Determination of intracellular fludarabine triphosphate in human peripheral blood mononuclear cells by LC–MS/MS

• Direct analysis of fludarabine triphosphate using hypercarb column.
• Highly sensitive with an LLOQ at 1.52 nM.
• The 1st report of a clinical application from pediatric patients.
• Provided valuable data for the stability of fludarabine triphosphate.

Fludarabine is a nucleoside analog routinely used in conditioning regimens of pediatric allogeneic stem cell transplantation to promote stem cell engraftment. In children, it remains a challenge to accurately and precisely quantify the active intracellular triphosphate species of fludarabine in vivo, primarily due to limitations on blood volume and inadequate assay sensitivity. Here we report a liquid chromatography tandem mass spectrometry (LC–MS/MS) method for determination of fludarabine triphosphate in human peripheral blood mononuclear cells (PBMC). PBMC (∼5 million cells) were collected and lysed in 1 mL 70% methanol containing 1.2 mM tris buffer (pH 7.4). The lysate (80 μL) was mixed with internal standard (2-chloro-adenosine triphosphate, 150 ng/mL, 20 μL) and injected onto an API5000 LC–MS/MS system. Separation was achieved on a hypercarb column (100 mm × 2.1 mm, 3 μm) eluted with 100 mM ammonium acetate (pH 9.8) and acetonitrile in a gradient mode at a flow rate of 0.4 mL/min. Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI−) were used for detection. The ion pairs 524.0/158.6 for the drug and 540.0/158.8 for the IS were selected for quantification and 524.0/425.7 used for confirmation. Retention time was 3.0 and 3.4 min for fludarabine triphosphate and the IS, respectively. The concentration range for the calibration curve was 1.52–76 nM. Our method is simple, fast, and has been successfully applied in a clinical dose–concentration study in children to quantify intracellular fludarabine in low volume clinical samples. The median concentration was 1.03 and 3.19 pmole/million PBMC at trough and peak time points, respectively. Fludarabine triphosphate is degraded in water within hours but relatively stable in 70% methanol–tris (1.2 mM, pH 7.4). One limitation is that the hypercarb column takes a longer time to equilibrate than conventional reverse phase columns, and peaks become broad and distorted if the column is not washed and stored properly.

Figure optionsDownload as PowerPoint slide