Development and Validation of an HPLC-MS/MS Method for Pharmacokinetic Evaluation of Tofacitinib/Simvastatin Compound...

Oxidative stress and immune dysfunction are closely associated with the onset and progression of vitiligo. Therefore, simultaneously regulating these two key pathological processes to achieve synergistic therapy is a practically feasible strategy. Based on this rationale, we previously developed a topical ointment co-loaded with antioxidant simvastatin and immunosuppressant tofacitinib, and evaluated its therapeutic potential for vitiligo. However, the lack of a bioanalytical method for simultaneous in vivo quantification of the two drugs has hindered further pharmacodynamic and pharmacokinetic studies of the formulation. The present study aimed to establish and validate an HPLC-MS/MS method for simultaneous determination of their in vivo concentrations, to support pharmacokinetic investigations of the ointment in rats.

Chromatographic separation was performed on a ZORBAX Eclipse C₁₈ column (50.0 × 3.5 mm, 1.7 μm) using a gradient elution program. The mobile phase comprised phase A (20 mM ammonium acetate aqueous solution) and phase B (acetonitrile), with a constant flow rate of 0.4 mL/min. The method was fully validated in strict accordance with the bioanalytical method validation guidelines specified in the Chinese Pharmacopoeia, including evaluations of specificity, calibration curve and linear range, lower limit of quantification, carryover effect, precision, recovery, matrix effect, stability, and dilution integrity.

The results demonstrated that the method met all validation criteria and was suitable for pharmacokinetic studies. Pharmacokinetic data showed that, compared with oral gavage, the 24 h bioavailability of tofacitinib and simvastatin after topical administration was only 7.1% and 5.2%, respectively. This low systemic exposure indicated that the ointment formulation could effectively reduce the potential toxicity caused by excessive drug absorption into the circulation.