This standard specifies the determination of choline. This standard is applicable to the determination of choline in infant formula and milk powder. The minimum detection limit of this method is about 2mg/100g. GB/T 5413.20-1997 Determination of choline in infant formula and milk powder GB/T5413.20-1997 Standard download decompression password: www.bzxz.net

GB/T5413.20—1997
This standard refers to the method of Nestle Company of Switzerland.
This series of standards shall replace GB5413-85 from the date of implementation. This standard is proposed by China Light Industry General Association.
This standard is under the jurisdiction of National Dairy Standardization Center. The responsible drafting units of this standard are: Nestle (China) Investment Service Co., Ltd., National Dairy Quality Supervision and Inspection Center. The participating drafting units of this standard are: Food Hygiene Supervision and Inspection Institute of Ministry of Health, Zhejiang Light Industry Research Institute, Harbin Morinaga Dairy Co., Ltd.
The main drafter of this standard is Zhang Fucai.
National Standard of the People's Republic of China
Milk powder and formula foods for infant and young childrcn-Determination of choline
This standard specifies the determination of choline.
This standard is applicable to the determination of choline in infant formula and milk powder. The minimum detection limit of this method is about 2mg/100g. 2 Principle of the method
The sample is acid-lyzed, filtered, oxidized, and then colorimetrically determined. 3 Reagents
GB/T5413.20—1997
Replaces GB5413--85
All reagents, if the specifications are not specified, are analytically pure; all experimental water, if no other requirements are specified, refers to grade tertiary water. 3.10.05mol/IT buffer (pII8.0): Contains 0.05% phenol by mass. Dissolve 6.057g tris(hydroxymethyl)aminomethane and 0.5g phenol in a 100GmL volumetric flask filled with about 5001mL of distilled water, and then dilute to the scale with distilled water. If necessary, adjust the pH to 8.0 with 6 mol/L hydrochloric acid. 3.2 Coloring agent for enzyme reaction
Dissolve 7.81 mg choline oxidase (12.8 U/mg), 1.31 mg peroxidase (190 U/mg) and 7.5 mg 1-aminoantipyrine in a 50 mL volumetric flask and dilute to scale with Tris buffer solution (3.1). 3.3 Standard solution: containing 250 μg/mL choline oxidase. Dissolve 23 mg creatine bitartrate in a 100 mL volumetric flask and dilute to scale with distilled water. Use a graduated pipette to draw 10 mL of the solution into a 100 mL volumetric flask and dilute to scale with distilled water. 3.4 Sodium hydroxide solution: mass fraction is 60% 3.5 Hydrochloric acid: c (HCl) is 3 mol/L.
3.6 Hydrochloric acid: c (HCl) is 1 mol/L.
4 Apparatus
Bring laboratory instruments and:
4.1 Reflux extractor.
4.2 Chromatographic column, 25 cm long, 1 cm inner diameter. 4.3 Spectrophotometer.
Approved by the State Administration of Technical Supervision in 1997-0528
Implemented on 1998-09-01
5 Operating steps
GB/T5413.20-—1997
5.1 Preparation of samples
Use mixing or grinding to homogenize the sample, and weigh the sample accurately with an accuracy of 0.01 g, so that the choline content is about 1-~10 mg (calculated as hydroxide).
5.1.1 Solid sample
Weigh 5 g of the mixed sample into a 250 mL flat-bottomed ground flask, add 30 mL of hydrochloric acid (3.6), and stir. 5.7.2 Liquid sample
Weigh 20 g of the mixed sample into a 250 mL flat-bottomed ground flask, add 10 mL of hydrochloric acid (3.5), and stir. 5.2 Hydrolysis
Connect the container containing the sample to a reflux condenser and heat in a 70 water bath for 5 h with magnetic stirring. Cool. Adjust the pH to 3.4-~3.6 with sodium hydroxide solution (3.4). Cool again if necessary, transfer to a 50 mL volumetric flask, and dilute to 60° with distilled water. 5.3 Filtration
Filter the hydrolyzate (5.2). The filtrate should be clear: if it is not clear, filter again through a 0.45um filter membrane. If the filtrate cannot be clarified due to the nature of the sample, or if filtration is difficult, it is recommended to adjust the pH to 44.5 instead of 3.4~3.6 (see 5.2)
5.4 Enzyme reaction
Prepare 3 test tubes (A, B, C) for each test and add reagents according to Table 1. Table 1
Test tube A
Filtrate to be analyzed
Steamed stuffing water
Coloring agent
Reagent blank
Test tube B
Liquid blank
Cover the test tube with a sealing protective film and shake. Place the test tube in a 37C water bath and keep it warm for 10 minutes. 5.5 Colorimetric determination
Adjust the wavelength of the spectrophotometer to 505nm. Use distilled water as a blank. Test tube C
5.6 Standard curve
Use a graduated pipette to transfer 2, 4, 6, and 8 mL of the standard solution (3.3) into four 10 mL volumetric flasks and dilute to the mark with distilled water. ml
Prepare 6 test tubes, one for the reagent blank (A), and the other five test tubes, numbered 1 to 5, for the standard solution and the four dilutions of the standard solution. Add reagents according to Table 2. Reagents
Dilution 1 (50μg/mL)
Dilution 2 (100gg/ml)
Dilution 3 (150g/mL)
Dilution 4 (200mg/ml)
Standard solution (250ug/mL)
Distilled water
Coloring agent
GB/T 5413.201997
Cover the test tube with a sealed protective film, shake, place the test tube in a 37℃ water bath for 10 minutes, and then follow the steps after 5.5. 6 Expression of analytical results
6.1 Calculation of net transmittance
Usually, reagents that are not freshly prepared will produce a slight color, and the filtrate is not colorless due to hydrolysis. In order to eliminate these interfering factors, the respective blank transmittances (tube A and tube B) should be subtracted from the total transmittance. A-Ao-Au-Ar
Wherein A—net transmittance:
Aut—total transmittance (tube C):
A—reagent transmittance (tube A):
A—extract transmittance (tube B).
A and A should not be greater than 20% of the total transmittance. For the standard curve, A=0. 6.2 Calculation of results
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Find the position of the net absorbance value on the standard curve. And record the corresponding concentration. The choline content expressed in milligrams of choline hydroxide per 100g of sample is calculated according to formula (2): Choline content in sample (mg/100g)==×V×100mX1000
Where: c——Choline concentration found on the white standard curve·ug/mL; V—Volume of the diluted hydrolyzate (usually 50mL), mL: m
7Allowance difference
-mass of the sample, g.
(2)
The difference between two independent results obtained using the same method on the same sample under the same operating conditions (same operator, sample preparation instrument, same laboratory, short time interval) should not exceed 8% of the average of the two results.
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