Methylation Enables Sensitive LC-MS/MS Quantification of Ciclopirox in a Mouse Pharmacokinetics Study

Ciclopirox (CPX), a topical antifungal agent of the N-hydroxypyridone class, has gained renewed interest for its potential anticancer, antiviral, antibacterial, and neuroprotective effects. However, due to lack of reliable validated bioanalytical methods, current insights into its pharmacokinetics profile beyond topical use remain limited. To support therapeutic repurposing, we developed and validated a rapid, sensitive LC-MS/MS method for systemic pharmacokinetic evaluation in mice. The method employs methyl derivatization of CPX’s N-hydroxy group, producing methylated CPX (Me-CPX) for improved chromatographic performance which was subsequently retained on the Atlantis^TM T3 C18 reverse phase column. Concentration of CPX is determined indirectly based on the measured response of Me-CPX. The method achieved excellent recovery, a 4-min rapid runtime, sensitivity with LLOQ of 3.906 nM (0.81 ng/mL), and a linear range up to 1000 nM (r ≥ 0.9998). All validation parameters including intra- and inter-day accuracy, precision, matrix effects, stability and dilution integrity met the criteria defined by regulatory International Council for Harmonisation (ICH) M10 bioanalytical method validation guidelines. Application of the method to in vitro plasma protein binding studies revealed high protein binding (>99%) of CPX in both human and mice plasma. Preliminary PK analysis following intravenous and oral administration in CD-1 mice demonstrated moderate systemic exposure after oral dosing, with an estimated absolute bioavailability of 52.5%. These findings establish the method’s suitability and robustness for preclinical and future clinical development of CPX as a repurposed therapeutic agent.