A novel derivatization method for the determination of Fosfomycin in human plasma by liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometric detection via phase transfer catalyzed derivatization.

An analytical method employing novel sample preparation and liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometric detection (LC-APCI/MS) was developed for the determination of fosfomycin in human plasma. Sample preparation involves derivatization through phase transfer catalysis (PTC) which offers multiple advantages due to the simultaneous extraction, preconcentration and derivatization of the analyte. Using a PT catalyst, fosfomycin was extracted from plasma in an organic phase and, then converted to a pentafluorobenzyl ester with the use of pentafluorobenzyl bromide (PFBBr) derivatization reagent. The method was fully optimized by taking into account both PTC and derivatization parameters. Several catalysts, in a wide range of concentrations, with different counter ions and polarities were tested along with different extraction solvents and pH values. Thereafter, the derivatization procedure was optimized by altering the amount of the derivatization reagent, the temperature of the reaction and finally, the derivatization duration. As internal standard (I.S.) ethylphosphonic acid was chosen and underwent the same pretreatment. The derivatives were separated on a pentafluorophenyl (PFP)-C18 analytical column, which provides unique selectivity, using an isocratic elution with acetonitrile-water (70-30, v/v). The method was validated according to US Food and Drug Administration (FDA) guidelines and can be used for a bioequivalence study of fosfomycin in human plasma. The correlation coefficient (r(2)) of the calibration curve of spiked plasma solutions in the range of 50-12000 ng/mL was found greater than 0.999 with a limit of quantitation (LOQ) equal to 50 ng/ml (for 500 μL plasma sample).