title: Determination of intracellular fludarabine triphosphate in human peripheral blood mononuclear cells by LC-MS/MS.
abstract: 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 1mL 70% methanol containing 1.2mM tris buffer (pH 7.4). The lysate (80μL) was mixed with internal standard (2-chloro-adenosine triphosphate, 150ng/mL, 20μL) and injected onto an API5000 LC-MS/MS system. Separation was achieved on a hypercarb column (100mm×2.1mm, 3μm) eluted with 100mM ammonium acetate (pH 9.8) and acetonitrile in a gradient mode at a flow rate of 0.4mL/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 3.4min for fludarabine triphosphate and the IS, respectively. The concentration range for the calibration curve was 1.52-76nM. 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 3.19pmole/million PBMC at trough and peak time points, respectively. Fludarabine triphosphate is degraded in water within hours but relatively stable in 70% methanol-tris (1.2mM, 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.
authors.affiliation: Drug Research Unit, Department of Clinical Pharmacy, University of California at San Francisco, San Francisco, CA 94143, USA. Electronic address: huangl@sfghsom.ucsf.edu.
authors.forename: Liusheng
authors.initials: L
authors.lastname: Huang
authors.forename: Patricia
authors.initials: P
authors.lastname: Lizak
authors.forename: Francesca
authors.initials: F
authors.lastname: Aweeka
authors.forename: Janel
authors.initials: J
authors.lastname: Long-Boyle
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry* / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood
chemical_list: D014740:Vidarabine; C024352:fludarabine
keywords: 2-chloroadenosine triphosphate; Cl-ara-ATP; F-ara-ATP; Fludarabine triphosphate; Hypercarb column; LC–MS/MS; PBMC; PK; Stability; alloHCT; allogeneic hematopoietic cell transplantation; fludarabine triphosphate; peripheral blood mononuclear cells; pharmacokinetic