A method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection

Abstract

The present application relates to the technical field of blood sample drug detection, and specifically to a method for improving the accuracy of liquid chromatography-mass spectrometry detection of blood sample drugs, the method comprising: after pre-processing the blood sample drugs to be detected, the liquid chromatograph separates the blood sample drugs by preset flow rate parameters to generate a chromatogram; calculates the peak intensity corresponding to each data point in the chromatogram; obtains each chromatographic peak in the chromatogram, obtains each peak interval and its corresponding chromatographic peak curve; determines the separation degree, adjustment factor, and adjusted flow rate parameter corresponding to any peak interval, and obtains the optimal flow rate parameter through iterative calculation; based on the optimal flow rate parameter, separates the blood sample drugs by liquid chromatograph, performs mass spectrometry analysis on the separated drug components, and identifies the chemical composition of the blood sample drugs. The present application improves the effect of liquid chromatograph on blood sample drug separation and improves the accuracy of liquid chromatography-mass spectrometry detection of blood sample drugs.

Description

Technical Field

[0001] The present application relates to the technical field of blood sample drug detection, and in particular to a method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection. Background Art

[0002] Measuring drug concentration in blood samples is the basis for studying pharmacokinetics, and animal and human pharmacokinetic studies are important components of preclinical drug research and phase I clinical safety evaluation, as well as an important way to screen diseases and early fetuses. Since plasma contains complex components such as plasma proteins, salt ions, water and the drug to be tested, and the drug concentration is relatively low, liquid chromatography-mass spectrometry integrates separation and detection technologies, and can separate drugs from most complex substances in the body for determination. It is currently the main method for determining drug concentration in blood samples.

[0003] When the chromatograph is performing liquid chromatography analysis, it is necessary to set the flow rate parameters of the mobile phase. Improper setting of the flow rate parameters will cause the blood sample to stay in the chromatographic column for too short a time, resulting in incomplete drug separation, or increased baseline noise, affecting sensitivity and detection accuracy, resulting in low accuracy in drug component detection. Summary of the invention

[0004] In order to solve the above technical problems, a method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection is provided to solve the existing problems.

[0005] The solution to the technical problem of the present application is to provide a method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection, comprising the following steps:

[0006] After pre-processing the blood sample drugs to be tested, the liquid chromatograph separates the blood sample drugs by using preset flow rate parameters to generate a chromatogram;

[0007] By analyzing the difference in the local range of each data point on the curve of the chromatogram and the time interval between the time corresponding to each data point and the baseline of the chromatogram, the peak intensity corresponding to each data point in the chromatogram is calculated;

[0008] Based on the peak intensity, all data points in the chromatogram are screened to obtain each chromatographic peak in the chromatogram, and based on each chromatographic peak, each peak interval and its corresponding chromatographic peak curve are obtained;

[0009] Analyze the overlap between any peak interval and the remaining peak intervals in the chromatogram, as well as the change trend between the chromatographic peak curve corresponding to any peak interval and the chromatographic peak curve corresponding to the remaining peak intervals, to determine the separation degree corresponding to any peak interval; based on the separation degree of the overlapping peak intervals, obtain the adjustment factor corresponding to the flow rate parameter in the liquid chromatograph;

[0010] Based on the adjustment factor, the flow rate parameter in the liquid chromatograph is adjusted to obtain the adjusted flow rate parameter in the liquid chromatograph, the blood sample drug is separated with the adjusted flow rate parameter, and the optimal flow rate parameter is obtained through iterative calculation;

[0011] Based on the optimal flow rate parameters, the blood sample medicine is separated by a liquid chromatograph, and the separated components are subjected to mass spectrometry analysis to identify the chemical components of the blood sample medicine.

[0012] Preferably, the calculating the peak intensity corresponding to each data point in the chromatogram comprises:

[0013] Each time corresponding to the baseline in the chromatogram is recorded as the baseline time; taking each data point on the curve in the chromatogram as the center, a local area consisting of multiple data points on the curve is intercepted;

[0014] In the chromatogram The peak intensity corresponding to the data point The calculation formula is: ,in, The chromatogram is The signal strength corresponding to the data point is The chromatogram is In the local area of ​​the data point The signal strength corresponding to the data point is The chromatogram is The time interval between the moment corresponding to a data point and the nearest baseline moment on its left, The chromatogram is The time interval between the moment corresponding to a data point and the nearest baseline moment on its right, The chromatogram is The number of all data points in the local area of ​​data points, To preset a value greater than 0, is the normalization function.

[0015] Preferably, the step of obtaining each chromatographic peak in the chromatogram comprises:

[0016] The mean of the peak intensities corresponding to all data points in the chromatogram is calculated, and the data points whose peak intensities are greater than or equal to the mean are recorded as chromatographic peaks.

[0017] Preferably, the step of obtaining each peak interval and its corresponding chromatographic peak curve comprises:

[0018] The time period between the baseline moments on the left and right sides of the corresponding moment of each chromatographic peak in the chromatogram is recorded as the peak interval; the corresponding curve in each peak interval is recorded as the chromatographic peak curve.

[0019] Preferably, determining the separation degree corresponding to any peak interval comprises:

[0020] If there is no intersection between any peak interval and all other peak intervals, the separation degree corresponding to any peak interval is a preset value; otherwise, the further determination process of the separation degree corresponding to any peak interval is:

[0021] Record all other peak intervals that have intersections with any peak interval as overlapping intervals corresponding to any peak interval;

[0022] Calculate the overlap degree corresponding to any peak interval according to the proportion of the overlap between any peak interval and the corresponding overlapping intervals;

[0023] Calculate the metric distance and correlation between the chromatographic peak curve corresponding to any peak interval and the chromatographic peak curve corresponding to each overlapping interval; calculate the sum of the correlation degree and the value 1, which is recorded as the similarity;

[0024] The product of the similarity and the metric distance is calculated, the cumulative sum of the products between any peak interval and all corresponding overlapping intervals is calculated, and the ratio of the cumulative sum to the overlap is used as the separation degree corresponding to any peak interval.

[0025] Preferably, the calculation method of the overlap degree corresponding to any peak interval is: ,in, The chromatogram is The overlap corresponding to the peak intervals is The chromatogram is The number of all overlapping intervals corresponding to the peak intervals, The chromatogram is The peak interval and its corresponding The length of the interval of intersection between overlapping intervals, The chromatogram is The interval length of the peak interval.

[0026] Preferably, the adjustment factor corresponding to the flow rate parameter in the liquid chromatograph is the normalized result of the mean of the separation degrees corresponding to all peak intervals in the chromatogram that have intersections with the remaining peak intervals.

[0027] Preferably, the step of obtaining the adjusted flow rate parameter in the liquid chromatograph comprises:

[0028] The baseline noise is calculated based on the difference in signal intensity between all data points outside the chromatographic peak curve corresponding to all peak intervals in the chromatogram and the data points on the baseline;

[0029] Adjusted flow rate parameters in liquid chromatograph The calculation formula is: ,in, is the adjustment factor corresponding to the flow rate parameter in the liquid chromatograph, is the baseline noise, is the preset flow rate parameter before adjustment in the liquid chromatograph, is to preset a first threshold, A second threshold is preset.

[0030] Preferably, the calculation process of the baseline noise is:

[0031] The sum of the differences between the signal intensities corresponding to all data points outside the chromatographic peak curve corresponding to all peak intervals in the chromatogram and the signal intensities corresponding to the data points on the baseline is recorded as the noise difference; the normalized result of the ratio between the noise difference and the signal intensities corresponding to the data points on the baseline is recorded as the baseline noise degree.

[0032] Preferably, the mass spectrometry analysis of the separated components to identify the chemical components of the blood sample drug includes:

[0033] The liquid chromatograph separates the blood sample drugs at the optimal flow rate parameters, introduces the separated components of the blood sample drugs into the mass spectrometer, obtains the mass spectrum corresponding to the drugs, calculates the molecular formula corresponding to the components in the drugs through the mass spectrum, and identifies the chemical composition of the drugs according to the molecular formula.

[0034] This application has at least the following beneficial effects:

[0035] The present application analyzes the local differences of data points on the curve in the chromatogram, calculates the peak intensity, screens the data points, extracts the chromatographic peaks in the chromatogram, and obtains the peak interval and chromatographic peak curve corresponding to each chromatographic peak. The beneficial effect is that the data points are screened by peak intensity to obtain the chromatographic peaks, thereby avoiding the interference caused by noise when the chromatographic peaks are directly extracted in the chromatogram; secondly, the separation degree is calculated by the overlap of different peak intervals and the similarity of the trend changes between the chromatographic peak curves. The beneficial effect is that the overlap of the peak intervals corresponding to different chromatographic peaks and the similarity of the change trends of the chromatographic peak curves are taken into account to reflect the similarity of the drug properties represented by different chromatographic peaks, thereby explaining that the different drug properties are separated. Separation effect; based on the separation degree, calculate the adjustment factor corresponding to the flow rate parameter in the liquid chromatograph, so as to subsequently adjust the flow rate parameter in the liquid chromatograph to obtain the adjusted flow rate parameter in the liquid chromatograph, and obtain the optimal flow rate parameter through iterative calculation. Based on the optimal flow rate parameter, the blood sample drug is separated by the liquid chromatograph, and the separated components are subjected to mass spectrometry analysis to identify the chemical components of the blood sample drug. The beneficial effect is that by adjusting the flow rate parameter, the blood sample drug is separated, the effect of the liquid chromatograph on the separation of the blood sample drug is improved, and the overlap between the chromatographic peaks is reduced, thereby improving the accuracy of the identification of the components in the blood sample drug and improving the accuracy of the liquid chromatography-mass spectrometry detection of the blood sample drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The following is a further detailed description of a method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection in the present application in conjunction with the accompanying drawings.

[0037] Figure 1 A flowchart of the steps of a method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection provided in an embodiment of the present application;

[0038] Figure 2 A flow chart of the steps for determining the optimal flow rate parameters provided in an embodiment of the present application. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the following is a further detailed description of a method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection proposed by the present application in conjunction with the accompanying drawings and implementation examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application.

[0040] Unless defined otherwise, 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.

[0041] See also Figure 1, which shows a flow chart of the steps of a method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection provided by an embodiment of the present application, the method comprising the following steps:

[0042] Step 1: After pre-processing the blood sample drugs to be tested, the liquid chromatograph separates the blood sample drugs through preset flow rate parameters to generate a chromatogram.

[0043] Liquid chromatography is an efficient separation and analysis technology. Its basic principle is to use the difference in the distribution coefficient between the mobile phase and the stationary phase to achieve separation. Driven by a high-pressure pump, the sample passes through the chromatographic column with the mobile phase and interacts with the stationary phase to varying degrees, thereby achieving component separation.

[0044] In this embodiment, a blood sample containing 22 kinds of antidepressant drugs is tested. Therefore, a blood sample drug to be tested is obtained and pre-treated, specifically including: performing protein precipitation method on the blood sample drug, removing the protein in the blood sample drug and then centrifuging, taking 2mL of supernatant, adding 400uL of mixed internal standard working solution to the supernatant, shaking and mixing for 3min, centrifuging at 12000r / min at 4°C for 10min, taking 300uL of supernatant and centrifuging again for 10min, taking 100uL and putting it into a 96-well plate, and analyzing it on a liquid chromatograph.

[0045] The preparation process of the mixed internal standard working solution is as follows: 22 kinds of antidepressant drug internal standards are dissolved in methanol into a 1 mg / mL stock solution, and then an appropriate amount of the stock solution is put into a 5 mL volumetric flask, and the volume is fixed with 60% methanol solution, 20% acetonitrile solution, 10% isopropanol solution and 10% purified water. According to this method, a mixed internal standard working solution containing 22 kinds of internal standard substances is prepared.

[0046] The liquid chromatograph consisted of an Acquity UPLC system (Waters) with automatic sampling, and chromatographic separation was performed on a Waters BEH C18 (50 mm × 2.1 mm, 1.7 um) prepared column at 35 °C. The mobile phase consisted of mobile phase A (0.1% formic acid in water) and mobile phase B (methanol containing 0.1% formic acid). Due to the different affinities between the components in the blood sample and the stationary phase, the components interacted differently with the stationary phase when passing through the chromatographic column, resulting in different flow rates. Due to the different distribution coefficients between the mobile phase and the stationary phase, the components moved at different rates in the chromatographic column, thereby achieving separation. The separated components were detected one by one by the detector, and a chromatogram was generated.

[0047] In this embodiment, the preset flow rate parameter of the mobile phase of the liquid chromatograph is 0.4 mL / min. As other implementation methods, the implementer can set it according to the actual situation.

[0048] It should be noted that the chromatogram can reflect the relationship between the time and signal intensity when each component in the blood sample is separated and detected. The horizontal axis of the chromatogram is time, indicating the time it takes for different components in the blood sample to pass through the chromatographic column, and the vertical axis is signal intensity, indicating the intensity of the detector response.

[0049] At this point, a chromatogram is obtained.

[0050] Step 2, by analyzing the difference in the local range of each data point on the curve in the chromatogram, and the time interval between the moment corresponding to each data point and the baseline in the chromatogram, calculate the peak intensity corresponding to each data point in the chromatogram; based on the peak intensity, screen all data points in the chromatogram to obtain each chromatographic peak in the chromatogram, and based on each chromatographic peak, obtain each peak interval and its corresponding chromatographic peak curve.

[0051] When performing chromatographic analysis using a liquid chromatograph, it is necessary to set the flow rate parameters in the mobile phase. The flow rate parameters refer to the speed at which the solvent, carrier gas or mobile phase passes through the chromatographic column during the chromatographic process. Among them, if the flow rate is too high, the blood sample will stay in the chromatographic column for too short a time, resulting in incomplete separation, that is, there will be overlap between chromatographic peaks in the chromatogram. If the flow rate is too low, it may cause an increase in baseline noise, affecting sensitivity and detection accuracy.

[0052] Secondly, there may be overlap in the chromatogram peaks. When there are multiple compounds with similar properties in the blood sample, their retention times on the chromatographic column may be similar, resulting in overlapping chromatogram peaks, that is, there may be multiple data points corresponding to a certain moment. If the peaks in the chromatogram are directly extracted, there will be noise interference, and the peaks corresponding to the noise points may be mistakenly extracted. Therefore, each data point in the chromatogram is locally analyzed, and the peak intensity is calculated by analyzing the difference in signal intensity within the local range, so as to extract the peaks in the chromatogram. Specifically:

[0053] Each moment corresponding to the baseline in the chromatogram is recorded as each baseline moment;

[0054] It should be noted that the determination of the baseline in the chromatogram is a well-known technique and will not be described in detail here.

[0055] Taking each data point on the curve in the chromatogram as the center, intercept the local area consisting of multiple data points on the curve;

[0056] In this embodiment, each data point on the curve in the chromatogram is taken as the center, and a local area consisting of 10 data points adjacent to each data point on the curve is intercepted. As other implementation methods, the implementer can set it according to the actual situation.

[0057] The calculation formula for the peak intensity corresponding to each data point in the chromatogram is:

[0058]

[0059] in, The chromatogram is The peak intensity corresponding to the data point is The chromatogram is In the local area of ​​the data point The signal strength corresponding to the data point is The chromatogram is The time interval between the moment corresponding to a data point and the nearest baseline moment on its left, The chromatogram is The time interval between the moment corresponding to a data point and the nearest baseline moment on its right, The chromatogram is The number of all data points in the local area of ​​data points, To preset a value greater than 0 to avoid the denominator being 0, in this embodiment, The value is 0.01. As other implementation methods, the implementer can set it according to the actual situation. is a normalization function. In this embodiment, a sigmoid function is used for normalization processing. The sigmoid function is a well-known technology and will not be described in detail here.

[0060] Calculate the mean of the peak intensities corresponding to all data points in the chromatogram, and record the data points whose peak intensities are greater than or equal to the mean as chromatographic peaks;

[0061] It should be noted that The larger the The greater the difference between a data point and the rest of the data points in its local area, the The greater the possibility that a data point is a peak point, the The smaller the The smaller the difference between the time corresponding to the data point and the adjacent baseline time on its left and right sides, the The better the symmetry of a data point, the more likely it is a peak point, and the greater the peak intensity, indicating that the The greater the probability that a data point is a peak point.

[0062] The time period between the baseline moments on the left and right sides of the corresponding moment of each chromatographic peak in the chromatogram is recorded as the peak interval; the corresponding curve in each peak interval is recorded as the chromatographic peak curve;

[0063] It should be noted that each chromatographic peak corresponds to a peak interval, and each peak interval corresponds to a section of the chromatographic peak curve.

[0064] At this point, each peak interval and its corresponding chromatographic peak curve are obtained.

[0065] Step 3, analyze the overlap between any peak interval in the chromatogram and the remaining peak intervals, as well as the change trend between the chromatographic peak curve corresponding to any peak interval and the chromatographic peak curve corresponding to the remaining peak intervals, to determine the separation degree corresponding to any peak interval; based on the separation degree of the overlapping peak intervals, obtain the adjustment factor corresponding to the flow rate parameter in the liquid chromatograph.

[0066] Furthermore, when the components of different drugs in the blood sample are similar, or due to unreasonable setting of liquid chromatograph parameters, the separation degree of different drugs is low, and the obtained chromatographic peaks may overlap. The separation degree is calculated by the overlapping conditions of the peak intervals of different chromatographic peaks and the difference in the chromatographic peak curves, specifically:

[0067] If there is no intersection between any peak interval and all other peak intervals, the separation degree corresponding to any peak interval is a preset value; otherwise, the calculation process of the separation degree corresponding to any peak interval is:

[0068] Record the remaining peak intervals that intersect with any peak interval as overlapping intervals corresponding to any peak interval;

[0069] In this embodiment, the preset value is 1. As for other implementation modes, the implementer can set it according to the actual situation.

[0070] The calculation method of the overlap corresponding to any peak interval in the chromatogram is:

[0071]

[0072] in, The chromatogram is The overlap corresponding to the peak intervals is The chromatogram is The number of all overlapping intervals corresponding to the peak intervals, The chromatogram is The peak interval and its corresponding The length of the interval of intersection between overlapping intervals, The chromatogram is The interval length of the peak interval.

[0073] It should be noted that the greater the overlap, the more serious the overlap between different peak intervals.

[0074] The calculation method of the separation degree corresponding to any peak interval in the chromatogram is:

[0075] Calculate the The chromatographic peak curve corresponding to the peak interval is The DTW distance between the chromatographic peak curves corresponding to the overlapping intervals;

[0076] It should be noted that the calculation of the DTW distance is a well-known technique and will not be described in detail here.

[0077] Calculate the The chromatographic peak curve corresponding to the peak interval is The degree of correlation between the chromatographic peak curves corresponding to the overlapping intervals;

[0078] Calculate the sum of the correlation degree and the value 1, and record it as similarity;

[0079] Calculate the product of the similarity and the DTW distance, and calculate the The cumulative sum of the products between the peak intervals and all the overlapping intervals, and the ratio of the cumulative sum to the overlap degree is used as the first The resolution of the peak intervals.

[0080] In this embodiment, the correlation degree is calculated by The chromatographic peak curve corresponding to the peak interval is The Pearson correlation coefficient between the chromatographic peak curves corresponding to the overlapping intervals, wherein the Pearson correlation coefficient is a well-known technology and will not be repeated here; by calculating the sum of the correlation degree and the value 1, the range of similarity is , so that the similarity value is a non-negative number.

[0081] In this embodiment, The calculation formula for the resolution of the peak interval is:

[0082]

[0083] in, The chromatogram is The resolution of the peak intervals is The chromatogram is The chromatographic peak curve corresponding to the peak interval is The chromatogram is The chromatographic peak curves corresponding to the overlapping intervals are The chromatogram is The chromatographic peak curve corresponding to the first peak interval is The correlation degree between the chromatographic peak curves corresponding to the overlapping intervals is To calculate the DTW distance, The chromatogram is The number of all overlapping intervals corresponding to the peak intervals, The chromatogram is The overlap corresponding to the peak intervals; is the similarity.

[0084] It should be noted that the chromatographic peak curve is a process that changes with time, so it can be regarded as a time series. The DTW distance reflects the differences between different chromatographic peak curves. The larger the DTW distance, the greater the difference in the changing trends of the chromatographic peak curves corresponding to the two overlapping peak intervals. The greater the similarity, the closer the drug properties represented by the chromatographic peaks in the chromatographic peak curves corresponding to the two overlapping peak intervals are. The close drug properties will cause overlapping chromatographic peaks. At this time, in order to effectively characterize the drug properties, chromatographic peaks with very similar drug properties may not need to be completely separated. In order to avoid erroneous separation caused by the close drug properties, the separation degree is increased to reflect the better separation effect between the chromatographic peaks at this time, thereby reducing the separation error. The smaller the overlap, the less significant the overlap between the two peak intervals. The larger the obtained separation degree, the better the separation effect between the chromatographic peaks.

[0085] Further, based on the separation degree, the adjustment factor corresponding to the flow rate parameter in the liquid chromatograph is determined, specifically:

[0086] The normalized result of the mean value of the separation degree corresponding to all the peak intervals with intersection in the chromatogram is used as the adjustment factor corresponding to the flow rate parameter in the liquid chromatograph;

[0087] In this embodiment, the sigmoid function is used for normalization processing, wherein the sigmoid function is a well-known technology and will not be described in detail here. As other implementation methods, the implementer can adopt other methods of the prior art, such as the tanh function, etc. This embodiment does not impose any special restrictions on this.

[0088] It should be noted that the larger the adjustment factor is, the better the separation effect of the chromatographic peaks is under the flow rate parameters of the liquid chromatograph at this time. The smaller the adjustment factor is, the worse the separation effect of the chromatographic peaks is under the flow rate parameters of the liquid chromatograph at this time, and the more serious the overlapping phenomenon is, which means that the flow rate parameter is too large, resulting in the blood sample staying in the chromatographic column for too short a time and incomplete separation. The flow rate parameter should be reduced.

[0089] At this point, the adjustment factor corresponding to the flow rate parameter in the liquid chromatograph is obtained.

[0090] Step 4: Based on the adjustment factor, the flow rate parameter in the liquid chromatograph is adjusted to obtain the adjusted flow rate parameter in the liquid chromatograph, and the blood sample drugs are separated with the adjusted flow rate parameter, and the optimal flow rate parameter is obtained through iterative calculation; based on the optimal flow rate parameter, the blood sample drugs are separated by the liquid chromatograph, and the separated components are subjected to mass spectrometry analysis to identify the chemical components of the blood sample drugs.

[0091] Based on the above analysis, the flow rate parameters of the liquid chromatograph are adjusted by adjusting the factors, specifically:

[0092] The sum of the differences between the signal intensities corresponding to all data points outside the chromatographic peak curve corresponding to all peak intervals in the chromatogram and the signal intensities corresponding to the data points on the baseline is recorded as the noise difference; the normalized result of the ratio between the noise difference and the signal intensities corresponding to the data points on the baseline is recorded as the baseline noise degree;

[0093] In this embodiment, the sum of the absolute values ​​of the differences between the signal intensities corresponding to all data points outside the chromatographic peak curves corresponding to all peak intervals in the chromatogram and the signal intensities corresponding to the data points on the baseline is recorded as the noise difference; the sigmoid function is used for normalization, wherein the sigmoid function is a well-known technology and will not be described in detail here. As other implementation methods, the implementer may adopt other methods of the prior art, such as the tanh function, etc., and this embodiment does not impose any special restrictions on this.

[0094] It should be noted that the baseline reflects the signal curve measured by the detector over a period of time after the chromatographic column is flushed with the mobile phase and reaches equilibrium with the mobile phase. If the signal intensity corresponding to the data point outside the chromatographic peak curve corresponding to the peak interval is different from the signal intensity corresponding to the data point on the baseline, it means that there is noise interference, resulting in fluctuations in the signal intensity corresponding to the data point. Therefore, the greater the baseline noise, the greater the degree of noise interference.

[0095] The calculation formula for the adjusted flow rate parameter in the liquid chromatograph is:

[0096]

[0097] in, is the adjusted flow rate parameter in the liquid chromatograph, is the adjustment factor corresponding to the flow rate parameter in the liquid chromatograph, is the baseline noise, is the preset flow rate parameter before adjustment in the liquid chromatograph, is to preset a first threshold, A second threshold is preset.

[0098] In this embodiment, the preset flow rate parameters The value is 0.4ml / min, and the first threshold is preset The value is 0.5, and the second threshold is preset The value is 0.5. As other implementation methods, the implementer can set it according to the actual situation.

[0099] The blood sample drug is separated using the adjusted flow rate parameter as the flow rate parameter of the liquid chromatograph, and based on the chromatogram obtained by separation, steps 2-4 are repeated, and the optimal flow rate parameter is obtained by iterative calculation until the adjusted flow rate parameter tends to be stable, or the iteration is stopped when the preset number of iterations is reached;

[0100] In this embodiment, the preset number of iterations is set to 20. As other implementation methods, the implementer can set it according to the actual situation. The step flow chart of the optimal flow rate parameter provided in the embodiment of the present application is as follows: Figure 2 shown.

[0101] Furthermore, the chromatogram can reflect the concentration of the drug and the separation of each component, but it cannot provide detailed information about the molecular structure or chemical composition of these components. Therefore, the chemical composition of the drug is identified by analyzing the different components after separation, specifically:

[0102] The liquid chromatograph separates the blood sample drug at the optimal flow rate parameter, and introduces the separated components of the blood sample drug into the mass spectrometer. The mass spectrometer ionizes the components of the drug by ionization technology, converts the components into charged ions, and obtains a mass spectrum corresponding to the blood sample drug;

[0103] In this embodiment, detection was performed by a Quattro premier tandem mass spectrometer using an electrospray ionization (ESI) source, the detector was operated in multiple reaction mode (MRM), and the experiment was performed in positive ionization mode. The source temperature was 500°C. Nitrogen was heated to 350°C and used as a nebulizing and desolvation gas with a flow rate of 800 liters per hour. The temperature of the source block was 150°C and the capillary voltage was 3 kV. The acquisition of the mass spectrum is a well-known technique and will not be repeated here.

[0104] Based on the ion peaks corresponding to the mass spectrum, the molecular formulas corresponding to each component in the drug are calculated, and the chemical components of the drug are identified according to the molecular formulas.

[0105] In this embodiment, the molecular formula corresponding to each component in the drug is calculated by using Molecular Formula Calculator software, and the implementer can select other existing software, such as Mass Frontier software.

[0106] It should be understood that although Figure 1The steps in the flowchart are shown in sequence as indicated by the arrows, but these steps are not necessarily executed in the order indicated by the arrows. Unless otherwise specified in this document, there is no strict order restriction for the execution of these steps, and these steps can be executed in other orders. Moreover, Figure 1 At least part of the steps may include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily executed at the same time, but can be executed at different times. The execution order of these sub-steps or stages is not necessarily sequential, but can be executed in turn or alternately with other steps or at least part of the sub-steps or stages of other steps.

[0107] The technical features of the above embodiments may be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0108] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the present application. It should be pointed out that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present application. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application without departing from the content of the technical solution of the present application, shall fall within the protection scope of the technical solution of the present application.

Claims

1. A method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection, characterized in that: The method comprises the following steps: After pre-processing the blood sample drugs to be tested, the liquid chromatograph separates the blood sample drugs by using preset flow rate parameters to generate a chromatogram; By analyzing the difference in the local range of each data point on the curve of the chromatogram and the time interval between the time corresponding to each data point and the baseline of the chromatogram, the peak intensity corresponding to each data point in the chromatogram is calculated; Based on the peak intensity, all data points in the chromatogram are screened to obtain each chromatographic peak in the chromatogram, and based on each chromatographic peak, each peak interval and its corresponding chromatographic peak curve are obtained; Analyze the overlap between any peak interval and the remaining peak intervals in the chromatogram, as well as the change trend between the chromatographic peak curve corresponding to any peak interval and the chromatographic peak curve corresponding to the remaining peak intervals, to determine the separation degree corresponding to any peak interval; based on the separation degree of the overlapping peak intervals, obtain the adjustment factor corresponding to the flow rate parameter in the liquid chromatograph; Based on the adjustment factor, the flow rate parameter in the liquid chromatograph is adjusted to obtain the adjusted flow rate parameter in the liquid chromatograph, the blood sample drug is separated with the adjusted flow rate parameter, and the optimal flow rate parameter is obtained through iterative calculation; Based on the optimal flow rate parameters, the blood sample drug is separated by a liquid chromatograph, and the separated components are subjected to mass spectrometry analysis to identify the chemical components of the blood sample drug; The calculating the peak intensity corresponding to each data point in the chromatogram comprises: Each time corresponding to the baseline in the chromatogram is recorded as the baseline time; taking each data point on the curve in the chromatogram as the center, a local area consisting of multiple data points on the curve is intercepted; In the chromatogram The peak intensity corresponding to the data point The calculation formula is: ,in, The chromatogram is The signal strength corresponding to the data point is The chromatogram is In the local area of ​​the data point The signal strength corresponding to the data point is The chromatogram is The time interval between the moment corresponding to a data point and the nearest baseline moment on its left, The chromatogram is The time interval between the moment corresponding to a data point and the nearest baseline moment on its right, The chromatogram is The number of all data points in the local area of ​​data points, To preset a value greater than 0, is the normalization function.

2. A method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection as claimed in claim 1, characterized in that: The obtaining of each chromatographic peak in the chromatogram comprises: The mean of the peak intensities corresponding to all data points in the chromatogram is calculated, and the data points whose peak intensities are greater than or equal to the mean are recorded as chromatographic peaks.

3. A method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection as claimed in claim 1, characterized in that: The step of obtaining each peak interval and its corresponding chromatographic peak curve comprises: The time period between the baseline moments on the left and right sides of the corresponding moment of each chromatographic peak in the chromatogram is recorded as the peak interval; the corresponding curve in each peak interval is recorded as the chromatographic peak curve.

4. A method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection as claimed in claim 1, characterized in that: Determining the separation degree corresponding to any peak interval includes: If there is no intersection between any peak interval and all other peak intervals, the separation degree corresponding to any peak interval is a preset value; otherwise, the further determination process of the separation degree corresponding to any peak interval is: Record all other peak intervals that have intersections with any peak interval as overlapping intervals corresponding to any peak interval; Calculate the overlap degree corresponding to any peak interval according to the proportion of the overlap between any peak interval and the corresponding overlapping intervals; Calculate the metric distance and correlation between the chromatographic peak curve corresponding to any peak interval and the chromatographic peak curve corresponding to each overlapping interval; calculate the sum of the correlation degree and the value 1, which is recorded as the similarity; The product of the similarity and the metric distance is calculated, the cumulative sum of the products between any peak interval and all corresponding overlapping intervals is calculated, and the ratio of the cumulative sum to the overlap is used as the separation degree corresponding to any peak interval.

5. A method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection as claimed in claim 4, characterized in that: The calculation method of the overlap degree corresponding to any peak interval is: ,in, The chromatogram is The overlap corresponding to the peak intervals is The chromatogram is The number of all overlapping intervals corresponding to the peak intervals, The chromatogram is The peak interval and its corresponding The length of the interval of intersection between overlapping intervals, The chromatogram is The interval length of the peak interval.

6. A method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection as claimed in claim 1, characterized in that: The adjustment factor corresponding to the flow rate parameter in the liquid chromatograph is the normalized result of the mean value of the separation degree corresponding to all peak intervals that have intersections with the remaining peak intervals in the chromatogram.

7. A method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection as claimed in claim 1, characterized in that: The step of obtaining the adjusted flow rate parameter in the liquid chromatograph comprises: The baseline noise is calculated based on the difference in signal intensity between all data points outside the chromatographic peak curve corresponding to all peak intervals in the chromatogram and the data points on the baseline; Adjusted flow rate parameters in liquid chromatograph The calculation formula is: ,in, is the adjustment factor corresponding to the flow rate parameter in the liquid chromatograph, is the baseline noise, is the preset flow rate parameter before adjustment in the liquid chromatograph, To preset the first threshold, A second threshold is preset.

8. A method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection as claimed in claim 7, characterized in that: The calculation process of the baseline noise is: The sum of the differences between the signal intensities corresponding to all data points outside the chromatographic peak curve corresponding to all peak intervals in the chromatogram and the signal intensities corresponding to the data points on the baseline is recorded as the noise difference; the normalized result of the ratio between the noise difference and the signal intensities corresponding to the data points on the baseline is recorded as the baseline noise degree.

9. The method for improving the accuracy of blood sample drug liquid chromatography-mass spectrometry detection as claimed in claim 1, characterized in that: The separated components are subjected to mass spectrometry analysis to identify the chemical components of the blood sample drug, including: The liquid chromatograph separates the blood sample drugs at the optimal flow rate parameters, introduces the separated components of the blood sample drugs into the mass spectrometer, obtains the mass spectrum corresponding to the drugs, calculates the molecular formula corresponding to the components in the drugs through the mass spectrum, and identifies the chemical composition of the drugs according to the molecular formula.

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