GB 5413.20-2022National food safety standard - Determination of Choline in foods, milk and milk products for infants and young children (English PDF)

3.1 Reagents

3.1.1 Tris(hydroxymethyl)aminomethane [(CH2OH)3CNH2].

3.1.2 Phenol (C6H5OH).

3.1.3 Concentrated hydrochloric acid (HCl).

3.1.4 Sodium hydroxide (NaOH).

3.1.5 Choline oxidase. >=10U/mg; store at -20 degrees C.

3.1.6 Peroxidase. >=250U/mg; store at 2 degrees C～8 degrees C.

3.1.7 4-Aminoantipyrine (C11H13N3O).

3.1.8 Phospholipase D. >=60U/mg, store at -20°C.

3.2 Preparation of reagents

3.3 Standard product

Choline bitartrate standard product (C9H19NO7, relative molecular mass. 253.25, CAS number.

87-67-2). purity >= 99%, or the reference material certified by the state and awarded the

reference material certificate.

3.4 Preparation of standard solution

3.5 Materials

4.1 Analytical balance. The sensitivities are 0.1 mg and 0.001 g, respectively.

4.3 pH meter. Accuracy 0.01.

4.4 Spectrophotometer.

5.1 Sample preparation

5.1.1 Sample pretreatment

Accurately take 20g (accurate to 0.001g) of the mixed liquid specimen into a 100mL conical

flask; add 10mL of 3mol/L hydrochloric acid solution; stopper it and mix well.

Accurately take 5g (accurate to 0.001g) of the mixed semi-solid or solid specimen into a 100mL

conical flask; add 30mL of 1mol/L hydrochloric acid solution; stopper it and mix well.

5.1.3 Filtration

Filter the hydrolyzate with filter paper; if the filtrate is not clear; filter again with a 0.45µm

aqueous filter membrane syringe filter. The filtrate is collected for testing.

5.2 Determination

5.2.1 Drawing of standard curve

Respectively pipette 2.00mL, 4.00mL, 6.00mL, 8.00mL, and 10.00mL of choline standard

working solution (250mg/L) into a 10mL volumetric flask; make constant volume to the mark

with water; mix well; and prepare the standard series working solutions with concentrations of

50.0 mg/L, 100 mg/L, 150 mg/L, 200 mg/L and 250 mg/L, respectively. Prepare 6 colorimetric

tubes; one colorimetric tube is used as reagent blank (A); add 100µL of water; add 100µL of

standard series working solutions to the other 5 colorimetric tubes, respectively; and then add

3.00 mL of color developer, respectively; mix well; the colorimetric tube is placed in a water

bath at 37°C+/-2°C for 15 min for thermal-insulation reaction.

6.1 Calculation of net absorbance value

Usually formulated reagents shall produce a slight color; and the filtrate is not colorless due to

hydrolysis. In order to remove these interference factors, The respective blank values

(colorimetric tubes A and B) must be subtracted from the total absorbance value.

The net absorbance value of the specimen is calculated according to Formula (1).

6.2 Calculation of choline content

The choline content (by choline hydroxide) in the specimen shall be calculated according to

Formula (2).

10.1 Reagents

10.1.1 Concentrated hydrochloric acid (HCl).

10.1.2 Methanesulfonic acid (CH4O3S). chromatographically pure.

10.2 Preparation of reagents

10.2.1 Hydrochloric acid solution (1 mol/L). Pipette 85 mL of concentrated hydrochloric acid

and inject it into about 900 mL of water; and dilute to 1000mL.

10.2.2 Hydrochloric acid solution (1.7 mol/L). Pipette 145 mL of concentrated hydrochloric

acid and inject it into about 800 mL of water; and dilute to 1000mL.

10.3 Standard products

Choline bitartrate standard product (C9H19NO7, relative molecular mass. 253.25, CAS number.

87-67-2). purity >= 99%; or a standard substance certified by the state and granted a standard

substance certificate.

10.4 Preparation of standard solutions

10.5 Materials

10.5.1 0.45µm aqueous filter membrane syringe filter.

10.5.2 Purification column. C18 solid phase extraction cartridge 1.0mL or equivalent.

10.5.3 Syringe. 5mL or equivalent.

11.1 Ion chromatograph (IC). Equipped with a conductive detector.

11.2 Analytical balance. The sensitivity is 0.1mg and 0.001g, respectively.

11.3 Electrothermal constant temperature water bath device. The temperature can be controlled at 70 degrees C+/-2 degrees C.

11.4 Vortex mixer.

12.1 Sample preparation

12.1.1 Specimen extraction

12.1.2 Specimen purification

C18 solid phase extraction cartridge (1.0mL) is passed through successively with 10mL of

methanol and 15mL of water before use; and stand for activation for 30min. Dilute the

extraction solution 50 times (the dilution ratio can be adjusted appropriately according to the

concentration of choline in the specimen, and it shall be no less than 10 times) with water; take

about 15 mL of the diluted solution; pass through a 0.45µm aqueous filter membrane and a C18

solid phase extraction cartridge (1.0mL); discard the first 3mL; and collect the later eluate for

testing.

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