Development and validation of a LC-MS/MS method for quantitation of fosfomycin - Application to in vitro...

Extensive use and misuse of antibiotics over the past 50 years has contributed to the emergence and spread of antibiotic-resistant bacterial strains, rendering them as a global health concern. To address this issue, a dynamic in vitro hollow-fiber system, which mimics the in vivo environment more closely than the static model, was used to study the emergence of bacterial resistance of Escherichia coli against fosfomycin (FOS). To aid in this endeavor we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for quantitative analysis of FOS in lysogeny broth. FOS was resolved on a Kinetex HILIC (2.1 × 50 mm, 2.6 μm) column with 2 mm ammonium acetate (pH 4.76) and acetonitrile as mobile phase within 3 min. Multiple reaction monitoring was used to acquire data on a triple quadrupole mass spectrometer. The assay was linear from 1 to 1000 μg/mL. Inter- and intra-assay precision and accuracy were <15% and between ±85 and 115% respectively. No significant matrix effect was observed when corrected with the internal standard. FOS was stable for up to 24 h at room temperature, up to three freeze-thaw cycles and up to 24 h when stored at 4°C in the autosampler. In vitro experimental data were similar to the simulated plasma pharmacokinetic data, further confirming the appropriateness of the experimental design to quantitate antibiotics and study occurrence of antimicrobial resistance in real time. The validated LC-MS/MS assays for quantitative determination of FOS in lysogeny broth will help antimicrobial drug resistance studies.