Two-dimensional liquid chromatography method for determining lactoferrin in infant milk powder

Abstract

This invention discloses a two-dimensional liquid chromatography (HPLC) method for determining lactoferrin in infant formula. Based on online purification HPLC technology, the first dimension uses a heparin affinity column, and the second dimension uses a reversed-phase C4 column. This method achieves accurate qualitative and quantitative analysis of lactoferrin in infant formula. By adding Tween-20 and sodium chloride to the extract, non-specific adsorption and competitive binding in complex matrices are effectively inhibited, significantly improving the recovery rate of lactoferrin (93.5%–97.3%). The method exhibits good linearity in the range of 5–200 μg / mL, with a detection limit of 2 mg / 100g, a quantitation limit of 5 mg / 100g, and an RSD of 1.2%–3.9%, providing accurate and reliable results. Optimized flow path and cleaning procedures enable online two-dimensional purification and analysis, allowing the expensive heparin affinity column to be reused and reducing detection costs.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a method for determining the lactoferrin content in infant formula using online two-dimensional liquid chromatography. Background Technology

[0002] Lactoferrin (LF) is a non-heme iron-binding glycoprotein with multiple biological activities. With a molecular weight of 80 kDa, it is one of the most representative bioactive proteins in mammalian milk. Lactoferrin possesses functions such as iron transport, antibacterial activity, immune regulation, gut microbiota regulation, and antioxidant activity. Bovine lactoferrin and human lactoferrin are highly similar in protein structure and amino acid sequence, are easily absorbed by the human body, and are widely used in dairy products, infant formula, and other foods. my country's GB 14880-2012 standard stipulates that its maximum usage in modified milk, flavored fermented milk, milk-containing beverages, and infant formula is 1.0 g / kg. Therefore, establishing rapid and accurate detection methods is crucial for ensuring product quality and safety.

[0003] Currently, methods for detecting lactoferrin include liquid chromatography (LC), enzyme-linked immunosorbent assay (ELISA), optical biosensor methods, capillary electrophoresis, and liquid chromatography-tandem mass spectrometry (LC-MS / MS). However, isotope peptides and LC-MS / MS instruments are expensive and not suitable for widespread use; moreover, enzyme cleavage sites are easily affected by protein glycosylation, leading to incomplete digestion and increasing detection errors. Capillary electrophoresis is easily interfered with by other proteins in the sample, so this method is often used for the detection of lactoferrin tablets and raw materials with relatively simple matrices. Optical biosensor methods are simple to operate but have low sensitivity. ELISA and LC typically employ immunoaffinity purification before detection. Among these, ELISA is more cumbersome and easily affected by impurities in the sample.

[0004] The current national standard GB 5009.299-2024 adopts the heparin affinity column purification-high performance liquid chromatography method. However, this method has several shortcomings: First, it is explicitly unsuitable for flavored fermented milk, soy-based infant formula, especially infant formula containing hydrolyzed protein (such as special medical purpose infant formula) and goat milk-based infant formula, because the free amino acids and peptides in these matrices will compete with lactoferrin for binding sites on the affinity column, leading to lower detection results; second, the heparin affinity column used in this method is a disposable consumable, which is expensive, cumbersome to operate, time-consuming, and prone to clogging, making it unsuitable for the analysis of large batches of samples.

[0005] In existing technologies, there are also reports of using two-dimensional liquid chromatography (2D liquid chromatography) to analyze proteins. For example, the literature "Simultaneous Determination of Lactoferrin and Immunoglobulin G in Dairy Products by Online 2D Liquid Chromatography" (Chen Qin et al.) discloses a method that uses a Pharmacia HI-Trap Protein G column as a one-dimensional chromatographic column and a size exclusion column as a two-dimensional chromatographic column to simultaneously detect lactoferrin and immunoglobulin G. However, it is mainly aimed at conventional dairy products and does not solve the interference problem of special matrices (hydrolyzed protein, goat milk powder). It cannot detect special medical-purpose infant formula milk powder and infant formula goat milk powder containing hydrolyzed protein, and its mobile phase system and separation mode are different from those of this invention. The literature "Identification and Comparative Quantification of Proteins Using iTRAQ Reagent Combined with 2D Liquid Chromatography-Mass Spectrometry" (Hu Wei et al.) uses strong cation exchange (SCX) coupled with reversed-phase (RP) chromatography, but its purpose is qualitative and quantitative research in proteomics, rather than targeted quantitative detection of specific proteins in complex food matrices.

[0006] Therefore, developing a method that can accurately, efficiently, and cost-effectively determine lactoferrin in infant formula, including partially hydrolyzed protein formulas and goat milk-based formulas, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] This invention aims to overcome the shortcomings of existing technologies and provide a two-dimensional liquid chromatography method for determining lactoferrin in infant formula. This method significantly improves the detection accuracy of lactoferrin in complex matrices (especially those containing hydrolyzed proteins and sheep milk-based samples) by constructing a specific "affinity chromatography-reversed-phase chromatography" online two-dimensional liquid chromatography system and optimizing the composition of the sample extract, while simultaneously reducing detection costs and improving analytical efficiency.

[0008] To achieve the above objectives, the present invention adopts the following specific technical solution: The present invention provides a two-dimensional liquid chromatography method for determining lactoferrin in infant formula, comprising the following steps: (1) Sample pretreatment: Weigh the sample to be tested, add phosphate buffer I for extraction, vortex mix, centrifuge and take the intermediate liquid (the upper layer is the fat layer and the lower layer is the precipitate, both of which are discarded) to obtain the sample solution. The phosphate buffer I is a 100 mmol / L phosphate buffer containing Tween-20 and sodium chloride, with a pH of 8.0±0.2; preferably, the concentration of Tween-20 in the phosphate buffer I is 3 to 7 mL / L, preferably 5 mL / L, and the concentration of sodium chloride is 20 to 40 mmol / L, preferably 30 mmol / L. In the sample pretreatment step, the weight of the solid sample is 1-5g, and the volume of the extract is 50mL. (2) Two-dimensional liquid chromatography analysis: The sample solution obtained in step (1) is injected into an online two-dimensional liquid chromatography system for detection; The two-dimensional liquid chromatography system includes: a first-dimensional column, a second-dimensional column, and a six-way valve (with a sample quantification loop) for connecting the two dimensions; the first-dimensional column is a heparin affinity column for capturing and enriching lactoferrin from the sample solution, and the second-dimensional column is a reversed-phase C4 column for separating and quantifying lactoferrin. The system flow path switching procedure for the two-dimensional liquid chromatography includes: During the first preset time period (e.g., 0–5.6 min), the six-way valve is in the first state (e.g., channel 1 and channel 2 are connected), allowing the effluent from the first-dimensional chromatographic column to enter the waste liquid, while the second-dimensional chromatographic column is equilibrated or analyzed. During the second preset time period (e.g., 5.6–6.6 min), the six-way valve switches to the second state (e.g., channel 1 and channel 6 are connected), precisely cutting and enriching the lactoferrin component eluted from the first-dimensional chromatographic column in the quantitative loop; During the third preset time period (e.g., 6.6–22 min), the six-way valve switches back to the first state (e.g., channel 1 and channel 2 are connected), and at the same time, the mobile phase is switched to carry the lactoferrin component enriched in the quantitative loop into the second-dimensional chromatographic column for separation and detection. The elution conditions for the first-dimensional chromatography include: gradient elution using mobile phase A1 and mobile phase B1, wherein mobile phase A1 is the phosphate buffer I and mobile phase B1 is a 50 mmol / L phosphate buffer containing 0.8–1.2 mol / L sodium chloride (pH 7.5 ± 0.2); and, during the first-dimensional column regeneration phase (e.g., 7.01–21.5 min), washing with pure water (as mobile phase C1) at a low flow rate (e.g., 0.3 mL / min) to extend the affinity column life. The detection conditions for the second dimension chromatography include: using a diode array detector, a detection wavelength of 280 nm, and gradient elution with an aqueous solution containing 0.1% trifluoroacetic acid as mobile phase A2 and acetonitrile as mobile phase B2; (3) Calculation of results: A standard curve was plotted with the concentration of lactoferrin standard on the x-axis and the peak area on the y-axis. The lactoferrin content was calculated by substituting the peak area of ​​lactoferrin in the sample solution into the standard curve.

[0009] Preferably, the first preset time period is 0 to 5.6 min, the second preset time period is 5.6 to 6.6 min, and the third preset time period is 6.6 to 22 min; the capacity of the sample quantification loop is 1 mL.

[0010] Preferably, the gradient elution range of the second-dimensional chromatographic column is as follows: 0–4 min: mobile phase A2 volume ratio is 10%, mobile phase B2 is 90%; 4.01–7 min: mobile phase A2 volume ratio is 60%, mobile phase B2 is 40%; 7–22 min: mobile phase A2 volume ratio is 40%, mobile phase B2 is 60%.

[0011] Preferably, the heparin affinity column is a Cytiva HiTrap heparin affinity column (1 mL), and the reversed-phase C4 column has specifications of 250 mm × 4.5 mm, particle size of 5 μm, and pore size of 300 Å, such as a Waters C4 column.

[0012] Preferably, the infant formula includes regular infant formula, infant formula goat milk powder, and partially hydrolyzed milk protein infant formula.

[0013] Preferably, the heparin affinity column can be reused more than 50 times by controlling the injection volume and adding an online cleaning step, while the lactoferrin recovery rate remains above 95%.

[0014] The present invention also provides the application of the two-dimensional liquid chromatography method described above in the detection of lactoferrin content in infant formula, special medical foods or dairy products.

[0015] Compared with the prior art, the present invention has the following technical advantages: (1) Excellent matrix adaptability: This invention effectively inhibits non-specific adsorption and competitive binding in complex matrices (especially partially hydrolyzed protein and goat milk-based samples) by optimizing the addition of specific concentrations of Tween-20 and sodium chloride to the extraction solution (phosphate buffer I), significantly improving the recovery rate of lactoferrin (93.5%–97.3%). Compared with the national standard method, it solves the problem of low detection results for partially hydrolyzed formula milk powder and goat milk powder.

[0016] (2) High accuracy and precision: The method of the present invention has good linearity in the range of 5 to 200 μg / mL (r=0.99994), and the limit of detection (2 mg / 100g) and limit of quantitation (5 mg / 100g) are better than the national standard. The RSD is low (1.2% to 3.9%), and the results are accurate and reliable.

[0017] (3) Low cost and high efficiency: This invention realizes online two-dimensional purification and analysis without the need for expensive solid phase extraction columns and cumbersome manual pretreatment processes (such as column passing, washing, and elution); more importantly, by optimizing the flow path and cleaning process, the expensive heparin affinity column can be reused (such as no performance degradation after more than 50 injections), which greatly reduces the detection cost and is suitable for rapid screening of large batches of samples.

[0018] (4) High degree of automation: The online two-dimensional liquid chromatography system constructed by this method realizes the integration and automation of sample purification and analysis through valve switching, which reduces human error and improves analysis efficiency. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the flow path connection for two-dimensional liquid chromatography according to the present invention.

[0020] Figure 2 This is a one-dimensional chromatogram for determining lactoferrin using the method of this invention.

[0021] Figure 3 This is a two-dimensional chromatogram for determining lactoferrin using the method of this invention.

[0022] Figure 4 This is a graph showing the effect of Tween-20 concentration on lactoferrin recovery rate in the method of this invention.

[0023] Figure 5 This is a graph showing the effect of sodium chloride concentration on the recovery rate of lactoferrin in the method of this invention. Detailed Implementation

[0024] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1 1. Instruments and reagents An Agilent 1260 liquid chromatograph (equipped with two quaternary pumps, an autosampler, a column oven, two DAD detectors, and a six-way valve); a heparin affinity column (Cytiva HiTrap, 1 mL); and a reversed-phase C4 column (Waters, 250 mm × 4.5 mm, 5 μm, 300 Å).

[0026] Lactoferrin standard (purity ≥95%); trifluoroacetic acid, acetonitrile (chromatographic grade); Tween-20, phosphate, sodium chloride, etc. (analytical grade).

[0027] Phosphate buffer I: 100 mmol / L phosphate buffer containing 5 mL / L Tween-20 and 30 mmol / L sodium chloride, pH 8.0.

[0028] Phosphate buffer II: 100 mmol / L phosphate buffer, pH 8.0 (excluding Tween-20 and sodium chloride, used for comparative experiments).

[0029] Mobile phase B1: 50 mmol / L phosphate buffer containing 1 mol / L sodium chloride, pH 7.5.

[0030] 2. Sample pretreatment Weigh 5g (accurate to 0.01g) of infant formula (regular, goat milk-based, or partially hydrolyzed formula) into a 50mL centrifuge tube, add phosphate buffer I to 40mL, and vortex to dissolve the sample; transfer to a 50mL volumetric flask, bring to the mark with phosphate buffer I, and mix well; transfer to a centrifuge tube, centrifuge at 10000 r / min for 8min, and take the intermediate liquid (the upper layer is the fat layer, and the lower layer is the precipitate, both of which should be discarded) to obtain the sample solution.

[0031] 3. Chromatographic conditions and flow path switching The connection diagram of the two-dimensional liquid chromatography system is shown below. Figure 1 The total run time was 22 minutes, the injection volume was 50 μL, the DAD detection wavelength was 280 nm, and the column temperature was 35 °C.

[0032] First dimension (pump 1): flow rate 1.0 mL / min (0.3 mL / min during regeneration); Second dimension (pump 2): flow rate 1.0 mL / min.

[0033] Six-way valve switching procedure: 0–5.6 min: Valve positions 1 and 2 connected (first dimension effluent enters waste liquid, second dimension equilibration / analysis); 5.6–6.6 min: Valve positions 1 and 6 connected (first dimension eluent is collected into the quantitative loop, volume 1 mL); 6.6–22 min: Valve positions 1 and 2 connected (sample in the quantitative loop is carried into the C4 column by the second dimension mobile phase for separation).

[0034] The gradient elution procedures are shown in Tables 1 and 2.

[0035] Table 1 Gradient elution program for pump 1 Table 2 Gradient elution program for pump 2 4. Method Validation (1) Linear range and sensitivity Lactoferrin standard solutions with concentrations of 5, 10, 20, 50, 100, and 200 μg / mL were prepared. The limits of detection (MLODs) and limits of quantitation (MLOQs) were calculated using a signal-to-noise ratio of 3 and a dilution factor. These were defined as the minimum spiking levels required to meet the recovery and precision requirements of lactoferrin, according to SANTE / 11312 / 2021.

[0036] Plotting peak area (y) against concentration (x) yielded the regression equation y = 2.89347x + 0.745008 for lactoferrin in the range of 5–200 μg / mL, with a correlation coefficient r = 0.99994. The limit of detection (LOD) for solid samples was 2 mg / 100g, and the limit of quantitation (LOQ) was 5 mg / 100g.

[0037] (2) Recovery rate and precision Spiked recovery experiments were conducted at three levels (5, 100, and 300 mg / 100g) on ​​infant formula, infant goat milk formula, and partially hydrolyzed infant formula. Each level was repeated 6 times. The results are shown in Table 3. The recovery rates were between 93.5% and 97.3%, and the RSDs were between 1.2% and 3.9%.

[0038] Table 3. Method recovery and precision (n=6) (3) Comparison with actual sample testing The present method and GB 5009.299-2024 were used to test 15 batches of commercially available samples (infant formula, goat milk formula, and partially hydrolyzed infant formula). The results are shown in Table 4. The results of the two methods were consistent for ordinary milk powder, but the national standard results for partially hydrolyzed milk powder and goat milk powder were significantly lower than those of the method of the present invention (the deviation for partially hydrolyzed infant formula was 10%–24%, and the deviation for goat milk powder was 10%–15%), proving that the method of the present invention is more accurate in these special matrices.

[0039] Table 4. Test results of actual milk powder samples (4) Service life of affinity column In GB 5009.299-2024, heparin immunoaffinity columns are designated as single-use consumables. This invention, under optimized conditions (reduced sample loading volume, increased water washing and regeneration steps), significantly increases the number of uses for heparin immunoaffinity columns. After 50 injections, the recovery rate of both standards and actual samples using the same heparin affinity column remains above 95%, far exceeding the single-use requirement of the national standard and significantly reducing costs. This demonstrates that this method not only improves detection accuracy, precision, and efficiency but also greatly reduces detection costs.

[0040] Comparative Example 1 (The extract did not contain Tween-20 and sodium chloride) Using partially hydrolyzed milk protein infant formula as the matrix, the lactoferrin spike concentration was 200 mg / 100 g. Except for the sample extraction buffer, which used phosphate buffer II (100 mmol / L phosphate buffer, pH 8.0±0.2, without Tween-20 and sodium chloride), the other pretreatment, chromatographic conditions, and flow path switching procedures were the same as in Example 1.

[0041] Results: The recovery rate of lactoferrin determined by the method in Comparative Example 1 was only about 75%. However, the recovery rate of Example 1 of this invention (using phosphate buffer I containing 5 mL / L Tween-20 and 30 mmol / L sodium chloride) reached 96.7% in the same matrix. This indicates that the addition of Tween-20 and sodium chloride can effectively inhibit the competition between impurities such as amino acids and peptides in the sample and lactoferrin for affinity column binding sites, significantly improving the recovery rate. Figure 4 This is a graph showing the effect of Tween-20 concentration on lactoferrin recovery rate in the method of this invention. Figure 5 This is a graph showing the effect of sodium chloride concentration on the recovery rate of lactoferrin in the method of this invention.

[0042] Comparative Example 2 (The first mobile phase A1 uses high-concentration phosphate buffer) A 200 μg / mL lactoferrin standard solution was used as the sample. Except for the first-dimensional chromatographic mobile phase A1, which was changed to phosphate buffer (200 mmol / L phosphate buffer, pH 8.0±0.2) as specified in the national standard GB5009.299-2023, all other conditions (including the use of phosphate buffer I of the present invention for the extraction solution, the flow path switching program, etc.) were the same as in Example 1.

[0043] Results: Under the conditions of Comparative Example 2, the peak width of lactoferrin reached 1.5 min (see Comparative Example 2). Figure 2 The peak width was greater than the collection capacity of the quantitative loop (1 mL), resulting in incomplete collection of the target analyte and severely affecting the accuracy of quantification. In contrast, the peak width in Example 1 of this invention (mobile phase A1 was 100 mmol / L phosphate buffer I) was 0.9 min (see [reference]). Figure 3 The target analyte peak can be completely collected by the quantitative loop. This indicates that appropriately reducing the phosphate buffer concentration in the one-dimensional mobile phase can decrease the peak width of the target analyte, ensuring complete collection by the quantitative loop.

Claims

1. A two-dimensional liquid chromatography method for determining lactoferrin in infant formula, characterized in that, Includes the following steps: (1) Weigh the sample to be tested, add the extraction solution for extraction, centrifuge and take the intermediate liquid to obtain the sample solution; the extraction solution is phosphate buffer I, which is a 100 mmol / L phosphate buffer containing Tween-20 and sodium chloride, and its pH is 8.0±0.2; (2) The sample solution is injected into an online two-dimensional liquid chromatography system for detection; the system includes a heparin affinity column for the first dimension chromatography, a reversed-phase C4 column for the second dimension chromatography, and a six-way valve with a quantitative loop; wherein, the elution program of the first dimension chromatography column includes gradient elution using mobile phase A1 and mobile phase B1, and after elution, pure water is used as mobile phase C1 to regenerate and clean the first dimension chromatography column at a low flow rate; The mobile phase A1 is the phosphate buffer I, and the mobile phase B1 is a 50 mmol / L phosphate buffer containing sodium chloride with a pH of 7.5 ± 0.

2. (3) The content of lactoferrin in the sample was calculated by using the external standard method based on the lactoferrin standard curve.

2. The two-dimensional liquid chromatography method according to claim 1, characterized in that, In the phosphate buffer I, the concentration of Tween-20 is 3-7 mL / L, and the concentration of sodium chloride is 10-60 mmol / L; the mobile phase B1 is a 50 mmol / L phosphate buffer containing 0.7-1.2 mol / L sodium chloride.

3. The two-dimensional liquid chromatography method according to claim 1, characterized in that, The following steps are achieved by switching the six-way valve: (a) In the first period, the effluent from the first-dimensional chromatographic column is prevented from entering the second-dimensional chromatographic column, while the lactoferrin in the sample is captured and enriched on the first-dimensional chromatographic column. (b) In the second period, the lactoferrin fraction eluted from the first-dimensional chromatographic column is collected in the quantitative loop; (c) In the third phase, the lactoferrin fraction enriched in the quantitative loop is injected into the second-dimensional chromatographic column for separation and detection.

4. The two-dimensional liquid chromatography method according to claim 2, characterized in that, The first period is 0–5.6 min, the second period is 5.6–6.6 min, and the third period is 6.6–22.0 min; the capacity of the quantitative loop is 1 mL.

5. The two-dimensional liquid chromatography method according to claim 1, characterized in that, In the first-dimensional column regeneration and cleaning step, pure water is used as the mobile phase C1, the flow rate is 0.3 mL / min, and the cleaning time is 7.01–21.5 min.

6. The two-dimensional liquid chromatography method according to claim 1, characterized in that, The mobile phase A2 of the second-dimensional chromatographic column is an aqueous solution containing 0.1% trifluoroacetic acid, and the mobile phase B2 is acetonitrile; Gradient elution is used, and the procedure includes: 0–4 min: Mobile phase A2 volume ratio is 10%, mobile phase B2 is 90%; 4.01–7 min: Mobile phase A2 volume ratio is 60%, mobile phase B2 is 40%; 7–22 min: Mobile phase A2 volume ratio is 40%, mobile phase B2 is 60%; The second-dimensional chromatography used a diode array detector with a detection wavelength of 280 nm and a column temperature of 35 °C.

7. The two-dimensional liquid chromatography method according to claim 1, characterized in that, The infant formula milk powder is selected from one or more of the following: regular infant formula milk powder, infant formula goat milk powder, and partially hydrolyzed milk protein infant formula milk powder.

8. The two-dimensional liquid chromatography method according to claim 1, characterized in that, The heparin affinity column can be reused at least 50 times when detected using the method described, and the recovery rate of lactoferrin is not less than 95%.

9. The two-dimensional liquid chromatography method according to claim 1, characterized in that, In the sample pretreatment step, the weight of the solid sample is 1-5g, and the volume of the extract is 50mL.

10. The application of the method according to any one of claims 1 to 9 in detecting the lactoferrin content in infant formula, special medical foods, or dairy products.

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