Determination of fosfomycin in pus by capillary zone electrophoresis

Abstract

A method is described for the determination of fosfomycin in pus by capillary zone electrophoresis with reversed electroosmotic flow, and indirect UV absorbance detection. Sample pre-treatment is limited to removal of proteins and cell debris by adding the double volume of methanol, followed by vortexing for few seconds, and centrifugation at 15,000 × g for 2 min. The supernatant is directly injected into the instrument. Fosfomycin is separated from sample constituents with a background electrolyte at pH 7.25 (25 mM benzoate buffer with 0.5 mM hexadecyltrimethylammonium bromide added, adjusted to pH with tris(hydroxymethyl)-aminomethane (TRIS)). Separation is carried out in a capillary with 50 μm I.D., 64.5 cm total length, 56.0 cm to the detector, at 25 °C with −25 kV voltage applied. Due to the low absorbance of the analyte, indirect UV detection was performed at 254 nm using a bubble cell capillary. Sample was injected by pressure (450 mbar s). Repeatability for fosfomycin in spiked pus (from 8 or 10 consecutive injections of three different series at concentrations of 100 μg/mL of the antibiotic) was between 2.4 and 8.2% relative standard deviation (RSD). Accuracy (expressed as recovery of fosfomycin determined by three independent analysis at 10, 100 and 300 μg/mL fosfomycin added to plain pus) was between 75 and 102%. Intermediate reproducibility ( n = 9 at three different days) was between 2 and 12% RSD. Limit of detection and limit of quantitation were 4.5 and 15 μg/mL, respectively. The concentration of fosfomycin in pus of patients treated with the antibiotic ranged up to 240 μg/mL. The concentration of other anionic pus constituents identified beside chloride (acetate, succinate, lactate, phosphate) ranged between 20 and 7800 μg/mL.