A method and system for simultaneous quantitative detection of multiple vitamins

Abstract

The present application relates to the technical field of column chromatography detection, in particular to a multi-vitamin synchronous quantitative detection method and system, which comprises the following steps: determining the chromatographic symmetry of the separated components of the sample to be detected, combining the peak distance of the adjacent separated components to obtain the interference degree of the interference components on the separated components; based on the difference between the retention time of the separated components and the preset standard retention time, obtaining the influence degree of the retention time on the polarity determination of the separated components, combining the difference between the detector response value of each retention time and its adjacent retention time to obtain the polarity of the water-soluble vitamin component in the separated components, and comprehensively determining the proportion of the organic solvent in the mobile phase required by the separated components by comprehensively considering the interference degree and the polarity, which can reduce the co-elution of water-soluble vitamins and other interference components, thereby reducing the matrix effect, helping to better separate the water-soluble vitamins, improving the separation effect of various vitamins, and further improving the multi-vitamin detection accuracy.

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 synchronous quantitative detection of multi-vitamins in the sample to be tested, the content of each vitamin contained therein can be accurately evaluated, which is of great significance. The sample to be tested is usually various foods.

[0003] The existing method usually uses high performance liquid chromatography tandem mass spectrometry to perform synchronous quantitative detection of multi-vitamins. The principle is to separate the multi-vitamins in the sample to be tested by high performance liquid chromatography (HPLC). In the chromatographic column, the sample to be tested 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 separated by chromatography enters mass spectrometry. Mass spectrometry converts the separated components separated by ionization technology into charged particles, 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 perform quantitative analysis by comparison with the standard. In separating the multi-vitamins, the same proportion of organic solvent is used in the mobile phase. However, in the prior art, multi-vitamins are usually derived 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 multi-vitamins using the same proportion of organic solvent in the mobile phase, the separation effect of vitamins is affected, thereby affecting the detection accuracy of multi-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 multi-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:

[0005] In the first aspect of the present application, a multi-vitamin synchronous quantitative detection method is provided, comprising:

[0006] obtaining a chromatographic symmetry of the separation component based on a peak symmetry of a chromatogram of the sample to be tested;

[0007] determining a peak distance of an adjacent separation component, and combining the chromatographic symmetry to obtain an interference degree of other interference components on the separation component;

[0008] obtaining an influence degree of each retention time of the separation component on the polarity determination of the separation component based on the each retention time of the separation component and a difference between the each retention time and a preset standard retention time;

[0009] determining a difference between the each retention time of the separation component and a detector response value of an adjacent retention time of the separation component, and combining the influence degree to obtain a polarity of a water-soluble vitamin component in the separation component;

[0010] determining a proportion of an organic solvent in a mobile phase required by the separation component according to the interference degree and the polarity.

[0011] In an exemplary embodiment, the process of obtaining the chromatographic symmetry comprises:

[0012] obtaining a symmetry difference of each retention time of the separation component; the symmetry difference is a difference between the retention time and a detector response value of a symmetric position of the retention time in a retention time range;

[0013] fusing the symmetry difference of each retention time in the retention time range of the separation component to obtain the chromatographic symmetry; the chromatographic symmetry is inversely related to the symmetry difference.

[0014] In an exemplary embodiment, the process of determining the peak distance of the adjacent separation component comprises:

[0015] determining a first peak distance and a second peak distance of the separation component; the first peak distance is a distance between a highest peak of the separation component and a highest peak of an adjacent previous separation component, and the second peak distance is a distance between a highest peak of the separation component and a highest peak of an adjacent next separation component;

[0016] determining a minimum value of the first peak distance and the second peak distance as a final peak distance of the separation component.

[0017] In an exemplary embodiment, the process of obtaining the interference degree comprises:

[0018] obtaining the interference degree of the separation component from the final peak distance of the separation component and the chromatographic symmetry; the interference degree is inversely related to both the final peak distance and the chromatographic symmetry.

[0019] 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:

[0020] obtaining a difference between any retention time of the separated component and a preset standard retention time of the vitamin, and determining a minimum difference as the difference between the any retention time and the preset standard retention time.

[0021] In an exemplary embodiment, the obtaining process of the influence degree comprises:

[0022] obtaining the influence degree corresponding to the any retention time 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 inversely related to both the difference between the any retention time and the preset standard retention time and the any retention time.

[0023] In an exemplary embodiment, the determining of the difference between the detector response value of each retention time of the separated component and the detector response value of its adjacent retention time comprises:

[0024] determining a first detector response value difference and a second detector response value difference of any retention time of the separated component; the first detector response value difference is a difference between the detector response value of the any retention time and the detector response value of its adjacent previous retention time, and the second detector response value difference is a difference between the detector response value of the any retention time and the detector response value of its adjacent next retention time;

[0025] calculating an average value of the first detector response value difference and the second detector response value difference as the difference between the detector response value of the any retention time and the detector response value of its adjacent retention time.

[0026] In an exemplary embodiment, the obtaining process of the polarity comprises:

[0027] obtaining a weight of each retention time according to the influence degree of each retention time of the separated component;

[0028] performing weighted summation on the difference between the detector response value of each retention time and the detector response value of its adjacent retention time based on the weight of each retention time to obtain the polarity of the separated component.

[0029] In an exemplary embodiment, the obtaining process of the proportion of the organic solvent in the mobile phase required by the separated component comprises:

[0030] 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 related to both the interference degree and the polarity.

[0031] 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.

[0032] In a second aspect of the present application, a multi-vitamin synchronous quantitative detection system is provided, comprising: a memory and a processor; the memory is connected with the processor; the memory is used for storing program instructions; and the processor is used for implementing the multi-vitamin synchronous quantitative detection method when the program instructions are executed.

[0033] The present application has the following beneficial effects: by analyzing the chromatographic related characteristics of each separated component separated from the chromatogram of the sample to be measured, the interference degree of other interference components on each separated component and the polarity of the water-soluble vitamin component in each separated component are obtained, and then the proportion of the organic solvent in the mobile phase required by each separated component is determined according to the two aspects of data information. Therefore, the proportion of the organic solvent in the mobile phase required by different separated components may be different. Compared with the existing method 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

[0034] Figure 1 is a chromatogram provided by an embodiment of the present application;

[0035] Figure 2 is a flow chart of a multi-vitamin synchronous quantitative detection method provided by an embodiment of the present application;

[0036] Figure 3 is a flow chart for obtaining chromatographic symmetry provided by an embodiment of the present application;

[0037] Figure 4 is a flow chart for obtaining the peak distance of a separated component provided by an embodiment of the present application;

[0038] Figure 5 is a flow chart for 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 provided by an embodiment of the present application;

[0039] Figure 6 is a flow chart for obtaining the proportion of the organic solvent provided by an embodiment of the present application. DETAILED DESCRIPTION

[0040] In order to further clarify the technical means and effects taken by the present application to achieve the predetermined inventive objectives, the specific embodiments, structures, features and effects of the present application are described in detail below in conjunction 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.

[0041] 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 this application belongs. The data information collected in this application is obtained with the full consent of the authorization.

[0042] The present embodiment provides a multi-vitamin synchronous quantitative detection method for detecting multiple vitamins in a sample to be tested. The sample to be tested is taken as an example of food, such as food made of one or more fruits.

[0043] First, if the sample to be tested 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 tested. It should be understood that for the same object to be tested, multiple samples to be tested are prepared to facilitate subsequent multi-vitamin synchronous quantitative detection.

[0044] An initial mobile phase (a mixture of water and organic solvent) is set, and the proportion of the organic solvent in the mobile phase is set as the initial organic solvent proportion as the initial flow condition. Then, a C18 reversed-phase chromatographic column suitable for analysis is selected, and combined with the initial mobile phase for separation. By setting appropriate flow rate (such as 1.0 mL / min), temperature (such as 35°C) and detection wavelength (such as 280 nm), one of the samples to be tested is separated, and each compound in the sample to be tested will be distributed in the chromatographic column according to its physical and chemical properties, forming different retention times. The sample to be tested is preliminarily separated by high performance liquid chromatography to obtain each separated component. In the process of chromatographic separation, the detector is used to monitor each separated component in the sample to be tested in real time, and a chromatogram as shown in Figure 1 is generated. Figure 1 In the chromatogram, the abscissa is the time axis, representing the retention time (the unit can be minutes), and the ordinate is the detector response value (i.e. signal intensity, such as absorbance, unit AU). According to the length of the retention time, i.e. in time sequence, each separated component corresponds to a peak, and each peak has a highest peak, i.e. the largest detector response value in the peak.

[0045] As shown in Figure 2 , the multi-vitamin synchronous quantitative detection method provided by the present embodiment includes the following steps:

[0046] Step S1: obtaining the chromatographic symmetry of the separated component based on the peak symmetry of the chromatogram of the sample to be tested;

[0047] Step S2: determining the peak distance of the adjacent separated components, and obtaining the interference degree of the other interfering components on the separated component in combination with the chromatographic symmetry;

[0048] Step S3: obtaining the influence degree of the retention time of the separated component on the polarity determination of the separated component based on the retention time of the separated component and the difference between the retention time and the preset standard retention time;

[0049] Step S4: determining the difference between the detector response value of the retention time of the separated component and the adjacent retention time, and obtaining the polarity of the water-soluble vitamin component in the separated component in combination with the influence degree;

[0050] Step S5: determining the proportion of the organic solvent in the mobile phase required by the separated component according to the interference degree and the polarity.

[0051] The specific steps are described below in combination with the drawings.

[0052] Step S1: obtaining the chromatographic symmetry of the separated component based on the peak symmetry of the chromatogram of the sample to be tested.

[0053] In high performance liquid chromatography, the symmetry of the wave peak (the peak represents the value of the ordinate, that is, 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 front sharp 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 component is obtained. The separated component is each component separated in the chromatographic analysis of the sample to be tested by high performance liquid chromatography. It can be understood as a vitamin component, that is, each separated component represents 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.

[0054] In an exemplary embodiment, as shown in Figure 3 A specific acquisition process of the chromatographic symmetry is given as follows:

[0055] Step S11: obtaining the symmetric difference of the retention time of the separated component.

[0056] Since each separated component is separated out in sequence, each separated component has different retention time, and each separated component has multiple retention times. The data processing process of each separated component is the same, and for the convenience of description, the following is an example of any one separated component. Each retention time of the separated component constitutes the retention time range of the separated component in the chromatogram. Thus, the retention time range of each separated component in the chromatogram is determined. The retention time ranges of different separated components in the chromatogram are different.

[0057] Each retention time in the retention time range of the separated component is determined. For any one retention time, the position of the retention time (i.e. the value of the horizontal coordinate in the chromatogram) is determined at the symmetrical position in the retention time range. The symmetrical position is obtained in the following manner, for example: the symmetrical 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 symmetrical 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, as well as the detector response value of the retention time at the symmetrical position of the retention time.

[0058] Then, the difference between the detector response value of the retention time and the detector response value of the retention time at the symmetrical position of the retention time is obtained as the symmetry difference of the retention time. The difference between the detector response values is the absolute value of the difference between the detector response values. Thus, the symmetry difference of each retention time in the retention time range of the separated component is obtained.

[0059] Step S12: fuse the symmetry differences of each retention time in the retention time range of the separated component to obtain the chromatographic symmetry.

[0060] From the above analysis, the smaller the symmetry 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 symmetry 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 symmetry difference. Then, the symmetry difference of each retention time in the retention time range of the separated component is fused to obtain the chromatographic symmetry of the separated component.

[0061] Based on the above logical analysis, a specific quantification method of chromatographic symmetry is given as follows:

[0062] ;

[0063] wherein, chromatographic symmetry of the i-th separated component, represents the number of retention times within the retention time range of the i-th separated component, 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, represents the detector response value of the j-th retention time of the i-th separated component, represents the j-th retention time within the retention time range of the i-th separated component.

[0064] In this way, the chromatographic symmetry of each separated component is obtained.

[0065] Step S2: determining the peak distance of the adjacent separated components, and combining the chromatographic symmetry to obtain the interference degree of the other interfering components on the separated component.

[0066] On the basis of the chromatographic asymmetry of the separated components, and if the resolution is weak (i.e., the distance between the peaks of the two adjacent separated components is small), it usually means that the interference degree of the other interfering 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 separated components and the other interfering components are not sufficiently separated. At this time, the other interfering components may overlap with the vitamin components, causing the shape of the peak in the chromatogram to become irregular, appearing tailing or front-sharp phenomenon, which indicates the existence of interfering components and affects the separation of the vitamin components. The weak resolution under asymmetry is usually an important indication of the influence of matrix effect on the analysis results of the vitamin.

[0067] First, the peak distance of the adjacent separated components is determined, and in an exemplary embodiment, as shown in FIG. 2, a specific implementation process is given as follows: Figure 4

[0068] Step S21: determining the first peak distance and the second peak distance of the separated component.

[0069] Taking the i-th separated component as an example, the i-1-th separated component adjacent to the i-th separated component in time sequence is determined, and the i+1-th separated component adjacent to the i-th separated component in time sequence is determined.

[0070] The position of the highest peak corresponding to the i-th separated component is obtained, i.e., the abscissa value corresponding to the maximum detector response value in the peak of the i-th separated component, i.e., the abscissa 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 separated component is obtained, and the position of the highest peak corresponding to the i+1-th separated component is obtained.

[0071] ​The distance between the highest peak of the i-th separated component and the highest peak of the i-1-th separated component, i.e. the absolute value of the difference between the retention time corresponding to the highest peak of the i-th separated component and the retention time corresponding to the highest peak of the i-1-th separated component, is defined as the first peak distance of the i-th separated component; similarly, the distance between the highest peak of the i-th separated component and the highest peak of the i+1-th separated component, i.e. the time interval between the retention time corresponding to the highest peak of the i-th separated component and the retention time corresponding to the highest peak of the i+1-th separated component, is defined as the second peak distance of the i-th separated component.

[0072] Step S22: determining the minimum value in the first peak distance and the second peak distance as the final peak distance of the separated component.

[0073] The minimum value in the first peak distance and the second peak distance of the i-th separated component is obtained as the final peak distance of the i-th separated component, i.e. the peak distance between the i-th separated component and the adjacent separated component.

[0074] The smaller the final peak distance of the i-th separated component, the more insufficient the separation between the i-th separated component and other interfering components, which means that the other interfering components have greater interference on the vitamin component, and thus the other interfering components have a higher degree of interference on the i-th separated component. Therefore, there is an inverse correlation between the final peak distance of the i-th separated component and the degree of interference of the other interfering components on the i-th separated component. By using the above process, the final peak distance of each separated component is obtained.

[0075] Since the worse the chromatographic symmetry of the i-th separated component, the higher the degree of interference of the other interfering components on the i-th separated component, and the two are inversely related, the degree of interference of the i-th separated component is obtained according to the final peak distance of the i-th separated component and the chromatographic symmetry. Based on the above logical analysis, one specific quantification method of the degree of interference of the i-th separated component is given as follows:

[0076] ;

[0077] wherein, represents the degree of interference of the i-th separated component, i.e. the degree of interference of the other interfering components on the i-th separated component, min represents the minimum value function, represents the first peak distance of the i-th separated component, i.e. the distance between the highest peak corresponding to the i-th separated component and the highest peak corresponding to the i-1-th separated component, represents the second peak distance of the i-th separated component, i.e. the distance between the highest peak corresponding to the i-th separated component and the highest peak corresponding to the i+1-th separated component.

[0078] f represents a max-min normalization function, and the implementation process is as follows: the calculation results in the brackets in the above interference degree calculation formula of each separated component are obtained, the maximum value and the minimum value are found therefrom, and then the calculation results in the brackets of each separated component are normalized in a max-min normalization manner.

[0079] In the above manner, the interference degree of other interference components on each separated component is obtained.

[0080] Step S3: Based on 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 of the separated component on the polarity determination of the separated component is obtained.

[0081] 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 polarity is relatively high, and they usually have a shorter 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 shorter 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 matching degree of the retention time of the separated component with the standard is higher, which means that the separated component is more likely to be the target water-soluble vitamin, and therefore the influence degree of the separated component on the polarity determination is higher, and a greater weight can be given.

[0082] First, the retention time of each separated component and the difference between the retention time and the preset standard retention time are determined. Taking the i-th separated component as an example, taking the j-th retention time of the i-th separated component as an example.

[0083] 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, and high performance liquid chromatography separation and detection is performed, so as to obtain the retention time of the plurality of known vitamins 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.

[0084] 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.

[0085] 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.

[0086] 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 as follows:

[0087] ;

[0088] 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.

[0089] 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.

[0090] Step S4: determining the difference between the detector response value of each retention time of the separated component and its adjacent retention time, combining the influence degree, to obtain the polarity of the water-soluble vitamin component in the separated component.

[0091] The separated component with higher polarity has stronger interaction with the stationary phase on the chromatographic column, and thus presents more significant peak shape difference in the chromatogram. Especially when the retention time is in a shorter position, the separated component with stronger polarity will increase the interaction with the mobile phase through the chromatographic column, resulting in a larger difference in the detector response value, which is manifested as the detector response value difference. The increase in the detector response value difference indicates that the interaction between the separated component and the stationary phase is stronger, and the elution time is shorter.

[0092] In high performance liquid chromatography, water-soluble vitamins usually have higher polarity, which makes their elution time shorter and retention time shorter in the chromatographic column. When analyzing each separated component, the greater the difference between the detector response value corresponding to the retention time and the detector response values on both sides thereof, usually means that the polarity of the water-soluble vitamin component in the separated component is higher. Therefore, first, the difference between the detector response value of each retention time of the separated component and its adjacent retention time is determined, and in an exemplary embodiment, as shown in Figure 5 , a specific process is given as follows:

[0093] Step S41: determining the first detector response value difference and the second detector response value difference of any retention time of the separated component.

[0094] 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 vertical coordinate 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.

[0095] 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, and specifically, the absolute value of the difference between the two detector response values is calculated, and the obtained absolute value of the difference between the detector response values is taken as the first detector response value difference of the jth retention time of the ith separated component.

[0096] 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 value difference of the jth retention time of the ith separated component.

[0097] Step S42: The average of the first detector response value difference and the second detector response value difference is calculated as the difference between the detector response values of any retention time and its adjacent retention time.

[0098] 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 is calculated as the difference between the detector response values of the jth retention time of the ith separated component and its adjacent retention time. As known from the foregoing, the greater the difference between 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.

[0099] By using the above process, the difference between the detector response values of each retention time of the ith separated component and its adjacent retention time is obtained.

[0100] 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 of the influence degrees of all 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 numerical range of the weight of each retention time of the ith separated component is 0-1, and the sum of the weights of all retention times of the ith separated component is 1.

[0101] Finally, based on the weight of each retention time of the ith separated component, the difference between 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:

[0102] ;

[0103] wherein, represents the polarity of the water-soluble vitamin component in the ith separated component, represents the difference between the detector response values of the jth retention time of the ith separated component and its adjacent retention time, The weight of the jth retention time of the ith separated component. Here, f also represents a maximum minimum value 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 in the maximum minimum value normalization manner.

[0104] By using the above process, the polarity of the water-soluble vitamin component in each separated component is obtained.

[0105] Step S5: Determine the proportion of organic solvent in the mobile phase required by the separated component according to the interference degree and the polarity.

[0106] The interference of non-target components (i.e. interference components such as proteins, fats, etc.) in the separated 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 interference degree of the separated component is large, other interference components can interact with the water-soluble vitamins, causing chromatographic peak asymmetry or tailing of the interference components, thereby affecting the accuracy of the mass spectrometry signal.

[0107] In this case, if the polarity of the water-soluble vitamin component in the separated 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 reduce 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, so that it is eluted faster, thereby effectively reducing the interference of matrix effect on the separation result and mass spectrometry.

[0108] Therefore, according to the polarity of the water-soluble vitamin component in the ith separated component and the interference degree of other interference components to the ith separated component, the proportion of organic solvent in the mobile phase required by the ith separated component is determined. The stronger the polarity, the higher the proportion of organic solvent in the mobile phase required, and the stronger the interference degree, the higher the proportion of organic solvent in the mobile phase required. In an exemplary embodiment, as shown in Figure 6 A specific acquisition process of the proportion of organic solvent is given as follows:

[0109] Step S51: Fuse the interference degree and the polarity of the separated component to obtain an adjustment coefficient of the separated component.

[0110] According to the polarity of the water-soluble vitamin component in the ith separated component and the interference degree of other interfering components to the ith separated component, an adjustment coefficient of the ith separated component is obtained, wherein the adjustment coefficient is positively correlated with both the interference degree and the polarity. In an exemplary embodiment, the product of the polarity of the water-soluble vitamin component in the ith separated component and the interference degree of other interfering components to the ith separated component is taken as the adjustment coefficient of the ith separated component.

[0111] Step S52: 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.

[0112] The embodiment requires that the proportion of the organic solvent in the initial mobile phase is positively adjusted according to the adjustment coefficient, that is, the larger the adjustment coefficient, the more the proportion of the organic solvent needs to be increased. In an exemplary embodiment, a specific adjustment method is given as follows:

[0113] ;

[0114] wherein, represents the proportion of the organic solvent in the mobile phase of the ith separated component after adjustment, that is, the proportion of the organic solvent in the mobile phase required by the ith separated component, represents the proportion of the organic solvent in the initial mobile phase. In the above manner, the proportion of the organic solvent in the mobile phase required by each separated component can be obtained.

[0115] It should be understood that the proportion of organic solvent in the mobile phase required for each separated component can be different, and the values can be high or low. In an exemplary embodiment, a specific application is provided: the proportion of organic solvent in the mobile phase required for 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 organic solvent proportion in the organic solvent proportion sequence, gradually adding organic solvent according to the preset adjustment program, the essence is to use a gradient elution control strategy, so that the proportion of organic solvent increases according to the organic solvent proportion sequence, thereby achieving 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 more clear, thereby improving the purity of the sample. Ultimately, such adjustments 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.

[0116] After obtaining more accurate separated components, mass spectrometry is used for simultaneous quantitative detection of multiple vitamins. First, each separated component is directly introduced into the mass spectrometer for further analysis. The mass spectrometer generates a mass-to-charge ratio (m / z) spectrum for each vitamin by detecting ionized molecular fragments. Combined with the retention time information in the chromatogram, mass spectrometry can accurately identify and quantify each vitamin. In addition, techniques such as multiple reaction monitoring can be used to achieve simultaneous quantitative detection of multiple vitamins, thereby improving the throughput and accuracy of detection.

[0117] The embodiment also provides a multi-vitamin simultaneous quantitative detection system, including a memory and a processor; the memory is connected with the processor, and the memory is used to store program instructions; the processor is used to implement the steps in the multi-vitamin simultaneous quantitative detection method embodiment described above when the program instructions are executed.

[0118] In an exemplary embodiment, the present application provides a computer-readable storage medium storing a computer program, which, when executed by a processor, implements the steps in the multi-vitamin simultaneous quantitative detection method embodiment described above.

[0119] It is to be noted that the progressive order of the above-mentioned embodiments of the present application is only for the purpose of 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, multi-task processing and parallel processing are also possible or can be advantageous.

[0120] 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 focuses on 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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