1. This experiment refers to GB 5009.299-2024, NY/T 4439-2023, as well as relevant group and enterprise standards, to determine the content of lactoferrin in pasteurized milk, ultra-high temperature sterilized milk, and infant formula milk powder.

2. To achieve good peak shape and separation, the sample pretreatment and liquid chromatography conditions were appropriately optimized in the experiment. Under the conditions of column temperature of 30 ℃ and flow rate of 1.0 mL/min0.1% trifluoroacetic acid water -0.1% trifluoroacetic acid acetonitrileAs a mobile phase for gradient elution, the maximum pressure value of the system does not exceed 106 bar.

3. The experimental results showed that the chromatographic peak asymmetry factor of lactoferrin standard solution (50.0 μ g/mL) was 1.00, the peak shape was good, the theoretical plate number was 30549, and the column efficiency was excellent; The RSD of retention time and peak area for three consecutive injections is less than 1%, indicating good reproducibility of the method; The linear low point (1.00 μ g/mL) has a signal-to-noise ratio of 8.2, indicating high sensitivity of the method; Linear range 1.00-200 μ g/mL, linear correlation coefficient r²≥ 0.9998, with good linearity.

4. In the experiment of determining lactoferrin content in dairy products by reverse phase liquid chromatography, the BioBasic 4 column (pore size 300 Å) combined with the Vanquish Core high-performance liquid chromatography system showed good reproducibility and excellent stability. The detection limit and quantification limit of the method met the standard requirements, which can be used as a reference for dairy companies and food testing laboratories.

1.1 Sample extraction

1.1.1Liquid samples: Low temperature milk (raw milk, pasteurized milk)/room temperature milk (ultra-high temperature (UHT) sterilized milk)

Weigh 5 g (accurate to 0.1 mg) of the sample into a 50 mL graduated centrifuge tube, dilute to 25.0 mL with phosphate buffer solution II, extract by vortex oscillation for 3 minutes, and centrifuge at 12000 rpm at 4 ℃ for 15 minutes. Let it stand at room temperature, remove the upper layer of floating fat, and take the supernatant for purification.

1.1.2Solid sample: Milk powder (infant formula milk powder)

Weigh 2 g (accurate to 0.1 mg) of the sample and transfer it to a 50 mL graduated centrifuge tube. Add an appropriate amount of phosphate buffer solution II and shake to dissolve. Dilute to 50.0 mL with phosphate buffer solution II, vortex and shake for 3 minutes, let it stand for more than 30 minutes, and centrifuge at 12000 rpm at 4 ℃ for 15 minutes. Let it stand at room temperature, remove the upper layer of floating fat, and take the supernatant for purification.

1.2 Sample Purification

1.2.1Activation: The lower end of the HyperSep heparin affinity column (P/N: 60105-110-B) is connected to a solid phase extraction device via a Luer connector, and the upper end is connected in series with a 10 mL syringe empty cylinder through an adapter (P/N: 60104-259). 2 × 5 mL of phosphate buffer solution II is added to activate heparin affinity, and the drip rate is controlled at 2d/3s;

1.2.2Sample loading: When the liquid level of the activation step approaches the sieve plate of the small column, accurately transfer 10.0 mL of the above sample solution to the empty cylinder of the syringe (P/N: S7510-10), control the drip rate for 2d/3s, and discard the effluent;

1.2.3Rinsing: When the liquid level of the sample is close to the sieve plate of the small column, add 2 × 5 mL of phosphate buffer solution II to rinse the small column, discard the effluent, remove the adapter and empty syringe, and vacuum dry the small column for 2 minutes;

1.2.4Elution: Add 2 × 2.5 mL of phosphate buffer solution I for elution, collect all elution solutions, and dilute to 5.00 mL with phosphate buffer solution I. Pass through a 0.2 μ m hydrophilic PTFE needle filter (P/N: 42213-NPL), and the filtrate is ready for machine testing.

Thermo Scientific ™ Vanquish Core ™ High performance liquid chromatography system, configuration: pump Quaternary Pump C (VC-P20-A), automatic sampler Split Sampler CT (VC-A12-A), column temperature box Column Comparison C (VC-C10-A), detector Diode Array Detector CG (VC-D11-A), data processing Chromeleon ™ seven point three

Column: BioBasic 4,5 μ m, 4.6 × 250 mm (P/N: 72305-254630)

Mobile phase: A: 0.1% trifluoroacetic acid water B: 0.1% trifluoroacetic acid acetonitrile

Flow rate: 1.0 mL/min

Gradient elution program:

Injection volume: 50 µ L

Column temperature: 30 ℃

Detector: DAD, acquisition wavelength: 280 nm; acquisition frequency: 5 Hz; spectral acquisition: 190-400 nm