GB/T 5413.17-1997 Determination of pantothenic acid in infant formula and milk powder
Some standard content:
GB/T 5413.17—1997
This standard provides two methods. Method 1 is the microbiological method. For equivalence, the method of the American Association of Analytical Chemists (AOAC) is adopted: although the operation steps are complicated, the determination results are highly accurate. Method 2 is the high pressure liquid chromatography method, which is a rapid and accurate method determined by experiments.
Method 1 of this standard is the arbitration method.
From the date of implementation, this series of standards will replace GB5413—85. This standard is proposed by the China Light Industry Federation.
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 are the Food Hygiene Supervision and Inspection Institute of the Ministry of Health, Zhejiang Light Industry Research Institute, Harbin Morinaga Dairy Co., Ltd., and Nestle (China) Investment Service Co., Ltd. The main drafters of this standard are: Zhang Yujie, Wang Yun, Yin Xiaohong, Yang Jinbao, and Lv Weiqun. 292
National Standard of the People's Republic of China
Infant formula foods and milk powder
Determination of pantothenic acid
Milk powder and formula foods for infant and young children-Determination of pantothenic acidScope
This standard specifies the method for determining pantothenic acid by microbiological method and high pressure liquid chromatography. GB/T5413.17-—1997
Replaces GB5413—85
This standard method is applicable to the determination of pantothenic acid in infant formula foods and milk powder, and method 2 is applicable to the determination of pantothenic acid in milk powder. Method Microbiological method
2 Principle of the method
The pantothenic acid content in the sample can be calculated by using the relationship that the reproduction amount of Lactobacillus plantarum is proportional to the pantothenic acid content in the sample.
3 Reagents, strains and culture media
All reagents, if not specified, are of analytical grade. All experimental water, if not specified otherwise, is grade III water. 3.1 Physiological saline: 9g sodium chloride is dissolved in a 1000mL volumetric flask. 3.2 Calcium pantothenate: standard.
3.3 Acetic acid: c(HAc) is 0.2mol/L. Take 12mL glacial acetic acid and dilute to 1L with distilled water. 3.4 Toluene.
3.5 Sodium acetate: c(NaAc) is 0.2mol/L. Dissolve 16.4g anhydrous sodium acetate in water and dilute to 1000mL. 3. 6 Strains - Lactobacillus plantarum. 3.7 Culture medium
3.7.1 Lactobacillus agar culture medium: 15g photolysis, 5g yeast extract, 10g glucose, 100mL tomato juice; 2g potassium dihydrogen phosphate, 1g polysorbate monooleate, 10g agar, add distilled water to 1000mL, pH 6.8±0.2 (25℃). 3.7.2 Lactobacillus broth culture medium: 15g photolysis, 5g yeast extract, 10g glucose, 100mL tomato juice, 2g potassium dihydrogen phosphate, 1g polysorbate monooleate, add distilled water to 1000mL, pH 6.8±0.2 (25℃). 3.7.3 Culture medium for pantothenic acid determination: 40 g glucose, 20 g sodium acetate, 10 g vitamin-free acid hydrolyzed casein, 1 g dipotassium hydrogen phosphate, 1 g potassium dihydrogen phosphate, 0.4 g L-cystine, 0.1 g L-tryptophan, 0.4 g magnesium sulfate, 20 mg sodium chloride, 20 mg ferrous sulfate, 20 mg manganese sulfate, 20 mg adenine sulfate, 20 mg guanine hydrochloride, 20 mg uracil, 400 μg carotene, 200 μg thiamine hydrochloride, 0.8 μg biotin, 200 μg p-aminobenzoic acid, 1 mg niacin, 800 μg pyridoxine hydrochloride, 0.1 g sorbitan monooleate, add distilled water to 1000 mL, pH 6.7±0.1 (25°C).
Approved by the State Administration of Technical Supervision on May 28, 1997, and implemented on September 1, 1998
4 Instruments
Common laboratory instruments and spectrophotometer. 5 Preparation
5.1 Preparation of bacterial strains
GB/T5413.17--1997
5.1.1 Inoculate the plant lactobacillus stored in the straight column solid culture medium into a new lactobacillus agar culture medium, subculture it once after cultivation, and then transfer it to the lactobacillus broth culture medium for cultivation and use. 5.1.2 Centrifuge the bacterial suspension in 5.1.1 at 2000r/min for 2~~3min, pour out the supernatant, add 10mL of physiological saline (3.1), stir well, centrifuge again for 2~~3min, wash it 3 times, and aspirate 1.0ml of the bacterial suspension into 10mL of physiological saline (3.1). 5.1.3 Use a 721 spectrophotometer at a wavelength of 550nm, with physiological saline (3.1) as a control, to measure the turbidity value of the bacterial suspension in 5.1.2. This value should be between 60% and 80%.
5.2 Preparation of standard solution
5.2.1 Pantothenic acid standard stock solution concentration 40μg/mL. Accurately weigh 45~~55mg of calcium pantothenate standard (3.2), dissolve in 500mL of distilled water, add 10mL of acetic acid (3.3), add 100mL of sodium acetate (3.5) and dilute with water to the exact concentration of calcium pantothenate of 43.47μg/mL (i.e. the concentration of pantothenic acid is 40μg/mL), add toluene (3.4) and store in a refrigerator.
5. 2.2 Pantothenic acid intermediate stock solution: concentration 1μg/mL. Take 25mL of standard stock solution (5.2.1), add about 500mL of distilled water, 10mL of acetic acid (3.3), 100mL of sodium acetate (3.5), and dilute to 1L with water. Add toluene (3.4) and store in a refrigerator. 5.2.3 Pantothenic acid standard working solution: high concentration is 10ng/mL, low concentration is 5ng/mL. Aspirate 5.0mL of intermediate stock solution (5.2.2), dilute to 500.0mL with distilled water. Prepare before each measurement. Aspirate 5.0mL of intermediate stock solution (5.2.2), dilute to 1000mL with distilled water. Prepare before each measurement. .6 Determination
6.1 Sample treatment
Weigh a certain amount of sample, add 10mL buffer (24.2g Trizma Base dissolved in 200mL water), then add enough water, hydrolyze at 121℃ for 15min, cool, adjust pH to 4.5, then dilute to 250mL, filter, aspirate 4mL filtrate, dilute to the concentration of pantothenic acid is about 5ng/ml, and set aside.
6.2 Preparation of standard curve
Add distilled water, standard solution and pantothenic acid determination culture base to the culture tube in the order of Table 1, in triplicate. Table 1
Test tube No.
Distilled water, mL
Standard solution\, mL
Culture medium.mL
1), add low concentration standard working solution to test tube No. 3~7, and high concentration standard working solution to test tube No. 8~10. 6.3 Determination liquid sample
Add distilled water, sample solution and pantothenic acid determination medium to the culture tube in the order of Table 2, in triplicate. 294
Test tube No.
Distilled water, mL
Sample solution, mLwww.bzxz.net
Culture medium, mL
6.4 Inoculation
GB/T 5413.17—1997
Sterilize all the test tubes in 6.2 and 6.3 at 121℃ for 5 minutes, and then cool quickly. Use a capillary dropper to add one drop of the bacterial suspension in 5.1.2 to each of the above test tubes (except test tube No. 1 in the standard solution), mix well, and culture at 37℃±0.5℃ for 19~20h. 6.5 Determination
Use the blank tube as a control, measure the transmittance of the standard sample tube with the highest concentration, and read again after 2 hours. If the second result shows a transmittance of 2%, take out all the test tubes to measure their transmittance and record it. 7 Calculation
Use the pantothenic acid content of the standard sample as the horizontal axis and the transmittance as the vertical axis to make a working curve. According to the transmittance measured by the sample, the average value of the pantothenic acid content is obtained from the standard curve, and then calculated according to formula (1): Pantothenic acid content in the sample (mg/10g or mg/10mL) = required × 1000 × 100…m
Where: the average content of pantothenic acid in the sample is obtained from the curve, μg; F
dilution factor,
m--—the mass (or volume) of the sample, g (or mL). 8 Allowable difference
The difference between the two measured values of the same sample shall not exceed 10% of the average value of the two measurements. Method 2 High-pressure liquid chromatography
9 Method summary
The sample is extracted with water, filtered, and after removing the protein, the ultraviolet absorption of the pantothenic acid carboxyl group in the filtrate is determined by high-pressure liquid chromatography. 10 Reagents
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. 10.1 Silicone resin: food additive.
10. 2 Hydrochloric acid: c(HCI) is 0. 1 mol/L. 10.3 Zinc sulfate: c(ZnSO) is 15 g/L. 10.4 Acetonitrile: spectrally pure.
10.5 Potassium dihydrogen phosphate: c(KH,PO,) is 0.02 mol/L. Weigh 2.72 g of potassium dihydrogen phosphate and dissolve it in water to 1000 mL. 10.6 Acetonitrile-0.02 mol/L potassium dihydrogen phosphate solution: volume ratio 1:9. Add 9 volumes of 0.02 mol/L potassium dihydrogen phosphate to 1 volume of acetonitrile and adjust the pH to 3 with hydrochloric acid.
11 Instruments
Common laboratory instruments and:
11.1 Centrifuge.
11.2 Membrane filter: 1.0 μm membrane. 11.3 Phosphorus pentoxide desiccator.
11.4 High pressure liquid chromatograph: with UV detector. 12 Operation steps
12.1 Preparation of sample solution
GB/T 5413. 171997
12.1.1 Accurately weigh about 10g of sample, add 60mL of water and 1 drop of silicone resin (10.1), transfer to a centrifuge tube, adjust the pH to 4-5 with hydrochloric acid solution (10.2), add 10mL of zinc sulfate solution (10.3), mix thoroughly, and centrifuge. 12.1.2 Filter the supernatant with filter paper, collect the filtrate with a 100mL volumetric flask, rinse the residue in the centrifuge tube with a small amount of water on the same filter paper, wash the filter paper with a small amount of water, combine the washing liquid and the filtrate, and add water to 100mL. Filter through a membrane filter (11.2) as the sample solution. 12.2 Preparation of standard solution
12.2.1 Pantothenic acid standard stock solution: concentration is 1mg/mL. Weigh 1.0918 g of calcium pantothenate dried in the desiccator (11.3) and dissolve it in water to 1000 mL. 12.2.2 Pantothenic acid standard working solution
Accurately pipette 1, 2, 4, 8, and 12 mL of the standard stock solution (12.2.1) and add water to make 100 mL respectively as standard working solution. The pantothenic acid concentrations are 10, 20, 40, 80, and 120 μg/mL respectively. 12.3 Determination conditions
Filling agent: chemically bonded stearic silane. Chromatographic column: inner diameter 6.2 mm, length 25 cm.
Eluent: acetonitrile-0.02 mol/L potassium dihydrogen phosphate (1:9). Flow rate: 1 mL/min.
Determination wavelength: 200 nm.
1 The inner diameter and length of the column can be freely selected.
2 The eluent composition ratio and flow rate can be changed according to the conditions of the chromatographic column, and there is no uniform regulation. The retention time of pantothenic acid can be set to 8 to 12 minutes. 3 The maximum absorption of pantothenic acid is near the wavelength of 194nm, but the mobile phase also absorbs here, so the measurement wavelength is set to 200nm. 12.4 Standard curve
Pipette 10μL of the standard working solution (12.2.2) respectively, inject it into the high pressure liquid chromatography, and draw a standard curve based on the peak height. Note: The injection volume and sensitivity are related to the injection method of the high pressure liquid chromatography, so the injection volume can vary between 3 and 20μL. 12.5 Sample determination
Accurately pipette 10μL of the sample solution (12.1), inject it into the high pressure liquid chromatograph, and calculate the concentration of pantothenic acid in the sample solution from the obtained peak height from the standard curve.
13 Expression of analysis results
100××100
Content of pantothenic acid in the sample (mg/100g)
Where: c—mass concentration of pantothenic acid in the sample solution, μg/100g;
Content of calcium pantothenate (mg/kg) = content of pantothenic acid (mg/100g) × 1.087Content of sodium pantothenate (mg/kg) = content of pantothenic acid (mg/100g) × 1.10014 Allowable difference
The difference between the two measured values of the same sample shall not exceed 10% of the average value of the two measurements. 296
(2)1) 10μL, injected into the high pressure liquid chromatograph, and the peak height obtained was used to calculate the concentration of pantothenic acid in the sample solution from the standard curve.
13 Expression of analysis results
100××100
Pantothenic acid content in the sample (mg/100g)-
Where: c-mass concentration of pantothenic acid in the sample solution, μg/100g;-Weigh the mass of the sample.
Calcium pantothenate content (mg/kg) = pantothenic acid content (mg/100g) × 1.087Sodium pantothenate content (mg/kg) = pantothenic acid content (mg/100g) × 1.10014Allowable difference
The difference between the two measured values of the same sample shall not exceed 10% of the average value of the two measurements. 296
(2)1) 10μL, injected into the high pressure liquid chromatograph, and the peak height obtained was used to calculate the concentration of pantothenic acid in the sample solution from the standard curve.
13 Expression of analysis results
100××100
Pantothenic acid content in the sample (mg/100g)-
Where: c-mass concentration of pantothenic acid in the sample solution, μg/100g;-Weigh the mass of the sample.
Calcium pantothenate content (mg/kg) = pantothenic acid content (mg/100g) × 1.087Sodium pantothenate content (mg/kg) = pantothenic acid content (mg/100g) × 1.10014Allowable difference
The difference between the two measured values of the same sample shall not exceed 10% of the average value of the two measurements. 296
(2)
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