LC/MS/MS Method with HILIC Separation for Quantitation of Fosfomycin in Human Plasma

Significance of the Topic

Fosfomycin is a small, highly hydrophilic broad-spectrum antibiotic reintroduced to combat multidrug-resistant pathogens in urinary tract infections. Accurate quantification in human plasma supports pharmacokinetic profiling and therapeutic dose optimization.

Study Objectives and Overview

This work aimed to develop and validate a reliable LC-MS/MS method with hydrophilic interaction liquid chromatography (HILIC) for quantifying fosfomycin in human plasma, addressing sensitivity, linearity, accuracy, precision, recovery, and matrix effects.

Instrumentation Used

• Shimadzu LCMS-8060 triple quadrupole mass spectrometer
• Shimadzu Shim-pack GIS HILIC column (150 mm × 3.0 mm I.D., 3 μm)

Methodology and Instrumentation

Sample preparation involved protein precipitation with acetonitrile/methanol (1:1, v/v) at a 4:1 solvent-to-plasma ratio, centrifugation, 0.22 μm filtration, and dilution with 5 mM ammonium acetate. Chromatography used isocratic elution (90% aqueous 5 mM ammonium acetate/10% acetonitrile), 0.3 mL/min flow, 40 °C oven, and 5 μL injection. Mass spectrometry operated in negative heated ESI with MRM transitions: fosfomycin 137.05→79.00 (quantifier), 137.05→63.00 and 137.05→81.05 (qualifiers); internal standard fosfomycin-13C3 benzylamine 140.00→79.00.

Main Results and Discussion

• Calibration was linear from 0.02 to 6 ppm (R2 ≥ 0.999); LLOQ of 0.02 ppm achieved S/N ≥ 10 and RSD < 8%.
• Accuracy ranged from 93% to 102% across low, medium, and high quality control levels; precision RSD < 5% (n = 6).
• Recovery in two plasma lots ranged 60.7%–81.2%; matrix effects spanned 52.2%–76.5%, demonstrating acceptable robustness.

Benefits and Practical Applications of the Method

The rapid, sensitive assay facilitates pharmacokinetic and therapeutic drug monitoring studies of fosfomycin, supporting clinical decision-making in multidrug-resistant infections and quality control in research and diagnostic laboratories.

Future Trends and Potential Applications

• Extension of HILIC-MRM workflows to other hydrophilic antibiotics and metabolites.
• Automation of sample preparation and integration into routine clinical monitoring systems.
• Coupling with high-resolution mass spectrometry for comprehensive metabolomic profiling.
• Development of multiplex assays for simultaneous quantitation of antibiotic combinations.

Conclusion

The established LC-MS/MS method with HILIC separation delivers robust, accurate, and precise quantification of fosfomycin in human plasma down to 0.02 ppm. Validation parameters meet regulatory criteria, making it suitable for pharmacokinetic research and therapeutic monitoring.

References

• Hee DH, Ting ZW, Lee LSU, Xing J, Cheng KL, Zhan Z. ASMS 2017, ThP 079.
• Sime FB, Johnson A, Whalley S, Santoyo-Castelazo A, Montgomery AB, Walters KA, Lipman J, Hope WW, Roberts JA. Antimicrob Agents Chemother. 2017;61(1):e01763-16.