title: Liquid chromatography tandem mass spectrometry quantitation of intracellular concentrations of ganciclovir and its phosphorylated forms.
abstract: Ganciclovir (GCV) is prescribed for cytomegalovirus infection which is a major issue in immunodepressed patients. It is however characterized by hematological toxicity. A better understanding of GCV concentration-effects relationships implies the measurement of intracellular forms. The objective of this study was to develop a method to measure GCV and its derivatives in cells. A four-stage procedure was developed with the following strategy: (1) to separate into different fractions the different intracellular forms of GCV (GCV itself and its phosphorylated forms) by solid-phase extraction (SPE) from blood cells, (2) to dephosphorylate the different phosphorylated forms into GCV, (3) to perform a second SPE to desalt samples and concentrate GCV, and (4) to measure GCV concentrations in the different extracts using a triple-quadrupole, linear ion trap mass spectrometer. Finally, the procedure was tested in 17 patients receiving GCV. From lysed cells, the different forms of GCV were fractionated, the phosphorylated forms were eluted with different KCl solutions, and the obtained fractions were treated with acid phosphatase to transform the phosphorylated metabolites back into GCV. The method was validated from 5 to 500 μg L(-1) with a limit of detection of 1 μg L(-1). The whole procedure was validated according to the US Food and Drug Administration guidelines and successfully applied in 17 patients receiving GCV. The method liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allowing the measurement of GCV and its phosphorylated forms in blood cells was developed and can be used in developing clinical studies to explore the role of these biomarkers in the event of toxicity.
authors.affiliation: INSERM UMR 850, University of Limoges, 2, Rue Docteur Raymond Marcland, 87025, Limoges, France.
authors.forename: Pierre-André
authors.initials: PA
authors.lastname: Billat
authors.forename: François-Ludovic
authors.initials: FL
authors.lastname: Sauvage
authors.forename: Nicolas
authors.initials: N
authors.lastname: Picard
authors.forename: Naїma
authors.initials: N
authors.lastname: Tafzi
authors.forename: Sophie
authors.initials: S
authors.lastname: Alain
authors.forename: Marie
authors.initials: M
authors.lastname: Essig
authors.forename: Pierre
authors.initials: P
authors.lastname: Marquet
authors.forename: Franck
authors.initials: F
authors.lastname: Saint-Marcoux
mesh_terms: D000135:Acid Phosphatase / Q000737:chemistry; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015774:Ganciclovir / Q000032:analysis* / Q000097:blood / Q000378:metabolism* / Q000627:therapeutic use; D006801:Humans; D016030:Kidney Transplantation; D010766:Phosphorylation; D011189:Potassium Chloride / Q000737:chemistry; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: D011189:Potassium Chloride; D000135:Acid Phosphatase; D015774:Ganciclovir