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GB 5413.10-2010: National food safety standard -- Determination of vitamin K1 in foods for infants and young children, milk and milk products
GB 5413.10-2010
National food safety standard - Determination of vitamin K1 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
Determination of infant foods and dairy products of vitamin K1
National food safety standard
Determination of vitamin K1 in foods for infants and young children,
milk and milk products
Foreword
This standard is identical with the International Society of Analysts (AOAC) AOAC 999.15 Vitamin K in Milk and Infant Formulas
Liquid Chromatographic Method.
This standard replaces GB/T 5413.10-1997 "infant formula and milk powder - Determination of vitamin K1."
This standard compared with GB/T 5413.10-1997, main changes are as follows.
- The standard name changed to "infant foods and dairy products - Determination of vitamin K1";
- Sample processing to the sample after enzymatic hydrolysis, saponification with NaOH, and extracted with n-hexane;
- After HPLC Columns Determination to restore fluorescence quantitative determination of vitamin K1;
- The instrument will be "high pressure liquid chromatography with UV detection" to "high performance liquid chromatography with fluorescence detector."
The Standard Appendix A, Appendix B and Appendix C are informative appendices.
This standard replaces the standards previously issued as follows.
--GB 5413-1985, GB/T 5413.10-1997.
National Food Safety Standard
Determination of infant foods and dairy products of vitamin K1
1 Scope
This standard specifies the method for determination of infant foods and dairy products of vitamin K1.
This standard applies to the determination of infant foods and dairy products of vitamin K1.
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.
Principle 3
Lipase sample degradation of fats and unsaturated fatty acids required for the starch-containing sample in the sample using starch to amylase degradation by
After alkaline saponification, extraction with hexane vitamin K1. Separating liquid chromatography, post-column by reducing vitamin K1, fluorescence detection, external
Standard method.
4 Reagents and materials
Unless otherwise specified, the reagents used in this method are analytically pure water GB/T 6682 provided a water.
4.1 Sodium hydroxide solution (10 mol/L). prepared before use.
4.2 95% ethanol.
4.3 with saturated sodium chloride solution.
4.4 hexane. chromatographically pure.
4.5 over anhydrous sodium sulfate.
4.6 Methanol. chromatographically pure.
4.7 dichloromethane. chromatography.
4.8 acetic acid.
4.9 zinc chloride.
4.10 of anhydrous sodium acetate.
4.11 Mobile phase. Methanol (4.6) 900 mL, dichloromethane (4.7) 100 mL, glacial acetic acid (4.8) 0.3 mL, zinc chloride (4.9)
1.5 g, anhydrous sodium acetate (4.10) 0.5 g, and dissolved with 0.45 μm membrane filter.
4.12 Amylase. enzyme activity ≥1.5 U/mg.
4.13 lipases. enzyme activity ≥700 U/mg.
4.14 zinc powder. particle size 50 μm ~ 70 μm.
4.15 Vitamin K1 standard solution. Standard solution concentration correcting method in Appendix A.
4.15.1 vitamin K1 standard stock solution (2 mg/mL). Weigh 0.05 g of vitamin K1 standard (accurate to 0.1 mg), in 25 mL
Flask, dissolve to volume with n-hexane.
4.15.2 vitamin K1 intermediate standard solution (20 μg/mL). take the standard stock solution (4.15.1) 1 mL hexane added volume to 100 mL.
5. Apparatus
5.1 high performance liquid chromatography with fluorescence detector.
5.2 Balance. a sense of the amount of 0.1 mg.
5.3 funnel. 250 mL.
5.4 rotary evaporator.
5.5 thermostatic air bath oscillator.
5.6 Centrifuge. ≥3000 rpm rev/min.
5.7 nitrogen blowing instrument.
Step 6 Analysis
6.1 Sample Processing
6.1.1 samples containing starch
Weigh-mixed solid sample or liquid sample of about 2.5 g to about 10 g (accurate to 0.1 mg) in the flask was added 0.5 g of starch
Enzyme (4.12), with 30 mL of warm water to dissolve.
6.1.2 No Starch
Weigh-mixed solid sample or liquid sample of about 2.5 g to about 10 g (accurate to 0.1 mg) in the flask with 30 mL warm aqueous
solution.
6.2 Preparation of the test liquid
To the processed sample solution was added 1 g of lipase (4.13) at 37 ℃ ± 5 ℃ constant temperature air bath shaker shaken overnight,
To make it fully digested. Taken good enzymatic sample, add 2 mL of sodium hydroxide solution (4.1) with 50 mL of ethanol (4.2) and the solution was transferred
Into a 250 mL separatory funnel, and mix well (plus, if necessary, a small amount of saturated sodium chloride solution (4.3)). Added 50 mL of n-hexane (4.4).
Fully shaking 2 min, standing layer. Release lower aqueous phase to another 250 mL separating funnel, leaving the organic phase. To the aqueous phase again
Added 50 mL of n-hexane extracted again. The combined organic phases. With the amount of organic phase was washed twice with distilled water. The organic phase was dried over anhydrous sodium sulfate (4.5)
Dehydration filtered into a 500 mL flask on a rotary evaporator to evaporate at 2 ℃ 40 ℃ ± to near dryness. (4.4) Transfer with n-hexane
The residue to a 10 mL tube, on the home nitrogen blowing instrument at 40 ℃ ± 2 ℃ for drying, accurately 1 mL of n-hexane (4.4), fully
The residue was dissolved by shaking. The tube was placed in the refrigerator freezer for 1 h Alternate below 0 ℃.
6.3 Determination
Chromatographic conditions 6.3.1 Reference
Column. C18 column, 150 mm × 4.6 mm, 5 μm, or equivalent performance of the column;
Zinc reduction column. 4.6 mm × 50 mm (see Appendix B);
Detection wavelength. excitation wavelength of 243 nm, emission wavelength of 430 nm;
Mobile phase. Methanol (4.6) 900 mL, dichloromethane (4.7) 100 mL, glacial acetic acid (4.8) 0.3 mL, zinc chloride (4.9) 1.5 g,
Anhydrous sodium acetate (4.10) 0.5 g, dissolved with 0.45 μm membrane filter;
Flow rate. 1 mL/min;
Injection volume. 10 μL.
6.3.2 Standard curve drawing vitamin K1
Imbibe respectively intermediate standard solution (4.15.2) 0.0 mL, 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL plus normal hexane
The volume to 25 mL, this series of standard working solution of vitamin K1 concentrations were 0.00 μg/mL, 0.40 μg/mL, 0.80 μg/mL, 1.20 μg/mL,
1.60 μg/mL, 2.00 μg/mL.
The vitamin K1 series standard working solution were injected into the high performance liquid chromatograph to measure the corresponding peak area or peak height, peak area or
Peak height for the vertical axis, the standard assay concentration as the abscissa standard curve.
6.3.3 Determination of the test liquid
Determination of the prepared solution (6.2) to room temperature in the dark, in a centrifuge at 3000 r/min centrifugation 5 min, the supernatant
High performance liquid chromatography, determining the corresponding peak area or peak height (standard chromatogram see Appendix C). From the standard curve obtained sample
The concentration in the solution of vitamin K1 or vitamin K1 concentration in the sample solution is calculated by the regression equation (μg/mL).
7 expression analysis
Vitamin K1 content of the sample according to equation (1).
100 ××× =
nVCX (1)
Where.
X-- vitamin K1 content, in micrograms per hundred grams (μg/100 g);
Ci-- concentration of the sample solution, in...