Hyphenated technique for the extraction and determination of isoflavones in algae: ultrasound-assisted supercritical fluid extraction followed by fast chromatography with tandem mass spectrometry.

New hyphenated technique for the extraction and determination of isoflavones in sea and freshwater algae and cyanobacteria was developed. The method consists of sonication sample pretreatment, extraction by supercritical CO(2) modified by 3% (v/v) of MeOH/H(2)O mixture (9:1, v/v) at 35 MPa and 40°C for 60 min, fast chromatography analysis by the means of Agilent 1200 Series Rapid Resolution and MS/MS determination. Agilent 1200 Series RRLC was used with Zorbax SB-CN chromatographic column (100 mm × 2.1mm, particle size 3.5 µm), 3µl injection volume, mobile phase consisting of 0.2% (v/v) acetic acid in water (solvent A) and acetonitrile (solvent B) and used with linear gradient (30% B at 0 min, from 0 min to 3 min up to 50% B, from 3 to 6 min up to 80% B and from 6 to 10 min down to 30% B). The flow-rate was 0.4 mL/min, column oven temperature 35°C. MS detector Agilent Technologies 6460 Triple quadrupole LC/MS with Agilent Jet Stream was used in a negative ESI mode under following conditions: gas temperature 350°C, gas flow 13 L/min, nebulizer gas pressure 50 psi, sheath gas temperature 400°C, sheath gas flow 12L/min, capillary voltage was 4 kV. Samples were analysed in the multiple reaction monitoring (MRM) mode. Eight isoflavone compounds were found for the first time in seven real samples of sea algae and in three control samples of freshwater algae and cyanobacteria. Usual optimisation study of extraction parameters was performed. Pressure and temperature optima for algae matrix are different from those obtained sooner for other matrices for most of the analytes, but the results of modifier optimisation study are in good accordance with those obtained sooner for spiked samples and red clover matrix. It seems that matrix has very small or no effect on the modifier selection. Two different approaches of sonication pretreatment were tested: sonication bath and the thorn instrument. In longer extraction time experiments, thorn sonication was more efficient and recovery of following supercritical fluid extraction was higher.

Bioactive phenols in algae: the application of pressurized-liquid and solid-phase extraction techniques.

A new extraction technique based on the off-line combination of pressurized-liquid with solid-phase extraction (PLE-SPE) is described. The method was used for the extraction of bioactive phenolic acids (protocatechuic, p-hydroxybenzoic, 2,3-dihydroxybenzoic, chlorogenic, vanillic, caffeic, p-coumaric, salicylic acid), cinnamic acid and hydroxybenzaldehydes (p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, vanillin) from in vitro culture of two freshwater algae (Anabaena doliolum and Spongiochloris spongiosa) and from food products of marine macroalgae Porphyra tenera (nori) and Undaria pinnatifida (wakame). For the identification and quantification of the compounds the molecular ions [M-H](-) and specific fragments were analyzed by quadrupole mass spectrometry analyzer connected on-line with a reversed-phase HPLC system. Our analysis showed that the freshwater algae and marine algal products contained submicrogram or microgram level of above-mentioned phenols per gram of lyophilized sample. In addition, the total phenol content (Folin-Ciocalteu assay) and antioxidant activity (TEAC assay, Trolox equivalent antioxidant capacity assay) of the PLE-SPE extracts were determined and discussed.

Solid-phase/supercritical-fluid extraction for liquid chromatography of phenolic compounds in freshwater microalgae and selected cyanobacterial species.

In the present paper a new extraction technique based on the combination of solid-phase/supercritical-fluid extraction (SPE/SFE) with subsequent reversed-phase HPLC is described. The SPE/SFE extractor was originally constructed from SPE-cartridge incorporated into the SFE extraction cell. Selected groups of benzoic acid derivatives (p-hydroxybenzoic, protocatechuic, gallic, vanillic and syringic acid), hydroxybenzaldehydes (4-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde) and cinnamic acid derivatives (o-coumaric, p-coumaric, caffeic, ferulic, sinapic and chlorogenic acid) were extracted. Cyclic addition of binary extraction solvent system based on methanol:water (1:1, v/v) and methanol/ammonia aqueous solution was used for extraction at 40MPa and 80 degrees C. The p-hydroxybenzoic, protocatechuic, vanillic, syringic, caffeic and chlorogenic acid; 4-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde were identified by HPLC-electrospray mass spectrometry in SPE/SFE extracts of acid hydrolyzates of microalga (Spongiochloris spongiosa) and cyanobacterial strains (Spirulina platensis, Anabaena doliolum, Nostoc sp., and Cylindrospermum sp.). For the identification and quantification of the compounds the quasi-molecular ions [M-H](-) and specific fragments were analysed by quadrupole mass spectrometry analyzer. Our analysis showed that the microalgae and cyanobacteria usually contained phenolic acids or aldehydes at microg levels per gram of lyophilized sample. The proposed SPE/SFE extraction method would be useful for the analysis of different plant species containing trace amount of polar fraction of phenols.

[Modern instrumentation for the studies of isoflavones]. / Moderní instrumentální metody studia isoflavonu.

Isoflavones belong to the natural substances exhibiting 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. The review discusses modern trends in the studies of isoflavones in plant materials and foodstuffs and procedures for chemical analyses of isoflavones in human body fluids and plant tissues. Highly effective extraction and purification techniques, i.e. solid-phase extraction (SFE), accelerated-solvent extraction (ASE), and Soxhlet extraction, are presented. Latest procedures in chromatographic separation of isoflavones that apply different types of stationary phases are described. Immunochemical analysis, electrochemical sensing of isoflavones, spectrometric and other analytical techniques and their applications are also mentioned. Special attention is focused on a 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 reason for the presentation of the review was not to give a full overview of the presented topics but mainly to show modern and the most recent methods in the 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.

Rapid-resolution HPLC with spectrometric detection for the determination and identification of isoflavones in soy preparations and plant extracts.

A rapid-resolution HPLC/UV-VIS DAD separation method (which takes <1 min) for the determination and identification of genistin, genistein, daidzein, daidzin, glycitin, glycitein, ononin, formononetin, sissotrin and biochanin A in fmol quantities in submicroliter sample volumes was optimized. A linear gradient elution (0 min 22% B, 1.0 min 80% B, 1.4 min 100% B, 1.8 min 22% B) using a mobile phase containing 0.2 % (v/v) acetic acid (solvent A) and methanol (solvent B) was applied on a Zorbax SB C18 column (1.8 microm particle size) at 80 degrees C. The method was verified using samples of bits of soy and methanolic extracts from Trifolium pratense, Iresine herbstii and Ononis spinosa plants. Pseudobaptigenin glucoside, irilone, prunetin, texasin, tlatlancuayin and other isoflavones, in addition to aglycones of isoflavones and their beta-glucosides and malonyl and acetyl derivatives, were identified by UV-VIS DAD and electrospray mass spectrometric (ESI-MS) detection in the extracts.

Hypericin and hyperforin production in St. John's wort in vitro culture: influence of saccharose, polyethylene glycol, methyl jasmonate, and Agrobacterium tumefaciens.

Influence of saccharose in the presence or absence of polyethylene glycol (PEG), methyl jasmonate, and an inactivated bacterial culture of Agrobacterium tumefaciens in cultivation medium on morphology of Hypericum perforatum L. and production of hypericin and hyperforin was studied under in vitro conditions. Production of hypericin and hyperforin was influenced by the presence of different concentrations of saccharose (10-30 g L(-1)) in cultivation medium. Addition of PEG (1.25-5 g L(-1)) in the presence of saccharose (10-30 g L(-1)) increased production of hypericin and hyperforin in the H. perforatum in vitro culture. Synthesis of hypericin and hyperforin was unchanged or reduced for most of the experimental plants at higher contents of PEG (10 and 15 g L(-1)). Concentrations of hypericin and hyperforin in the H. perforatum were on the order 100 and 103 microg g(-1) of dry plant material, respectively. Production of hypericin and hyperforin was stimulated either in the presence of a chemical elicitor (methyl jasmonate) or an inactivated bacterial culture of A. tumefaciens. Morphological changes induced by the abovementioned substances were observed and described in detail. The obtained results will be applied in experimental botany and in the technology of H. perforatum cultivation for pharmaceutical applications.

[Hypericin and hyperforin: bioactive components of St. John's Wort (Hypericum perforatum). Their isolation, analysis and study of physiological effect]. / Hypericin a hyperforin: biologicky aktivní komponenty trezalky teckované (Hypericum perforatum). Jejich izolace, analýza a studium fyziologických úcinku.

St. John's Wort (Hypericum perforatum L.) is commonly accepted as a medicinal plant. The data on the physiological activities of the individual substances that are produced in different organs of H. perforatum are well known at present. The highest attention is focused on the characterization and phytochemical properties of hypericin and hyperforin. These organic compounds are used as antidepressant, anticarcinogenic (photodynamic), antimicrobial and virostatic agents. The review paper surveys the present knowledge of chemical and analytical methods for their identification and quantification, physiological activity, and pharmacological and biomedical applications of hypericin and hyperforin.

Disposition of sanguinarine in the rat.

Sanguinarine is an alkaloid with known antibiotic and anti-inflammatory activity and its pharmacokinetics have been studied in the rat after a single oral dose (10 mg kg(-1) body weight). Alkaloid determination in the plasma and liver was carried out by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS). The pharmacokinetic parameters (t(max), c(max), AUC(0-->t) and AUC(0-->infinity)) were determined for sanguinarine and dihydrosanguinarine, the major components detected in plasma. The first step in sanguinarine metabolism in the rat was the reduction of the iminium bond resulting in formation of the less toxic dihydrosanguinarine. Both compounds were completely eliminated from the plasma and liver after 24 h and not detected in urine. After a single oral dose of (3)H-sanguinarine, more than 42% of the ingested radioactivity was present in gastrointestinal tract. Benz[c]acridine, up to date the only sanguinarine metabolite referred to in the literature, was not detected in the plasma, liver or urine.

Determination of phenolic compounds and their antioxidant activity in fruits and cereals.

Three methods, FCM (with Folin-Ciocalteu reagent), PBM (Price and Butler) and AAPM (with 4-aminoantipyrine) for assessment of phenolic compounds and three commonly used methods, TEAC (Trolox equivalent antioxidant capacity), DPPH (with diphenyl-picrylhydrazyl radical), and FRAP (ferric reducing antioxidant power) for evaluation of antioxidant capacity, were modified to a semimicroscale (total volume 1ml) with minimum consumption (to 100mul) of a sample and thereby applicable for fast screening. Appropriate standards and extracts of 17 kinds of fruit and six kinds of cereal were assessed for total content of phenolic compounds and total antioxidant capacity by each of these methods. The results of analyses of commonly used standards (gallic, caffeic and ferulic acids, (+)-catechin, Trolox, fenol and FeSO(4)) for these methods and identical plant extract showed different reactivity of principal reagent of the methods with individual standards and therefore with phenolic substances of extracts as well. However, the trends of the measured values of extracts could be compared, though their absolute values differ proportionally. At assessments of phenolic compounds it is important to determine content of ascorbic acid at roughly the same time and correct the obtained values according to its contribution to the increase in absorbance calculated on the basis of absorbance equations, especially for samples with a higher content. The same is true for reducing saccharides; they can significantly "elevate" values of contents of phenolic compounds and antioxidant activities (by even more than 50%), especially in samples of sweeter fruits. The saccharides should therefore be removed or a correction applied reflecting their concentration.

The effect of extractants on degradation of L-glutamate and L-arginine in the course of shaking and filtration at low temperature.

The effects of demineralized water (DEMI H(2)O) and 0.5 M ammonium acetate (0.5 M AAc) on losses of L-glutamic acid and L-arginine in the course of shaking and filtration at low temperature (6 degrees C) were tested. The concentration of L-glutamic acid decreased by 6.3% in DEMI H(2)O and by 4.9% in 0.5 M AAc, whereas the L-arginine concentration decreased by 6.0% (DEMI H(2)O) and 10.7% (0.5 M AAc). We found a significantly (P < 0.05) higher degradation of L-arginine in 0.5 M AAc compared with that of DEMI H(2)O.

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 x 2.1 mm, 3 microm) 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 > or = 98 and > or = 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.

Isoflavonoids are present in Arabidopsis thaliana despite the absence of any homologue to known isoflavonoid synthases.

Extracts from Arabidopsis thaliana leaves and inflorescence stalks and from Lepidium sativa (Brassicaceae) seedlings were analysed by HPLC-MS-SIM and by five isoflavonoid-specific ELISA methods after the HPLC fractionation of samples, in order to determine presence of isoflavonoids. Individual ELISAs were specific for daidzein, genistein, biochanin A and for their derivatives substituted either at the 4'- or at the 7- positions. Both analytical approaches revealed homologous spectra of isoflavonoids in both plant species. As the ononin specific immunoassay was not available this compound was only detected by HPLC-MS. Formononetin and prunetin represented the main aglycones followed by biochanin A, daidzein and genistein; sissotrin was the most abundant isoflavonoid glycoside followed by ononin, daidzin and genistin. The content of individual compounds ranged from a few micrograms up to 2.2 mg kg(-1) (dry weight). Expression profiles of A. thaliana genes homologous to enzymes involved in isoflavonoid synthesis and metabolism were extracted from publicly available transcriptomic datasets for various tissues. Genes likely to be involved in important steps of the isoflavonoid metabolism in A. thaliana were identified. However, in accord with the previously published data, no homologue to isoflavone synthases known from the Fabaceae plants was found. These aryl migrating enzymes belong to the CYP93C family that is absent in A. thaliana. We conclude that another gene must be responsible for biosynthesis of the isoflavone skeleton in Brassicaceae.

Bio-available amino acids extraction from soil by demineralized water and 0.5 M ammonium acetate.

The extraction and comparison of soil bio-available amino acids using either demineralised water (DEMI H(2)O) or 0.5 M ammonium acetate (0.5 M AAc) solution is reported. Results show that the extraction by 0.5 M AAc is a better method to assess the concentration of bio-available amino acids in soil than DEMI H(2)O due to higher extraction efficiency and better amino acid protection against microbial degradation during processing.

Effect of starter culture, spice mix and storage time and temperature on biogenic amine content of dry fermented sausages.

Two types of dry fermented sausage differing in spicing mixture and the diameter (low content of red pepper+diameter 80 mm, H-sausage; high content of red pepper+diameter 55 mm, P-sausage, respectively) were produced in parallel with two different starter cultures (Pediococcus pentosaceus+Staphylococcus carnosus, B-samples and S. carnosus+Staphylococcus xylosus+Lactobacillus farciminis, F-samples, respectively). The sausages were ripened 21 days and subsequently stored 91 days at the room temperature. Concentration of both most abundant amines, putrescine and tyramine (y; mg/kg DM) increased significantly (P<0.01) in HB-sausage during ripening (x; days): y=2.5+18.13x-0.3144x(2) (R(2)=0.99) and y=0.7+8.17x-0.1130x(2) (R(2)=0.99), and also during storage: y=127.3+5.123x (R(2)=0.79) and y=26.0+3.211x (R(2)=0.74), respectively. At the end of ripening, putrescine (247 mg/kg DM) and tyramine (123 mg/kg DM) content in the HB-sausage was higher (P<0.05) than in the PB-sausage (12 and 9 mg/kg DM, respectively), concentration of either of these amines was negligible (1 mg/kg DM) in either type of F-inoculated sausage. Both starter culture and sausage type influenced significantly (P<0.001) both putrescine and tyramine content in the sausage; starter accounted for 57% and 55% of total variability in putrescine and tyramine content, respectively. Due to the significant (P<0.05) increase of total aerobic counts in the HB-sausage between the end of ripening and the 7th day of storage, followed by the significant (P<0.01) increase of the sum of total biogenic amines between the end of ripening (425 mg/kg DM) and the end of storage (1029 mg/kg DM), the storage of the dry fermented sausages at the room temperature should not be recommended.

Application of solid-phase extraction for determination of phenolic compounds in barrique wines.

A fast, selective and sensitive chromatographic method has been developed for determination of gallic, protocatechuic, p-hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, benzoic, ferulic, sinapic, cinnamic, and ellagic acids and p-hydroxybenzaldehyde, vanillin, syringaldehyde, 2-furfural, 5-methylfurfural, and 5-methoxyfurfural. The compounds from untreated wine samples were pre-concentrated and cleaned using solid-phase extraction on RP-105 polymeric sorbent. The cartridge was conditioned with methanol and water. Co-extracted ballast substances were rinsed from the sorbent with 0.1 mol L(-1) hydrochloric acid-methanol, 1:4 (v/ v). Retained phenolic compounds were selectively eluted with diethyl ether. A linear mobile phase gradient containing 0.3% acetic acid and methanol was used for final baseline chromatographic separation on a Hypersil BDS C18 column. Limits of detection (LOD=3 s(bl)) in the range 5.2 to 181.2 microg L(-1), resolution (R) better than 1.7, and repeatability of 2.7-5.1% (RSD for real samples) were achieved. The method was applied for quantification of individual phenolic compounds in barrique wines.