Crucial aspects of high performance thin layer chromatography quantitative validation. The case of determination of rosmarinic acid in different matrices

A new HPTLC method was envisaged to determine rosmarinic acid (RA) in different matrices with the aim of testing the influence of optimizing the main HPTLC operative parameters in view of a more stringent validation process. HPTLC LiChrospher silica gel 60 F254s, 20 cm × 10 cm, plates with toluene:ethyl formate:formic acid (6:4:1, v/v) as the mobile phase were used. Densitometric determinations were performed in reflectance mode at 330 nm. The method was validated giving rise to a dependable and high throughput procedure well suited to routine applications. RA was quantified in the range of 132–660 ng with RSD of repeatability and intermediate precision not exceeding 2.0% and accuracy within the acceptance limits. The method was tested on several commercial preparations containing RA in different amounts.

► A new HPTL method validated for determination of rosmarinic acid in complex matrices. ► Influence of optimizing HPTLC operative parameters in view of a strict validation process. ► Usefulness of a pre-validation step based on the accuracy profiles. ► RSD repeatability and intermediate precision <2.0%, accuracy in the 5% acceptance limits.