Development of a sensitive enzyme-linked immunosorbent assay for the determination of ochratoxin A

Polyclonal antibodies for ochratoxin A (OTA) were generated from rabbits after the animals had been immunized with either OTA-gamma-globulin or OTA- keyhole limpet hemocyanin (KLH). A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA (ciELISA) were used for the characterization of the antibodies and for analysis of OTA in various agricultural commodities. The antibody titers in the serum of rabbits immunized with OTA-gamma-globulin were considerably higher than those in rabbits immunized with OTA-KLH. The antibodies from the rabbits immunized with OTA-gamma-globulin were further characterized. In the cdELISA, the concentrations causing 50% inhibition (IC(50)) of binding of OTA-horseradish peroxidase to the antibodies by OTA, ochratoxin B (OTB), and ochratoxin C (OTC) were found to be 0.90, 110, and 0.54 ng/mL, respectively. When 10 to 250 ng/g of standard OTA was spiked to soybean samples and then extracted with 50% aqueous methanol, the recovery rate of OTA was found to be 85.9% in the cdELISA. Analysis of OTA in various agricultural commodities showed that 12 of the 20 examined samples were contaminated with OTA at levels from 16 to 160 ng/g. The efficacy of cdELISA was also confirmed by the high-performance liquid chromatography method.     

Development of an enzyme-linked immunosorbent assay for the detection of pentachloronitrobenzene residues in environmental samples

A competitive enzyme-linked immunosorbent assay (ELISA) for pentachloronitrobenzene (PCNB), a fungicide and chemical intermediate, was developed using a polyclonal antiserum produced against a hapten-protein conjugate of pentachlorophenoxypropionic acid-bovine serum albumin (BSA). An indirect competitive ELISA of PCNB showed an IC50 of 37 ng/mL and a limit of detection (LOD) of 7 ng/mL. The ELISA can tolerate up to 10% (v/v) methanol, 5% (v/v) acetonitrile, or 5% (v/v) acetone without significant fluctuation of Amax and IC50. The assay sensitivity showed little change in a range of pH from 6 to 8 and concentrations of 0.05-0.2 M NaCl in the assay buffer. Very low cross-reactivities were observed for some structurally related compounds except for hexachlorobenzene (12%). The average recoveries of PCNB from fortified well water, river water, and soil samples were in ranges of 88-94, 80-91, and 70-81%, respectively. The correlations between the gas chromatographic and ELISA results were excellent (r 2 >or= 0.97, slopes from 0.86 to 1.10) for those fortified samples. The ELISA is a good alternative tool for monitoring PCNB residues in environmental samples.     

Development of a chemiluminescent ELISA for determining chloramphenicol in chicken muscle

An indirect competitive enzyme-linked immunosorbent assay (ELISA) with chemiluminescent (CL) detection for chloramphenicol (CAP) in chicken muscle was developed. CAP-specific polyclonal antibody was raised in rabbit with a CAP-succinate derivative conjugated with bovine serum albumin. Luminol solution was used as the substrate of horseradish peroxidase. The detection limit was 6 ng/L. The CL-ELISA was 10 times more sensitive compared to the colorimetric-ELISA. When CAP was spiked in chicken muscle at levels of 0.05-5 microg/kg, recoveries ranged from 97 to 118% with coefficients of variation of 6-22%. In an actual residue study, the results obtained by CL-ELISA correlated well with those obtained by gas chromatography with microcell electron capture detector. The residue levels of CAP in treated chicken decreased with time and dropped rapidly after the first 6 h from around 50 to 10 microg/kg. After 3 days, CAP was not detected in chicken muscle. The developed method is therefore suitable for screening of CAP in chicken muscle samples.     

Automated on-line solid-phase extraction-liquid chromatography-electrospray tandem mass spectrometry method for the determination of ochratoxin A in wine and beer

An automated on-line solid-phase extraction-liquid chromatography-electrospray tandem mass spectrometry (SPE-LC-ESI-MS/MS) method was developed for the determination of ochratoxin A (OTA) in alcoholic beverages. Mean recoveries for wine and beer were, respectively, 75 and 82%. Detection was achieved in negative ionization with a Q TRAP mass spectrometer operating in multiple-reaction monitoring (MRM) mode or enhanced product ion (EPI) mode, using the third quadrupole as linear ion trap. The MRM mode turned out to be more sensitive; the method allowed accurate determination of OTA in the range of 0.01-25 ng mL(-1) using external calibration. Within-day and between-day relative standard deviation percentages were <6.2 and <9.1%, respectively. In EPI mode, fragmentation spectra at the limit of quantification (0.03 ng mL(-1)) and good linearity could be obtained. Application of the method (MRM mode) to the analysis of several wine and beer samples purchased in local stores revealed OTA levels in the ranges of 0.03-1.44 ng mL(-1) for wines and 0.02-0.14 ng mL(-1) for beers.     

Production of a monoclonal antibody against ochratoxin A and its application to immunochromatographic assay

A monoclonal antibody (Mab) against ochratoxin A (OTA) was produced from the hybridoma cell line C7G25, which was established by the fusion of Sp2/0-Ag14 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with the OTA-bovine serum albumin conjugate. This Mab belongs to the IgG(2a) heavy-chain subclass with a kappa-type light chain. The level of 50% inhibition concentration was 1.20 ng/mL in a competitive direct enzyme-linked immunosorbent assay (cdELISA), and the detection limit was 0.12 ng/mL. This antibody is specific for OTA but also shows cross-reactivity with ochratoxin B (31.7%) in a cdELISA. On the basis of the sandwich format using the produced Mab against OTA, a rapid immunochromatographic assay was developed to efficiently detect OTA. This method was able to detect up to 500 ng/mL of OTA in <10 min.     

Soybean peroxidase-catalyzed oxidation of luminol by hydrogen peroxide

Anionic soybean peroxidase Glycine max (SbP) is shown to efficiently catalyze luminol oxidation by hydrogen peroxide. Contrary to horseradish peroxidase, the presence of p-iodophenol in the reaction medium affects slightly the efficiency of SbP catalysis. A maximal intensity of chemiluminescence, produced through this enzymatic reaction, was detected at pH 8.4-8.6. Contrary to anionic palm tree peroxidase, in the presence of SbP, chemiluminescence intensity increases with the reaction buffer concentration. The detection limit of SbP in the reaction of luminol oxidation is 0.3 x 10(-12) M. Therefore, high sensitivity in combination with the long-term chemiluminescent signal is indicative of good prospects for application of this enzyme in enzyme immunoassay with chemiluminescent detection.     

Residue depletion of ractopamine and its metabolites in swine tissues, urine, and serum

Ractopamine hydrochloride is a beta-adrenergic leanness-enhancing agent approved for use in swine in the United States. Depletion of ractopamine and its metabolites from animal tissues, urine, and serum is of interest for the detection of illegal use. The objectives of this study were to measure the residues of ractopamine in swine incurred samples after treatment with dietary ractopamine for 28 consecutive days. An efficient and sensitive analytical method was developed for the detection of parent ractopamine and its metabolites in swine tissues, urine, and serum by HPLC-FLD. After extraction, enzymatic digestion, and solid-phase cleanup of the samples, ractopamine residues were determined by liquid chromatography (LC) with fluorescence detector. The limits of detection (LOD) for tissues, urine, and serum were 1 ng g(-1), 0.5 ng mL(-1), and 0.5 ng mL(-1), respectively. Recoveries ranged from 70.5 to 94.5% for samples fortified at 1-50 ng g(-1) or ng mL(-1). Sixty pigs were fed twice daily for 28 consecutive days with feeds containing 18 mg kg(-1) ractopamine HCl. The residue concentrations in urine, liver, and kidney were 650.06 ng mL(-1), 46.09 ng g(-1), and 169.27 ng g(-1), respectively, compared with those in muscle, fat, and serum (4.94 ng g(-1), 3.28 ng g(-1), and 7.48 ng mL(-1), respectively) at the feeding period of 7 days. The residue concentrations at withdrawal period of 0 days in all edible tissues were lower than tolerance values established by the FDA and MRL values listed by the JECFA. These data support the withdrawal time of 0 days established by the FDA for ractopamine used as feed additive in swine.     

Development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide bromophos-ethyl

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of the organophosphorus insecticide bromophos-ethyl. Four bromophos-ethyl derivatives (haptens) were synthesized and were coupled to carrier proteins through the pesticide thiophosphate group to use as an immunogen or as a coating antigen. Rabbits were immunized with either one of two haptens coupled to bovine serum albumin for production of polyclonal antibodies, and the sera were screened against one of the haptens coupled to ovalbumin. Using the serum with highest titer, an antigen-coated ELISA was developed, which showed an IC(50) of 3.9 ng/mL with a detection limit of 0.3 ng/mL (20% inhibition). An antibody-coated ELISA using an enzyme tracer was also developed, which showed an IC(50) of 6.5 ng/mL with a detection limit of 1.0 ng/mL (20% inhibition). The antibodies showed negligible cross-reactivity with other organophosphorus pesticides except with the insecticides bromophos-methyl and chlorpyrifos in the antibody-coated assay only. Recoveries of bromophos-ethyl from fortified crop and water samples ranged from 82 to 128% and from 95 to 127%, respectively.     

Development of an enhanced chemiluminescence ELISA for the rapid detection of acrylamide in food products

In this work, a polyclonal antibody for acrylamide (AA) was obtained by immunization of rabbits with N-acryloxysuccinimide (NAS) and keyhole limpet hemocyanin (KLH) conjugate. A direct enzyme-linked immunosorbent assay (ELISA) based on this antibody was developed with enhanced chemiluminescent (ECL) detection of AA in food samples. Assay conditions, such as concentrations of antibody and enzyme conjugate and competition time, were optimized. The effects of ionic strength and pH value were investigated. The optimized ECL-ELISA system allowed AA determination in a linear working range of 26.3-221.1 ng mL(-1) with an IC(50) value of 60.6 ng mL(-1) and a limit of detection of 18.6 ng mL(-1). Good recoveries with spiked food samples were obtained with a recovery range from 74.4 to 98.1%, and these results correlated well with those obtained using an HPLC method. This indicates that ECL-ELISA is applicable to the specific detection and routine monitoring of AA in food samples.     

Robust and sensitive monoclonal enzyme-linked immunosorbent assay for the herbicide molinate

This paper reports on the generation of monoclonal antibodies and the development of a new enzyme-linked immunosorbent assay (ELISA) for the detection of molinate (S-ethyl hexahydroazepine-1-carbothioate). Hybridoma cells were generated using spleen and lymph node cells from a mouse immunized with S-2-carboxyethyl hexahydroazepine-1-carbothioate conjugated to keyhole limpet hemocyanin. After screening with a competitive ELISA, two monoclonal antibodies, mAbs 16C11 and 14D7, with IC(50) values of 82 +/- 2 and 173 +/- 8 ng/mL, respectively, were selected. MAb 16C11 can detect molinate concentrations of 1 ng/mL with no cross-reactivity to any other thiocarbamate pesticides; however, it was susceptible to the presence of organic solvents and to variation in buffer ionic strength. MAb 14D7 tolerated concentrations up to 5% of propylene glycol and 12.5% of acetonitrile in the assay buffer. The sensitivity of mAb 14D7 was further improved by decreasing the amount of coating antigen in the ELISA; the final inhibition assay showed an IC(50) of 69.2 +/- 1.4 ng/mL. In summary, mAb14D7 provided a more sensitive and robust assay, as compared with previous polyclonal antibody-based assays, with the additional advantage of being based upon a consistent and unlimited source of a defined reagent.     

Production and characterization of a monoclonal antibody against the beta-adrenergic agonist ractopamine

A monoclonal antibody was generated toward the beta-adrenergic agonist ractopamine hydrochloride ?(1R,3R),(1R, 3S)-4-hydroxy-alpha-[[[3-(4-hydroxyphenyl)-1-methylpropyl]amino]methy l]benzenemethanol hydrochloride?. Ractopamine-glutarate-keyhole limpet hemocyanin (KLH) was used as the antigen for antibody generation in mice. Clone 5G10, secreted antibody with isotype IgG1kappa, was used for the development of an immunoassay. The selected antibody was specific for racemic ractopamine with an IC(50) of 2.69 +/- 0.36 ng/mL (n = 15). Antibody binding toward ractopamine was stereoselective with (1R,3R)-ractopamine having an IC(50) of 0.55 +/- 0.09 ng/mL (n = 3). IC(50) values for the (1S, 3R)-, (1S,3S)-, and (1R,3S)-ractopamine stereoisomers were 2.00 +/- 0.37, 140 +/- 23, and 291+/- 32 ng/mL (n = 3), respectively. Phenethanolamine beta-agonists showed low cross-reactivity. Studies using a series of ractopamine metabolites and ractopamine analogues demonstrated structural requirements for the antibody binding. A free phenolic group on the N-butylphenol moiety was required for high-affinity binding because methoxylated analogues and metabolites glucuronidated at this phenol generally had IC(50) values greater than 200 ng/mL. Ractopamine analogues methoxylated or glucuronidated at the ethanolamine phenol had IC(50) values of 0.7-2.6 ng/mL. Lack of a benzylic hydroxyl group was of less importance to antibody binding than was the correct stereochemical orientation (3R) of ractopamine's N-phenylalkyl group. In conclusion, a highly specific monoclonal antibody to ractopamine hydrochloride was developed that could be of potential utility in screening assays.     

Development of an enzyme-linked immunosorbent assay for the detection of glyphosate

A competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed to quantitate the herbicide glyphosate [N-(phosphonomethyl)glycine] in water. The ELISA has a detection limit of 7.6 microg mL(-1) and a linear working range of 10-1000 microg mL(-1) with an IC(50) value of 154 microg mL(-1). The glyphosate polyclonal antisera did not cross-react with a number of other herbicides tested but did cross-react with the glyphosate metabolite aminomethylphosphonic acid and a structurally related herbicide, glyphosine [(N,N-bis(phosphonomethyl)glycine]. The assay was used to estimate, quantitatively with accuracy and precision, glyphosate concentrations in water samples. Water samples were analyzed directly, and no sample preparation was required. To improve detection limits, water samples were concentrated prior to analysis, resulting in the increase of the detection limits by 100-fold. After the sample preconcentration step, the detection limit improved to 0.076 microg mL(-1) with an IC(50) value of 1.54 microg mL(-1), and a linear working range was 0.1-10 microg mL(-1). Glyphosate concentrations determined by ELISA correlated well with those determined by high-pressure liquid chromatography (r(2) = 0.99). This assay contributes to reducing the costs associated with conventional residue analysis techniques for the quantitation of glyphosate in water.     

Use of soybean peroxidase in chemiluminescent enzyme-linked immunosorbent assay

A procedure for the production of conjugates of soybean peroxidase (SbP) oxidized by sodium periodate and anti-mouse IgG antibody (Ab) was optimized. A sandwich chemiluminescent enzyme-linked immunosorbent assay (ELISA) for determination of mouse IgG using SbP and specific Ab was developed, and SbP-catalyzed oxidation of luminol was carried out in the absence of any enhancer. Comparison of conjugates produced by labeling Ab by soybean and horseradish peroxidases in the chemiluminescent ELISA showed that in the case of SbP a rate of emission decay formed through luminol oxidation was significantly lower. Application of the soya enzyme allowed the development of the immunoassay with improved sensitivity and a wider linear range.     

Development of an enzyme-linked immunosorbent assay for the detection of the fungicide fenarimol

To develop an enzyme-linked immunosorbent assay for the fungicide fenarimol, two synthesized haptens, haptens-1 and -2, and the purchased 4,4'-DDA were conjugated to carrier proteins (BSA, KLH, and OVA). Polyclonal antibodies raised against hapten-1,2-KLH conjugates in rabbits and the coating antigens of hapten-1,2-BSA conjugates, hapten-2-OVA conjugate, and 4,4'-DDA-BSA conjugate were screened and selected for the homologous and/or heterologous ELISA formats. Two competitive indirect ELISAs were selected: assays I and II. The optimized ciELISAs of assays I and II showed average IC(50) values of fenarimol of 5.4 and 9.4 ng/mL, detection ranges of 1.1-25.9 and 1.1-82.7 ng/mL, and lowest detection limits of 0.3 and 0.3 ng/mL, respectively. The cross-reactivities with several structurally related compounds indicated the importance of the steric fitness in the antigen-antibody interaction. Recoveries of fenarimol from apple and pear samples spiked with the analyte by assay I were in the range of 93-113% by simple extraction, concentration, and dilution. This assay could be a convenient and supplemental analytical tool for monitoring fenarimol residues in environmental and agricultural samples.     

Assay of ochratoxin A in wine and beer by high-pressure liquid chromatography photodiode array and gas chromatography mass selective detection.

To routinely assay the concentrations of ochratoxin A (OTA) in wines and beers, two new methods were developed and evaluated. The first utilized solid-phase extraction on a C(18) cartridge to achieve a 100-fold sample concentration followed by high-performance liquid chromatography on a C(18) column with gradient elution and quantitation at 333 nm by means of a photodiode array detector. Positive confirmation can be carried out by purity and match-factor analysis as well as peak shift following esterification with BF(3). Total run time is 28 min. The limits of detection (LOD) and quantitation (LOQ) are 0.05 and 0.10 microg/L, respectively. Recovery and imprecision ranged from 83 to 94% and from 4.0 to 8.9%, respectively. With a throughput of 35 assays per working day, this method is ideal for routine OTA analysis. It was used to survey the concentrations of OTA in 942 wines (2 of which gave values between 0.1 and 0.2 microg/L) and 107 beers (2 of which gave values between 0.05 and 0.1 microg/L). OTA was detected more frequently in red than white wines, with the highest incidence in red wines from Spain and Argentina. There was no association between OTA and country of origin or beverage type among the beers analyzed. The second method utilized gas chromatography with mass selective detection monitoring eight specific ions, preceded by extraction in dichloromethane and derivatization with bis[trimethylsilyl]trifluoroacetamide. LOD and LOQ were 0.1 and 2 microg/L, respectively; recovery and imprecision were 69-75 and 9.0-11.1%, respectively. The method is not suitable for routine quantitation but is potentially useful as a confirmatory tool for samples with OTA > or =0.1 microg/L.     

Lunasin concentration in different soybean genotypes, commercial soy protein, and isoflavone products

Lunasin is a unique and novel cancer preventive peptide originally isolated from soy. Information on lunasin concentration of soybean cultivars and commercial soy proteins would be useful in developing lunasin-enriched cultivars and soy products. We report the development of an enzyme-linked immunosorbent assay (ELISA) method to identify lunasin and quantify the variations in concentration in 144 selected, diverse soybean accessions from the U.S. Department of Agriculture Soybean Germplasm Collection, several commercially available soy protein fractions and isoflavone-enriched products. With synthetic lunasin and monoclonal antibody, ELISA shows a linear concentration range of 24-72 ng/mL, good reproducibility, a detection limit of 8 ng/mL, and a recovery of 90% on spiked soy samples. Lunasin concentrations in the tested materials range from 0.10 to 1.33 g/100 g flour. Differences that exceeded 100% have been observed among accessions of similar maturity that were grown in the same environment, indicating that genetic differences in soybeans exist for lunasin. The mean of 23 major ancestral lines of U.S. cultivars is similar to the mean of 16 modern cultivars selected to represent the current diversity of the crop, but the highest values were found within the ancestral and exotic accessions. Soy protein concentrate, isolate, and hydrolyzate contain 2.81 +/- 0.30, 3.75 +/- 0.43, and 4.43 +/- 0.59 g lunasin/100 g flour, respectively, while soy flour and soy flakes contain 1.24 +/- 0.22 g lunasin/100 g flour. Isoflavone-enriched products contain very little or no lunasin. The relative mass (M(r)) of lunasin in the samples is 5.45 +/- 0.25 kDa. The wide range of lunasin concentrations within the Glycine max species indicates that the levels of this important bioactive peptide can be genetically manipulated. Furthermore, soy isolates and hydrolyzed soy proteins contain the highest concentrations of lunasin.     

Ochratoxin a determination in beer by solid-phase microextraction coupled to liquid chromatography with fluorescence detection: a fast and sensitive method for assessment of noncompliance to legal limits

A solid-phase microextraction-liquid chromatography-fluorescence detection (SPME-LC-FD) method for the determination of ochratoxin A (OTA) in commercial beer samples was developed for the first time using a 60 microm thick poly(dimethylsiloxane)/divinylbenzene (PDMS/DVB) fiber. The procedure required a very simple sample pretreatment, an isocratic elution, and provides a selective extraction. All of the factors influencing fiber adsorption (extraction time, temperature, pH, and salt addition) and desorption of the analyte (desorption and injection time and desorption solvent mixture composition) have been investigated. The linear range investigated in beer was 0.03-2 ng/mL; within-day and between-days relative standard deviation in beer were 4.3 and 5.9%, respectively. The limit of quantification in spiked beer was 53 pg mL(-)(1), well below all European regulatory levels.     

Development of monoclonal ELISAs for azinphos-methyl. 2. Assay optimization and water sample analysis.

Two enzyme-linked immunosorbent assays (ELISA) for the insecticide azinphos-methyl have been optimized and characterized. Both ELISAs are based on monoclonal antibodies produced from an immunogen with a hapten containing a phthalimido moiety and on protein conjugates of heterologous ligands containing a 1,2,3-benzotriazine group. Assay I was performed in the conjugate-coated ELISA format and assay II in the antibody-coated format. Several physicochemical factors (ionic strength, pH, incubation times, and Tween 20 and BSA concentrations) that influence assay performance were studied and optimized. Regarding specificity, both monoclonal immunoassays highly cross-reacted with azinphos-ethyl and phosmet. Finally, both assays were applied to the analysis of azinphos-methyl in spiked real water samples. For assay I the sensitivity, estimated as the I(50) value, was 0.40 nM, with a practical working range between 0.10 and 1.75 ng/mL and a limit of detection of 0.05 ng/mL. For assay II the sensitivity was 1.01 nM, with a practical working range between 0.32 and 2.54 ng/mL and a limit of detection of 0.08 ng/mL.     

Development of an immunoassay for the residues of the herbicide bensulfuron-methyl

To develop a competitive indirect enzyme-linked immunosorbent assay based on polyclonal antibodies for the detection of the sulfonylurea herbicide bensulfuron-methyl, seven structurally related haptens were synthesized. Four of them mimicking the target analyte were conjugated to keyhole limpet hemocyanin by the N-hydroxysuccinimide activated ester method to use as immunogens, and all of them were conjugated to bovine serum albumin to use as plate-coating antigens. Polyclonal antibodies raised in rabbits and the coating antigens were screened and selected for the assay in simple homologous and heterologous ELISA formats. Three sensitive heterologous ELISAs were selected and optimized, showing the average IC(50) values of bensulfuron-methyl as low as 0.17, 0.09, and 0.09 ng/mL, the detection ranges of 0.04-0.60, 0.01-0.60, and 0.04-0.25 ng/mL, and the lowest detection limits of 0.03, 0.002, and 0.03 ng/mL, respectively. The cross-reactivities of other sulfonylurea herbicides and metabolites of bensulfuron-methyl to the antibodies were less than 15% in the two assays. Recoveries from the analyte-fortified water samples in assay I were in the range of 81-125% by simple dilution. The correlation between the ELISA and HPLC was 0.999 (n = 15) with a slope of 1.37 in the analysis of groundwater samples fortified with bensulfuron-methyl. The results obtained strongly indicate that the ELISA can be a highly sensitive and convenient tool for detecting bensulfuron-methyl residues in agricultural and environmental samples.     

LC/UV/ESI-MS analysis of isoflavones in Edamame and Tofu soybeans

High-performance liquid chromatography coupled with ultraviolet and electrospray ionization mass spectrometry (HPLC/UV/ESI-MSD) was applied to the study of isoflavones in both Edamame and Tofu soy varieties, from which the immature fresh soybeans or the mature soybean seeds are consumed, respectively. Positive atmospheric pressure interface (API) MS and MS/MS were used to provide molecular mass information and led to the identification of a total 16 isoflavones, including three aglycones, three glycosides, two glycoside acetates, and eight glycoside malonates. The major isoflavones in soybean seeds were daidzein and genistein glycoside and their malonate conjugates. Trace levels of daidzein and genistein acetyl glycosides were found only in the mature dry soybean seeds. To facilitate quantitative analysis, acid hydrolysis during extraction of soy samples was selected to convert the various phytoestrogen conjugates into their respective isoflavone aglycones, allowing accurate quantitation of total phytoestrogens as aglycones. On the basis of HPLC combined with UV and MS detection, all three targeted soy isoflavone aglycones, daidzein, genistein and glycitein in hydrolyzed extracts were successfully quantified within 25 min with formononetin used as the internal standard. The standard curves of UV detection were fitted in the range of 14.16-29000 ng/mL for daidzein, 15.38-31500 ng/mL for genistein, and 11.72-24000 ng/mL for glycitein. For MS detection, the standard curves were established in the range of 3.54-1812.5 ng/mL for daidzein, 3.85-1968.75 ng/mL for genistein, and 2.93-1500 ng/mL for glycitein. Good linearities (r(2) > 0.999 for UV and r(2) > 0.99 for MS) for standard curves were achieved for each isoflavone. The accuracy and precision (RSD) were within 10% for UV detection and 15% for MS detection (n = 10). Using this method, the phytoestrogen levels of total isoflavone aglycones from 30 soybean seed varieties were then evaluated for confirmation of the technique. Total isoflavones ranged across the varieties from 0.02 to 0.12% in the Edamame varieties, which are harvested while the seeds are still immature, and from 0.16 to 0.25% in Tofu varieties, harvested when the seeds are physiologically mature. While the literature has focused on the isoflavone content of soy products and processing soy, this report provides a reliable analytical technique for screening of authenticated fresh immature Edamame soybeans and Tofu soybeans.