GB 5009.157-2016National food safety standard - Determination of Organic Acid in Foods (English PDF)

3.1 Reagents

3.1.1 Methanol (CH3OH). chromatographic purity.

3.1.2 Anhydrous ethanol (CH3CH2OH). chromatographic purity.

3.1.3 Phosphoric acid (H3PO4).

3.2 Preparation of Reagents

3.2.1 Phosphoric acid solution (0.1%). weigh-take 0.1 mL of phosphoric acid; add

water to dilute to 100 mL; mix it up.

3.2.2 Phosphoric acid - methanol solution (2 %). weigh-take 2 mL of phosphoric acid;

add methanol to dilute to 100 mL; mix it up.

3.3 Standards

5.2 Sample Processing

5.2.1 Fruit juice beverage, fruit juice and fruity carbonated beverage

Weigh-take 5 g (accurate to 0.01 g) of homogenized sample (heat up the sample to

remove carbon dioxide if the sample contains carbon dioxide), place it in 25 mL

volumetric flask; add water to the constant volume; adopt 0.45 µm aqueous

membrane to filter the sample; inject it into high-performance liquid chromatograph for

analysis.

5.2.2 Jelly and canned fruit

Weigh-take 10 g (accurate to 0.01 g) of homogenized sample, place it in 50 mL plastic

centrifuge tube, then, add 20 mL of water; homogenize and extract at 15,000 r/min for

0.45 µm aqueous membrane to filter the sample; inject it into high-performance liquid

chromatograph for analysis.

5.2.3 Gum-based candy

Weigh-take 1 g (accurate to 0.01 g) of homogenized sample, place it in 50 mL plastic

centrifuge tube which is equipped with a plug; add 20 mL of water, then, start

oscillation extraction on a swirling mixer for 5 min; start centrifugation for 3 min at

4,000 r/min, then, transfer the supernatant into 100 mL volumetric flask; add 20 mL of

water to re-extract the remains; combine the extract in the sample volumetric flask.

Use anhydrous ethanol (3.1.2) to dilute to the constant volume, shake it well.

Accurately remove-take 10 mL of supernatant and place it in 100 mL heart-shaped

bottle; add 10 mL of anhydrous ethanol (3.1.2) to the bottle, then, rotate and

concentrate it under 80°C±2°C till it approaches dryness; add 5 mL of anhydrous

ethanol (3.1.2) to continue the concentration, till it reaches absolute dryness. Use 1

mL x 1 mL of water to rinse the heart-shaped bottle twice. Transfer all the liquid to be

purified to solid phase extraction column (SAX) that's pre-activated, control the flow

rate at 1 mL/min~2 mL/min, then, remove the effluent. Use 5 mL of water to rinse the

purification column; use 5 mL of phosphoric acid - methanol solution (3.2.2) to elute it,

control the flow rate at 1 mL/min~2 mL/min; gather the eluent and place it into 50 mL

heart-shaped bottle; rotate the eluent under 45 °C and evaporate till it approaches

dryness; add 5 mL of anhydrous ethanol (3.1.2) to continue the concentration, till it

reaches absolute dryness. Use 1.0 mL of phosphoric acid solution (3.2.1) to oscillate

and dissolve the remains; adopt 0.45 µm membrane to filter it; inject it into

high-performance liquid chromatograph for analysis.

5.2.4 Solid beverage

Weigh-take 5 g (accurate to 0.01 g) of homogenized sample, place it in 50 mL beaker.

Add 40 mL of water to dissolve it, and transfer it into 100 mL volumetric flask. Adopt

anhydrous ethanol (3.1.2) to dilute to the constant volume; shake it well, place it

evenly for 10 min.

Accurately remove-take 20 mL of supernatant and place it in 100 mL heart-shaped

the balance for 5 min.

5.3.1.3 Column temperature. 40 °C.

5.3.1.4 Inlet volume. 20 µL.

5.3.1.5 Detection wavelength. 210 nm.

5.3.2 Determination of adipic acid

5.3.2.1 Chromatographic column. CAPECELL PAK MG S5 C18 column, 4.6 mm x 250

mm, 5 µm, or other chromatographic columns with equivalent performance.

5.3.2.2 Mobile phase. use 0.1% phosphoric acid solution - methanol=75+25 (volume

ratio) equivalent flow to elute for 10 min.

5.3.2.3 Column temperature. 40 °C.

5.3.2.4 Inlet volume. 20 µL.

5.3.2.5 Detection wavelength. 210 nm.

5.4 Draw a Standard Curve Line

Respectively inject standard series of working fluid into high-performance liquid

chromatograph; determine the corresponding peak height or peak area. Take the

concentration of standard working fluid as x-coordinate, take the peak height or peak

area as y-coordinate, and draw a standard curve line (please refer to Appendix A for

the standard curve line).

5.5 Determination of Sample Solution

Inject the sample solution into high-performance liquid chromatograph, obtain the

peak height or peak area. Obtain the concentration of organic acid in the test solution

in accordance with the standard curve line.