A Simple and Sensitive LC‐MS/MS Method for Quantification of Capsaicin in Human Plasma and Its Application to a Clinical Pharmacokinetic Study

Capsaicin, a transient receptor potential vanilloid 1 (TRPV1) agonist, is used topically for neuropathic pain. Its minimal systemic absorption necessitates highly sensitive quantification methods to accurately assess systemic exposure. This study aimed to develop and validate a simple, rapid, and highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of capsaicin in human plasma. Plasma samples (50 μL) were prepared by a simple protein precipitation using acetonitrile, with capsaicin-d3 as the internal standard (IS). Chromatographic separation was performed on a Waters Xbridge C₁₈ column (2.1 × 50 mm, 3.5 μm) with a 3-min gradient elution using 1% formic acid in water and acetonitrile. Detection was conducted on a triple quadrupole tandem mass spectrometer in positive electrospray ionization (ESI) mode, utilizing multiple-reaction monitoring (MRM). The assay was validated over the linear range of 0.05-50 ng/mL. The method demonstrated excellent precision (CV% ≤ 11.62%) and accuracy (RE% within ±13.60%), with high recovery (>95%). Stability was confirmed under various conditions relevant to clinical sample handling. The validated method was successfully applied to a pharmacokinetic study involving 24 patients with knee osteoarthritis receiving a topical capsaicin liniment, demonstrating its suitability for clinical applications.