Simultaneous determination of 1-β-d-Arabinofuranosylcytosine and two metabolites, 1-β-d-Arabinofuranosyluracil and 1-β-d-Arabinofuranosylcytosine triphosphate in leukemic cell by HPLC-MS/MS and the application to cell pharmacokinetics

• The concentration of ara-C, ara-U and ara-CTP in human leukemic cells was determined simultaneously by HPLC-MS/MS for the first time.
• NFPA was added to the mobile phase to retain the analytes in the column.
• The method was applied to support cell pharmacokinetics.

A specific and reliable HPLC-MS/MS method was developed and validated for the simultaneous determination of 1-β-d-Arabinofuranosylcytosine (ara-C), 1-β-d-Arabinofuranosyluracil (ara-U) and 1-β-d-Arabinofuranosylcytosine triphosphate (ara-CTP) in the leukemic cells for the first time. The analytes were separated on a C18 column (100 mm × 2.1 mm, 1.8 μm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was used for detection. The ion-pairing reagent, NFPA, was added to the mobile phase to retain the analytes in the column. The cell homogenates sample was prepared by the simple protein precipitation. The calibration curves were linear over a concentration range of 3.45–3450.0 ng/mL for ara-C, 1.12–1120.0 ng/mL for ara-U and 4.13–4130.0 ng/mL for ara-CTP. The intra-day and inter-day precision was less than 15% and the relative error (RE) were all within ±15%. The validated method was successfully applied to assess the disposition characteristics of ara-C and support cell pharmacokinetics after the patients with leukemia were intravenously infused with SDAC and HiDAC. The result of the present study would provide the valuable information for the ara-C therapy.