The accumulation and species composition of anthocyanins in the flowers of several varieties of double-feathered cosmea (Cosmos bipinnatus) and in the flowers of red and orange coloration are more sulfur-yellow (C. sulphureus). And the level of accumulation of lutein diesters in the flowers of C. sulphureus of lemon yellow, yellow, orange and red colors is also determined. It was found that according to the level of anthocyanin accumulation, the flowers of C. bipinnatus of intensely red color are comparable to the fruits of black currant, and the species composition differs due to the different effectiveness in different varieties, primarily O-methyltransferase - from 15 to 60%, which ensures the transfer of cyanidin derivatives to peonidin derivatives. The effectiveness of rhamnosyltransferase in all studied cases is about 90 percent or more, which affects the fact that the main components of anthocyanins of flowers are cyanidin and peonidin rhamnosides. In the case of C. sulphureus the effectiveness of O-methyltransferase has not been detected, therefore cyanidin derivatives are synthesized in the complete absence of peonidin derivatives. But the peculiarity of the species is the biosynthesis of 3-deoxyanthocyanin - 4'-glucoside cosmonidin, which, along with xanthophylls, provides a yellow color due to a strong hypsochromic shift of the absorption maximum. The belonging of this substance to anthocyanins (as substances having a positive charge on the aglycone) is confirmed by an increase in retention under conditions of ion-pair reversed-phase HPLC with an increase in the concentration of sodium heptanesulfonate in the mobile phase. It was found that in all the studied samples of C. sulphureus flowers, lutein diesters are synthesized with a practically color-independent ratio of diester species, among which dimyristate is predominant. The level of accumulation of xanthophylls increases sharply during the transition from lemon-yellow to yellow flowers. The increase in the color of the petals from yellow to orange and red is accompanied by a relatively small increase in the accumulation level, reaching 0.5 g per 100 g of dry petals. Thus, the flowers of the plant are good sources of traditional anthocyanins, as dyes for the food and medical industry, as well as yellow water-soluble and fat-soluble dyes.

The chromatographic behavior (change in the logarithm of the retention factor with a change in the composition of the mobile phase) of dicaffeoylquinic (DCQ) acids is determined by the chromatographic behavior of monocaffeoylquinic acids. The most efficient combination is the conventional C18 stationary phase (Kromasil) and environmentally friendly mobile phases, in which acetonitrile is replaced by 2-propanol, with the mobile phase acidified with 0.25 vol % of phosphoric acid to stabilize the charge form of acids. The retention of dicaffeoylquinic acids can be explained by the simultaneous adsorption of caffeic acid substituents by two aromatic rings for both studied organic modifiers of the mobile phase. The replacement of the C18 phase by the phenylhexyl phase did not lead to significant changes in the selectivity of the separation of isomeric DCQ acids. At the same time, not all isomers were separated using mobile phases based on both organic modifiers: acetonitrile and 2-propanol. The developed conditions for the separation of DCQ in the gradient mode with “green” eluents A (10 vol % of isopropanol and 0.25 vol % of phosphoric acid in water) and B (20 vol % of isopropanol and 0.25 vol % of phosphoric acid in water) were used to determine the species composition of caffeoylquinic acids in coffee, mate, and artichoke tea.

В работе определен видовой состав антоцианов цветков гвоздики традиционной красной и необычных темно-красной и фиолетовой окрасок, а также определен вклад заместителей яблочной кислоты в общее удерживание антоцианов при разделении в условиях обращенно-фазовой ВЭЖХ с использованием диодно-матричного и масс-спектрометрического детектирования. Установлены основные компоненты экстрактов цветков двух сортов гвоздики садовой – алой и темно-красной окраски, – как 3-гликозиды пеларгонидина и цианидина (Pg3Glu и Cy3Glu), соответственно, в основном ацилированные яблочной кислотой в положении 6"-Pg3(6"MalylGlu) и Сy3(6"MalylGlu) При этом антоцианы гвоздики садовой третьего сорта (с фиолетовой окраской) были построены на иной основе и состояли в основном из производных цианидин-3,5-диглюкозида (Cy3,5diGlu), также главным образом ацилированных яблочной кислотой, но уже в иной форме, соединяющей оба глюкозидных радикала в цикл. Во всех случаях электронные спектры поглощения соединений лишь незначительно (на 0.5-1 нм) батохромно сдвигаются при ацилировании. Антоцианы сухих цветков сорта «Мираж» гвоздики турецкой состояли из производных Cy3Glu и Cy3,5diGlu, также ацилированных яблочной кислотой. Последовательность элюирования всех соединений не зависит от составов подвижных фаз системы «ацетонитрил-муравьиная кислота-вода», приемлемых для ВЭЖХ-анализа. Отмечено, что фактор удерживания, k(i), антоцианов увеличивается в 2.5-3.5 раза в зависимости от состава подвижной фазы при ацилировании яблочной кислотой в положение 6" Cy3Glu и Pg3Glu и только на 25-40% для Cy3,5diGlu, что позволяет анализировать состав антоцианов в изократическом режиме элюирования. Впервые сравниваются два варианта карт разделения как logk(i) vs ϕ (объемная доля CH3CN) с использованием квадратичной зависимости и как logk(i) vs logk(Cy3Glu). Показано, что экстраполяция удерживания на подвижные фазы с нулевым содержанием воды приводит к сходным результатам в обоих случаях.

By reverse-phase HPLC with diode-matrix and mass spectrometric detection the species composition of anthocyanins of cornflower petals (Centaureacyanus) of the three colors: blue, red and maroon was determined. For the first time, the anthocyanin composition of cornflower flower extract of dark maroon color, was found to include including the same anthocyanins as cornflower flowers of traditional blue color, but with another ratio. The main component is cyanidin-3-O-(6”-malonylglucoside)-5-glucoside (37-58 mol. %) at outstanding overall anthocyanin accumulation reaching 3.5 ±0.4 and even 9.5 ± 1.0 g per 100 g of fresh and dried plant material, correspondingly. Meanwhile the anthocyanin set of blue cornflowers includescyanidin-3,5-diglucoside (23-25 mol.%), cyanidin-3-O-(6”-malonylglucoside)-5-glucoside (15 – 17 mol.%), cyanidin-3-O-(6”-succinylglucoside)-5-glucoside (49 – 56 mol. % ) and cyanidin-3-glucoside (0.3-4.1 mol.%), which corresponds to the literature data, but additionally also isomeric malonated cyanidin-3,5-diglucoside (0.5 – 1.1 mol. % ) and cyanidin-3-O-(6”-malonylglucoside) (3.3 – 6.7 mol.%) were found. The anthocyanin composition of red flowers is formed by similar substituents of anthocyanins, built on a pelargonidinebachbone with a different ratio between the components: pelargonidin-3,5-diglucoside (25 – 36 mol. %), pelargonidin-3-O-(6”-malonylglucoside)-5-glucoside (37 – 47 mol. %), pelargonidin-3-O-(6”-succinylglucoside)-5-glucoside (7 – 31 mol. % ) aa well as two products with unknown malonation position of pelargonidin-3,5-diglucoside (in total up to 5 mol.%). It was found that the stationary phase of Kromasil 100-5C4 is more effective for separation of all components compared to the stationary phase of Symmetry C18 and hydrophilic chromatography withKromasil 60-5DIOL phase.

Определены закономерности статистической сорбции антоцианов на бентонитовой глине.Установлено, что метод, применимый в статическом варианте, является альтернативой методу динамической твердофазной очистки на концентрирующих патронах ДИАПАК С18.Предложен способ оценки относительной сорбционной способности антоцианов различного строения на бентонитовой глине в условиях выполнения уравнения Ленгмюра.Показано, что сорбционная способность уменьшается при усложнении строения гликозидных радикалов (от пентозида к гексозиду и далее -к ди

В работе методом обращенно-фазовой ВЭЖХ был определен набор антоцианов, ответствен-ных за окраску цветков популярного садового растения штокрозы (Altea roseum). 15 антоцианов тем-ноокрашенных цветков представлены 3-глюкозидами, 3-рутинозидами и 3-(6"-малонилглюкозидами) пяти агликонов – трех из дельфинидинового ряда (дельфинидин, петунидин и мальвидин) и двух (ци-анидин и пеонидин) из цианидинового ряда. Было обнаружено, что цветы накапливают в основном производные дельфинидинового или цианидинового рядов. Количественное соотношение в накопле-нии различных антоцианов в цветках зависит от сорта штокрозы; установлено, что общее количество антоцианов превышает 1 г на 100 г свежих лепестков для сортов с темной окраской цветков. Зависи-мость удерживания от структуры веществ исследовали специальными методами относительного ана-лиза удерживания на примере добавления рамнозильной части к 3-глюкозидам и ацилирования мало-новой кислотой тех же 3-глюкозидов. Показано, что изменение липофильности (как miLogP), обу-словленное усложнением структуры антоцианов, не коррелирует с изменением удерживания раство-ренного вещества, что, вероятно, связано со специфическим “поплавковым” механизмом удержива-ния антоцианов. Установлено, что изменение температуры разделения имеет большое значение для контроля селективности разделения антоцианов. И именно изменение температуры от 40 до 27.5оС позволило добиться разделения всех 15 антоцианов, в то время как градиентный режим элюирования в этом случае позволяет осуществить разделение менее чем за 20 мин.

Patterns of the static sorption of anthocyanins on bentonite clay are determined. It is found that the approach used for static sorption is an alternative to dynamic solid phase purification on DIAPAK C18 concentrating cartridges. A way of determining the relative sorption capacity of anthocyanins characterized by different structures on bentonite clay using the Langmuir equation is proposed. It is shown that the sorption capacity falls as the structure of the glycoside radicals becomes more complicated (from pentoside to hexoside and then to di- and trisaccharides). The acylation of glycoside radicals by malonic acid has a weak effect on sorptive activity, while acylation by substituted cinnamic acids improves sorption.

The anthocyanin composition of five purple leaves cultivars of Ocimum basilicum L. was investigated by reversed-phase HPLC with mass-spectrometric detection by ESI mode with ion partial fragmentation as well as preparation of dried differently colored forms of anthocyanins encapsulated into maltodextrin matrix. Analysis of the mass spectra revealed that according to the chromatographic profile the set of basil cultivar anthocyanins under investigation may be divided into two groups with the common feature being a high level of acylation with (mainly) p -coumaric, ferulic and malonic acids of the same base: cyanidin-3-dihexoside-5-hexoside. The presence of acylation with substituted cinnamic acids permits us to obtain solutions not only with a red color (the property of the flavylium form) but also with blue shades of coloration due to quinonoid and negatively charged quinonoid forms. All forms except that of flavylium are not stable in solution but stable enough to prepare dried encapsulated forms by lyophilization. Although the loss of anthocyanins with drying is not negligible, the final product is characterized with high stability for storage in a refrigerator.

Karapet’yants comparative analysis is applied to anthocyanin retention using the example of an extract of Adonis aestivalis petals. The attribution of anthocyanin peaks according to the comparative analysis is confirmed by the parameters of the mass spectra. A set of purple maize anthocyanins is used as a reference sample.

В работе методом обращенно-фазовой ВЭЖХ определен видовой состав антоциановых комплексов лепестков цветков нескольких сортов астры китайской Callistephus chinensis. Найдено, что в лепестках всех исследованных образцов антоцианы построены на трех агликонах: дельфинидине, цианидине и пеларгонидине. Установлено, что для большинства сортов характерен биосинтез либо 3-глюкозидов, либо 3,5-диглюкозидов этих антоцианидинов, включая ацилированные малоновой кислотой, хотя для одного из образцов получена сложная смесь, включающая оба типа гликозилирования. Показано, что среди малонированных антоцианов один является доминирующим; ему обычно сопутствуют (в небольших количествах) изомеры по положению ацилирования глюкозидных радикалов, а дималонированные производные не были обнаружены ни в одном из исследованных экстрактов.

В работе методом обращенно-фазовой ВЭЖХ с диодно-матричным и масс-спектроскопическим детектированиями исследован состав антоцианового комплекса плодов популярного местного сорта винограда неизвестной селекции. По появлению производных 3,5-диглюкозидов установлен гибридный характер данного сорта винограда. Показано, что вследствие сольватохромного эффекта количественная оценка видового состава по площадям пиков осложнена, однако при сопоставлении спектров, записанных при различном содержании в подвижной фазе ацетонитрила, найдена особая точка, при которой величина поглощения при СФ-детектировании не зависит от состава растворителя. Для выполнения анализа антоцианов винограда в изократических условиях предложен вариант флэш-хроматографии, позволяющий отделить неацилированные компоненты от ацилированных с использованием насадочных картриджей для твердофазной экстракции

The dependence of the degree of fragmentation of anthocyanin “molecular” ions on the cone voltage of a mass spectrometric electrospray ionization detector was studied. It was found that the voltage required for the fragmentation of 50% of original “molecular” ions, E f (0.5), increased with the number of glycoside residues. The fragmentation of glycosides proceeds with the removal of the entire residue regardless of their structures. In the case of 3,5-diglycosides, two types of fragment ions formed due to the loss of glycosidic residues from different positions; the ratio of their peak intensities is reciprocal to the ratio of the masses of residues eliminated. The values of E f (0.5) for monoglycosides (190 V), diglycosides (229 V), triglycosides (267 V), and for some acylated cyanidin-3,5-diglycosides are determined. These results were given for the gradient separation of anthocyanins by reversed-phase HPLC in aqueous acetonitrile containing 10 vol % of formic acid using quadrupole mass spectrometric detection.

Reversed-phase HPLC with mass spectrometric and diode array detection as well as some literature data were used to reveal the individual types of solutes in anthocyanin complexes of tulip flower petals that are responsible for tulip flower petals coloration of the samples available in the local flower market. It has been found that the main components of the complexes are 3-rutinosides and their 2”’ and 3”’ acylated with acetic acid derivatives of the three anthocyanidins - delphinidin, cyanidin and pelargonidin in the color dependent ratios, though trace quantities of 3-glucosides were found in some cases. For the anthocyanin structure confirmation a correlation analysis of solute retentions of cyanidin or pelargonidin derivatives vs that of delphinidin was proposed based upon equivalence of structures alteration in the solute pairs for each series. The specificity of solutes retention modes was revealed by relative retention analysis, the trend parameters reflected particularities of chromatographic behavior as well as that of electron spectra of the solutes. The difference of acylated anthocyanins retentions was proposed to disclose the conformation states of solutes in the sorbent interface.

Методом обращенно-фазовой ВЭЖХ c диодно-матричным, масс-спектрометрическим и амперометрическим детекторами исследованы комплексы антоцианов ряда сортов винограда с традиционным для Vitis vinifera составом, содержащие в качестве основных компонентов 3-глюкозиды пяти антоцианидинов – дельфинидина (Dp3G), цианидина (Cy3G), петунидина (Pt3G), пеонидина (Pn3G) и мальвидина (Mv3G). Разработана методика определения антиоксидантной активности индивидуальных антоцианов амперометрическим методом, выявлены особенности использования прибора Цвет Яуза-01-АА. Показано, что чувствительность амперометрического детектирования зависит не только от строения антоцианов, но и от напряжения на аноде. Предложена схема расчета антиоксидантной активности антоциановых комплексов по результатам амперометрического детектирования смеси индивидуальных антоцианов.

Anthocyan complexes of a series of grape varieties with the composition traditional for Vitis vinifera, containing 3-glucosides of five anthocyanidins, delphinidin (Dp3G), cyanidin (Cy3G), petunidin (Pt3G), peonidin (Pn3G), and malvidin (Mv3G) as the major components have been studied by reversed-phase HPLC with diode-array, mass spectrometric, and amperometric detectors. A procedure has been developed for the determination of the antioxidant activity of individual anthocyans using an amperometric method; the peculiarities of using a Tsvet Yauza-01-AA system have been evaluated. It has been shown that the sensitivity of amperometric detection depends not only on the structure of anthocyans, but also on the anode voltage. A scheme of the calculation of the antioxidant activity of anthocyan complexes by the results of amperometric detection of a mixture of individual anthocyans has been proposed.

By means of RP HPLC with spectrophotometric and mass-spectrometric detection the anthocyaninpatterns of red colored currant varieties fruits harvested in Kursk have been determined. The separation mapof all main components in eluent system of «acetonitrile – water - 10 vol. % of HCOOH» was built revealingpossibilities of the system to determine the individual compounds. Particularities of solute differentiation bymeans of relative retention parameters, electron spectra as well as mass-spectra for ESI-mode are discussed.All the solutes were found to be composed by a single aglicon – cyanidin; the full spectrum of derivativeswas described by cyanidine-3-glucoside transformation under activity of three specific enzymes giving rise tothree types of anthocyanin complexes. The first type includes cultivars with all of six possible glycosideswitch are synthesized by RT (6”-rhamnosyltransferase), 2”XT (2”-xylosyltransferase) and 2”GT (2”-glucosyltransferase). For the second type of complexes a loss of 2”GT is a characteristic fixture, while thethird type has a simplest composition due to activity of the lonely RT. The Viksne variety had a largestanthocyanin accumulation (above 140 mg per 100 g of fresh fruits).

A float mechanism of retention in reversed-phase HPLC is proposed as an alternative to the known mechanisms of the distribution and hydrophobic expulsion of sorbate to the surface of a sorbent. Experimental data that the sorption of a flavylium structure is poorly sensitive to the position of OH groups, and that the retention of anthocyanins depends on the length of bonded alkyl radicals of reversed phase, form the basis of the proposed hypothesis. It is noted that the retention of anthocyanins depends on the orientation of hydroxyl groups in carbohydrate radicals, due to which the chromatographic behavior of anthocyanins is different for glucosides and galactosides, for arabinosides and xylosides, and so on. In other words, retention is a reliable indicator of the composition of a carbohydrate fragment. It is concluded that carbohydrate radicals serve as unique floats, while flat flavilic ions penetrate into the bonded phase. The existence of floats is the main reason for the lower efficiency (of the number of theoretical plates) of the peaks of anthocyanins. It is shown that if two carbohydrate radicals are present at different sites of aglycone (a two-float sorbate), the peaks of the substance are characterized by substantial additional broadening.