•Effect of germination on the folate composition of three legume seeds was investigated.•A novel HPTLC method was developed and validated for fingerprinting and quantitation of folates.•High ...resolution plate imaging was hyphenated to mass spectrometry for fingerprint analysis.•Germination of all seeds resulted in a 2.5–4 fold increase in the content of total folates.•5-CH3-H4 folate had maximum content in day 5 sprouts of black-eyed peas.
Legumes are the main sources of folates which are not synthesized in the human body. The five folate species: 5-methyl tetrahydrofolate, tetrahydrofolate, pteroyl glutamate, 5-formyl tetrahydrofolate and 10-formyl tetrahydrofolate were quantitatively determined in legumes seeds and sprouts by a newly developed and validated high performance thin layer chromatography method. High resolution plate imaging hyphenated to mass spectrometry was exploited for fingerprint analysis of tested samples. Results indicated that germination of all seeds resulted in a 2.5–4 fold increase in the content of total folates as well as the individual vitamers. The total amount of folate reached a maximum on the fifth day in the case of black-eyed peas (861 μg/100 g Fresh Weight), white beans (755 μg/100 g FW) and brown lentils (681 μg/100 g FW). 5-CH3-H4 folate was found to be the most dominating folate species reaching its maximum content in day 5 sprouts of black-eyed peas (490 μg/100 g FW).
•Separation of five triterpenes by HPTLC using pre-chromatographic derivatization.•2D-HPTLC separation of triterpenes with intermediate derivatization by iodine.•(2D)-HPTLC-MS confirmation of ...presence of triterpenes.•Separation of ursolic, oleanolic, betulinic, maslinic and corosolic acids in silica.•HPTLC-bioassay of native separated compounds made possible by temporary derivatization.
Prunus armeniaca leaf extract was screened for antibacterial compounds by high-performance thin-layer chromatography (HPTLC)-direct bioautography using a Gram-positive Bacillus subtilis bacterium. Six chromatographic zones exhibited characteristic bioactivity. Five of them also appeared after derivatization with vanillin-sulfuric acid reagent and could be characterized with HPTLC-electrospray ionization (ESI)-mass spectrometry (MS), suggesting the presence of triterpenoids and the fatty acids linolenic and palmitic acid. To confirm the identification of triterpenoids an HPTLC method using in situ pre-chromatographic derivatization with iodine was developed to separate the closely related triterpenoids. After development, the iodine could be eliminated from the chromatogram (verified by HPTLC-MS), making it suitable for the B. subtilis assay. Ursolic acid, oleanolic acid, betulinic acid, corosolic acid, and maslinic acid were discovered for the first time as antibacterial components of P. armeniaca leaves. Their presence was proved also by 2D-HPTLC combined with intermediate in situ derivatization by iodine.
The authentication of ingredients in formulas is crucial yet challenging, particularly for constituents with comparable compositions but vastly divergent efficacy. Rehmanniae Radix and its ...derivatives are extensively utilized in food supplements, which contain analogous compositions but very distinct effects. Rehmanniae Radix, also a difficult-to-detect herbal ingredient, was chosen as a case to explore a novel HPTLC-QDa MS technique for the identification of herbal ingredients in commercial products. Through systematic condition optimization, including thin layer and mass spectrometry, a stable and reproducible HPTLC-QDa MS method was established, which can simultaneously detect oligosaccharides and iridoids. Rehmannia Radix and its processed products were then analyzed to screen five markers that could distinguish between raw and prepared Rehmannia Radix. An HPTLC-QDa-SIM method was further established for formula detection by using the five markers and validated using homemade prescriptions and negative controls. Finally, this method was applied to detect raw and prepared Rehmannia Radix in 12 commercial functional products and supplements.
•Novel device for extracting substances from a chromatographic plate.•Simple, cost-effective, reliable solid-liquid phase extraction method.•The extraction method ensures no cross-contamination ...between samples occurs.•Sample preparation technique for LC-MS analysis of coccidiostats.
A new approach to extracting substances from a spot on a chromatographic plate for subsequent liquid chromatography-mass spectrometry analysis is described. This method involves extraction in a solid phase (an adsorbent layer of a chromatographic plate) - a liquid system using a simple device. For a single extraction of six selected coccidiostats from the adsorbent layer on the chromatographic plate with silica gel, 50 µL of methanol was used for 5 min. The data from the extraction experiments and liquid chromatography-mass spectrometry measurements demonstrated a good correlation between the ratio of the peak areas of the coccidiostats to the internal standard and the concentration of the substances in the range of two orders of magnitude. The coefficients of determination for the mentioned correlations range from 0.962 to 0.999. Moreover, the repeatability and reproducibility, expressed as the percentage values of relative standard deviation, do not exceed 7.5 % for any of the coccidiostats.
Display omitted
•Untargeted HPTLC-image analyses chemical profiling applied to classify Hedera helix subspecies.•PCA revealed separation between samples using data set of phenolics ...profiling.•Imortnat variables were identified using HPTLC/MS.•HPTLC method developed for quantification of six phenolics and three saponins.•OPLS model used to screen bioactive markers in samples using antimicrobial data.
A strategy combining untargeted and targeted chemical profiling for discrimination of the different subspecies of Hedera helix L (Ivy) based on HPTLC fingerprints and antioxidant and antimicrobial activity testing followed by chemometric analysis has been implemented for the first time for the holistic profiling of bioactive secondary metabolites in the different subspecies. Principal component analysis (PCA) based on HPTLC-image analysis fingerprints of secondary metabolites loading plots were utilized for determination of chemical markers responsible for the classification which were then identified using HPTLC/MS technique as the six phenolics; caffeic acid, kaempferol-3-O-glucoside, quercetin-3-O-glucoside, kaempferol-3-O-rutinoside, rutin and chlorogenic acid and the three saponins; alpha-hederin, hederasaponin B and hederacoside C. The identified markers were then quantified using a HPTLC-high resolution plate imaging technique for targeted profiling. Biomarkers were determined from the coefficients plot of orthogonal projection to latent structures (OPLS) model based on the secondary metabolites quantitative data together with the biological activity data which revealed that the phenolic metabolites were strongly correlated to the antioxidant and antimicrobial activity data points compared to saponins. According to targeted hierarchical clustering analysis (HCA)-heat-map, samples could be divided into three main clusters; H. helix subsp. helix cluster, H. helix subsp. rhizomatifera and H. helix subsp. hiberinica sub-clusters and H. helix subsp. poetrum and H. helix subsp. canariensis sub-clusters, and such clustering pattern was directly related to the variation in the content of bioactive phenolics among the different subspecies rather than their saponins content, providing an insight into the unsettled taxonomy of the ivy subspecies and successful precise efficacy-directed discrimination of the different samples.
The
genus is one of India's prominent botanical classes of traditional medicinal culture comprising medicinally and agronomically important plants. Morphological resemblances, overlapping ...geographical distribution, and history of traditional nomenclature have necessitated a comprehensive qualitative report for effective quality control and removing the species ambiguity pertaining to this genus. This paper provides detailed morpho-micrometric characteristics used to differentiate between six indigenous
species of India. Among them,
was distinguished as the only shrub with a fleshy petiole. In green and purple forms,
leaves had serrate margins and showed no particular anatomical differences except for the anthocyanins containing epidermal cells of the latter.
had glabrous leaves except for the veins, which were puberulous.
had tenuous anther filaments and was the least aromatic while
had a citrusy odour, which along with the number of xylary rows, size of mesophyll cells, and epidermal cell wall architecture, distinguished it from
. An HPTLC method was developed using experimental design and validated for quantification of multi-class compounds from terpenoic, phenolic acids, and flavonoids in
leaves. It was found linear (
> 0.99) with recoveries between 95 - 100% for all compounds. The eluted bands of marker compounds were subjected to HPTLC-MS analysis as a confirmative tool. This is the first anatomical and analytical report of
Forssk. The obtained results could be effectively used for species identification using vegetative characters alone with the anatomical-HPTLC data backing up the former as a rapid and economical tool.
•An easy characterization for E 472 food emulsifiers was developed.•HPTLC screening with sensitive fluorescence detection was performed.•Visual detection of emulsifier patterns was assessed.•Elegant ...comparison of samples and batches through fingerprints was introduced.•HPTLC–MS offered the identification of constituents of interest.
Esters of fruit acids including acetic acid and mono- and diacylglycerols (MAG and DAG), also known as E 472 emulsifiers, are used in the food industry as food additives to adjust techno-functional properties like viscosity, emulsion stability and foaming stability in various products, mainly dairy products. Based on the respective acids, E 472 emulsifiers are classified in several categories with acetic acid esters (ACETEM, E 472a), lactic acid esters (LACTEM, E 472b), citric acid esters (CITREM, E 472c), and mono- and diacetyl tartaric acid esters (DATEM, E 472e) as the most prominent representatives. Besides fruit acid esters, E 472 emulsifiers mainly comprise MAG, DAG, triacylglycerols, free fatty acids, free fruit acids and free glycerol in different amounts. Here we present an innovative and sensitive method for the characterization of the composition of E 472 emulsifiers by high-performance thin-layer chromatography with fluorescence detection (HPTLC–FLD). For HPTLC–FLD, technical emulsifiers were simply dissolved and analyzed on HPTLC silica gel plates after a two-fold development and derivatization with primuline, enabling the easy characterization and direct visual comparison of the emulsifier pattern (fingerprint) through the fluorescent lipid components under UV 366 nm. Thus, the HPTLC–FLD fingerprint approach represents a simple tool for the comparison of different samples, batches and categories of E 472. Coupling of HPTLC to mass spectrometry moreover enabled the identification of constituents of interest.
•First on-line HPTLC-MS methods on silica gel for proanthocyanidins up to decamers.•Ionization suppression was solved by two pre-developments of HPTLC plates.•Stability testing after development ...revealed a phenomenon of enhanced absorption.•Free and galloylated B-type proanthocyanidins were identified in Japanese knotweed.•HPTLC-MS methods enabled also identification of stilbenes and anthraquinones.
On-line elution based TLC-MS is now a well-established technique, but the quality of the data obtained can sometimes be hampered by a severe spectral background or by strong ion suppression, especially when silica gel plates are used in combination with an acidic modifier in the developing solvent. We solved this issue simply and efficiently using two pre-developments of the plates, firstly with methanol–formic acid (10:1, v/v) and secondly with acetonitrile-methanol (2:1, v/v). This solution resulted in significant improvement in the sensitivity of HPTLC-MS methods. The applicability of this approach was proven on analysis of flavan-3-ols and proanthocyanidins in crude extracts of Japanese knotweed (Fallopia japonica Houtt.) rhizomes. Separations on HPTLC silica gel and HPTLC silica gel MS grade plates using developing solvents toluene-acetone-formic acid (3:3:1, 6:6:1, 3:6:1, v/v) and dichloromethane-acetone-formic acid (1:1:0.1, v/v) were followed by post-chromatographic derivatization with 4-dimethylaminocinnamaldehyde (DMACA) detection reagent. Examination of the stability of the analytes on the start confirmed that the plates should be developed immediately after the application of standards and sample test solutions. In a five hours stability testing after development we discovered an unexpected phenomenon of enhanced absorption at 280nm. However, based on an experiment with post-chromatographic derivatization with DMACA detection reagent, the analytes were proven to be sufficiently stable in the time frame of an HPTLC-MS analysis. This was important for development of the first HPTLC-MS and HPTLC-MSn methods for identification of flavan-3-ols and B-type proanthocyanidins from monomers up to decamers. For the first time, based on this research methodology, trimers, trimer gallates, tetramer gallates, pentamers, pentamer gallates, hexamers, hexamer gallates, heptamers, octamers, nonamers and decamers were tentatively identified in Japanese knotweed rhizomes. Additionally, all developed HPTLC-MS methods have enabled simultaneous identification of stilbenes (resveratrol, piceatannol hexoside, piceid) and anthraquinones (emodin, emodin-O-hexoside, emodin-O-(acetyl)-hexoside and emodin-O-(6′-O-malonyl)-hexoside).
•HPTLC analysis of Serbian Salicaceae bud extracts targetedly combined with HRMS•Chemotypical profiling of Serbian poplars by PCA of HPTLC images•Hyphenation of HPTLC to cholinesterase assays for ...Alzheimer’s disease•Hyphenation of HPTLC to biological assays for antimicrobial and estrogenic substances•Identification by HPTLC-MS of chrysin, caffeic acid and galangin as active molecules
The buds of poplars (Populus L.) and willows (Salix L.), both from the same family (Salicaceae Mirbel), are increasingly used in gemmotherapy and importantly contribute to the production of the physiologically active propolis by European bee Apis mellifera L. In order to study their phenolic profiles, polar extracts of buds from P. nigra L. were compared to those of P. alba L. and S. alba L. through high-performance thin-layer chromatography (HPTLC). Five chemotypical patterns were distinguished after derivatisation with the Natural Product reagent and confirmed by principal component analysis. The HPTLC analysis was directly hyphenated to various microbiological and biochemical assays as well as spectrometric techniques, directly linking to active molecules in the chromatograms. At a glance, polyvalent compounds were evident when all derivatisation and activity assays, to which HPTLC was hyphenated at ease, were combined together. In Populus buds, at least three antimicrobial compound zones were detected using Aliivibrio fischeri and Bacillus subtilis bioassays, and one phyto-œstrogen with the planar yeast œstrogen screen. In all samples, several inhibitors of acetyl- and butyrylcholinesterase and rabbit liver esterase were detected. Hyphenation to high resolution mass spectrometry supported the assignment of bioactive compounds, as shown for chrysin as selective cholinesterase inhibitor as well as caffeic acid and galangin as antimicrobials in P. nigra and P. alba. This fast and cost-efficient method can be appropriately extended and applied to the botanical origin determination and quality control of bud extracts and propolis samples.
