The use of high performance thin-layer chromatography (HPTLC) in combination with high resolution time of flight mass spectrometry (MS) for the detection, identification and imaging (HPTLC/MSI) of ...ecdysteroids (insect moulting hormones) present in a number of plant extracts obtained from members of the
Silene
family is demonstrated. DESI is shown to be a convenient method for the recovery of these polar polyhydroxylated steroids from the silica gel of the HPTLC plate for subsequent MS detection and imaging. The incorporation of an ion mobility separation (IMS) to the system to give HPTLC/IMS/MS provided additional drift time data which enabled more confident identification. Using HPTLC/DESI/IMS/MS, a range of ecdysteroids were detected and characterized in extracts of
S. otitis, S nutans
,
S. maritime, S. viridiflora
and
S. fimbriata.
A simple and rapid high-performance thin-layer chromatography-based autographic assay was established to screen plant extracts for the presence of tyrosinase-inhibiting substances. Three mobile ...phases were selected for the chromatographic separation of different types of extracts. After development, the plate was sprayed with the substrate solution Levodopa followed by a solution of the enzyme tyrosinase. Several known tyrosinase inhibitors were tested simultaneously as positive controls. They were detected as white spots with white light in remission from the plate as well as with white light transmitted through the plate. Some of the investigated extracts included spots showing a different behaviour; some lipophilic substances appeared as white spots in white light remission but were black in white light transmission. This behaviour, which could lead to false-positive results, was due to poor wettability of the corresponding spots. False-positive results were eliminated by adding Triton X-100 to the Levodopa solution and drying the plate after 10 minutes incubation with a molecular sieve. Tyrosinase inhibitors can be clearly identified as white spots against a dark background in white light remission as well as in white light transmitted through the plate. The established high-performance thin-layer chromatography autographic assay was validated and can be used as a standard method for the detection of tyrosinase inhibitors in plant extracts without causing false-positive results.
One of the most important contributions to the analysis of mixtures using TLC/HPTLC has been the interface device allowing for direct elution of the sample spots from the developed TLC/HPTLC plate ...directly into a mass spectrometer. This is a review of more recent papers using this technology in various fields since 2015. Applications covered include caffeine in energy drinks, the anti-cancer drug ifosfamide, estrogens, lichen metabolites, lipids, traditional medicinals & phytochemicals, triterpenoids & phytosterols in vegetables, saponins, synthetic pharmaceutical products, and peptides in wines. New and different interface designs and uses are detailed in additional sections. Different techniques for producing ions (ionization) found suitable for TLC/MS applications are shown. Lastly, a summary and the future prospects of this useful analytical tool are discussed.
Introduction
Many species within Combretaceae are traditionally used for the treatment of bacterial infections. The similarity in chemistry and antimicrobial activities within the family pose a ...challenge in selecting suitable species for herbal drug development.
Objective
This study aimed at rapidly identifying antimicrobial compounds using bioautography‐guided high‐performance thin‐layer chromatography coupled with mass spectrometry (HPTLC–MS).
Methods
Hierarchical cluster analysis of ultra‐performance liquid chromatography‐mass spectrometry data from the methanol extracts of 77 samples, representing four genera within Combretaceae, was carried out. Based on groupings on the dendrogram, 15 samples were selected for bioautography analysis against four pathogens (Staphylococcus aureus, Bacillus cereus, Escherichia coli and Salmonella typhimurium). Active compounds were identified using HPTLC–MS analysis of bands corresponding to the inhibition zones.
Results
Bioautography revealed 15 inhibition zones against the four pathogens, with the most prominent present for Combretum imberbe. Analysis of the active bands, using HPTLC–MS indicated that flavonoids, triterpenoids and combretastatin B5 contributed to the antibacterial activity. The compounds corresponding to molecular ions m/z 471 (Combretum imberbe) and 499 (Combretum elaeagnoides) inhibited all four pathogens, and were identified as imberbic acid and jessic acid, respectively. Chemotaxonomic analysis indicated that arjunic acid, ursolic acid and an unidentified triterpenoid (m/z 471) were ubiquitous in the Combretaceae species and could be responsible for their antibacterial activities.
Conclusion
Application of HPTLC–MS enabled the rapid screening of extracts to identify active compounds within taxonomically related species. This approach allows for greater efficiency in the natural product research workflow to identify bioactive compounds in crude extracts.
This study was aimed at rapidly identifying antimicrobial compounds within Combretaceae using bioautography‐guided high‐performance thin‐layer chromatography‐mass spectrometry (HPTLC‐MS). Representative samples were subjected to bioautography against bacterial pathogens, and compounds were identified from the active bands using HPTLC‐MS. Chemotaxonomic analysis revealed that arjunic acid, ursolic acid, and an incompletely identified triterpenoid (m/z 471) were ubiquitous in the Combretaceae species and associated with antibacterial activity. This approach enables rapid extract screening to identify active compounds within taxonomically related species.
This work is a review on HPTLC contribution to lipid analysis in complex matrices, in the period from 2010 to now, lapse of time where hyphenation with other techniques, especially Mass Spectrometry, ...has experienced an important growth. Scanning densitometry (SD)-UV/FL of separated lipids, as the core of detection and centerpiece for hyphenation, and its coupling with MS using soft ionization techniques (ESI, APCI, MALDI, DESI and others), are the central axis of this work. The occasional intercalation in this coupling of an on-plate biological assay for effect-direct analysis (EDA) of lipids, as well as the combination of SD with radio-densitometry (RD) using isotopically labeled-lipids are also covered topics. The described techniques make possible to develop strategies for obtaining qualitative and different levels of quantitative information, including untargeted lipids species. HPTLC of lipids has been used for: comparative purposes; fingerprinting; semi-quantitative determination, identification of species; or quantitative determination of a given individual lipid. Goals in molecular biology and biochemistry-related samples have been: preparative isolations; control of purity; verification of metabolic products; on-plate biological assays; profiles in cells; analysis of products from the cellular metabolism; measurement of enzymatic activities; monitoring lipid transport across membranes or at biological interfaces; or monitoring lipid dynamics.
Abbreviations: AcH: acetic acid; AMD: Automated Multiple Development; APCI: Atmospheric Pressure Chemical Ionization; Cer: Ceramides; Chol: Cholesterol; CholE: Cholesteryl esters: CholS: Cholesteryl sulfate; DCM: Dichloromethane; DESI: Desorption Electrospray Ionization; DG: Diacylglycerides; E1: estrone; E2: 17-β estradiol; E3: estriol; EE2: 17-α ethynylestradiol; EDA: Effect-Direct Analysis; ESI: Electrospray Ionization; EtOH: ethanol; FA: Fatty Acids; FAME: Fatty acid-methyl esters; FFA: Free Fatty Acids; GalCer: Galactosyl-Ceramides; Gb
3
: Globotriaosylceramides; Gb
4
: Globotetraosylceramides; GC: Gas Chromatography; GL: Glyco-lipids; GlcCer: Glucosyl-Ceramides; GSL: Glycosphingolipids; HPTLC: High-Performance Thin-Layer Chromatography; IMS: Ion-Mobility Separation; LacCer: Lactosyl-Ceramides; LC: Liquid Chromatography; LESA: Liquid Extraction Surface Analysis; LPC: Lyso-Phosphatidylcholines; LPG: Lyso-Phosphatidylglycerols; MALDI: Matrix Assisted Laser Desorption Ionization; m.d.: Migration distance; MeOH: Methanol; MG: Monoacylglycerides; NL: Neutral Lipids; PA: Phosphatidic Acids; PC: Phosphatidylcholines; PE: Phosphatidylethanolamines; PG: Phosphatidylglycerols; PI: Phosphatidylinositols; PL: Glycerophospholipids; PMA: Phosphomolybdic acid; PS: Phosphatidylserines; SD: Scanning Densitometry; SL: Sphingolipids; SM: Sphingomyelins; SQ: Semi-quantitative; Sq: squalene; SterylE: Steryl-esters; TG: Triacylglycerides
•RGP-pYES was successfully applied on ethanolic hops extracts.•Prenylnaringenin was identified and confirmed as estrogen active compound (EAC) in hops by means of pYES and HPTLC-MS.•Heat-induced ...formation of estrogen active prenylnaringenin was shown.•The option to estimate estradiol equivalent concentrations of EAC in liquid samples using logit-log plots was demonstrated.•The estimation of the estrogenic potential of EAC in solid samples by means of estradiol equivalent amounts was shown.
Hops used in the brewing process of beer for flavoring are known to contain estrogen active compounds (EAC) and to be the source of EAC in beer. The recently developed planar yeast estrogen screen (pYES) with the substrate resorufin-β-d-galactopyranoside (RGP) successfully was applied for the detection of EAC in ethanolic extracts of hops pellet samples. The only pYES positive compound was identified as the hop flavanone prenylnaringenin (PN) by thin-layer chromatography–mass spectrometry. The heat-induced formation of estrogen active PN from the inactive hop flavonoid desmethylxanthohumol was confirmed by simulation of wort boiling, extraction of both the hops’ remainder and the supernatant water, and subsequent investigation of the extracts by pYES. By means of the dose-response curve of PN of a hops’ remainder extract, the estradiol equivalent concentration (EEQ) and thus the estradiol equivalent amount (EEA) of PN in the hops’ remainder after simulation of the wort boiling was determined to 39 μg/L and 52 μg/kg, respectively.
This mini-review summarizes the protocol we have developed for the analysis of neutral glycosphingolipids (GSLs) by high-performance thin layer chromatography (HPTLC)–mass spectrometry (MS). We also ...present results obtained using this glycolipidomic approach to study neutral GSLs from mouse kidney, spleen, and small intestine. Finally, we discuss what is required for further development of this method, as well as what is expected for the future of glycolipid biology. This article is part of a Special Issue entitled Lipodomics and Imaging Mass Spectrometry.
► The protocol for the analysis of neutral glycosphingolipids (GSLs) by high-performance thin layer chromatography (HPTLC)-mass spectrometry (MS). ► Mass spectra of neutral GSLs prepared from mouse kidney, spleen, and small intestine. ► Coupling of the bird eye overview of GSLs by HPTLC with precise structural information by MS. ► Discussion on other MS methods for GSLs and further development of HPTLC-MS.
•HPTLC-bioautography-MS is demonstrated for rapid detection of antimicrobial compounds from propolis.•Pinobanksin was identified as the antimicrobial compound.•Pinocembrin was identified as the ...antifungal compound.•The anti-quorum potential of caffeic acid was confirmed and quantified.
The well-known anti-infective properties of propolis are determined by its chemical composition, which in turn is influenced by geographical factors and reflects the botanical diversity in the vicinity of the beehive. Although there are several reports on the anti-infective properties of crude propolis, few are aimed at identifying specific compound(s) responsible for the observed activities. Using South African propolis as an example, the application of high performance thin layer chromatography-bioautography in tandem with mass spectrometry was investigated for the rapid identification of antimicrobial and anti-quorum sensing (anti-QS) compounds. Pinocembrin was found to be responsible for the observed antifungal activity of the propolis against Candida albicans. Three compounds were found to be active against all of the evaluated Gram-positive and Gram-negative bacteria. The identity of the first was confirmed as pinobanksin, one remains unidentified, while the third corresponds to either pinobanksin 3-O-pentanoate or 2-methylbutyrate. The identification of caffeic acid as the anti-QS component was confirmed quantitatively using the violacein inhibitory assay.
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