Cow's milk can be used as a potential source of equol in the human diet. In order to study human intake, however, it is necessary to develop a reliable and sensitive analytical method. This paper reports on the validation of an analytical method using ultra-performance liquid chromatography coupled with a tandem mass spectrometry detector to quantify the equol in commercial milks (raw, whole, semi-skimmed, and skimmed milk). The equol was initially released using enzymatic hydrolysis, and it was then extracted using a double liquid/liquid extraction. The analytical method produced a linear calibration curve with a high correlation coefficient (R 2 ≥0.996) between 5 and 1,000 ng.mL -1 . Good intra-and inter-day precision (≤5.3% and≤5.2%, respectively) and accuracy (≤8.6%) were achieved. The recovery rate differed slightly among the different types of milk, ranging between 60.6± 1.09% and 82.3±5.21%. Good method repeatability was observed (≤15%). There was neither matrix effect nor carry-over effect, and the sample extracts were stable for at least 7 days of storage at -21 °C and 5 °C. The method proved to be specific, sensitive, precise, and accurate and was used for the first time to quantify the equol content in Belgian commercial cow's milk. In all the samples analyzed, equol was present at a concentration ≥10 ng.mL -1 and had a significantly higher content in organic than in conventional milk. The study also found that the mean concentrations of equol were similar for each type of commercial conventional cow's milk.
This paper provides an update and comprehensive review of the analytical methods used for quantifying isoflavones and their metabolites in cow’s milk. Isoflavones are secondary plant metabolites that are similar to 17 β-estradiol in chemical structure. They form one of the most common categories of phytoestrogens. Numerous health benefits have been attributed to isoflavones, but many of these compounds are also considered to be endocrine disruptors, with adverse effects on health. These contradictory trends offer an attractive prospect for future research, and therefore, sensitive and reliable analytical methods are required to clarify various issues about isoflavones. For this review, a structured methodology was used to select 26 relevant articles published between 2005 and 2015 from the Scopus and CAB Abstract databases. The review discusses individual steps of the analytical procedures described in these articles, including sample preparation, instrumental analysis and validation. The most commonly used analytical procedure is sample preparation involving liquid-liquid extraction and an enzymatic hydrolysis step followed by liquid chromatography with mass spectrometry analysis. Currently, however, there is no standardized procedure for the sample preparation and analysis of isoflavones in milk.
A performant method for the simultaneous quantification of daidzein, genistein, formononetin, and biochanin A in forages using an UPLC ® -MS/MS was developed and fully validated. The ultrasound-assisted extraction and enzymatic hydrolysis used in the sample preparation step were optimized using the Box-Behnken experimental design. The optimal extraction conditions used for a representative mix of forage plants were 80 °C, 10 min, and 55 % methanol, and for hydrolysis, they were 20 °C, 18 h, and pH = 6. The chromatographic separation was achieved using an Acquity UPLC ® HSS T3 column, with a water/ methanol linear gradient containing 0.01 % of formic acid at a 0.55 mL min -1 flow rate. The four isoflavones were detected by ESI mass spectrometry in positive ion MRM mode. The method allows high throughput analyses of samples and showed an adequate linear regression model for all isoflavones over a range from 5 to 125 ng mL -1 . There were good intra-and interday precisions (≤8.2 and ≤7.6 %) and accuracy (≤11.4 and ≤7.1 %). The recovery rates were in an acceptable range of 70-120 %, except for biochanin A, where the rate was about 50 %. Good method repeatability was also observed, and there was no matrix effect or carryover problem. The sample extracts were stable for at least 6 days of storage at -21 and 6 °C. The method proved to be sensitive, precise, and accurate for discriminating a wide variety of forages likely to be grazed by ruminants according to their isoflavone contents and to observe the impact of storage process on isoflavone content in forages.formononetin, and biochanin A in forages silages originating from three different meadows. This development is part of a larger study on the metabolization of the biomolecules of interest and the potential accumulation of metabolites such equol in milk [15,16]. Materials and Methods Chemicals and ReagentsA freeze dried forage sample containing six plants (Trifolium pratense L., Trifolium repens L., Medicago sativa L., Lotus corniculatus L., Medicago lupulina L, and Lolium perenne L.; 1:1:1:1:1:1, w/w/w/w/w/w) was mixed with flaxseed meal [4:1, w/w; 93.7 ± 0.1 % dry matter). Formononetin 'FO' (CAS number: 485-72-3), biochanin A 'BA' (491-80-5), genistein 'GE' (446-72-0), and flavone 'IS' (525-82-6) were bought from Sigma-Aldrich (Diegem, Belgium). Daidzein 'DA' (486-66-8) was acquired from Cayman Europe (Tallinn, Estonia). Daidzeind 4 (1219803-57-2) was purchased from C/D/N ISOTOPES (Pointe-Claire, Canada). Individual molecule stock solutions (100 µg mL -1 ) were prepared in methanol and stored at -20 °C in the dark. β-Glucosidase (from almonds, ≥6 U rng -1 , 9001-22-3), β-glucuronidase (type H-2 from Helix pomatia, ≥ 85000 units mL -1 , 9001-45-0), and cellulase (from Aspergillus niger, ≥0.3 units mg -1 , 9012-54-8) were bought from Sigma-Adlrich (Diegem, Belgium). Sodium acetate (127-09-3) was obtained from Merck KGaA (Darmstadt, Germany). Throughout the study, the powder enzymes and Helix pomatia juice were dissolved in different proportions of 0.2-M sodium ace...
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