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

Abstract

Fosfomycin-based combination therapy has emerged as an attractive option in our armamentarium due to its synergistic activity against carbapenem-resistant Gram-negative bacteria (CRGNB). The ability to simultaneously measure fosfomycin and other antibiotic drug levels will support in vitro and clinical investigations to develop rational antibiotic combination dosing regimens against CRGNB infections. We developed an analytical assay to measure fosfomycin with nine important antibiotics in human plasma and cation-adjusted Mueller–Hinton II broth (CAMHB). We employed a liquid-chromatography tandem mass spectrometry method and validated the method based on accuracy, precision, matrix effect, limit-of-detection, limit-of-quantification, specificity, carryover, and short-term and long-term stability on U.S. Food & Drug Administration (FDA) guidelines. Assay feasibility was assessed in a pilot clinical study in four patients on antibiotic combination therapy. Simultaneous quantification of fosfomycin, levofloxacin, meropenem, doripenem, aztreonam, piperacillin/tazobactam, ceftolozane/tazobactam, ceftazidime/avibactam, cefepime, and tigecycline in plasma and CAMHB were achieved within 4.5 min. Precision, accuracy, specificity, and carryover were within FDA guidelines. Fosfomycin combined with any of the nine antibiotics were stable in plasma and CAMHB up to 4 weeks at −80 °C. The assay identified and quantified the respective antibiotics administered in the four subjects. Our assay can be a valuable tool for in vitro and clinical applications.

Fast and Sensitive Analysis of Fosfomycin in Human Plasma Microsamples Using Liquid Chromatography–Tandem Mass Spectrometry for Therapeutic Drug Monitoring

“…This may be a practical alternative to the HILIC approach used for fosfomycin determination in previous studies. 16,17,19 The relatively low intra-and inter-day CVs (,10% for LQC, MQC, and HQC) demonstrate the precision of our method in the determination of fosfomycin concentrations. This enables the provision of reliable values for implementing real-time TDM of fosfomycin in different clinical settings.…”

“…Analytical sensitivity assessed through the S/N ratio of the 1 mg/L LLOQ sample is valuable for TDM. 6 Thus, obtaining this level of sensitivity in microsamples may be considered a remarkable achievement in the analysis of fosfomycin using LC-MS/MS, 17,19 which is the most sensitive technique for drug analysis. Sample preparation consisted of plasma dilution with water (1:17 v/v), followed by protein precipitation with methanol (1:3 v/v).…”

“…However, they have reported slightly lower extraction yields. 16,17,19 We employed the same parameters used in previous studies for fosfomycin quantification 16,17,19,32,33 in the negative ionization mode of MRM transitions. Absolute specificity and selectivity of these transitions were confirmed by the absence of interfering peaks in MRM chromatograms relative to .100 plasma samples from hundreds of patients under various antimicrobial therapies, except for fosfomycin.…”

“…These include methods based on LC-MS/MS, which is the most sensitive. 14,16,19 Some LC-MS/MS methods recently showed better sensitivity and shorter run times. 14,16,17,19 However, they were based on a HILIC approach rather than on the standard reverse-phase chromatographic approach, which usually offers a wider drug spectrum of applicability.…”

“…14,16,19 Some LC-MS/MS methods recently showed better sensitivity and shorter run times. 14,16,17,19 However, they were based on a HILIC approach rather than on the standard reverse-phase chromatographic approach, which usually offers a wider drug spectrum of applicability. 20 LC-MS/MS methods are characterized by high sensitivity and specificity and may enable the direct analysis of very small biological samples.…”