Development and validation of a HILIC–MS/MS method for quantification of decitabine in human plasma by using lithium adduct detection

• The assay demonstrated high sensitivity (0.44–440 pg injected on column) with a simple protein precipitation extraction.
• HILIC separation of anomers and two other degradation isomers was achieved over a 3-min gradient elution.
• [M+Li]+ replaced other alkali adducts with 2 mM of lithium acetate in mobile phase and was used for SRM detection.
• The assay was successfully validated and applied for clinical sample analysis.

A highly sensitive, selective, and rugged quantification method was developed and validated for decitabine (5-aza-2′-deoxycytidine) in human plasma treated with 100 μg/mL of tetrahydrouridine (THU). Chromatographic separation was accomplished using hydrophilic interaction liquid chromatography (HILIC) and detection used electrospray ionization (ESI) tandem mass spectrometry (MS/MS) by monitoring lithiated adducts of the analytes as precursor ions. The method involves simple acetonitrile precipitation steps (in an ice bath) followed by injection of the supernatant onto a Thermo Betasil Silica-100, 100 × 3.0 mm, 5 μm LC column. Protonated ([M+H]+), sodiated ([M+Na]+), and lithiated ([M+Li]+) adducts as precursor ions for MS/MS detection were evaluated for best sensitivity and assay performance. During initial method development abundant sodium [M+Na]+ and potassium [M+K]+ adducts were observed while the protonated species [M+H]+ was present at a relative abundance of less than 5% in Q1. The alkali adducts were not be able to be minimized by the usual approach of increasing acid content in mobile phases. Significant analyte/internal standard (IS) co-suppression and inter-lot response differences were observed when using the sodium adduct as the precursor ion for quantification. By adding 2 mM lithium acetate in aqueous mobile phase component, the lithium adduct effectively replaced other cationic species and was successfully used as the precursor ion for selected reaction monitoring (SRM) detection. The method demonstrated the separation of anomers and from other endogenous interferences using a 3-min gradient elution. Decitabine stock, working solution stabilities were investigated during method development. Three different peaks, including one from anomerization, were observed in the SRM transition of the analyte when it was in neutral aqueous solution. The assay was validated over a concentration range of 0.5–500 ng/mL (or 0.44–440 pg injected on column) in 50 μL of human plasma. The accuracy and precision were within 8.6% relative error and 6.3% coefficient of variation, respectively. Decitabine was stable in THU treated human plasma for at least 68 days and after 5 freeze–thaw cycles when stored at −70 °C. Stability of decitabine in THU treated human whole blood, matrix factor and recovery were also evaluated during method validation. The method was successfully used for clinical sample analysis.