Unveiling Adulteration in Herbal Markets: MassARRAY iPLEX Assay for Accurate Identification of Plumbago indica L.

The root of <i>Plumbago indica</i> L. is commercially available in herbal markets in both crude and powdered forms. <i>P. indica</i> root is a key ingredient in numerous polyherbal formulations. However, <i>P. indica</i> has two closely related species, <i>P. zeylanica</i> L. and <i>P. auriculata</i> Lam. Since only <i>P. indica</i> is traditionally used in Thai polyherbal products, adulteration with other species could potentially compromise the therapeutic efficacy and overall effectiveness of these formulations. To address this issue, a MassARRAY iPLEX assay was developed to accurately identify and differentiate <i>P. indica</i> from its closely related species. Five single nucleotide polymorphism (SNP) sites-positions 18, 112, 577, 623, and 652-within the internal transcribed spacer (ITS) region were selected as genetic markers for species identification. The assay demonstrated high accuracy in identifying <i>P. indica</i> and was capable of detecting the species at DNA concentrations as low as 0.01 ng/µL. Additionally, the assay successfully identified <i>P. zeylanica</i> in commercial crude drug samples, highlighting potential instances of adulteration. Furthermore, it was able to distinguish <i>P. indica</i> in mixed samples containing <i>P. indica,</i> along with either <i>P. zeylanica</i> or <i>P. auriculata</i>. The developed MassARRAY iPLEX assay proves to be a reliable and effective molecular tool for authenticating <i>P. indica</i> raw materials. Its application holds significant potential for ensuring the integrity of herbal products by preventing misidentification and adulteration.