Sustainable UHPLC-MS/MS assay for ultra-sensitive simultaneous quantification of amoxicillin and flucloxacillin with application to a bioequivalence study.

A novel, highly sensitive and robust ultra-performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) method was developed, optimized, and validated for the simultaneous quantification of amoxicillin (AMOX) and flucloxacillin (FLUX) in human plasma. The proposed method employs stable isotope-labelled isotopes, AMOX-d4 and FLUX‑13C4 sodium as an ideal internal standard of the studied compounds. The method is considered the first bioanalytical assay designed to concurrently measure both antibiotics in plasma, offering significant value for clinical and pharmacokinetic studies. Protein precipitation using acetonitrile was selected as a simple and effective approach for sample preparation. Chromatographic separation was achieved on an Inert Sustain Swift C18 column with an isocratic mobile phase of 0.1% formic acid and acetonitrile (55:45, v/v), delivered at a flow rate of 0.3 mL/min. Detection was carried out using positive electrospray ionization in multiple reaction monitoring mode with the following mass transitions: AMOX (m/z 366.284 → 114.000), AMOX-d4 (m/z 370.178 → 114.000), FLUX (m/z 454.206 → 160.100), and FLUX‑13C4 (m/z 458.135 → 160.100). Full validation, in accordance with FDA bioanalytical guidelines, was conducted over a linearity range of 0.05-6.00 µg/mL for AMOX and 0.05-15.00 µg/mL for FLUX, adequately covering their respective Cmax values. The method's sustainability was comprehensively evaluated by the recently introduced Sustainability Assessment Method for Instrumental Analysis (SAMI), highlighting the study's alignment with the United Nations Sustainable Development Goals (UN-SDGs). Furthermore, the developed method was successfully applied in a bioequivalence study involving 33 healthy volunteers following oral administration of a single 5 mL dose of Flucamox® 250 mg, enabling comprehensive pharmacokinetic evaluation while promoting sustainable and resource-efficient analytical practices.