This standard supersedes Determination of Lutein in Milk Powder - HPLC-UV Method (GB/T 23209-2008).
Compared with GB/T23209-2008, the main changes in this standard are as follows:
——the standard name is revised as "National Food Safety Standard - Determination of Lutein in Foods";
——the scope of application is extended, and the liquid chromatography method used for determining lutein in frozen drinks, rice & flour products, bakery products, jam, jelly and beverage is added.
——the principle used in the former standard is modified, with the original principle of adopting acetone as the solvent for extraction replaced by the solvent system of ethyl ether - normal hexane - cyclohexane for extraction.
——the precautions for the operation processes are increased;
——the saponification procedure for samples (including milk powder) with high fat content is increased, and different pretreatment methods are provided to adapt to the analysis requirements of various sample matrixes;
——the calibration requirements for the concentration of lutein standard solution are added;
——in case that the lutein may be isomerized during the operation process of the test, the qualitative and quantitative requirements for cis-lutein generated due to isomerization are added.
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
中华人民共和国国家标准
GB 5009.248-2016
National Standard of Food Safety
Determination of Lutein in Foods
食品安全国家标准
食品中叶黄素的测定
1 Scope
This standard specifies the liquid chromatography method for the determination of lutein in foods.
This standard is applicable to the liquid chromatography method for the determination of lutein in infant formula, milk products, frozen drinks, rice & flour products, bakery products, jam, jelly and beverage.
2 Principle
Saponify the foods with high fat content (the fat content, calculated in dry basis, is not less than 3%) at room temperature by using potassium hydroxide solution to dissociate the lutein; extract it with ethyl ether -normal hexane -cyclohexane (volume ratio: 40+40+20), separate with liquid chromatography method, examine it with ultraviolet detector or diode array detector, then quantify with external standard method.
Extract the lutein in the samples of other foods directly with ethyl ether - normal hexane - cyclohexane (volume ratio: 40+40+20). After purifying the extracting solution with Alumina N neutral alumina solid phase extraction column, separate it with liquid chromatography method, examine it with ultraviolet detector or diode array detector, and quantify it with external standard method.
During the extraction and analysis of the samples, the trans-lutein may be isomerized and transformed to cis-lutein. The cis-lutein generated by such transformation may be qualified according to the retention time and quantified by peak area adduction.
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 Cyclohexane (C6H12): chromatographically pure.
3.1.2 Ethyl ether [(C2H5)2O]: chromatographically pure.
3.1.3 Normal hexane (C6H10): chromatographically pure.
3.1.4 Absolute alcohol (C2H5OH): chromatographically pure.
3.1.5 Methyl tertlary butyl ether [CH3OCC(CH3)3, MTBE]: chromatographically pure.
3.1.6 Butylated hydroxytoluene (C15H24O, BHT).
3.1.7 Potassium hydroxide (KOH).
3.1.8 Iodine (I2).
3.2 Reagent preparation
3.2.1 10% potassium hydroxide solution: weigh 10g of potassium hydroxide (3.1.7), then dissolve and dilute it with water to 100mL.
3.2.2 20% potassium hydroxide solution: weigh 20g of potassium hydroxide (3.1.7), then dissolve and dilute it with water to 100mL.
3.2.3 Extraction solvent: weigh 1g of BHT (3.1.6), dissolve it with 200mL of cyclohexane, and add 400mL of ethyl ether (3.1.2) and 400mL of normal hexane (3.1.3) into the solution, then mix uniformly.
3.2.4 0.1% BHT ethanol solution: weigh 0.1g of BHT (3.1.6), dissolve it with 100mL of ethanol (3.1.4), and mix uniformly.
3.2.5 Iodine ethanol solution: weigh 1mg of iodine (3.1.8), dissolve it with ethanol (3.1.4) and dilute the solution to 1L.
3.3 Standard substance
Lutein (CAS No.: 127-40-2) with purity not less than 98.0%.
3.4 Preparation of standard solution
3.4.1 Standard stock solution (50μg/mL): accurately weigh 5mg (with an accuracy of 0.01mg) of lutein (3.1.3), dissolve it with 0.1% BHT ethanol solution (3.2.4) and dilute to the scale of 100mL. This standard stock solution may be preserved for six months if it is filled with nitrogen and stored away from light in the refrigerator of -20℃ or below.
Note: prior to use, the standard stock solution of lutein shall be calibrated according to the specific operation detailed in Appendix A.
3.4.2 Standard working solution: accurately transfer 0.050mL, 0.100mL, 0.200mL, 0.400mL, and 1.00mL of lutein standard stock solution into 5 brown 25mL volumetric flasks, then dilute the solution in those flasks to the scale with 0.1% BHT ethanol solution (3.2.4) to obtain a series of standard working solution with 0.100μg/mL, 0.200μg/mL, 0.400μg/mL, 0.800μg/mL and 2.00μg/mL in concentration respectively. This standard working solution may be preserved for one month if it is filled with nitrogen and stored away from light in the refrigerator of -20℃ or below.
3.5 Alumina-N solid phase extraction column (500mg /3mL), which is rinsed with 5mL of extraction solvent (3.2.3) before use so as to keep the column moist.
3.6 0.45μm organic membrane filter.
4 Instruments and Apparatus
4.1 Liquid chromatography equipped with diode array or ultraviolet detector.
4.2 Ultraviolet and visible spectrophotometer
4.3 Analytical balance: with sensibility of 0.01mg and 0.01g.
4.4 Tissue blender.
4.5 Vortex shaker.
4.6 Oscillator.
4.7 Rotary evaporator.
4.8 Solid-phase extraction device.
4.9 Centrifuge with the rotation speed not less than 4,500r/min.
5 Analysis Steps
Note: due to the light sensitiveness of lutein, unless otherwise specified, all test operations shall be carried out in the environment with yellow or red light source without ultraviolet light of 500nm bellow.
5.1 Specimen preparation
Store a certain number of samples in the sample bottles after pulverizing, homogenizing and splitting them as required. The prepared specimen shall be subjected to nitrogen sealing before being preserved in the refrigerator of -20℃ or below.
5.2 Extraction
5.2.1 Foods with high fat content (e.g. infant formula, milk powder, ice cream and baked nut fruits)
Accurately weigh 2g (with an accuracy of 0.01 g) of uniform specimen, place it in a 50mL polypropene centrifugal tube, add approximately 0.2g of BHT (3.1.6) and 10mL of ethanol (3.1.4) and mix it uniformly; add 10mL of 10% potassium hydroxide solution (3.2.1) into the solution and mix it uniformly through vortex shaking for 1 min; shake and saponify the solution without light for 30min at room temperature, extract the solution for 3min with 10mL of extraction solvent (3.2.3) away from light through vortex shaking and centrifuge it for 3min at 4,500r/min; repeat the extraction for twice and combine the extraction solution; rinse it with 10mL of water, centrifuge for 3 min at 4,500r/min for separation, then rinse it once again; combine the organic phase and concentrate it to nearly dry with reduced pressure; dissolve the residue with 0.1% BHT (3.2.4) through vortex shaking and scale the volume to 5mL; filter the solution with 0.45μm membrane filter (3.6) to carry out liquid chromatographic determination.
Liquid milk: accurately weigh 10g (with an accuracy of 0.01 g) of sample, place it in a 50mL centrifugal polypropene tube, add approximately 0.2g of BHT (3.1.6) and 10mL of ethanol (3.1.4) and mix it uniformly; add 2mL of 20% potassium hydroxide solution (3.2.2) into the solution and mix it uniformly through 1 min of vortex shaking; shake and saponify the solution without light for 30min at room temperature, extract the solution for 3 min with 10mL of extraction solvent (3.2.3) away from light through vortex shaking, and centrifuge it for 3min at 4,500r/min; repeat the extraction for twice and combine the extraction solution; rinse it with 10mL of water, centrifuge for 3 min at 4,500r/min for separation, then rinse it once again; combine the organic phase and concentrate it to nearly dry with reduced pressure; dissolve the residue with 0.1% BHT (3.2.4) through vortex shaking and scale the volume to 5mL; filter the solution with 0.45μm membrane filter (3.6) to carry out liquid chromatographic determination.
5.2.2 Other foods (e.g., rice, flour products and jam)
Accurately weigh 5g (with an accuracy of 0.01g) of uniform sample and place it into a 50mL centrifugal polypropene tube; extract it for 3min away from light with 10mL of extraction solvent (3.2.3) through vortex shaking and centrifuge for 3min at 4,500r/min; repeat the extraction for twice and combine the extraction solution; concentrate it to nearly dry at room temperature with reduced pressure; dissolve it with 3mL of extraction solvent (3.2.3) through vortex shaking; repeat the operation once again, combine the extraction solvent and mix it uniformly for purify.
Filter the above-mentioned solution with activated alumina-N solid phase extraction column (3.5) at the flow velocity of approximately 1mL /min; elute it with 3mL of extraction solvent (3.2.3), then combine the effluent and eluent and concentrate the solution to nearly dry with reduced pressure at room temperature; dissolve the residue with 0.1% BHT ethanol solution (3.2.4) through vortex shaking and scale the volume to 10 mL; then filter it through a 0.45μm membrane filter (3.6) for liquid chromatographic determination.
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Apparatus
5 Analysis Steps
6 Expression of Analysis Result
7 Precision
8 Others
Appendix A Calibration Method for Concentration of Standard Solution
Appendix B Liquid Chromatogram of Standard Solution
This standard supersedes Determination of Lutein in Milk Powder - HPLC-UV Method (GB/T 23209-2008).
Compared with GB/T23209-2008, the main changes in this standard are as follows:
——the standard name is revised as "National Food Safety Standard - Determination of Lutein in Foods";
——the scope of application is extended, and the liquid chromatography method used for determining lutein in frozen drinks, rice & flour products, bakery products, jam, jelly and beverage is added.
——the principle used in the former standard is modified, with the original principle of adopting acetone as the solvent for extraction replaced by the solvent system of ethyl ether - normal hexane - cyclohexane for extraction.
——the precautions for the operation processes are increased;
——the saponification procedure for samples (including milk powder) with high fat content is increased, and different pretreatment methods are provided to adapt to the analysis requirements of various sample matrixes;
——the calibration requirements for the concentration of lutein standard solution are added;
——in case that the lutein may be isomerized during the operation process of the test, the qualitative and quantitative requirements for cis-lutein generated due to isomerization are added.
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
中华人民共和国国家标准
GB 5009.248-2016
National Standard of Food Safety
Determination of Lutein in Foods
食品安全国家标准
食品中叶黄素的测定
1 Scope
This standard specifies the liquid chromatography method for the determination of lutein in foods.
This standard is applicable to the liquid chromatography method for the determination of lutein in infant formula, milk products, frozen drinks, rice & flour products, bakery products, jam, jelly and beverage.
2 Principle
Saponify the foods with high fat content (the fat content, calculated in dry basis, is not less than 3%) at room temperature by using potassium hydroxide solution to dissociate the lutein; extract it with ethyl ether -normal hexane -cyclohexane (volume ratio: 40+40+20), separate with liquid chromatography method, examine it with ultraviolet detector or diode array detector, then quantify with external standard method.
Extract the lutein in the samples of other foods directly with ethyl ether - normal hexane - cyclohexane (volume ratio: 40+40+20). After purifying the extracting solution with Alumina N neutral alumina solid phase extraction column, separate it with liquid chromatography method, examine it with ultraviolet detector or diode array detector, and quantify it with external standard method.
During the extraction and analysis of the samples, the trans-lutein may be isomerized and transformed to cis-lutein. The cis-lutein generated by such transformation may be qualified according to the retention time and quantified by peak area adduction.
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 Cyclohexane (C6H12): chromatographically pure.
3.1.2 Ethyl ether [(C2H5)2O]: chromatographically pure.
3.1.3 Normal hexane (C6H10): chromatographically pure.
3.1.4 Absolute alcohol (C2H5OH): chromatographically pure.
3.1.5 Methyl tertlary butyl ether [CH3OCC(CH3)3, MTBE]: chromatographically pure.
3.1.6 Butylated hydroxytoluene (C15H24O, BHT).
3.1.7 Potassium hydroxide (KOH).
3.1.8 Iodine (I2).
3.2 Reagent preparation
3.2.1 10% potassium hydroxide solution: weigh 10g of potassium hydroxide (3.1.7), then dissolve and dilute it with water to 100mL.
3.2.2 20% potassium hydroxide solution: weigh 20g of potassium hydroxide (3.1.7), then dissolve and dilute it with water to 100mL.
3.2.3 Extraction solvent: weigh 1g of BHT (3.1.6), dissolve it with 200mL of cyclohexane, and add 400mL of ethyl ether (3.1.2) and 400mL of normal hexane (3.1.3) into the solution, then mix uniformly.
3.2.4 0.1% BHT ethanol solution: weigh 0.1g of BHT (3.1.6), dissolve it with 100mL of ethanol (3.1.4), and mix uniformly.
3.2.5 Iodine ethanol solution: weigh 1mg of iodine (3.1.8), dissolve it with ethanol (3.1.4) and dilute the solution to 1L.
3.3 Standard substance
Lutein (CAS No.: 127-40-2) with purity not less than 98.0%.
3.4 Preparation of standard solution
3.4.1 Standard stock solution (50μg/mL): accurately weigh 5mg (with an accuracy of 0.01mg) of lutein (3.1.3), dissolve it with 0.1% BHT ethanol solution (3.2.4) and dilute to the scale of 100mL. This standard stock solution may be preserved for six months if it is filled with nitrogen and stored away from light in the refrigerator of -20℃ or below.
Note: prior to use, the standard stock solution of lutein shall be calibrated according to the specific operation detailed in Appendix A.
3.4.2 Standard working solution: accurately transfer 0.050mL, 0.100mL, 0.200mL, 0.400mL, and 1.00mL of lutein standard stock solution into 5 brown 25mL volumetric flasks, then dilute the solution in those flasks to the scale with 0.1% BHT ethanol solution (3.2.4) to obtain a series of standard working solution with 0.100μg/mL, 0.200μg/mL, 0.400μg/mL, 0.800μg/mL and 2.00μg/mL in concentration respectively. This standard working solution may be preserved for one month if it is filled with nitrogen and stored away from light in the refrigerator of -20℃ or below.
3.5 Alumina-N solid phase extraction column (500mg /3mL), which is rinsed with 5mL of extraction solvent (3.2.3) before use so as to keep the column moist.
3.6 0.45μm organic membrane filter.
4 Instruments and Apparatus
4.1 Liquid chromatography equipped with diode array or ultraviolet detector.
4.2 Ultraviolet and visible spectrophotometer
4.3 Analytical balance: with sensibility of 0.01mg and 0.01g.
4.4 Tissue blender.
4.5 Vortex shaker.
4.6 Oscillator.
4.7 Rotary evaporator.
4.8 Solid-phase extraction device.
4.9 Centrifuge with the rotation speed not less than 4,500r/min.
5 Analysis Steps
Note: due to the light sensitiveness of lutein, unless otherwise specified, all test operations shall be carried out in the environment with yellow or red light source without ultraviolet light of 500nm bellow.
5.1 Specimen preparation
Store a certain number of samples in the sample bottles after pulverizing, homogenizing and splitting them as required. The prepared specimen shall be subjected to nitrogen sealing before being preserved in the refrigerator of -20℃ or below.
5.2 Extraction
5.2.1 Foods with high fat content (e.g. infant formula, milk powder, ice cream and baked nut fruits)
Accurately weigh 2g (with an accuracy of 0.01 g) of uniform specimen, place it in a 50mL polypropene centrifugal tube, add approximately 0.2g of BHT (3.1.6) and 10mL of ethanol (3.1.4) and mix it uniformly; add 10mL of 10% potassium hydroxide solution (3.2.1) into the solution and mix it uniformly through vortex shaking for 1 min; shake and saponify the solution without light for 30min at room temperature, extract the solution for 3min with 10mL of extraction solvent (3.2.3) away from light through vortex shaking and centrifuge it for 3min at 4,500r/min; repeat the extraction for twice and combine the extraction solution; rinse it with 10mL of water, centrifuge for 3 min at 4,500r/min for separation, then rinse it once again; combine the organic phase and concentrate it to nearly dry with reduced pressure; dissolve the residue with 0.1% BHT (3.2.4) through vortex shaking and scale the volume to 5mL; filter the solution with 0.45μm membrane filter (3.6) to carry out liquid chromatographic determination.
Liquid milk: accurately weigh 10g (with an accuracy of 0.01 g) of sample, place it in a 50mL centrifugal polypropene tube, add approximately 0.2g of BHT (3.1.6) and 10mL of ethanol (3.1.4) and mix it uniformly; add 2mL of 20% potassium hydroxide solution (3.2.2) into the solution and mix it uniformly through 1 min of vortex shaking; shake and saponify the solution without light for 30min at room temperature, extract the solution for 3 min with 10mL of extraction solvent (3.2.3) away from light through vortex shaking, and centrifuge it for 3min at 4,500r/min; repeat the extraction for twice and combine the extraction solution; rinse it with 10mL of water, centrifuge for 3 min at 4,500r/min for separation, then rinse it once again; combine the organic phase and concentrate it to nearly dry with reduced pressure; dissolve the residue with 0.1% BHT (3.2.4) through vortex shaking and scale the volume to 5mL; filter the solution with 0.45μm membrane filter (3.6) to carry out liquid chromatographic determination.
5.2.2 Other foods (e.g., rice, flour products and jam)
Accurately weigh 5g (with an accuracy of 0.01g) of uniform sample and place it into a 50mL centrifugal polypropene tube; extract it for 3min away from light with 10mL of extraction solvent (3.2.3) through vortex shaking and centrifuge for 3min at 4,500r/min; repeat the extraction for twice and combine the extraction solution; concentrate it to nearly dry at room temperature with reduced pressure; dissolve it with 3mL of extraction solvent (3.2.3) through vortex shaking; repeat the operation once again, combine the extraction solvent and mix it uniformly for purify.
Filter the above-mentioned solution with activated alumina-N solid phase extraction column (3.5) at the flow velocity of approximately 1mL /min; elute it with 3mL of extraction solvent (3.2.3), then combine the effluent and eluent and concentrate the solution to nearly dry with reduced pressure at room temperature; dissolve the residue with 0.1% BHT ethanol solution (3.2.4) through vortex shaking and scale the volume to 10 mL; then filter it through a 0.45μm membrane filter (3.6) for liquid chromatographic determination.
Contents of GB 5009.248-2016
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Apparatus
5 Analysis Steps
6 Expression of Analysis Result
7 Precision
8 Others
Appendix A Calibration Method for Concentration of Standard Solution
Appendix B Liquid Chromatogram of Standard Solution
