Simultaneous determination of fludarabine and clofarabine in human plasma by LC-MS/MS

Abstract

A method for quantification of fludarabine (FDB) and clofarabine (CFB) in human plasma was developed with an API5000 LC-MS/MS system. FDB and CFB were extracted from EDTA plasma samples by protein precipitation with trichloroacetic acid. Briefly, 50 μL plasma sample was mixed with 25 μL internal standard (50 ng/mL aqueous 2-Cl-adensosine) and 25 μL 20% trichloroacetic acid, centrifuged at 25,000 × g (20,000 rpm) for 3 min, and then transfered to an autosampler vial. The extracted sample was injected onto an Eclipse extend C18 column (2.1 mm×150 mm, 5 μm) and eluted with 1mM NH4OH (pH 9.6) - acetonitrile in a gradient mode. Electrospray ionization in positive mode (ESI(+)) and multiple reaction monitoring (MRM) were used, and ion pairs 286/134 for FDB, 304/170 for CFB and 302/134 for the internal standard were selected for quantification. The retention times were typically 3.72 min for FDB, 4.34 min for the internal standard, 4.79 min for CFB. Total run time was 10 min per sample. Calibration range was 0.5-80 ng/mL for CFB and 2-800 ng/mL for FDB. The method was applied to a clinical pharmacokinetic study in pediatric patients.

Keywords: Clofarabine; Fludarabine; LC–MS/MS; Plasma.

Figure 1

Chemical structures of fludarabine, clofarabine, and 2-chloro-adenosine (IS).

Figure 2

Representative chromatograms of FDB and CFB at LLOQ and ULOQ levels.

Figure 3

Mean plasma concentration-time profile of FDB and CFB. The dash line with triangle markers represents FDB alone from 24 subjects (n=24); solid lines represent combination of FDB (square markers) and CFB (circle markers). The error bar represents one standard deviation.