Method for detecting glucocorticoids based on in situ oximation derivatization and desi-pi mass spectrometry imaging

Abstract

The application discloses a glucocorticoid detection method based on in-situ oxime derivatization and DESI-PI mass spectrum imaging in the field of mass spectrum imaging analysis, and steps include: applying a derivatization reagent to the surface of a tissue sample slice containing glucocorticoid, and contacting the tissue sample with the derivatization reagent; under the in-situ oxime derivatization reaction of the tissue sample slice, the carbonyl in the glucocorticoid molecule reacts with the hydroxylamine of the derivatization reagent to generate an oxime product containing a C=NOH group; the tissue sample slice is detected by using desorption electrospray ionization (DESI) and secondary photoionization (PI) mass spectrum imaging technology, the mass spectrum signal of the glucocorticoid oxime product is obtained, and in-situ detection of the glucocorticoid is realized. Through two-step signal enhancement strategies: derivatization enhances the ionization efficiency of DESI, and secondary photoionization enhances the signal intensity of the derivatization product, the detection sensitivity of the glucocorticoid is significantly improved (three orders of magnitude), and high-sensitivity in-situ molecular imaging of trace glucocorticoid on the surface of the tissue is realized.

Description

Technical Field

[0001] This invention relates to the field of mass spectrometry imaging analysis, specifically to a method for detecting glucocorticoids based on in-situ oxime derivatization and DESI-PI mass spectrometry imaging. Background Technology

[0002] Glucocorticoids are a class of steroidal anti-inflammatory drugs widely used in clinical practice. Research on their distribution, metabolism, and mechanisms of action in vivo is crucial for guiding rational drug use. Traditional liquid chromatography-mass spectrometry (LC-MS) is currently a common method for studying drugs and metabolites in blood and tissue homogenates, used to assess drug efficacy and toxicity, and to study drug regulatory mechanisms. However, these methods cannot detect the distribution and concentration of drugs in tissues and lesion micro-areas in situ. Determining the distribution of drugs and their metabolites in tissues is a crucial step in drug development and also an important approach to studying the molecular mechanisms of drug action.

[0003] Mass spectrometry imaging is a powerful, label-free, high-coverage, high-sensitivity, and high-specificity in-situ molecular imaging technique based on mass spectrometry analysis. It can simultaneously present the spatial distribution and relative abundance of hundreds or thousands of known and unknown compounds on the surface of a tissue sample in a single experiment. In-situ mass spectrometry imaging techniques, such as desorption electrospray ionization (DESI), can directly image tissue surfaces under ambient pressure and in an open environment without any pretreatment. However, due to the influence of its ionization mechanism, DESI ion sources have very low sensitivity for weakly polar and neutral compounds and are easily affected by matrix effects in the sample. Because glucocorticoids are weakly polar, their ionization efficiency is extremely low under traditional DESI sources, making them almost undetectable. Therefore, this severely limits the application of DESI in the in-situ detection of trace glucocorticoids on tissue surfaces. Photoionization (PI) is a soft ionization source with high sensitivity for detecting nonpolar and weakly polar compounds. At the same time, it has a strong ability to interfere with the matrix effects of endogenous substances such as inorganic salts and phospholipids in complex biological tissues.

[0004] Therefore, developing an in-situ mass spectrometry imaging method that can significantly improve the detection sensitivity of weakly polar glucocorticoid drugs on the surface of complex biological tissues is of great scientific significance and application value for drug distribution, metabolism and toxicology studies. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting glucocorticoids based on in situ oxime derivatization and DESI-PI mass spectrometry imaging, in order to solve the problem that glucocorticoid drugs are difficult to detect with high sensitivity in complex biological tissue imaging due to their weak polarity and low ionization efficiency in the prior art.

[0006] The present invention achieves the above objectives through the following technical solutions: A glucocorticoid detection method based on in situ oxime derivatization and DESI-PI mass spectrometry imaging includes the following steps: S1. Apply the derivatization reagent to the surface of a tissue sample section containing glucocorticoids, so that the tissue sample comes into contact with the derivatization reagent; S2. In the in-situ oxime derivatization reaction of tissue sample sections, the carbonyl group in the glucocorticoid molecule reacts with the hydroxylamine of the derivatization reagent to generate an oxime product containing a C=NOH group. S3. Desorption electrospray ionization (DESI) and secondary photoionization (PI) mass spectrometry imaging techniques are used to detect tissue sample sections and obtain mass spectrometry signals (molecular ion peaks or quasi-molecular ion peaks) of glucocorticoid oxime products, thereby achieving in-situ detection of glucocorticoids.

[0007] A further improvement is that the derivatizing reagent is hydroxylamine hydrochloride.

[0008] A further improvement is that the glucocorticoid is cortisone.

[0009] A further improvement is that the derivatizing reagent is sprayed onto the surface of the tissue sample section using an HTX matrix sprayer, with the following spraying parameters: nozzle temperature 40 ℃, high-purity nitrogen pressure 10 psi, derivatizing reagent stock solution concentration 1 mg / mL, nozzle flow rate 0.05 mL / min, nozzle moving speed 1200 mm / min, spraying interval 2 mm, nozzle operating mode CC, gas flow rate 3 L / min, drying time 20 s, nozzle height 40 mm, and spraying times 20 times.

[0010] A further improvement is that the in-situ oxime derivatization reaction is carried out at room temperature.

[0011] A further improvement is that the concentration of the derivatizing reagent is 0.5-2 mg / mL, and the amount of derivatizing reagent on the surface of the tissue sample section after application is 0.0002-0.0010 mg / mm², and the in-situ oxime derivatization reaction time is 1-12 hours.

[0012] A further improvement is that the concentration of the derivatizing reagent is 1 mg / mL, and the amount of derivatizing reagent on the surface of the tissue sample slice after application is 0.000417 mg / mm², and the in-situ oxime derivatization reaction time is 4 hours.

[0013] A further improvement is that the desorption electrospray ionization (DESI) and secondary photoionization (PI) mass spectrometry imaging technologies are performed using a DESI / PI-MSI device, and the parameters of the DESI / PI-MSI device are set as follows: distance between the nozzle and the sample surface 1-3 mm, angle 40-65°, distance between the nozzle and the transfer tube 1-2 cm, angle between the transfer tube and the sampling platform 10-30°, spray solvent flow rate 1-3 μL / min, nebulizing gas pressure 1-3 MPa, spray voltage 2-5 kV, photoionization using a vacuum ultraviolet discharge lamp, and the mass spectrometer selected in positive ion mode.

[0014] A further improvement is that the spray solution used in the DESI / PI-MSI device is methanol, toluene, and trifluoroacetic acid in a volume ratio of 70:30:0.1.

[0015] A further improvement is that the tissue sample section is a whole-body zebrafish tissue section or a mammalian tissue section.

[0016] The principle of this invention is as follows: A polar C=NOH group is introduced onto the carbonyl group of glucocorticoids through in-situ oxime reaction, significantly increasing the polarity of the compound and thus enhancing its ionization efficiency under a DESI ion source (first-step enhancement). The ions and neutral molecules after DESI desorption and ionization further enter the photoionization chamber and are re-ionized by vacuum ultraviolet light, resulting in a secondary enhancement of the signal intensity of the derivatized product (second-step enhancement). Through the synergistic effect of in-situ derivatization and secondary photoionization, highly sensitive detection and imaging of trace glucocorticoid drugs on tissue surfaces are achieved.

[0017] The beneficial effects of this invention are as follows: (1) For the first time, in situ oxime derivatization reaction was combined with DESI / PI-MSI technology, and a two-step signal enhancement strategy was proposed, which improved the detection sensitivity of cortisone in complex biological tissues by three orders of magnitude; (2) The derivatization reaction can be carried out at room temperature, and the operation is simple and does not require complicated pretreatment steps; (3) This method has good linear quantitative ability (R²>0.99) and is suitable for qualitative and quantitative analysis of trace glucocorticoids on tissue surface; (4) Spatial distribution information of exogenous drugs and multiple endogenous metabolites can be obtained in the same experiment, providing highly sensitive in situ molecular imaging technology for drug metabolism mechanism and toxicology research. Attached Figure Description

[0018] Figure 1 DESI and DESI / PI mass spectra of rat brain tissue homogenate containing cortisone before and after derivatization; Figure 2A bar chart showing the average signal intensity of cortisone and its derivatives under different spraying times of the derivatizing reagent; Figure 3 A bar chart showing the average signal intensity of cortisone parent compound and derivatives at different oxime reaction times; Figure 4 Standard curves of the parent compound and derivatives of cortisone before and after derivatization; Figure 5 DESI / PI images of cortisone matrix, oxime products, and representative endogenous compounds in zebrafish tissue sections after drug administration. Detailed Implementation

[0019] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0020] Example 1: Cortisone oxime derivatization and DESI / PI detection of tissue homogenate Take 100 μL of 0.1 mg / mL cortisone methanol solution, add 100 μL of 2 mg / mL hydroxylamine hydrochloride aqueous solution and 100 μL of mouse brain tissue homogenate, and mix and react at room temperature for 2 hours. Take 1.2 μL of the reaction solution and add it dropwise into the well of a PVC membrane. After drying at room temperature, add another 1.2 μL, for a total of 2.4 μL. After drying, detect the solution in DESI and DESI / PI modes, respectively.

[0021] See Figure 1 Without the addition of derivatizing reagents, cortisone parent compound ( m / z 361.20) was almost undetectable in DESI mode. After adding the derivatizing reagent, the cortisone oxime product signal peak could be clearly detected in DESI mode. m / z (376.21); In DESI / PI mode, the signal of this product is further enhanced, and the peak of the parent compound basically disappears.

[0022] Example 2: Optimization of Derivatized Reagent Spraying Amount A 0.05 mg / mL cortisol-containing tissue homogenate was prepared, dropped into the wells of a PVC membrane, and dried. A 1 mg / mL hydroxylamine hydrochloride solution was sprayed onto the tissue homogenate surface using an HTX matrix sprayer. The spraying times were set to 10, 20, and 30 times, with corresponding derivatization reagent densities of 0.000208, 0.000417, and 0.000625 mg / mm², respectively. After reacting at room temperature for 4 hours, DESI / PI was measured.

[0023] See Figure 2 The results showed that when the number of sprayings increased from 10 to 20, the signal intensity of the derivative increased significantly from 12780 to 19209; when the number of sprayings increased to 30, the signal intensity only increased slightly to 20056, reaching saturation. Therefore, the optimal number of sprayings was determined to be 20, corresponding to a derivatizing reagent density of 0.000417 mg / mm².

[0024] Example 3: Optimization of Derivatization Reaction Time See Figure 3 Under optimal spraying conditions, DESI / PI measurements were performed at 1, 2, 4, 6, 9, and 12 hours after spraying the derivatizing reagent. The results showed that the signal intensity of the derivatized product increased rapidly within 1-4 hours, reaching its maximum at 4 hours; the signal remained relatively stable from 4-6 hours, and there was no significant increase after 12 hours. Considering both time cost and signal gain, the optimal reaction time was determined to be 4 hours.

[0025] Example 4: Sensitivity Enhancement Effect and Quantitative Ability Verification Prepare a series of brain tissue homogenates with final cortisone concentrations of 0, 9, 30, 60, 120, and 240 μg / mL. Spray one homogenate with hydroxylamine hydrochloride derivatizing reagent, and leave the other unsprayed. Detect in DESI and DESI / PI modes, respectively.

[0026] See Figure 4 The results showed that the signal intensity of the cortisone derivative in DESI mode was 5.4 times higher than that of the underrendered product (standard curve slope 3.105 / 0.576 = 5.4). After derivatization, the standard curve R² in DESI / PI mode was 0.995, and the standard curve slope was 563.538, which was 181.5 times higher than the slope in the derivatized DESI mode (standard curve slope 563.538 / 3.105 = 181.5). Through these two enhancement steps, the detection sensitivity of cortisone was improved by approximately three orders of magnitude.

[0027] Example 5: In-situ derivation and imaging analysis of cortisol in zebrafish Zebrafish were exposed to an aqueous solution containing 40 μg / mL cortisone for 1 hour, washed, euthanized, and then subjected to whole-body frozen sections (16 μm thick). A 1 mg / mL hydroxylamine hydrochloride solution was sprayed onto the sections using an HTX matrix sprayer under optimal conditions, and DESI / PI-MSI imaging was performed after 4 hours of reaction.

[0028] See Figure 5 The results showed that cortisone oxime products ( m / z 376.21) is mainly distributed in the stomach tissue of zebrafish, with a weak distribution in the oral cavity, consistent with the pharmacological characteristics of cortisone absorption in the digestive tract after oral administration. Simultaneously, endogenous choline ( m / z104.07) is distributed in the eye and brain regions, creatine ( m / z 132.08) and cholesterol ( m / z (369.35) was mainly distributed in the brain and spinal cord, verifying the compatibility of the method in simultaneous imaging of endogenous metabolites.

[0029] As can be seen from the above, the method used in this invention is simple to operate and has good repeatability. It can be widely applied in the fields of tissue distribution analysis, efficacy and toxicity evaluation, and drug metabolism mechanism research in the drug development stage, providing a feasible technical solution for in-situ high-sensitivity detection and imaging of trace glucocorticoids and other carbonyl drugs.

[0030] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for detecting glucocorticoids based on in situ oxime derivatization and DESI-PI mass spectrometry imaging, characterized in that the steps are as follows: include: S1. Apply the derivatization reagent to the surface of a tissue sample section containing glucocorticoids, so that the tissue sample comes into contact with the derivatization reagent; S2. In the in-situ oxime derivatization reaction of tissue sample sections, the carbonyl group in the glucocorticoid molecule reacts with the hydroxylamine of the derivatization reagent to generate an oxime product containing a C=NOH group. S3. Desorption electrospray ionization (DESI) and secondary photoionization (PI) mass spectrometry imaging techniques are used to detect tissue sample sections, obtain mass spectrometry signals of glucocorticoid oxime products, and realize in-situ detection of glucocorticoids.

2. The method for detecting glucocorticoids based on in situ oxime derivatization and DESI-PI mass spectrometry imaging according to claim 1, characterized in that, The derivatizing reagent is hydroxylamine hydrochloride.

3. The method for detecting glucocorticoids based on in-situ oxime derivatization and DESI-PI mass spectrometry imaging according to claim 1, characterized in that, The glucocorticoid mentioned is cortisone.

4. The method for detecting glucocorticoids based on in-situ oxime derivatization and DESI-PI mass spectrometry imaging according to claim 1, characterized in that, The derivatizing reagent was sprayed onto the surface of tissue sample sections using an HTX matrix sprayer, with the following spraying parameters: nozzle temperature 40 ℃, high-purity nitrogen pressure 10 psi, derivatizing reagent stock solution concentration 1 mg / mL, nozzle flow rate 0.05 mL / min, nozzle movement speed 1200 mm / min, spraying interval 2 mm, nozzle operation mode CC, gas flow rate 3 L / min, drying time 20 s, nozzle height 40 mm, and 20 spraying cycles.

5. The method for detecting glucocorticoids based on in-situ oxime derivatization and DESI-PI mass spectrometry imaging according to claim 1, characterized in that, The in-situ oxime derivatization reaction is carried out at room temperature.

6. The method for detecting glucocorticoids based on in situ oxime derivatization and DESI-PI mass spectrometry imaging according to claim 1, characterized in that, The concentration of the derivatizing reagent is 0.5-2 mg / mL, and the amount of derivatizing reagent on the surface of the tissue sample section after application is 0.0002-0.0010 mg / mm. 2 The in-situ oxime derivatization reaction takes 1-12 hours.

7. The method for detecting glucocorticoids based on in situ oxime derivatization and DESI-PI mass spectrometry imaging according to claim 6, characterized in that, The concentration of the derivatizing reagent is 1 mg / mL, and the amount of derivatizing reagent on the surface of the tissue sample section after application is 0.000417 mg / mL. 2 The in-situ oxime derivatization reaction time is 4 hours.

8. The method for detecting glucocorticoids based on in situ oxime derivatization and DESI-PI mass spectrometry imaging according to claim 1, characterized in that, The desorption electrospray ionization (DESI) and secondary photoionization (PI) mass spectrometry imaging techniques are performed using a DESI / PI-MSI device. The DESI / PI-MSI device parameters are set as follows: distance between the nozzle and the sample surface 1-3 mm, angle 40-65°, distance between the nozzle and the transfer tube 1-2 cm, angle between the transfer tube and the sampling platform 10-30°, spray solvent flow rate 1-3 μL / min, nebulizing gas pressure 1-3 MPa, spray voltage 2-5 kV, photoionization using a vacuum ultraviolet discharge lamp, and the mass spectrometer selected in positive ion mode.

9. The method for detecting glucocorticoids based on in-situ oxime derivatization and DESI-PI mass spectrometry imaging according to claim 8, characterized in that, The spray solution used in the DESI / PI-MSI device is methanol, toluene, and trifluoroacetic acid in a volume ratio of 70:30:0.

1.

10. The method for detecting glucocorticoids based on in-situ oxime derivatization and DESI-PI mass spectrometry imaging according to claim 1, characterized in that, The tissue sample sections are zebrafish whole-body tissue sections or mammalian tissue sections.

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