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 17β-estradiol in feeds for experimental animals

[Outline]
17β-Estradiol (estradiol, E2) in feeds is extracted with methanol, cleaned up in a graphite carbon/aminopropyl silica gel column for solid phase extraction, and analyzed by an LC/MS/MS qualitatively and quantitatively.

[Reagents]
Confirm by chromatography that all the reagents present no problem in E2 analysis.
(i) Standard material: Standard E2 (≥97.0%)
(ii) Preparation of standard solutions: Place 20 mg standard E2 in a 20 ml measuring flask, and add methanol to 20 ml to obtain a concentrated standard solution of 1000 μg/ml. Dilute appropriately with methanol, as necessary, to prepare standard solutions.
(iii) Organic solvents: Residual pesticide analysis, environmental analysis or LC-MS grade.
(iv) Graphite carbon/aminopropyl silica gel for solid phase extraction¹: Wash with 20 ml dichloromethane/methanol (50:50) and 20 ml methanol before use.
(v) Internal standard: Place 20 mg standard 17β-Estradiol (16, 16, 17-d3) (E2-d3) in a 20-ml measuring flask, and add methanol to 20 ml to obtain a concentrated solution of 1000 μg/ml. Dilute appropriately with methanol, as necessary, to prepare internal standard solutions.

[Tools]
Confirm beforehand that the tools used for sample preparation do not affect E2 analysis. It is sufficient to wash glassware, previously washed thoroughly with water, with acetone immediately before use, because contamination from the environment is usually not expected.
　

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