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GB 5009.248-2016 PDF English

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GB 5009.248-2016: National food safety standard - Determination of lutein in foods
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GB 5009.248-2016: National food safety standard - Determination of lutein in foods

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GB NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA National Food Safety Standard - Determination of lutein in foods Issued on. AUGUST 31, 2016 Implemented on. MARCH 01, 2017 Issued by. National Health and Family Planning Commission of the PRC

Table of Contents

Foreword... 3 1 Scope... 4 2 Principles... 4 3 Reagents and materials... 4 4 Instruments and equipment... 6 5 Analytical procedures... 6 6 Analysis results expression... 9 7 Precision... 9 8 Other... 9 Appendix A Method for correction of standard solution concentration... 11 Appendix B Liquid chromatogram of standard solution... 12

Foreword

This Standard replaces GB/T 23209-2008 “Determination of lutein in milk powder - HPLC-UV method”. As compared with GB/T 23209-2008, the main changes of this Standard are as follows. - CHANGE the standard’s name to “National Food Safety Standard - Determination of lutein in foods”; - EXPAND the scope of application; ADD the liquid chromatography for the determination of lutein in frozen drinks, rice and flour products, bakery products, jams, jellies, and beverages; - MODIFY the “Principles” of the former standard. The former “USE acetone as a solvent to extract” is changed to “USE diethyl ether-n-hexane- cyclohexane solvent system to extract”; - ADD the points worthy of notice for the operation process; - ADD a saponification step for samples with high fat content including milk powder; and PROVIDE different pretreatment methods to accommodate various sample matrix analysis needs; - ADD the correction requirement for the concentration of lutein standard solution; - For the phenomenon that lutein may be isomerized during the test operation, ADD the qualitative and quantitative requirements for cis-lutein produced due to isomerization. National Food Safety Standard - Determination of lutein in foods

1 Scope

This Standard specifies the liquid chromatography for the determination of lutein in foods. This Standard applies to liquid chromatography determination of lutein in infant formula, dairy products, frozen drinks, rice and flour products, bakery products, jams, jellies, and beverages.

2 Principles

After foods with high fat content (fat content of not less than 3% on a dry basis) are saponified with potassium hydroxide solution at room temperature to free lutein, then USE diethyl ether-n-hexane-cyclohexane (40+40+20, volume ratio) to extract; USE liquid chromatography to separate; USE UV-detector or diode array detector to detect; and USE external standard method for quantification. Lutein in the samples of the other foods is directly extracted by diethyl ether-n- hexane-cyclohexane (40+40+20, volume ratio). After the extract is purified by a neutral alumina solid phase extraction cartridge, USE liquid chromatography to separate; USE UV-detector or diode array detector to detect; and USE external standard method for quantification.

3 Reagents and materials

Unless otherwise stated, the reagents used in this method are of analytically pure; the water is Grade I water specified in GB/T 6682. 3.1 Reagents 3.1.1 Cyclohexane (C6H12). chromatographically pure. 3.1.2 Diethyl ether [(C2H5)2O]. chromatographically pure. 3.1.7 Potassium hydroxide (KOH). 3.1.8 Iodine (I2). 3.2 Preparation of reagents 3.2.1 10% potassium hydroxide solution. WEIGH 10 g of potassium hydroxide (3.1.7); ADD water to dissolve and dilute to 100 mL. 3.2.2 20% potassium hydroxide solution. WEIGH 20 g of potassium hydroxide (3.1.7); ADD water to dissolve and dilute to 100 mL. 3.2.3 Extraction solvent. WEIGH 1 g of BHT (3.1.6); USE 200 mL of cyclohexane (3.1.1) to dissolve; ADD 400 mL of diethyl ether (3.1.2) and 400 mL of n-hexane (3.1.3), and MIX well. 3.3 Standard Lutein (CAS number. 127-40-2), with a purity of not less than 98.0%. 3.4 Preparation of standard solutions 3.4.1 Standard stock solution (50 μg/mL). Accurately WEIGH 5 mg (accurate to 0.01 mg) of lutein (3.1.3); USE 0.1% BHT ethanol solution (3.2.4) to dissolve and dilute to 100 mL. This standard stock solution can be stored for six months by filling nitrogen and avoiding light in a refrigerator of -20 °C or below. 3.4.2 Standard working solutions. accurately PIPETTE 0.050 mL, 0.100 mL, 0.200 mL, 0.400 mL, 1.00 mL of standard stock solution of lutein (3.4.1) into 5 25 mL brown volumetric flasks; USE 0.1% BHT ethanol solution (3.2.4) to dilute to volume; and OBTAIN a series of standard working solutions having concentrations of 0.100 μg/mL, 0.200 μg/mL, 0.400 μg/mL, 0.800 μg/mL, and 2.00 μg/mL.

4 Instruments and equipment

4.1 Liquid chromatograph, with diode array detector or UV-detector. 4.2 UV-visible spectrophotometer. 4.3 Analytical balance. The sensitivity is 0.01 mg and 0.01 g. 4.7 Decompression concentrator. 4.8 Solid phase extraction plant. 4.9 Centrifuge. The speed is not less than 4500 r/min.

5 Analytical procedures

Note. Because lutein is sensitive to light, unless otherwise stated, all test operations shall be performed in a yellow or red light source environment with no UV light below 500 nm. 5.1 Sample preparation A certain number of samples are pulverized, homogenized, and divided as required, and stored in a sample bottle. The prepared sample shall be filled with nitrogen and sealed; and stored in a refrigerator at -20 °C or below. 5.2 Extraction 5.2.1 Foods with high fat content (such as infant formula, milk powder, ice cream, bakery nuts, etc.) 5.3 Instrument reference conditions 5.3.1 Chromatographic column. C30 column, 5 μm, 250 mm×4.6 mm (inner diameter) or equivalent; 5.3.2 Column temperature. 30 °C; 5.3.3 Mobile phase. methanol/water (88+12, volume ratio, containing 0.1% BHT)-methyl tert-butyl ether (containing 0.1% BHT), gradient elution. 0 min~18 min, methanol/water is changed from 100% to 10%; 18.1 min, methanol/water is changed from 10% to 100%, retained for 10 min; 5.4 Making of standard curve The series of standard working solutions are separately injected into the liquid chromatograph, to determine the corresponding peak area. USE the concentration of standard working solution as the abscissa. USE the peak area as the ordinate. The standard curve is drawn. 5.5 Determination of sample solution The response value of lutein in the sample solution to be determined shall be within the linear response range of the instrument, otherwise it shall be diluted or concentrated appropriately.

6 Analysis results expression

The content of lutein in the sample shall be calculated according to formula (1).

7 Precision

The absolute difference between the two independent determination results, obtained under repeated conditions, shall not exceed 15% of the arithmetic mean.

8 Other

The detection limit of this method. For infant formula, milk powder, ice cream, bakery nuts, and other foods, when the sampling amount is 2 g and the constant volume is 5 mL, the detection limit is 3 μg/100 g. For liquid milk, etc., when the sampling amount is 10 g and the constant volume is 25 mL, the detection limit is 3 μg/100 g. For rice and flour products, jams, and other foods, when the sampling amount is 5 g and the constant volume is 10 mL, the detection limit is 3 μg/100 g.

Appendix A

Method for correction of standard solution concentration The lutein standard solution, after preparation, needs to be calibrated. TAKE 1 mL of standard stock solution; USE ethanol to dilute to 25 mL. PIPETTE this solution into a 1 cm quartz cuvette. USE ethanol as a blank; USE a spectrophotometer, under a wavelength of 445 nm, to determine the absorbance A. According to formula (A.1), calculate the standard solution concentration.

Appendix B

Liquid chromatogram of standard solution B.1 Liquid chromatogram of lutein (trans) standard solution The liquid chromatogram of lutein (trans) standard solution is shown in Figure B.1. ......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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