Conventional sample enrichment strategies combined with high-performance liquid chromatography–solid phase extraction–nuclear magnetic resonance analysis allows analyte identification from a single minuscule Corydalis solida plant tuber

Identification of putative biomarker molecules within the genus Corydalis (Papaveraceae) was pursued by combining conventional off-line sample enrichment with high-performance liquid chromatography–solid phase extraction–nuclear magnetic resonance (HPLC–SPE–NMR) based structure elucidation. Off-line reversed phase solid phase extraction (SPE) was used to enrich the desired analytes from a methanolic extract (93 mg dry weight) of a miniscule single tuber (233 mg dry weight) of C. solida. An aliquot of the SPE fraction (2.1 mg) was subjected to separation in the HPLC–SPE–NMR hyphenation. Chromatographic peaks bearing the metabolites under investigation were trapped in the SPE device in a single experiment and transferred to a 600 MHz NMR spectrometer equipped with a 30 μl cryofit insert fed into a 3 mm cryoprobe. Recorded homo- and heteronuclear 1D and 2D NMR data allowed the identification of the three analytes under investigation as protopine, allocryptopine, and N-methyl-laudanidinium acetate. The latter is a rare alkaloid, which has been isolated only once before.