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Liquid chromatography tandem mass spectrometry quantitation of intracellular concentrations of ganciclovir and its phosphorylated forms

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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.

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Abbreviations

ACV:

Acyclovir

AK:

Enzyme acetate phosphotransferase

AUC:

Area under the curve

C max :

Maximal concentration observed after drug intake

C min :

Residual concentration

CMV:

Cytomegalovirus

CV:

Coefficient of variation

GCV:

Ganciclovir

GCV-DP:

Ganciclovir diphosphate

GCV-MP:

Ganciclovir monophosphate

GCV-TP:

Ganciclovir triphosphate

IQC:

Internal quality control

IS:

Internal standard

LC-MS/MS:

Liquid chromatography coupled to tandem mass spectrometry

LLOQ:

Lower limit of quantification

LOD:

Limit of detection

PBMC:

Peripheral blood mononuclear cell

SPE:

Solid-phase extraction

SRM:

Selected reaction monitoring

T max :

Time at which appears the maximal concentration

UPW:

Ultrapure water

VGCV:

Valganciclovir

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Author information

Authors and Affiliations

  1. INSERM UMR 850, University of Limoges, 2, Rue Docteur Raymond Marcland, 87025, Limoges, France

    Pierre-André Billat, François-Ludovic Sauvage, Nicolas Picard, Marie Essig, Pierre Marquet & Franck Saint-Marcoux

  2. Department of Pharmacology, Toxicology and Pharmacovigilance, Limoges University Hospital, 87042, Limoges, France

    François-Ludovic Sauvage, Nicolas Picard, Naїma Tafzi, Pierre Marquet & Franck Saint-Marcoux

  3. Centre National de Référence CMV, Limoges University Hospital, 2, Avenue Martin Luther King, 87042, Limoges, France

    Sophie Alain

  4. Department of Bacteriology, Virology and Hygiene, Limoges University Hospital, 2, Avenue Martin Luther King, 87042, Limoges, France

    Sophie Alain

  5. Department of Nephrology, Dialysis and Transplantation, Limoges University Hospital, 2, Avenue Martin Luther King, 87042, Limoges, France

    Marie Essig

  6. Toxicology Laboratory, Faculty of Pharmacy, University of Limoges, 2, Rue Docteur Raymond Marcland, 87042, Limoges, France

    Franck Saint-Marcoux

Authors
  1. Pierre-André Billat
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  2. François-Ludovic Sauvage
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  3. Nicolas Picard
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  4. Naїma Tafzi
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  5. Sophie Alain
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  8. Franck Saint-Marcoux
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Correspondence to Franck Saint-Marcoux.

Additional information

Parts of this work “Discussed Poster Abstracts (2014). Fundamental & Clinical Pharmacology vol 28. doi: 10.1111/fcp.12065” were presented at the joint 18th Annual Meeting of French Society of Pharmacology and Therapeutics and 81th Annual Meeting of Society of Physiology [1].

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Billat, PA., Sauvage, FL., Picard, N. et al. Liquid chromatography tandem mass spectrometry quantitation of intracellular concentrations of ganciclovir and its phosphorylated forms. Anal Bioanal Chem 407, 3449–3456 (2015). https://doi.org/10.1007/s00216-015-8554-0

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  • DOI: https://doi.org/10.1007/s00216-015-8554-0

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