HPLC-MS/MS determination of amoxicillin in blood plasma using derivatization
Abstract
Amoxicillin (AMOX) is an antibiotic widely used both in general medicine and veterinary medicine that is why it is very important to enable its reliable determination in biological matrices, such as blood plasma. AMOX is an unstable analyte, its degradation in solutions and matrices is influenced by many parameters: temperature, the nature of the solvent, the pH of the medium, salts or acids added to the extracts. These factors impair sample preparation and the quantification of amoxicillin in complex matrices since analyte stability plays an important role in the pharmacokinetic study of a large number of samples or a long-term analysis. The developed method allows reliably determining amoxicillin in blood plasma by HPLC-MS/ MS. It involves increasing amoxicillin stability in blood plasma extracts and increasing the retention time on reverse-phase sorbents by derivatisation with a secondary cyclic amine (pyrrolidine). The developed methodology considers the influence of many factors on the completeness of derivatisation and extraction. For this, we varied the pH of the extracting phosphate buffer, the added volume of pyrrolidine, the derivatisation time, and the pH of the extract before it was purified by solid-phase extraction. The method's conditions were optimised to achieve a good recovery of at least 80%. During HPLC-MS/MS analysis, the detectable substance was a derivative, with a molar mass of 436 g/mol. The final analyte was detected in the negative ionisation mode with m/z=435.3, during fragmentation it produced product ions with m/z=263.1, 357.2, and 340.1. Derivatisation allowed improving the retention of the determined analyte on the reverse-phase sorbent. Chromatographic separation took 9 minutes and took place in a gradient elution mode. At the same time, amoxicillin in blood plasma extracts in the autosampler degraded by more than 20% in 24 hours and its derivative remained stable under these conditions.
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