Method for detecting lactoferrin in breast milk

Abstract

The present application belongs to the technical field of food detection, and particularly relates to a method for detecting lactoferrin in breast milk, which comprises the following steps: a sample processing step: weighing a breast milk sample, adding an extraction solution to form an extraction system, mixing and centrifuging, and taking the supernatant as a to-be-detected sample solution; a capillary electrophoresis step: sequentially injecting a first sodium hydroxide solution, water and a running buffer into a capillary to clean the capillary, then injecting an alkaline solution into the capillary to form an alkali dam, then injecting the to-be-detected sample solution into the capillary, and then injecting the running buffer into the capillary to perform capillary zone electrophoresis separation, and detecting by using an ultraviolet detector; the extraction solution is a weak acid solution containing sodium chloride, and the running buffer is an acid phosphate system containing polysorbate 20. The method provided by the present application has the advantages of high accuracy, high sensitivity, stable peak shape and batch detection applicability.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a method for detecting lactoferrin in breast milk. Background Technology

[0002] Lactoferrin (LF) is an iron-binding glycoprotein widely found in human milk, cow's milk, and the milk of other mammals. LF possesses a variety of biological functions, including binding iron ions, participating in immune regulation, antibacterial activity, antiviral activity, promoting intestinal development, and participating in the body's defense mechanisms. It is one of the important indicators for evaluating the bioactive components of breast milk, the nutritional value of breast milk, and the functional design of infant formula.

[0003] The LF content in breast milk is affected by lactation stage, individual differences, sampling time, sample storage conditions, freeze-thaw cycles, and sample pretreatment methods. When conducting research on the construction of breast milk nutrition databases, the stability of breast milk bioactive proteins, and the development of milk-based nutritional products, a quantitative LF detection method that requires low sample volume, simple pretreatment, good reproducibility, and can be adapted to complex breast milk matrices is needed.

[0004] Currently, methods for detecting lactalbuminuria (LF) include immunoassay, liquid chromatography, mass spectrometry, gel electrophoresis, and capillary zone electrophoresis. Among these, capillary zone electrophoresis is suitable for rapid analysis of protein components in breast milk due to its advantages of small sample volume, high separation efficiency, fast analysis speed, and low reagent consumption. However, breast milk matrix contains fats, casein, whey protein, salts, and other small molecule components. When directly detecting breast milk LF using conventional capillary zone electrophoresis, problems such as insufficient target peak response, peak broadening, migration time drift, interference from adjacent protein peaks, and decreased quantitative repeatability can easily occur.

[0005] Reference 1 discloses a method for detecting human lactoferrin (LF) content in human milk using a high-voltage capillary electrophoresis analyzer. This method establishes a LF standard curve and analyzes the human milk sample to calculate the LF content. This method demonstrates that capillary electrophoresis can be used for LF detection in human milk, and the sample processing mainly employs a protein dispersion / denaturation system.

[0006] Reference 2 discloses a method for detecting lactoferrin (LF) content in bovine milk using high-voltage capillary electrophoresis, including sample pretreatment, sample loading, detection, and calibration readings. The detection conditions involve capillary column size, injection pressure, detector, separation voltage, and electrophoresis buffer. This method is primarily aimed at detecting LF in bovine milk and dairy products.

[0007] Reference 3 discloses a capillary electrophoresis method for detecting LF in dairy products. The method involves treating samples with sample buffer, separating them using a coated neutral capillary column and citrate buffer, and detecting at a wavelength of 214 nm. The reported average LF recovery rate is 75%–91%, with a relative standard deviation of 1.1%–2.8%, and a limit of detection of 0.003 mg / mL. This reference demonstrates that capillary electrophoresis can be used for the detection of LF in dairy products, primarily targeting the dairy matrix.

[0008] Reference 4 discloses a method for determining milk LF in infant formula using CE-UV. This reference points out that sample pretreatment and the addition of a running buffer additive are crucial for accurate LF determination, and that acetic acid is used for pretreatment of infant formula samples. The target sample is milk LF in infant formula.

[0009] Reference 5 discloses a method for LF analysis based on uncoated capillary electrophoresis, focusing on optimizing the background electrolyte, pH, surfactant, and buffer system; its optimal system involves adding urea to phosphate buffer and achieving LF separation under specific pH conditions. This reference mainly addresses the problem of LF adsorption and separation behavior in uncoated capillaries.

[0010] Reference 6 discloses a method for extracting lactoferrin from milk using a weakly acidic cation exchange resin and detecting it using dynamic coated capillary electrophoresis. This method can reduce the adsorption of LF on the inner wall of the capillary and improve the detection effect, but it requires ion exchange resin extraction and dynamic coating treatment, making the operation steps relatively complex; this method is mainly for bovine LF in milk.

[0011] Furthermore, dynamic pH interfaces, pH-mediated stacking, and related online pre-concentration techniques have been used to improve the detection sensitivity of capillary electrophoresis. Reference 7 indicates that dynamic pH interfaces are a simple online pre-concentration method for capillary electrophoresis, suitable for amphoteric analytes, and can improve sample loading and detection sensitivity. The above studies provide a theoretical basis for online enrichment by capillary electrophoresis. However, the samples used are not breast milk, and the methods for addressing LF co-precipitation loss, peak migration matching, and insufficient detection sensitivity with low sample volumes in the complex matrix of breast milk are not disclosed.

[0012] In summary, existing literature has demonstrated that LF can be detected by capillary electrophoresis, but the following shortcomings remain: First, conventional acid precipitation, simple dilution, or protein dispersion treatments do not adequately address the issue of LF forming complexes with casein or other oppositely charged components in breast milk and being lost during precipitation; Second, conventional capillary zone electrophoresis lacks an alkaline dam focusing or dynamic pH / conductivity interface enrichment step before sample introduction, which can easily lead to insufficient target peak response, peak broadening, migration position drift, and interference from adjacent matrix peaks under complex breast milk matrices and low sample volume conditions; Third, although ion exchange extraction or dynamic coating capillary electrophoresis can improve some separation problems, the pretreatment steps are relatively complex, which is not conducive to small sample volumes and batch detection of breast milk.

[0013] Therefore, it is necessary to establish a method for detecting LF in the complex matrix of breast milk, reduce electrostatic recombination and co-precipitation loss of LF with casein or other impurities, and improve LF peak response, peak shape stability and migration matching, so as to achieve rapid, accurate and batch quantitative detection of LF in breast milk.

[0014] References:

[0015] Reference 1: CN101799449A

[0016] Reference 2: CN101290322A

[0017] Reference 3: Sun Guoqing, Kang Xiaohong, Liu Weixing, Hu Xinyu. Study on the determination of lactoferrin in dairy products by capillary electrophoresis [J]. Food Industry Technology, 2008, 29(12):241-242. DOI:10.13386 / j.issn1002-0306.2008.12.058.

[0018] Cited literature 4: Li J., Ding X., Chen Y., Song B., Zhao S., Wang Z. Determination of bovine lactoferrin in infant formula by capillaryelectrophoresis with ultraviolet detection[J]. Journal of Chromatography A, 2012, 1244:178-183. DOI:10.1016 / j.chroma.2012.05.004.

[0019] Reference 5: Chen H., Wang Z., Fan F., Shi P., Xu X., Du M., Wang C. Analysis Method of Lactoferrin Based on Uncoated Capillary Electrophoresis[J]. eFood, 2021, 2:147-153. DOI:10.2991 / efood.k.210720.001.

[0020] Reference 6: Zhao Lingguo, You Jun, Liang Zhaohai, Li Ke, Qiu Fen, Zeng Shengbo. Determination of lactoferrin content in milk by cation exchange chromatography and dynamic coated capillary electrophoresis [J]. Journal of Analytical Testing, 2014, 33(3):339-343.

[0021] Reference 7: Zhu G., Sun L., Dovichi NJ Dynamic pH junctionpreconcentration in capillary electrophoresis-electrospray ionization-massspectrometry for proteomics analysis[J]. Analyst, 2016, 141:5216-5220. DOI:10.1039 / C6AN01140C Summary of the Invention

[0022] The problem the invention aims to solve

[0023] Although existing literature has demonstrated that LF can be detected by capillary electrophoresis, several limitations remain. For example, while conventional acid precipitation can remove casein and some fatty impurities, LF is a charged protein and easily forms complexes with casein or other oppositely charged components, leading to LF loss during precipitation and lower detection results. Conventional capillary zone electrophoresis lacks an effective online desalting and enrichment mechanism; when salt, weak acid, whey protein, and residual fat are present in breast milk samples, LF bands tend to diffuse, the target peak broadens, and the signal-to-noise ratio decreases. Other LF detection methods are also not perfect. Immunoassays rely on antibodies or kits, which have high costs and batch stability risks; chromatographic and mass spectrometric methods typically require complex purification steps, which are not conducive to high-throughput routine detection of breast milk samples. Currently, there is still a lack of a highly accurate, sensitive, peak-shape-stable, and batch-scale LF capillary electrophoresis detection method suitable for the complex matrix of breast milk.

[0024] Based on this, the present invention aims to provide a quantitative detection method for LF suitable for the complex matrix of breast milk. The method reduces LF pretreatment loss by extracting with a weak acid containing salt, and performs online desalting and enrichment of LF by focusing the alkaline dam before capillary injection to form a moving pH / conductivity interface. Stable separation is achieved in an acidic phosphate running buffer containing polysorbate 20, thereby comprehensively improving the accuracy, sensitivity, peak shape stability and batch detection applicability of LF detection.

[0025] Solution for solving the problem

[0026] [1]. A method for detecting lactoferrin in breast milk, characterized in that the method comprises the following steps:

[0027] Sample processing steps: Weigh the breast milk sample, add the extraction solution to form an extraction system, mix well and centrifuge, and take the supernatant as the sample solution to be tested; wherein, the extraction solution is prepared by sodium chloride, weak acid and water;

[0028] The steps of capillary electrophoresis are as follows: A first sodium hydroxide aqueous solution, water, and running buffer are sequentially injected into the capillary. Then, an alkaline solution is injected into the capillary, followed by the sample solution to be tested. Finally, the running buffer is injected into the capillary for capillary zone electrophoresis separation, and detection is performed using a UV detector. The running buffer is prepared from sodium dihydrogen phosphate, phosphoric acid, polysorbate 20, and water. The alkaline solution includes at least one of a second sodium hydroxide aqueous solution, ammonia, and Tris-base aqueous solution, with the first and second sodium hydroxide aqueous solutions having different concentrations.

[0029] [2]. The method according to [1] is characterized in that the concentration of sodium chloride in the extract is 70-150 mmol / L, and / or the weak acid in the extract includes at least one of acetic acid, citric acid and lactic acid.

[0030] [3]. The method according to [1] or [2] is characterized in that the pH value of the extraction system is 4.0-4.8.

[0031] [4]. The method according to any one of [1]-[3] is characterized in that the concentration of sodium dihydrogen phosphate in the running buffer is 40-70 mmol / L, and / or the concentration of polysorbate 20 in the running buffer is 0.05% (v / v)-0.3% (v / v).

[0032] [5]. The method according to any one of [1]-[4] is characterized in that the pH value of the running buffer is 1.7-2.4.

[0033] [6]. The method according to any one of [1]-[5] is characterized in that the pressure of injecting the alkaline solution into the capillary is 5-20 psi, and / or the time of injecting the alkaline solution into the capillary is 0.05-0.4 min.

[0034] [7]. The method according to any one of [1]-[6] is characterized in that the pressure of injecting the sample solution into the capillary is 0.1-1.0 psi, and / or the time of injecting the sample solution into the capillary is 5-15 s.

[0035] [8]. The method according to any one of [1]-[7] is characterized in that the voltage for injecting the running buffer into the capillary for capillary zone electrophoresis separation is 8-14 kV, and / or the time for injecting the running buffer into the capillary for capillary zone electrophoresis separation is 12-18 min.

[0036] [9]. The method according to any one of [1]-[8] is characterized in that the capillary is a fused silica capillary.

[0037]

[10] . The method according to any one of [1]-[9] is characterized in that the method further includes a quantitative analysis step, the quantitative analysis step including: preparing a series of standard working solutions with lactoferrin standards, establishing a linear relationship between the calibration peak area and the lactoferrin concentration, and calculating the lactoferrin content in the breast milk sample based on the lactoferrin peak area of ​​the sample to be tested.

[0038] The effects of the invention

[0039] By implementing the above technical solution, the present invention has achieved the following technical effects:

[0040] This invention optimizes a continuous combined system for LF capillary electrophoresis detection of emulsions, using a weak acid extraction with salt, alkali dam focusing, and an acidic phosphate running buffer containing polysorbate 20. The pretreatment stage reduces LF loss, the electrophoresis injection stage improves enrichment and desalting capabilities, and the separation stage improves peak shape and migration stability, resulting in a synergistic effect. Experimental data show that using a weak acid extraction buffer containing NaCl can remove casein and fatty impurities through acid precipitation while reducing the formation of complex precipitates between LF and casein or other impurities by adjusting the ionic strength, thus reducing pretreatment loss of the target protein. The spiked recovery rate is between 95.7% and 101.7%, which meets the quantitative requirements for LF in the complex matrix of breast milk. In the control without alkali dam or with only water, the LF peak tends to exhibit a low response, a large peak width, and migration position significantly affected by the matrix. After forming an alkali dam with the alkali solution, the LF peak is more concentrated, has a higher response, and a narrower peak shape, indicating that the moving pH / conductivity interface can achieve online enrichment and partial online desalting. The acidic phosphate system provides a stable buffer environment, and polysorbate 20, as a nonionic surfactant, reduces the nonspecific adsorption of proteins on the inner wall of the capillary, thereby improving the peak shape, migration time, and batch injection stability of LF.

[0041] The capillary electrophoresis method for detecting LF in emulsions provided by this invention exhibits good linearity and repeatability. Experimental data show that within the range of 0.045-0.54 mg / mL, the LF correction peak area shows good linearity with concentration (R² = 0.9957); the peak area RSD is 1.8%, and the migration time RSD is 0.03%. Furthermore, the capillary electrophoresis method for detecting LF in emulsions provided by this invention is highly applicable. This method requires low sample volume, consumes few reagents, and has a single separation time of approximately 15 minutes. It can be integrated with automated sample loading and sample tray detection workflows, making it suitable for batch detection of breast milk samples, construction of breast milk nutrition databases, and evaluation of LF content in the research and development of milk-based nutritional products. Attached Figure Description

[0042] Figure 1 Effect of NaH2PO4 concentration on the separation efficiency of breast milk matrix LF.

[0043] Figure 2 The effect of buffer pH on the separation efficiency of breast milk matrix LF.

[0044] Figure 3 The effect of alkaline solution type on the separation effect of breast milk matrix LF.

[0045] Figure 4 The effects of no added alkali and different NaOH concentrations on the separation efficiency of breast milk matrix LF.

[0046] Figure 5 The effect of alkali introduction time on the separation effect of breast milk matrix LF.

[0047] Figure 6 The effect of NaCl concentration in the extract on the LF extraction efficiency of breast milk samples.

[0048] Figure 7 The effect of the final pH of the extraction system on the LF extraction efficiency of breast milk samples. Detailed Implementation

[0049] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0050] <Terminology Definition>

[0051] In this invention, "comprising," "having," "including," or "containing" can mean included or open-ended, and does not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0052] In this invention, the meaning of "may" includes both performing a certain process and not performing a certain process.

[0053] In this invention, "optional" or "optionally" means that certain substances, components, execution steps, application conditions, etc., are used or not used.

[0054] In this invention, the numerical ranges represented by "value A ~ value B", "value A - value B", and "value A above / below" refer to the ranges including the endpoint values ​​A and B.

[0055] In this invention, the term "about" is used to define that the numerical ranges and parameters of this invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified by the term "about". Here, "about" generally means that the actual value is within ±5%, ±3%, ±1%, or ±0.5% of a specific value or range. Furthermore, the values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0056] In this invention, terms such as "some specific / preferred embodiments," "other specific / preferred embodiments," and "implementation" refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to a particular embodiment that are included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0057] In this invention, "breast milk" refers to human milk.

[0058] In this invention, "weak acid" can refer to an acid with an ionization constant (Ka) of less than 0.0001.

[0059] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0060] Methods for detecting lactoferrin in breast milk

[0061] This invention provides a method for detecting lactoferrin in breast milk. The method treats breast milk samples with a weakly acidic extraction solution containing sodium chloride. While acid precipitation removes casein and fat impurities, the electrostatic binding between lactoferrin and casein or other impurity proteins is weakened by adjusting the ionic strength, reducing the risk of lactoferrin loss during precipitation. Furthermore, in capillary electrophoresis, before sample introduction, a certain length of alkaline solution is injected into the capillary to form an alkaline dam, creating a moving pH gradient interface and a conductivity difference interface between the dam and the acidic running buffer. After the sample enters the capillary, lactoferrin undergoes a transient migration velocity difference and bandwidth compression at this interface, thus achieving online focusing. Simultaneously, the small molecule salt and weakly acidic components exhibit different migration behaviors than the protein, playing a role in online desalting and reducing matrix interference. The running buffer used in capillary electrophoresis is an acidic phosphate system containing phosphate, sodium dihydrogen phosphate, and polysorbate 20 (Tween 20). Polysorbate 20, as a nonionic surfactant, can reduce the nonspecific adsorption between lactoferrin and other whey proteins and the capillary wall, improve the target peak shape and migration stability, and facilitate the identification and quantification of lactoferrin peaks in the complex matrix of breast milk.

[0062] In some specific implementations, the method includes the following steps:

[0063] Sample processing steps: Weigh the breast milk sample, add the extraction solution to form an extraction system, mix well and centrifuge, and take the supernatant as the sample solution to be tested; wherein, the extraction solution is prepared by sodium chloride, weak acid and water;

[0064] The steps of capillary electrophoresis are as follows: A first sodium hydroxide aqueous solution, water, and running buffer are sequentially injected into the capillary. Then, an alkaline solution is injected into the capillary, followed by the sample solution to be tested. Finally, the running buffer is injected into the capillary for capillary zone electrophoresis separation, and detection is performed using a UV detector. The running buffer is prepared from sodium dihydrogen phosphate, phosphoric acid, polysorbate 20, and water. The alkaline solution includes at least one of a second sodium hydroxide aqueous solution, ammonia, and Tris-base aqueous solution, with the first and second sodium hydroxide aqueous solutions having different concentrations.

[0065] <Sample preparation steps>

[0066] This invention does not simply remove impurities through acid precipitation, but introduces an appropriate amount of NaCl into a weak acid system to adjust the ionic strength and reduce electrostatic recombination and co-precipitation loss between lactoferrin and casein or other proteins.

[0067] In some embodiments, the concentration of sodium chloride in the extract is 70-150 mmol / L; for example, it can be 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L, 110 mmol / L, 120 mmol / L, 130 mmol / L, 140 mmol / L, or 150 mmol / L, etc.; preferably 80-120 mmol / L; more preferably 100 mmol / L. By adjusting the sodium chloride concentration in the extract, the ionic strength is adjusted, reducing the precipitation of lactoferrin with casein or other proteins.

[0068] In some embodiments, the weak acid in the extract includes at least one of acetic acid, citric acid, and lactic acid; preferably acetic acid, citric acid, or lactic acid; more preferably acetic acid, and even more preferably an acetic acid concentration of 4 mmol / L. The weak acid in the extract provides acid precipitation conditions and maintains the extractability of lactoferrin.

[0069] In some embodiments, the pH of the extraction system is 4.0-4.8; for example, it can be 4.0, 4.1, 4.2, 4.3, 4.5, 4.6, 4.7, or 4.8, etc.; preferably 4.3-4.6; more preferably 4.5. By adjusting the pH of the extraction system, casein interference and lactoferrin loss can be reduced.

[0070] <Steps of capillary electrophoresis>

[0071] In some embodiments, the present invention uses a fused silica capillary; preferably an uncoated fused silica capillary; more preferably, the capillary has an inner diameter of 50 μm, a total length of 30 cm, and an effective length of 20 cm.

[0072] In some implementations, the capillary needs to be cleaned and activated before injecting the alkaline solution and the sample to be tested into it. Preferably, the capillary is pressure-washed by sequentially injecting a first sodium hydroxide aqueous solution, water, and a running buffer solution into it.

[0073] In some specific implementations, the concentration of the first sodium hydroxide aqueous solution is about 1 mol / L, the injection pressure is about 20 psi, and the injection time is about 3 min.

[0074] In some specific implementations, the water is injected at a pressure of about 20 psi for about 3 minutes.

[0075] In some specific implementations, the run buffer is injected at a pressure of approximately 40 psi for approximately 3 minutes.

[0076] The running buffer used in this invention is an acidic phosphate system with a pH value of 1.7-2.4; for example, it can be 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3 or 2.4, etc.; preferably 1.9-2.2; more preferably 2.2.

[0077] In some embodiments, the concentration of sodium dihydrogen phosphate in the running buffer is 40-70 mmol / L; for example, it can be 40 mmol / L, 45 mmol / L, 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L, or 70 mmol / L, preferably 45-55 mmol / L; more preferably 50 mmol / L. An appropriate sodium dihydrogen phosphate concentration can regulate buffering capacity, conductivity, and migration stability.

[0078] In some embodiments, the concentration of polysorbate 20 in the running buffer is 0.05% (v / v) to 0.3% (v / v); for example, it can be 0.05% (v / v), 0.1% (v / v), 0.15% (v / v), 0.2% (v / v), 0.25% (v / v), or 0.3% (v / v), etc.; preferably 0.05% (v / v) to 0.2% (v / v); more preferably 0.1% (v / v). An appropriate concentration of polysorbate 20 can improve peak shape, reduce inner wall adsorption, and reduce tailing of adjacent peaks.

[0079] In some implementations, after cleaning and activating the capillary, the present invention injects an alkaline solution into the capillary to form an alkaline dam. The moving pH / conductivity interface formed between the alkaline dam and the acidic running buffer is used to focus and desalt lactoferrin online, thereby improving peak response, reducing matrix interference, and improving target peak recognition.

[0080] In some embodiments, the alkaline solution comprises at least one of a second aqueous sodium hydroxide solution, ammonia, and a Tris-base aqueous solution; preferably a second aqueous sodium hydroxide solution, ammonia, or a Tris-base aqueous solution; more preferably a second aqueous sodium hydroxide solution, and even more preferably a sodium hydroxide concentration of 1-50 mmol / L, more preferably 10-40 mmol / L, and more preferably 30 mmol / L. An appropriate sodium hydroxide concentration can control the interface strength and focusing efficiency.

[0081] In some embodiments, the pressure at which the alkaline solution is injected into the capillary is 5-20 psi, preferably 7-13 psi, more preferably 10 psi; and the time for injecting the alkaline solution into the capillary is 0.05-0.4 min, preferably 0.05-0.15 min, more preferably 0.1 min. By controlling the pressure and time of injecting the alkaline solution, the length of the alkali dam can be adjusted, balancing enrichment and separation.

[0082] After the alkali dam is formed, the sample solution to be tested is injected into the capillary. In some embodiments, the pressure at which the sample solution to be tested is injected into the capillary is 0.1-1.0 psi, preferably about 0.5 psi; and the time is 5-15 s, preferably about 10 s.

[0083] After the sample to be tested is injected, a running buffer is injected under a certain pressure to perform capillary zone electrophoresis separation. In some embodiments, the voltage for injecting the running buffer into the capillary for capillary zone electrophoresis separation is 8-14 kV, preferably about 11 kV; the time is 12-18 min, preferably about 15 min.

[0084] This invention optimizes a continuous combination system of salt-containing weak acid extraction, alkaline dam focusing, and acidic phosphate running buffer containing polysorbate 20. The sample processing stage reduces lactoferrin loss, the electrophoresis injection stage improves enrichment and desalting capabilities, and the separation stage improves peak shape and migration stability. The three components work synergistically.

[0085] In some embodiments, the present invention uses an ultraviolet detector with a detection wavelength of approximately 220 nm.

[0086] In some embodiments, the capillary electrophoresis of the present invention is performed at approximately 25°C.

[0087] In some implementations, in order to quantify lactoferrin in breast milk samples, the method further includes a quantitative analysis step, which includes: preparing a series of standard working solutions with lactoferrin standards, establishing a linear relationship between the calibration peak area and the lactoferrin concentration, and calculating the lactoferrin content in the breast milk sample based on the lactoferrin peak area of ​​the sample to be tested.

[0088] The method for detecting lactoferrin in breast milk provided by this invention does not rely on antibodies or kits, avoiding quantitative deviations caused by batch-to-batch antibody differences, cross-reactions, and changes in antigenic epitopes. It offers superior detection cost and batch testing consistency. Furthermore, this invention simplifies breast milk sample pretreatment, requires small sample volumes, and has short single-session separation times. It is not dependent on chromatographic columns or high-cost mass spectrometry equipment, making it more suitable for routine laboratory or enterprise R&D testing scenarios.

[0089] Example

[0090] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all materials and instruments used are commercially available conventional products.

[0091] Example 1: Optimization of Run Buffer Composition

[0092] 1. Samples and reagents

[0093] The samples and reagents used in the experiment are shown in Table 1 below.

[0094] Table 1 Samples and Reagents

[0095]

[0096] 2. Reagent preparation

[0097] NaOH solution (1.0 mol / L): Weigh 4.00 g NaOH, dissolve in water, and bring the volume to 100 mL. Mix well.

[0098] NaOH solution (30 mmol / L): Take 3 mL of 1.0 mol / L NaOH solution into a 100 mL volumetric flask, add water to make up to the mark, and mix well.

[0099] Phosphoric acid solution (5 mol / L): Take an appropriate amount of phosphoric acid solution, dilute it to the target concentration, and mix well.

[0100] Sodium dihydrogen phosphate solution (100 mmol / L): Weigh an appropriate amount of sodium dihydrogen phosphate, add water to make up to the volume, and mix well.

[0101] LF running buffer: Pipette 25 mL of 100 mmol / L sodium dihydrogen phosphate solution, 0.4 mL of 5 mol / L phosphate solution, and 50 μL of polysorbate 20, add water to a final volume of 50 mL, and mix well; the resulting running buffer contains 50 mmol / L sodium dihydrogen phosphate, 40 mmol / L phosphate and 0.1% (v / v) polysorbate 20, with a pH of approximately 2.2.

[0102] Acetic acid solution (100 mmol / L): Add about 580 μL of acetic acid to a 100 mL volumetric flask containing 85 mL of water, dilute to 100 mL with water, and mix well.

[0103] LF extract: Weigh 584.4 mg NaCl into a 100 mL volumetric flask, add about 80 mL of water, then add 4 mL of 100 mmol / L acetic acid solution, and dilute to 100 mL with water to obtain a weak acid extract containing 100 mmol / L NaCl and 4 mmol / L acetic acid.

[0104] LF standard stock solution (1 mg / mL): Weigh 5 mg of LF standard into a centrifuge tube, and dilute to 5 mL with LF extract to obtain 1 mg / mL LF standard stock solution. Prepare fresh before use.

[0105] LF standard working solution: Accurately pipette 0.05 mL, 0.10 mL, 0.20 mL, 0.40 mL, and 0.60 mL of 1 mg / mL LF standard stock solution into centrifuge tubes, respectively. Make up to 1.0 mL with LF extraction buffer, and then add 100 μL of water and mix well to obtain LF standard working solutions with concentrations of 0.045 mg / mL, 0.09 mg / mL, 0.18 mg / mL, 0.36 mg / mL, and 0.54 mg / mL, respectively.

[0106] 3. Sample Preparation

[0107] Accurately weigh or transfer 100 μL of breast milk sample into a 1.5 mL centrifuge tube and record the sample mass m (g). Add 1 mL of LF extraction buffer to the sample, vortex to mix, and bring the mixture to a weakly acidic condition; centrifuge at 10000 r / min for 10 min, and collect approximately 100 μL of the supernatant as the sample solution to be tested.

[0108] 4. Capillary electrophoresis conditions

[0109] The capillary electrophoresis conditions are shown in Table 2 below.

[0110] Table 2 Capillary Electrophoresis Conditions

[0111]

[0112] 5. Capillary electrophoresis separation procedure

[0113] The capillary electrophoresis separation procedure is shown in Table 3 below.

[0114] Table 3 Capillary Electrophoresis Separation Procedure

[0115]

[0116] 6. Data Processing

[0117] The LF content X (mg / 100 g) in a breast milk sample can be calculated using the following formula:

[0118] X = C × V × P × 100 / m

[0119] In the formula: C is the LF concentration in the sample solution obtained from the calibration curve, in mg / mL; V is the total volume after adding the extract, in mL; P is the purity correction coefficient of the LF standard, in decimal form; m is the mass of the breast milk sample, in g.

[0120] 7. To determine the optimal conditions for phosphate concentration and pH in the LF running buffer, unless otherwise specified, sample pretreatment, capillary type, detection wavelength, temperature, injection method, and separation voltage shall be performed according to the conditions described in Sections 1-6.

[0121] Using 40 mmol / L H3PO4 and 0.1% (v / v) polysorbate 20 as the basic system, the effects of 30, 50, 100, and 200 mmol / L NaH2PO4 on the peak shape, peak height, migration time, and separation of adjacent protein peaks in the LF reaction were investigated. The results are as follows: Figure 1 As shown, with increasing NaH2PO4 concentration, the ionic strength and conductivity of the system increase, and the overall LF migration time is delayed. Excessively high salt concentrations enhance sample band diffusion, leading to peak broadening and reduced response. At 30 mmol / L, the LF peak response is high, but interference from adjacent matrix peaks remains significant. At 100 mmol / L and 200 mmol / L, migration is significantly delayed and peak height decreases. At 50 mmol / L, LF shows good separation from other protein peaks, and the peak response remains at a high level. Therefore, a NaH2PO4 concentration of 50 mmol / L is preferred.

[0122] Further, based on a system of 50 mmol / L NaH2PO4 and 0.1% (v / v) polysorbate 20, the pH of the running buffer was adjusted to 1.7, 2.2, and 2.4 using H3PO4. The results are as follows: Figure 2As shown, with decreasing pH, the positive charge of LF increases under low pH conditions, resulting in improved mobility and peak response. However, when the pH drops to 1.7, the acidity of the system becomes too high, increasing the risk of current fluctuations and electrolytic side reactions, and decreasing the stability of the method. Considering the separation degree, peak response, current stability, and repeatability, a pH of approximately 2.2 is selected as the optimal condition.

[0123] In summary, this embodiment determines that the preferred LF running buffer is 40 mmol / L H3PO4, 50 mmol / L NaH2PO4 and 0.1% (v / v) polysorbate 20, with a pH of approximately 2.2.

[0124] Example 2: Optimization of Alkali Dam Focusing Conditions and Control Without Alkali Dam

[0125] To illustrate the necessity and optimal conditions of pre-injection alkaline dam focusing for LF detection, unless otherwise specified, the running buffer consisted of 40 mmol / L H3PO4, 50 mmol / L NaH2PO4, and 0.1% (v / v) polysorbate 20, with a pH of approximately 2.2; the separation voltage was 11 kV; the detection wavelength was 220 nm; the injection pressure was 0.5 psi; the injection time was 10 s; and all other conditions were the same as those described in sections 1-6 of Example 1.

[0126] First, the LF separation performance was compared using 10 mmol / L NaOH, 10 mmol / L Tris-base, and 10 mmol / L ammonia as alkaline dam solutions. The 10 mmol / L Tris-base solution was prepared as follows: accurately weigh 0.061 g of tris(hydroxymethyl)aminomethane (Tris-base) into a 50 mL volumetric flask, add water to the final volume, and mix thoroughly. The 10 mmol / L ammonia solution was prepared as follows: pre-measure 35 mL of water into a volumetric flask, accurately pipette 0.035 mL of ammonia solution (NH3·H2O, analytical grade, concentration 25% (w / w, g ammonia / 100 g ammonia solution), ρ = 0.91 g / mL) into a 50 mL volumetric flask, add water to the final volume, and mix thoroughly. The results are as follows: Figure 3 The results show that the LF peak response is higher and the peak shape is more concentrated in the NaOH system, indicating that the strongly alkaline NaOH can form a more obvious pH gradient interface and conductivity difference interface with the acidic separation buffer in a shorter capillary segment, which is conducive to the accumulation and focusing of LF at the interface. Therefore, NaOH is preferred as the alkaline dam solution.

[0127] Further experiments were conducted with a control group (no alkali dam added) or an influent control group, and with alkali dam conditions of 1 mmol / L, 4 mmol / L, 10 mmol / L, 30 mmol / L, and 50 mmol / L NaOH. The results are as follows: Figure 4As shown, without the addition of an alkaline dam, the LF peak appears at approximately 8 min, indicating that in the absence of a pre-injection zone, LF directly undergoes electrophoretic migration after entering the acidic running buffer, resulting in a short migration time. However, due to the lack of an online focusing interface, LF migrates synchronously with residual whey proteins and other charged components in the breast milk supernatant. With only water added, the LF peak is delayed to approximately 26 min. This is because the conductivity of the water plug is significantly lower than that of the sample extract and running buffer, forming a non-reactive low-conductivity zone within the capillary. This causes LF to undergo changes in electric field intensity and a redistribution of migration velocity among the sample zone, water plug zone, and running buffer zone. Simultaneously, the water plug locally dilutes the running buffer, leading to instantaneous changes in buffer capacity, pH, and electroosmotic flow state, thus significantly delaying LF migration. In contrast, after the addition of the NaOH alkaline dam, the LF peak shows a significant forward shift, narrowing, and enhanced response with increasing NaOH concentration, indicating that the moving pH / conductivity interface formed by the dam can compress the sample band, reduce the influence of matrix salinity, and improve the signal-to-noise ratio of the target peak.

[0128] At 1 mmol / L and 4 mmol / L NaOH concentrations, some interfacial focusing effect was observed, but the interfacial strength was insufficient, resulting in limited improvement in LF peak response and shape. At 10 mmol / L, the LF peak advanced significantly and its shape improved. At 30 mmol / L, the LF peak was more concentrated, the response was higher, and the separation from adjacent protein peaks was better, representing the optimal condition for overall performance. When the NaOH concentration increased to 50 mmol / L, the local alkaline segment became too strong, the interface became too steep, or the transient migration state of the protein was disturbed, leading to a decrease in peak shape and sensitivity. Therefore, 30 mmol / L NaOH was preferred.

[0129] The alkali injection times of 0.1 min, 0.2 min, and 0.4 min were further investigated under conditions of 30 mmol / L NaOH and 10 psi. The results are as follows: Figure 5 As shown, at 0.1 min, the alkali dam length is moderate, the LF peak is narrow and well separated from adjacent peaks; when extended to 0.2 min or 0.4 min, the alkali dam becomes too long, causing the LF migration to shift excessively forward, and the sample band is compressed, shortening the distance to other matrix peaks and reducing the resolution. Therefore, an alkali injection pressure of 10 psi and an alkali injection time of 0.1 min are preferred.

[0130] This embodiment demonstrates that alkaline dam focusing is not a routine rinsing step, but a key feature of the present invention that improves detection sensitivity and resistance to matrix interference: it constructs a controlled alkaline segment before sample introduction, which forms a moving interface with the acidic running buffer during electrophoresis, allowing LF to accumulate online at the interface and simultaneously achieving partial online desalting, thereby making the LF peak in complex breast milk samples narrower, with higher response and more stable identification.

[0131] Example 3: Optimization of Sample Pretreatment Conditions

[0132] To determine the NaCl concentration in the salt-containing weak acid extract and the final pH of the extraction system, unless otherwise specified, the running buffer, capillary electrophoresis conditions, and alkali dam conditions were performed according to the conditions described in sections 1-6 of Example 1.

[0133] Accurately weigh 100 μL of breast milk sample into a 1.5 mL centrifuge tube, and add 1 mL of extraction buffer with NaCl concentrations of 20 mmol / L, 50 mmol / L, 100 mmol / L, and 200 mmol / L and acetic acid concentration of 4 mmol / L, respectively. Vortex to mix, centrifuge at 10000 r / min for 10 min, and take the supernatant for analysis. The results are as follows. Figure 6 The results showed that the peak height of LF increased significantly under 100 mmol / L NaCl conditions, indicating that the appropriate salt concentration can weaken the electrostatic binding between LF and casein or other impurity proteins, reducing coprecipitation losses. When the NaCl concentration was increased to 200 mmol / L, the excessively high ionic strength may affect subsequent electrophoretic migration and interfacial enrichment, resulting in a decrease in peak response. Therefore, a NaCl concentration of 100 mmol / L is preferred.

[0134] Under 100 mmol / L NaCl conditions, the final pH of the breast milk-extract mixture was further adjusted to 4.0, 4.3, and 4.5. The results are as follows: Figure 7 The results showed little difference in LF extraction efficiency under the three pH conditions, indicating that LF maintains good extractability within this weakly acidic range under suitable salt concentration protection. Considering operational convenience and the stability of batch processing of breast milk samples, quantitative addition of extraction buffer to achieve a final pH of approximately 4.5 in the mixed system was selected as the preferred sample pretreatment condition.

[0135] Example 4: Methodological Validation

[0136] Methodological validation was performed according to the preferred conditions in Examples 1-3: the LF running buffer consisted of 40 mmol / L H3PO4, 50 mmol / L NaH2PO4 and 0.1% (v / v) polysorbate 20, with a pH of approximately 2.2; the alkaline dam conditions were 30 mmol / L NaOH, 10 psi, and 0.1 min; sample pretreatment was performed using an LF extraction solution containing 100 mmol / L NaCl and 4 mmol / L acetic acid, with a final pH of approximately 4.5; and the remaining capillary electrophoresis conditions and separation procedures were the same as those described in sections 4-5 of Example 1.

[0137] Spiking recovery experiments were conducted on three groups of breast milk samples (low, medium, and high concentrations), with 0.5 mg / mL, 1.0 mg / mL, and 1.5 mg / mL LF standard solutions added, respectively, and compared with a weak acid precipitation system without added salt. In the weak acid system without added salt, the migration positions of the LF standard peak and the actual LF peak in the breast milk sample deviated. Matrix ionic strength and protein precipitation behavior affected the accuracy of target peak identification and quantification. After extraction using a weak acid system containing 100 mmol / L NaCl, the matching of the migration positions of the LF peak in the standard and the sample improved, and the spiked recovery rate was between 95.7% and 101.7%.

[0138] Standard curves were established using LF standard working solutions at concentrations of 0.045 mg / mL, 0.09 mg / mL, 0.18 mg / mL, 0.36 mg / mL, and 0.54 mg / mL. The corrected peak area Ac showed good linearity with the concentration C, and the linear equation was Ac = 144056.5C - 3442.3, with a correlation coefficient R. 2 =0.9957; Repeated injection studies showed that the RSD of the corrected peak area was 1.8% and the RSD of the migration time was 0.03%, indicating that the method has good linearity, repeatability and migration stability.

[0139] The validation results show that the salt-containing weak acid system not only reduces the loss caused by the precipitation of LF and casein complex, but also improves the migration matching of LF peaks in the standard and actual breast milk samples; alkaline dam focusing further improves peak shape and the signal-to-noise ratio of the target peak. Together with acidic phosphate / polysorbate 20 running buffer, these two components constitute a combined detection scheme suitable for the complex matrix of breast milk.

[0140] Meanwhile, this embodiment also used the same method to detect lactoferrin in cow's milk. Although the method also responded to lactoferrin in cow's milk, its recovery rate in breast milk samples was superior. This indicates that the method has stronger tolerance and compatibility with the complex matrix of breast milk, and after targeted optimization, it better meets the detection needs of breast milk samples, thereby ensuring the accuracy and reliability of the quantitative results of lactoferrin in breast milk.

Claims

1. A method for detecting lactoferrin in breast milk, characterized in that, The method includes the following steps: Sample processing steps: Weigh the breast milk sample, add the extraction solution to form an extraction system, mix well and centrifuge, and take the supernatant as the sample solution to be tested; wherein, the extraction solution is prepared by sodium chloride, weak acid and water; The steps of capillary electrophoresis are as follows: first, sodium hydroxide aqueous solution, water, and running buffer are sequentially injected into the capillary; then, an alkaline solution is injected into the capillary; then, the sample solution to be tested is injected into the capillary; and finally, running buffer is injected into the capillary for capillary zone electrophoresis separation, and detection is performed using an ultraviolet detector. The running buffer is prepared from sodium dihydrogen phosphate, phosphoric acid, polysorbate 20 and water, and the alkaline solution includes at least one of a second sodium hydroxide aqueous solution, ammonia and Tris-base aqueous solution, wherein the first sodium hydroxide aqueous solution and the second sodium hydroxide aqueous solution have different concentrations.

2. The method according to claim 1, characterized in that, The concentration of sodium chloride in the extract is 70-150 mmol / L, and / or the weak acid in the extract includes at least one of acetic acid, citric acid and lactic acid.

3. The method according to claim 1 or 2, characterized in that, The pH value of the extraction system is 4.0-4.

8.

4. The method according to any one of claims 1-3, characterized in that, The concentration of sodium dihydrogen phosphate in the running buffer is 40-70 mmol / L, and / or the concentration of polysorbate 20 in the running buffer is 0.05% (v / v)-0.3% (v / v).

5. The method according to any one of claims 1-4, characterized in that, The pH of the running buffer is 1.7-2.

4.

6. The method according to any one of claims 1-5, characterized in that, The pressure at which the alkaline solution is injected into the capillary is 5-20 psi, and / or the time for injecting the alkaline solution into the capillary is 0.05-0.4 min.

7. The method according to any one of claims 1-6, characterized in that, The pressure at which the sample solution is injected into the capillary is 0.1-1.0 psi, and / or the time for injecting the sample solution into the capillary is 5-15 s.

8. The method according to any one of claims 1-7, characterized in that, The voltage for injecting the running buffer into the capillary for capillary zone electrophoretic separation is 8-14 kV, and / or the time for injecting the running buffer into the capillary for capillary zone electrophoretic separation is 12-18 min.

9. The method according to any one of claims 1-8, characterized in that, The capillary is a fused silica capillary.

10. The method according to any one of claims 1-9, characterized in that, The method also includes a quantitative analysis step, which includes: preparing a series of standard working solutions with lactoferrin standards, establishing a linear relationship between the calibration peak area and the lactoferrin concentration, and calculating the lactoferrin content in the breast milk sample based on the lactoferrin peak area of ​​the sample to be tested.

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