Analysis of Softwood Lignans by Comprehensive Two-Dimensional Liquid Chromatography.

Lignans constitute a large group of phenolic plant secondary metabolites possessing high bioactivity. Their accurate determination in plant extracts with a complex chemical composition is challenging and requires advanced separation techniques. In the present study, a new approach to the determination of lignans in coniferous knotwood extracts as the promising industrial-scale source of such compounds based on comprehensive two-dimensional liquid chromatography separation and UV spectrophotometric detection is proposed. First and second-dimension column screening showed that the best results can be obtained using a combination of non-polar and polar hydroxy group embedded octadecyl stationary phases with moderate (~40%) "orthogonality". The optimization of LC × LC separation conditions allowed for the development of a new method for the quantification of the five lignans (secoisolariciresinol, matairesinol, pinoresinol, 7-hydroxymatairesinol, and nortrachelogenin) in knotwood extracts with limits of quantification in the range of 0.27-0.95 mg L-1 and a linear concentration range covering at least two orders of magnitude. Testing the developed method on coniferous (larch, fir, spruce, and pine) knotwood extracts demonstrated the high selectivity of the analysis and the advantages of LC × LC in the separation and accurate quantification of the compounds co-eluting in one-dimensional HPLC.

Comprehensive Characterization of Secondary Metabolites in Fruits and Leaves of Cloudberry (Rubus chamaemorus L.).

Cloudberry (Rubus chamaemorus L.) is a circumpolar boreal plant rich in bioactive compounds and is widely used in food and in folk medicine. In this study, a combination of two-dimensional NMR spectroscopy and liquid chromatography-high-resolution mass spectrometry was used for the comprehensive characterization of secondary metabolites in cloudberry lipophilic and hydrophilic extracts. Special attention was paid to the leaf extractives, which are highly enriched in polyphenolic compounds, the content of which reaches 19% in the extract (in gallic acid equivalent). The chemical composition of the polyphenolic fraction is represented mainly by the glycosylated derivatives of flavonoids, hydroxycinnamic (primarily caffeic), gallic (including the structure of galloyl ascorbate) and ellagic acids, catechin, and procyanidins. The contents of aglycones in the polyphenolic fraction were 64 and 100 mg g-1 for flavonoids and hydroxycinnamic acids, respectively, while the content of free caffeic acid was 1.2 mg g-1. This determines the exceptionally high antioxidant activity of this fraction (750 mg g-1 in gallic acid equivalent) and the ability to scavenge superoxide anion radicals, which is 60% higher than that of Trolox. The lower polar fractions consist mainly of glycolipids, which include polyunsaturated linolenic acid (18:3), pentacyclic triterpenic acids, carotenoid lutein, and chlorophyll derivatives, among which pheophytin a dominates. Along with the availability, the high antioxidant and biological activities of cloudberry leaf extracts make them a promising source of food additives, cosmetics, and pharmaceuticals.

New Oligomeric Dihydrochalcones in the Moss Polytrichum commune: Identification, Isolation, and Antioxidant Activity.

One of the most widespread representatives of mosses in the temperate and boreal latitudes of the Northern Hemisphere is common haircap (Polytrichum commune), which is known as the largest moss in the world and widely used in traditional herbal medicine. Polyphenolic compounds constitute one of the most important groups of biologically active secondary metabolites of P. commune, however, the available information on their chemical composition is still incomplete and contradictory. In the present study, a group of dihydrochalcone polyphenolic derivatives that were not previously found in mosses was isolated from P. commune biomass using pressurized liquid extraction with aqueous acetone. The combination of two-dimensional NMR spectroscopy and high-performance liquid chromatography-high-resolution mass spectrometry allowed for identifying them as 3-hydroxyphloretin oligomers formed through a carbon-carbon bond between phloroglucinol and pyrocatechol moieties ("head-to-tail" coupling), with a polymerization degree of 2-5. The individual compounds isolated by preparative reverse-phase HPLC had a purity of 71 to 97% and demonstrated high radical scavenging activity (17.5-42.5% with respect to Trolox) determined by the photochemiluminescence method. Along with the low toxicity predicted by QSAR/QSTR algorithms, this makes 3-hydroxyphloretin oligomers a promising source for the production of biologically active food additives and pharmaceuticals.

Supercritical Fluid Chromatography-Tandem Mass Spectrometry for Rapid Quantification of Pentacyclic Triterpenoids in Plant Extracts.

Pentacyclic triterpenoids (PCTs) are a widely distributed class of plant secondary metabolites. These compounds have high bioactive properties, primarily antitumor and antioxidant activity. In this study, a method was developed for the quantitative analysis of pentacyclic triterpenoids in plants using supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS). Separation of ten major PCTs (friedelin, lupeol, ß-amyrin, α-amyrin, betulin, erythrodiol, uvaol, betulinic, oleanolic and ursolic acids) was studied on six silica-based reversed stationary phases. The best results (7 min analysis time in isocratic elution mode) were achieved on an HSS C18 SB stationary phase using carbon dioxide-isopropanol (8%) mobile phase providing decisive contribution of polar interactions to the retention of analytes. It was shown that the use of atmospheric pressure chemical ionization (APCI) is preferred over atmospheric pressure photoionization (APPI). The combination of SFC with APCI-MS/MS mass spectrometry made it possible to achieve the limits of quantification in plant extracts in the range of 2.3-20 µg·L-1. The developed method was validated and tested in the analyses of birch outer layer (Betula pendula) bark, and licorice (Glycyrrhiza glabra) root, as well as lingonberry (Vaccinium vitis-idaea), cranberry (Vaccinium oxycoccos), apple (Malus domestica "Golden Delicious" and Malus domestica "Red Delicious") peels.

Rapid simultaneous determination of pentacyclic triterpenoids by mixed-mode liquid chromatography-tandem mass spectrometry.

Pentacyclic triterpenoids (PCTs) possess high biological activity, including antitumor, anti-inflammatory, antiviral and hepatoprotective properties and are widespread in a plant biomass. Due to significant differences in polarity and other physicochemical properties, the simultaneous determination of different classes of PCTs by the methods of reversed phase liquid chromatography is difficult. In the present study, we proposed a new approach to chromatographic separation of such compounds based on the use of a stationary phase with a mixed retention mechanism combining hydrophobic, weak anion exchange and hydrophilic interactions. The use of the Acclaim Mixed-Mode WAX-1 column and tuning the selectivity by changing the contributions of different types of analyte-stationary phase interactions allowed the separation of 10 PCTs (betulin, erythrodiol, uvaol, friedelin, lupeol, ß-amyrin, α-amyrin, betulinic, oleanolic and ursolic acids) belonging to four different classes (monools, diols, ketones and triterpenic acids) during 7.5 min in isocratic elution mode. The combination of this approach with atmospheric pressure chemical ionization tandem mass spectrometric detection and pressurized liquid extraction of analytes with methanol allowed to develop a rapid, accurate and highly sensitive method for analyzing PCTs in plant tissues with a total duration of the analytical cycle (including sample preparation steps) of not more than 40 min. It provides the detection limits in plant biomass extracts of 3-12 µg L-1 (44 µg L-1 for friedelin). The developed method was validated and successfully tested in the analyses of real birch bark and lingonberry peels.

Negative ion mode atmospheric pressure ionization methods in lignin mass spectrometry: A comparative study.

RATIONALE: Mass spectrometry with atmospheric pressure ionization is the most promising method for studying the structure of natural lignin, which is the second most abundant biopolymer in nature. The goal of this study is to compare the efficiency and characteristics of different types of ionization techniques (ESI, APCI, and APPI) in the negative ion mode by the example of softwood lignin. METHODS: As the subjects of the study, we selected a preparation of spruce dioxane lignin and several phenols, simulating the basic structural fragments of the lignin macromolecule. High-resolution mass spectra were recorded using an Orbitrap mass spectrometer. Acetone was used as a solvent for samples and a dopant in photoionization mode. The ionization conditions were optimized to achieve the maximum intensity of the mass spectra. RESULTS: The formation of deprotonated lignin molecules is characteristic of all the studied types of ionization; partial fragmentation of the biopolymer occurs in all ionization modes. ESI in the presence of ammonia yields low-intensity signals, leads to a significant decrease in ionization efficiency with increasing molecular weight of lignin oligomers, gives high-intensity impurity peaks in the mass spectra, and demonstrates selectivity for more polar structures. The ionization efficiency increases sharply in the order of ESI < APCI < APPI. The two latter methods are characterized by similar mechanisms of ionization; they ensure detection of approximately 1900 spruce lignin oligomers in the range of molecular weights up to 1.8 kDa. The determination of the elemental composition of oligolignols enabled the four main groups of compounds to be distinguished. CONCLUSIONS: Photoionization using acetone as a dopant is distinguished by a significantly higher intensity of signals and the lowest sensitivity to contaminants present in the lignin preparation. This ionization method can be considered as preferred for studying the dioxane lignin preparations of woody plants. Copyright © 2016 John Wiley & Sons, Ltd.