Stoichiometric simultaneous quantification of mycophenolate mofetil and mycophenolic acid in paired culture medium and cell extracts by common product ion LC-MS/MS

Mycophenolate mofetil (MMF) is an immunosuppressive prodrug whose efficacy depends on conversion to mycophenolic acid (MPA). Quantitative, stoichiometry-ready measurement of MMF-to-MPA conversion in matched extracellular and intracellular matrices remains limited, restricting interpretation of prodrug activation and cellular exposure beyond plasma MPA. A common expectation is that a lipophilic prodrug rapidly equilibrates across membranes; however, extracellular concentrations alone cannot resolve intracellular accumulation, activation, and retention. Here, we developed a common product ion (m/z 207.1) LC-MS/MS workflow that enables stoichiometrically comparable quantification of MMF and MPA across matched extracellular and intracellular matrices from microliter-scale samples and applied it to paired medium/cell sampling in murine Hepa1-6 cells as a proof-of-application system. In Hepa1-6 cells, intracellular MMF transiently exceeded extracellular MMF, peaking at ∼5.5 µM at 15 min (≈1.4-fold above the 4 µM dosing level); in contrast, with direct MPA dosing, intracellular MPA did not exceed extracellular MPA, consistent with equilibration. With carboxylesterase inhibition by bis-nitrophenyl phosphate (BNPP), intracellular MMF reached ∼9.2 µM and exceeded extracellular MMF by ∼2.4-fold at 1 h (∼9.2 vs ∼3.9 µM), consistent with intracellular accumulation and/or retention not readily explained by simple passive equilibration alone. Integrating the data with a mechanistic medium-cell model separated intracellular conversion from bidirectional exchange and parameterized these processes. This compartment-resolved workflow provides a proof-of-application framework for cellular MMF/MPA measurements in matched medium/cell samples.