A validated bioanalytical method for the simultaneous estimation of telmisartan and gallic acid in rat plasma samples by high-performance thin-layer chromatography-mass spectrometry: Application to an oral pharmacokinetic study.

A novel and cost-effective high-performance thin-layer chromatography (HPTLC) method, combined with densitometric quantification, was developed for the biomedical analysis of telmisartan (TEL) and gallic acid (GA). Recent research indicates that when used in combination, these compounds offer improved therapeutic efficacy for the treatment of cardiovascular diseases with reduced side effects. The study focused on the simultaneous quantification and pharmacokinetic analysis of drugs in rat plasma. The separation was conducted using HPTLC silica gel 60 F254 plates with dimensions of 20 × 10 cm and a thickness of 0.2 mm. The mobile phase used for separation consisted of a mixture of ethyl acetate, methanol, chloroform, and acetic acid in the ratio of 4:2:2:0.2 (v/v). GA and TEL were analyzed using ultraviolet detection at specific wavelengths, with GA at 280 nm and TEL at 296 nm. Peak purity was assessed through spectral correlation analysis using Vision CATS software. The method underwent validation following the guidelines of the US Food and Drug Administration (US FDA). Calibration plots demonstrated linearity in the concentration range of 200-1200 ng/spot, with high correlation coefficients (R2 ). The retention factors (Rf ) were 0.67 for TEL and 0.60 for GA. The identity of the separated compounds was further confirmed using MS, with GA having a mass-to-charge ratio (m/z) of 168.9 in negative mode and TEL with m/z 515.2 in positive mode. In the pharmacokinetic study, the maximum peak plasma concentration (Cmax ) for GA was 899.7 ng/mL, and for TEL, it was 1013 ng/mL. The time to reach maximum concentration (Tmax ) was 2 h for GA and 6 h for TEL. This simultaneous qualitative and quantitative determination of the drugs in an oral pharmacokinetic study involving Wistar rats can serve as a valuable tool for future investigations into pharmacokinetic interactions, quality control, and routine analysis of these drugs, both in their pure forms and in novel formulations.

TLC and HPTLC-APCI-MS for the rapid discrimination of plant resins frequently used for lacquers and varnishes by artists and conservators.

INTRODUCTION: Depending on their terpenoid and phenolic constituents plant resins can be classified as diterpenoid, triterpenoid or phenolic resins; thereby the profile of diterpenes and triterpenes is considered as genus- or even species-specific. OBJECTIVES: We aimed to develop a simple, rapid, inexpensive, sensitive and specific method for the identification of resin-specific triterpenoid and phenolic compounds in plant resins using (HP)TLC [(high-performance) thin-layer chromatography] combined with APCI-MS (atmospheric pressure chemical ionisation mass spectrometry) and post-chromatographic detection reactions. METHODS: Twenty resin samples from different plant species were analysed. Different extraction procedures, post-chromatographic detection reagents as well as various sorbents and solvents for planar chromatography were tested. To evaluate the potential of the optimised (HP)TLC-APCI-MS methods, parameter such as limit of detection (LOD) was determined for selected marker compounds. RESULTS: Our protocol enabled qualitative analyses of chemotaxonomic molecular markers in natural resins such as dammar, mastic, olibanum and benzoin. For the first time, the application of thionyl chloride-stannic chloride reagent for a specific post-chromatographic detection of triterpenes is reported, sometimes even allowing discrimination between isomers based on their characteristic colour sequences. For triterpene acids, triterpene alcohols and phenolic compounds, detection limits of 2-20 ng/TLC zone and a system precision with a relative standard deviation (RSD) in the range of 3.9%-7.0% were achieved by (HP)TLC-APCI-MS. The applicability of the method for the analysis of resin-based varnishes was successfully tested on a mastic-based varnish. Thus, the method we propose is a helpful tool for the discrimination of resins and resin-based varnishes with respect to their botanical origin.

Bioautography-guided HPTLC-MS as a rapid hyphenated technique for the identification of antimicrobial compounds from selected South African Combretaceae species.

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.

High-Performance Thin-Layer Chromatography-Densitometry-Tandem ESI-MS to Evaluate Phospholipid Content in Exosomes of Cancer Cells.

The question of whether exosome lipids can be considered as potential cancer biomarkers faces our current limited knowledge of their composition. This is due to the difficulty in isolating pure exosomes, the variability of the biological sources from which they are extracted, and the uncertainty of the methods for lipid characterization. Here, we present a procedure to isolate exosomes and obtain a deep, repeatable, and rapid phospholipid (PL) composition of their lipid extracts, from embryonic murine fibroblasts (NIH-3T3 cell line) and none (B16-F1) and high (B16-F10) metastatic murine skin melanoma cells. The analytical method is based on High Performance Thin-Layer Chromatography with Ultraviolet and fluorescence densitometry and coupled to Electrospray (ESI)-tandem Mass Spectrometry (MS). Under the conditions described in this work, separation and determination of PL classes, (sphingomyelins, SM; phosphatidylcholines, PC; phosphatidylserines, PS; and phosphatidylethanolamines, PE) were achieved, expressed as µg PL/100 µg exosome protein, obtained by bicinchoninic acid assay (BCA). A detailed structural characterization of molecular species of each PL class was performed by simultaneous positive and negative ESI-MS and MS/MS directly from the chromatographic plate, thanks to an elution-based interface.

Detection of azo dyes and aromatic amines in women undergarment.

Women are exposed to several chemical additives including azo dyes that exist in textile materials, which are a potential health hazard for consumers. Our objective was to analyze suspected carcinogenic azo dyes and their degradation aromatic amines in women underwear panties using a fast and simple method for quantification. Here, we evaluated 120 different samples of women underwear for their potential release of aromatic amines to the skin. Seventy-four samples yielded low level mixtures of aromatic amines; however eighteen samples were found to produce greater than 200 mg/kg (ppm) of aromatic amines. Azo dyes in these 18 samples were extracted from the fabrics and analyzed by reverse phase thin layer chromatography in tandem with atmospheric pressure chemical ionization mass spectrometry. Eleven azo dyes were identified based on their mass spectral data and the chemical structure of the aromatic amine produced from these samples. We demonstrate that planar chromatography and mass spectrometry can be really helpful in confirming the identity of the azo dyes, offering highly relevant molecular information of the responsible compounds in the fabrics. With the growing concern about the consumer goods, analysis of aromatic amines in garments has become a highly important issue.

High-Performance Thin-Layer Chromatography Coupled with Single Quadrupole: Application the Identification and Differentiation of Rehmanniae Radix and Its Different Processing Products from Raw Materials to Commercial Products.

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.

A solid-liquid microextraction approach from a spot in an adsorbent layer of a chromatographic plate.

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.

Quantitation of Usnic acid from three Usnea species, its commercial formulations Using High-Performance Thin-Layer Chromatography-Mass Spectrometry (HPTLC-MS) and their metabolite profiling using UPLC-QTof-MSE.

BACKGROUND: The genus Usnea (Parmeliaceae; lichenized Ascomycetes) is pale grayish-green fruticose lichens which grow as leafless mini-shrubs and comprise about 360 species. Most of the Usnea species are edible and is utilized in preparation of traditional foods as well as in medicines to combat wide range of ailments. OBJECTIVE: The goal of this work was to quantify usnic acid in three Usnea spp. [Usnea ghattensis (UG), Usnea orientalis (UO) and Usnea undulata (UU)] using HPTLC-MS and chemical profiling of acetone extracts using UPLC-QTof-MSE resulted in the identification of sixteen compounds based on their MS/MS fragmentation patterns. METHODS: Hyphenated techniques, HPTLC-MS and UPLC-QTof-MSE have been proposed to quantify usnic acid and analysis of metabolites in the crude extracts qualitatively. This method allowed tentative characterization of metabolites from Usnea spp. RESULTS: The quantification study showed the excellent linearity of the usnic acid at 0.25-1 µg/band with a correlation coefficient r  2>0.99, and LOD, LOQ was found to be 51.7 and 156.6 ng/band, respectively. Further, UPLC-QTof-MSE analysis of crude extract led identification of lichen substances through their exact molecular masses and MS/MS fragmentation studies. CONCLUSIONS: The present study summarizes HPTLC method for quantification of usnic acid in three different Usnea spp. Along with two herbal formulations containing Usnea spp. as the ingredient and developed method was validated as per the ICH guidelines and further UPLC-QTof-MSE analysis provides characterization of the sixteen different secondary metabolites based on their mass fragmentation studies. HIGHLIGHTS: Rapid HPTLC method for quantification of usnic acid in three different Usnea spp. along with two herbal formulations and metabolite profiling using UPLC-QTof-MSE.

Fermentation dynamics of bile salt hydrolase production in Heyndrickxia coagulans ATCC 7050 and Lactiplantibacillus plantarum ATCC 10012: Addressing ninhydrin assay limitations with a novel HPTLC-MS method.

Bile salt hydrolase (BSH), a pivotal enzyme in cholesterol management, holds significant promise in both human and animal subjects. This study investigated the effect of fermentation dynamics in Heyndrickxia coagulans ATCC 7050 and Lactiplantibacillus plantarum ATCC 10012 to enhance BSH production. Cultivation of cultures in MRS and M17 media revealed that MRS medium enhanced BSH production by 235.98 % in H. coagulans ATCC 7050 and 147.37 % in L. plantarum ATCC 10012, compared to M 17 medium. Additionally, varying oxygen concentration levels indicated that H. coagulans ATCC 7050 exhibited its minimum doubling time of 79.8 ± 0.64 min in anaerobic conditions, whereas L.plantarum ATCC 10012 demonstrated its minimum doubling time of 85.5 ± 1.2 min under microaerophilic conditions. However, their highest BSH activity was observed during the stationary phase under anaerobic conditions, yielding 17.14 ± 0.78 U/mL by H. coagulans ATCC 7050 and 19.04 ± 0.81 U/mL by L.plantarum ATCC 10012. Furthermore, it was observed that both organisms did not retain BSH within their cells. BSH activity was assessed using ninhydrin assay that detected free taurine liberated from sodium taurocholate. However, ninhydrin can yield false-positive results owing to its interaction with other free amino acids. To subjugate this limitation, the study introduced a novel and sensitive HPTLC-MS method capable of accurately detecting taurine. By comprehending fermentation dynamics and selecting appropriate conditions, BSH production increased 2.1-fold in both organisms. These findings illuminate critical insights, offering a pathway for novel strategies to enhance the BSH-producing capabilities of these LAB strains.

Elucidating the anti-obesity potential of bioactive fractions of kalanchoe pinnata (lam.) leaves extract using a combination of in vitro, in vivo and in silico methods along with characterisation of lead compounds through an HPTLC ms-MSn analytical study.

Fractions were isolated from the leaves extract of Kalanchoe pinnata and subjected to scrutiny for their prospective anti-obesity properties. An array of preliminary phytochemical, invitro antioxidant, and enzyme inhibition assays were executed, which discerned fractions F1 and F2 as the most effective fractions. These fractions were subsequently studied through invivo experiments, affirming that F2 as the most potent fraction. Further characterisation of F2 was conducted via HPTLC-Mass spectrometry (MS-MSn) techniques. The outcomes demonstrated that F2 produced a notable anti-obesity effect in obese mice, reducing their body weight and lipid metrics, and leading to advantageous changes in their organs. An analytical examination of F2 revealed the existence of four principal compounds, which were subsequently subjected to insilico molecular docking and dynamic analysis, confirming their aptitude for binding to selected proteins. These findings imply that the utilisation of Kalanchoe pinnata leaves could provide a promising therapeutic strategy for the treatment of obesity.

Anti-obesity potential of bioactive guided fractions of Annona squamosa linn. leaves extract: a combination of in-vitro, in-vivo and in-silico studies along with profiling of lead compounds by HPTLC MS-MSn method.

The antiobesity potential of bioactive fractions derived from Annona squamosa was approached using a combination of in vitro, in silico and in-vivo studies. The study was analyzed to validate and select the potent bioactive fractions of A. squamosa leaves extract through in vitro and in vivo activities targeting obesity. The phytochemical properties of the bioactive fractions were investigated utilizing total flavonoid, total phenolic and total steroidal content. Further, in vitro antioxidant assays such as nitric oxide (NO2), DPPH, ABTS, and Hydrogen peroxide (H2O2) scavenging assays were performed whereas pancreatic lipase, Alpha-amylase and Alpha glucosidase assays were carried out for enzyme inhibition activities. The overall study revealed that fractions F2 and F3 had shown significant in vitro activities targeting obesity. The selected potent fractions (F2 and F3) were orally bio-screened for efficacy in MSG-HFD-induced obese mice at 80 mg/kg/bw. The invivo study confirmed that fractions 2 and 3 with a dose of 80 mg/kg/bw had a significant potency compared to obese control and standard for various parameters. Body weight and lipid metrics were significantly reduced, and histological examinations revealed considerable beneficial alterations in the organs of the animals. Further HPTLC MS-MSn was used to characterize and identify the major compounds in the potent bioactive fractions, which confirmed the presence of seven major compounds: Ascorbic acid, Gallic acid, Quercetin, ß-sitosterol, Stigmasterol, Caffeine and Epigallocatechin gallate. An in silico model was then employed to determine the best binding activity of the identified compound towards the specific receptors targeting obesity, confirming the most effective docking score towards stigmasterol and sitosterol. The in vitro and in vivo studies of derived bioactive fractions of A. squamosa leaves extract revealed a possible therapeutic approach towards anti-obesity activity for the first time.

Eco-friendly stability-indicating HPTLC micro-determination of the first FDA approved SARS-CoV-2 antiviral prodrug Remdesivir: Study of degradation kinetics and structural elucidation of the degradants using HPTLC-MS.

The worldwide spread coronavirus (covid-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a global health crisis. The world was forced to face a great challenge to control and overcome this health disaster through various containment measures including efficient vaccination side by side with effective medication. Remdesivir (RMD) is the first FDA approved antiviral agent for treatment of covid-19 pandemic and hence regarded as the first-in-class medication of this highly contagious respiratory disease. The current study represents the first stability indicating HPTLC method for the estimation of RMD in bulk form and pharmaceutical formulation. The method employed TLC silica gel aluminum plates 60 F254 as stationary phase and green mobile phase composed of ethyl acetate and ethanol (96: 4, v/v) with densitometric detection at 245 nm. Comprehensive validation of the adopted method was accomplished according to the ICH guidelines regarding linearity, ranges, detection and quantification limits, precision, accuracy and robustness. The developed method offered a neat separation of the drug in presence of pharmaceutical excipients as well as in presence of acidic, alkaline, neutral hydrolytic, oxidative and photolytic degradants. Additionally, structural elucidation of alkaline and hydrolytic oxidation degradation products was carried out using HPTLC-MS. Furthermore, for the first time the acidic and alkaline degradation kinetics of RMD were studied and its degradation rate constants and half-lives were calculated. Moreover, greenness appraisal of the developed method as well as comparison with previously published stability indicating HPLC methods were performed using analytical Eco-scale, GAPI and AGREE metrics.

High performance Reversed-Phase Thin-Layer Chromatography-Desorption electrospray ionisation - time of flight high resolution mass spectrometric detection and imaging (HPTLC/DESI/ToFMS) of phytoecdysteroids.

Reversed-phase high performance thin-layer chromatography (RP-HPTLC) on C18 bonded silica gel was combined with desorption electrospray ionization (DESI) and high resolution time of flight mass spectrometry (HRToFMS) to detect, characterize and image (MSI) phytoecdysteroids (plant-derived insect moulting hormones) in ethanolic extracts of members of the Silene plant family. As seen previously for silica gel, DESI provided a simple and convenient method for recovering polar polyhydoxysteroids from RP-HPTLC plates for the purposes of both the MS and MSI of extracts obtained from three species of the Silene family (Silene otites, S. nutans and S. viridiflora). Using RP-HPTLC/DESI/MSI/HRToFMS a number of ecdysteroids, including 20-hydroxyecdysone, polypodine-B, 2-deoxy-20-hydroxyecdysone and 2-deoxyecdysone were identified in these extracts. Differences were noted in the mass spectra obtained depending upon both the stationary phase on which they were separated, and the temperatures used in the heated transfer line used for introduction into the ion source. Ecdysteroids detected after chromatography on C18 bonded silica showed increased fragmentation due to water loss compared to those imaged from silica. In addition, the benefits of the additional resolution provided by 2-dimensional TLC for increasing spectral quality compared to a 1-dimensional separation are demonstrated.

Non-target bioactive compound profiles of coffee roasts and preparations.

Coffee is an inherent part of our daily nutrition and seems to have protective effects against diseases, whereby it is often not fully understood, which ingredients are responsible for the observed effect. Hence, a non-targeted bioactivity profiling was developed to investigate 27 hand-filtered coffee brews of differently roasted coffee beans and 14 differently prepared and stored coffee brews. After separation, multi-imaging, and densitometry, six planar effect-directed assays were performed to reveal individual antioxidative, antibacterial, anti-cholinesterase, anti-diabetic, and estrogenic effects. Individual compounds were mainly responsible for the observed effects, e.g. 5-O-caffeoylquinic acid regarding antioxidative potential and α-glucosidase inhibition, while coffee brews made by a fully automated coffee machine showed the highest antioxidative potential. Unlike preparation and storage conditions, applied roasting conditions and origin of coffee samples played a less important role. Therefore, the way we daily consume our coffee has an impact on the magnitude of potential health effects.

Separation and detection of apricot leaf triterpenes by high-performance thin-layer chromatography combined with direct bioautography and mass spectrometry.

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.

Determination of the effect of germination on the folate content of the seeds of some legumes using HPTLC-mass spectrometry-multivariate image analysis.

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).

Flavan-3-ols and Proanthocyanidins in Japanese, Bohemian and Giant Knotweed.

Flavan-3-ols and proanthocyanidins of invasive alien plants Japanese knotweed (Fallopia japonica Houtt.), giant knotweed (Fallopia sachalinensis F. Schmidt) and Bohemian knotweed (Fallopia × bohemica (Chrtek & Chrtkova) J.P. Bailey) were investigated using high performance thin-layer chromatography (HPTLC) coupled to densitometry, image analysis and mass spectrometry (HPTLC-MS/MS). (+)-Catechin, (-)-epicatechin, (-)-epicatechin gallate and procyanidin B2 were found in rhizomes of these three species, and for the first time in Bohemian knotweed. (-)-Epicatechin gallate, procyanidin B1, procyanidin B2 and procyanidin C1 were found in giant knotweed rhizomes for the first time. Rhizomes of Bohemian and giant knotweed have the same chemical profiles of proanthocyanidins with respect to the degree of polymerization and with respect to gallates. Japanese and Bohemian knotweed have equal chromatographic fingerprint profiles with the additional peak not present in giant knotweed. Within the individual species giant knotweed rhizomes and leaves have the most similar fingerprints, while the fingerprints of Japanese and Bohemian knotweed rhizomes have additional peaks not found in leaves. Rhizomes of all three species proved to be a rich source of proanthocyanidins, with the highest content in Japanese and the lowest in Bohemian knotweed (based on the total peak areas). The contents of monomers in Japanese, Bohemian and giant knotweed rhizomes were 2.99 kg/t of dry mass (DM), 1.52 kg/t DM, 2.36 kg/t DM, respectively, while the contents of dimers were 2.81 kg/t DM, 1.09 kg/t DM, 2.17 kg/t DM, respectively. All B-type proanthocyanidins from monomers to decamers (monomers-flavan-3-ols, dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers and decamers) and some of their gallates (monomer gallates, dimer gallates, dimer digallates, trimer gallates, tetramer gallates, pentamer gallates and hexamer gallates) were identified in rhizomes of Bohemian knotweed and giant knotweed. Pentamer gallates, hexamers, hexamer gallates, nonamers and decamers were identified for the first time in this study in Bohemian and giant knotweed rhizomes.

Leaves of Invasive Plants-Japanese, Bohemian and Giant Knotweed-The Promising New Source of Flavan-3-ols and Proanthocyanidins.

This is the first report on identification of all B-type proanthocyanidins from monomers to decamers (monomers-flavan-3-ols, dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, and decamers) and some of their gallates in leaves of Japanese knotweed (Fallopia japonica Houtt.), giant knotweed (Fallopia sachalinensis F. Schmidt) and Bohemian knotweed (Fallopia × bohemica (Chrtek & Chrtkova) J.P. Bailey). Flavan-3-ols and proanthocyanidins were investigated using high performance thin-layer chromatography (HPTLC) coupled to densitometry, image analysis, and mass spectrometry (HPTLC-MS/MS). All species contained (-)-epicatechin and procyanidin B2, while (+)-catechin was only detected in Bohemian and giant knotweed. (-)-Epicatechin gallate, procyanidin B1 and procyanidin C1 was only confirmed in giant knotweed. Leaves of all three knotweeds have the same chemical profiles of proanthocyanidins with respect to the degree of polymerization but differ with respect to gallates. Therefore, chromatographic fingerprint profiles of proanthocyanidins enabled differentiation among leaves of studied knotweeds, and between Japanese knotweed leaves and rhizomes. Leaves of all three species proved to be a rich source of proanthocyanidins (based on the total peak areas), with the highest content in giant and the lowest in Japanese knotweed. The contents of monomers in Japanese, Bohemian and giant knotweed were 0.84 kg/t of dry weight (DW), 1.39 kg/t DW, 2.36 kg/t, respectively, while the contents of dimers were 0.99 kg/t DW, 1.40 kg/t, 2.06 kg/t, respectively. Giant knotweed leaves showed the highest variety of gallates (dimer gallates, dimer digallates, trimer gallates, tetramer gallates, pentamer gallates, and hexamer gallates), while only monomer gallates and dimer gallates were confirmed in Japanese knotweed and monomer gallates, dimer gallates, and dimer digallates were detected in leaves of Bohemian knotweed. The profile of the Bohemian knotweed clearly showed the traits inherited from Japanese and giant knotweed from which it originated.

Extraction of Anthraquinones from Japanese Knotweed Rhizomes and Their Analyses by High Performance Thin-Layer Chromatography and Mass Spectrometry.

Anthraquinones (yellow dyes) were extracted from Japanese knotweed rhizomes with twelve extraction solvents (water; ethanol(aq) (20%, 40%, 60%, 70% and 80%), ethanol, 70% methanol(aq), methanol, 70% acetone(aq), acetone and dichloromethane). The obtained sample test solutions (STSs) were analyzed using high-performance thin-layer chromatography (HPTLC) coupled to densitometry and mass spectrometry (HPTLC-MS/MS) on HPTLC silica gel plates. Identical qualitative densitometric profiles (with anthraquinone aglycones and glycosylated anthraquinones) were obtained for STSs in all the solvents except for the STS in dichloromethane, which enabled the most selective extractions of anthraquinone aglycones emodin and physcion. The highest extraction efficiency, evaluated by comparison of the total peak areas in the densitograms of all STSs scanned at 442 nm, was achieved for 70% acetone(aq). In STS prepared with 70% acetone(aq), the separation of non-glycosylated and glycosylated anthraquinones was achieved with developing solvents toluene-acetone-formic acid (6:6:1, 3:6:1 and 3:3:1 v/v) and dichloromethane-acetone-formic acid (1:1:0.1, v/v). Non-glycosylated anthraquinones were separated only with toluene-acetone-formic acid, among which the best resolution between emodin and physcion gave the ratio 6:6:1 (v/v). This solvent and dichloromethane-acetone-formic acid (1:1:0.1, v/v) enabled the best separation of glycosylated anthraquinones. Four HPTLC-MS/MS methods enabled the identification of emodin and tentative identification of its three glycosylated analogs (emodin-8-O-hexoside, emodin-O-acetyl-hexoside and emodin-O-malonyl-hexoside), while only the HPTLC-MS/MS method with toluene-acetone-formic acid (6:6:1, v/v) enabled the identification of physcion. Changes of the shapes and the absorption maxima (bathochromic shifts) in the absorption spectra after post-chromatographic derivatization provided additional proof for the detection of physcion and rejection of the presence of chrysophanol in STS.

HPTLC-DESI-HRMS-Based Profiling of Anthraquinones in Complex Mixtures-A Proof-of-Concept Study Using Crude Extracts of Chilean Mushrooms.

High-performance thin-layer chromatography (HPTLC) coupled with negative ion desorption electrospray ionization high-resolution mass spectrometry (DESI-HRMS) was used for the analysis of anthraquinones in complex crude extracts of Chilean dermocyboid Cortinarii. For this proof-of-concept study, the known anthraquinones emodin, physcion, endocrocin, dermolutein, hypericin, and skyrin were identified by their elemental composition. HRMS also allowed the differentiation of the investigated anthraquinones from accompanying compounds with the same nominal mass in the crude extracts. An investigation of the characteristic fragmentation pattern of skyrin in comparison with a reference compound showed, exemplarily, the feasibility of the method for the determination of these coloring, bioactive and chemotaxonomically important marker compounds. Accordingly, we demonstrate that the coupling of HPTLC with DESI-HRMS represents an advanced and efficient technique for the detection of anthraquinones in complex matrices. This analytical approach may be applied in the field of anthraquinone-containing food and plants such as Rheum spp. (rhubarb), Aloe spp., Morinda spp., Cassia spp. and others. Furthermore, the described method can be suitable for the analysis of anthraquinone-based colorants and dyes, which are used in the food, cosmetic, and pharmaceutical industry.