A Novel Method for the Determination of Ganciclovir Triphosphate in Human Red Blood Cells by LC-MS/MS.

Abstract

INTRODUCTION: Ganciclovir (GCV) and its prodrug valganciclovir (VGCV) are commonly used to prevent and treat cytomegalovirus (CMV) infections in solid organ transplant recipients. Because of high interindividual pharmacokinetic variability, therapeutic drug monitoring (TDM) is important for optimizing therapy. Ganciclovir-triphosphate (GCV-TP), the active intracellular metabolite, is linked to both efficacy and toxicity; however, clinical monitoring is limited by analytical challenges. OBJECTIVES: To develop and validate a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying GCV-TP in human red blood cells (RBCs). METHODS: GCV-TP was extracted using protein precipitation, separated on a BioBasic AX column, and detected with tandem mass spectrometry using guanosine-13C10-5'-triphosphate as the internal standard. RESULTS: The assay was linear over 0.01-2.00 μg/mL (r 2 ≥ 0.99), with intra- and inter-assay variability ≤7.99%. GCV-TP in RBC lysate was stable when stored at room temperature for 4 h or at 4 °C for 24 h. This method was applied to RBC samples from 27 renal transplant recipients receiving VGCV, with a median GCV-TP concentration of 180.8 pmol/8 × 109 RBC (IQR: 113.6-297.6 pmol/8 × 109 RBC). GCV-TP levels showed a significant correlation with VGCV dose (p = 0.020, r = 0.31) but not with plasma GCV concentrations (p > 0.05). CONCLUSION: This validated method enables reliable measurement of intracellular GCV-TP and may serve as a useful tool to support individualized VGCV therapy and pharmacodynamic monitoring in clinical practice. Ganciclovir and valganciclovir prevent and treat CMV in transplant patients; however, standard blood tests may not accurately reflect intracellular drug efficacy. This study developed a simple, reliable method to measure ganciclovir-triphosphate—the active form of the drug—inside red blood cells. When the method was applied to kidney transplant patient samples, the results showed that intracellular levels correlate with the prescribed valganciclovir dose but not with plasma ganciclovir concentrations. This method may help clinicians better individualize therapy and improve monitoring.

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