GB 5009.299-2024National food safety standards--Determination of lactoferrin in food (English PDF)

3.1.1 Acetonitrile (CH3CN). chromatographically pure.

3.1.5 Disodium hydrogen phosphate (Na2HPO4).

3.2 Preparation of reagents

3.2.1 Phosphate buffer I. Weigh 7.1 g of disodium hydrogen phosphate and 58.4 g of

sodium chloride, add 900 mL of water to dissolve, adjust the pH to 8.0 +/- 0.2 with phosphoric acid, add water to make the volume to 1000 mL, and freshly prepare each time before use.

3.3 Reference material

Bovine lactoferrin reference material (CAS number. 146897-68-9). purity >= 95 %, iron content <= 35 mg/100 g, or a reference material certified by the country and awarded a reference material certificate.

3.4 Preparation of standard solutions

3.4.1 Bovine lactoferrin standard stock solution (5.0 mg/mL). Weigh 50 mg (accurate

to 0.1 mg) of bovine lactoferrin reference material, dissolve it in water, transfer to a 10 mL volumetric flask and dilute to the mark, and mix well. Store at 0 degrees C ~ 4 degrees C, it can be stored for 3 months.

3.5 Materials

3.5.1 Heparin affinity column. 1 mL, column capacity >= 2 mg, or one with equivalent

performance (see Annex B for verification method).

3.5.2 Filter membrane, aqueous phase, 0.45 um.

3.5.3 Aqueous syringe filter. polyethersulfone material, 0.45 um.

4.1 High-performance liquid chromatograph. equipped with a UV detector or

equivalent.

4.5 Vortex oscillator.

4.6 Constant temperature water bath.

4.7 Solid phase extraction column device.

5.1 Pre-treatment of samples

5.1.1 Preparation of samples

Take at least 200 g of a representative sample. If it is a liquid sample, shake well and store it in a container as a sample, store it at 0 degrees C ~ 4 degrees C for later use. If it is a solid sample, mix well and store it in a container as a sample, store it dry at room temperature for later use.

5.1.2 Extraction of samples

5.1.3 Purification of samples

Before use, add 5.0 mL of phosphate buffer II to activate the heparin affinity column.

Accurately pipette 10.0 mL of the above sample solution to pass through the column, control the flow rate at 1 mL/min ~ 3 mL/min.

5.2 Instrument reference conditions

5.2.1 Chromatographic column. C4, 5 um, 300Å, 250 mm x 4.6 mm (inner diameter) or

equivalent.

5.3 Plotting of standard curve

Respectively inject the bovine lactoferrin standard series working solutions into the high-performance liquid chromatograph, determine the corresponding peak areas, and plot a standard curve with the concentrations of bovine lactoferrin in the standard working solutions as the abscissa and the peak areas as the ordinate.

5.4 Determination of sample solution

Inject the sample solution into the high-performance liquid chromatograph to obtain the peak area, then obtain the concentration of bovine lactoferrin in the sample according to the standard curve.