Lea Lojková

Current trends in isolation, separation, determination and identification of isoflavones: A review

Isoflavones are natural substances which elicit a number of physiological effects in living organisms. The substances are synthesized in plant tissues as protective agents against biotic stress (i. e. bacterial infection). Isoflavones are also an important dietary constituent in human nutrition. Modern trends in studies of isoflavones in plant materials and foodstuffs and procedures for chemical analyses of isoflavones in human body fluids and plant tissues are discussed in this review. Highly effective extraction and purification techniques, i. e. solid-phase extraction, accelerated-solvent extraction, and Soxhlet extraction, are presented. Latest procedures in chromatographic separation of isoflavones that apply different types of sorbents are described. Immunochemical analysis, electrochemical sensing of isoflavones, and spectrometric and other analytical techniques and their applications are also mentioned. Special attention is paid to the highly selective and sensitive technique of mass spectrometry and its application for identification of isoflavones and their glucosides in plants. Studies of interactions of isoflavones with cell receptors and a number of biologically active substances such as DNA and proteins are described. The review does not intend to give a complete overview of the topics considered but rather to present modern and most recent methods used in studies of isoflavones.

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.

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.

Supercritical fluid extraction of amino acids from birch (Betula pendula Roth) leaves and their liquid chromatographic determination with fluorimetric detection

A method for supercritical fluid extraction (SFE) of amino acids was adapted and optimal experimental conditions were selected for a matrix consisting of dry leaves. The matrix-dependent SFE method uses a mixture of MeOH-H(2)O-acetonitrile (10:10:1 v/v/v) as a modifier (0.5 mL in situ, 300 muL on-line) at 70 degrees C and 40 MPa and no HCl is needed as an entrainer. The amino acids were quantified using high-performance liquid chromatography with fluorimetric detection (HPLC/FLD) after gradient elution on Zorbax Eclipse AAA columns (4.6x150 mm, 3.5 mum) with aqueous Na(2)HPO(4 )buffer of pH 7.8 and ACN-MeOH-water as a mobile phase. In comparison with Soxhlet extraction, SFE gave higher recovery and selectivity, but it required longer extraction time (90 min) and it was more labor-intensive (clean-up step after the pre-concentration). Both methods should be used separately or in combination according to the matrix, number of samples, and levels of ballast compounds.

Liquid chromatographic/electrospray mass spectrometric determination (LC/ESI-MS) of chelerythrine and dihydrochelerythrine in near-critical CO2 extracts from real and spiked plasma samples

Two polar benzo[c]phenanthridine alkaloids, chelerythrine (CHE) and dihydrochelerythrine (DHCHE), were extracted at 35 degrees C and 10MPa (15MPa for real samples) from real and spiked plasma samples with acceptable recoveries (95.1% and 81.0%, respectively) using near-critical CO(2) modified with aqueous (1:1, v/v) methanol. The alkaloids were quantified by a liquid chromatographic/electrospray mass spectrometric (LC/ESI-MS) method on a Zorbax SB-CN column (75mmx4.6mm, 3.5mum particle size) using methanol (organic phase) and 50mM ammonium formiate (aqueous phase) as a mobile phase. A linear gradient 0-1min, isocratic at 60% organic phase (v/v); from 1.0 to 7.0min, 60-71% organic phase (v/v); from 7.0 to 18.0min, 71-60% organic phase (v/v) was applied. The limit of detection was 1.22ng (3.50pmol) for CHE and 0.95ng (2.72pmol) for DHCHE per 1ml of the sample. The linearity of the calibration curves was satisfactory as indicated by coefficients of determination 0.9979 and 0.9995 for CHE and DHCHE, respectively. Repeatability and intermediate precision (average R.S.D.s) were 1.0-1.5%, accuracy was in the range 99.7-100.3%. Average recovery was 100.1% for both, standard solutions and spiked plasma extracts. Three samples of real rat plasma were extracted and analysed to test the method.

Supercritical fluid extraction as a tool for isolation of monoterpenes from coniferous needles and walnut-tree leaves

Several monoterpenes, i.e., (+)-α-pinene, (−)-camphene, sabinene, (−)-β-pinene, myrcene, R-(+)-limonene, (−)-bornylacetate, (−)-trans-caryophyllene and a-humulene were identified and determined by gas chromatography-mass spectrometry (GC-MS) in needles of Pinaceae (Picea abies, P. omorika, P. pungens, P. Breweriana, Pinus nigra, P. mungo turra, P. black, P. sylvestris, Abies pinsapo, A. holophylla, A. Bronmuelleris, A. alba, Larix Kaempferi L. decidua) and tree-leaves of Juglandaceae (Juglans regia, J. nigra, J. sieboldiana var. Cordiformis) families. Supercritical fluid extraction (SFE) was found to be very useful for their isolation at optimised conditions (needles/leaves: pressure 20/30 MPa, temperature 80/130°C, time of extraction 60/60 min, modifier chloroform/chloroform). Their seasonal distribution, evaluation of differences in concentrations and relative amounts in different trees and their varieties grown in different localities were evaluated.