Development and Validation of an LC–MS/MS Method for Quantitative Determination of Tacrolimus in Mouse Serum

Tacrolimus, a potent immunosuppressant with a narrow therapeutic index, is a known substrate of P-glycoprotein (P-gp), a key efflux transporter involved in drug disposition. Accurate and sensitive quantification of tacrolimus in small-volume mouse serum is essential not only for pharmacokinetic studies but also for evaluating in vivo P-gp functional activity, particularly in pregnancy-related drug transport research. Tacrolimus was extracted using methyl tert-butyl ether after alkalization of the serum with NH₄OH. Chromatographic separation was achieved on a C₁₈ HPLC column with a mobile phase of acetonitrile and water containing 0.2% NH₄OH. Detection was performed in negative ion mode using multiple reaction monitoring (MRM) transitions of m/z 802.5 → 560.6 for tacrolimus and m/z 805.5 → 563.6 for the internal standard, tacrolimus-¹³C,d₂. The calibration range for tacrolimus was 0.26–44.8 ng/mL. The method demonstrated good precision, with intra- and inter-day relative standard deviations below 12%, and accuracy ranging from 94% to 103%. Compared to previously published methods, this approach requires only 200 μL of mouse serum and provides high sensitivity, with a lower limit of quantification of 0.26 ng/mL. The assay showed high sensitivity, minimal matrix interference, and good stability across tested conditions.