Recent advances in analysis of new psychoactive substances by means of liquid chromatography coupled with low-resolution tandem mass spectrometry.

The number of methods for the analysis of new psychoactive substances (NPS) is continually increasing, and there is no indication that this trend will change in the near future. The constantly growing market of "designer drugs" makes it necessary to develop new methods of their analysis. The aim of this review is to present the multi-component methods of detection and identification of NPS using low-resolution tandem mass spectrometry coupled with liquid chromatography. For this purpose, 36 articles were selected by applying strictly defined search criteria. Due to the large differences in the matrices and physicochemical properties of the analytes, the described research methods are diverse. These differences are visible in sample preparation methods, chromatographic columns, mobile phases, gradients, or additives to mobile phases used. This work collects and organizes the existing information on the subject of NPS screening analysis methods and will be helpful to forensic scientists working on this topic.

Study on the effect of blackcurrant extract - based preservative on model membranes and pathogenic bacteria.

In this work the cosmetic preservative based on a Ribes Nigrum (blackcurrant) plant extract (PhytoCide Black Currant Powder abbr. BCE) was investigated to evaluate its antibacterial effect and to gain an insight into its mechanism of action. The influence of this commercially available formulation on model Escherichia coli and Staphylococcus aureus lipid membranes was studied to analyze its interactions with membrane lipids at a molecular level. The mixed lipid monolayers and one component bacteria lipid films were used to investigate the effect of BCE on condensation and morphology of model systems and to study the ability of BCE components to penetrate into the lipid environment. The in vitro tests were also done on different bacteria species (E. coli, Enterococcus faecalis, S. aureus, Salmonella enterica, Pseudomonas aeruginosa) to compare antimicrobial potency of the studied formulation. As evidenced the in vitro studies BCE formulation exerts very similar antibacterial activity against E. coli and S. aureus. Moreover, based on the collected data it is impossible to indicate which bacteria: Gram-positive or Gram-negative are more susceptible to this formulation. Model membrane experiments evidenced that the studied preservative affects organization of both E. coli and S. aureus model system by decreasing their condensation and altering their morphology. BCE components are able to penetrate into the lipid systems. However, all these effects depend on the lipid composition and monolayer organization. The collected results were analyzed from the point of view of the mechanism of action of blackcurrant extract and the factors, which may determine the activity of this formulation.

Antioxidant and Antimicrobial Effects of Baby Leaves of Amaranthus tricolor L. Harvested as Vegetable in Correlation with Their Phytochemical Composition.

Amaranth is used as a spinach replacement; therefore, it is sometimes called Chinese Spinach. So far, the activity of the plant has not been associated with the presence of specific compounds. Three cultivars of Amaranthus tricolor L. were investigated for their antioxidant and antimicrobial activities. The correlation between the bioactivity and metabolite profiles was investigated in order to indicate active compounds in A. tricolor. The phytochemical profile of a total of nine extracts was studied by HPLC-DAD-ESI/HRMS, revealing the presence of 52 compounds. The highest antioxidant activity was noticed in the Red cultivar (0.06 mmol TE/g DE (Trolox Equivalent/Dry Extract Weight) and was related to the presence of amino acids, flavonoids and phenolic acids, as well as individual compounds such as tuberonic acid hexoside. All studied extracts revealed antimicrobial activity. Gram-positive bacteria were more susceptible to N-(carboxyacetyl) phenylalanine, phenylalanine, tuberonic acid and succinic acid and Gram-negative bacteria to dopa, tryptophan, norleucine, tuberonic acid hexoside, quercetin-O-hexoside, luteolin-O-rhamnosylhexoside, luteolin-6-C-hexoside succinic acid, gallic acid-O-hexoside, dihydroxybenzoic acid and hydroxybenzoic acid. Maleic acid showed promising antifungal activity. In summary, A. tricolor is a good source of antioxidant and antimicrobial compounds.

Dehydrogenation of Betacyanins in Heated Betalain-Rich Extracts of Red Beet (Beta vulgaris L.).

Betacyanins are a group of water-soluble red-violet compounds containing nitrogen in their structure. These are biosynthesized in red beetroot (Beta vulgaris L.), a widely consumed vegetable that contains significant amounts of nutritious and bioactive compounds which are also found in dietary supplements. This contribution presents results of betacyanin thermal oxidation (resulting in dehydrogenation) interrelated with decarboxylation in selected acetate/phosphate buffers at pH 3-8 and at 85 °C, which may be of particular significance for formulation and performance of foods. Most of the reaction products were detected at the highest concentrations in the acidic solutions (pH 3-4). The main dehydrogenation reaction pathways were monitored by LC-DAD-MS/MS and were associated with decarboxylation of the principal extract pigments, betanin/isobetanin and neobetanin, at carbon positions C-2 and C-17. Additional reactions are accompanied by the 2,15-decarboxylation processes at different dehydrogenation levels with 15-decarboxy-betanin and 2,15-bidecarboxy-betanin, structurally elucidated by NMR analysis, as the distinct indicators of this route type. For other novel pigments detected, 2,15-bidecarboxy-xanbetanin, 2,15-bidecarboxy-xanneobetanin and 2,15,17-tridecarboxy-neobetanin, additional high resolution mass spectrometric analyses were performed and confirmed their molecular formulas.

Structural Studies on Diverse Betacyanin Classes in Matured Pigment-Rich Fruits of Basella alba L. and Basella alba L. var. 'Rubra' (Malabar Spinach).

Identification of betacyanins in Basella alba L. and Basella alba L. var. 'Rubra' fruits was performed by low- and high-resolution mass spectrometry (LRMS and HRMS) as well as 1H, 13C and two-dimensional NMR which revealed hitherto completely not known betacyanin classes in the plant kingdom. Especially, the presence of unique nitrogenous acyl moieties in the structures of the pigments was ascertained by the HRMS Orbitrap detection. Except for detected polar betacyanin glycosylated derivatives, presence of a series of previously not reported pigments such as malonylated betanidin 6-O-ß-glusosides with their acyl migration isomers along with the evidence of the 3''-hydroxy-butyrylated betacyanins is reported. The first complete NMR data were obtained for novel and principal acylated gomphrenins with hydroxycinnamic acids: 6'-O-E-caffeoyl-gomphrenin (malabarin), 6'-O-E-sinapoyl-gomphrenin (gandolin), 6'-O-E-4-coumaroyl-gomphrenin (globosin) and 6'-O-E-feruloyl-gomphrenin (basellin).

Influence of Citrates and EDTA on Oxidation and Decarboxylation of Betacyanins in Red Beet (Beta vulgaris L.) Betalain-Rich Extract.

The influence of stabilizing activity of citric buffers on betacyanins, as well as their thermal dehydrogenation and decarboxylation in a beetroot betalain-rich extract (BRE), was studied at pH 3-8 and temperature 30, 50 and 85 °C with an additional effect of EDTA. In acetate/phosphate buffers, the highest stability is observed at pH 5 and it decreases toward pH 3 as well as pH 8, which is more remarkable at 85 °C. For the citrates, a contradictory effect was observed. Citric buffers tend to stabilize the substrate pigments and their intermediary products in acidic solutions, although increase their reactivity at pH 6-8. The highest impact of EDTA addition on pigment retention in acetate buffers is observed at 85 °C and pH 3-5 as well as 8, reflecting the preserving activity of EDTA at the most unfavorable conditions. At lower temperatures, pigment stability in more acidic conditions is still at higher levels even without addition of citrates or EDTA. The most striking effect on generation of betanin derivatives during heating is 2-decarboxylation which preferentially proceeds in the most acidic environment and this generation rate at 85 °C is much higher in the citrate buffers compared to acetates.

Acridine as an Anti-Tumour Agent: A Critical Review.

This review summarized the current breakthroughs in the chemistry of acridines as anti-cancer agents, including new structural and biologically active acridine attributes. Acridine derivatives are a class of compounds that are being extensively researched as potential anti-cancer drugs. Acridines are well-known for their high cytotoxic activity; however, their clinical application is restricted or even excluded as a result of side effects. The photocytotoxicity of propyl acridine acts against leukaemia cell lines, with C1748 being a promising anti-tumour drug against UDP-UGT's. CK0403 is reported in breast cancer treatment and is more potent than CK0402 against estrogen receptor-negative HER2. Acridine platinum (Pt) complexes have shown specificity on the evaluated DNA sequences; 9-anilinoacridine core, which intercalates DNA, and a methyl triazene DNA-methylating moiety were also studied. Acridine thiourea gold and acridinone derivatives act against cell lines such as MDA-MB-231, SK-BR-3, and MCF-7. Benzimidazole acridine compounds demonstrated cytotoxic activity against Dual Topo and PARP-1. Quinacrine, thiazacridine, and azacridine are reported as anti-cancer agents, which have been reported in the previous decade and were addressed in this review article.

Multi-colored shades of betalains: recent advances in betacyanin chemistry.

Covering: 2001 to 2021Betacyanins cover a class of remarkable natural red-violet plant pigments with prospective chemical and biological properties for wide-ranging applications in food, pharmaceuticals, and the cosmetic industry. Betacyanins, forming the betalain pigment group together with yellow betaxanthins, have gained much attention due to the increasing social awareness of the positive impact of natural products on human health. Betalains are commercially recognized as natural food colorants with preliminarily ascertained, but to be further investigated, health-promoting properties. In addition, they exhibit a remarkable structural diversity based on glycosylated and acylated varieties. The main research directions for natural plant pigments are focused on their structure elucidation, methods of their separation and analysis, biological activities, bioavailability, factors affecting their stability, industrial applications as a plant-based food, natural colorants, drugs, and cosmetics as well as methods for high-yield production and stabilization. This review covers period of the last two decades of betacyanin research. In the first part of the review, we present an updated classification of all known betacyanins and their derivatives identified by chemical means as well as by mass spectrometric and NMR techniques. In the second part, we review the current research reports focused on the chemical properties of the pigments (decarboxylation, oxidation, conjugation, and chlorination reactions as well as the acyl group migration phenomenon) and describe the semi-synthesis of natural and artificial fluorescent betalamic acid conjugates, showing various prospective research directions.

Fluorescence assay for the determination of glutathione based on a ring-fused 2-pyridone derivative in dietary supplements.

Herein, a novel fluorescent method for the determination of GSH levels in aqueous solutions involving the utilization of citric acid as a derivatization reagent was developed. Therefore, the crucial parameters of the derivatization process were established from what has resulted in the development of a sensitive, reproducible, and accurate GSH assay. The method was validated, and its applicability in the characterization of the GSH concentration in dietary supplements concerning the selectivity in the determination of GSH over GSSG was both confirmed. The chemical structure of the new fluorophore 3-[(carboxymethyl)carbamoyl]-5-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2-a]pyridine-7-carboxylic acid - CTPC was elucidated using detailed NMR: one-dimensional (1H, 13C), as well as two-dimensional NMR spectra (1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC, 1H-15N HSQC, 1H-15N HMBC) experiments. Besides, the essential optical, biological and antioxidative properties of CTPC were investigated.

High-speed countercurrent chromatography for isolation and enrichment of betacyanins from fresh and dried leaves of Atriplex hortensis L. var. "Rubra".

Betacyanins and their decarboxylated derivatives from fresh and dried edible leaves of Atriplex hortensis L. var. "Rubra" were fractionated for the first time by high-speed countercurrent chromatography. Pigments present in fresh leaf extract were separated in systems: ethanol - acetonitrile - n-propanol - ammonium sulphate - water (0.5:0.5:0.5:1.2:1.0, v/v/v/v/v) (tail-to-head mode) and tert-butyl methyl ether - n-butanol - acetonitrile - water with 0.7% heptafluorobutyric acid (2:2:1:5, v/v/v/v) (head-to-tail mode). The mobile phase flow rate was 2 mL/min and the retention of the stationary phase was 79.8 and 75.2%, respectively. Pigments from dried leaves were separated in a similar ion-pair system with heptafluorobutyric acid in different volume proportions 1:3:1:5 (head-to-tail mode) and the flow rate of the mobile phase 3 mL/min. The stationary phase retention was 64.0%. The application of the countercurrent chromatography for the fractionation of betacyanins from leaves of Atriplex hortensis enabled to isolate and pre-concentrate the pigments for further low- and high-resolution liquid chromatographic-tandem mass spectrometric detection. This study revealed the presence of 10 betacyanins in fresh and 16 in dried leaves of Atriplex hortensis. Two compounds were not previously identified in the whole Amaranthaceae family. Additionally, instead of (iso)amaranthin, celosianin and its epimer were dominant betacyanins in the Atriplex hortensis.

The Responses of Bioactive Betanin Pigment and Its Derivatives from a Red Beetroot (Beta vulgaris L.) Betalain-Rich Extract to Hypochlorous Acid.

Neutrophils produce hypochlorous acid (HOCl) as well as other reactive oxygen species as part of a natural innate immune response in the human body; however, excessive levels of HOCl can ultimately be detrimental to health. Recent reports suggest that betacyanin plant pigments can act as potent scavengers of inflammatory factors and are notably effective against HOCl. Comparison of the in vitro anti-hypochlorite activities of a novel betalain-rich red beetroot (Beta vulgaris L.) extract with its pure betalainic pigments revealed that the extract had the highest anti-hypochlorite activity, far exceeding the activity of all of the betalainic derivatives and selected reference antioxidants. This suggests that it may be an important food-based candidate for management of inflammatory conditions induced by excessive HOCl production. Among all pigments studied, betanidin exhibited the highest activity across the pH range.

Phytochemical Molecules from the Decarboxylation of Gomphrenins in Violet Gomphrena globosa L.-Floral Infusions from Functional Food.

Herein, the generation of decarboxylated derivatives of gomphrenin pigments exhibiting potential health-promoting properties and the kinetics of their extraction during tea brewing from the purple flowers of Gomphrena globosa L. in aqueous and aqueous citric acid solutions were investigated. Time-dependent concentration monitoring of natural gomphrenins and their tentative identification was carried out by LC-DAD-ESI-MS/MS. The high content of acylated gomphrenins and their principal decarboxylation products, 2-, 15-, 17-decarboxy-gomphrenins, along with minor levels of their bidecarboxylated derivatives, were reported in the infusions. The identification was supported by the determination of molecular formulas of the extracted pigments by liquid chromatography coupled with high-resolution mass spectrometry (LCMS-IT-TOF). The influence of plant matrix on gomphrenins' stability and generation of their derivatives, including the extraction kinetics, was determined by studying the concentration profiles in the primary and diluted infusions. Isolated and purified acylated gomphrenins from the same plant material were used for the preliminary determination of their decarboxylated derivatives. The acylated gomphrenins were found to be more stable than nonacylated ones. Citric acid addition had a degradative influence on natural gomphrenins mainly during the longer tea brewing process (above 15 min); however, the presence of plant matrix significantly increased the stability for betacyanins' identification.

Structural Study on Hypochlorous Acid-Mediated Chlorination of Betanin and Its Decarboxylated Derivatives from an Anti-Inflammatory Beta vulgaris L. Extract.

Hypochlorous acid (HOCl) produced by neutrophils is a part of the natural innate immune response system in the human body, but excessive levels of HOCl can ultimately be detrimental to health. Recent reports suggest that betacyanin plant pigments can act as potent scavengers of inflammatory factors and are notably effective against HOCl. In this contribution, chlorination mechanism and position of the electrophilic substitution in betacyanins was studied by high-resolution mass spectrometry and further structural analyses by NMR techniques, which completed the identification of the chlorinated betacyanins. For the study on the influence of the position of decarboxylation on the chlorination mechanism, a comparison of the chlorination position between betanin as well as 17-, and 2,17-decarboxylated betanins was performed. The structural study confirmed that the chlorination position in betanin occurs within the dihydropyridinic moiety at carbon C-18. Therefore, out of the aqueous free chlorine equilibrium species: HOCl, OCl-, Cl2, and Cl2O, the most potent chlorinating agents are HOCl and Cl2O postulated previously and the attack of the Cl⁺ ion on the carbon C-18 with a cyclic intermediate version is considered.

Separation of betacyanins from flowers of Amaranthus cruentus L. in a polar solvent system by high-speed counter-current chromatography.

Betacyanin extract of Amaranthus cruentus L. flowers was fractionated by semi-preparative high-speed counter-current chromatography in a highly polar solvent system: propan-1-ol/acetonitrile/(NH4 )2 SO4satd. soln /H2 O (1.0:0.5:1.2:1.0, v/v/v/v) in tail-to-head mode with 76% retention of the stationary phase. The crude extract as well as the fractions containing betacyanins were analyzed by liquid chromatography with tandem mass spectrometry as well as by high-resolution ion-trap time-of-flight mass spectrometry detection technique for the molecular formulae and multi-step fragmentation pattern elucidation. Four betacyanins; namely, amaranthin, betanin, 6'-O-formyl-amaranthin, and 6'-O-malonyl-amaranthin as well as their diastereomeric forms differing in the configuration of the C-15 carbon atom were identified in the fractions. Amaranthin was the dominant pigment in the extract and was additionally analyzed by nuclear magnetic resonance correlation techniques after the counter-current chromatographic and high-performance liquid chromatographic isolation. Betacyanins were highly enriched during a single high-speed counter-current chromatographic step; therefore, the tentative identification of new compounds for the whole Amaranthaceae family, 6'-O-formyl-amaranthin and 6'-O-malonyl-amaranthin was possible. Different elution profiles of the pigments observed in the counter-current chromatographic system in comparison to high-performance liquid chromatography system confirm a complementarity of both the techniques especially in the separation of diastereomeric pairs of betacyanins.

Fluorescence Quenching-Based Mechanism for Determination of Hypochlorite by Coumarin-Derived Sensors.

A fluorescence quenching-based mechanism for the determination of hypochlorite was proposed based on spectroscopic and chromatographic studies on the hypochlorite-sensing potency of three structurally similar and highly fluorescent coumarins. The mode of action was found to rely upon a chlorination of the coumarin-based probes resulting from their reaction with sodium hypochlorite. Importantly, the formation of chlorinated derivatives was accompanied by a linear decrease in the fluorescence intensities of the probes tested. The results obtained suggest the applicability of a coumarin-dependent hypochlorite recognition mechanism for the detection of, as well as for quantitative determination of, hypochlorite species in vitro.

Effect of Solvent Polarizability on the Keto/Enol Equilibrium of Selected Bioactive Molecules from the 1,3,4-Thiadiazole Group with a 2,4-Hydroxyphenyl Function.

Three novel 1,3,4-tiadiazole-derived compounds with biological-activity, i.e., 4-(5-(methylamino)-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (MDFT), 4-(5-(phenylamino)-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (PhATB), and 4-(5-(4-chlorophenylamino)-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (4-CIPhATB) were characterized with the use of several spectroscopic methods. Detailed UV-vis studies revealed keto/enol tautomerism of the examined compounds. The absorption spectra recorded in nonpolar solvents exhibited bands that were characteristic of keto tautomers, while in polar solvents the enol form is predominant. A number of spectra revealed the presence of both tautomeric forms in the solution. The keto/enol equilibria observed were both solvent- and temperature-dependent. The keto/enol equilibrium was also observed using FTIR spectroscopy. A detailed analysis of the spectroscopic data leads to a conclusion that the solvent-induced tautomerism of the selected compounds from the 1,3,4-thiadiazole group does not depend on the electric dipole moment of the solvent but more likely on its average electric polarizability. Additionally, a clear effect of the substituent present in the molecule on the tautomeric equilibrium in the selected 1,3,4-thiadiazole analogues was noted.

Chemical quenching of singlet oxygen by betanin.

Betanin is the best known natural dye belonging to the betacyanin family. In this work, efficient singlet oxygen quenching by betanin in deuterated water with the rate constant 1.20 ± 0.15 × 10(8) M(-1) s(-1) is reported, deduced from the (1)O2 phosphorescence decays measured as a function of betanin concentration. The quenching occurs by a chemical mechanism, as confirmed by the analysis of the transient absorption kinetics at the probe λ ∼ 535 nm, by comparison of the initial triplet signal amplitude of perinaphthenone acting as the (1)O2 photosensitizer with the final bleaching signal of betanin. The main betanin oxidation product is 2-decarboxy-2,3-dehydrobetanin, with its formation observed as the transient absorption signal at λ ∼ 445 nm. LC-MS/MS analysis of the photolyzed solutions supports the product identification as 2-decarboxy-2,3-dehydrobetanin, based on the molecular ion [M](+) observed at m/z 505. Isobetanin also undergoes a similar photooxidation reaction.

Time-resolved spectroscopy of the singlet excited state of betanin in aqueous and alcoholic solutions.

The photophysical properties of betanin in aqueous and alcoholic solutions were determined at room temperature using ultrafast UV-vis-NIR transient absorption spectroscopy (λexc = 535 nm). Its S1 → Sn (n > 1) absorption bands appear with maxima at about λ ∼ 450 and 1220 nm. The short betanin S1 state lifetime (6.4 ps in water) is mainly determined by the efficient S1 → S0 radiationless relaxation, probably requiring a strong change in geometry, since the S1 lifetime grows to 27 ps in the more viscous ethylene glycol. The fluorescence quantum yield is very low (Φf ∼ 0.0007 in water), therefore this deactivation path is of minor importance. Other processes, such as S1 → T1 intersystem crossing or photoproduct formation, are virtually absent, since full S0 ← S1 ground state recovery is observed within tens of picoseconds after photoexcitation. The observed fast light-to-heat conversion in the absence of triplet excited state formation supports the idea that betanin is a photoprotector in vivo.

Identification and reactivity of pigments in prominent vegetable leaves of Basella alba L. var. 'Rubra' (Malabar spinach).

The unique profiles of betacyanins as well as their stability and antioxidant activity in purple leaf extracts of the fast-growing, soft-stemmed vine Basella alba L. var. 'Rubra', known as Malabar spinach, are partly characterized for the first time. The distribution of gomphrenin and its acylated derivatives in the leaves is completely different from the profiles of the pigments in the fruits. The most abundant acylated pigment in leaves (24%) turned out 6'-O-E-sinapoyl-gomphrenin (gandolin), however, the most significant difference in the pigment profiles is a presence of two novel pigments tentatively identified as highly abundant 6'-O-(3,4-dimethoxy-E-cinnamoyl)-gomphrenin and 6'-O-(3,4,5-trimethoxy-E-cinnamoyl)-gomphrenin as well as their isoforms. Significant degradation of the pigments in the fruit extracts under the impact of selected metal cations and UV-Vis irradiation as well as high protective activity of the leaf extract matrix were observed. Partial chromatographic purification of the leaf extract resulted in an increase of the pigment concentration which was correlated positively with the increased antioxidant activity of obtained fractions.

Basella alba L. (Malabar Spinach) as an Abundant Source of Betacyanins: Identification, Stability, and Bioactivity Studies on Natural and Processed Fruit Pigments.

The antioxidant and anti-inflammatory activities of acylated and decarboxylated gomphrenins, as well as Basella alba L. fruit extract, were investigated in relation to gomphrenin, known for its high biological potential. The most abundant natural acylated gomphrenins, namely, 6'-O-E-caffeoyl-gomphrenin (malabarin) and 6'-O-E-4-coumaroyl-gomphrenin (globosin), were isolated from B. alba extract for the studies. In addition, controlled thermal decarboxylation of gomphrenin in the purified B. alba extract at 65-75 °C resulted in the formation of the most prevalent decarboxylated products, including 17-decarboxy-gomphrenin and 2,17-bidecarboxy-gomphrenin, along with their isoforms. The structures of the decarboxylated pigments were confirmed by NMR analyses. Exploring the matrix effect on pigment reactivity revealed a tremendous increase in the stability of all betacyanins after the initial stage of extract purification using a cation exchanger under various conditions. This indicates the removal of a substantial portion of the unfavorable matrix from the extract, which presumably contains reactive species that could otherwise degrade the pigments. Furthermore, the high concentration of citrates played a significant role in favoring the formation of 2-decarboxy-gomphrenin to a considerable extent. In vitro screening experiments revealed that the tested compounds demonstrated strong anti-inflammatory properties in lipopolysaccharide (LPS)-activated human macrophages. This effect encompassed the selective inhibition of cytokine and chemokine release from activated macrophages, modulation of the chemotactic activity of immune cells, and the regulation of tissue remodeling mediators' release.