GB 5009.292-2023National food safety standard - Determination of 8'-apo-beta,psi-caroten-8'-al in foods (English PDF)

Foreword

This document was issued on 6 September 2023 by the State Administration for Market Regulation; Standardization Administration of the PRC and takes effect on 6 March 2024.

It is a GB standard without the /T suffix: compliance is mandatory in China.

1 Scope

GB 5009.292-2023 covers the liquid chromatographic determination of beta-apo-8'-carotenal in food, the carotenoid colorant written in full as 8'-apo-beta,psi-caroten-8'-al. The sample is saponified with potassium hydroxide solution and the freed colorant taken up in n-hexane; the extract is concentrated, made up again in acetonitrile, then separated by high-performance liquid chromatography and measured on a diode array or ultraviolet-visible detector. That opening step is what the matrix forces: the compound is fat soluble and sits in the fat of the food, so it has to be released from the fat before any chromatography is worth running. The standard gives the principle, the reagents and materials, the instruments and equipment, the analysis steps, the calculation and presentation of results, the precision and the detection figures, with Appendix A on calibrating the mass concentration of the standard solution and Appendix B showing the standard chromatogram. Repeat determinations are to agree within 15 percent of their arithmetic mean, and on a two gram test portion the detection limit is 0.0002 grams per kilogram against a quantification limit of 0.0005. A mandatory national food safety standard for inspection laboratories, regulators and manufacturers checking declared colorant levels.

This standard specifies the liquid chromatography method for the determination of beta-

Apo-8'-carotene aldehyde in food.

This standard is applicable to the determination of beta-Apo-8'-carotene aldehyde in

flavored fermented milk, processed cheese, frozen drinks, candies, baked goods, semi-

solid compound seasonings, and beverages.

2 Principle

The sample is saponified with potassium hydroxide solution, and the free beta-Apo-8'-

carotene aldehyde is extracted with n-hexane. After the extract is concentrated and

reconstituted with acetonitrile, it is separated by high-performance liquid

chromatography, detected with a UV-visible light detector or diode array detector, and

quantified by an external standard method.

3 Reagents and materials

Unless otherwise stated, the reagents used in this method are of analytical grade and the

water is first-grade water specified in GB/T 6682.

3.1 Reagents

3.1.1 Acetonitrile (C2H3N): chromatographically pure.

3.1.2 Absolute ethanol (C2H6O).

3.1.3 n-hexane (C6H14).

3.1.4 Potassium hydroxide (KOH).

3.1.5 2,6-Di-tert-butyl-p-cresol (C15H24O, butylated hydroxytoluene, referred to as

BHT).

3.1.6 Formic acid (CH2O2): chromatographically pure.

3.2 Reagent preparation

3.2.1 Potassium hydroxide solution (200 g/L): Weigh 200 g of potassium hydroxide,

add water to dissolve and dilute to 1000 mL.

3.2.2 0.1% BHT-acetonitrile solution: Weigh 0.1 g of BHT, dissolve it in 100 mL

acetonitrile, and mix well.

3.2.3 0.1% formic acid (volume fraction) solution: Pipette 1 mL of formic acid and

dilute to 1 L with water.

3.3 Standard product

beta-Apo-8'-carotene aldehyde (C30H40O, CAS number: 1107-26-2): purity is >=98%, or a

reference material with national authentication and a Reference Material Certificate.

3.4 Preparation of standard solution

3.4.1 beta-Apo-8'-carotene aldehyde standard stock solution (10 ug/mL): Accurately

weigh 1 mg (accurate to 0.01 mg) of beta-apo-8'-carotene aldehyde standard, dissolve it

with 0.1% BHT-acetonitrile solution, and transfer it to a 100 mL brown volumetric flask;

make the volume up to 100 mL with 0.1% BHT-acetonitrile solution, and mix well;

store it at -18 degrees C, and the period of validity is 3 months.

NOTE: beta-Apo-8'-carotene aldehyde standard stock solution needs to be calibrated before use. See

4 Instruments and equipment

4.1 Liquid chromatograph: equipped with a diode array detector or UV-visible light

detector.

4.2 Balance: The sensitivity is 0.01 g and 0.01 mg, respectively.

4.3 Spectrophotometer.

4.4 Constant temperature oscillating water bath device.

4.5 Tissue masher.

4.6 Rotary evaporator.

5.1.3 Blank test

Except for adding no sample, conduct a blank test according to the measurement steps

in 5.1.2.

5.2 Instrument reference conditions

5.2.1 Chromatographic column: C18 column, 150 mmx4.6 mm (i.d.), the particle size

of 5 um, or a chromatographic column with equivalent performance.

5.2.2 Mobile phase: acetonitrile + 0.1% formic acid solution (95:5, volume ratio).

5.2.3 Flow rate: 1.0 mL/min.

5.2.4 Detection wavelength: 460 nm.

5.2.5 Column temperature: 40 degrees C.

5.2.6 Injection volume: 20 uL.

5.3 Preparation of standard curve

Inject the standard series working solutions into the high-performance liquid

chromatograph respectively, and measure the corresponding peak areas. Taking the

mass concentration of beta-Apo-8'-carotene aldehyde in the standard series working

solutions as the abscissa, and the peak area of beta-Apo-8'-carotene aldehyde as the

ordinate, draw a standard curve. See Appendix B for the chromatogram of the standard

solution of beta-Apo-8'-carotene aldehyde.

5.4 Determination of sample solution

The sample solution is injected into the high-performance liquid chromatograph to

obtain the peak area of the object to be measured; it is put into the standard curve to

calculate the mass concentration of beta-Apo-8'-carotene aldehyde in the test solution.

Compared with the standard solution, the change range of the retention time of the

compound chromatographic peak in the sample to be tested shall be within +/-2.5%.