradation. The proposed HPLC method was utilized to investigate the kinetics of acidic, alkaline, and oxidative degradation processes of PZ at different temperatures and the apparent pseudo-first-order rate constant, half-life, and activation energy were calculated. The pH-rate profile of degradation of PZ in Britton-Robinson buffer solutions within the pH range 1.8–11.4 was studied.

Keywords:

• Arrhenius plot
• HPLC
• HPTLC
• kinetics of degradation
• pH-rate profile
• pipazethate

Notes

^a Y = a + b × for DG₁ and Y = a + b X + C X² for PZ where X is the concentration of drug in µg/spot, and Y is peak area of the band.

^b 95% confidence limit.

^a Y = a + bC, where C is the concentration in µg mL⁻¹ and Y is the peak area.

^b 95% confidence limit.

The retention time of unretained peak is 0.697 min.

a₁, b₁: are α and R_s calculated for DG₃ – DG₄.

a₂, b₂: are α and R_s calculated for DG₄ – PZ.

a₃, b₃: are α and R_s calculated for PZ – DG₁.

^a Mean and S.D., percentage recovery from the label claim amount.

^b Theoretical values for t and F.

^c For standard addition of different concentrations of PZ, DG₁, DG₃, and DG₄.