Multi-vitamin synchronous quantitative detection method and system

Abstract

The invention relates to the technical field of column chromatography detection, in particular to a multi-vitamin synchronous quantitative detection method and system.The method comprises the steps that the chromatographic symmetry of separated components of a sample to be detected is determined, and the interference degree of interference components on the separated components is obtained by combining the peak distance of the adjacent separated components; on the basis of each retention time of the separated component and the difference between the retention time and the preset standard retention time, the influence degree of each retention time on the polarity determination of the separated component is obtained, and the polarity of the water-soluble vitamin component in the separated component is obtained by combining the difference between the detector response values of each retention time and the adjacent retention time. According to the present invention, the interference degree and the polarity are integrated, and the organic solvent ratio in the mobile phase required by component separation is determined, such that the co-elution of the water-soluble vitamins and other interference components can be reduced so as to reduce the matrix effect, such that the water-soluble vitamins can be well separated, the separation effect of various vitamins can be improved, and the multi-vitamin detection accuracy can be improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of column chromatography detection, in particular to a multi-vitamin synchronous quantitative detection method and system. BACKGROUND

[0002] Through the multi-vitamin synchronous quantitative detection of the to-be-detected sample, the content of each vitamin contained in the to-be-detected sample can be accurately evaluated, which is of great significance. The to-be-detected sample is usually various foods.

[0003] The existing multi-vitamin synchronous quantitative detection is usually carried out by high performance liquid chromatography tandem mass spectrometry. The principle is to separate multiple vitamins in the to-be-detected sample by high performance liquid chromatography (HPLC). In the chromatographic column, the to-be-detected sample is separated under the action of the mobile phase, and each vitamin will interact differently with the mobile phase on the chromatographic column according to its chemical properties, so as to flow out at different time points and form different chromatographic peaks. Then, each separated component after chromatographic separation enters mass spectrometry. Mass spectrometry converts the separated components into charged particles through ionization technology, and then detects and analyzes them according to the mass-to-charge ratio (m / z) of these ions. Mass spectrometry can accurately measure the concentration of each vitamin and quantitatively analyze it by comparison with the standard. When separating multiple vitamins, the same proportion of organic solvents is used in the mobile phase. However, in the prior art, multiple vitamins usually come from complex matrices such as food, which usually contains other components such as proteins, fats, salts, etc. They may interact with vitamin molecules, affecting the ionization efficiency of vitamins, and thus causing matrix effects. Matrix effect refers to the interference of other substances in the matrix with the ionization process of the analyte (such as vitamins), which may cause signal suppression or enhancement. Specifically, the signal of some vitamins is suppressed and difficult to accurately measure, or due to the interference of the matrix, the signal of some vitamins is excessively enhanced, thereby affecting the accuracy of the detection result. Therefore, when separating multiple vitamins using the same proportion of organic solvents in the mobile phase, the separation effect of vitamins is affected, thereby affecting the detection accuracy of multiple vitamins. SUMMARY

[0004] In order to solve the technical problem that the setting method of the proportion of organic solvents in the existing mobile phase affects the detection accuracy of multiple vitamins, the purpose of the present application is to provide a multi-vitamin synchronous quantitative detection method and system, and the technical scheme adopted is as follows: In the first aspect of the present application, a multi-vitamin synchronous quantitative detection method is provided, comprising: Based on the peak symmetry of the chromatogram of the to-be-detected sample, the chromatographic symmetry of the separated components is obtained; determining a peak distance of the adjacent separated separation component, combining the symmetry of the chromatogram, to obtain an interference degree of other interference components to the separation component; based on the each retention time of the separation component and its difference from the preset standard retention time, obtaining an influence degree of the each retention time of the separation component to the polarity determination of the separation component; determining the difference between the each retention time of the separation component and the detector response value of its adjacent retention time, combining the influence degree, to obtain the polarity of the water-soluble vitamin component in the separation component; determining the proportion of the organic solvent in the mobile phase required by the separation component according to the interference degree and the polarity.

[0005] In an exemplary embodiment, the process of obtaining the symmetry of the chromatogram comprises: obtaining the symmetry difference of the each retention time of the separation component; the symmetry difference is the difference between the retention time and the detector response value of its symmetric position in the retention time range; fusing the symmetry difference of the each retention time in the retention time range of the separation component, to obtain the symmetry of the chromatogram; the symmetry of the chromatogram is inversely related to the symmetry difference.

[0006] In an exemplary embodiment, the process of determining the peak distance of the adjacent separated separation component comprises: determining the first peak distance and the second peak distance of the separation component; the first peak distance is the distance between the highest peak of the separation component and the highest peak of the adjacent previous separation component, and the second peak distance is the distance between the highest peak of the separation component and the highest peak of the adjacent next separation component; determining the minimum value of the first peak distance and the second peak distance as the final peak distance of the separation component.

[0007] In an exemplary embodiment, the process of obtaining the interference degree comprises: obtaining the interference degree of the separation component from the final peak distance of the separation component and the symmetry of the chromatogram; the interference degree is inversely related to both the final peak distance and the symmetry of the chromatogram.

[0008] In an exemplary embodiment, the process of obtaining the difference between the each retention time of the separation component and the preset standard retention time comprises: obtaining the difference between any retention time of the separation component and the preset standard retention time of various vitamins, and determining the minimum difference as the difference between the any retention time and the preset standard retention time.

[0009] In an exemplary embodiment, the process of obtaining the influence degree comprises: According to the difference between the any retention time and the preset standard retention time, and the any retention time, the influence degree corresponding to the any retention time is obtained; the influence degree corresponding to the any retention time is inversely related to the difference between the any retention time and the preset standard retention time, and the any retention time.

[0010] In an exemplary embodiment, the determination of the difference between the detector response value of each retention time of the separated component and the adjacent retention time thereof includes: The first detector response value difference and the second detector response value difference of any retention time of the separated component are determined; the first detector response value difference is the difference between the detector response value of the any retention time and the adjacent previous retention time thereof, and the second detector response value difference is the difference between the detector response value of the any retention time and the adjacent next retention time thereof; The average value of the first detector response value difference and the second detector response value difference is calculated as the difference between the detector response value of the any retention time and the adjacent retention time thereof.

[0011] In an exemplary embodiment, the obtaining process of the polarity includes: According to the influence degree of each retention time of the separated component, the weight of each retention time is obtained; Based on the weight of each retention time, the weighted sum of the difference between the detector response value of each retention time and the adjacent retention time thereof is obtained, and the polarity of the separated component is obtained.

[0012] In an exemplary embodiment, the obtaining process of the proportion of the organic solvent in the mobile phase required by the separated component includes: The interference degree and the polarity of the separated component are fused to obtain the adjustment coefficient of the separated component; the adjustment coefficient is positively related to the interference degree and the polarity; According to the adjustment coefficient, the proportion of the organic solvent in the initial mobile phase is positively adjusted to obtain the proportion of the organic solvent in the mobile phase required by the separated component.

[0013] In the second aspect of the present application, a multi-vitamin synchronous quantitative detection system is provided, including: a memory and a processor; the memory is connected with the processor; the memory is used for storing program instructions; the processor is used for implementing the above-mentioned multi-vitamin synchronous quantitative detection method when the program instructions are executed.

[0014] The present application has the following beneficial effects: the present application analyzes the chromatographic correlation characteristics of each separated component separated from the chromatogram of the sample to be tested, obtains the interference degree of other interference components on each separated component and the polarity of the water-soluble vitamin component in each separated component, and determines the proportion of the organic solvent in the mobile phase required by each separated component according to the two aspects of data information. Then, the proportion of the organic solvent in the mobile phase required by different separated components may be different. Compared with the existing way of using the same proportion of organic solvent in the mobile phase, by optimizing the proportion of the organic solvent in the mobile phase required by each separated component, the co-elution of water-soluble vitamins and other interference components can be reduced, the matrix effect can be reduced, the separation of water-soluble vitamins can be better, the separation effect of various vitamins can be improved, and the detection accuracy of multi-vitamins can be improved. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a chromatogram provided by one embodiment of the present application; Figure 2 is a flow chart of a multi-vitamin synchronous quantitative detection method provided by one embodiment of the present application; Figure 3 is a flow chart of obtaining chromatographic symmetry provided by one embodiment of the present application; Figure 4 is a flow chart of obtaining the peak distance of a separated component provided by one embodiment of the present application; Figure 5 is a flow chart of obtaining the difference between the detector response value of each retention time of a separated component and the detector response value of the adjacent retention time of the separated component provided by one embodiment of the present application; Figure 6 is a flow chart of obtaining the proportion of the organic solvent provided by one embodiment of the present application. DETAILED DESCRIPTION

[0016] In order to further illustrate the technical means and effects adopted by the present application to achieve the predetermined object of the present application, the specific embodiments, structures, features and effects of the present application are described in detail below in combination with the drawings and preferred embodiments. In the following description, different "one embodiment" or "another embodiment" do not necessarily refer to the same embodiment. In addition, the specific features, structures or characteristics in one or more embodiments can be combined in any suitable form.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The data information collected in the present application is obtained with the consent of the authorized person.

[0018] The embodiment provides a multi-vitamin synchronous quantitative detection method for detecting multiple vitamins in a sample to be detected. The sample to be detected is exemplified by food, such as food made of one or more fruits.

[0019] First, if the sample to be detected is solid, it needs to be prepared into a sample solution. In an exemplary embodiment, a protein precipitant or a filter can be used to remove macromolecular impurities in the sample to be detected. It should be understood that the embodiment is directed to the same object to be detected, and multiple samples to be detected are prepared to facilitate subsequent synchronous quantitative detection of multiple vitamins.

[0020] An initial mobile phase (a mixture of water and an organic solvent) is set, and the proportion of the organic solvent in the mobile phase is set as an initial organic solvent proportion, as an initial flow condition. Then, a C18 reversed-phase chromatographic column suitable for analysis is selected, and separation is performed in combination with the initial mobile phase. Then, by setting a suitable flow rate (such as 1.0 mL / min), a temperature (such as 35°C), and a detection wavelength (such as 280 nm), one of the samples to be detected is separated, and each compound in the sample to be detected will be distributed in the chromatographic column according to its physical and chemical properties, forming different retention times. The sample to be detected is preliminarily separated by high-performance liquid chromatography, and each separated component is obtained. In the chromatographic separation process, each separated component in the sample to be detected is monitored in real time by using an ultraviolet or other detector, and a chromatogram as shown in Figure 1 is generated. Figure 1 In the chromatogram, the abscissa represents the time axis, indicating the retention time (the unit can be minutes), and the ordinate represents the detector response value (that is, the signal intensity, such as absorbance, the unit is AU). According to the length of the retention time, that is, in the time sequence, each separated component corresponds to a peak, and each peak has a highest peak, that is, the largest detector response value in the peak.

[0021] As shown in Figure 2 , the multi-vitamin synchronous quantitative detection method provided by the embodiment includes the following steps: Step S1: obtaining the chromatographic symmetry of the separated components based on the peak symmetry of the chromatogram of the sample to be detected; Step S2: determining the peak distance of the adjacent separated separated components, and obtaining the interference degree of other interference components on the separated components in combination with the chromatographic symmetry; Step S3: obtaining the influence degree of the retention time of the separated components on the polarity determination of the separated components based on the retention time of the separated components and the difference between the retention time and the preset standard retention time; Step S4: determining the difference between the detector response value of the retention time of the separated components and the adjacent retention time, and obtaining the polarity of the water-soluble vitamin component in the separated components in combination with the influence degree; Step S5: determining the proportion of organic solvent in the mobile phase required for separating the components according to the degree of interference and polarity.

[0022] The various steps are described in detail below in conjunction with the accompanying drawings.

[0023] Step S1: obtaining the chromatographic symmetry of the separated components based on the peak symmetry of the chromatogram of the sample to be tested.

[0024] In high performance liquid chromatography, the symmetry of the wave peak (the value of the peak representing the ordinate, i.e. the detector response value) in the chromatogram can reflect the degree of matrix effect in the sample to be tested. For any separated component in the chromatogram, the wave peak in the ideal chromatogram should be symmetrical, indicating that the interaction between the separated component and the chromatographic column is normal and the separation effect is good. When the matrix effect exists, other interfering components (such as proteins, fats, etc.) may interact with the vitamin molecules, causing the elution behavior of these interfering components to change, resulting in tailing or fronting phenomenon, so that the wave peak in the chromatogram becomes asymmetrical. The asymmetry of the wave peak usually indicates that there may be interfering components in the separated component, which affects the analysis signal of the vitamin. Therefore, based on the peak symmetry of the chromatogram of the sample to be tested (i.e. the symmetry of the wave peak), the chromatographic symmetry of the separated components is obtained. The separated components are the components separated one by one in the chromatographic analysis of the sample to be tested by high performance liquid chromatography. It can be understood that the separated components represent various vitamin components in the sample to be tested, and the vitamin type of each separated component needs to be determined in mass spectrometric analysis.

[0025] In an exemplary embodiment, as shown in FIG. 1, a specific process for obtaining the chromatographic symmetry is as follows: Figure 3 Step S11: obtaining the symmetry difference of each retention time of the separated component.

[0026] Since each separated component is separated one by one, each separated component has different retention times, and each separated component has multiple retention times. The data processing process for each separated component is the same, and for the sake of convenience, an arbitrary separated component is taken as an example. Each retention time of the separated component forms a retention time range of the separated component in the chromatogram. Thus, the retention time range of each separated component in the chromatogram is determined. Different separated components have different retention time ranges in the chromatogram.

[0027] ​determining each retention time in the retention time range of the separated component. For any one retention time, a symmetric position of the retention time (i.e. the value of the horizontal coordinate in the chromatogram) in the retention time range is determined. The symmetric position is obtained in the following manner, for example: the symmetric position of the first retention time in the retention time range of the separated component is the last retention time in the retention time range, the symmetric position of the second retention time in the retention time range of the separated component is the second last retention time in the retention time range, and so on. The detector response value of the retention time is obtained, and the detector response value of the retention time at the symmetric position of the retention time is obtained.

[0028] Then, the difference between the detector response value of the retention time and the detector response value of the retention time at the symmetric position of the retention time is obtained as the symmetric difference of the retention time. The difference between the detector response values is specifically the absolute value of the difference between the detector response values. Thus, the symmetric difference of each retention time in the retention time range of the separated component is obtained.

[0029] Step S12: fusing the symmetric differences of each retention time in the retention time range of the separated component to obtain the chromatographic symmetry of the separated component.

[0030] From the above analysis, it can be seen that the smaller the symmetric difference of each retention time in the retention time range of the separated component, the more symmetrical the peak corresponding to each retention time, and the better the separation effect of the separated component. Therefore, the smaller the symmetric difference of each retention time in the retention time range of the separated component, the better the chromatographic symmetry of the separated component, and the chromatographic symmetry is inversely related to the symmetric difference. Then, the symmetric differences of each retention time in the retention time range of the separated component are fused to obtain the chromatographic symmetry of the separated component.

[0031] Based on the above logical analysis, a specific quantification method of the chromatographic symmetry is given as follows: ; wherein, represents the chromatographic symmetry of the i-th separated component, represents the number of retention times in the retention time range of the i-th separated component, and exp represents the exponential function with the natural constant as the base, represents the detector response value of the j-th retention time of the i-th separated component, represents the detector response value of the j-th retention time of the i-th separated component, and the symmetric position of the j-th retention time in the retention time range of the i-th separated component is the k-th retention time.

[0032] In the above manner, the chromatographic symmetry of each separated component is obtained. ​​

[0033] Step S2: determining the peak distance of adjacent separated-out separation components, combining the chromatographic symmetry, to obtain the interference degree of other interference components on the separation component.

[0034] On the basis of the appearance of chromatographic asymmetry of the separated-out separation component, and if the resolution is weak (i.e. the distance between the peaks of the two adjacent separated-out separation components is small), it usually means that the interference degree of other interference components on the vitamin component is large. The resolution reflects the separation effect between the chromatographic peaks, and when the resolution is weak, it means that the separation component and other interference components are not sufficiently separated. At this time, other interference components may overlap with the vitamin component, causing the shape of the peak in the chromatogram to become irregular, appearing tailing or front-sharping, which indicates the existence of interference components and affects the separation of the vitamin component. The weak resolution under asymmetry is usually an important indication of the influence of matrix effect on the analysis results of the vitamin.

[0035] First, the peak distance of adjacent separated-out separation components is determined, and in an exemplary embodiment, as shown in FIG. 2, a specific implementation process is given as follows: Figure 4 Step S21: determining the first peak distance and the second peak distance of the separation component.

[0036] Taking the i-th separation component as an example, the previous separation component adjacent to the i-th separation component, i.e. the i-1-th separation component in time sequence, is determined, and the next separation component adjacent to the i-th separation component, i.e. the i+1-th separation component in time sequence, is determined.

[0037] The position of the highest peak corresponding to the i-th separation component, i.e. the maximum detector response value in the peak of the i-th separation component, is obtained, i.e. the maximum detector response value in the peak of the i-th separation component, i.e. the position of the maximum detector response value in the chromatogram, i.e. the retention time. Similarly, the position of the highest peak corresponding to the i-1-th separation component is obtained, and the position of the highest peak corresponding to the i+1-th separation component is obtained.

[0038] The distance between the highest peak of the i-th separation component and the highest peak of the i-1-th separation component, i.e. the absolute value of the difference between the retention time corresponding to the highest peak of the i-th separation component and the retention time corresponding to the highest peak of the i-1-th separation component, i.e. the time interval between the two retention times, is defined as the first peak distance of the i-th separation component; similarly, the distance between the highest peak of the i-th separation component and the highest peak of the i+1-th separation component, i.e. the time interval between the retention time corresponding to the highest peak of the i-th separation component and the retention time corresponding to the highest peak of the i+1-th separation component, is defined as the second peak distance of the i-th separation component.

[0039] ​Step S22: Determine the minimum value of the first peak distance and the second peak distance of the first peak distance and the second peak distance of the separated component as the final peak distance of the separated component.

[0040] The minimum value of the first peak distance and the second peak distance of the ith separated component is obtained as the final peak distance of the ith separated component, that is, the peak distance between the ith separated component and the adjacent separated component.

[0041] The smaller the final peak distance of the ith separated component, the more insufficient the separation between the ith separated component and other interfering components, which means that the other interfering components have greater interference on the vitamin component, and the higher the interference degree of the other interfering components on the ith separated component. Therefore, there is an inverse correlation between the final peak distance of the ith separated component and the interference degree of the other interfering components on the ith separated component. The above process is used to obtain the final peak distance of each separated component.

[0042] Since the worse the chromatographic symmetry of the ith separated component, the higher the interference degree of the other interfering components on the ith separated component, and the two are inversely related. Therefore, the interference degree of the ith separated component is obtained according to the final peak distance of the ith separated component and the chromatographic symmetry. Based on the above logic analysis, a specific quantification method of the interference degree of the ith separated component is given as follows: ; Wherein, represents the interference degree of the ith separated component, that is, the interference degree of the other interfering components on the ith separated component, min represents the minimum value function, represents the first peak distance of the ith separated component, that is, the distance between the highest peak corresponding to the ith separated component and the highest peak corresponding to the ith-1 separated component, represents the second peak distance of the ith separated component, that is, the distance between the highest peak corresponding to the ith separated component and the highest peak corresponding to the ith+1 separated component.

[0043] f represents the maximum minimum value normalization function, and the implementation process is as follows: obtain the calculation results in the brackets in the above interference degree calculation formula of each separated component, find the maximum value and the minimum value from the calculation results, and then normalize the calculation results in the brackets of each separated component by using the maximum minimum value normalization method.

[0044] By using the above method, the interference degree of each separated component by the other interfering components is obtained.

[0045] Step S3: Based on the retention time of each separated component and the difference between the retention time and the preset standard retention time, the influence degree of each retention time of the separated component on the polarity determination of the separated component is obtained.

[0046] In high performance liquid chromatography, the polarity of water-soluble vitamin components is usually closely related to their retention time. For water-soluble vitamins, the higher the polarity, the shorter the retention time in the chromatogram. On this basis, by analyzing the chromatogram of each separated component, if the retention time of a certain separated component is short and close to the retention time of the standard vitamin under the flow condition, it indicates that the separated component has similar polarity characteristics, and its elution behavior is consistent with that of the standard vitamin. At this time, the retention time of the separated component has a higher matching degree with the standard, which means that the separated component is more likely to be the target water-soluble vitamin, so the separated component has a higher degree of influence on the determination of polarity and can be given a greater weight.

[0047] First, the retention time of each separated component and the difference between the retention time and the preset standard retention time are determined. Take the ith separated component as an example, and take the jth retention time of the ith separated component as an example.

[0048] In this embodiment, the standard retention time of various vitamins under the initial mobile phase is obtained in advance. The standard retention time of various vitamins under the initial mobile phase can be obtained by experimental means in the laboratory, and is used as the preset standard retention time of various vitamins. In an exemplary embodiment, in the laboratory, a standard sample is prepared, the standard sample includes a plurality of known vitamins, the initial mobile phase in this embodiment is used, high performance liquid chromatography separation and detection is performed, and the retention time of the plurality of known vitamins is obtained as the standard retention time. Further, this embodiment can also perform multiple experiments, and the average value of the retention time of a certain vitamin obtained in all experiments is used as the standard retention time of the vitamin.

[0049] The difference between the jth retention time of the ith separated component and the preset standard retention time of each vitamin is obtained, where the difference is specifically the time interval between the two retention times, and the smaller the difference, the closer the jth retention time is to the preset standard retention time of a certain vitamin, and the higher the matching degree with the vitamin. Then, the smallest difference is determined from the differences between the jth retention time of the ith separated component and the preset standard retention time of each vitamin, and the smallest difference is taken as the difference between the jth retention time of the ith separated component and the preset standard retention time. The smaller the difference between the jth retention time of the ith separated component and the preset standard retention time, the more likely the ith separated component is the target water-soluble vitamin, and therefore the jth retention time of the ith separated component has a higher degree of influence on the polarity determination. Therefore, the difference between the retention time and the preset standard retention time is inversely related to the degree of influence. In the above manner, the differences between the preset standard retention time and each retention time of the ith separated component are obtained. It should be understood that the preset standard retention times of different retention times may correspond to different types of vitamins, i.e., the preset standard retention times of different retention times may be different.

[0050] Since water-soluble vitamins generally have a shorter retention time in the chromatogram, the smaller the jth retention time of the ith separated component, the higher the degree of influence of the jth retention time of the ith separated component on the polarity determination, and the two are inversely related.

[0051] Therefore, according to the difference between the jth retention time of the ith separated component and the preset standard retention time, and the jth retention time, the degree of influence of the jth retention time of the ith separated component on the polarity determination is obtained. Based on the above logical analysis, one specific quantification method of the degree of influence is given as follows: ; Wherein, represents the degree of influence of the jth retention time of the ith separated component on the polarity determination, represents the time corresponding to the jth retention time of the ith separated component (i.e., the horizontal coordinate value), represents the difference between the jth retention time of the ith separated component and the preset standard retention time.

[0052] In the above process, the degree of influence of each retention time of the ith separated component on the polarity determination is obtained, and thus the degree of influence of each retention time of each separated component on the polarity determination is obtained.

[0053] Step S4: Determine the difference between the retention time of the separated component and the detector response value of its adjacent retention time, and combine the degree of influence to obtain the polarity of the water-soluble vitamin component in the separated component.

[0054] The more polar separated components interact more strongly with the stationary phase on the column and therefore show more pronounced peak shape differences in the chromatogram. In particular, at shorter retention times, the more polar separated components will have increased interaction with the mobile phase as they pass through the column, resulting in a larger difference in detector response, which is manifested as a difference in detector response. An increase in the difference in detector response indicates that the separated component interacts more strongly with the stationary phase and elutes more quickly.

[0055] In HPLC, water-soluble vitamins are typically more polar, which makes them elute more quickly, i.e. have a shorter retention time, in the column. When analyzing each separated component, the greater the difference between the detector response at the retention time and the detector responses on either side of it, typically means that the water-soluble vitamin component of the separated component is more polar. Therefore, first the difference in detector response between each retention time of a separated component and its adjacent retention times is determined, in one exemplary embodiment, as shown in FIG. 1, a specific procedure is given as follows: Figure 5 Step S41: determining the first detector response difference and the second detector response difference of any retention time of the separated component.

[0056] Taking the jth retention time of the ith separated component as an example, the adjacent previous retention time of the jth retention time of the ith separated component is the (j-1)th retention time of the ith separated component, and the adjacent next retention time of the jth retention time of the ith separated component is the (j+1)th retention time of the ith separated component. The detector response value corresponding to the jth retention time of the ith separated component (i.e. the ordinate value in the chromatogram) is obtained, as well as the detector response value corresponding to the (j-1)th retention time of the ith separated component and the detector response value corresponding to the (j+1)th retention time of the ith separated component.

[0057] The difference between the detector response value corresponding to the jth retention time of the ith separated component and the detector response value corresponding to the (j-1)th retention time of the ith separated component is calculated, specifically the absolute value of the difference between the two detector response values, and the absolute value of the difference between the detector response values obtained is taken as the first detector response difference of the jth retention time of the ith separated component.

[0058] The difference between the detector response value corresponding to the jth retention time of the ith separated component and the detector response value corresponding to the (j+1)th retention time of the ith separated component is calculated, specifically the absolute value of the difference between the two detector response values, and the absolute value of the difference between the detector response values obtained is taken as the second detector response difference of the jth retention time of the ith separated component.

[0059] ​Step S42: Calculate the average of the first detector response value difference and the second detector response value difference as the difference of the detector response values of any retention time and its adjacent retention time.

[0060] Calculate the average of the first detector response value difference and the second detector response value difference of the jth retention time of the ith separated component as the difference of the detector response values of the jth retention time of the ith separated component and its adjacent retention time. As known from the above, the greater the difference of the detector response values of the jth retention time of the ith separated component and its adjacent retention time, the stronger the polarity of the water-soluble vitamin component in the ith separated component.

[0061] By the above process, the difference of the detector response values of each retention time of the ith separated component and its adjacent retention time is obtained.

[0062] Then, the weight of each retention time of the ith separated component is obtained according to the influence degree of each retention time of the ith separated component. In an exemplary embodiment, the sum value of the influence degrees of all the retention times of the ith separated component is calculated as the overall influence degree of the ith separated component, and then the ratio of the influence degree of each retention time of the ith separated component to the overall influence degree is calculated to obtain the weight of each retention time of the ith separated component. In this way, the weight of each retention time of the ith separated component ranges from 0 to 1, and the sum of the weights of all the retention times of the ith separated component is 1.

[0063] Finally, based on the weight of each retention time of the ith separated component, the difference of the detector response values of each retention time of the ith separated component and its adjacent retention time is weighted and summed to obtain the polarity of the water-soluble vitamin component in the ith separated component, and the calculation formula is as follows: ; Wherein, Polarity of the water-soluble vitamin component in the ith separated component, Difference of the detector response values of the jth retention time of the ith separated component and its adjacent retention time, Weight of the jth retention time of the ith separated component. Here, f also represents the maximum-minimum normalization function, and the implementation process is as follows: obtain the calculation results in the brackets in the above polarity calculation formula of each separated component, find the maximum value and the minimum value therefrom, and then normalize the calculation results in the brackets of each separated component by the maximum-minimum normalization method.

[0064] By the above process, the polarity of the water-soluble vitamin component in each separated component is obtained.

[0065] Step S5: determining the proportion of organic solvent in the mobile phase required for separating the component according to the degree of interference and the polarity.

[0066] The interference of non-target components (i.e. interfering components such as proteins, fats, etc.) in the separating component to the target analyte (i.e. water-soluble vitamins) can cause errors in the mass spectrometry results. In high-performance liquid chromatography, water-soluble vitamins usually have high polarity and strong interaction with the stationary phase during chromatographic separation. Therefore, when the degree of interference of the separating component is large, other interfering components can interact with the water-soluble vitamins, causing chromatographic peak asymmetry or tailing of the interfering components, thereby affecting the accuracy of the mass spectrometry signal.

[0067] In this case, if the polarity of the water-soluble vitamin component in the separating component is high, the proportion of organic solvent in the mobile phase can be increased to reduce the interaction between the water-soluble vitamin and the stationary phase and the influence of the matrix component. Since the water-soluble vitamin has high polarity, appropriately increasing the proportion of organic solvent helps to strengthen its affinity with the mobile phase, making it elute faster and effectively reducing the interference of matrix effects on the separation results and mass spectrometry.

[0068] Therefore, according to the polarity of the water-soluble vitamin component in the i-th separating component and the degree of interference of other interfering components to the i-th separating component, the proportion of organic solvent in the mobile phase required for the i-th separating component is determined. The stronger the polarity, the higher the proportion of organic solvent in the mobile phase required, and the stronger the degree of interference, the higher the proportion of organic solvent in the mobile phase required. In an exemplary embodiment, as shown in FIG. 2, a specific process for obtaining the proportion of organic solvent is given as follows: Figure 6 Step S51: fusing the degree of interference and the polarity of the separating component to obtain an adjustment coefficient of the separating component.

[0069] According to the polarity of the water-soluble vitamin component in the i-th separating component and the degree of interference of other interfering components to the i-th separating component, the adjustment coefficient of the i-th separating component is obtained, wherein the adjustment coefficient is positively correlated with both the degree of interference and the polarity. In an exemplary embodiment, the product of the polarity of the water-soluble vitamin component in the i-th separating component and the degree of interference of other interfering components to the i-th separating component is taken as the adjustment coefficient of the i-th separating component.

[0070] Step S52: positively adjusting the proportion of organic solvent in the initial mobile phase according to the adjustment coefficient to obtain the proportion of organic solvent in the mobile phase required for separating the component.

[0071] ​The embodiment requires positive adjustment of the proportion of organic solvent in the initial mobile phase according to the adjustment coefficient, that is, the larger the adjustment coefficient, the greater the proportion of organic solvent needs to be increased. In an exemplary embodiment, a specific adjustment method is given as follows: ; wherein, represents the proportion of organic solvent in the adjusted mobile phase of the ith separated component, that is, the proportion of organic solvent in the mobile phase required by the ith separated component, represents the proportion of organic solvent in the initial mobile phase. In this way, the proportion of organic solvent in the mobile phase required by each separated component can be obtained.

[0072] It should be understood that the proportion of organic solvent in the mobile phase required by each separated component may be different, with high and low values. Then, in an exemplary embodiment, a specific application is provided: the obtained proportion of organic solvent in the mobile phase required by each separated component is sorted in order from small to large, obtaining an organic solvent proportion sequence. And re-detecting other samples to be tested using high performance liquid chromatography, during the separation of the sample to be tested using high performance liquid chromatography, starting from the lowest proportion of organic solvent in the organic solvent proportion sequence, gradually adding organic solvent according to the preset adjustment program, the essence is to use the control strategy of gradient elution, so that the proportion of organic solvent increases according to the organic solvent proportion sequence, thereby realizing the separation of various vitamin components. This embodiment can also be continuously optimized and adjusted to adjust the elution behavior of water-soluble vitamins and other components, reducing their co-elution on the chromatographic column. Through this optimization, the interference of the matrix effect can be effectively reduced, ensuring that the separation between water-soluble vitamins and interfering components is clearer, thereby improving the purity of the sample. Ultimately, such adjustment will help improve the detection effect of mass spectrometry, making the signal of water-soluble vitamins more accurate and reliable. In high performance liquid chromatography, the proportion of organic solvent in the mobile phase is continuously optimized through the above process, effectively reducing the co-elution of water-soluble vitamins and other interfering components, thereby obtaining purer water-soluble vitamin components. This optimization process ensures that the detection results of water-soluble vitamins in each separated component are more accurate, and significantly reduces the influence of other interfering components on vitamin quantitative analysis.

[0073] After the more accurate separated components are obtained, the multi-vitamin is synchronously quantitatively detected by using mass spectrometry. First, each separated component is directly introduced into a mass spectrometer for further analysis. The mass spectrometer generates a mass-to-charge ratio (m / z) spectrum of each vitamin by detecting ionized molecular fragments. In combination with the retention time information in the chromatogram, the mass spectrometry can accurately identify and quantify each vitamin. In addition, multiple reaction monitoring and other technologies can be used to synchronously quantitatively detect multiple vitamins, thereby improving the throughput and accuracy of detection.

[0074] The embodiment also provides a multi-vitamin synchronous quantitative detection system, comprising a memory and a processor; the memory is connected with the processor, and is used for storing program instructions; the processor is used for implementing the steps in the multi-vitamin synchronous quantitative detection method embodiment when the program instructions are executed.

[0075] In one exemplary embodiment, the present application provides a computer readable storage medium storing a computer program, which is executed by a processor to implement the steps in the multi-vitamin synchronous quantitative detection method embodiment.

[0076] It should be noted that the above-mentioned sequence of the embodiments of the present application is only for description, and does not represent the advantages and disadvantages of the embodiments. The processes depicted in the drawings do not necessarily require the specific order or continuous order shown to achieve the desired results. In some embodiments, multitasking and parallel processing are possible or can be advantageous.

[0077] Each of the embodiments in the specification is described in a progressive manner, and the same or similar parts between the embodiments can be referred to each other. Each embodiment mainly describes the difference from other embodiments.

Claims

1. A method for simultaneous quantitative determination of multivitamins, characterized in that, The method comprises the following steps: Based on the peak symmetry of the chromatogram of the sample to be tested, the chromatographic symmetry of the separated component is obtained; The peak distance of the adjacent separated component is determined, and the interference degree of other interfering components on the separated component is obtained in combination with the chromatographic symmetry; Based on the retention time of the separated component and the difference between the retention time and the preset standard retention time, the influence degree of the retention time of the separated component on the polarity determination of the separated component is obtained; The difference between the detector response value of the retention time of the separated component and the adjacent retention time is determined, and the polarity of the water-soluble vitamin component in the separated component is obtained in combination with the influence degree; According to the interference degree and the polarity, the proportion of the organic solvent in the mobile phase required by the separated component is determined.

2. The method for simultaneous quantitative determination of vitamins according to claim 1, characterized in that, The process of obtaining the chromatographic symmetry comprises: Obtain the symmetry difference of each retention time of the separated component; the symmetry difference is the difference between the detector response value of the retention time and the symmetric position in the retention time range; Fuse the symmetry difference of each retention time in the retention time range of the separated component to obtain the chromatographic symmetry; the chromatographic symmetry is inversely related to the symmetry difference.

3. The method for simultaneous quantitative determination of vitamins according to claim 1, characterized in that, The determination of the peak distance of the adjacent separated component comprises: Determine the first peak distance and the second peak distance of the separated component; the first peak distance is the distance between the highest peak of the separated component and the highest peak of the adjacent previous separated component, and the second peak distance is the distance between the highest peak of the separated component and the highest peak of the adjacent next separated component; Determine the minimum value of the first peak distance and the second peak distance as the final peak distance of the separated component.

4. The method for simultaneous quantitative detection of multiple vitamins as described in claim 3, characterized in that, The process of obtaining the interference degree comprises: Obtain the interference degree of the separated component from the final peak distance of the separated component and the chromatographic symmetry; the interference degree is inversely related to the final peak distance and the chromatographic symmetry.

5. The method for simultaneous quantitative determination of vitamins according to claim 1, characterized in that, The process of obtaining the difference between the retention time of the separated component and the preset standard retention time comprises: Obtain the difference between any retention time of the separated component and the preset standard retention time of various vitamins, and determine the minimum difference as the difference between the any retention time and the preset standard retention time.

6. The method for simultaneous quantitative determination of vitamins according to claim 5, characterized in that, The process of obtaining the influence degree comprises: According to the difference between the any retention time and the preset standard retention time, and the any retention time, the influence degree corresponding to the any retention time is obtained; the influence degree corresponding to the any retention time is inversely related to the difference between the any retention time and the preset standard retention time, and the any retention time.

7. The method for simultaneous quantitative detection of multiple vitamins as described in claim 1, characterized in that, The determination of the difference between the detector response value of the retention time of the separated component and the adjacent retention time comprises: Determine the first detector response value difference and the second detector response value difference of any retention time of the separated component; the first detector response value difference is the difference between the detector response value of the any retention time and the adjacent previous retention time, and the second detector response value difference is the difference between the detector response value of the any retention time and the adjacent next retention time; The average of the first detector response value difference and the second detector response value difference is calculated as the difference of the detector response values of any retention time and its adjacent retention time.

8. The method for simultaneous quantitative determination of vitamins according to claim 1, characterized in that, The obtaining process of the polarity comprises: obtaining the weight of each retention time according to the influence degree of each retention time of the separated component; weighting and summing the difference of the detector response values of each retention time and its adjacent retention time based on the weight of each retention time to obtain the polarity of the separated component.

9. The method for simultaneous quantitative determination of vitamins according to claim 1, characterized in that, The obtaining process of the required proportion of the organic solvent in the mobile phase of the separated component comprises: fusing the interference degree and the polarity of the separated component to obtain an adjustment coefficient of the separated component; the adjustment coefficient is positively correlated with the interference degree and the polarity; positively adjusting the proportion of the organic solvent in the initial mobile phase according to the adjustment coefficient to obtain the required proportion of the organic solvent in the mobile phase of the separated component.

10. A multi-vitamin simultaneous quantitative detection system, comprising: a memory and a processor; the memory is connected with the processor; the memory is used for storing program instructions; the processor is used for realizing the multi-dimensional vitamin synchronous quantitative detection method in any one of claims 1-9 when the program instructions are executed.

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