To address multi-drug resistance (MDR) among harmful substances, liquid chromatography–tandem mass spectrometry was used to test fosfomycin as a means of bypassing growing MDR amongst microorganisms.
Researchers from the Free University of Berlin (Germany) worked to bypass multi-drug resistance in antibacterials using fosfomycin (FOS), an underutilized antibiotic. Its potential was validated using a liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based technique, and the subsequent results were published in the Journal of Chromatography B (1).
Multi-drug resistance (MDR) is a type of acquired resistance microorganisms can face when proteins are overexpressed (2). Multi-drug-resistant bacterial infections are a major concern to public health, with a promising strategy for combatting them being the reassessment of neglected antibiotics. Fosfomycin (FOS), which is used to treat urinary tract infections and cystitis (bladder infection) in women, is one antibiotic that is being assessed, as it is said to remain effective against MDR Gram-positive and Gram-negative pathogens (3). That said, current in vitro and clinical data on this antibiotic, which is used to treat urinary tract infections and cystitis (a type of bladder infection) in women, are mostly insufficient at identifying optimized dosing strategies. Investigations in in vitro infection models (IVIMs) can generate crucial knowledge for treatment optimization. However, reliable bioanalytical assays for FOS in Mueller-Hinton broth (MHB), a standard bacterial growth medium used for IVIM experiments, are essential.
In this study, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for quantifying FOS in MHB was developed and validated. The assay was created according to the criteria of the European Medicines Agency (EMA) guidelines on bioanalytical method validation. The calibration range of 10–2000 mg/L FOS (found in the original samples, corresponding to 0.04–8 mg/L after sample preparation) covered clinically relevant concentrations. The assay used a sample preparation process made up of precipitation and dilution, making the process both simple and rapid. Additionally, the scientists investigated the thermal stability of FOS in MHB before applying the assay to samples from IVIM studies with clinical E. coli isolate.
Within-run mean accuracy ranged from 91.3–108%. Within-run and between-run precision were investigated with the coefficient of variation, which represents the ratio of the standard deviation to the expected return (4). According to the scientists, the CV was required to be ≤15% (and ≤ 20% at the lower limit of quantification [LLOQ]). Within-run precision fulfilled the set criteria with a maximum CV of 11.7% across all concentration levels and analytical runs (n = 126) and no trend was observed across the concentration range. Between-run accuracy (n = 18 per concentration level), ranging from 95.9–104% and precision, ranging from 4.72–8.48%, met the specified criteria.
When applied to quantify FOS concentration in samples from IVIM experiments, one clinical E. coli isolate (ST95) was exposed to FOS concentration between 16 and 1024 mg/L for 24 h. Recovery ranged from 85.7–106% at the start of the analysis period and from 80.1–115% at the 24-hour mark. Overall, the method was shown to be feasible when applied to bacteria-containing samples, with acceptable recovery of all measured QC samples recorded across the entire concentration range. The scientists did not expect matrix effects from bacterial by-products in MHB to influence routing batch processing. Further, the method is believed to successfully transferable to samples with MHB from different suppliers.
Overall, the assay was deemed a valuable tool for supporting further studies with FOS in IVIM set-ups, to gain a deeper understanding of the pharmacokinetic and pharmacodynamic relationship of FOS. FOS continues to be a promising treatment option for life-threatening MDR infections.
(1) Zimmermann, N.; Schumacher, F.; Bredendiek, F.; Kloft, C. Development and Validation of an LC-MS/MS Assay for Quantification of Fosfomycin in Mueller-Hinton Broth for Application in Profiling Antibacterial Efficacy Against Clinical Bacterial Isolates. J. Chromatogr. B 2025, 1256, 124561. DOI:
(2) Catalano, A.; Iacopetta, D.; Ceramella, J.; Scumaci, D.; et al. Multidrug Resistance (MDR): A Widespread Phenomenon in Pharmacological Therapies. Molecules 2022, 27 (3), 616. DOI:
(3) Fosfomycin (Oral Route). Mayo Clinic 2025.
(4) Hayes, A. Coefficient of Variation: Definition and How to Use It. Investopedia 2025.
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