This standard specifies the method for the determination of vitamin B6 by reverse phase liquid chromatography. This standard is applicable to the determination of vitamin B6 in infant formula and milk powder. GB/T 5413.13-1997 Determination of vitamin B6 in infant formula and milk powder GB/T5413.13-1997 Standard download decompression password: www.bzxz.net

GB/T5413.13-1997
Vitamin B. The determination of vitamin B includes chemical method, microbiological method, spectrophotometry, gas chromatography, etc. The reversed phase high pressure liquid chromatography method given in this standard is determined after repeated experiments based on reference to relevant domestic and foreign data. It has the advantages of rapidity, economy and high accuracy. This series of standards will replace GB5413-85 from the date of implementation. This standard is proposed by the China Light Industry General Association.
This standard is under the jurisdiction of the National Dairy Standardization Center. The responsible drafting unit of this standard: National Dairy Quality Supervision and Inspection Center. The participating drafting units of this standard: Food Sanitation Supervision and Inspection Institute of the Ministry of Health, Zhejiang Light Industry Research Institute, Harbin Morinaga Dairy Co., Ltd., Nestle (China) Investment Service Co., Ltd. The main drafters of this standard: Yang Jinbao, Lai Ming, Wang Yun, Liu Bo. 273
National Standard of the People's Republic of China
Infant and young children formula foods and milk powder
Determination of vitamin B6
Milk powder and formula foods for infant and young children-Determination of vitamin B, contentScope
This standard specifies the method for the determination of vitamin B6 by reversed phase liquid chromatography. This standard is applicable to the determination of vitamin B6 in infant and young children formula foods and milk powder. 2 Method Summary
GB/T 5413.13—1997
Replaces GB5413--85
After pretreatment such as hot water extraction, the sample is separated by C1. chromatographic column and detected by fluorescence detector. The content of vitamin B6 (pyridoxine, pyridoxal, pyridoxamine) is quantified by external standard method.
3 Reagents
All reagents, if the specifications are not specified, are of analytical grade; all experimental water, if no other requirements are specified, is grade tertiary water. 3.1 Taka-Diastase. 3.2 Hydrochloric acid solution, c(HCI) is 5.0 mol/L, 0.1 mol/L. 3.3 Sodium hydroxide solution, c(NaOH) is 5.0 mol/L, 0.1 mol/L. 3.4 Glacial acetic acid: high grade.
3.5 Anhydrous methanol: chromatographic grade.
3.6 Sodium octane sulfonate: high grade.
3.7 Triethylamine: volume fraction ≥ 99.9%. 3.8 Standard solution
3.8.1 Pyridoxine, pyridoxal, pyridoxamine standard stock solutions, concentration is 200 μg/mL. Accurately weigh 20.00 mg of pyridoxine, pyridoxal, pyridoxamine standard products, dissolve in water and dilute to 100 mL volumetric flask. 3.8.2 Standard working solutions of pyridoxine, pyridoxal and pyridoxamine, all with a concentration of 2.0 μg/mL. Take 1.0 mL of the standard stock solution (3.8.1) and dilute to 100 mL with water. 4 Instruments
Common laboratory instruments and:
4.1 Ultrasonic oscillator.
4.2 High pressure liquid chromatograph: with fluorescence detector. 4.3 Chromatographic column: Varian MCH-10μm 30cm×4mm, Cia or chromatographic column with equivalent performance. 4.4 Acidity meter.
Approved by the State Administration of Technical Supervision on May 28, 1997 274
Implemented on September 1, 1998
5 Operating procedures
5.1 Sample pretreatment
5.1.1 Samples containing starch
GB/T 5413.13-—1997Www.bzxZ.net
Accurately weigh 5.0 g of sample and put it into a 150 mL conical flask. Add 0.5 g of Taka amylase (3.1) and 25 mL of 45-50 ℃ distilled water. Mix well, remove the air in the flask with nitrogen, cover the flask with a stopper, and place it in a 45 ℃ oven for 30 min. Take it out and cool it to room temperature. 5.1.2 Starch-free samples
Weigh 5.0 g of sample accurately, put it into a 150 mL conical flask, add 25 mL of 60°C distilled water, shake, and let it stand for 5 to 10 minutes to allow the sample to fully dissolve and cool to room temperature.
5.2 Preparation of test solution
5.2.1 Slowly adjust the pH value of the sample solution to 1.70 with hydrochloric acid solution (3.2), let it stand for about 1 minute, and then adjust the pH value of the sample solution to 4.50 with sodium hydroxide solution (3.3).
5.2.2 Transfer the sample solution to a 50 mL volumetric flask, rinse the conical flask repeatedly with distilled water, combine the washings in a 50 mL volumetric flask, and dilute to 50 mL with water.
5.2.3 Place the above volumetric flask in an ultrasonic water bath (4.1) and shake for 10 minutes. 5.2.4 Take another 50mL volumetric flask, put a triangular funnel and filter paper on it, pour the sample solution into it, and filter naturally. The filtrate is then filtered under pressure through a 0.45um microporous filter membrane and collected in a test tube, which is the sample solution to be tested. 5.3 Chromatographic conditions
Detection sensitivity: 0.002AU/MV.
Detector conditions: fluorescence excitation wavelength E: 293nm, emission wavelength Em: 395nm. Column temperature: 30℃.
Flow rate: 1.00mL/min
Mobile phase: 5.0% methanol by volume, 0.20g/100mL sodium octane sulfonate, 0.25% triethylamine aqueous solution by volume, pH adjusted to 3.00 with acetic acid.
5.4 Quantitative analysis (external standard method)
Inject a certain amount of standard working solution (3.8.2) into the chromatograph to obtain the peak height (or peak area) A of component i; inject an equal volume of sample solution into the chromatograph to obtain the peak height (or peak area) B of component i. 6 Expression of analysis results
Content of vitamin B12 in the sample (mg/100g) = X+ X+ Xamine Note: The content of vitamin B12 in the sample is the sum of the contents of each component. The content of each component in the sample (mg/100g) = ×B:×V×100m × A, X 1000
Where: B is the peak height or area of ​​component i in the sample; A, ———the peak height or area of ​​component i in the standard working solution; Cc. The mass concentration of component i in the standard working solution, ug /mLm—-the mass of the sample, g;
The volume of the sample solution, mL.
（1）
Allowable difference
GB/T5413.13-1997
The difference between two measured values ​​of the same sample shall not exceed 10% of the average value of the two measurements. 276
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