Orthogonal Hyphenation of Planar and Liquid Chromatography for Mass Spectrometry of Biomarkers out of the Bioassay Matrix (NP-HPTLC-UV/vis/FLD-Bioassay-RP/IEX-HPLC-UV/vis-ESI-MS)

Abstract

Bioprofiling on the planar chromatogram with in situ biological/enzymatic assays is a powerful bioanalytical screening tool for the nontargeted detection of known and especially unknown/unidentified bioactive compounds directly in multicomponent mixtures (e.g., foods, spices, and botanicals). However, together with the bioactive zone, the adsorbed bioassay medium is eluted into the mass spectrometer (MS) and interfering with evaluation. Another sample track without bioassay has thus been handled in parallel. Hence, for a direct zone elution from the bioautogram, different setups were investigated to reduce the impact of the bioassay medium load. A biocompatible filter, orthogonal reversed-phase/cation-exchange columns (RP/IEX-HPLC), UV/vis detector, and a Rheodyne valve were installed between the zone eluting interface (after normal-phase high-performance thin-layer chromatography-multi-imaging-bioassay, NP-HPTLC-UV/vis/FLD-bioassay) and the MS. For the negative electrospray ionization mode (ESI–), an RP-18e-HPLC column and valve switch were exploited. After gradient optimization, the RP-column retarded the eluted polar compounds and split-off the salts of the bioassay medium in the first minutes. This reduced the bioassay load and separated analyte signals thereof. However, most bioassay medium mass signals were predominantly detectable in ESI+-MS. Here, the reduction of bioassay matrix signals was achieved by integrating a mixed-mode RP/IEX column. Finally, two different superhyphenations were successfully proven: NP-HPLC-UV/vis/FLD-bioassay-RP-HPLC-UV/vis-ESI–-MS with a valve switch and NP-HPLC-UV/vis/FLD-bioassay-RP/IEX-HPLC-UV/vis-ESI±-MS with or without it. Although the original bioprofiling (NP-HPTLC-UV/vis/FLD-bioassay) was prolonged from 3 to 13 min per sample, such superhyphenations covering chemistry/biology/mass spectrometry are considered as an efficient nontarget bioanalytical tool for fast evaluation of complex samples.

Schisandra rubriflora Fruit and Leaves as Promising New Materials of High Biological Potential: Lignan Profiling and Effect-Directed Analysis

“…Bioassays, which are the EDA tools, are performed directly on a TLC plate by so-called thin layer chromatography—direct bioautography (TLC-DB), which can be also named effect-directed detection (EDD). Detected biologically active compound can be further identified by spectroscopic methods such as MS, IR, and NMR [ 12 , 13 , 14 , 15 , 16 , 17 ]. TLC-DB can be based on any biological activity: antibacterial, antioxidant, estrogenic, or enzymatic.…”

Effects of the Probiotic Activity of Bacillus subtilis DSM 29784 in Cultures and Feeding Stuff

“…The NP–HPTLC plate was connected via an elution head-based interface [TLC–MS Interface 2, CAMAG] to the ultra-HPLC–photodiode array detector–electrospray ionization (ESI) mass spectrometry system (UPLC–PDA–ESI–MS; Acquity H Class with PDA and QDa, Waters, Eschborn, Germany). A desalting device and an orthogonal reversed-phase (RP)-HPLC column (Accucore RP–MS 100 × 2.1 mm, 2.6 μm, Thermo Scientific) were installed to reduce the matrix components of the culture medium or the derivatization reagent and to prevent ion suppression of the low-concentrated metabolites. , …”

Development of an On-Line Two-dimensional Normal Phase Liquid Chromatography System for Analysis of Weakly Polar Samples

“…Each of these RPLC based combination has its application scope. NP × RP [7] and RP × RP [8] systems are commonly used to separate medium and strong polar compounds, while IEX × RP [9], HILIC × RP [10] and SEC × RP [4] systems are suitable to separate strong polar compounds. According to research it has de cient in separating of weakly polar samples.…”