Quantitative determination of azacitidine triphosphate in peripheral blood mononuclear cells using liquid chromatography coupled with high-resolution mass spectrometry.

Azacitidine is a cytidine analog used in the treatment of myelodysplastic syndromes, chronic myelomonocytic leukemia and acute myeloid leukemia. The pharmacological effect of azacitidine arises after incorporation into the DNA and RNA. To this end, the drug first has to be converted into its triphosphate forms. This paper describes the development of an assay for quantitative determination of azacitidine triphosphate (aza-CTP) in peripheral blood mononuclear cells (PBMCs). To quantify aza-CTP, separation from the endogenous nucleotides cytidine triphosphate (CTP) and uridine triphosphate (UTP) is required. This was a challenge as the structures of these nucleotides are highly similar and the monoisotopic molecular masses of aza-CTP, UTP and the naturally occurring [(13)C]- and [(15)N]-isotopes of CTP differ less than 0.02 Da. Efforts to select a specific MS(2)-fragment for aza-CTP using a triple quadrupole mass spectrometer remained without success. Therefore, we investigated the feasibility to separate these highly resembling nucleotides based on accurate mass spectrometry using a linear trap quadrupole (LTQ) coupled with an Orbitrap. The LTQ-Orbitrap was able to differentiate between aza-CTP and the endogenous nucleotides UTP and [(13)C]-CTP. There was no baseline resolution between aza-CTP and [(15)N]-CTP, but the [(15)N]-CTP interference was low. For quantification, extracted ion chromatograms were obtained for the accurate m/z window of the aza-CTP product ion. The assay was able to determine aza-CTP concentrations in PBMC lysate from 40.7 to 281 nM. Assuming that an average cell suspension extracted from 16 mL blood contains 10 to 42 million PBMCs per mL, this range corresponds with 2.58/10.9-17.8/74.9 pmol aza-CTP per million PBMCs. Intra-assay accuracies were between -1.1 and 9.5% deviation and coefficient of variation values were ≤13.2%. The assay was successfully applied to quantify aza-CTP in samples from two patients treated with azacitidine. Aza-CTP concentrations up to 19.0 pmol per million PBMCs were measured. This is the first time that aza-CTP concentrations were quantified in PBMCs from patients treated with azacitidine.