HPTLC method for the simultaneous determination of six bioactive terpenoids in Putranjiva roxburghii Wall.

High-throughput and sensitive determination of triterpenoids is challenging by high-performance liquid chromatographic methods due to their non-chromophoric nature, lipophilic properties, and sequential run. The present study demonstrates the development of a validated high-performance thin-layer chromatography (HPTLC) method for the simultaneous determination of bioactive terpenoids in the bark of Putranjiva roxburghii with uncertainty measurement to fulfill the technical requirement of ISO 17025:2017. Optimum separation of friedelin (1), methylputranjate (2), putrone (3), roxburghonic acid (4), putranjivadione (5), and roxburghonol (6) was achieved on silica gel 60 F254 plates using n-hexane–ethyl acetate (90:10, v/v). The sensitive estimation of targeted terpenoids was achieved by screening 15 different post-chromatographic derivatization reagents. The method was validated as per the ICH guidelines. Additionally, an uncertainty measurement for reliable and accurate determination was established following EURACHEM/CITAC guide CG-4 statistical procedure. The expanded uncertainty of terpenoids (1–6) measurement in P. roxburghii bark was in the range of 0.44–2.17%, which confirmed that at a confidence level of 95%, the unknown true value was within ± 5% range of the measured value. The compound methylputranjate showed the minimum uncertainty (0.4424), while putranjivadione showed the maximum uncertainty (2.1722). This is the first validated HPTLC method with new derivatization reagent providing the simultaneous, sensitive, and high-throughput analysis of six bioactive triterpenes in the bark of P. roxburghii with defined traceability and accuracy profile.