An LC–MS–MS method for the simultaneous quantification of amoxicillin and clavulanic acid in human plasma and its pharmacokinetic application

Objective

To develop a simple, sensitive and selective LC–MS–MS method for the simultaneous determination of amoxicillin and clavulanic acid in human plasma using amoxicillin D4 and ampicillin as internal standard (IS).

Method

Amoxicillin and clavulanic acid in plasma were concentrated by solid phase extraction and chromatographed on a C18 column using a mobile phase of acetonitrile: 2 mM ammonium acetate (80:20 v/v) under isocratic condition. Quantitation was performed on a triple quadrupole mass spectrometer by employing electrospray ionization technique and operating in multiple reaction monitoring (MRM) and negative ion mode with mass transitions 364.00/223.00, 198.00/136.00, 368.00/227.00 and 347.90/304.00 for Amoxicillin, Clavulanic acid, Amoxicillin D4 and Ampicillin respectively.

Results

The method was validated over a linear range of 50.43–31500.68 and 25.28–6185.18 ng/mL for amoxicillin and clavulanic acid respectively. The Lower Limit of Quantitation (LLOQ) were 50.43 and 25.28 ng/mL for amoxicillin and clavulanic acid respectively. Inter-batch and intra-batch coefficient of variation across three validation runs (LLOQ, LQC, MQC1, MQC and HQC) was less than 3.55% and 3.07% for amoxicillin and clavulanic acid respectively.

Conclusion

The method was validated and was suitable for the quantitation of amoxicillin and clavulanic acid from plasma samples in a pharmacokinetic study.