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GB 5413.17-2010 English PDF

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GB 5413.17-2010: National food safety standard -- Determination of pantothenic acid in foods for infants and young children, milk and milk products
Status: Obsolete

GB 5413.17: Evolution and historical versions

Standard IDContents [version]USDSTEP2[PDF] delivered inStandard Title (Description)StatusPDF
GB 5413.17-2010English359 Add to Cart 3 days [Need to translate] National food safety standard -- Determination of pantothenic acid in foods for infants and young children, milk and milk products Obsolete GB 5413.17-2010
GB/T 5413.17-1997English199 Add to Cart 2 days [Need to translate] Milk powder and formula foods for infant and young children--Determination of pantothenic acid Obsolete GB/T 5413.17-1997

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Basic data

Standard ID GB 5413.17-2010 (GB5413.17-2010)
Description (Translated English) National food safety standard -- Determination of pantothenic acid in foods for infants and young children, milk and milk products
Sector / Industry National Standard
Classification of Chinese Standard C53;X82
Classification of International Standard 67.100.10
Word Count Estimation 9,979
Date of Issue 2010-03-26
Date of Implementation 2010-06-01
Older Standard (superseded by this standard) GB/T 5413.17-1997
Adopted Standard AOAC 945.74, IDT
Regulation (derived from) Circular of the Ministry of Health (2010)7
Issuing agency(ies) Ministry of Health of the People's Republic of China
Summary This Chinese standard specifies the infant foods and milk products Determination of pantothenic acid. This standard applies to infant foods and milk products Determination of pantothenic acid.

GB 5413.17-2010: National food safety standard -- Determination of pantothenic acid in foods for infants and young children, milk and milk products


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National food safety standard.Determination of pantothenic acid in foods for infants and young children, milk and milk products National Standards of People's Republic of China People's Republic of China Ministry of Health issued Issued on. 2010-03-26 2010-06-01 implementation National Food Safety Standard Infant food and dairy products Determination of pantothenic acid National food safety standard Determination of pantothenic acid in foods for infants and young children, milk and milk products

Foreword

This standard is equivalent to the first law of international analysts Institute (AOAC) 945.74 Pantothenic Acid in Vitamin Preparations. This standard replaces GB/T 5413.17-1997 "infant formula milk powder and pantothenic acid determination." This standard compared with GB/T 5413.17-1997, the first major change in law as follows. - Added tris buffer preparation methods; - Determine the measurement wavelength; - Added text description of the standard curve plotted. The second major change in law as follows. - Replacement of the column; - Changed the mobile phase; - Increasing the enzymatic treatment of starch-containing samples. Appendix A of this standard is an informative annex. This standard replaces the standards previously issued as follows. --GB 5413-1985, GB/T 5413.17-1997. National Food Safety Standard Infant food and dairy products Determination of pantothenic acid

1 Scope

This standard specifies the method for determination of infant foods and dairy pantothenic acid. This standard applies to infant food and dairy products Determination of pantothenic acid.

2 Normative references

The standard file referenced in the application of this standard is essential. For dated references, only the edition date of the note Apply to this standard. For undated references, the latest edition (including any amendments) applies to this standard. The first method microbiological method Principle 3 Use of Lactobacillus (Lactobacillus plantarum) ATCC 8014 Dui specific pantothenic acid, pantothenic acid sample containing the growth in productivity Health acidity and the formation of the optical density to determine the content of pantothenic acid.

4 Reagents and materials

Unless otherwise specified, the reagents used in this method are analytical reagents water GB/T 6682 provisions of secondary water. 4.1 0.9% saline. 9.0 g sodium chloride are dissolved in 1000 mL of water, packed in a stoppered test tube, each tube 10 mL, 121 ℃ sterilization 15 min. Ready to once a week. 4.2 calcium pantothenate standard. 4.3 acetic acid solution (0.2 mol/L). draw 12 mL of glacial acetic acid diluted with water to 1000 mL. 4.4 toluene (C7H8). 4.5 sodium acetate solution (0.2 mol/L). 16.4 g of anhydrous sodium acetate was dissolved in water and diluted to 1000 mL. 4.6 strains. Lactobacillus (Lactobacillus plantarum) ATCC 8014. 4.7 Medium 4.7.1 Lactobacillus agar medium. light tryptone 15 g, yeast extract 5 g, glucose 10 g, tomato juice, 100 mL, potassium dihydrogen phosphate 2 g, polyethylene sorbitan monooleate 1 g, agar 10 g, add distilled water to 1000 mL, adjust pH to 6.8 ± 0.2 (20 ℃ ~ 25 ℃). 4.7.2 Lactobacillus broth. light tryptone 15 g, yeast extract 5 g, glucose 10 g, tomato juice, 100 mL, potassium dihydrogen phosphate 2 g, polyethylene sorbitan monooleate 1 g, add distilled water to 1000 mL, adjust pH to 6.8 ± 0.2 (20 ℃ ~ 25 ℃). 4.7.3 pantothenic acid measurement medium. Glucose 40 g, sodium acetate, 20 g, no vitamin acid hydrolysis of casein 10 g, dipotassium phosphate 1 g, Potassium dihydrogen phosphate 1 g, L- cystine 0.4 g, L- tryptophan 0.1 g, magnesium sulfate 0.4 g, sodium chloride 20 mg, ferrous sulfate 20 mg, sulfuric acid Manganese 20 mg, adenine sulfate 20 mg, guanine hydrochloride 20 mg, uracil, 20 mg, carotene, 400 μg, thiamin hydrochloride 200 μg, Biotin 0.8 μg, p- amino acid 200 μg, nicotinic acid 1 mg, pyridoxine hydrochloride 800 μg, polyethylene sorbitan monooleate 0.1 g, plus steamed Distilled water to 1000 mL, adjust pH to 6.7 ± 0.1 (20 ℃ ~ 25 ℃). 4.8 Tris buffer solution. Weigh 24.2 g Trizma Base in the beaker, add 200 mL of water to dissolve. 4.9 hydrochloric acid (0.1 mol/L). draw 8.3 mL of hydrochloric acid, diluted with water to 1000 mL. 4.10 standard solution 4.10.1 pantothenic acid stock standard solution (40 μg/mL). Weigh 45 mg ~ 55 mg calcium pantothenate standard (4.2), dissolved in 500 mL Distilled water, add 10 mL of acetic acid solution (4.3), added to 100 mL of sodium acetate (4.5), diluted with water to a precise concentration of calcium pantothenate 43.47 μg/mL (ie, pantothenic acid at a concentration of 40 μg/mL), add 0.5 mL of toluene (4.4) stored at 2 ℃ ~ 4 ℃ refrigerator shelf life of 4 Months. 4.10.2 pantothenic acid intermediate stock solution (1 μg/mL). 25 mL of stock standard solution (4.10.1), distilled water was added 500 mL, in acetic acid Solution 10 mL (4.3), sodium acetate 100 mL (4.5), and diluted with water to 1 L. Add 0.5 mL of toluene (4.4) stored in a refrigerator (2 ℃ ~ 4 ℃), storage period of one month. 4.10.3 pantothenic acid working standard solution (10 ng/mL, 5 ng/mL). 5.0 mL twice withdrawing intermediate stock solution (4.10.2) were given water The volume to 500 mL and 1000 mL, prepared before use. 5. Apparatus 5.1 Spectrophotometer. 5.2 pH meter. accuracy of 0.01. 5.3 Vortex. 5.4 Balance. a sense of the amount of 0.1 mg. 5.5 Incubator. 36 ℃ ± 0.5 ℃. 5.6 Centrifuge. ≥2000 rpm rev/min. Step 6 Analysis 6.1 Preparation of test broth 6.1.1 The Lactobacillus (Lactobacillus plantarum) ATCC 8014 lyophilized powder into Lactobacillus broth (4.7.2) Tubes, 36 ℃ ± 1 ℃ cultured 24 h. Then transfer to Lactobacillus agar medium (4.7.1) in a test tube, 36 ℃ ± 1 ℃ cultured 24 h. Develop good Lactobacillus agar medium (4.7.1) in vitro cultures of bacteria as a reserve. 6.1.2 from stock strains were transferred to three medium Lactobacillus agar medium (4.7.1) in a test tube, into an incubator 36 ℃ ± 1 ℃ cultured 24 h. Subculture monthly, as the monthly tube stored in the refrigerator. Monthly from monthly tube re-inoculated 3 adapter Save the new strain of tubes. 6.1.3 dated from culture tubes inoculated in a revaccination one Lactobacillus agar medium (4.7.1) tube, 36 ± 1 ℃ ℃ cultured 24 h, As a Japanese daily inoculated tube measurement. 6.1.4 inoculated a Lactobacillus broth (4.7.2) from the date of inoculation tube, 36 ℃ ± 1 ℃ cultured 24 h. From under sterile conditions The heart of the culture medium 10 min (2000 rev/min), the supernatant was discarded. With 10 mL saline (4.1) oscillation washed cells, centrifuged 10 min (2000 rev/min), the supernatant was discarded, with 10 mL of normal saline (4.1) oscillation cleaning. As before centrifugation, the supernatant was discarded. again Add 10 mL of normal saline (4.1), and mix. Suction 1 mL bacterial suspension in 10 mL of normal saline (4.1), and mix into the test bacteria. 6.1.5 saline (4.1) as control at 550 nm wavelength spectrophotometer measured test broth (6.1.4) of the optical density, This value should be between 60% to 80%. 6.2 sample handling Weigh 2 g (accurate to 0.0001 g) or solid sample 5 g (accurate to 0.0001 g) a liquid sample (containing about pantothenic acid 0.1 mg) in 250 Triangle mL beaker. Was added 10 mL Tris buffer (4.8), then add a small amount of water, 121 ℃ hydrolysis 15 min, cooled. With hydrochloric acid (4.9) PH was adjusted to 4.5 ± 0.2, into 250 mL volumetric flask to volume with water. Filtered, the filtrate was suction 4 mL, diluted to a concentration of pantothenic acid is about 5 ng/mL. 6.3 standard curve Add distilled water in Table 1, the standard solution and the medium in tubes, Table 1, each of the numbers required to make three. Tube S2 to S10 , A considerable amount of pantothenic acid 0 ng, 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 40 ng, 50 ng. Table 1 Standard curve tube production Tube No. S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Distilled water (mL) 5 5 4 3 2 1 0 2 1 0 Standard solution (mL) 0 0 1 2 3 4 5 3 4 5 Medium (mL) 5 5 5 5 5 5 5 5 5 5 Note 1. The tubes plus low concentration of the standard solution S3 ~ S7. Note 2. The test tubes S8 ~ S10 in heightening the concentration of the standard solution. 6.4 sample tube production Add distilled water in Table 2, the sample and the medium in tubes, in triplicate. Table 2 Sample tubes production Tube No. 1234 Distilled water (mL) 4 3 2 1 Sample (mL) 1 2 3 4 Medium (mL) 5 5 5 5 6.5 Sterilization The standard curve tubes and sterilized sample tubes 121 ℃ 5 min, rapidly cooled to room temperature (Commercially available media according to label directions sterilized). Note. to ensure that the heating and cooling process conditions uniform, too sterile or too close to the number of tubes, can adversely affect the sterilization pot. 6.6 inoculation Under sterile conditions to each tube was added one drop (about 50 μL) test bacteria solution (6.1.4), capped, thoroughly shaken mix all tubes (superscript Standard curve except unvaccinated blank tube S1). 6.7 Training 36 ℃ ± 0.5 ℃ cultured 16 h ~ 24 h. Visual inspection of each tube, the tube unvaccinated broth should be clear, standard curve Ray tube and the sample tube broth turbidity due gradient. Uninoculated tube if turbidity and the result is invalid. 6.8 Determination Blank inoculate tube (Table 1 test tube number S2) as control, remove the highest concentration of the standard curve tube S7, oscillation 5 s, at a wavelength of 550 nm Read the optical density conditions, culture back again. 2 h after the same conditions to re-measure the optical density of the tube, if the optical density of the absolute difference between the two Results ≤2%, then remove all the tubes measuring optical density of the sample and standard solutions. Draw 6.9 standard curve Pantothenic acid content of a standard curve for the horizontal tube to the optical density value for the vertical standard curve. 6.10 Calculation of pantothenic acid content of the sample tube 6.8 optical density values in accordance with each sample tube assay, Richard corresponding pantothenic acid content from the standard curve. Three samples of each tube numbered It should be calculated per ml tube pantothenic acid content was measured and compared with the average of the three. Relative standard deviation of less than 15% of the tube is effectively tube, Invalid sample tube should be discarded. The total number of valid sample tube should be greater than two-thirds of the total number of sample tubes. Recalculated effective sample tube in each numbered Determination of the average per milliliter of liquid pantothenic acid content, calculated as the average of the average of the total number of all sample tubes Cx. Note. The sample tubes value pantothenic acid content of less than 5 ng, 50 ng of the above should be discarded.

7 expression analysis

Pantothenic acid content of the sample according to equation (1). X = 100 ×× f Cx (1) Where. X-- pantothenic acid content of the sample, in micrograms per hundred grams (μg/100 g); Cx - 6.10 overall average calculated in micrograms (μg); f-- dilution; m-- sample mass, in grams (g). The arithmetic mean of two under the same condition of independent determination results indicated that the results to three significant figures.

8 Precision

Two independent determination results under the absolute difference in repeatability condition must not exceed 10% of the arithmetic mean. The second method HPLC Principle 9 After the samples were extracted with hot water pre-treatment, the C18 column, UV detector, pantothenic acid content of external standard. 10 Reagents and materials Unless otherwise specified, the reagents used in this method are analytically pure water GB/T 6682 provided a water. 10.1 amylase. enzyme activity ≥1.5 U/mg. 10.2 methanol (CH4O). chromatography. 10.3 hydrochloric acid. 10.4 zinc sulfate (ZnSO4). 10.5 hydrochloric acid (0.1 mol/L). Pipette 8.3 mL of hydrochloric acid (10.3) in 1000 mL volumetric flask, and water volume. 10.6 zinc sulfate solution (15 g/100mL). Weigh 15 g zinc sulfate (10.4) dissolved in water and dilute to 100 mL. 10.7 potassium dihydrogen phosphate solution (0.05 mol/L). Weigh 6.8 g potassium dihydrogen phosphate, dissolved in water and dilute to 1000 mL. Adjusted with phosphoric acid To pH 3.0 with 0.45 μm membrane filter. 10.8 pantothenic acid standard solution 10.8.1 pantothenic acid standard stock solution (1 mg/mL). Weigh accurately calcium pantothenate 1.087 g, dissolved in water and dilute to 1000 mL. Pantothenic acid concentration of calcium pantothenate concentration × 0.920 = 10.8.2 pantothenic acid intermediate standard solution (0.1 mg/mL). draw standard stock solution (10.8.1) 10 mL to 100 mL volumetric flask, add water Volume. Pro preparation before use. 11 instruments and equipment 11.1 balance. a sense of the amount of 0.1 mg. 11.2 HPLC with UV detector. 11.3 ultrasound. 11.4 pH meter. accuracy of 0.01. 11.5 incubator. 55 ℃ ± 2 ℃. 12 analysis steps 12.1 sample processing 12.1.1 free starch sample processing Weigh mixed solid sample of about 5 g (accurate to 0.0001 g) or a liquid sample of about 20 g (accurate to 0.0001 g) in 150 mL Flask, solid sample was added about 30 mL 40 ℃ ~ 50 ℃ warm water, dissolve after shaking ultrasonic extraction 20 min. 12.1.2 starch-containing sample processing If the sample contains starch, mixing solid weighed sample of about 5 g (accurate to 0.0001 g) or a liquid sample of about 20 g (accurate To 0.0001 g) in 150mL flask, adding amylase (10.1) approximately 0.2 g, solid sample was added about 30 mL 40 ℃ ~ 50 ℃ warm water Shaking dissolved, covered with cork, hydrolysis 30 min at 50 ℃ ~ 60 ℃ conditions. 12.2 Preparation of the test liquid After the sample solution was cooled to room temperature, with hydrochloric acid (10.5) adjusting the pH to 4.5 ± 0.1, add 5 mL zinc sulfate solution (10.6), fully mixing. Transferred to 50 mL volumetric flask to volume with water and mix thoroughly with a filter paper. The filtrate after 0.45 μm filter membrane, That is, the sample test solution. 12.3 reference chromatographic conditions Column. ODS-C18 (particle size 5 μm, 250 mm × 4.6 mm) or equivalent in performance to the column. Mobile phase. take potassium dihydrogen phosphate solution (10.7) 900 mL, Methanol (10.2) 100 mL, and mix through 0.45 μm microporous membrane Pressure filtration. Flow rate. 1.0 mL/min. Detection wavelength.200 nm. Column temperature. 30 ℃ ± 1 ℃. Injection volume. 10 μL. 12.4 Determination 12.4.1 Determination of a standard curve Imbibe respectively pantothenic acid intermediate standard solution (10.8.2) 1.0 mL, 2.0 mL, 4.0 mL, 8.0 mL, 12.0 mL in 100 mL capacity Bottle, add water to volume, to give concentrations of 1.0 μg/mL, 2.0 μg/mL, 4.0 μg/mL, 8.0 μg/mL, 12.0 μg/mL of Pantothenic acid working standard solution, prepared before use. The above-described pantothenic acid standard working sequentially chromatography (standard sample chromatogram in Appendix A in Figure A.1), recording chromatographic peak height (or Peak area). Peak height (or peak area) for the vertical axis, the standard working solution concentration as the abscissa standard curve. 12.4.2 Determination of the sample solution Draw a sample test solution (12.2) 10 μL, the sample to be tested liquid chromatography, Richard pantothenic acid concentration in the sample solution from the standard curve degree. 13 analysis results presentation Pantothenic acid content of the sample according to equation (2). 100 ××× = KCVX (2) Where. X-- pantothenic acid content of the sample, in micrograms per hundred grams (μg/100 g) The concentration of the sample solution C-- pantothenic acid, in micrograms milliliter (μg/mL); m - said the quality of the sample taken, in grams (g); V-- sample solution measured total volume in milliliters (mL of); K-- sample liquid dilution factor. The arithmetic mean of two under the same condition of independent determination results indicated that the results to three significant figures. 14 Precision Two independent determination results under the absolute difference in repeatability condition must not exceed 10% of the arithmetic mean. Other 15 The second standard method detection limit of 100 μg/100 g.

Appendix A

(Informative) Pantothenic acid liquid chromatogram of the standard solution LC Figure A.1 pantothenic acid standard solution FIG pantothenic HPLC standard solution is shown in Figure A.1. Figure A.1 liquid chromatogram of standard solution of pantothenic acid

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