Vlastimil Kubáň

Identification of isoflavone conjugates in red clover (Trifolium pratense) by liquid chromatography-mass spectrometry after two-dimensional solid-phase extraction

A high-performance liquid chromatography–mass spectrometry (LC–MS) combined with a two-dimensional solid-phase extraction (2D-SPE) enabled the identification of fourteen isoflavone glycoside malonates (daidzin-6″-O-malonate, genistin-6″-O-malonate, orobol-7-O-β-d-glucoside-6″-O-malonate, 3-methylorobol-7-O-β-d-glucoside-6″-O-malonate, calycosin-7-O-β-d-glucoside-6″-O-malonate, pratensein-7-O-β-d-glucoside-6″-O-malonate, pseudobaptigenin-7-O-β-d-glucoside-6″-O-malonate, formononetin-7-O-β-d-glucoside-6″-O-malonate, irilone-4′-O-β-d-glucoside-6″-O-malonate, afrormosin-7-O-β-d-glucoside-6″-O-malonate, biochanin A-7-O-β-d-glucoside-6″-O-malonate, texasin-7-O-β-d-glucoside-6″-O-malonate, 5,7,2′-trihydroxy-6-methoxyisoflavone-7-O-β-d-glucoside-6″-O-malonate, prunetin-4′-O-β-d-glucoside-6″-O-malonate) and six acetyl glycosides (daidzin-6″-O-acetate, formononetin-7-O-β-d-glucoside-6″-O-acetate, pseudobaptigenin-7-O-β-d-glucoside-6″-O-acetate, irilone-4′-O-β-d-glucoside-6″-O-acetate, biochanin A-7-O-β-d-glucoside-6″-O-acetate and prunetin-4′-O-β-d-glucoside-6″-O-acetate) isolated from red clover (Trifolium pratense). Eight isoflavone glycoside malonates and six acetyl glycosides were firstly identified in red clover. The analyses of crude extracts with those using 2D-SPE extracts were compared. UV spectra, mass spectra of protonated molecules and their fragmentation and subsequent conversion to known flavonoid glycosides were the basis for the identification of the substances.

Application of a contactless conductometric detector for the simultaneous determination of small anions and cations by capillary electrophoresis with dual-opposite end injection.

A contactless conductometric detection (CCD) system for capillary electrophoresis (CE) with a flexible detection cell was applied for the simultaneous determination of small anions and/or cations in rain, surface and drainage water samples. The applied frequency, the amplitude of the input signal, the electrolyte conductivity and electrode distance were found to be the most significant factors affecting the detection sensitivity. 2-(N-Morpholino)ethanesulfonic acid/histidine-based (MES/His) electrolytes were used for direct conductivity detection of anions and cations, while ammonium acetate was selected for indirect conductivity determination of alkylammonium salts. For the simultaneous separation procedure, involving dual-opposite end injection, an electrolyte consisting of 20 mM MES/His, 1.5 mM 18-crown-6 and 20 microM cetyltrimethylammonium bromide provided baseline separation of 13 anions and cations in less than 6 min. The detection limits achieved were 7-30 micrograms/l for direct conductometric detection of various common inorganic cations and anions, excluding F- (62 micrograms/l) and H2PO4- (250 micrograms/l), and 35-178 micrograms/l for indirect conductometric detection of alkyl ammonium cations. The developed electrophoretic method with conductometric detection was compared to ion chromatography.

Simultaneous determination of inorganic and organic anions, alkali, alkaline earth and transition metal cations by capillary electrophoresis with contactless conductometric detection.

Simultaneous separation of up to 22 inorganic and organic anions, alkali, alkaline earth and transition metal cations was achieved in less than 3 min in the capillary electrophoresis system with contactless conductometric detector. The sample was injected from both capillary ends (dual opposite end injection) and anionic and cationic species were detected in the center of the separation capillary. The parameters of the separation electrolyte, such as pH, concentration of the electrolyte, concentration of complexing agents and concentration of 18‐crown‐6 were studied. Best results were achieved with electrolytes consisting of 8 mM L‐histidine, 2.8 mM 2‐hydroxyisobutyric acid, 0.32 mM 18‐crown‐6 at pH 4.25 or 9 mM L‐histidine, 4.6 mM lactic acid, 0.38 mM 18‐crown‐6 at pH 4.25. Other electrolytes containing complexing agents such as malic or tartaric acid at various concentrations could also be used. The detection limits achieved for most cations and anions were 7.5 – 62 νgL−1 except for Ba2+ (90 νgL−1), Cd2+, Cr3+ and F− (125 νgL−1), and fumarate (250 νgL−1). The repeatability of migration times and peak areas was better than 0.4% and 5.9%, respectively. The developed method was applied for analysis of real samples, such as tap, rain, drainage and surface water samples, plant exudates, plant extracts and ore leachates.

Ultrahigh-pressure liquid chromatography of isoflavones and phenolic acids on different stationary phases

Complete separation of aglycones and glucosides of selected isoflavones (genistin, genistein, daidzin, daidzein, glycitin, glycitein, ononin, sissotrin, formononetin, and biochanin A) was possible in 1.5 min using an ultrahigh-pressure liquid chromatography (U-HPLC) on a different particular chemically modified stationary phases with a particle size under 2 microm. In addition, selected separation conditions for simultaneous determination of isoflavones together with a group of phenolic acids (gallic, protocatechuic, p-hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, and sinapic acid) allowed separation of all 19 compounds in 1.9 min. Separations were conducted on a non-polar reversed phase (C(18)) and also on more polar phases with cyanopropyl or phenyl groups using a gradient elution with a mobile phase consisting of 0.3% aqueous acetic acid and methanol. Chromatographic peaks were characterised using parameters such as resolution, symmetry, selectivity, etc. Individual substances were identified and quantified using UV-vis diode array detector at wavelength 270 nm. Limits of detection (3S/N) were in the range 200-400 pg ml(-1). Proposed U-HPLC technique was used for separation of isoflavones and phenolic acids in samples of plant materials (Trifolium pratense, Glycine max, Pisum sativum and Ononis spinosa) after acid hydrolysis of the samples and modified Soxhlet extraction.

Reversed-phase high-performance liquid chromatographic determination of isoflavones in plant materials after isolation by solid-phase extraction

Abstract A fast, sensitive (LODs ∼9–17.6 n M ) and precise (RSDs ≤8%) method combining solid-phase extraction (SPE) and RP-HPLC for isolation, purification as well as qualitative and quantitative determination of isoflavones (0.06–520 μ M ) in plant materials is described. Plant extracts were purified and isoflavones isolated on various SPE columns. Classical sorbents were compared with new polymer sorbents as well as the wet/dry ways of extraction. Extraction efficiencies ( R >90%) for determination of isoflavones (daidzein, genistein, formononetin and biochanin A) were evaluated and optimized on an Spe-ed ABN cartridge. Eluates were analyzed by RP-HPLC on C 18 bonded silica using an acetonitrile–0.1% TFA gradient and UV detection at 280 nm. The method can be used for fast determination of isoflavones in plants, foods and other biological materials.

A hydrophilic interaction chromatography coupled to a mass spectrometry for the determination of glutathione in plant somatic embryos

An electrospray ionization mass spectrometric (ESI-MS) determination of glutathione (GSH), a sulfur-containing tripeptide (gamma-Glu-Cys-Gly) with regulation and detoxication functions in metabolisms of most living organisms, from nanomolar to micromolar levels is described. A hydrophilic interaction chromatography (HILIC) with an isocratic elution using a mobile phase containing acetonitrile and an aqueous 0.00005% solution of trifluoroacetic acid (60/40%, v/v) was applied for the separation of GSH. The peptide detection was achieved in the presence of L-ascorbic acid which significantly enhanced the signal intensity of the molecular ion GSH [M+H]+ (m/z 308). The calibration curve was linear (R2=0.9995) in the concentration range from 2 nM to 10 microM with a detection limit (LOD, S/N=3) of 0.5 nM. The excellent detection limit, and the excellent selectivity and high reproducibility of this method enabled determination of GSH in a single plant somatic embryo of a Norway spruce (Picea abies). The average amount of GSH in the single somatic embryos (n=18) was 9 pmol per embryo. Owing to our results, it can be supposed that the proposed HILIC/ESI-MS analysis might be used for GSH determination in microscopic cell structures and in single cell analyses.

Supercritical fluid extraction (SFE) of 4(5)-methylimidazole (4-MeI) and 2-acetyl-4(5)-(1,2,3,4)-tetrahydroxybutyl-imidazole (THI) from ground-coffee with high-performance liquid chromatographic-electrospray mass spectrometric quantification (HPLC/ESI-MS)

Two polar analytes, 4(5)-methylimidazole (4-MeI) and 2-acetyl-4(5)-(1,2,3,4)-tetrahydroxybutyl-imidazole (THI), were extracted with supercritical carbon dioxide (CO2) modified with aqueous methanol. The method was applied to a roasted coffee powder with good recovery rates. Method efficiency was compared with that of solid-phase extraction using SCX Disc cartridges and validated for spiked solid matrix. The analytes were determined using isocratic liquid chromatography-mass spectrometry (LC/MS) on an Atlantis HILIC Silica column (150 × 2.1 mm, 3 µm) with 80% methanol and 20% 0.01 mol l−1 ammonium formate as the mobile phase. The limit of quantification was around 1.5 pg for 4-MeI and 2.0 pg for THI. The linearity of the calibration curves was satisfactory as indicated by correlation coefficients of >0.999. The coefficient of variation for the intra-day and inter-day precisions was <4% (n = 6). Accuracy was in the range 98–101%; recovery rates were ≥ 98 and ≥ 99% for THI and 4-MeI, respectively. Several samples of Arabica coffee from various locations and commercially available ‘off-the-shelf’ coffee products (Arabica/Robusta mixtures) were analysed to test the method.

Enhancing sensitivity of liquid chromatographic/ion-trap tandem mass spectrometric determination of estrogens by on-line pre-column derivatization.

A rapid and sensitive liquid chromatographic/ion-trap mass spectrometric method has been developed for the simultaneous determination of estriol, alpha-, beta-estradiol, 17alpha-ethynylestradiol and estrone. The substances were extracted from river water samples (typically 250 ml) and cleaned-up using two-stage solid-phase extraction (SPE). 12-(Difluoro-1,3,5-triazinyl)-benz[f]isoindolo-[1,2b][1,3]benzothiazolidine was applied as a derivatizing agent. The recovery for each compounds ranged from 75 to 88% and the repeatability represented as RSDs ranged from 5.6 to 8.4%. Limits of detection (LOD, 3 x S/N) were 67-285 pg/l.

Capillary electrochromatography of inorganic cations in open tubular columns with a controllable capacity multilayered stationary phase architecture

In this paper capillary electrochromatography of alkali and alkaline-earth metal cations in open tubular capillary columns is described. Capillary columns are prepared by coating fused silica capillaries of 75 microm I.D. with poly(butadiene-maleic acid) copolymer (PBMA) in multiple layers. Thermally initiated radical polymerization is used to crosslink the stationary phase. Capillary columns with different number of stationary phase layers can be prepared and allow for the adjustment of separation selectivity in the electrochromatographic mode. Fast and sensitive separations of common inorganic cations are achieved in less than 6 min in a 60 cm capillary column with on-column capacitively coupled contactless conductivity detector. Limits of detection (S/N=3) for the determination of alkali and alkaline-earth metal cations range from 0.3 to 2.5 microM and repeatability is better than 0.5, 4.5 and 6.1% for migration times, peak heights and peak areas, respectively.

Determination of octan-1-ol-water partition coefficients by flow-injection extraction without phase separation

Abstract Single-channel coaxial segmenters were used for the introduction of an aqueous or octan-1-ol solution of an organic substance directly into a continuous flow of the other solvent. The analytical signal was measured simultaneously on both aqueous and organic phase segments by an “on-tube” fast-reading spectrophotometric detector (ca. 3 ms time resolution) and treated mathematically. The octan-1-ol-water phase signal ratio corresponds to the partition coefficient of the organic substances. The applicability of the method is demonstrated by the determination of partition coefficients of phenol, citric acid, acetylsalicylic acid and sodium salicylate.