Millimeter‑segmental analysis of individual equine mane hairs by LC/HRMS for revealing chronological drug distribution in anti‑doping investigations.

Millimeter‑segmental analysis offers substantially improved temporal resolution over conventional bulk hair testing, yet no validated workflow has been established for equine mane hair. This proof‑of‑concept study presents the first liquid chromatography/high‑resolution mass spectrometry (LC/HRMS)‑based millimeter‑segmental workflow optimized for equine anti‑doping investigations. Mane hairs collected up to six months after a single 50‑mg oral administration of osilodrostat were cut into 2‑mm segments, extracted under standard conditions, and quantified using a fully-validated LC/HRMS method. The assay exhibited excellent linearity (r ≥ 0.999), negligible matrix effects, and high extraction recovery. Accuracy and precision consistently satisfied all predefined in-house acceptance criteria. Segmental concentration profiles revealed sharply localized osilodrostat incorporation peaks that migrated distally over time, yielding Average Highest‑Concentration Positions (AHCPs) of 6, 28, 56, and 118 mm at 1, 2, 3, and 6 months respectively. A literature-based mane hair growth rate of approximately 25 mm/month was used solely as a reference for temporal interpretation. The narrow peak widths indicate rapid follicular uptake during a short systemic exposure window and minimal post‑incorporation diffusion. In silico physicochemical predictions, in which osilodrostat exhibits low molecular weight, moderate lipophilicity, weak basicity, and high permeability, support passive follicular uptake and stable retention, consistent with established biochemical models of drug incorporation into hair. This validated workflow enables high‑resolution reconstruction of drug exposure timing and represents a significant methodological advancement for equine anti‑doping analysis, with potential applicability to other compounds exhibiting suitable physicochemical properties.