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This article is the first report describing a new validated method to determine the content of HupA in Huperzia selago (Huperziaceae) from wild population and obtained in in vitro culture using the chaotropic mobile phase. An aqueous-organic (acetonitrile) mobile phase with an added chaotropic salt (NaPF6) was used. The system of mobile phases ensured very high selectivity and efficiency at up to N = 6683 ± 963 theoretical plates calculated for isocratic mode. A Hypercosil GOLD column, C18 250 × 4.6 mm, and a Hypercosil GOLD precolumn, 5UM 10 × 4 mm, were employed for detection at four wavelengths, 230 nm being analytical. The regression coefficient (R2) of the calibration was 0.9993 over the range 25–1252 μg mL -1. The recovery rates were 98.36–105.1% with RSD <2.9%. The intra- and inter-day precisions, expressed as RSD, ranged from 1.2% to 2.7%. LOD for HupA was 14 ng mL -1 for a signal-to-noise ratio of 3:1. The limit of quantification was 140 ng mL. -1. The huperzine A (HupA) content of the plant material ranged from 0.65 mg g−1 dry weight (d.w.) to 1.59 mg g−1 d.w. (material from wild plants) and from 0.44 to 1.10 mg g−1 d.w. (material from in vitro cultures). Interestingly, in our study, plants of H. selago derived from wild population had one of the highest HupA concentrations recorded for a club moss (1.59 mg g -1 d.w.). The findings demonstrate that H. selago, found in Europe and North America, is an alternative source of HupA, richer than H. serrata. In order to confirm that HupA was present in the alkaloid extracts, HPLC-ESI-MS/MS analyses of the patterns were performed in the positive ion mode. The fragmentation quasi-molecular ion of the standard HupA (m/z = 243, [M+H]+) and the ion with m/z = 243 found in the samples were identical, confirming the compound as HupA.
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