GB 5009.287-2022National food safety standard - Determination of bixinin foods (English PDF)

3.1 Reagents

3.1.1 Methanol (CH4O). chromatographically pure.

3.1.2 Anhydrous ethanol (C2H6O). chromatographically pure.

3.1.3 Acetonitrile (C2H3N). chromatographically pure.

3.1.4 petroleum ether. with a boiling range of 30 C ~ 60 C.

3.1.5 Dichloromethane (CH2Cl2). chromatographically pure.

3.1.6 2,6-di-tert-butyl-4-methylphenol (C15H24O, BHT for short).

3.1.7 Concentrated ammonia water (NH3  H2O), with a concentration of 25% ~ 28%.

3.1.8 Glacial acetic acid (C2H4O2).

3.2 Preparation of Reagents

3.2.1 BHT-methanol solution. weigh-take 0.5 g of BHT (3.1.6); use 500 mL of methanol to

dissolve it.

3.2.2 BHT-ethanol solution. weigh-take 0.5 g of BHT (3.1.6); use 500 mL of anhydrous ethanol

to dissolve it.

3.2.3 70% aqueous acetonitrile. measure-take 350 mL of acetonitrile and 150 mL of water, mix them up; add 0.5 g of BHT (3.1.6); shake to dissolve it.

3.2.4 5% ammonia ethanol solution. measure-take 50 mL of ammonia water and 950 mL of anhydrous ethanol, mix them up; add 1 g of BHT (3.1.6); shake to dissolve it.

3.2.5 BHT-petroleum ether solution. weigh-take 0.5 g of BHT (3.1.6); use 500 mL of petroleum

ether to dissolve it.

3.2.6 BHT-dichloromethane solution. weigh-take 0.5 g of BHT (3.1.6); use 500 mL of

dichloromethane to dissolve it.

3.2.7 2% acetic acid aqueous solution. measure-take 20 mL of glacial acetic acid; add it to 980 mL of water; mix it up.

3.3 Reference Substances

3.3.1 Norbixin (C24H28O4, CAS. 542-40-5). purity  98.0%, or reference substances certified

by the state and awarded with a reference substance certificate.

3.3.2 Bixin (C25H30O4, CAS. 6983-79-5). purity  98.0%, or reference substances certified by

the state and awarded with a reference substance certificate.

3.4 Preparation of Standard Solutions

3.4.1 Standard stock solution (100 mg/L). accurately weigh-take 10.0 mg of norbixin reference

substance (3.3.1) with the converted content into a 100 mL brown volumetric flask; add BHT-methanol solution (3.2.1) to dissolve and reach a constant volume, then, mix it up. Accurately weigh-take 10.0 mg of bixin reference substance (3.3.2) with the converted content into a 100 mL brown volumetric flask; add BHT-ethanol solution (3.2.2) to dissolve and reach a constant volume, then, mix it up. Store the standard stock solution at 18 C in the dark. The shelf life is 6 months.

3.4.2 Mixed standard working solution. respectively absorb-take 0.05 mL, 0.1 mL, 0.2 mL, 0.5

mL, 1.0 mL, 2.0 mL and 5.0 mL of the norbixin and bixin standard stock solution (3.4.1) into a

10 mL brown volumetric flask. Add 70% aqueous acetonitrile (3.2.3) and reach a constant volume; mix it up. The concentration of the norbixin and bixin mixed standard series working solutions is respectively. 0.5 mg/L, 1.0 mg/L, 2.0 mg/L, 5.0 mg/L, 10 mg/L, 20 mg/L and 50 mg/L. Prepare them right before use.

Weigh-take 0.5 g ~ 5 g (accurate to 0.01 g) of specimen into a 50 mL plastic centrifuge tube with a stopper. Add 2 g ~ 5 g of diatomaceous earth; use a glass rod to thoroughly mix the sample and diatomaceous earth; stir, until it becomes loose and granular. Add 20 mL of 5% ammonia ethanol solution (3.2.4). The following steps are the same as 5.1.2.1.

5.1.2.3 Liquid samples Weigh-take 0.5 g ~ 5 g (accurate to 0.01 g) of specimen into a 50 mL plastic centrifuge tube with a stopper. Add 5 mL of 5% ammonia ethanol solution (3.2.4). Reserve it for purification.

5.1.3 Sample purification Add 2 mL of ammonia water to the specimen extract; use water to dilute to about 20 mL. Add

20 mL of BHT-petroleum ether solution (3.2.5); perform vortex oscillation for 1 min; centrifuge it and discard the upper layer of solution. Repeatedly add 20 mL of BHT-petroleum ether solution (3.2.5); perform vortex oscillation for 1 min; centrifuge it and discard the upper layer of solution. Add 2 mL of glacial acetic acid and 25 mL of BHT-dichloromethane solution (3.2.6)

to the lower layer of solution; perform vortex oscillation for 1 min; centrifuge it and discard the upper layer of solution. Add 1 mL of BHT-ethanol solution (3.2.2) to the lower layer of dichloromethane solution. At 35 c, adopt the nitrogen concentration device to concentrate to the volume of about 1 mL. Use BHT-ethanol solution (3.2.2) to assist the transferring of the residual solution to a 5 mL brown volumetric flask; reach a constant volume and shake it well.

Use 0.45 m filter membrane (3.5.2) to filter it into an injection vial. Reserve it for liquid chromatographic determination.

NOTE. avoid light during the pre-processing.

5.2 Instrument Reference Conditions

5.2.1 Chromatographic column. C18 chromatographic column, column length. 150 mm, inner diameter. 4.6 mm, particle size. 5 m, or equivalent chromatographic column.

5.2.2 Mobile phase. acetonitrile + 2% aqueous acetic acid = 70 + 30.

5.2.3 Flow rate. 1.0 mL/min.

5.2.4 Column temperature. 30 C.

5.2.5 Injection volume. 10 L.

5.2.6 Detection wavelength. 458 nm.

5.3 Drawing of Standard Curve Respectively inject the mixed standard working solution prepared in 3.4.2 into the liquid chromatograph (see Figure A.1 in Appendix A for the liquid chromatogram of the cis-norbixin and cis-bixin standard solutions). Use the retention time for qualitative determination to determine the corresponding peak area; take the mass concentration of the mixed standard