Method for detecting lactoferrin in dairy product

Abstract

The invention relates to the field of detection, in particular to a method for detecting lactoferrin in dairy products, which can adaptively adjust the elution gradient according to the influence condition of wave crests of interfering substances on wave crests of the lactoferrin in the chromatographic detection process, so that the wave crests of the lactoferrin are not overlapped with wave crests of other interfering substances. Furthermore, the concentration of the lactoferrin can be accurately determined according to the wave crest of the lactoferrin, and the content of the lactoferrin is calculated, so that the detection deviation caused by interfering substances is greatly reduced, and the detection precision of the lactoferrin is remarkably improved.

Description

Technical Field

[0001] The invention relates to the detection field, and in particular to a method for detecting lactoferrin in dairy products. Background Art

[0002] Lactoferrin is an important ingredient in dairy products. It plays an important role in antibacterial, antiviral and antioxidant effects, and can regulate immune function and promote iron absorption.

[0003] The content of lactoferrin in dairy products is an important indicator for evaluating the quality of dairy products. In the prior art, liquid chromatography is commonly used to detect the lactoferrin content of dairy products, and in order to prevent the peaks of the chromatogram from overlapping, two-dimensional chromatographic detection is often performed, that is, two chromatographic detections are performed on the test liquid, and the elution gradient (determining the position of the peaks in the chromatogram) of the second chromatographic detection is different from that of the first chromatographic detection, so that the substances with overlapping peaks in the first chromatographic detection will not have overlapping peaks in the second chromatographic detection process; however, the elution gradients of the prior art are all preset elution gradients, and since a large number of unknown interfering substances are often present in the tested dairy samples, the preset elution gradients are difficult to effectively separate the interfering substances from the peaks of lactoferrin, thereby affecting the detection accuracy of lactoferrin. Summary of the invention

[0004] Based on this, it is necessary to provide a method for detecting lactoferrin in dairy products to address the above-mentioned problems.

[0005] The embodiment of the present invention is implemented by providing a method for detecting lactoferrin in dairy products, the method comprising: S1: Prepare a test solution using the test sample, and perform two-dimensional chromatography on the test solution using a liquid chromatograph; S2: during the first-dimension chromatography detection process, generating a first-dimension chromatography coordinate system, and generating a chromatography curve in real time in the first-dimension chromatography coordinate system, wherein the elution gradient of the first-dimension chromatography detection is a preset elution gradient; S3: Determine the appearance time of the first peak corresponding to lactoferrin based on historical data; S4: when a second peak corresponding to any other substance is generated before the appearance time, determining the extension trend of the second peak to judge whether the second peak will extend after the appearance time, and if not, continuing to elute with the preset elution gradient; S5: If yes, adjust the elution gradient in real time according to the historical data, so that the second peak extends to before the appearance time or the appearance time is delayed to after the end point of the second peak, so that the second peak does not overlap with the first peak; S6: after the occurrence time, determining whether a second peak overlapping the first peak is generated at the same time as the first peak is generated, if not, not performing the second-dimensional chromatographic detection, and determining the lactoferrin content according to the peak area of ​​the first peak; S7: If yes, determine the elution gradient of the second-dimension chromatographic detection based on the historical data so that the first peak in the second-dimension chromatographic detection does not overlap with any second peak; S8: performing second-dimension chromatography detection according to the determined elution gradient, and determining the content of lactoferrin based on the peak area of ​​the obtained first wave peak.

[0006] Preferably, the step of preparing a test solution using the test sample comprises: Weigh 5 g of the sample to be tested in a 50 mL volumetric flask, add 30 mL of disodium hydrogen phosphate buffer solution, place the volumetric flask on a vortex oscillator and vortex until the sample is completely dissolved, then use an ultrasonic cleaner to ultrasonically treat the volumetric flask for 10 min, adjust the pH of the solution to 4.6 with phosphoric acid, and then make up to 50 mL with water. After fully mixing, transfer it to a centrifuge tube, place the centrifuge tube in a centrifuge, and centrifuge it at 4°C, 10000 rpm for 10 min. Immediately remove the middle layer solution with a syringe, and pass it through a 0.45 μm filter membrane to obtain the solution to be tested.

[0007] Preferably, determining the appearance time of the first peak corresponding to lactoferrin based on historical data comprises: Retrieving a historical chromatogram of chromatographic detection of lactoferrin under a preset elution gradient from a chromatogram database, wherein the chromatogram database includes chromatograms of each substance corresponding to different substance concentrations under each elution gradient; Identify the starting point of the first peak in the historical chromatogram; The time corresponding to the identified starting point is taken as the appearance time, and the appearance time is marked on the horizontal axis of the first-dimensional chromatographic coordinate system.

[0008] Preferably, when a second peak corresponding to any other substance is generated before the appearance time, determining the extension trend of the second peak to judge whether the second peak will extend after the appearance time comprises: When a second wave peak begins to be generated before the appearance time, the spectral information corresponding to the second wave peak is monitored, and the acquired spectral information is compared with the spectral information in the spectral information library, so as to determine the interfering substance corresponding to the second wave peak; When the second wave peak reaches the peak top, the first half of the second wave peak is obtained, the concentration of the interfering substance is determined according to the peak area of ​​the first half of the second wave peak, and the historical chromatogram of the interfering substance of the concentration under the preset elution gradient is determined in the chromatogram database, the second half of the peak in the determined historical chromatogram is intercepted, and the intercepted second half of the peak is connected with the first half of the second wave peak to obtain the predicted complete second wave peak; It is determined whether the time corresponding to the end point of the complete second wave peak is after the appearance time. If so, the second wave peak will extend after the appearance time. Otherwise, the second wave peak will not extend after the appearance time.

[0009] Preferably, adjusting the elution gradient in real time according to historical data comprises: S51: retrieve all elution gradients of mobile phases that are the same as the preset elution gradient from the chromatographic database; S52: determining an alternative elution gradient from the retrieved elution gradients that can make the end point of the second peak and the starting point of the first peak move away from each other; S53: selecting an alternative elution gradient, determining a first moment corresponding to the starting point of the first peak when the elution gradient is adjusted to the alternative elution gradient, and determining a second moment corresponding to the end point of the second peak when the elution gradient is adjusted to the alternative elution gradient; S54: determining whether the second moment is earlier than the first moment, and if so, determining the candidate elution gradient as the target elution gradient; S55: If not, repeat steps S53 to S54 until the target elution gradient is obtained; S56: After the preset elution gradient is converted into the target elution gradient, the first dimension chromatographic detection is continued, the time of converting the elution gradient is recorded as the adjustment time, and the target elution gradient is recorded.

[0010] Preferably, determining from the retrieved elution gradient an alternative elution gradient that can distance the end point of the second peak and the starting point of the first peak from each other comprises: S521: selecting an elution gradient from the retrieved elution gradients; S522: Retrieving a historical chromatogram of chromatographic detection of the interfering substance under the elution gradient, identifying the width of the peak in the historical chromatogram, and determining whether the width is smaller than the width of the second peak; if not, the elution gradient is not an alternative elution gradient; S523: If yes, retrieve the historical chromatogram of lactoferrin chromatographically detected under the elution gradient, identify the time corresponding to the peak starting point in the historical chromatogram, and judge whether the time is later than the appearance time. If yes, the elution gradient is the alternative elution gradient, and if no, the elution gradient is not the alternative elution gradient; S524: Repeat steps S521 to S523 until all the alternative elution gradients are obtained.

[0011] Preferably, determining the first moment corresponding to the starting point of the first peak when adjusting to the alternative elution gradient comprises: Divide the value corresponding to the current moment by the value corresponding to the appearance moment to obtain the elution progress of lactoferrin; Subtract the elution progress from 1 to get the remaining progress; Retrieving a historical chromatogram of lactoferrin under a target elution gradient, and then identifying the time corresponding to the starting point of the first peak in the historical chromatogram as the third time; Multiply the value corresponding to the third moment by the remaining progress to obtain the first cumulative duration; The first moment is obtained by adding the current moment to the first accumulated duration.

[0012] Preferably, determining the second moment corresponding to the end point of the second peak when adjusting to the alternative elution gradient comprises: Retrieve the historical chromatogram of the interfering substance under the target elution gradient; Identify the duration between the start and end points of the peak in the retrieved historical chromatogram; Multiply the determined duration by 1 / 2 to obtain a second accumulated duration; The second time is obtained by adding the current time to the second accumulated duration.

[0013] Preferably, determining the elution gradient of the second-dimensional chromatographic detection based on historical data comprises: S71: Retrieve a complete chromatogram of the first-dimensional chromatographic detection, identify the span duration of overlapping peaks including the first peak on the complete chromatogram, and obtain spectral information of all peaks; S72: determining all interfering elements in the solution to be tested based on the acquired spectral information; S73: generating a time axis, and retrieving from the chromatogram database all elution gradients of mobile phases that are the same as the preset elution gradient; S74: selecting an elution gradient from the retrieved elution gradients as a simulated elution gradient; S75: for each interfering substance, determining whether the second peak of the interfering substance in the complete chromatogram overlaps with the first peak; S76: If yes, determine the starting time of the peak of the interfering substance under the simulated elution gradient, determine the starting time on the time axis, and add the span time to the starting time to obtain the corresponding end time, thereby obtaining the second peak time period of the interfering substance; S77: If not, determine the starting time and the ending time of the peak of the interfering substance under the simulated elution gradient, determine the starting time and the ending time on the time axis, and obtain the second peak time period of the interfering substance; S78: determining the starting point of the simulated elution gradient of lactoferrin as the comparison time, and judging whether the comparison time is in any second peak period, and if so, excluding the simulated elution gradient; S79: If not, determine whether the free time period after the comparison time is longer than the span time, and if so, determine the simulated elution gradient as the elution gradient of the second-dimensional chromatographic detection; S710: If not, the simulated elution gradient is excluded, and steps S74 to S79 ​​are repeated until the elution gradient of the second-dimensional chromatographic detection is determined.

[0014] The present invention provides a method for detecting lactoferrin in dairy products, comprising preparing a test solution with a test sample, and performing two-dimensional chromatographic detection on the test solution by a liquid chromatograph; in the first-dimensional chromatographic detection process, generating a first-dimensional chromatographic coordinate system, and generating a chromatographic curve in real time in the first-dimensional chromatographic coordinate system; determining the appearance time of a first wave peak corresponding to lactoferrin according to historical data; when a second wave peak corresponding to any other substance is generated before the appearance time, determining the extension trend of the second wave peak to judge whether the second wave peak will extend after the appearance time, and if not, continuing to elute with a preset elution gradient; if so, adjusting the elution gradient in real time according to the historical data to make the second wave peak extend before the appearance time or to make the appearance time delayed to after the end point of the second wave peak, so that the second wave peak does not overlap with the first wave peak; after the appearance time, determining the second wave peak at the time when the first wave peak is generated. At the same time, whether a second peak overlapping with the first peak is generated, if not, the second-dimensional chromatographic detection is not performed, and the content of lactoferrin is determined according to the peak area of ​​the first peak; if so, the elution gradient of the second-dimensional chromatographic detection is determined according to the historical data, so that the first peak in the second-dimensional chromatographic detection does not overlap with any second peak; the second-dimensional chromatographic detection is performed according to the determined elution gradient, and the content of lactoferrin is determined according to the peak area of ​​the obtained first peak; in the present application, the elution gradient can be adaptively adjusted according to the influence of the peak of the interfering substance on the peak of lactoferrin during the chromatographic detection, so that the peak of lactoferrin does not overlap with the peak of other interfering substances, and then the concentration of lactoferrin can be accurately determined according to the peak of lactoferrin, and then its content can be calculated, which greatly reduces the detection deviation caused by the interfering substance and significantly improves the detection accuracy of lactoferrin. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a flow chart of a method for detecting lactoferrin in dairy products provided in one embodiment; Figure 2 A schematic diagram of an implementation environment of a method for detecting lactoferrin in dairy products provided in an embodiment; Figure 3 A schematic diagram of a complete chromatogram of a method for detecting lactoferrin in dairy products provided in an embodiment; Figure 4 The figure is a schematic timeline diagram of a method for detecting lactoferrin in dairy products provided in an embodiment. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0017] It is understood that the terms "first", "second", etc. used in the present invention may be used herein to describe various elements, but unless otherwise specified, these elements are not limited by these terms. These terms are only used to distinguish a first element from another element. For example, a first xx script may be referred to as a second xx script, and similarly, a second xx script may be referred to as a first xx script without departing from the scope of the present invention.

[0018] like Figure 1 As shown, in one embodiment, a method for detecting lactoferrin in dairy products is proposed, the method comprising: S1: Prepare a test solution using the test sample, and perform two-dimensional chromatography on the test solution using a liquid chromatograph; S2: during the first-dimension chromatography detection process, generating a first-dimension chromatography coordinate system, and generating a chromatography curve in real time in the first-dimension chromatography coordinate system, wherein the elution gradient of the first-dimension chromatography detection is a preset elution gradient; S3: Determine the appearance time of the first peak corresponding to lactoferrin based on historical data; S4: when a second peak corresponding to any other substance is generated before the appearance time, determining the extension trend of the second peak to judge whether the second peak will extend after the appearance time, and if not, continuing to elute with the preset elution gradient; S5: If yes, adjust the elution gradient in real time according to the historical data, so that the second peak extends to before the appearance time or the appearance time is delayed to after the end point of the second peak, so that the second peak does not overlap with the first peak; S6: after the occurrence time, determining whether a second peak overlapping the first peak is generated at the same time as the first peak is generated, if not, not performing the second-dimensional chromatographic detection, and determining the lactoferrin content according to the peak area of ​​the first peak; S7: If yes, determine the elution gradient of the second-dimension chromatographic detection based on the historical data so that the first peak in the second-dimension chromatographic detection does not overlap with any second peak; S8: performing second-dimension chromatography detection according to the determined elution gradient, and determining the content of lactoferrin based on the peak area of ​​the obtained first wave peak.

[0019] In this embodiment, if Figure 2As shown, in step S1, 'preparing the test solution with the test sample' is performed by the staff, and 'performing two-dimensional chromatography detection on the test solution by the liquid chromatograph' is performed by the staff through the liquid chromatograph; steps S2 to S8 are performed by a computer device, and the computer device can be an independent physical server or terminal, or a server cluster composed of multiple physical servers, and can be a cloud server that provides basic cloud computing services such as cloud servers, cloud databases, cloud storage and CDN; the computer device is connected to the liquid chromatograph, and after the detection results of the liquid chromatograph are input into the computer device, the computer device can calculate the content of the substance to be detected according to the detection results; the computer device can also control the liquid chromatograph to adjust its elution parameters.

[0020] In this embodiment, two chromatographic columns are installed in the liquid chromatograph (for performing first-dimensional chromatographic detection and second-dimensional chromatographic detection, respectively), and each chromatographic column is equipped with a UV detector, a quaternary pump and a valve switching system, so that the solution can flow smoothly through the chromatographic column and be detected by the detector; the chromatographic conditions include: Temperature: 30℃; Injection volume: 50 μL; Mobile phase A: 50mmol / L Na2HP04 solution; Mobile phase B: 50mmol / L Na2HP04+1.0mol / L NaCl solution (pH7.00±0.5); Flow rate: 1.0 mL / min; In this embodiment, the elution gradient is a method in which the concentration ratio of two mobile phases is continuously changed according to a certain procedure in chromatographic detection, so that different components in the sample are gradually separated on the chromatographic column according to their respective elution characteristics; the difference in elution gradients mainly refers to the difference in the concentration ratio of the mobile phases of the two elution gradients and / or the difference in the time interval for changing the concentration ratio of the mobile phases; a preset elution gradient is set for the first-dimensional chromatographic detection, and no elution gradient is pre-set for the second-dimensional chromatographic detection, and its elution gradient is determined by the chromatogram of the first-dimensional chromatographic detection (if the second-dimensional chromatographic detection is required).

[0021] In this embodiment, chromatographic detection utilizes the difference in distribution coefficients of components in the test solution between the mobile phases. When the mixture passes through the chromatographic column with the mobile phase, the distribution coefficients of the components between the two mobile phases are different, resulting in different retention times in the chromatographic column, thereby achieving separation. The ordinate of the first-dimensional chromatographic coordinate system is the response signal value. For example, when an ultraviolet detector is used, the ordinate is the absorbance, and the abscissa is the time, which corresponds to the retention time. The origin of the abscissa is time 0, which corresponds to the time when the test solution enters the chromatographic column, and the time when a peak on the coordinate system begins to appear. That is, the moment when the substance corresponding to the peak begins to flow out of the chromatographic column, and the time difference between this moment and time 0 is the retention time of the substance; the moment of peak appearance is the moment corresponding to the starting point of the peak, the starting point is the first intersection of the peak and the chromatographic baseline (sorted in time order), and the end point is the last intersection of the peak and the chromatographic baseline; for each substance, under different elution gradients, the elution efficiency is different, which will change its retention time, and finally change its position and shape on the chromatogram (the peak may become wider and shorter, or taller and narrower, and the total area remains unchanged); In this embodiment, during the first-dimension chromatographic detection (i.e., the first round of chromatographic detection, the process of passing the test solution through the first chromatographic column and detecting), elution is first performed with a preset elution gradient, and then it is monitored whether a peak of an interfering substance (i.e., a second peak) appears before the peak of lactoferrin (i.e., the first peak) appears (i.e., before the appearance moment). If not, there is no need to adjust the elution gradient. If so, its extension trend is predicted, and it is determined whether it will overlap with the first peak. If so, the elution gradient is adjusted so that the second peak changes position and shape with the first peak so that the two do not overlap. If not, the preset elution gradient is maintained for elution. When the appearance moment is reached, that is, the retention time of lactoferrin is reached, and the first peak begins to be generated in the first-dimension chromatographic coordinate system. Since the components in the test solution are uncertain, at the same time as the first peak is generated, A second peak may be generated (i.e., an overlapping peak with two peaks may be formed), or the second peak may not be generated; therefore, in the case of no overlapping peaks, the first peak can be independently displayed in the coordinate system and can be directly used to calculate the content of lactoferrin without the need for second-dimensional chromatographic detection; in the case of overlapping peaks, since the first peak and the second peak have overlapped and cannot be separated, the two can only be separated in the second-dimensional chromatographic detection. After the first-dimensional chromatographic detection, all interfering substances in the solution to be tested can be known based on the peaks of each peak. Based on these known interfering substances and the peak distribution of these substances under different elution gradients in history, the elution gradient that makes the peak of the interfering substance not overlap with the first peak can be determined. The second-dimensional chromatographic detection is performed using this elution gradient to obtain an independent first peak, and then the content of lactoferrin can be determined; In the present application, it is possible to adaptively adjust the elution gradient according to the influence of the peak of the interfering substance on the peak of lactoferrin during the chromatographic detection process, so that the peak of lactoferrin does not overlap with the peak of other interfering substances, and then the concentration of lactoferrin can be accurately determined based on the peak of lactoferrin, and then its content can be calculated, which greatly reduces the detection deviation caused by the interfering substance and significantly improves the detection accuracy of lactoferrin.

[0022] As a preferred embodiment, the step of preparing a test solution using a test sample comprises: Weigh 5 g of the sample to be tested in a 50 mL volumetric flask, add 30 mL of disodium hydrogen phosphate buffer solution, place the volumetric flask on a vortex oscillator and vortex until the sample is completely dissolved, then use an ultrasonic cleaner to ultrasonically treat the volumetric flask for 10 min, adjust the pH of the solution to 4.6 with phosphoric acid, and then make up to 50 mL with water. After fully mixing, transfer it to a centrifuge tube, place the centrifuge tube in a centrifuge, and centrifuge it at 4°C, 10000 rpm for 10 min. Immediately remove the middle layer solution with a syringe, and pass it through a 0.45 μm filter membrane to obtain the solution to be tested.

[0023] In this embodiment, the measurement solution is measured by a liquid chromatograph, and the chromatogram generated during the measurement can be presented in real time in a computer device. The computer device determines the concentration of lactoferrin based on the peak area of ​​lactoferrin in the chromatogram, and then calculates the content of lactoferrin based on the concentration.

[0024] In this embodiment, the computer device calculates the content of lactoferrin in the sample to be tested (such as milk powder, cow's milk, etc.) by the following formula: Wherein, X is the content of lactoferrin, is the concentration of lactoferrin in the test solution, m is the sampling mass, and V is the fixed volume of the test solution.

[0025] In this embodiment, the unit of C is g / mL, the unit of V is mL, and the unit of m is g; to detect lactoferrin, an isotope internal standard working solution of lactoferrin needs to be prepared accordingly, and the preparation steps are as follows: accurately weigh 10 mg of lactoferrin standard (accurate to 0.00001 g), place it in a 10 mL brown volumetric flask, dissolve it with 0.1% formic acid methanol solution and make it up to the scale, prepare a standard stock solution with a concentration of 1000 μg / mL, and store it below 0-4°C; In this embodiment, before the solution to be tested is measured on the machine, a standard curve series of solutions of lactoferrin to be measured is required to be prepared, including a plurality of solutions of lactoferrin with standard concentrations from small to large (such as 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, etc.), and the solutions are measured on the machine in order from small to large concentrations, and a standard curve is drawn with the lactoferrin concentration as the abscissa and the lactoferrin isotope internal standard peak area as the ordinate; then the solution to be tested is measured to obtain the peak area of ​​lactoferrin in the solution to be tested, and the peak area is compared with the peak area corresponding to the standard curve, so as to determine the concentration of lactoferrin in the solution to be tested.

[0026] As a preferred embodiment, the step of determining the appearance time of the first peak corresponding to lactoferrin based on historical data includes: Retrieving a historical chromatogram of chromatographic detection of lactoferrin under a preset elution gradient from a chromatogram database, wherein the chromatogram database includes chromatograms of each substance corresponding to different substance concentrations under each elution gradient; Identify the starting point of the first peak in the historical chromatogram; The time corresponding to the identified starting point is taken as the appearance time, and the appearance time is marked on the horizontal axis of the first-dimensional chromatographic coordinate system.

[0027] When a second wave peak corresponding to any other substance is generated before the appearance time, determining the extension trend of the second wave peak to judge whether the second wave peak will extend after the appearance time includes: When a second wave peak begins to be generated before the appearance time, the spectral information corresponding to the second wave peak is monitored, and the acquired spectral information is compared with the spectral information in the spectral information library, so as to determine the interfering substance corresponding to the second wave peak; When the second wave peak reaches the peak top, the first half of the second wave peak is obtained, the concentration of the interfering substance is determined according to the peak area of ​​the first half of the second wave peak, and the historical chromatogram of the interfering substance of the concentration under the preset elution gradient is determined in the chromatogram database, the second half of the peak in the determined historical chromatogram is intercepted, and the intercepted second half of the peak is connected with the first half of the second wave peak to obtain the predicted complete second wave peak; It is determined whether the time corresponding to the end point of the complete second wave peak is after the appearance time. If so, the second wave peak will extend after the appearance time. Otherwise, the second wave peak will not extend after the appearance time.

[0028] In the present embodiment, when no substance is detected, the chromatographic line is a horizontal line (i.e., the chromatographic baseline). When a substance is detected, the chromatographic line extends upward, then slides downward until it reaches the height of the chromatographic baseline, and forms a horizontal line again, thereby forming a peak. The intersection of the line extending upward and the chromatographic baseline is the starting point of the peak, and the intersection of the peak line extending downward and the chromatographic baseline is the focus of the peak. In the present embodiment, although the concentration of lactoferrin is not yet determined, since its concentration only affects the shape of the first peak (without affecting the peak width and peak time), and the starting point of the first peak is only determined by the elution gradient, the starting point of the first peak can be determined by selecting any elution gradient as the historical chromatogram of the preset elution gradient.

[0029] In this embodiment, the historical data includes data in a chromatogram database; the chromatograms in the chromatogram database are chromatograms stored in historical tests; the chromatograms include a chromatogram coordinate system and a corresponding chromatogram curve; a historical chromatogram of a substance includes only the peak of the substance (other peaks are hidden); for each substance, its position on the chromatogram coordinate system is different under different elution gradients, and its peak shape is also different corresponding to different substance concentrations, therefore, for each substance, the chromatogram database stores its historical chromatogram corresponding to each elution gradient under each concentration condition for analysis and call; In this embodiment, the spectrum information library includes ultraviolet spectra of standard solutions of various substances; when the second wave peak is generated, the ultraviolet detector starts to detect the substance of the second wave peak. The ultraviolet detector can detect the ultraviolet spectrum (i.e., spectrum information) of the substance while detecting the response value of the substance. Since each substance has a unique ultraviolet spectrum, the detected ultraviolet spectrum is compared with the ultraviolet spectrum of each substance in the spectrum information library to determine the interfering substance corresponding to the second wave peak; In this embodiment, since the independent peaks show a normal distribution, when the second peak reaches its peak, that is, the first half of the peak has been generated, the peak area of ​​the first half of the peak is half of the area of ​​the entire second peak, and the area of ​​the entire peak can be calculated, so that the concentration of the corresponding interfering substance can be calculated, and the historical chromatogram of the interfering substance under the same working conditions (consistent substance concentration, consistent elution gradient) can be retrieved from the chromatogram database, and the second half of the historical chromatogram is combined with the generated first half of the peak to determine its extension trend.

[0030] As a preferred embodiment, adjusting the elution gradient in real time according to historical data includes: S51: retrieve all elution gradients of mobile phases that are the same as the preset elution gradient from the chromatographic database; S52: determining an alternative elution gradient from the retrieved elution gradients that can make the end point of the second peak and the starting point of the first peak move away from each other; S53: selecting an alternative elution gradient, determining a first moment corresponding to the starting point of the first peak when the elution gradient is adjusted to the alternative elution gradient, and determining a second moment corresponding to the end point of the second peak when the elution gradient is adjusted to the alternative elution gradient; S54: determining whether the second moment is earlier than the first moment, and if so, determining the candidate elution gradient as the target elution gradient; S55: If not, repeat steps S53 to S54 until the target elution gradient is obtained; S56: After the preset elution gradient is converted into the target elution gradient, the first dimension chromatographic detection is continued, the time of converting the elution gradient is recorded as the adjustment time, and the target elution gradient is recorded.

[0031] Alternative elution gradients that can be determined from the retrieved elution gradient to move the end point of the second peak and the start point of the first peak away include: S521: selecting an elution gradient from the retrieved elution gradients; S522: Retrieving a historical chromatogram of chromatographic detection of the interfering substance under the elution gradient, identifying the width of the peak in the historical chromatogram, and determining whether the width is smaller than the width of the second peak; if not, the elution gradient is not an alternative elution gradient; S523: If yes, retrieve the historical chromatogram of lactoferrin chromatographically detected under the elution gradient, identify the time corresponding to the peak starting point in the historical chromatogram, and judge whether the time is later than the appearance time. If yes, the elution gradient is the alternative elution gradient, and if no, the elution gradient is not the alternative elution gradient; S524: Repeat steps S521 to S523 until all the alternative elution gradients are obtained.

[0032] Determining the first time corresponding to the starting point of the first peak when adjusting to the alternative elution gradient comprises: Divide the value corresponding to the current moment by the value corresponding to the appearance moment to obtain the elution progress of lactoferrin; Subtract the elution progress from 1 to get the remaining progress; Retrieving a historical chromatogram of lactoferrin under a target elution gradient, and then identifying the time corresponding to the starting point of the first peak in the historical chromatogram as the third time; Multiply the value corresponding to the third moment by the remaining progress to obtain the first cumulative duration; The first moment is obtained by adding the current moment to the first accumulated duration.

[0033] Determining a second time point corresponding to the end point of the second peak when adjusting to the alternative elution gradient comprises: Retrieve the historical chromatogram of the interfering substance under the target elution gradient; Identify the duration between the start and end points of the peak in the retrieved historical chromatogram; Multiply the determined duration by 1 / 2 to obtain a second accumulated duration; The second time is obtained by adding the current time to the second accumulated duration.

[0034] In this embodiment, different mobile phases will also cause different elution gradients. Since the mobile phase cannot be changed during the detection process, the alternative elution gradient must be an elution gradient with the same mobile phase as the preset elution gradient; the alternative elution gradient can make the first peak and the second peak away from each other, but the degree of distance varies, so it is necessary to calculate each alternative elution gradient to determine the target elution gradient that can prevent the first peak and the second peak from overlapping; if multiple second peaks appear before the appearance time, all target elution gradients corresponding to each interfering substance can be determined through this embodiment, and finally the target elution gradient shared by each interfering substance is used for adjustment; In this embodiment, the width of the peak is the width from the starting point to the end point of the peak; if an elution gradient can reduce the peak width of the second peak, the end point of the second peak will move forward and away from the first peak; further, if an elution gradient can move the starting point of the first peak backward, the first peak will move away from the second peak; therefore, this embodiment can move the first peak away from the second peak by determining the elution gradient that reduces the peak width of the second peak and moves the starting point of the first peak backward as an alternative elution gradient.

[0035] In this embodiment, for example, if the current moment is 3 minutes (the starting moment is 0 minutes), the appearance moment is 5 minutes, then the elution progress is 3 / 5=60%, and the remaining progress is 1-60%=40%; if the third moment is 10 minutes, then the first cumulative duration is 10 minutes*40%=4 minutes, and the first moment is 3+4=7 minutes; if the duration corresponding to the peak width of the historical chromatogram of the interfering substance under the target elution gradient is 6 minutes, then the second moment is 3+6 / 2=6 minutes.

[0036] like Figure 3-Figure 4 As shown, as a preferred embodiment, determining the elution gradient of the second-dimensional chromatographic detection based on historical data includes: S71: Retrieve a complete chromatogram of the first-dimensional chromatographic detection, identify the span duration of overlapping peaks including the first peak on the complete chromatogram, and obtain spectral information of all peaks; S72: determining all interfering elements in the solution to be tested based on the acquired spectral information; S73: generating a time axis, and retrieving from the chromatogram database all elution gradients of mobile phases that are the same as the preset elution gradient; S74: selecting an elution gradient from the retrieved elution gradients as a simulated elution gradient; S75: for each interfering substance, determining whether the second peak of the interfering substance in the complete chromatogram overlaps with the first peak; S76: If yes, determine the starting time of the peak of the interfering substance under the simulated elution gradient, determine the starting time on the time axis, and add the span time to the starting time to obtain the corresponding end time, thereby obtaining the second peak time period of the interfering substance; S77: If not, determine the starting time and the ending time of the peak of the interfering substance under the simulated elution gradient, determine the starting time and the ending time on the time axis, and obtain the second peak time period of the interfering substance; S78: determining the starting point of the simulated elution gradient of lactoferrin as the comparison time, and judging whether the comparison time is in any second peak period, and if so, excluding the simulated elution gradient; S79: If not, determine whether the free time period after the comparison time is longer than the span time, and if so, determine the simulated elution gradient as the elution gradient of the second-dimensional chromatographic detection; S710: If not, the simulated elution gradient is excluded, and steps S74 to S79 ​​are repeated until the elution gradient of the second-dimensional chromatographic detection is determined.

[0037] In this embodiment, the spectral information corresponding to each peak is stored during the first round of chromatographic detection, and the interfering substances corresponding to each second peak can be determined based on the corresponding spectral information. The determination method is consistent with the above and is not limited here. The time of the generated time axis corresponds to the time of the abscissa of the first-dimensional chromatographic coordinate system. The starting time and the ending time of the peak of the interfering substance under the simulated elution gradient are determined as described above, and the historical chromatogram under the simulated elution gradient is retrieved, and the starting time and the ending time of the peak in the historical chromatogram are identified. When the concentration of the substance cannot be determined, the starting time is determined in the same way as the above-mentioned method of determining the occurrence time. If the second peak overlaps with the first peak in the complete chromatogram, the concentration of the substance corresponding to the second peak cannot be determined, and then only the starting time of the substance under other elution gradients can be determined, and its peak width and the ending time cannot be determined. At this time, the width of the overlapping peak is directly regarded as the width of the second peak (because the width of the overlapping peak is often larger than the width of the single second peak, This method can make the separation arrangement of the first peak and the second peak have higher redundancy. In this case, the first peak does not overlap with other second peaks, and the two will not overlap in actual circumstances); if the second peak does not overlap with the first peak in the complete chromatogram, the concentration of the substance corresponding to the second peak can be determined, and the starting time and the end time of the substance under other elution gradients can be determined; in addition, for the first peak, the width of the overlapping peak is also regarded as the width of the first peak, which can also improve the redundancy of the arrangement; in this embodiment, for each elution gradient, the starting time and the end time of each corresponding second peak and the first peak are marked on the time axis, and it can be intuitively determined whether the time period occupied by the second peak and the time period occupied by the first peak intersect. If there is no intersection, the two will not overlap, so that the elution gradient that makes the first peak and each second peak not overlap can be determined, and the second-dimensional chromatographic detection is performed using the elution gradient, that is, an independent first peak can be obtained, and then the content of lactoferrin can be determined.

[0038] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0039] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for detecting lactoferrin in dairy products, characterized in that: The method comprises: S1: Prepare a test solution using the test sample, and perform two-dimensional chromatography on the test solution using a liquid chromatograph; S2: during the first-dimension chromatography detection process, generating a first-dimension chromatography coordinate system, and generating a chromatography curve in real time in the first-dimension chromatography coordinate system, wherein the elution gradient of the first-dimension chromatography detection is a preset elution gradient; S3: Determine the appearance time of the first peak corresponding to lactoferrin based on historical data; S4: when a second peak corresponding to any other substance is generated before the appearance time, determining the extension trend of the second peak to judge whether the second peak will extend after the appearance time, and if not, continuing to elute with the preset elution gradient; S5: If yes, adjust the elution gradient in real time according to the historical data, so that the second peak extends to before the appearance time or the appearance time is delayed to after the end point of the second peak, so that the second peak does not overlap with the first peak; S6: after the occurrence time, determining whether a second peak overlapping the first peak is generated at the same time as the first peak is generated, if not, not performing the second-dimensional chromatographic detection, and determining the lactoferrin content according to the peak area of ​​the first peak; S7: If yes, determine the elution gradient of the second-dimension chromatographic detection based on the historical data so that the first peak in the second-dimension chromatographic detection does not overlap with any second peak; S8: performing second-dimension chromatography detection according to the determined elution gradient, and determining the content of lactoferrin based on the peak area of ​​the obtained first wave peak.

2. The method according to claim 1, characterized in that: The method of preparing the solution to be tested by using the tested sample comprises: Weigh 5 g of the sample to be tested in a 50 mL volumetric flask, add 30 mL of disodium hydrogen phosphate buffer solution, place the volumetric flask on a vortex oscillator and vortex until the sample is completely dissolved, then use an ultrasonic cleaner to ultrasonically treat the volumetric flask for 10 min, adjust the pH of the solution to 4.6 with phosphoric acid, and then make up to 50 mL with water. After fully mixing, transfer it to a centrifuge tube, place the centrifuge tube in a centrifuge, and centrifuge it at 4°C, 10000 rpm for 10 min. Immediately remove the middle layer solution with a syringe, and pass it through a 0.45 μm filter membrane to obtain the solution to be tested.

3. The method according to claim 1, characterized in that Determining the appearance time of the first peak corresponding to lactoferrin according to historical data includes: Retrieving a historical chromatogram of chromatographic detection of lactoferrin under a preset elution gradient from a chromatogram database, wherein the chromatogram database includes chromatograms of each substance corresponding to different substance concentrations under each elution gradient; Identify the starting point of the first peak in the historical chromatogram; The time corresponding to the identified starting point is taken as the appearance time, and the appearance time is marked on the horizontal axis of the first-dimensional chromatographic coordinate system.

4. The method according to claim 3, characterized in that When a second wave peak corresponding to any other substance is generated before the appearance time, determining the extension trend of the second wave peak to judge whether the second wave peak will extend after the appearance time includes: When a second wave peak begins to be generated before the appearance time, the spectral information corresponding to the second wave peak is monitored, and the acquired spectral information is compared with the spectral information in the spectral information library, so as to determine the interfering substance corresponding to the second wave peak; When the second wave peak reaches the peak top, the first half of the second wave peak is obtained, the concentration of the interfering substance is determined according to the peak area of ​​the first half of the second wave peak, and the historical chromatogram of the interfering substance of the concentration under the preset elution gradient is determined in the chromatogram database, the second half of the peak in the determined historical chromatogram is intercepted, and the intercepted second half of the peak is connected with the first half of the second wave peak to obtain the predicted complete second wave peak; It is determined whether the time corresponding to the end point of the complete second wave peak is after the appearance time. If so, the second wave peak will extend after the appearance time. Otherwise, the second wave peak will not extend after the appearance time.

5. The method according to claim 4, characterized in that Real-time adjustment of elution gradient based on historical data includes: S51: retrieve all elution gradients of mobile phases that are the same as the preset elution gradient from the chromatographic database; S52: determining an alternative elution gradient from the retrieved elution gradients that can make the end point of the second peak and the starting point of the first peak move away from each other; S53: selecting an alternative elution gradient, determining a first moment corresponding to the starting point of the first peak when the elution gradient is adjusted to the alternative elution gradient, and determining a second moment corresponding to the end point of the second peak when the elution gradient is adjusted to the alternative elution gradient; S54: determining whether the second moment is earlier than the first moment, and if so, determining the candidate elution gradient as the target elution gradient; S55: If not, repeat steps S53 to S54 until the target elution gradient is obtained; S56: After the preset elution gradient is converted into the target elution gradient, the first dimension chromatographic detection is continued, the time of converting the elution gradient is recorded as the adjustment time, and the target elution gradient is recorded.

6. The method according to claim 5, characterized in that Alternative elution gradients that can be determined from the retrieved elution gradient to move the end point of the second peak and the start point of the first peak away include: S521: selecting an elution gradient from the retrieved elution gradients; S522: Retrieving a historical chromatogram of chromatographic detection of the interfering substance under the elution gradient, identifying the width of the peak in the historical chromatogram, and determining whether the width is smaller than the width of the second peak; if not, the elution gradient is not an alternative elution gradient; S523: If yes, retrieve the historical chromatogram of lactoferrin chromatographically detected under the elution gradient, identify the time corresponding to the peak starting point in the historical chromatogram, and judge whether the time is later than the appearance time. If yes, the elution gradient is the alternative elution gradient, and if no, the elution gradient is not the alternative elution gradient; S524: Repeat steps S521 to S523 until all the alternative elution gradients are obtained.

7. The method according to claim 5, characterized in that Determining the first time corresponding to the starting point of the first peak when adjusting to the alternative elution gradient comprises: Divide the value corresponding to the current moment by the value corresponding to the appearance moment to obtain the elution progress of lactoferrin; Subtract the elution progress from 1 to get the remaining progress; Retrieving a historical chromatogram of lactoferrin under a target elution gradient, and then identifying the time corresponding to the starting point of the first peak in the historical chromatogram as the third time; Multiply the value corresponding to the third moment by the remaining progress to obtain the first cumulative duration; The first moment is obtained by adding the current moment to the first accumulated duration.

8. The method according to claim 5, characterized in that Determining a second time point corresponding to the end point of the second peak when adjusting to the alternative elution gradient comprises: Retrieve the historical chromatogram of the interfering substance under the target elution gradient; Identify the duration between the start and end points of the peak in the retrieved historical chromatogram; Multiply the determined duration by 1 / 2 to obtain a second accumulated duration; The second time is obtained by adding the current time to the second accumulated duration.

9. The method according to claim 5, characterized in that The elution gradient for the second-dimension chromatography detection was determined based on historical data including: S71: Retrieve a complete chromatogram of the first-dimensional chromatographic detection, identify the span duration of overlapping peaks including the first peak on the complete chromatogram, and obtain spectral information of all peaks; S72: determining all interfering elements in the solution to be tested based on the acquired spectral information; S73: generating a time axis, and retrieving from the chromatogram database all elution gradients of mobile phases that are the same as the preset elution gradient; S74: selecting an elution gradient from the retrieved elution gradients as a simulated elution gradient; S75: for each interfering substance, determining whether the second peak of the interfering substance in the complete chromatogram overlaps with the first peak; S76: If yes, determine the starting time of the peak of the interfering substance under the simulated elution gradient, determine the starting time on the time axis, and add the span time to the starting time to obtain the corresponding end time, thereby obtaining the second peak time period of the interfering substance; S77: If not, determine the starting time and the ending time of the peak of the interfering substance under the simulated elution gradient, determine the starting time and the ending time on the time axis, and obtain the second peak time period of the interfering substance; S78: determining the starting point of the simulated elution gradient of lactoferrin as the comparison time, and judging whether the comparison time is in any second peak period, and if so, excluding the simulated elution gradient; S79: If not, determine whether the free time period after the comparison time is longer than the span time, and if so, determine the simulated elution gradient as the elution gradient of the second-dimensional chromatographic detection; S710: If not, the simulated elution gradient is excluded, and steps S74 to S79 ​​are repeated until the elution gradient of the second-dimensional chromatographic detection is determined.

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