National Food Safety Standard
Determination of Vitamin K1 in Foods
食品安全国家标准
食品中维生素K1的测定
1 Scope
This standard specifies determination methods of Vitamin K1 in foods.
Method I in this standard is high performance liquid chromatography-fluorescence detection method and Method II is liquid chromatography-tandem mass spectrometry, which are applicable to the determination of Vitamin K1 in all kinds of formula foods, vegetable oils, fruits and vegetables.
Method I High Performance Liquid Chromatography-Fluorescence Detection Method
2 Principle
After samples like foods for infants and young children, milk and milk products and vegetable oils are subjected to enzymolysis with lipase and amylase, Vitamin K1 in these samples is extracted by n-hexane, separated from other impurities with C18 liquid chromatographic column, subjected to zinc column reduction, detected with fluorescence detector and quantified by external standard method.
For low fat plant samples like fruit and vegetable, Vitamin K1 in them is extracted with isopropanol and n-hexane, subjected to purification by neutral alumina column to remove interfering substances like chlorophyl. Then Vitamin K1 is separated from other impurities with C18 liquid chromatographic column, subjected to zinc column reduction, detected with fluorescence detector and quantified by external standard method.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Grade I water (defined in GB/T 6682) are adopted for the purposes of this method.
3.1 Reagents
3.1.1 Absolute ethanol (CH3CH2OH).
3.1.2 Potassium carbonate (K2CO3).
3.1.3 Anhydrous sodium sulfate (Na2SO4).
3.1.4 Isopropanol (C3H8O).
3.1.5 n-hexane (C6H14).
3.1.6 Methanol (CH3OH): chromatographically pure.
3.1.7 Tetrahydrofuran (C4H8O): chromatographically pure.
3.1.8 Ethyl acetate (C4H8O2).
3.1.9 Glacial acetic acid (CH3COOH): chromatographically pure.
3.1.10 Zinc chloride (ZnCl2): chromatographically pure.
3.1.11 Anhydrous sodium acetate (CH3COONa).
3.1.12 Potassium hydroxide (KOH).
3.1.13 Lipase: enzyme activity ≥700U/mg.
3.1.14 Amylase: enzyme activity ≥1.5U/mg.
3.1.15 Zinc powder: particle size of 50μm~70μm.
3.2 Preparation of reagents
3.2.1 40% potassium hydroxide solution: weigh 20g of potassium hydroxide into a 100mL beaker and dissolve it with 20mL of water, after cooling, add into water to 50mL and store the solution in a polyethylene bottle.
3.2.2 Phosphate buffer solution (pH 8.0): dissolve 54.0g of potassium dihydrogen phosphate into 300mL of water, adjust the pH value to 8.0 with 40% potassium hydroxide solution and add into water to 500mL.
3.2.3 n-hexane-ethyl acetate mixed solution (90+10): measure 90mL of n-hexane, add into 10mL of ethyl acetate and mix well.
3.2.4 Mobile phase: measure 900mL of methanol, 100mL of tetrahydrofuran and 0.3mL of glacial acetic acid; after mixing them well, add into 1.5g of zinc chloride and 0.5g of anhydrous sodium acetate; and filter with 0.22μm organic filter membrane after dissolving them by ultrasound.
3.3 Standards
Vitamin K1 (C31H46O2, CAS No.: 84-80-0): purity ≥99%, or reference material approved and awarded with reference material certificate by the nation.
3.4 Preparation of standard solutions
3.4.1 Vitamin K1 standard stock solution (1mg/mL): accurately weigh 50mg (accurate to 0.1mg) of Vitamin K1 standard into a 50mL volumetric flask, dissolve it with methanol solution and bring the volume to the scale. Transfer the solution into a brown glass vessel, store it in a dark place at below -20℃ for a storage life of two months. Carry out concentration calibration before using the standard stock solution and refer to Appendix A for the calibration method.
3.4.2 Vitamin K1 standard intermediate solution (100μg/mL): accurately pipet 10.00mL of standard stock solution into a 100mL volumetric flask, add into methanol to the scale and shake well. Transfer the solution into a brown glass vessel, store it in a dark place at below -20℃ for a storage life of two months.
3.4.3 Vitamin K1 standard working solution (1.00μg/mL): accurately pipet 1.00mL of standard intermediate solution into a 100mL volumetric flask, add into methanol to the scale and shake well.
3.4.4 Standard series working solution: accurately pipet 0.10mL, 0.20mL, 0.50mL, 1.00mL, 2.00mL and 4.00mL of the Vitamin K1 standard working solution respectively into 10mL volumetric flasks, bring the volumes to the scale with methanol; the concentration of this standard series of Vitamin K1 is 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 200ng/mL and 400ng/mL respectively.
3.5 Materials
3.5.1 Neutral alumina: particle size of 50μm~150μm.
3.5.2 Neutral alumina column: 2g/6mL, 10% of water is contained in filler, it is allowed to purchase commercial column directly or fill the column by oneself.
Notes: Filling methods for neutral alumina column:
a) Filler treatment: take 200g of neutral alumina into a 500mL wide-mouth bottle, bake for 2h in a 150℃ drying oven, cover the bottle and cool to room temperature in dryer, slowly add into 20mL of pure water while shaking; after that, cover with the bottle cap and put the bottle inside a 80℃ drying oven for 3min ~ 5min; and then take out, fiercely shake until the alumina inside the bottle freely flows and has no caking, and place it into dryer to cool for 30min for future use.
b) Filling for chromatographic column: take a 6mL needle cylinder column sleeve, put on a sieve plate, weigh 2.00g of the above filler which has been through deactivation, put on another piece of sieve plate and compact with filling tools.
3.5.3 Zinc column: 50mm in column length and 4.6mm in inner diameter; it is allowed to purchase commercial column directly or fill the column by oneself.
Notes:
a) Filling method for zinc column: fill the zinc powder in stainless steel column sleeve (50mm in column length and 4.6mm in inner diameter) intensively. During filling, small amounts of zinc powder shall be filled in column frequently while patting to make the filled zinc powder compact.
b) Water in pipes for liquid chromatograph must be drained clearly before connecting the zinc column with apparatus.
3.5.4 Microporous filter head: equipped with 0.22μm organic microporous membrane.
4 Apparatuses
4.1 High performance liquid chromatograph: equipped with fluorescence detector.
4.2 Refiner.
4.3 High speed disintegrator.
4.4 Tissue blender.
4.5 Vortex oscillator.
4.6 Thermostatic waterbath oscillator.
4.7 pH meter: with the accuracy of 0.01.
4.8 Balance: with the sensibility of 1mg and 0.1mg.
4.9 Centrifuge: rotation speed ≥6 000r/min.
4.10 Rotary evaporator.
4.11 Nitrogen blower.
4.12 Ultrasonic vibrator.
5 Analysis Procedures
5.1 Preparation of specimen
As for powdery samples like rice flour and milk powder, carry out direct sampling after mixing well; as for flaky and granular samples, pulverize them into powder with sample pulverizer and store them in sample sack for future use; as for liquid samples like liquid milk and vegetable oil, carry out direct sampling after shaking well; as for fruits and vegetables, take edible part of them, wash with water, wipe off the surface moisture with gauze, homogenate with homogenizer and store in sample bottle for future use. Carry out determination after specimen preparation as soon as possible.
5.2 Specimen treatment
Warning: Direct radiation of ultraviolet light shall be avoided in treatment process and operation shall be carried out in a dark place as possible.
5.2.1 Foods for infants and young children, milk and milk products and vegetable oils
5.2.1.1 Enzymolysis
Accurately weigh 1g~5g (accurate to 0.01g and with Vitamin K1 content not less than 0.05μg) of the homogenized specimen into a 50mL centrifugal tube, add into 5mL of warm water to dissolve it (for liquid samples, directly pipet 5mL; vegetable oils need not to be diluted with water); after that, add into 5mL of phosphate buffer solution (pH of 8.0) and mix well, add into 0.2g of lipase and 0.2g of amylase (amylase is not needed if the sample does not contain starch), cover the cap and apply vortex for 2min~3min; after mixing well, place the centrifugal tube in 37℃±2℃ thermostatic waterbath oscillator to oscillate for above 2h and make the enzymolysis thorough.
5.2.1.2 Extraction
Take out the specimen which has been through enzymolysis, add into 10mL of ethanol and 1g of potassium carbonate respectively, mix them well and add into 10mL of n-hexane and 10mL of water, extract for 10min by vortex or oscillation and centrifuge for 5min at 6 000r/min, or transfer the enzymatic hydrolysis solution into a 150mL separating funnel for extraction; after that, keep the solution still for layering (if emulsification phenomenon occurs, more n-hexane or water may be added appropriately to avoid emulsification), transfer the supernatant into a 100mL rotary evaporator and add another 10mL of n-hexane into the subnatant, repeat the operation once, and then combine the supernatant to the above-mentioned rotary evaporator.
5.2.1.3 Concentration
Evaporate the above-mentioned n-hexane extracting solution to dry in a rotary way (if residual liquid exists, gently blow to dry with nitrogen), transfer with methanol and scale the volume in a 5mL volumetric flask, shake well and filter with 0.22μm filter membrane to obtain filtrate for sample injection.
Carry out blank test by adopting the same operating method except for adding specimen.
5.2.2 Fruit and vegetable samples
5.2.2.1 Extraction
Accurately weigh 1g~5g (accurate to 0.01g and with Vitamin K1 content not less than 0.05μg) of homogenized sample into a 50mL centrifugal tube, add into 5mL of isopropanol, apply vortex for 1min and ultrasonic for 5min; and then add into 10mL of n-hexane, extract for 3min by vortex and oscillation and centrifuge for 5min at 6 000r/min; after that, transfer the supernatant into a 25mL brown volumetric flask, add 10mL of n-hexane into the lower layer solution, repeat the extraction once, combine supernatant in the above-mentioned volumetric flask and bring the volume to the scale with n-hexane; at last, accurately pipette 1mL~5mL (determined according to the content of Vitamin K1 in sample) of the supernatant to a 10mL test tube, gently blow to dry with nitrogen and dissolve with 1mL of n-hexane for purification.
5.2.2.2 Purification and concentration
Transfer 1mL of the above-mentioned extracting solution with a small amount of n-hexane to the neutral alumina column which is activated with 5mL of n-hexane beforehand; when the extracting solution flows to nearly dry, elute with 5mL of n-hexane and 6mL of n-hexane-ethyl acetate mixed solution to a 10mL test tube; after blowing to dry with nitrogen, bring the volume to 5mL with methanol, filter with 0.22μm filter membrane to obtain filtrate for analytic determination.
Carry out blank test by adopting the same operating method except for adding specimen.
5.3 Reference conditions for chromatograph
a) Chromatographic column: C18 column, 250mm in column length, 4.6mm in inner diameter and 5μm in particle size or chromatographic column with equivalent performance;
b) Zinc reduction column: 50mm in column length and 4.6mm in inner diameter;
c) Mobile phase: prepared in accordance with 3.2.4;
d) Flow rate: 1mL/min;
e) Detection wavelength: excitation wavelength of 243nm and emission wavelength of 430nm;
f) Injection volume: 10μL;
g) See Appendix B for chromatogram of standard solutions.
5.4 Plotting of standard curve
Carry out quantification by adopting external standard curve method. Inject the standard series working solution of Vitamin K1 into high performance liquid chromatograph respectively to determine the corresponding peak areas. Plot the standard curve by taking peak area as the longitudinal coordinate and the concentration of standard series working solution as horizontal coordinate and calculate the linear regression equation.
5.5 Determination of specimen solution
Inject the prepared blank solution and specimen solution respectively under the same chromatographic conditions and carry out high performance liquid chromatographic analysis. Carry out qualitative analysis by retention time and quantitative analysis by peak area external standard method, and calculate the concentration of Vitamin K1 in specimen solution by linear regression equation.
Contents
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Apparatuses
5 Analysis Procedures
6 Expression of Analysis Results
7 Accuracy
8 Others
9 Principle
10 Reagents and Materials
11 Apparatuses
12 Analysis Procedures
13 Expression of Analysis Results
14 Accuracy
15 Others
Appendix A Calibration Method for Standard Concentration of Vitamin K
Appendix B High Performance Liquid Chromatogram
Appendix C Reference Mass Spectrogram
National Food Safety Standard
Determination of Vitamin K1 in Foods
食品安全国家标准
食品中维生素K1的测定
1 Scope
This standard specifies determination methods of Vitamin K1 in foods.
Method I in this standard is high performance liquid chromatography-fluorescence detection method and Method II is liquid chromatography-tandem mass spectrometry, which are applicable to the determination of Vitamin K1 in all kinds of formula foods, vegetable oils, fruits and vegetables.
Method I High Performance Liquid Chromatography-Fluorescence Detection Method
2 Principle
After samples like foods for infants and young children, milk and milk products and vegetable oils are subjected to enzymolysis with lipase and amylase, Vitamin K1 in these samples is extracted by n-hexane, separated from other impurities with C18 liquid chromatographic column, subjected to zinc column reduction, detected with fluorescence detector and quantified by external standard method.
For low fat plant samples like fruit and vegetable, Vitamin K1 in them is extracted with isopropanol and n-hexane, subjected to purification by neutral alumina column to remove interfering substances like chlorophyl. Then Vitamin K1 is separated from other impurities with C18 liquid chromatographic column, subjected to zinc column reduction, detected with fluorescence detector and quantified by external standard method.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Grade I water (defined in GB/T 6682) are adopted for the purposes of this method.
3.1 Reagents
3.1.1 Absolute ethanol (CH3CH2OH).
3.1.2 Potassium carbonate (K2CO3).
3.1.3 Anhydrous sodium sulfate (Na2SO4).
3.1.4 Isopropanol (C3H8O).
3.1.5 n-hexane (C6H14).
3.1.6 Methanol (CH3OH): chromatographically pure.
3.1.7 Tetrahydrofuran (C4H8O): chromatographically pure.
3.1.8 Ethyl acetate (C4H8O2).
3.1.9 Glacial acetic acid (CH3COOH): chromatographically pure.
3.1.10 Zinc chloride (ZnCl2): chromatographically pure.
3.1.11 Anhydrous sodium acetate (CH3COONa).
3.1.12 Potassium hydroxide (KOH).
3.1.13 Lipase: enzyme activity ≥700U/mg.
3.1.14 Amylase: enzyme activity ≥1.5U/mg.
3.1.15 Zinc powder: particle size of 50μm~70μm.
3.2 Preparation of reagents
3.2.1 40% potassium hydroxide solution: weigh 20g of potassium hydroxide into a 100mL beaker and dissolve it with 20mL of water, after cooling, add into water to 50mL and store the solution in a polyethylene bottle.
3.2.2 Phosphate buffer solution (pH 8.0): dissolve 54.0g of potassium dihydrogen phosphate into 300mL of water, adjust the pH value to 8.0 with 40% potassium hydroxide solution and add into water to 500mL.
3.2.3 n-hexane-ethyl acetate mixed solution (90+10): measure 90mL of n-hexane, add into 10mL of ethyl acetate and mix well.
3.2.4 Mobile phase: measure 900mL of methanol, 100mL of tetrahydrofuran and 0.3mL of glacial acetic acid; after mixing them well, add into 1.5g of zinc chloride and 0.5g of anhydrous sodium acetate; and filter with 0.22μm organic filter membrane after dissolving them by ultrasound.
3.3 Standards
Vitamin K1 (C31H46O2, CAS No.: 84-80-0): purity ≥99%, or reference material approved and awarded with reference material certificate by the nation.
3.4 Preparation of standard solutions
3.4.1 Vitamin K1 standard stock solution (1mg/mL): accurately weigh 50mg (accurate to 0.1mg) of Vitamin K1 standard into a 50mL volumetric flask, dissolve it with methanol solution and bring the volume to the scale. Transfer the solution into a brown glass vessel, store it in a dark place at below -20℃ for a storage life of two months. Carry out concentration calibration before using the standard stock solution and refer to Appendix A for the calibration method.
3.4.2 Vitamin K1 standard intermediate solution (100μg/mL): accurately pipet 10.00mL of standard stock solution into a 100mL volumetric flask, add into methanol to the scale and shake well. Transfer the solution into a brown glass vessel, store it in a dark place at below -20℃ for a storage life of two months.
3.4.3 Vitamin K1 standard working solution (1.00μg/mL): accurately pipet 1.00mL of standard intermediate solution into a 100mL volumetric flask, add into methanol to the scale and shake well.
3.4.4 Standard series working solution: accurately pipet 0.10mL, 0.20mL, 0.50mL, 1.00mL, 2.00mL and 4.00mL of the Vitamin K1 standard working solution respectively into 10mL volumetric flasks, bring the volumes to the scale with methanol; the concentration of this standard series of Vitamin K1 is 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 200ng/mL and 400ng/mL respectively.
3.5 Materials
3.5.1 Neutral alumina: particle size of 50μm~150μm.
3.5.2 Neutral alumina column: 2g/6mL, 10% of water is contained in filler, it is allowed to purchase commercial column directly or fill the column by oneself.
Notes: Filling methods for neutral alumina column:
a) Filler treatment: take 200g of neutral alumina into a 500mL wide-mouth bottle, bake for 2h in a 150℃ drying oven, cover the bottle and cool to room temperature in dryer, slowly add into 20mL of pure water while shaking; after that, cover with the bottle cap and put the bottle inside a 80℃ drying oven for 3min ~ 5min; and then take out, fiercely shake until the alumina inside the bottle freely flows and has no caking, and place it into dryer to cool for 30min for future use.
b) Filling for chromatographic column: take a 6mL needle cylinder column sleeve, put on a sieve plate, weigh 2.00g of the above filler which has been through deactivation, put on another piece of sieve plate and compact with filling tools.
3.5.3 Zinc column: 50mm in column length and 4.6mm in inner diameter; it is allowed to purchase commercial column directly or fill the column by oneself.
Notes:
a) Filling method for zinc column: fill the zinc powder in stainless steel column sleeve (50mm in column length and 4.6mm in inner diameter) intensively. During filling, small amounts of zinc powder shall be filled in column frequently while patting to make the filled zinc powder compact.
b) Water in pipes for liquid chromatograph must be drained clearly before connecting the zinc column with apparatus.
3.5.4 Microporous filter head: equipped with 0.22μm organic microporous membrane.
4 Apparatuses
4.1 High performance liquid chromatograph: equipped with fluorescence detector.
4.2 Refiner.
4.3 High speed disintegrator.
4.4 Tissue blender.
4.5 Vortex oscillator.
4.6 Thermostatic waterbath oscillator.
4.7 pH meter: with the accuracy of 0.01.
4.8 Balance: with the sensibility of 1mg and 0.1mg.
4.9 Centrifuge: rotation speed ≥6 000r/min.
4.10 Rotary evaporator.
4.11 Nitrogen blower.
4.12 Ultrasonic vibrator.
5 Analysis Procedures
5.1 Preparation of specimen
As for powdery samples like rice flour and milk powder, carry out direct sampling after mixing well; as for flaky and granular samples, pulverize them into powder with sample pulverizer and store them in sample sack for future use; as for liquid samples like liquid milk and vegetable oil, carry out direct sampling after shaking well; as for fruits and vegetables, take edible part of them, wash with water, wipe off the surface moisture with gauze, homogenate with homogenizer and store in sample bottle for future use. Carry out determination after specimen preparation as soon as possible.
5.2 Specimen treatment
Warning: Direct radiation of ultraviolet light shall be avoided in treatment process and operation shall be carried out in a dark place as possible.
5.2.1 Foods for infants and young children, milk and milk products and vegetable oils
5.2.1.1 Enzymolysis
Accurately weigh 1g~5g (accurate to 0.01g and with Vitamin K1 content not less than 0.05μg) of the homogenized specimen into a 50mL centrifugal tube, add into 5mL of warm water to dissolve it (for liquid samples, directly pipet 5mL; vegetable oils need not to be diluted with water); after that, add into 5mL of phosphate buffer solution (pH of 8.0) and mix well, add into 0.2g of lipase and 0.2g of amylase (amylase is not needed if the sample does not contain starch), cover the cap and apply vortex for 2min~3min; after mixing well, place the centrifugal tube in 37℃±2℃ thermostatic waterbath oscillator to oscillate for above 2h and make the enzymolysis thorough.
5.2.1.2 Extraction
Take out the specimen which has been through enzymolysis, add into 10mL of ethanol and 1g of potassium carbonate respectively, mix them well and add into 10mL of n-hexane and 10mL of water, extract for 10min by vortex or oscillation and centrifuge for 5min at 6 000r/min, or transfer the enzymatic hydrolysis solution into a 150mL separating funnel for extraction; after that, keep the solution still for layering (if emulsification phenomenon occurs, more n-hexane or water may be added appropriately to avoid emulsification), transfer the supernatant into a 100mL rotary evaporator and add another 10mL of n-hexane into the subnatant, repeat the operation once, and then combine the supernatant to the above-mentioned rotary evaporator.
5.2.1.3 Concentration
Evaporate the above-mentioned n-hexane extracting solution to dry in a rotary way (if residual liquid exists, gently blow to dry with nitrogen), transfer with methanol and scale the volume in a 5mL volumetric flask, shake well and filter with 0.22μm filter membrane to obtain filtrate for sample injection.
Carry out blank test by adopting the same operating method except for adding specimen.
5.2.2 Fruit and vegetable samples
5.2.2.1 Extraction
Accurately weigh 1g~5g (accurate to 0.01g and with Vitamin K1 content not less than 0.05μg) of homogenized sample into a 50mL centrifugal tube, add into 5mL of isopropanol, apply vortex for 1min and ultrasonic for 5min; and then add into 10mL of n-hexane, extract for 3min by vortex and oscillation and centrifuge for 5min at 6 000r/min; after that, transfer the supernatant into a 25mL brown volumetric flask, add 10mL of n-hexane into the lower layer solution, repeat the extraction once, combine supernatant in the above-mentioned volumetric flask and bring the volume to the scale with n-hexane; at last, accurately pipette 1mL~5mL (determined according to the content of Vitamin K1 in sample) of the supernatant to a 10mL test tube, gently blow to dry with nitrogen and dissolve with 1mL of n-hexane for purification.
5.2.2.2 Purification and concentration
Transfer 1mL of the above-mentioned extracting solution with a small amount of n-hexane to the neutral alumina column which is activated with 5mL of n-hexane beforehand; when the extracting solution flows to nearly dry, elute with 5mL of n-hexane and 6mL of n-hexane-ethyl acetate mixed solution to a 10mL test tube; after blowing to dry with nitrogen, bring the volume to 5mL with methanol, filter with 0.22μm filter membrane to obtain filtrate for analytic determination.
Carry out blank test by adopting the same operating method except for adding specimen.
5.3 Reference conditions for chromatograph
a) Chromatographic column: C18 column, 250mm in column length, 4.6mm in inner diameter and 5μm in particle size or chromatographic column with equivalent performance;
b) Zinc reduction column: 50mm in column length and 4.6mm in inner diameter;
c) Mobile phase: prepared in accordance with 3.2.4;
d) Flow rate: 1mL/min;
e) Detection wavelength: excitation wavelength of 243nm and emission wavelength of 430nm;
f) Injection volume: 10μL;
g) See Appendix B for chromatogram of standard solutions.
5.4 Plotting of standard curve
Carry out quantification by adopting external standard curve method. Inject the standard series working solution of Vitamin K1 into high performance liquid chromatograph respectively to determine the corresponding peak areas. Plot the standard curve by taking peak area as the longitudinal coordinate and the concentration of standard series working solution as horizontal coordinate and calculate the linear regression equation.
5.5 Determination of specimen solution
Inject the prepared blank solution and specimen solution respectively under the same chromatographic conditions and carry out high performance liquid chromatographic analysis. Carry out qualitative analysis by retention time and quantitative analysis by peak area external standard method, and calculate the concentration of Vitamin K1 in specimen solution by linear regression equation.
Contents of GB 5009.158-2016
Contents
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Apparatuses
5 Analysis Procedures
6 Expression of Analysis Results
7 Accuracy
8 Others
9 Principle
10 Reagents and Materials
11 Apparatuses
12 Analysis Procedures
13 Expression of Analysis Results
14 Accuracy
15 Others
Appendix A Calibration Method for Standard Concentration of Vitamin K
Appendix B High Performance Liquid Chromatogram
Appendix C Reference Mass Spectrogram
