Non-target bioanalytical eight-dimensional hyphenation including bioassay, heart-cut trapping, online desalting, orthogonal separations and mass spectrometry.

It is still a challenge to discover and identify individual bioactive compounds directly in multicomponent mixtures. Current workflows are too tedious for routine use. Hence, the hyphenation of separation and detection techniques is a powerful tool to maximize the information obtained by a single sample run. A robust eight-dimensional (8D) hyphenation was developed. Orthogonal separations, biological assay detection, analyte trapping, desalting, and physico-chemical detections were arranged in the following order, i.e. 1) normal phase high-performance thin-layer chromatography (NP-HPTLC) separation, 2) Vis detection, 3) UV detection, 4) fluorescence detection (FLD), 5) bioassay for effect-directed analysis (EDA), 6) heart-cut trapping/desalting/elution to reversed phase high-performance liquid chromatography (RP-HPLC) separation, 7) photodiode array (PDA) and 8) mass spectrometry (MS) detection. For the first time, the hyphenation exploited online analyte trapping to desalt the eluted bioactive zone from the plate containing highly salted bioassay media. Subsequent valve switching guided the trapped analyte(s) to the main column, followed by multiple detection. As proof-of-principle, cinnamon samples were analyzed by NP-HPTLC-UV/Vis/FLD-EDA-RP-HPLC-PDA-MS, whereby a bioactive zone was separated into two distinct peaks detected by PDA and MS to be 2-methoxy cinnamaldehyde and cinnamaldehyde. The developed 8D hyphenation is applicable for routine, allowing the non-target high-throughput screening of complex samples for individual bioactive compounds.