GB 5009.32-2016Determination of propyl gallate in oils and fats (English PDF)

3.1 Reagents

3.1.1 Formic acid (HCOOH).

3.3.8 Tert-butylhydroquinone. purity >=98%.

3.3.9 2,6-Di-tert-butyl-4-hydroxymethylphenol. purity >=98%.

NOTE. See Annex A for English names, molecular formulas, and CAS numbers.

3.4 Preparation of standard solutions

3.4.1 Mixed stock solution of antioxidant reference materials. Accurately weigh 0.1 g

(to the nearest 0.1 mg) of solid antioxidant reference materials. Dissolve the materials in acetonitrile in a 100 mL amber volumetric flask and dilute to the mark to prepare a mixed stock solution with a concentration of 1000 mg/L. Store at 0°C~4°C, protected from light.

3.4.2 Antioxidant mixed working standard solutions. Pipette appropriate volumes of

the mixed antioxidant standard stock solution (1000 mg/L) and dilute them to prepare mixed working standard solutions with concentrations of 20 mg/L, 50 mg/L, 100 mg/L,

200 mg/L, and 400 mg/L, respectively.

3.5 Materials

3.5.1 C18 solid-phase extraction column. 2000 mg/12 mL.

3.5.2 Organic membrane filter. pore size is 0.22 µm.

5.1 Specimen preparation

Pulverize and homogenize solid or semi-solid samples. Then, using the diagonal method, take two-quarters or two-sixths of the material, or otherwise obtain a representative sample based on the specific nature of the specimen. Seal and store. For liquid samples, mix thoroughly, take a representative sample, and seal for storage.

5.2 Determination steps

5.2.1 Extraction

5.2.1.1 For solid samples. Weigh 1 g (to the nearest 0.01 g) of the specimen (5.1) into a 50 mL centrifuge tube. Add 5 mL of acetonitrile-saturated n-hexane. Vortex for 1 min to mix thoroughly, then let soak for 10 min. Add 5 mL of saturated sodium chloride solution. Vortex with 5 mL of n-hexane-saturated acetonitrile for 2 min and centrifuge at 3000 r/min for 5 min. Collect the acetonitrile layer in a test tube. Repeat the extraction twice more using 5 mL of n-hexane-saturated acetonitrile each time. Combine the three extracts. Adjust the pH to 4 using 0.1% formic acid solution, and set aside for cleanup.

Conduct a blank test in parallel.

5.2.1.2 For oil samples. Weigh 1 g (to the nearest 0.01 g) of the specimen (5.1) into a

50 mL centrifuge tube. Add 5 mL of acetonitrile-saturated n-hexane to dissolve the sample. Vortex for 1 min and let stand for 10 min. Extract by vortexing for 2 min with

5 mL of n-hexane-saturated acetonitrile. Centrifuge at 3000 r/min for 5 min and collect the acetonitrile layer in a test tube. Repeat the extraction twice more using 5 mL of n- hexane-saturated acetonitrile each time. Combine the three extracts for subsequent cleanup. Conduct a blank test in parallel.

5.2.2 Purification

Pack a C18 solid-phase extraction (SPE) column with approximately 2 g of anhydrous sodium sulfate. Condition the column with 5 mL of methanol, then equilibrate it with 5 mL of acetonitrile. Discard the effluent. Pour the entire extract (from 5.2.1) into the column and discard the effluent. Elute with 5 mL of an acetonitrile-methanol mixture and collect the eluate in a test tube. Evaporate to dryness by rotary evaporation at 40°C.

Reconstitute the residue to a final volume of 2 mL with acetonitrile. Filter the solution through a 0.22 µm organic-compatible membrane filter prior to liquid chromatographic analysis.

5.2.3 Gel permeation chromatography (optional for pure oil samples)

Weigh 10 g (to the nearest 0.01 g) of the specimen (5.1) into a 100 mL volumetric flask.

Dilute to the mark with a mixture of ethyl acetate and cyclohexane to serve as the stock solution. Transfer 5 mL of the stock solution into a 10 mL volumetric flask. Dilute to the mark with the ethyl acetate-cyclohexane mixture. Set aside for cleanup.

Transfer 10 mL of the sample solution into the injection vial for gel permeation chromatography (GPC). Perform GPC cleanup (refer to Annex B for GPC cleanup conditions) and collect the eluate. Evaporate the eluate to dryness using a rotary evaporator at 40°C. Reconstitute the residue with acetonitrile to a final volume of 2 mL.

Filter the solution through a 0.22 µm organic-compatible membrane filter for liquid chromatographic analysis. Conduct a blank test in parallel.

5.3 Liquid chromatography conditions

a) Chromatographic column. C18 column, length of 250 mm, inner diameter of 4.6 mm, particle size of 5 µm, or equivalent;

b) Mobile phase A. 0.5% aqueous formic acid solution; Mobile phase B. methanol;

c) Elution gradient. 0 min~5 min mobile phase (A) 50%, 5~15 min. mobile phase (A) decreases from 50% to 20%; 15 min~20 min mobile phase (A) 20%, 20 min~25 min. mobile phase (A) decreases from 20% to 10%; 25 min~27 min.

mobile phase (A) increases from 10% to 50%; 27 min~30 min. mobile phase (A)

50%;

d) Column temperature. 35°C;

e) Injection volume. 5 µL;

f) Detection wavelength. 280 nm.

5.4 Preparation of the standard curve

Inject standard working solutions of various concentrations into the liquid chromatograph. Determine the corresponding antioxidants. Plot a standard curve with the concentration of the standard working solution on the x-axis and the response value (e.g., peak area, peak height, absorbance) on the y-axis. Standard liquid chromatograms for the nine antioxidants are shown in Annex C.

5.5 Determination of the specimen solution

Inject the specimen solution into the high-performance liquid chromatograph to obtain the response values of the corresponding chromatographic peaks. Determine the concentration of the antioxidant in the test solution based on the standard curve.

10.1 Reagents

Same as 3.1.

10.2 Preparation of reagents

Same as 3.2.

10.3 Reference products

10.3.1 Octyl gallate. purity >=98%.

10.3.2 Dodecyl gallate. purity >=98%.

10.3.3 Propyl gallate. purity >=98%.

10.3.4 Nordihydroguaiaretic acid. purity >=98%.

10.3.5 2,4,5-Trihydroxybutyrophenone. purity >=98%.

NOTE. See Annex A for English names, molecular formulas, and CAS numbers.

10.4 Preparation of standard solutions

10.4.1 Standard stock solution. Accurately weigh 0.1 g (to the nearest 0.1 mg) of the solid antioxidant reference material. Dissolve it in acetonitrile in a 100 mL amber volumetric flask and dilute to the mark to prepare a standard stock solution with a concentration of 1000 mg/L. Store at 0°C~4°C, protected from light.

10.4.2 Intermediate standard solution. Pipette 1.0 mL of the standard stock solution into

a 100 mL volumetric flask and dilute to volume with acetonitrile to prepare a mixed intermediate standard solution with a concentration of 10 mg/L. Store at 0°C~4°C, protected from light.

10.4.3 Working standard solutions. Pipette appropriate volumes of the intermediate

standard solution and dilute them separately to prepare mixed working standard solutions with concentrations of 0.01 mg/L, 0.02 mg/L, 0.05 mg/L, 0.1 mg/L, 0.2 mg/L,

0.5 mg/L, 1 mg/L, and 2 mg/L.

10.5 Materials

Same as 3.5.