Simultaneous quantification of five penicillins and detection of penicilloic acid in single and/or multi– component flucloxacillin pharmaceutical formulations

Background: The role of β-lactams among antibacterial agents in the treatment and management of infectious diseases cannot be under-estimated. Swift actions regarding quality assessment of penicillins (most frequently prescribed β-lactam) supplied to patients especially in circumstances where the products do not conform to specifications remain a challenge, i.e. inability to detect quality products from counterfeit/substandard ones.

Objectives: The goals of this study were to develop a simple RP-HPLC method for the simultaneous quantification of amoxicillin trihydrate (AXT), ampicillin trihydrate (APT), benzylpenicillin sodium (BPS), flucloxacillin sodium (FXS) and phenoxymethyl penicillin sodium (PMS) in single and combination dosage forms, detect substandard penicillin antibacterial agents and also detect penicilloic acid, a major breakdown product of penicillins and a major semi-synthetic precursor of penicillins.

Methods: Separation was achieved on a Phenomenex® LICHROSORB 10 RP-1 C18 column (250 × 4.60 mm I.D., 5 μm particle size) with methanol: 0.02M KH2PO4(pH = 3.70) (4:6v/v) in isocratic mode as mobile phase. A flow rate of 1.00 mL/min with UV detection at 225 nm was optimized, validated, and employed to assay samples from the Ghanaian market.

Results: The calibration plots for the determination of the penicillins showed correlation values (r2) between 0.9984 - 0.9995 in the concentration range of 0.005 - 0.1%w/v. Accuracy for the analytical method was confirmed with mean recoveries (n=3) in the range of 99.88 - 101.33%, 100.01 - 100.66% and 99.10 - 100.79% at concentration levels of 80, 100 and 120% respectively. Method precision with relative standard deviation (RSD) obtained for intra-day precision were 1.62 - 4.03% and the inter-day precision were 1.22 – 3.97%. The developed method was found suitable for the detection of penicilloic acid and could distinguish between good quality and substandard penicillin antibacterial products sampled from Kumasi metropolis.

Conclusion: The method is suitable for the simultaneous assay of AXT, APT, BPS, PMS and FXS in either single/multi – component dosage forms. It is also applicable for routine screening of penicillin antibiotics  for adulteration and counterfeiting and the detection of penicilloic acid, a major decomposition product in penicillins.