Determination of the pyrrolo[2,3-d] pyrimidine antifolate AGF94 from a preclinical murine study using a simple UHPLC-MS/MS assay

A UHPLC-MS/MS method for the detection and quantification of the 6-substituted pyrrolo[2,3-d] pyrimidine AGF94, a novel antifolate, in mouse plasma and tissue was developed and validated. The developed method relies on a simple protein precipitation with methanol, followed by separation on a C18 column using a gradient solvent system of acetonitrile and water with 0.1 % formic acid in both. Detection and quantification of AGF94 were achieved by multiple reaction monitoring using a Sciex QTRAP 5500 mass spectrometer operated in positive electrospray ionization mode. The transitions for AGF94 and the internal standard were m/z 448 to 137 and 442 to 295, respectively. The calibration curve ranged from 5 to 500 ng/mL in mouse plasma with a linearity of R² = 0.99611 ± 0.00280 across multiple days. Accuracy of the assay ranged from −6.22 to 5.56 % and precision was less than 11.58 % off from nominal concentrations. Benchtop, freeze/thaw cycling, and autosampler stabilities did not indicate any substantial changes in concentrations during processing. The overall process efficiency was greater than 96 % for both the analyte and internal standard. The precision and accuracy of the assay were established, and the assay was utilized to analyze preclinical samples from a pharmacokinetic study using AGF94 in a murine pancreatic cancer model.