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

ABSTRACT 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 C 18 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‐ 13 C , d 2 . 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.