Compositional changes induced by UV-B radiation treatment of common bean and soybean seedlings monitored by capillary electrophoresis with diode array detection

In this work, a new CE method with diode array detection (DAD) was developed for the monitoring and quantitation of flavonoids in different beans treated and untreated with UV-B radiation. Flavonoid concentration was monitored in UV-B-treated and untreated sprouts of three common beans (Zolfino ecotype, cv. Verdone, cv. Lingua di Fuoco) and one soybean (cv. Pacific). After acid hydrolysis of extracts, the CE-DAD method provides reproducible quantitative determinations of daidzein, glycitein, genistein, and kaempferol at ppm level in these natural matrices within a relatively short time (less than 16 min). Total flavonoid content determined by CE-DAD was 159 +/- 8, 26 +/- 2, 13 +/- 1, and 1.3 +/- 0.3 microg/g fresh weight for untreated sprouts of Pacific soybean, Verdone bean, Zolfino bean, and Lingua di Fuoco bean, respectively. UV-B treatment caused no significant quantitative effect on Pacific soybean sprouts, whereas it enhanced the total isoflavone content by 1.5, 1.8, and 3.2-fold in Verdone, Zolfino, and Lingua di Fuoco beans, respectively. The proposed method shows (i) the potentialities of bean sprouts as a natural source of bioactive compounds (antioxidants); (ii) the technological role of UV-B treatment for sprout isoflavone enrichment; and (iii) the good capabilities of CE-DAD to monitor this process.     

Quantitative analysis of isoflavone aglycones in human serum by solid phase extraction and liquid chromatography-tandem mass spectrometry

This paper describes a liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) for the qualitative and quantitative analysis of three isoflavone aglycones (glycitein, daidzein and genistein) in human serum. Positive ion mode was used for the detection of these compounds and selective reaction monitoring (SRM) was employed for quantitative measurement. The SRM transitions monitored were as 285.0 → 242.0, 270.0 for glycitein, 255.0 → 137.0, 153.0, 181.0, 199.0 for daidzein and 271.0 → 153.0, 215.0 for genistein. d₃-Daidzein was used as an internal standard for quantitative measurement. The linearity was good from 0.5 to 500 ng/ml. The detection limit based on a signal-to-noise ratio of three was 0.27, 0.38 and 0.29 ng/ml for glycitein, daidzein and genistein, respectively. A newly developed solid phase extraction (SPE) procedure was developed for sample pre-treatment. Good recovery, 92.3-103.2%, for three isoflavone aglycones were obtained. This newly developed method was successfully applied to evaluate isoflavone pharmacokinetic in human serum after oral administration.     

Biomolecules and Nutritional Quality of Soymilk Fermented with Probiotic Yeast and Bacteria

Soymilk was fermented with five isolates of probiotic lactic acid bacteria and in combination with probiotic yeast Saccharomyces boulardii. Nutritional profile like fat, protein, ash, pH, acidity, polyphenol, and protein hydrolysis were analyzed. Polyphenol content decreased from 265.88 to 119 microg/ml with different cultures. Protein hydrolysis ranged from 2.46 to 2.83 mmol l(-1) with different cultures. The antioxidant activity was assessed using different methods like 1, 1-diphenyl-2-picrylhydrazyl free radical-scavenging assay, inhibition of ascorbate autoxidation, and measurement of reducing activity. The activities varied with the starters used but, nevertheless, were significantly higher than those found in unfermented soymilk. Bioconversion of the isoflavone glucosides (daidzin + genistin) into their corresponding bioactive aglycones (daidzein + genistein) was observed during soymilk fermentation. Total glucosides in soyamilk were 26.35 mg/100 ml. In contrast, aglycones genistein and daidzein were quantitatively lesser accounting 2.91 mg/100 ml (genistein 1.17 mg/100 ml and daidzein 1.19 mg/100 ml). Soymilk fermented with probiotic cultures resulted in the reduction of glycosides ranging from 0.40 mg to 1.36 mg/100 ml and increase in aglycones ranging from 6.32 mg to 13.66 mg/100 ml.     

Direct determination of free methionine in soy-based infant formula

A method was developed for the direct determination of free methionine in soy-based infant formula, with analyte separation and quantitation by reversed-phase liquid chromatography (LC), and UV absorbance at 214 nm, respectively. Sample preparation required only dilution with mobile phase and syringe filtration. Using a 0.02M KH2PO4 mobile phase (pH adjusted to 2.9 with 85% o-phosphoric acid) and 0.7 mL/min flow rate, methionine eluted at approximately 8 min, and total run time was 14 min after column regeneration with acetonitrile-water. System linearity was demonstrated as peak area versus analyte concentration, ranging from 80 to 120% of the formula specification for free methionine (r > 0.999, and all residuals < 0.45%). Intermediate precision relative standard deviation values were < 1.5% for ready-to-feed and reconstituted powder samples, and recoveries ranged from 98.0 to 103.5% for inter-method comparison with an amino acid analyzer method. The limit of quantitation was 3 mg methionine/L in the "as fed" infant formula. Despite the relatively weak UV absorptivity of methionine, the 214 nm signal was sufficiently intense in the 30-65 mg/L (201-436 microM) range to afford quantitation by peak area proportionation versus a 2-point external standard calibration. This direct UV detection after reversed-phase LC separation provides a simple and accurate method for determining free methionine without derivatization.     

Quantitative determination of acetyl glucoside isoflavones and their metabolites in human urine using combined liquid chromatography-mass spectrometry

To investigate whether the bioavailability of isoflavones could be an alternative to fermented soy foods, the conjugated forms of soy nutritional supplement (containing 98% acetyl glucoside isoflavones) were consumed by eight human volunteers (three were Asian people and five were British). Their daily urine samples were collected before and after a 5-week consumption of supplementation period. Conjugated isoflavones of genistein, daidzein and glycitein were hydrolyzed by enzyme, extracted with methyl tert-butyl ether and analysed using liquid chromatography coupled with electrospray tandem mass spectrometry. Daidzein, genistein, glycitein, dihydrogenistein, dihydrodaidzein and O-desmethylangolensin were identified and quantified simultaneously with high recoveries. The levels of free isoflavones and total isoflavones were compared, and isoflavone glucuronides were identified much higher than the corresponding sulfates or aglycone isoflavones. This method provided the measurement of isoflavones with high sensitivity and specificity and simplified the sample pre-treatment procedure. The limitation of detections of dihydrodaidzein, 3'-hydroxydaidzein, glycitein, daidzein, genistein, dihydrogenistein and O-desmethylangolensin were 37, 23.5, 12.2, 15.4, 14.8, 2.20 and 0.31 pmol, respectively. Only 0.5 ml of urine sample was needed.     

An accurate and reproducible method for the quantitative analysis of isoflavones and their metabolites in rat plasma using liquid chromatography/mass spectrometry combined with photodiode array detection

To study the safety and potential health benefits of soy isoflavones, a rapid and simple method based on liquid chromatography combined with mass spectrometry (LC/MS) and photodiode array detector (PDA) was developed for the determination of isoflavones in rat plasma. The analytes included daidzein, genistein, glycitein, equol, 4-ethyl phenol, and biochanin A over a concentration range of 1.0-4320.0 nM using 75 microL of rat plasma. Rat plasma samples were hydrolyzed by adding an enzyme mixture from Helix pomatia containing glucuronidase and sulfatase to convert the isoflavone beta-glycosides daidzin, genistin, and glycitin to their active aglycone forms. A liquid-liquid extraction method using ethyl acetate as the extraction solvent was used to extract aglycones and the internal standards (phenolphthalein beta-D glucuronide, 4-methylumbelliferyl sulfate, and apigenin) from digested plasma samples. The extract was evaporated to dryness under a nitrogen stream, reconstituted with 0.1% formic acid in water-acetonitrile (85 + 15), and injected into a Zorbax SB-CN reversed-phase column (4.6 x 75 mm, 3.5 microm particle size). The Micromass ZQ detector was operated in the positive ion selected-ion monitoring mode. The flow rate for LC was 1.0 mL/min, with a split where 25% of the effluent was introduced into the electrospray ionization probe of the MS instrument and 75% into the PDA. The chromatographic run time was 16.0 min, with delay of 10 min/injection. The interday precision and accuracy of the standard samples were <2.6% relative standard deviation and <10% relative error, respectively. Recovery of the reported isoflavones with this method varied from 86 to 100%.     

Analysis of isoflavones in soybeans employing direct analysis in real-time ionization-high-resolution mass spectrometry

A direct analysis in real‐time (DART) ion source coupled to a high‐resolution orbitrap mass spectrometer was used for the quantitative analysis of isoflavones isolated from soybeans. For the isolation of genistein, daidzein, glycitein, and their respective acetyl, malonyl, and glucoside forms, an extraction employing 80% aqueous MeOH enhanced by sonication was used. As far as the total isoflavones (expressed as aglycones) were to be determined, an acid hydrolysis with 80% aqueous EtOH and refluxing had to be employed, while in the latter case a good agreement of the results with the data generated by the UHPLC‐orbitrap MS method was achieved, in the case of the analysis of non‐hydrolyzed extracts, some overestimation of the results as compared with those generated by UHPLC‐orbitrap MS was observed. A careful investigation of this phenomenon showed that the free aglycones originated from the conjugated forms of isoflavones in the DART ion source, thus contributing significantly to the “free” genistein/daidzein/glycitein signals during the DART analysis. Good recoveries (95–102%) and repeatabilities (RSD: 7–15%) were obtained at the spiking levels of 0.5, 1, and 0.05 g/kg, for daidzein, genistein, and glycitein, respectively. The limits of detection estimated for the respective analytes were 5 mg/kg.     

The presence of three isoflavonoid compounds in Psoralea corylifolia.

The optimization of a high-performance liquid chromatographic method to determine three isoflavonoids (daidzein, genistein, and biochanin A) in the fruit of Psoralea corylifolia is developed and validated. Dried psoralea fruit powder is extracted with aqueous methanol followed by the hydrolysis of the analytes' conjugated glycosides with hydrochloric acid. The HPLC assay is performed on a reverse-phase C18 column with gradient elution using acetonitrile and 10% acetic acid as the mobile phase at a flow rate of 0.8 mL/min. Flavone is used as the internal standard and the substances are detected at 260 nm. Calibrations are linear (correlation coefficient > or = 0.995) for all three analytes. The limits of detection are 0.01 microg/mL for daidzein and genistein and 0.1 microg/mL for biochanin A. The overall intra- and interassay precision range from 2.5% to 4.9% and from 0.5% to 4.7%, respectively. The method proved to be sensitive, specific, accurate, and precise for the determination of daidzein, genistein, and biochanin A in Psoralea corylifolia.     

Flavonoid profile in soybeans by high-performance liquid chromatography/tandem mass spectrometry

Flavonoid profiling of a soybean sample has been performed by liquid chromatography/positive electrospray tandem mass spectrometry (LC/ESI(+)-MS/MS) using a quadrupole-linear ion trap (QLIT) instrument with an information-dependent data acquisition (IDA) protocol that looped, in a single run, an enhanced MS scan and an enhanced product ion scan. As compromise between time and obtainable information, spectra acquisition was split into two distinct runs: 220-450 Th and 400-800 Th, respectively. The isoflavones daidzein and genistein were identified as aglycones, monoglycosides, diglycosides, triglycosides, acetylglycosides, malonylglycosides, malonyl diglycosides, and dimalonyl diglycosides, whereas glycitein triglycosides, acetylglycosides, and dimalonyl diglycosides were not detected. Also kaempferol di- and triglycosides, malonylglycosides and malonyl diglycosides, previously reported in soy leaves and pods, and four naringenin malonylglycosides were identified.     

Trace determination of bisphenol A and phytoestrogens in infant formula powders by gas chromatography-mass spectrometry

This investigation describes a reliable and sensitive method for simultaneously determining bisphenol A (BPA) and two major phytoestrogens, daidzein and genistein, in powdered milks and infant formulas by gas chromatography-mass spectrometric analysis after trimethylsilylation. To reduce the matrix interference associated with the constituents of the formulas, the dissolved formula solutions were firstly ultra-centrifuged and the analytes in the supernatant were then extracted using a C18 solid-phase extraction column. The accuracy and precision of the method were determined and the technique was successfully employed to measure trace concentrations of BPA, daidzein and genistein in powdered formulas. The results show that BPA, daidzein and genistein were detected in all the testing samples (n = 6) at concentrations from 45 to 113 ng/g (except one infant formula), 20 to 2050 ng/g and 21 to 6510 ng/g, respectively. The highest concentrations of daidzein and genistein (i.e., 2050 and 6510 ng/g) were detected in a soy-based powdered infant formula. The quantitation limits were 1.0 ng/g for BPA, and 10 ng/g for daidzein and genistein using 0.5 g powdered milk samples.     

Determination of the levels of isoflavonoids in soybeans and soy-derived foods and estimation of isoflavonoids in the Japanese daily intake

The levels of 6 kinds of isoflavonoids found in 11 domestic and imported soybeans, and 12 kinds of soybean-based processed foods in Japan were systematically analyzed, and the Japanese daily intake of isoflavonoids from those foods was estimated. The total isoflavonoids (daidzein, glycitein, and genistein) were analyzed with acid hydrolysis and the intact isoflavonoids (daidzein, glycitein, genistein, daidzin, glycitin, and genistin) were analyzed without hydrolysis. This was followed by cleanup with an ODS cartridge column and determined by liquid chromatography with a diode array detector. The highest content of isoflavonoids was found in kinako (a roasted soybean powder) and the lowest was found in soy sauce. The contents and composition of the isoflavonoids in the 11 soybeans varied by species and country of origin. The level of isoflavonoids found in the processed foods varied by manufacturing method or ingredients. The percentage of aglycone tended to be higher in miso (fermented soybean paste) and soy sauce, which are heated and fermented during the manufacturing process. Japanese daily intake of isoflavonoids from soybeans and soybean-based processed foods was estimated as 27.80 mg per day (daidzein 12.02 mg, glycitein 2.30 mg, and genistein 13.48 mg).     

Determination of isoflavones in red clover and related species by high-performance liquid chromatography combined with ultraviolet and mass spectrometric detection

High-performance liquid chromatography-UV-electrospray ionization-mass spectrometric detector (HPLC-UV-ESI-MSD) method for determination of isoflavones in red clover (Trifolium pratense L.) and related species has been developed. The separated isoflavones including aglycones, glycosides and glycoside malonates, were individually analyzed and identified by their molecular ions and characteristic fragment ion peaks using LC-MSD under MS and MS-MS mode, and in comparison with the standard isoflavones. A total of 31 isoflavones were detected in red clover. Several isoflavones were also identified for the first time in related species, T. repense L. (white clover), T. hybridum L. (alsike clover) and T. campestre Schreber (hop trefoil). Based on reversed phase HPLC, all 10 isoflavone aglycones, daidzein, formononetin, genistein, pseudobaptigenin, glycitein, calycosin, prunetin, biochanin A, irilone and pratensein in acidic hydrolyzed extracts were successfully separated within 40 min and quantified individually by UV and MS detectors. For the 10 target compounds, the investigated concentrations ranged from approximately 24 to approximately 12500 ng/ml for UV detection and approximately 6 to approximately 3125 ng/ml for MS detection, and good linearities (r2 > 0.999 for UV and r2 > 0.99 for MS) for standard curves were achieved for each isoflavone. The accuracy and repeatability (n = 10) were within 15% for these 10 compounds. This is the first method reported that enables the simultaneous quantitation of all 10 isoflavone aglycones in red clover and related species.     

Optimization of a rapid microwave assisted extraction method for the liquid chromatography-electrospray-tandem mass spectrometry determination of isoflavonoid aglycones in soybeans

A very fast chromatographic separation of isoflavonoids genistein, daidzein, formononetin and biochanin A was developed on a C18 high-speed column under isocratic conditions. The method was validated in terms of detection limits, quantitation limits (LOQs), linearity and precision. LOQs in 0.04-0.2 microg/g range were calculated, making feasible the determination of these compounds of nutritional concern at trace levels. Good linearity was demonstrated over three concentration orders of magnitude for each analyte (r2 0.990-1.000). The intra-day and inter-day repeatability was evaluated in terms of relative standard deviation (RSD%) at two concentration levels for each analyte (RSD% <9%). An optimization strategy was adopted to find the best conditions for the extraction of isoflavonoid aglycones from yellow soybeans using microwave-assisted extraction. The most relevant parameters resulted to be the microwave power, the extraction time and the acid concentration, optimal values being 600 W, 1 min and 12 M, respectively. When performing sample treatment on a fortified soybean sample, high recovery percentage was obtained for both compounds (94+/-8% for daidzein and 97+/-5% (n = 4) for genistein). The concentration level at which daidzein and genistein were found in the soybean sample were 1.21+/-0.15 mg/g and 2.38+/-0.09 mg/g (n=4), respectively.     

Quantitative determination of isoflavones and coumestrol in soybean by column liquid chromatography

Four different stationary phases and a variety of solvents in varying proportions were examined in this study. Daidzein, genistein, formononetin, biochanin A and coumestrol were separated within 24 min on a phenyl column with acetonitrile-water (33:67, v/v) as eluent. The proposed method showed an acceptable repeatability with a RSD of quantitation <6%. The mean recoveries of daidzein, genistein, formononetin, biochanin A and coumestrol from soybean ranged from 89 to 104%. The identity of the individual analytes was confirmed by LC-MS-MS. The four isoflavones and coumestrol were isolated from soybean by hydrolysis with acid and heat. Neutralization of the soybean samples prior to identification did not alter the concentration of daidzein and genistein in soybean.     

Determination of choline in infant formula by ion chromatography.

Choline was determined in infant formula by ion chromatography with suppressed conductivity detection. Samples were digested with 1M hydrochloric acid, filtered, diluted, and injected into the chromatographic system. Choline and the alkali and alkaline earth metals were separated on a high-resolution cation-exchange column and detected by suppressed conductivity. The method was linear between 2 and 200 mg/L (r2 = 0.9999), the concentration range of the diluted samples. This method accurately determined choline in powdered, concentrated, and ready-to-feed infant formulas. Recoveries of choline spikes into powdered infant formula at approximately 1, 0.8, 0.5, and 0.2 times the labeled value ranged from 85 to 114%. This method had good agreement for 8 blind duplicates. The values determined for these samples, which were used in an AOAC collaborative study of an enzymatic method, were consistent with the values determined by the enzymatic method.     

Supercritical fluid extraction of isoflavones from biological samples with ultra-fast high-performance liquid chromatography/mass spectrometry

An efficient method of modifier addition for supercritical fluid extraction (SFE) of polar isoflavones was developed and yielded extraordinarily high recoveries. To find the optimal extraction conditions, a temperature and pressure optimization and modifier impact study was performed in naturally contaminated and spiked samples. Ultra-fast high-performance liquid chromatography/mass spectrometry (HPLC/MS) was used for the determination of isoflavones on an Atlantis dC18 high-speed reversed phase chromatographic column (20 x 2.1 mm, 3 microm particle size). A newly elaborated supercritical fluid extraction (SFE) procedure allowed more accurate (< 5%) and precise (< 4-7%) determination of isoflavones in biological materials. The HPLC/MS method significantly reduced analysis time with simultaneous improvement of sensitivity and detection limits. The on-column limits of detection LOD (S/N = 3) for isoflavone glycosides (daidzin, genistin, glycitin, ononin, and sissotrin) were 1.3-3.6 fmol and 0.2-1.0 fmol for aglycones (daidzein, glycitein, genistein, formononetin, and biochanin A), respectively.     

Liquid chromatographic analysis of vitamin B6 in soy-based infant formula

A liquid chromatographic (LC) method is described for determination of total vitamin B6 in soy-based infant formula. Total vitamin B6 is quantitated by using ion-pair LC after precolumn transformation of phosphorylated and free vitamers into pyridoxol. The limit of detection is 0.3 ng and the limit of quantitation is 1.0 ng on-column (injection volume = 100 microL). Linear response ranged from 39 to 616 ng/mL (r2 = 0.99986). Analysis of a soy-based infant formula control fortified at 6 different concentration levels gave recoveries that averaged 104%. Assay of SRM 1846 gave results within the certified range (8.6 +/- 0.086 mg/kg versus the certified value of 8.4 +/- 1.0 mg/kg). The method provides a rapid and specific assay for the analysis of total vitamin B6 in fortified soy-based infant formula.     

Concurrent measurement of unbound genistein in the blood, brain and bile of anesthetized rats using microdialysis and its pharmacokinetic application

Genistein, the major isoflavone in soybeans, has been shown to have a wide range of effects. We used an HPLC-UV combined with microdialysis method to detect unbound genistein in rat blood, brain and bile. Genistein dialysates were eluted with a mobile phase containing acetonitrile-water (40:60, v/v, pH 3.5 adjusted by 0.1% acetic acid). Samples were separated using a phenyl (5 microm) column maintained at ambient temperature. The UV detector wavelength was set at 259 nm. The flow rate was 1.0 m/min. The limit of quantitation for genistein was 50 ng/ml. The in vitro recoveries of genistein were 31 +/- 1, 13 +/- 1 and 59 +/- 4% in microdialysis probes of blood, brain and bile, respectively (n = 4). Inter- and intra-assay accuracy and precision of the analysis were less than 10% in the concentration ranges of 0.05-5.0 microg/ml. A small ratio of genistein penetrates the blood-brain barrier (BBB) and goes through hepatobiliary excretion after genistein administration (10 or 30 mg/kg, i.v.). The brain-to-blood (AUC(brain)/AUC(blood)) and bile-to-blood (AUC(bile)/AUC(blood)) distribution ratios were 0.04 +/- 0.01 and 1.85 +/- 0.42, respectively for the dosage of genistein 30 mg/kg. After co-administration of cyclosporine, a P-glycoprotein (P-gp) inhibitor, the distribution ratios of genistein in brain and bile were not significantly altered. These results suggest that the BBB penetration and hepatobiliary excretion of genistein may not regulated by P-gp.     

Liquid chromatography with mass spectrometry--detection of lipophilic shellfish toxins

A rapid multiple toxin method based on liquid chromatography with mass spectrometry (LC/MS) was developed for the detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1), DTX-2, yessotoxin (YTX), homoYTX, 45-hydroxy-YTX, 45-hydroxyhomo-YTX, pectenotoxin-1 (PTX-1), PTX-2, azaspiracid-1 (AZA-1), AZA-2, and AZA-3. Toxins were extracted from shellfish using methanol-water (80%, v/v) and were analyzed using a C8 reversed-phase column with a 5 mM ammonium acetate-acetonitrile mobile phase under gradient conditions. The method was validated for the quantitative detection of OA, YTX, PTX-2, and AZA-1 in 4 species (mussels, Mytilus edulis; cockles, Cerastoderma edule; oysters, Crassostrea gigas; king scallop, Pecten maximus) of shellfish obtained from United Kingdom (UK) waters. Matrix interferences in the determination of the toxins in these species were investigated. The validated linear range of the method was 13-250 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. Recovery and precision ranged between 72-120 and 1-22%, respectively, over a fortification range of 40-160 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. The limit of detection, reproducibility, and repeatability of analysis showed acceptable performance characteristics. A further LC/MS method using an alkaline hydrolysis step was assessed for the detection of OA, DTX-1, and DTX-2 in their esterified forms. In combination with the LC/MS multiple toxin method, this allows detection of all toxin groups described in Commission Decision 2002/225/EC.     

Quantitative determination and structural characterization of isoflavones in nutrition supplements by liquid chromatography-mass spectrometry

In this paper, an accurate and route method was developed to quantitative determine daidzein, genistein, glycitein, daidzin, glycitin, 6"-O-acetyldaidzin, 6"-O-acetylglycitin and 6"-O-acetylgenistin contents in selected high and low isoflavones in nutrition supplements by on line liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry (LC-APCI-MS). Improved extraction and hydrolysis methods of the isoflavones from three nutrition supplements were also studied and a rapid extraction method was developed. Comparison of different MS2 and MS3 spectra of isoflavones and some unknown compounds were also explored and proposed pathway fragments of nine isoflavones were first systematically suggested.