National Standards of the People's Republic of China
gb 5009.292-2023
National standards for food safetyin foodApuDetermination of carotene aldehyde
Scope
This standard specifies a liquid chromatography method for the determination of -apo-8'-carotene aldehyde in food.
This standard applies to flavored fermented milk, processed cheese, frozen drinks, confectionery, baked goods, semi-solid compound seasonings and beverages-AP-8'Determination of carotene aldehyde
Principle
The sample was saponified with potassium hydroxide solution, and the free -Apo-8 was extracted from n-hexane'- Carotene aldehyde, the extract is concentrated and reconstituted with acetonitrile, separated by high performance liquid chromatography, detected by ultraviolet-visible light detector or diode array detector, and quantified by external standard method.
Reagents and materials
Unless otherwise stated, the reagents used in this method are analytically pure, and the water is the first-class water specified in GB T 6682.
3.1 Reagent.
3.1.1 Acetonitrile (C2H3N): chromatographically pure.
3.1.2 absolute ethanol (C2H6O).
3.1.3 n-hexane (C6H14).
3.1.4 potassium hydroxide (KOH).
3.1.5 2,6-di-tert-butyl-p-cresol (C15H24O, butylated hydroxytoluene, BHT).
3.1.6 formic acid (CH2O2): chromatographically pure.
3.2. Reagent preparation.
3.2.1 potassium hydroxide solution (200 g l): weigh 200 g of potassium hydroxide and dilute it to 1 000 ml with water.
3.2.2 0.1% BHT-acetonitrile solution: weigh 01 g BHT, dissolved in 100 ml acetonitrile, mix well.
3.2.3 0.1% formic acid (volume fraction) solution: Pipette 1 ml of formic acid and dilute to 1 L with water.
3.3 standard products.
Apo-8'-carotene aldehyde (C30H40O, CAS number: 1107-26-2): 98% purity, or a reference material that has been certified by the state and awarded a sub-reference material certificate.
3.4. Preparation of standard solution.
3.4.1-Apo-8'-carotene aldehyde standard stock solution (10 g ml): Weigh 1 mg accurately (accurate to 0.).01 mg) - Apo-8'- Carotene aldehyde standard with 0Dissolve in 1% BHT-acetonitrile solution and transfer to a 100 ml brown volumetric flask with 01% BHT-acetonitrile solution to 100 mL and mix well. Stored on -18 and valid for 3 months.
Note: Apo-8'-carotene aldehyde standard stock solution needs to be calibrated before use, see Appendix A for specific operations.
3.4.2 - Apo-8'- Carotene aldehyde standard series working solution: Absorb an appropriate amount of -Apo-8'-carotene aldehyde standard stock solution in a 10ml brown volumetric flask, add acetonitrile to the scale, and prepare the mass concentration of 00500μg/ml、0.100μg/ml、0.200μg/ml、0.500μg/ml、1.00 g ml of working solution. Temporary use.
Instruments and equipment
4.1 Liquid chromatograph: equipped with diode array detector or ultraviolet-visible light detector.
4.2 balances: the inductance is 001 g and 001 mg。
4.3 spectrophotometer.
4.4. Constant temperature shaking water bath device.
4.5. Tissue masher.
4.6. Rotary evaporator.
4.7. Tissue homogenizer.
4.8 Centrifuge: 4 000 r min.
4.9. Vortex mixer.
4.10Ultrasonic cleaner: operating frequency 40 kHz, power 500 W.
4.11. Organic phase microporous membrane: 045 μm。
Analyze the steps
Note: Due to -Apo-8'- Carotene aldehyde is light-sensitive, unless otherwise stated, all test operations should be performed in an environment free of yellow light source or red light source below 500 nm ultraviolet light.
5.1. Sample preparation.
5.1.1. Specimen preparation.
Solid samples (candy, baked goods, solid beverages, etc.) are fully crushed and homogenized by a tissue mascher; Semi-solid samples (flavored fermented milk, semi-solid compound seasonings, etc.) were homogenized by a tissue homogenizer and mixed; Liquid samples (liquid beverages, etc.) are mixed directly and evenly; Mix the frozen drink well after melting in a 40 water bath and avoiding light.
After a certain number of samples are prepared according to the requirements, they should be stored in a sample bottle, protected from light and refrigerated, and tested as soon as possible.
5.1.2. Specimen processing.
Weigh 2 g accurately (accurate to 0.).01 g) Specimen in a 50 ml centrifuge tube, add about 02 gbht, 10 ml absolute ethanol, 10 ml potassium hydroxide solution, vortex and shake for 1 min, mix well, and place in a 30 2 constant temperature water bath device to avoid light and shake for saponification for 30 min.
The saponification solution was transferred to the separating funnel, and 10 ml of water was added to the separating funnel twice, 10 ml of n-hexane was added, and the shaking extraction was carried out for 5 min, the lower solution was transferred to another 100 ml separating funnel, 10 ml of n-hexane was added to extract again, the lower aqueous solution was discarded, the n-hexane layer was combined into a 50 ml evaporation flask, and the rotary evaporation was concentrated to near dry at room temperature. Accurately add 20 ml of acetonitrile to dissolve the residue, vortex and mix for 30s, over 045 m organic membrane for liquid chromatography.
5.1.3 Blank experiments.
Except without a sample, in accordance with 51.2. Perform blank experiments for the assay step.
5.2 Instrument Reference Conditions.
5.2.1 Column: C18 column, 150 mm 46 mm（i.d.), particle size 5 m, or a column with equivalent performance.
5.2.2 mobile phase: acetonitrile +01% formic acid solution (95:5, volume ratio).
5.2.3 Flow Rate: 10 ml/ min。
5.2.4 Detection wavelength: 460 nm.
5.2.5. Column temperature: 40.
5.2.6. Injection volume: 20 l.
5.3. Production of standard curves.
The standard series working solution was injected into the high-performance liquid chromatograph respectively, and the corresponding peak area was determined, and the standard series working solution was used in -Apo-8'The mass concentration of -carotene aldehyde was taken as the abscissa, and the peak area of -Apo-8'-carotene aldehyde was used as the ordinate to plot the standard curve. - The chromatogram of the Apo-8'-carotene aldehyde standard solution is shown in Appendix B.
5.4. Determination of sample solution.
The sample solution was injected into the high-performance liquid chromatograph to obtain the peak area of the analyte, and the standard curve was substituted to calculate -Apo-8 in the test solution'- Mass concentration of carotene aldehyde.
The retention time of the compound chromatographic peaks in the sample to be tested should vary by 2 compared to the standard solutionWithin 5%.
Calculation and presentation of results
In the specimen -AP-8'- The content of carotene aldehyde is calculated according to equation (1).
Where: x - in the specimen - Apo-8'- carotene aldehyde content in grams per kilogram (g kg);
The mass concentration of -apo-8'-carotene aldehyde in the sample solution obtained from the standard curve in micrograms per milliliter (g ml);
0 - in the blank experiment obtained from the standard curve -Apo-8'- The mass concentration of carotene aldehyde in micrograms per milliliter.
g/ml）；
v - the reconstituted volume of the sample, in milliliters (ml);
m - the sample size of the sample, in grams (g);
1 000 - unit conversion factor.
The calculation result retains 2 significant digits.
Precision
The absolute difference between the results of two independent measurements obtained under repeatability conditions must not exceed 15% of the arithmetic mean.
Miscellaneous
When the sample size is 2 g, the detection limit is 0000 2 g kg, the limit of quantification is 0000 5 g/kg。
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