Development of a validated ultra-fast UPLC-MS/MS method for famciclovir quantification in human liver microsomes:...

An ultra-fast UPLC-MS/MS method was developed and validated for measuring famciclovir (Famvir®; FMV), the orally bioavailable prodrug of the antiherpetic nucleoside analog penciclovir, in human liver microsomes (HLMs). Chromatographic separation was conducted utilizing a Luna 3 µm HILIC column (200 Å: 50 × 2 mm), with the mobile phase comprising 0.1% formic acid in ACN (90%) and 10 mM ammonium formate in water (10% at pH 3.2 by adding a few drops of HCOOH), and the total run time was 1.0 min at a 0.4 mL min^-1 flow rate. The method was linear over the range of 1 to 4000 ng mL^-1 (R² = 0.9932; y = 1.2854x + 0.41327, 1/x weighting), with an LLOQ of 1 ng mL^-1. Intra-day accuracy ranged from -2.84% to 8.67%, with precision (RSD) of 1.11-5.07%; inter-day accuracy ranged from -3.34% to 11.0%, with precision of 0.59-4.50%. Extraction recovery was 102.92 ± 4.16% (FMV) and 101.96% ± 5.99% (fedratinib). Three independent greenness metrics indicated the method's greenness: AGREEprep 0.64/1.00, ComplexMoGAPI 65.0/100, and AGSA 62.50/100. Applying this validated method to substrate depletion in HLMs + NADPH yielded an in vitro t_1/2 of 15.72 min and a Cl_int of 51.56 mL min^-1 kg^-1, and in HLMs -NADPH (t_1/2: 18.18 min and Cl_int: 44.59 mL min^-1 kg^-1), confirming its usefulness for high-clearance DMPK studies. FMV depletion in HLM is esterase-driven and NADPH-independent (as it has a minor effect on FMV metabolic stability). To our knowledge, this is the first validated, sustainability-assessed UPLC-MS/MS method for FMV in HLMs.