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

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

  • J Pharm Biomed Anal. 2026 Oct 15:279:117552. doi: 10.1016/j.jpba.2026.117552.
Ushio W Ishikawa 1,  Hirotaka X Kanoh 2,  Kei Irie 3,  Yusei Ota 4,  Tomoharu Yasuda 5,  Akiyoshi Hirayama 6,  Tomoyuki Mizuno 7,  Atsuo T Sasaki 8
Affiliations

Affiliations

  • 1 Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan; Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa, Japan; Division of Hematology and Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267, United States.
  • 2 Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan; Division of Hematology and Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267, United States.
  • 3 Division of Translational and Clinical Pharmacology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States.
  • 4 Division of Hematology and Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267, United States; Department of Immunology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima 734-8551, Japan.
  • 5 Department of Immunology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima 734-8551, Japan.
  • 6 Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan; Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa, Japan.
  • 7 Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, United States; Division of Translational and Clinical Pharmacology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States.
  • 8 Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan; Division of Hematology and Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267, United States; Department of Clinical and Molecular Genetics, Hiroshima University Hospital, Hiroshima 734-8551, Japan. Electronic address: [email protected].
PMID: 42202755 DOI: 10.1016/j.jpba.2026.117552
Abstract

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.

Keywords

Common product ion; Intracellular pharmacokinetics; LC-MS/MS; Mycophenolate mofetil; Mycophenolic acid; Pharmacokinetic modeling; Prodrug activation.

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