High-sensitivity quantitative drug mass spectrum imaging method and application thereof

Abstract

The invention provides a high-sensitivity quantitative drug mass spectrum imaging method. The invention also provides a preparation method of the matrix simulation sample for quantitative drug mass spectrum imaging. The invention further provides an analysis condition screening method for quantitative drug mass spectrum imaging. According to the high-sensitivity quantitative drug mass spectrum imaging method and the application thereof, the sensitivity and the detection coverage of a drug mass spectrum analysis method can be efficiently improved, imaging analysis can be carried out on tissue endogenous metabolites, and the high-sensitivity quantitative drug mass spectrum imaging method is an innovative drug DMPK research method; the method has a wide application prospect in the field of life science, pharmaceutical enterprises, cosmetic industries and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and relates to a highly sensitive quantitative drug mass spectrometry imaging method and application thereof. Background Art

[0002] Drug metabolism and pharmacokinetics (DMPK) research aims to understand the metabolic process of drugs in the body, the interaction between drugs and targets, and the distribution of drugs in tissues. It runs through the entire life cycle of drug development and plays a vital role. At present, the mainstream technologies for drug DMPK research include liquid chromatography-mass spectrometry (LCMS), quantitative whole body autoradiography (QWBA) and mass spectrometry imaging technology, etc. However, LCMS technology is difficult to accurately characterize the in situ distribution of drugs, while QWBA is expensive and has certain safety risks, and it cannot distinguish between drugs and their metabolites. Mass spectrometry imaging technology is an ideal technology that can simultaneously perform qualitative, quantitative, and localized analysis of drugs and their metabolites in different types of tissues and endogenous metabolites affected by drugs, especially for highly heterogeneous tissue samples with fine micro-region structures, such as tumors, brain, skin, etc. Through drug mass spectrometry imaging technology, researchers can directly observe and quantitatively analyze the distribution and metabolism of drugs in biological tissues, thereby providing important information about the mechanism of action and efficacy evaluation of drugs in vivo.

[0003] The detection sensitivity of mass spectrometry imaging is a major challenge of this technology. Therefore, it is urgent to improve the sensitivity of mass spectrometry imaging technology through method optimization. Summary of the invention

[0004] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a highly sensitive quantitative drug mass spectrometry imaging method and its application, by preparing a convenient and efficient alternative matrix and optimizing the analysis conditions, thereby improving the sensitivity and detection coverage of the drug mass spectrometry analysis method.

[0005] To achieve the above-mentioned and other related purposes, the present invention provides a highly sensitive quantitative drug mass spectrometry imaging method, comprising the following steps:

[0006] 1) After the mouse samples are administered, samples are taken at different time points, embedded and fixed, sliced, and adhered and fixed on a glass slide to obtain the samples to be tested;

[0007] 2) Performing mass spectrometry imaging analysis on the sample to be tested to determine the distribution of the drug administered in the sample to be tested at different sampling time points, and determining the content of the drug administered in the sample to be tested at different sampling time points according to the standard curve method.

[0008] The second aspect of the present invention provides a method for preparing a matrix simulation sample for quantitative drug mass spectrometry imaging, comprising: adding a mouse sample to an extraction solvent for grinding to homogenize, centrifuging the obtained homogenate, taking the supernatant and adding the compound solution to mix, and dropping the obtained mixed solution onto a glass slide and waiting for the droplets to evaporate completely to provide a matrix simulation sample.

[0009] The third aspect of the present invention provides a method for screening analytical conditions for quantitative drug mass spectrometry imaging, using a matrix simulation sample prepared by the method provided in the second aspect of the present invention, selecting different conditions for mass spectrometry imaging analysis, and comparing to determine the quantitative analysis conditions for drug mass spectrometry imaging.

[0010] As described above, the highly sensitive quantitative drug mass spectrometry imaging method and its application provided by the present invention have the following beneficial effects:

[0011] (1) The present invention provides a highly sensitive quantitative drug mass spectrometry imaging method and its application. By preparing a matrix simulation sample, the analysis conditions of the quantitative drug mass spectrometry imaging method, including the spray solvent composition, source parameters (spray voltage), and analysis method parameters, can be optimized. Compared with the conventional mass spectrometry imaging method, the present method can improve the sensitivity by at least one order of magnitude by optimizing the three aspects of spray solvent, source parameters, and mass spectrometry acquisition parameters, thereby effectively improving the sensitivity and detection coverage of the drug mass spectrometry analysis method.

[0012] (2) The present invention provides a highly sensitive quantitative drug mass spectrometry imaging method and its application, which can perform imaging analysis on endogenous metabolites in tissues. It has strong operability, wide detection coverage and high sensitivity. It is an innovative drug DMPK research method and has broad application prospects in the fields of life sciences, pharmaceutical companies, and the cosmetics industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Shown is the detection of paeoniflorin (PF) precursor ions in matrix-spiked samples.

[0014] Figure 2 Shown is the detection of amygdalin (AD) precursor ions in matrix-spiked samples.

[0015] Figure 3 Shown is the effect of different solvents on the detection of paeoniflorin (PF) in brain tissue.

[0016] Figure 4 Shown is the effect of different solvents on the detection of amygdalin (AD) in brain tissue.

[0017] Figure 5 Shown is the effect of different solvents on the detection of paeoniflorin (PF) in the liver.

[0018] Figure 6 Shown is the effect of different solvents on the detection of amygdalin (AD) in the liver.

[0019] Figure 7 Shown is the effect of different collection methods on the detection of amygdalin (AD) in brain tissue.

[0020] Figure 8 Shown is the effect of different collection methods on the detection of paeoniflorin (PF) in brain tissue.

[0021] Fig. 9 Shown is the effect of loop count on the detection of amygdalin (AD) in the liver.

[0022] Fig.10 Shown is the effect of MIT on the detection of paeoniflorin (PF) in the liver.

[0023] Fig.11 The graph shows the detection of different adduct ions of paeoniflorin (PF) under different spray voltages.

[0024] Fig.12 Shown is a standard curve graph for the quantification of paeoniflorin (PF).

[0025] Fig.13 Shown is the distribution of paeoniflorin (PF) in the kidney at different sampling time points.

[0026] Fig.14 Shown are typical endogenous component images. DETAILED DESCRIPTION

[0027] The first aspect of the present invention provides a highly sensitive quantitative drug mass spectrometry imaging method, comprising the following steps:

[0028] 1) After the mouse samples are administered, samples are taken at different time points, embedded and fixed, sliced, and adhered and fixed on a glass slide to obtain the samples to be tested;

[0029] 2) Performing mass spectrometry imaging analysis on the sample to be tested to determine the distribution of the drug administered in the sample to be tested at different sampling time points, and determining the content of the drug administered in the sample to be tested at different sampling time points according to the standard curve method.

[0030] In the above step 1), the mice are conventionally used mice, specifically SD rats.

[0031] In the above step 1), the mouse sample is selected from at least one of the liver or brain tissue of the mouse.

[0032] In the above step 1), the drug administered is selected from at least one of paeoniflorin (PF) and amygdalin (AD). Specific information of the paeoniflorin (PF) and amygdalin (AD) is shown in Table 1 below.

[0033] Table 1

[0034]

[0035] In the above step 1), the mass ratio of the administration dose to the mouse sample is 50-120:1, mg / kg, preferably 90:1, mg / kg.

[0036] In the above step 1), the different time points are selected from at least one of 10 min, 30 min, 1 h, 2 h, and 5 h after administration.

[0037] In the above step 1), the embedding is a conventional embedding process step. Specifically, the embedding is to add embedding glue into the embedding box to fix the mouse sample.

[0038] In a specific embodiment, the embedding gel is Cryo-Gel embedding gel (Leica Cryo-Gel Embedding Medium, Item No. 14020108926).

[0039] In a specific embodiment, the embedding includes: first adding embedding glue on the bottom support of the embedding box, then putting the mouse sample in and filling it with embedding glue to completely cover it, and adding dry ice to solidify the embedding glue to fix the mouse sample.

[0040] In a preferred embodiment, the embedding glue is laid with a thickness of 2-4 mm.

[0041] In a preferred embodiment, the embedding gel covering height is 1-3 mm higher than the top surface of the mouse sample.

[0042] In the above step 1), the slicing is a conventional slicing process step. Specifically, the slicing is to slice the embedded and fixed mouse sample using a microtome.

[0043] In a specific embodiment, the slicer is a conventionally used slicer. Specifically, the slicer is a Leica CM1950 cryostat. The cryostat needs to be turned on one day in advance, and different slice temperatures are set according to the conditions of the brain tissue sample, and precooled in advance.

[0044] In a preferred embodiment, the slicing conditions of the microtome are: the box temperature is -22 to -18°C; the sample head temperature is -20.5 to -20°C.

[0045] In the above step 1), the slice thickness after slicing is 8-10 μm. When the slicer is slicing, the embedded and fixed mouse sample is placed on the sample head of the slicer to cut out frozen tissue slices of a set thickness.

[0046] In the above step 1), the slice size after slicing is 70-80 mm 2 .

[0047] In the above step 1), the glass slide is a positively charged anti-shedding glass slide.

[0048] In a specific embodiment, the positively charged anti-shedding glass slide is a Superfrost Plus positively charged anti-shedding glass slide.

[0049] In the above step 1), the sample to be tested is placed in a slide box, covered with plastic wrap, and stored in an ultra-low temperature refrigerator at -85 to -75°C, preferably -80°C, for subsequent imaging analysis.

[0050] In the above step 2), the mass spectrometry imaging analysis is performed using an imaging platform tandem mass spectrometer.

[0051] In a specific embodiment, the imaging platform is an AFAI-MSI imaging platform. The AFAI-MSI imaging platform is produced by Vicoto (Beijing) Technology Co., Ltd.

[0052] In a specific embodiment, the mass spectrometer is a Q-Orbitrap mass spectrometer, which is produced by Thermo Fisher, USA.

[0053] In the above step 2), in the mass spectrometry imaging analysis, the data acquisition scanning conditions of the imaging platform include: scanning mode: line-by-line scanning mode; the X-axis scanning length of the scanning area is 6-9mm, preferably 7-8mm; the Y-axis scanning length of the scanning area is 8-11mm, preferably 9-10mm; the scanning speed in the X-axis direction (horizontal displacement rate, Vx) is 0.18-0.21mm / s, preferably 0.19-0.2mm / s; the step spacing between two adjacent scanning lines in the Y-axis direction (vertical displacement distance, Dy) is 0.098-0.12mm, preferably 0.099-0.11mm; the spatial resolution is 99-101μm*99-101μm, preferably 100μm*100μm. The scanning parameters are set by the mass spectrometry platform control program software.

[0054] In the above step 2), in the mass spectrometry imaging analysis, the measurement conditions of the mass spectrometer include: the spray solvent is an aqueous solution of 70-100% acetonitrile, preferably 85-95%, more preferably 90%; the spray voltage is 4900-5100V, preferably 5000V; the scanning acquisition mode is selected from at least one of PRM, Full scan or t-SIM.

[0055] In a specific embodiment, the measurement conditions of the mass spectrometer also include: capillary temperature of 345-355°C, preferably 350°C; S Lens pressure of 50-60V, preferably 55V; ion polarity mode: positive ion mode; resolution: 59000-61000, preferably 60000; scanning acquisition mode is Full scan and t-SIM.

[0056] In a preferred embodiment, when the scanning acquisition mode is Full scan and t-SIM, the automatic gain of Full scan is 4-6e 6 , preferably 5e 6 , the automatic gain of t-SIM is 4-6e 5 , preferably 5e 5 ; The mass range of Full scan is 70 to 1050, preferably 80 to 1000, and the mass range of t-SIM is 519.1±3, preferably 519.1±2; the maximum injection time of Full scan is 120±3ms, preferably 120±2ms, and the maximum injection time of t-SIM is 50±2ms, preferably 50±1ms.

[0057] In the above step 2), in the mass spectrometry imaging analysis, when the administered drug is paeoniflorin (PF) and / or amygdalin (AD), the adduct ion form of the administered drug is selected from [M+H] + 、[M+Na] + or [M+K] + At least one of the following, preferably selected from [M+Na] + or [M+K] + At least one of the following, more preferably [M+K] + .

[0058] In the above step 2), the determination of the content of the administered drug in the sample to be tested at different sampling time points according to the standard curve method comprises the following steps:

[0059] A) adding a drug standard to be administered to a first solvent for dissolution, adding a second solvent for dilution of the obtained standard stock solution, and spotting a series of standard solutions of different concentrations on a blank glass slide to obtain standard samples;

[0060] B) performing mass spectrometry imaging analysis on the standard sample and the sample to be tested in step 1), respectively, and determining the content of the administered drug in the sample to be tested at different sampling time points according to the standard curve method.

[0061] In the above step A), the administered drug standard is selected from at least one of a paeoniflorin (PF) standard or an amygdalin (AD) standard.

[0062] In the above step A), the first solvent is dimethyl sulfoxide (DMSO).

[0063] In the above step A), the drug concentration in the standard stock solution is 9-11 mM, preferably 10 mM.

[0064] In the above step A), the second solvent is water.

[0065] In the above step A), the concentration of the drug administered in the standard solution is 1-100 μM, preferably 3.125-100 μM.

[0066] In the above step A), the amount of the standard solution spotted on the blank glass slide is 0.4-0.6 μL, preferably 0.5 μL.

[0067] In the above step B), the standard curve method refers to: performing mass spectrometry imaging analysis on a series of standard samples respectively, obtaining a linear relationship between the concentration of the drug standard administered in the standard solution of a series of standard samples and the relative ion intensity (average drug peak intensity), drawing a corresponding standard working curve with each relative ion intensity corresponding to its corresponding concentration, calculating the regression equation of the standard working curve, and then performing mass spectrometry imaging analysis on the sample to be tested, substituting the relative ion intensity of the drug administered in the sample to be tested into the regression equation of the above standard working curve, and calculating the content of the drug administered.

[0068] In a specific embodiment, in the standard working curve, the concentration (μg / g) of the drug standard is used as the abscissa, and the relative ion intensity (average drug peak intensity) is used as the ordinate.

[0069] The second aspect of the present invention provides a method for preparing a matrix simulation sample for quantitative drug mass spectrometry imaging, comprising: adding a mouse sample to an extraction solvent for grinding to homogenize, centrifuging the obtained homogenate, taking the supernatant and adding the compound solution to mix, and dropping the obtained mixed solution onto a glass slide and waiting for the droplets to evaporate completely to provide a matrix simulation sample.

[0070] In the above method, the mice are conventionally used mice, such as SD rats.

[0071] In the above method, the mouse sample is selected from at least one of the liver or brain tissue of the mouse.

[0072] In the above method, the extraction solvent is water, and the water is pure water, which is used to homogenize the mouse sample.

[0073] In the above method, the ratio of the mass g of the mouse sample added to the volume mL of the extraction solvent added is 1:8-10, preferably 1:9.

[0074] In the above method, steel balls are added during grinding. The steel balls are conventional grinding steel balls with a diameter of 2-4 mm, preferably 3 mm.

[0075] In a specific embodiment, the ratio of the number of steel beads added to the mass of the mouse sample added is 3:49-51, preferably 3:50, pieces / mg.

[0076] In the above method, the grinding device is a commonly used grinding machine.

[0077] In the above method, the grinding temperature is 3-5°C, preferably 4°C.

[0078] In the above method, the grinding frequency is 3-4 Hz, preferably 3.60 Hz.

[0079] In the above method, the grinding time is 1-3 min, preferably 2 min.

[0080] In the above method, the homogenate should reach a uniform suspension state.

[0081] In the above method, the centrifugal device is a commonly used centrifuge.

[0082] In the above method, the centrifugal rotation speed is 1000-3000g, preferably 2000g.

[0083] In the above method, the centrifugation temperature is 3-5°C, preferably 4°C.

[0084] In the above method, the centrifugation time is 1-3 min, preferably 2 min.

[0085] In the above method, the compound solution is water, and the water is pure water.

[0086] In the above method, the volume ratio of the supernatant to the compound solution is 48-50:0.9-1.1.

[0087] In the above method, the mixing condition is vortex mixing at room temperature for 1-3 minutes, preferably 2 minutes. The room temperature is 20-30°C.

[0088] In the above method, the volume of the supernatant added dropwise to the slide glass is 0.5-2 μL, preferably 1 μL.

[0089] The third aspect of the present invention provides a method for screening analytical conditions for quantitative drug mass spectrometry imaging, using a matrix simulation sample prepared by the method provided in the second aspect of the present invention, selecting different conditions for mass spectrometry imaging analysis, and comparing to determine the quantitative analysis conditions for drug mass spectrometry imaging.

[0090] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0091] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0092] Example 1

[0093] Purchase 1 SD rat, weigh 50 mg of liver and brain tissue respectively as mouse samples. Add the mouse sample to a 1.5 mL EP tube, add 900 μL of extraction solvent (the ratio of the mass g of the mouse sample added to the volume mL of the extraction solvent added is 1:9), add 3 steel balls to the EP tube, grind at 3.60 Hz at 4 ° C for 2 minutes, and obtain a homogenate. Repeatedly observe the homogenization, and the homogenate should reach a uniform suspension state.

[0094] The homogenate was centrifuged at 2000g for 2 min at 4°C and the supernatant was taken. 49 μL of the supernatant was vortexed with 1 μL of water at room temperature for 2 min to obtain a mixed solution. 1 μL of the mixed solution was dropped onto a glass slide and the droplet was completely evaporated to obtain matrix simulation sample 1#.

[0095] Example 2

[0096] Purchase 1 SD rat, weigh 50 mg of liver and brain tissue respectively as mouse samples. Add the mouse sample to a 1.5 mL EP tube, add 800 μL of extraction solvent (the ratio of the mass g of the mouse sample added to the volume mL of the extraction solvent added is 1:8), add 3 steel balls to the EP tube, grind at 4 Hz at 3 ° C for 2.5 minutes, and obtain a homogenate. Repeatedly observe the homogenization, and the homogenate should reach a uniform suspension state.

[0097] The homogenate was centrifuged at 2100g for 2.5 min at 3°C ​​to obtain the supernatant. 48 μL of the supernatant was vortexed with 0.9 μL of water at room temperature for 3 min to obtain a mixed solution. 1 μL of the mixed solution was dropped onto a glass slide and the droplet was completely evaporated to obtain matrix simulation sample 2#.

[0098] Example 3

[0099] The matrix simulation sample 1# containing 10 μM drug prepared in Example 1 was subjected to mass spectrometry imaging analysis using an AFAI-MSI imaging platform connected in series with a Q-Orbitrap mass spectrometer. The data acquisition scanning conditions of the imaging platform include: scanning mode: line-by-line scanning mode; the X-axis scanning length of the scanning area is 7 mm; the Y-axis scanning length of the scanning area is 10 mm; the scanning speed in the X-axis direction (horizontal displacement rate, Vx) is 0.2 mm / s; the step spacing between two adjacent scanning lines in the Y-axis direction (vertical displacement distance, Dy) is 0.1 mm; and the spatial resolution is 100 μm*100 μm.

[0100] The measurement conditions of the mass spectrometer include: the spray solvent is 90% acetonitrile in water; the spray voltage is 5000V; the scanning acquisition mode is Full scan; the automatic gain of Full scan is 5e 6 ; Full scan mass range is 80 to 1000; Full scan maximum injection time is 120ms. Capillary temperature is 350℃; S Lens pressure is 55V; Ion polarity mode: positive ion mode; Resolution: 60000.

[0101] Full scan analysis shows that AD and PF exist in the matrix, and the adduct ion form of the administered drug is [M+H] + 、[M+Na] + or [M+K] + , mainly [M+Na] + and [M+K] + exists, and [M+K]+ has a higher intensity. See Table 2 below for details. Figure 1 , Figure 2 .

[0102] Table 2

[0103]

[0104] Example 4

[0105] The matrix simulation sample 1# containing 10 μM drug prepared in Example 1 was subjected to mass spectrometry imaging analysis using the AFAI-MSI imaging platform in series with a Q-Orbitrap mass spectrometer. In the mass spectrometry imaging analysis, 50% acetonitrile aqueous solution, 60% acetonitrile aqueous solution, 70% acetonitrile aqueous solution, 80% acetonitrile aqueous solution, 90% acetonitrile aqueous solution, and 100% acetonitrile aqueous solution were prepared as 5 mL of spray solvents. The effect on drug detection sensitivity was investigated, and the specific test results are shown in Figure 3-6 .

[0106] Among other conditions for mass spectrometry imaging analysis, the data acquisition scanning conditions of the imaging platform include: scanning mode: line-by-line scanning; the X-axis scanning length of the scanning area is 7 mm; the Y-axis scanning length of the scanning area is 10 mm; the scanning speed in the X-axis direction (horizontal displacement rate, Vx) is 0.2 mm / s; the step spacing between two adjacent scanning lines in the Y-axis direction (vertical displacement distance, Dy) is 0.1 mm; and the spatial resolution is 100 μm*100 μm.

[0107] The mass spectrometer measurement conditions include: the spray solvent is 90% acetonitrile in water; the spray voltage is 5000V; the scanning acquisition mode is Full scan and t-SIM; the automatic gain of Full scan is 5e 6 , the automatic gain of t-SIM is 5e 5 ; The mass range of Fullscan is 80 to 1000, and the mass range of t-SIM is 519.1; The maximum injection time of Fullscan is 120ms, and the maximum injection time of t-SIM is 50ms. The capillary temperature is 350℃; The S Lens pressure is 55V; Ion polarity mode: positive ion mode; Resolution: 60000.

[0108] Depend on Figure 3-6 It can be seen that when the water phase is higher than 30%, the sensitivity gradually increases with the increase of the water phase. However, when the water phase increases to 30%, droplets will appear in the spray, affecting the imaging effect. Therefore, considering the sensitivity and imaging effect, a 70-100% acetonitrile aqueous solution is selected, and a 90% acetonitrile aqueous solution is the best choice as the final spray solvent.

[0109] Example 5

[0110] The matrix simulation sample 1# containing 10 μM drug prepared in Example 1 was subjected to mass spectrometry imaging analysis using the AFAI-MSI imaging platform in series with a Q-Orbitrap mass spectrometer. In the mass spectrometry imaging analysis, the scanning acquisition modes used were fullscan, tSIM, PRM, and full scan+tSIM, respectively, to investigate the effects of target drug analysis detection sensitivity, tissue endogenous component detection coverage, and spatial resolution. The specific test results are shown in Figure 7-10 .

[0111] Among them, when the scanning acquisition mode is full scan, the corresponding condition is that the automatic gain is 5e 6 , the maximum injection time is 200ms, and the mass range is 80-1000. When the scanning acquisition mode is tSIM, the corresponding condition is that the automatic gain is 2e 5 , the maximum injection time is 50ms, and the mass range is 519.1±1.5Da. When the scanning acquisition mode is PRM, the corresponding condition is that the automatic gain is 5e 5 , the maximum injection time is 50ms, the mass range is 519.1±1.5Da, and the normalized collision energy is 10, 20, 40. When the scanning acquisition mode is Full scan and t-SIM, the automatic gain of Full scan is 5e 6 , the automatic gain of t-SIM is 5e 5 The mass range of Fullscan is 80-1000, and the mass range of t-SIM is 519.1±1.5Da; the maximum injection time of Fullscan is 120ms, and the maximum injection time of t-SIM is 50ms. Other mass spectrometry imaging analysis is the same as in Example 4.

[0112] Depend on Figure 7It can be seen that compared with the conventional Full scan, both PRM and t-SIM can improve the detection sensitivity of AD [M+K] + and [M+Na] +, and the improvement factor of t-SIM is higher, which is 3.5 times for [M+K] + and 4.6 times for [M+Na] +, while the improvement factor of PRM is slightly lower than that of t-SIM, which is 1.4 times for [M+K] + and 2.6 times for [M+Na] +. In addition, different acquisition methods also have a significant impact on spatial resolution. When PRM is used to acquire two adduct ions of AD at the same time, the spatial resolution is 75μm, and when t-SIM is used to acquire two adduct ions of AD at the same time, the spatial resolution is 36μm. Therefore, if these two acquisition methods are used, if the spatial resolution of 100μm is maintained, the entire analysis time can be shortened by 1.3 to 2.8 times. It is worth mentioning that although PRM and t-SIM can improve detection sensitivity and shorten analysis time, they lack analysis of endogenous components of the sample. Next, the effects of simultaneous Full scan and t-SIM on target ion detection, spatial resolution, and endogenous component detection were examined.

[0113] Depend on Figure 8 It can be seen that compared with conventional Fullscan, PRM and full scan+t-SIM can both improve the detection sensitivity of PF[M+K]+ and [M+Na]+, and the improvement of full scan+t-SIM is higher, which is 4.4 times for [M+K]+ and 4.5 times for [M+Na]+, while the improvement of PRM is slightly lower than that of t-SIM, which is 1.3 times for [M+K]+ and 2.6 times for [M+Na]+. In addition, different acquisition methods also have a certain impact on spatial resolution. PRM acquisition of two adduct ions of AD at the same time has a spatial resolution of 75μm, and full scan+t-SIM acquisition of two adduct ions of sample and target drug AD at the same time has a spatial resolution of 105μm. Therefore, if these two acquisition methods are used, if the spatial resolution of 100μm is maintained, PRM can shorten the analysis time by about 1.3 times, which is basically consistent with the above conclusion. Full scan+t-SIM will increase the analysis time by 1.05 times, which has little effect. In addition, full scan+t-SIM analyzes the endogenous components of the sample at the same time, and increases the number of m / z detections with intensities higher than 1000 to a certain extent, about 1.1 times. Therefore, comprehensively comparing these three acquisition methods, Full scan+t-SIM is the best, which can analyze endogenous components and drugs at the same time, and improves the detection sensitivity of drugs. In addition, it has little effect on the analysis time, which increases by about 1.05 times. Although the acquisition method is relatively clear, key parameters such as AGC, MIT, and Loop count in t-SIM are still worth further investigation and optimization.

[0114] Depend on Fig. 9 It can be seen that Full scan+t-SIM(2) is to perform a full mass spectrometry scan and acquire 2 target m / z at the same time, and Fullscan+t-SIM(4) is to perform a full mass spectrometry scan and acquire 4 target m / z at the same time. Compared with Full scan, these two methods can improve the detection sensitivity of target ions by 4.4 and 6.0 times for [M+K]+ and [M+Na]+, respectively, which are basically the same. Therefore, increasing the number of target ions has little effect on the sensitivity of target ions and the detection of endogenous components in tissues. The main effect is on the spatial resolution. Through the spatial resolution of the two, it is found that for every additional ion, the spatial resolution decreases by 15μm. Therefore, the subsequent experiments can adjust this parameter according to the actual number of drugs and experimental objectives. In this experiment, 1-2 target ions can be selected for t-SIM analysis.

[0115] Depend on Fig.10 It can be seen that increasing the MIT time can increase [M+K]+ and [M+Na]+ by 1.1 times and 1.31.4 times respectively. For every 50ms increase in MIT, the spatial resolution decreases by about 15μm. In addition, increasing the MIT time can also increase the number of endogenous components in the sample with m / z intensity above 1000, which is about 1.1 times (base 813). Considering the impact of the target m / z sensitivity increase (1.1, 1.31.4), sample analysis time (increased by 1.11.4 times) and the increase in the number of endogenous components (about 1.1 times, 6299), choosing 50ms is more cost-effective.

[0116] In summary, by comparing the effects of different acquisition methods and key parameters on the detection of AD and PF drugs in tissues, we can know that: (1) Among the different acquisition methods, the Full scan + t-SIM combination is better, taking into account the detection of endogenous components in tissue samples and improving the detection sensitivity of target ions. The improvement factor is 3.5 to 4.4 times for [M+K]+ and 4.5 to 6.2 times for [M+Na]+. It also increases the number of endogenous components with m / z intensities above 1000 to a certain extent. In addition, the impact on spatial resolution is small, and the data acquisition time will be increased by about 1.05-1.2 times. (2) The key parameter Loop count mainly affects the spatial resolution, that is, the data acquisition time. When other parameters are the same, the spatial resolution decreases by 15μm for each additional ion. In this experiment, 1-2 m / z can be selected for t-SIM analysis. (3) The key parameter MIT improves the detection sensitivity of the target m / z by about 1.1 to 1.4 times, and also affects the spatial resolution. When other parameters are the same, the spatial resolution decreases by about 15 μm for every 50 ms increase in MIT. In this experiment, 50 ms was selected.

[0117] Example 6

[0118] The matrix simulation sample 1# containing 10 μM drug prepared in Example 1 was subjected to mass spectrometry imaging analysis using the AFAI-MSI imaging platform in series with a Q-Orbitrap mass spectrometer. In the mass spectrometry imaging analysis, the spray voltage was investigated, and the detection of different PF adduct ions was investigated at 0, 2000, 4000, 4500, 5000, 6000 and 8000. At the same time, a 90% acetonitrile aqueous solution (with 1 ppm LE) was used as the spray solvent, full+t-SIM was used as the collection method, and Liver+PF was used as the test sample. Other mass spectrometry imaging analyses were the same as in Example 4. See the specific test results. Fig.11 .

[0119] Depend on Fig.11 It can be seen that according to the ion intensity trend diagram under different spray voltages, when the spray voltage is 5000V, the intensity is the highest, which is the optimal spray voltage.

[0120] Example 7

[0121] 1. Samples to be tested

[0122] Kidney samples of mice gavaged with paeoniflorin (PF) were collected, and the ratio of the dosage mg to the mass kg of the mouse sample was 90:1. The sampling time points were 10min, 30min, 1h, 2h, and 5h. Prepare an embedding box, add an appropriate amount of embedding glue to the bottom of the embedding box to fix the sample, place the fixed mouse sample on the sample head of the slicer, cut frozen tissue sections of a set thickness, adhere and fix them on the Superfrost Plus positively charged anti-shedding slide, and use them as the sample to be tested. The cut sample to be tested was stored in a -80℃ ultra-low temperature refrigerator for subsequent imaging analysis.

[0123] 2. Standard samples

[0124] The standard of the administered drug paeoniflorin (PF) was dissolved in dimethyl sulfoxide, and the obtained standard stock solution was diluted with water to obtain a series of standard solutions of different concentrations. The concentrations of the standard solutions were 3.125, 6.25, 12.5, 25, 50, and 100 μM, and 0.5 μL was taken and spotted on blank tissue sections to obtain a series of standard samples of different concentrations.

[0125] 3. Determination

[0126] The samples to be tested and the standard samples were subjected to mass spectrometry imaging analysis using the AFAI-MSI imaging platform in series with the Q-Orbitrap mass spectrometer, and the drug content in the samples to be tested at different sampling time points was determined according to the standard curve method. Specifically, a series of standard samples were subjected to mass spectrometry imaging analysis to obtain a linear relationship between the concentration of the drug standard administered in the standard solution of a series of standard samples and the relative ion intensity (average drug peak intensity), and the corresponding standard working curve was drawn with each relative ion intensity corresponding to its corresponding concentration, and the regression equation of the standard working curve was calculated. The specific standard working curve is shown in Fig.12 , Table 3.

[0127] Then, the sample to be tested is subjected to mass spectrometry imaging analysis, and the relative ion intensity of the drug administered in the sample to be tested is obtained and substituted into the regression equation of the above standard working curve to calculate the content of the drug administered. Fig.13 .Depend on Fig.13 It can be seen that the distribution of paeoniflorin in the kidney at different sampling time points (the blue area is the drug, and the green area is the outline of the kidney sample). Substituting the mean value of the drug expression area into the above-fitted standard working curve can calculate the concentration value of paeoniflorin in the kidney sample, as shown in Table 3 below, which is the calculated value of kidney concentration at different sampling time points.

[0128] Table 3

[0129] Sampling time point Concentration (ug / g) 10min 5.96 30min 4.42 1h 28.8 2h N / A 5h N / A

[0130] The data acquisition scanning conditions of the imaging platform include: scanning mode: line-by-line scanning; the X-axis scanning length of the scanning area is 7 mm; the Y-axis scanning length of the scanning area is 10 mm; the scanning speed in the X-axis direction (horizontal displacement rate, Vx) is 0.2 mm / s; the step spacing between two adjacent scanning lines in the Y-axis direction (vertical displacement distance, Dy) is 0.1 mm; the spatial resolution is 100 μm*100 μm.

[0131] The measurement conditions of the mass spectrometer include: the spray solvent is 90% acetonitrile aqueous solution; the spray voltage is 5000V; the scanning acquisition mode is selected from Full scan and t-SIM. The capillary temperature is 350°C; the S Lens pressure is 55V; the ion polarity mode: positive ion mode; and the resolution is 60000.

[0132] When the scan acquisition mode is Full scan and t-SIM, the automatic gain of Full scan is 5e 6 , the automatic gain of t-SIM is 5e 5; The mass range of Full scan is 80-1000, and the mass range of t-SIM is 519.1; The maximum injection time of Full scan is 120ms, and the maximum injection time of t-SIM is 50ms. The adduct ion form is [M+K] + .

[0133] Example 8

[0134] In addition to drug detection, the quantitative drug mass spectrometry imaging method of Example 7 can also take into account the detection of endogenous components of tissues. In this experiment, a total of 467 endogenous components were detected (only in positive ion mode), see Fig.14 .Depend on Fig.14 It can be seen that, as shown in some typical endogenous component imaging images (10 minutes on the left and 30 minutes on the right), L-valine, creatine, Elaidicarnitine, Cholesterol glucuronide, PC (22:5 / P-18:1) and other samples expressed differently at different administration time points indicate that drugs have an effect on the metabolism of endogenous components in the organism. This indicates that while performing drug quantification, endogenous metabolites in the organism can also be visualized for further study of the drug's mechanism of action.

[0135] In summary, the highly sensitive quantitative drug mass spectrometry imaging method and its application provided by the present invention can effectively improve the sensitivity and detection coverage of drug mass spectrometry analysis methods, and can perform imaging analysis on endogenous metabolites in tissues. It is an innovative drug DMPK research method and has broad application prospects in the field of life sciences, pharmaceutical companies, and the cosmetics industry. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0136] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A quantitative drug mass spectrometry imaging method comprising the following steps: 1) After the mouse samples are administered, samples are taken at different time points, embedded and fixed, sliced, and adhered and fixed on a glass slide to obtain the samples to be tested; 2) Performing mass spectrometry imaging analysis on the sample to be tested to determine the distribution of the drug administered in the sample to be tested at different sampling time points, and determining the content of the drug administered in the sample to be tested at different sampling time points according to the standard curve method.

2. The quantitative drug mass spectrometry imaging method according to claim 1, characterized in that: In step 1), any one or more of the following conditions are included: A1) the mouse sample is selected from at least one of the liver or brain tissue of a mouse; A2) the administered drug is selected from at least one of paeoniflorin and amygdalin; A3) The mass ratio of the administered dose to the mouse sample is 50-120:1, mg / kg; A4) the different time points are selected from at least one of 10 min, 30 min, 1 h, 2 h, and 5 h after administration; A5) The embedding is to add embedding glue into the embedding box to fix the mouse sample; A6) The slicing is performed by slicing the embedded and fixed mouse sample using a microtome; A7) the slice thickness after slicing is 8-10 μm; A8) The slice size after slicing is 70-80 mm 2 ; A9) the glass slide is a positively charged anti-shedding glass slide; A10) The sample to be tested is placed in a glass slide box, covered with plastic wrap, and stored at -85 to -75°C.

3. The quantitative drug mass spectrometry imaging method according to claim 2, characterized in that: Also includes any one or more of the following conditions: A51) the embedding gel is Cryo-Gel embedding gel; A52) The embedding comprises: first adding embedding glue on the bottom support of the embedding box, then putting the mouse sample in and filling the embedding glue to completely cover it, and adding dry ice to solidify the embedding glue to fix the mouse sample; A61) The slicing conditions of the microtome are: the box temperature is -22 to -18°C; the sample head temperature is -20.5 to -20°C.

4. The quantitative drug mass spectrometry imaging method according to claim 1, characterized in that: In step 2), any one or more of the following conditions are included: B1) the mass spectrometry imaging analysis is performed using an imaging platform tandem mass spectrometer; B2) In the mass spectrometry imaging analysis, the data acquisition scanning conditions of the imaging platform include: scanning mode: line-by-line scanning mode; the X-axis scanning length of the scanning area is 6-9mm; the Y-axis scanning length of the scanning area is 8-11mm; the scanning speed in the X-axis direction is 0.18-0.21mm / s; the step spacing between two adjacent scanning lines in the Y-axis direction is 0.098-0.12mm; the spatial resolution is 99-101μm*99-101μm; B3) In the mass spectrometry imaging analysis, the measurement conditions of the mass spectrometer include: the spray solvent is a 70-100% acetonitrile aqueous solution; the spray voltage is 4900-5100V; the scanning acquisition mode is selected from at least one of PRM, Full scan or t-SIM; B4) In the mass spectrometry imaging analysis, when the administered drug is paeoniflorin and / or amygdalin, the adduct ion form of the administered drug is selected from [M+H] + 、[M+Na] + or [M+K] + At least one of them.

5. The quantitative drug mass spectrometry imaging method according to claim 4, characterized in that: In item B3), the measurement conditions of the mass spectrometer also include: capillary temperature of 345-355°C; S Lens pressure of 50-60V; ion polarity mode: positive ion mode; resolution: 59000-61000; scanning acquisition mode is Full scan and t-SIM; Preferably, when the scanning acquisition mode is Full scan and t-SIM, the automatic gain of Full scan is 4-6e 6 , the automatic gain of t-SIM is 4-6e 5 ; The mass range of Full scan is 70 to 1050, and the mass range of t-SIM is 519.1±3; The maximum injection time for Full scan is 120±3ms, and the maximum injection time for t-SIM is 50±2ms.

6. The quantitative drug mass spectrometry imaging method according to claim 1, characterized in that: In step 2), determining the content of the administered drug in the sample to be tested at different sampling time points according to the standard curve method comprises the following steps: A) adding a drug standard to be administered to a first solvent for dissolution, adding a second solvent for dilution of the obtained standard stock solution, and spotting a series of standard solutions of different concentrations on a blank glass slide to obtain standard samples; B) performing mass spectrometry imaging analysis on the standard sample and the sample to be tested in step 1), respectively, and determining the content of the administered drug in the sample to be tested at different sampling time points according to the standard curve method.

7. The quantitative drug mass spectrometry imaging method according to claim 6, characterized in that: Includes any one or more of the following conditions: C1) In step A), the drug standard substance administered is selected from at least one of a paeoniflorin standard substance or amygdalin standard substance; C2) In step A), the first solvent is dimethyl sulfoxide; C3) In step A), the drug concentration in the standard stock solution is 9-11 mM; C4) In step A), the second solvent is water; C5) In step A), the drug concentration administered in the standard solution is 1-100 μM; C6) In step A), the amount of the standard solution spotted on the blank glass slide is 0.4-0.6 μL.

8. A method for preparing a matrix simulation sample for quantitative drug mass spectrometry imaging, comprising: The mouse sample is added to the extraction solvent and ground to homogenize, the obtained homogenate is centrifuged, the supernatant is added to the compound solution and mixed, and the obtained mixed solution is dropped onto a glass slide and waited for the droplet to evaporate completely to provide a matrix simulation sample.

9. The method for preparing a matrix simulation sample for quantitative drug mass spectrometry imaging according to claim 6, characterized in that: Includes any one or more of the following conditions: D1) the mouse sample is selected from at least one of the liver or brain tissue of the mouse; D2) the extraction solvent is water; D3) the ratio of the mass of the mouse sample added to the volume of the extraction solvent added is 1:8-10, g / mL; D4) steel beads are added during the grinding; preferably, the diameter of the steel beads is 2-4 mm; the ratio of the number of steel beads added to the mass of the mouse sample added is 3:49-51, pieces / mg; D5) the grinding temperature is 3-5°C; D6) the grinding frequency is 3-4 Hz; D7) The grinding time is 1-3min; D8) the centrifugal speed is 1000-3000g; D9) the centrifugation temperature is 3-5°C; D10) the centrifugation time is 1-3min; D11) the compound solution is water; D12) The volume ratio of the supernatant to the compound solution added is 48-50: 0.9-1.1； D13) The mixing condition is vortex mixing at room temperature for 1-3 minutes; D14) The volume of the supernatant added onto the glass slide is 0.5-2 μL.

10. A method for screening analytical conditions for quantitative drug mass spectrometry imaging, comprising: using a matrix simulation sample prepared by the method for preparing a matrix simulation sample for quantitative drug mass spectrometry imaging according to any one of claims 8 to 9, selecting different conditions for mass spectrometry imaging analysis, and comparing and determining the quantitative analysis conditions for drug mass spectrometry imaging.

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