Advisory Committee on Health Effects of Endocrine Disruptors
The Supplement II to the Intermediary Report
1.2.4.2

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Analysis of phytoestrogens in feeds for experimental animals (tentative)

[Outline]
Phytoestrogens extracted from animal feeds with 80% methanol is analyzed by a high performance liquid chromatograph/mass spectrometer (LC/MS) qualitatively and quantitatively.

[Reagents]
Confirm by chromatography that all the reagents present no problem in phytoestrogen analysis.
(i) Standard materials: Glycosides, malonylated forms and acetylated forms of daidzein, genistein and glyciten (12 in total)
(ii) Standard solutions: Dissolve an exact 10 mg of each standard substance in 100 ml ethanol. Dilute with 80% methanol as necessary.
Other reagents should be of the special grade or HPLC grade.

[Apparatus and conditions]
The high performance liquid chromatograph/mass spectrometer is typically operated under the conditions shown below.
Column for analysis: C18 Column¹ (2.1 mm i.d., 150 mm long, grain size 5 μm)
Column temperature: 40℃
Mobile phase: 0.003% acetic acid-acetonitrile (gradient elution)
Flow rate: 0.2 ml/min
Quantity injected: 5 μl
Ionization: Electrospray ionization (ESI), negative mode
Fragmenter voltage: 120 V
Monitor ions: m/z = 253, 415, 457, 269, 431, 473, 283, 445, 487

Condition for gradient elution: A/B = 100:0 to 25:75 in 20 min, where
A = 10% acetonitrile containing 0.003% acetic acid
B = 50% acetonitrile containing 0.003% acetic acid

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