Herbal medicines (HMs) have a long history of medicinal use with safety and efficacy against various diseases and have contributed as major sources of modern medicines. Traditional herbal medicines (THMs), the prescriptions consisted of various HMs, have long been used as cures for diseases. Some of them are still used as tonics or complementary medicines. Multiple components are one of the main advantages of THMs for multi-targeting, but considered as hurdles for the quality control of THMs in the regulatory framework of modern medicine. Ojeoksan (OJS), the second most prescribed herbal medicine in Korea, consisted of 16 herbal substances and are the THM used as pain-relieving agent. Until now, quantitative methods have been performed with only a few marker compounds of the HMs that make up OJS, but no method has been established to set and confirm marker compounds of all constituent HMs. The aim of this study is to establish an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for simultaneous determination of 22 marker compounds in OJS. The analytes were separated in liquid chromatography using an Infinitylab Poroshell 120 EC-C18 (2.1 × 150 mm, 2.7 μm) column and gradient elution of the mobile phases 0.1% formic acid and methanol containing 0.1% formic acid. Analytes were detected in a tandem mass spectrometry using both negative and positive electrospray ionization (ESI) mode and scheduled multiple reaction monitoring (sMRM) acquisition. Method validation study was carried out using the matrices of OJS tablet and soft-extract for three consecutive days to access intra- and inter-day variance. Validations results showed acceptable performance characteristics, including specificity, linearity (R2 ≥ 0.9956), repeatability (RSD ≤ 6.5%), LOD (0.3–3.6 ng/mL), LOQ (1.0–10.9 ng/mL), precision (RSD ≤ 12.1%), and accuracy (recovery 78.4–108.9%). Established method was applied to five commercial OJS products (1 tablet and 4 soft-extract dosage form). The 14 marker compounds indicating the 12 component HMs were determined in products; paeoniflorin, hesperidine, glycyrrhizin, albiflorin, ephedirine, neohesperidin, cinnamic acid, decursin, platycodin D, ferulic acid, 6-gingerol, atractylenolide III, magnolol, and honokiol. Alternative marker compounds for the 3 unidentified HMs should be tested in our future study. Fast and easy assay method was established with sufficient discrimination power. This method as a new assay can contribute to quality control for OJS products.
