Validating the use of an ultra-performance liquid chromatography with tandem mass spectrometry method to quantify equol in cow’s milk

Abstract

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.

Analytical methods used to quantify isoflavones in cow’s milk: a review

“… EQ ( commercial milks ) Frozen (–18 °C) - Thawed overnight at 4–6 °C, equilibrated to room T°, and homogenized (2 mL) - Enz. hydrolysis (0.1 mL β-glucuronidase/sulfatase from H. pomatia type H2 [≥45,000/3750 units.mL −1 ], 37 °C, and 2 h) - Cooled - Centrifugation - Double L/L extraction (n-hexane and ethyl acetate, 3 × 3 mL) - Evaporation to dryness - Reconstitution MeOH:H 2 O (80:20, v / v ) - Filtration (0.2 μm) Daems et al ( 2015 ) DA and GE ( commercial milks ) Frozen (−20 °C) or directly analyzed - Deproteinized (10 g of milk mixed with 12 mL of 1% ( v / v ) acetic acid in acetonitrile, 1 g NaCl, and 2.5 g MgSO 4 ) - Centrifugation - Evaporation to dryness - Reconstitution (5 mL water) - SPE (OASIS-HLB) - Evaporation - Reconstitution in 200 μL of H 2 O/MeOH (3:1, v / v ) - Filtration (0.2 μm) Wielogórska et al ( 2015 ) EQ ( technological processing ) N.C. - Deproteinized (10 mL of milk mixed with 3 mL of acetonitrile and centrifuged at 5 °C) - Supernatant stored at –80 °C - Filtration (0.2 μm) Tsen et al ( 2014 ) PU ( technological processing ) Frozen (≤−10 °C) - Freeze-drying - Defatted three times with hexane (1:5, w / v ) - Extraction with MeOH (1:3, w / v ) - Evaporation to dryness - Reconstitution in MeOH (30 g.mL −1 ) - Filtration (0.45 μm) Rastogi et al ( 2013 ) GE and GE-in ( commercial milks ) N.C. - Double UAE (10 and 5 mL of acetonitrile, 15 min) - Centrifugation - Filtration (0.45 μm) - Concentration and dilution - SPE (OASIS-HLB) - Concentration - Dilution Maggioni et al ( 2013 ) DA, EQ, FO, and GE ( feeding experiments ) Frozen (−18 °C) - L/L extraction (20 mL milk/20 mL acetone/aceta...…”

“…vol. : 10 μL MS/MS ESI + , MRM mode External calibration 5 ng.mL -1 0.3 ng.mL −1 V V V V V V Daems et al ( 2015 ) UHPLC Column: Acquity HSS T3 (100 × 2.1 mm, 1.8 μm) Mobile phases: (A) 5 mM ammonium acetate in H 2 O/acetonitrile (9:1, v / v ) (B) MeOH:acetonitrile (1:1, v / v ) Elution: gradient Column T°: 45 °C Flow: 0.4 mL.min −1 Run time: 10.5 min Inj. vol.…”

Quantification of Four Isoflavones in Forages with UPLC®-MS/MS, Using the Box–Behnken Experimental Design to Optimize Sample Preparation

“…Intra-day (five injections within 1 day) precision (expressed as RSD %) and accuracy (expressed as bias %) at four concentrations (15,30,50, and 80 ng mL -1 ) were determined. Inter-day (five injections over 3 days) precision and accuracy were also evaluated for the same standard concentrations (Table S2).…”

“…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].…”

“…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].…”