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Naphthoquinones are important class of molecules found as a natural red color pigments in roots of  Arnebia benthamii  (Wall. ex G. Don) L M. Johnston. The aim of present investigation is to develop and validates a simple, cost-effective and reliable method for quantification of these compounds. Therefore, a normal phase-high performance thin-layer chromatography (NP-HPTLC) method for concurrent determination of shikonin and β,β-dimethylacryl shikonin in  A. benthamii  was established. Method development of naphthoquinones in the methanol extract was done using hexane–ethyl acetate–methanol (40:7.5:2.5, v/v/v) solvent system at 520 nm. The developed method showed good band separation for shikonin ( R  f , 0.37) and β,β-dimethylacryl shikonin ( R  f , 0.58). The linearity ranged between 100 and 8,000 ng spot −1  with an average recovery of &gt;97% in both cases. The results showed reproducible intraday and interday precision (&lt;2.0% RSD) in quantification of naphthoquinones. The limits of detection are 12.96 and 14.65 ng spot −1  while the limits of quantification are 39.27 and 44.39 ng spot −1  for shikonin and β,β-dimethylacryl shikonin, respectively. The developed method is reliable, fast, easy to follow and economic in concurrent assessment of shikonin and β,β-dimethylacryl shikonin in  A. benthamii  root samples. In addition, it seems to be first report for identification and quantification of β,β-dimethylacryl shikonin from the  A. benthamii .

Concurrent NP-HPTLC Determination of Shikonin and β,β-Dimethylacryl Shikonin in Arnebia benthamii