Advisory Committee on Health Effects of Endocrine Disruptors
The Supplement II to the Intermediary Report
1.2.3.2

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[Preparation of sample solutions]
(i) Sample solutions for LC/MS
- Sample solution for determination of free BPA:
Take 1 ml serum or peritoneal fluid, or 2 ml urine. Add 5 ng BPA-16 as the internal standard and charge the multimode cartridge with the solution. After washing with 3 ml water and 3 ml 20% methanol, elute BPA with 3 ml methanol and evaporate to dryness under reduced pressure. Dissolve the residue in 1 ml 20% methanol to obtain the sample solution.
- Sample solution for determination of total BPA:
Take 1 ml serum or peritoneal fluid, or 2 ml urine. Add 1 ml of 0.2 M acetic acid buffer solution (pH 5) and 50μl of 6,500 units/ml β-glucuronidase (obtained by diluting β-glucuronidase reagent to 10 times volume by 0.2 M acetic acid buffer solution (pH 5)), and incubate for an hour at 37℃. Then operate as for the sample solution for free BPA described above to obtain the sample solution.

(ii) Sample solutions for GC/MS
- Sample solution for determination of free BPA:
Place 100 ml urine in a stoppered conical flask, add 0.1μg ¹³C-BPA and 1 ml (1+1) phosphoric acid to a pH less than 3. Charge the C18 cartridge, conditioned with 5 ml methanol and 10 ml purified water, with the solution. After washing 10 ml of 10% methanol, elute BPA with 3 ml methanol into a 100 ml pear-shaped flask, add 20 ml ethyl acetate, and concentrate to dryness using the rotary evaporator. Add 200μl BSTFA and 2 ml acetone to the flask, let stand overnight for trimethylsilylation, and remove acetone by distillation in the rotary evaporator. Add 2 ml n-hexane to dissolve ultrasonically, and charge a Florisil cartridge, previously washed with 5 ml n-hexane, with the solution, and collect the eluent in a test tube. Wash further the flask two times with 2 ml each of n-hexane, charge the cartridge with the wash liquid, and merge the eluent with the previously obtained eluent. Concentrate the eluent to 1 ml by nitrogen blowing to obtain the sample solution for GC/MS-SIM.
- Sample solution for determination of total BPA:
Place 100 ml urine in a stoppered conical flask, add 100μl β-glucuronidase solution and 0.1 ng ¹³C-BPA, and perform enzyme treatment for 90 min at 37℃. Then operate as for the sample solution for free BPA described above to obtain the sample solution.

[Detection/determination limit]
The detection limit is defined as 3 times, and the determination limit as 10 times, the standard deviation of the entire operation blank test results.

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