Development of a HILIC-MS/MS method for simultaneous quantification of lipophilic antitumor TriPPPro-prodrugs and their polar metabolites in HT29 cell extracts

Nucleoside analogues are among the most widely used antiviral and also antitumoral agents. The nucleoside analogues require intracellular metabolic activation through stepwise phosphorylation resulting in the bioactive nucleoside triphosphates. To directly deliver the active metabolite, our group developed a prodrug system where the nucleoside triphosphate (NTP) is masked by two lipophilic moieties which are enzymatically cleaved off after successful cellular uptake. To date, no data are available on the intracellular concentrations of the active metabolites responsible for the determined antiviral or antitumor activity. In this paper, we describe the development of a HILIC-MS/MS method for the quantification of TriPPPro-prodrugs, derived from the anticancer drug fluorouracil (5-FU) and all resulting metabolites, FdU, FdU-monophosphate (MP), FdU-diphosphate (DP), and FdU-triphosphate (TP), in cancer cell lysate. Because of the different chemical properties of the lipophilic prodrugs and the hydrophilic metabolites, sample preparation as well as liquid chromatography method development were challenging factors. A liquid-liquid extraction protocol was employed and with use of hydrophilic liquid chromatography, the simultaneous retention of all analytes was guaranteed. The method was validated for the following concentration ranges in cancer cell lysate and the associated supernatant: 2.0-1000 ng/mL. The method was successfully applied to quantify prodrugs and metabolites in HT29 cancer cell lysate and supernatant samples after cellular uptake studies with two different TriPPPro-prodrugs. The method can also be employed for the quantification of other lipophilic prodrugs, as well as nucleotides and nucleosides (derivatives).