GB 5009.305-2025National food safety standard - Determination of bisphenol A, bisphenol F and bisphenol S in foods (English PDF)

3.1 Reagents

3.1.1 Acetonitrile (CH3CN). Chromatographically pure.

3.1.2 Methanol (CH3OH). Chromatographically pure.

3.1.3 Sodium chloride (NaCl).

3.1.4 Disodium hydrogen phosphate (Na2HPO4).

3.1.5 Potassium dihydrogen phosphate (KH2PO4).

3.1.6 Potassium chloride (KCl).

3.1.7 Hydrochloric acid (HCl). Guaranteed reagent.

3.2 Preparation of reagents

3.2.1 Hydrochloric acid solution (10+90). Pipette 10 mL of hydrochloric acid; add to 90 mL of water; and mix well.

3.2.2 Phosphate buffer solution (hereinafter referred to as PBS). Weigh

8.0 g of sodium chloride;

1.2 g of disodium hydrogen phosphate,

0.2 g of potassium dihydrogen phosphate, and

0.2 g of potassium chloride; dissolve in 900 mL of water; adjust the pH to 7.4±

0.1 with hydrochloric acid solution (10+90); and then dilute to 1,000 mL with water.

3.2.3 Methanol-water solution (80+20). Pipette 80 mL of methanol; add 20 mL of water; and mix well.

3.2.4 Methanol-water solution (40+60). Pipette 40 mL of methanol; add 60 mL of water; and mix well.

3.3 Standard sample

3.3.1 Bisphenol A standard sample (C15H16O2, CAS. 80-05-7). Purity >= 99.0%; or a standard sample certified by the state and granted a standard substance certificate.

3.3.2 Bisphenol F standard sample (C13H12O2, CAS. 620-92-8). Purity >= 99.0%, or a standard sample certified by the state and granted a standard substance certificate.

3.3.3 Bisphenol S standard sample (C12H10O4S, CAS. 80-09-1). purity >= 99.0%, or a standard sample certified by the state and awarded a standard substance certificate. 3.3.4 13C12-Bisphenol A isotope internal standard (C313C12H16O2, CAS. 263261-65-0). 100 mg/L, or a solid standard sample with a purity of >= 98.0%. 3.3.5 13C12-Bisphenol F isotope internal standard (C13C12H12O2). 100 mg/L, or a solid standard sample with a purity of >= 98.0%. 3.3.6 13C12-bisphenol S isotope internal standard (13C12H10O4S). 100 mg/L, or solid standard sample with a purity of >=98.0%.

3.4.1 Standard stock solution (100 mg/L). Accurately weigh 10 mg (accurate to

0.1 mg) of bisphenol A, bisphenol F and bisphenol S standard samples; dissolve them in methanol and dilute to 100 mL, respectively; and mix well. Transfer the solution to a brown glass reagent bottle and store it at -18 °C away from light. The validity period is 12 months.

3.4.2 Standard intermediate solution (1 mg/L). Accurately pipette

0.10 mL of the standard stock solution of bisphenol A, bisphenol F and bisphenol S, respectively, into 10 mL volumetric flasks; make constant volume to the scale with methanol; and mix well. Transfer the solution to a brown glass reagent bottle and store it at -18 °C away from light. The validity period is 6 months.

3.4.3 Standard mixed solution (bisphenol A and bisphenol F. 100 µg/L; bisphenol S. 10 µg/L). Accurately pipette

1.00 mL of each standard intermediate solution of bisphenol A and bisphenol F, respectively, and

0.10 mL of the standard intermediate solution of bisphenol S; place them in a 10 mL volumetric flask; make constant volume to the scale with methanol-water solution (40+60); and mix well. Prepare immediately before use.

3.4.4 Isotope internal standard intermediate solution (1 mg/L). Accurately pipette

0.10 mL of each 13C12-bisphenol A standard solution, 13C12-bisphenol F standard solution, and 13C12- bisphenol S standard solution; respectively place them in 10 mL volumetric flasks; make constant volume to the scale with methanol; and mix well. Transfer the solution to a brown glass reagent bottle and store it at -18 °C away from light. The validity period is 6 months.

3.4.5 Mixed isotope internal standard solution (13C12-bisphenol A and 13C12-bisphenol F. 100 µg/L; 13C12-bisphenol S. 10 µg/L). Accurately pipette

1.00 mL of each 13C12-bisphenol A and 13C12-bisphenol F intermediate solution and

0.10 mL of 13C12-bisphenol S intermediate solution, respectively; place them in a 10 mL volumetric flask; make constant volume to the scale with methanol-water solution (40+60); and mix well. Prepare immediately before use.

3.4.6 Standard series working solution. Accurately and respectively pipette

0.10 mL,

0.20 mL,

0.50 mL,

0.80 mL,

2.00 mL of the standard mixed working solution into a 10 mL volumetric flask; add

0.50 mL of isotope internal standard mixed working solution; make constant volume to the scale with methanol-water solution (40+60); and mix well. The mass concentrations of bisphenol A and bisphenol F in the standard series working solutions are 1.00 µg/L, 2.00 µg/L, 5.00 µg/L, 8.00 µg/L, 10.0 µg/L, and 20.0 µg/L, respectively; the mass concentrations of bisphenol S are 0.100 µg/L, 0.200 µg/L, 0.500 µg/L, 0.800 µg/L, 1.00 µg/L, and 2.00 µg/L, respectively; the mass concentrations of 13C12-bisphenol A and 13C12-bisphenol F are 5.00 µg/L; and the mass concentration of 13C12-bisphenol S is 0.500 µg/L. Prepare immediately before use.

3.5 Materials

3.5.1 Bisphenol A, bisphenol F and bisphenol S composite immunoaffinity column. column capacity of each compound >=200 ng, column recovery >=90% (see Appendix A for verification method). NOTE. Different batches of immunoaffinity columns need to be quality verified before use.

3.5.2 Polypropylene centrifuge tubes. 2 mL and 50 mL.

3.5.3 Glass nitrogen blowpipe. 15 mL.

5.2.3 Cereals and infant cereal complementary foods Weigh 1 g of specimen (accurate to

0.01 g); place in a 50 mL polypropylene centrifuge tube; add 50 µL of isotope internal standard mixed solution; shake and mix; and stand for 30 min. Add 10 mL of methanol-water solution (80 +20); vortex for 30 s; ultrasonically extract for 15 min; and centrifuge at 10,000 r/min and at 4 °C for 10 min. Take the supernatant and place it in a glass nitrogen blowpipe; slowly blow it with nitrogen at 40 °C to about 2 mL; add 8 mL of PBS and mix well; and wait for purification.

5.2.4 Vegetables and fruits Weigh 1 g of specimen (accurate to

0.01 g); place in a 50 mL polypropylene centrifuge tube; add 100 µL of hydrochloric acid solution (10+90) and mix well. Then add 50 µL of isotope internal standard mixed solution; shake and mix; and stand for 30 min. Add 5 mL of acetonitrile; vortex for 30 s; ultrasonically extract for 15 min; and centrifuge at 10,000 r/min and at 4 °C for 10 min. Take the supernatant and place it in a glass nitrogen blowpipe; slowly blow it with nitrogen at 40 °C to about 2 mL; add 8 mL of PBS and mix well; and wait for purification.

5.2.5 Beverages Weigh 1 g of specimen (accurate to

0.01 g); place it in a 2 mL polypropylene centrifuge tube; add 50 µL of isotope internal standard mixed solution; shake and mix; and stand for 30 min. Centrifuge at 10,000 r/min for 10 min. Take the supernatant to a 50 mL polypropylene centrifuge tube; add 10 mL of PBS and mix well; and wait for purification. NOTE. Clarified specimens (such as purified water, mineral water, etc.) can be directly added to PBS and mixed well without centrifugation.

5.3 Purification of specimen Restore the immunoaffinity column stored at low temperature to room temperature; and then let the original liquid in the column flow out naturally. Take all the specimen extracts in

5.2 and pass them through the column; let them flow out naturally; discard all the effluent; and then rinse the immunoaffinity column with 10 mL of water. After the water drips out, use a vacuum pump to dry the immunoaffinity column. Add 1 mL of methanol for elution; collect the eluate in a glass nitrogen blowpipe; and slowly blow dry with nitrogen at 40 °C. Accurately add

0.40 mL of methanol; vortex and mix for 10 s; then accurately add