Analysis of Bioactive Components of Oilseed Cakes by High-Performance Thin-Layer Chromatography-(Bio)assay Combined with Mass Spectrometry

Abstract

Hemp, flax and canola seed cakes are byproducts of the plant oil extraction industry that have not received much attention in terms of their potential use for human food instead of animal feed. Thus, the bioactivity profiling of these oilseed cakes is of interest. For their effect-directed analysis, planar chromatography was combined with several (bio)assays, namely 2,2-diphenyl-1-picrylhydrazyl scavenging, acetylcholine esterase inhibition, planar yeast estrogen screen, antimicrobial Bacillus subtilis and Aliivibrio fischeri assays. The streamlined high-performance thin-layer chromatography (HPTLC)-bioassay method allowed the discovery of previously unknown bioactive compounds present in these oilseed cake extracts. In contrast to target analysis, the direct link to the effective compounds allowed comprehensive information with regard to selected effects. HPTLC-electrospray ionization-mass spectrometry via the elution-head based TLC-MS Interface was used for a first characterization of the unknown effective compounds. The demonstrated bioactivity profiling on the feed/food intake side may guide the isolation of active compounds for production of functional food or for justified motivation of functional feed/food supplements.

HPTLC-aptastaining – Innovative protein detection system for high-performance thin-layer chromatography

“…As described in the literature, various pre-treatments of the HPTLC-plate can be applied to improve overlay (detection) procedures 26 27 28 . In order to induce the best success in detecting specific proteins using HPTLC-AS, various procedures were investigated.…”

“…In order to induce the best success in detecting specific proteins using HPTLC-AS, various procedures were investigated. Due to the rapid process of HPTLC-AS, neither a fixation of the stationary phase material was necessary 26 nor a neutralization of the HPTLC-plate 27 led to an improved result. A renaturation of the proteins after chromatography, as described for an antibody-based assay detecting gangliosides 28 , led to band-broadening and an overall less satisfying result, consequently.…”

Bioassay-coupled Chromatographic Analysis of Medicinal Natural Products: A Review

“…Usually, TLC is hyphenated with appropriate bioassay to enable direct in vitro activity study of an extract that has previously been separated into its components on the plate. Coupling of TLC with bioassay and a spectroscopic technique like UV, using UV densitometric scanning, 39,40 MS using TLC-MS interface 41 will result in the identification of the targeted active substances in the plant extract. It has the advantage of good availability, ease of application and cost effectiveness.…”

“…42 Advantages of this technique include simplified sample preparation, short time of analysis, low cost of operation, simultaneous analysis of several samples, option for multiple detection, matrix-tolerance and compatibility to biological assays. 41,1 The HPTLC setup can be used for many project and bioassay at any time without additional capital cost. One other major advantage is the eluent-free detection made possible by this system since chromatography is distinctly separated from the detection step.…”

“…45 However, it may not be useful for high sensitive volatile and oxidation-prone bioactive components. 41 Useful detail information on the background and practical performance of HPTLC-MS has been reported. 46,47 Liquid Chromatography (LC) Offline and online separation of complex mixtures can be done by Liquid chromatography including Low pressure LC, high performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UPLC), counter current chromatography (CCC) and other variants.…”