Development and validation of a LC‐MS/MS method for quantitation of fosfomycin – Application to in vitro antimicrobial resistance study using hollow‐fiber infection model

Abstract

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.

Quantification of Fosfomycin in Combination with Nine Antibiotics in Human Plasma and Cation-Adjusted Mueller-Hinton II Broth via LCMS

“…Over the past five years, mass spectrometry-based methods to measure fosfomycin in various matrices such as plasma [ 9 , 10 , 11 ], plasma dried spots [ 12 ], growth media [ 13 ], and urine [ 11 , 14 , 15 ] have been reported to support PK/PD optimization of fosfomycin therapy. Interestingly, these methods solely measure fosfomycin.…”

A high-throughput bioanalytical assay to support pharmacokinetic interaction study of oxycodone and diazepam in Sprague Dawley rats

“…Also, this 96-well plate format is rapid and consumes minimum solvents. 36,37 Addition of 2 mM ammonium formate in 0.1% formic acid to the extracted samples helped in achieving good peak shapes for oxycodone and diazepam.…”

“…Internal standard selection is important in mass spectrometry analysis to avoid matrix effect related issues. 36,37,43 Use of stable labeled isotopes as internal standard (IS) increases the bioanalytical assay precision and accuracy. Hence, for the present work oxycodone-d 6 and diazepam-d 5 were employed as ISs, to quantitate oxycodone and diazepam, respectively.…”

“…The proposed high-throughput LC-MS/MS assay was developed and validated for its reproducibility by using deutated internal standards oxycodone-d 6 (IS1) and diazepam-d 5 (IS2) for the quantitation of oxycodone and diazepam, respectively, to avoid matrix effect problems and variability in recovery between analyte and the internal standard. 36,37 The method offers an efficient extraction procedure based on protein precipitation (PP) in a 96-well plate format, with minimum usage of organic solvents. The method was successfully applied to determine the pharmacokinetic (PK) prole of oxycodone/diazepam in SD rats treated with different doses.…”

Evaluation of Immunocompetent Urinary Tract Infected Balb/C Mouse Model For the Study of Antibiotic Resistance Development Using Escherichia Coli CFT073 Infection

“…Ciprofloxacin was quantified in urine and urinary tissues by LC-MS/MS as previously described [30]. Fosfomycin and ampicillin quantifications were similarly done as previously reported [31,32], with slight modifications. For plasma and urinary tissues, samples were extracted with solvent in the ratio of 1:5 (matrix: precipitation solvent).…”