Simultaneous determination and stability studies of linezolid, meropenem and vancomycin in bacterial growth medium by high-performance liquid chromatography.

For pharmacokinetic/pharmacodynamic (PK/PD) assessment of antibiotics combinations in in vitro infection models, accurate and precise quantification of drug concentrations in bacterial growth medium is crucial for derivation of valid PK/PD relationships. We aimed to (i) develop a high-performance liquid chromatography (HPLC) assay to simultaneously quantify linezolid (LZD), vancomycin (VAN) and meropenem (MER), as typical components of broad-spectrum antibiotic combination therapy, in bacterial growth medium cation-adjusted Mueller-Hinton broth (CaMHB) and (ii) determine the stability profiles of LZD, VAN and MER under conditions in in vitro infection models. To separate sample matrix components, the final method comprised the pretreatment of 100μL sample with 400μL methanol, the evaporation of supernatant and its reconstitution in water. A low sample volume of 2μL processed sample was injected onto an Accucore C-18 column (2.6μm, 100×2.1mm) coupled to a Dionex Ultimate 3000 HPLC+ system. UV detection at 251, 240 and 302nm allowed quantification limits of 0.5, 2 and 0.5μg/mL for LZD, VAN and MER, respectively. The assay was successfully validated according to the relevant EMA guideline. The rapid method (14min) was successfully applied to quantify significant degradation of LZD, VAN and MER in in vitro infection models: LZD was stable, VAN degraded to 90.6% and MER to 62.9% within 24h compared to t=0 in CaMHB at 37°C, which should be considered when deriving PK/PD relationships in in vitro infection models. Inclusion of further antibiotics into the flexible gradient-based HPLC assay seems promising.