Method for identifying drug carboxylic acid metabolite structure based on derivatization reagent and high-resolution mass spectrum

Abstract

The invention relates to the field of biological medicine, in particular to a method for identifying a drug carboxylic acid metabolite structure based on a derivatization reagent and high-resolution mass spectrometry. The method comprises the following steps: pre-treating a biological sample (such as plasma, urine, excrement and the like), extracting a solvent, precipitating protein, centrifuging and collecting supernate; drying the supernatant with liquid nitrogen and redissolving; reacting the carboxylic acid metabolite with a derivatization reagent trimethylsilyl diazomethane at 30-60 DEG C for 1-60 minutes to enable the carboxylic acid metabolite to generate a methylation product; the carboxylic acid metabolite is identified through molecular weight change (+ 14Da) by utilizing high-resolution mass spectrometry detection. The method is high in accuracy, simple and convenient to operate, low in cost and suitable for structure identification of various biological matrixes and conventional small molecules, polypeptides and other carboxylic acid-containing metabolites, has important application value in early drug research and development and metabolism research, and can be widely applied to the fields of early drug research and development, drug metabolism research and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a method for identifying the structure of drug carboxylic acid metabolites based on a derivatization reagent and high-resolution mass spectrometry. Background Art

[0002] The metabolic process of drugs in the body is closely related to the efficacy and safety of the drugs. During the drug development process, it is necessary to detect and identify the main metabolites of drugs in the body and determine the specific structure of the metabolites. As an exogenous substance, drugs enter the body and the body tends to metabolize them into molecules with greater polarity and better water solubility, such as carboxylic acid, glucuronic acid and sulfate-conjugated metabolites, which are then excreted from the body, thereby playing a detoxifying role. Among them, carboxylic acid is a metabolite structure form after the metabolism of many structural types. For example, fatty alcohols are oxidized and metabolized into aldehydes and then oxidized to form carboxylic acids, lactones are hydrolyzed and ring-opened to form carboxylic acids, and oxygen or nitrogen atoms are oxidized and dealkylated to form carboxylic acids. These are all common metabolic pathways.

[0003] However, in the process of drug development, it is challenging to determine the chemical structure of carboxylic acid metabolites. The pharmaceutical industry often uses high-resolution mass spectrometry to identify the structure of drug metabolites. The molecular weight information of the metabolites is obtained through the primary mass spectrometry information of the high-resolution mass spectrometry. The change in the elemental composition is inferred from the accurate molecular weight change of the metabolite relative to the parent drug, and finally the specific information of the structure is obtained through the secondary mass spectrometry information. For carboxylic acid metabolites, the elemental composition can be preliminarily determined using the molecular weight information of the primary mass spectrometry of the high-resolution mass spectrometry. For example, lactone or lactam is hydrolyzed into carboxylic acid (+H2O, the molecular weight increases by 18 Da) ( Figure 1 Case 1), oxidation of alcohol to form carboxylic acid (+O-2H, molecular weight increased by 14 Da) ( Figure 1 Case 2), amide hydrolysis to carboxylic acid ( Figure 1 Case 3), nitrogen or oxygen heterocyclic ring is oxidized to carboxylic acid (+2O, molecular weight increased by 32 Da) ( Figure 1 Case 4 in the above discussion). While it can be preliminarily determined that a carboxylic acid metabolite is formed, other structural metabolites are also possible. For example, the oxidation of an alcohol to form a carboxylic acid with a molecular weight increase of 14 Da could be due to oxidative dehydrogenation; the oxidative ring-opening of a nitrogen or oxygen heterocycle to a carboxylic acid with a molecular weight increase of 32 Da could be due to double oxidation; the hydrolysis of a lactone or lactam to a carboxylic acid with a molecular weight increase of 18 Da could be due to oxidative ring-opening to an alcohol, and so on. Further confirmation of the structure of the carboxylic acid metabolite based on secondary data from high-resolution mass spectrometry also presents some difficulties. In some cases, the presence of 44 or 46 Da of deoxygenation fragments on the mass spectrum can initially indicate a carboxylic acid, but in many cases, these typical fragments are not produced, making accurate confirmation of the carboxylic acid metabolite extremely difficult.

[0004] Several other approaches can confirm the results of carboxylic acid metabolites. One is to isolate the carboxylic acid metabolite using preparative liquid phase separation to obtain a standard of at least 1 mg, followed by structural confirmation using nuclear magnetic resonance spectroscopy. However, isolating and synthesizing metabolite standards is difficult in the early stages of drug development. This method in biological matrices requires obtaining large amounts of sample to maintain the sensitivity of the nuclear magnetic resonance instrument, which is time-consuming and expensive. Alternatively, derivatization can be used to remove the carboxylic acid group, thereby obtaining a specific mass spectrometric signal. David W. Johnson reported a derivatization method for carboxylic acid drugs. The specific process involves preparing alkyl dimethylaminoethyl ester iodide by quaternization of dimethylaminoethyl ester with alkyl iodide. Plasma samples are then processed, and the labeled fatty acid mixture is mixed with plasma, acetonitrile, and hydrochloric acid, followed by heating. The mixture is then extracted with hexane, evaporated, reacted with oxalyl chloride, and then with dimethylaminoethanol. Finally, the product is quaternized by the addition of methyl iodide to form the derivative. The residue is dissolved in an acetonitrile / water / formic acid mixture for analysis by electrospray tandem mass spectrometry. However, this method has a long process, requiring heating to a high temperature environment of 100°C and the use of some highly reactive toxic reagents such as iodomethane. Overall, this method is time-consuming and unsafe.

[0005] Currently, there is a lack of an efficient, accurate, effective, and low-cost method to identify the structures of drug carboxylic acid metabolites, which poses many challenges to the research of drugs that exhibit such metabolic reactions. Summary of the Invention

[0006] The main purpose of the present invention is to provide an efficient, low-cost, rapid and safe method for identifying the structure of drug carboxylic acid metabolites, so as to solve the problem in the prior art that it is difficult to accurately confirm carboxylic acid metabolites.

[0007] In order to achieve the above object, the present invention provides the following technical solutions: A method for identifying the structure of drug carboxylic acid metabolites based on chemical derivatization and high-resolution mass spectrometry comprises the following steps: Step 1: Sample pretreatment: Take a biological sample, add an extraction solvent, vortex and centrifuge, and collect the supernatant; the biological sample includes plasma, urine, feces, hepatocytes or liver microsomes; the extraction solvent is methanol or acetonitrile, and the volume of the extraction solvent is 3-4 times the volume of the biological sample; Step 2, drying with nitrogen and re-dissolving: drying the supernatant with nitrogen, adding a re-dissolving solvent for re-dissolving to obtain a re-solution solution; the re-dissolving solvent is one or a mixed solvent of methanol, acetonitrile, water or dimethyl sulfoxide; Step 3, carboxylic acid derivatization reaction: the reconstituted solution is mixed with a derivatization reagent, trimethylsilyldiazomethane, and reacted at 30-60° C. for 1-60 minutes to form a carboxylic acid methylation product; the concentration of the trimethylsilyldiazomethane is 0.1-1 mol / L; Step 4: High-resolution mass spectrometry detection: The solution after the reaction in step 3 is injected into a high-resolution mass spectrometer for detection to analyze the change in molecular weight. If a molecular weight change of +14 Da occurs, it is identified as a carboxylic acid metabolite.

[0008] Furthermore, when the biological sample is a plasma sample, the extraction solvent is acetonitrile, the added volume is 3 times the volume of the plasma sample, the sample is vortexed for 1 minute, and the centrifugation conditions are 15,000 rpm for 10 minutes.

[0009] Furthermore, the temperature of the nitrogen drying is 30-40°C.

[0010] Furthermore, in step 2, the reconstitution solvent is acetonitrile, and the reconstitution volume is adjusted according to the sample concentration and subsequent experimental requirements.

[0011] Furthermore, in step three, the mixing ratio of the complex solution and trimethylsilyldiazomethane is 1:1.

[0012] Furthermore, in the high-resolution mass spectrometry detection, the ion source is ESI (+), the ion source spray voltage is 3.8 kV, the capillary temperature is 320°C, the sheath gas is 40 units, the auxiliary gas is 10 units, the purge gas is 0 units, the S-lens is 55 units, the collision energy NCE is 25, 45, and 60, the scan mode is Full MS and MS2, and the resolution is Full Scan 35000 and MS2 17500.

[0013] Application of the above method in identifying carboxylic acid metabolites.

[0014] Beneficial effects of the present invention: 1. High Accuracy: The specific derivatization reagent trimethylsilyldiazomethane (TMSCHN2) reacts with carboxylic acid metabolites to generate characteristic methylated products (molecular weight increase of +14 Da). Combined with the precise molecular weight and fragment information of high-resolution mass spectrometry, this significantly improves the accuracy and sensitivity of structural identification of carboxylic acid metabolites, avoiding misidentification caused by the lack of typical fragments in traditional mass spectrometry.

[0015] 2. High efficiency: The pretreatment steps are simple, requiring only protein precipitation, centrifugation, air drying and resolubilization, and derivatization under mild conditions (30-60°C, 1-60 minutes). No complex separation or high-temperature operation is required. In addition, mass spectrometry detection is fast, allowing batch analysis of samples and significantly shortening the experimental cycle.

[0016] 3. Low cost: The reagent TMSCHN2 used is inexpensive and requires minimal dosage. Experimental equipment and solvents (methanol, acetonitrile, etc.) are all conventional consumables. There is no need to prepare high-purity standards or use toxic reagents (such as iodomethane), thus reducing experimental costs.

[0017] 4. The reaction conditions are mild, avoiding the high temperature of 100°C and the use of toxic reagents in existing derivatization methods, making the operation safer.

[0018] 5. Applicable to various matrices: The present invention is applicable to various biological samples, including plasma, urine, feces, etc., and can meet the needs of different drug metabolism studies.

[0019] 6. Applicable to carboxylic acid metabolites of various drugs: The present invention is applicable to carboxylic acid metabolites of various types of drugs, including carboxylic acid products of conventional small molecule drugs, as well as metabolites containing multiple carboxylic acids such as peptides, and has good applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Carboxylic acid metabolites produced by different metabolic reactions and the corresponding molecular weight changes.

[0021] Figure 2 Figure 2 High-resolution mass spectrometry primary spectra and corresponding chemical structures of plasma samples containing 10 μM diclofenac before and after TMSCHN2 treatment. A: without TMSCHN2 treatment; B: after TMSCHN2 treatment.

[0022] Figure 3 The high-resolution mass spectrometry primary spectra and corresponding chemical structures of 10 μM semaglutide plasma samples before and after TMSCHN2 treatment, A: without TMSCHN2 treatment; B: after TMSCHN2 treatment.

[0023] Figure 4 Flowchart for the identification of carboxylic acid metabolites using the derivatization method. DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected. Example 1

[0025] 1. Experimental Preparation: Two drugs containing carboxylic acid structures, diclofenac and semaglutide, were prepared in dimethyl sulfoxide and water, respectively, to obtain stock solutions with a concentration of 10 mM. The stock solutions were then diluted 1000-fold to 10 μM in plasma to simulate actual in vivo plasma samples containing carboxylic acid metabolites.

[0026] Prepare the methylation reagent TMSCHN2 at a concentration of 1 mol / L in a mixture of methanol / acetonitrile / water (1:1:1), the extraction solvent acetonitrile, the reconstitution solvent acetonitrile, and experimental equipment and reagents such as a high-resolution mass spectrometer.

[0027] Calibrate and debug the high-resolution mass spectrometer to ensure its stable performance and accurate molecular weight measurement.

[0028] 2. Pre-processing steps: The plasma sample was mixed with three volumes of the extraction solvent acetonitrile, shaken, and then centrifuged (15,000 rpm, 10 min) to remove the biological matrix such as plasma proteins in the lower layer and collect the upper layer containing the extract containing potential carboxylic acid metabolites.

[0029] 3. Nitrogen drying and re-dissolution: Transfer the extracted product to a nitrogen blow dryer and set the temperature to 30-40°C and the nitrogen flow rate to dry it. Ensure that the residual extraction solvent is completely removed while avoiding the loss of potential carboxylic acid metabolites.

[0030] After drying, add 200 μL of acetonitrile for reconstitution, depending on the nature of the sample and the requirements of subsequent experiments. The optimal reconstitution solvent and volume can be determined experimentally to ensure sufficient dissolution of potential carboxylic acid metabolites.

[0031] 4. Reaction with derivatization reagent TMSCHN2: Mix the reconstituted solution with 1 mol / L TMSCHN2 in a predetermined ratio of 1:1. Accurate mixing can be performed using a micropipette or autosampler.

[0032] Place the mixed solution in a suitable reaction vessel, such as a polypropylene test tube or glass vial, and then place it in a thermostatic water bath for reaction. Control the reaction temperature (1 min–60 min) and time to ensure adequate derivatization reaction.

[0033] High-resolution mass spectrometry detection: The reaction solution was injected into a high-resolution mass spectrometer via an autosampler. Appropriate mass spectrometry parameters, such as ion source type, scan range, and resolution, were set as follows.

[0034] Mass spectrometer: Q Exactive Plus (Thermo) Ion source: ESI (+) Ion source spray voltage: 3.8 kV Capillary temperature: 320°C Sheath air: 40 units Auxiliary gas: 10 units Purge gas: 0 units S-lens: 55 units Collision Energy (NCE): 25, 45, 60 Scan mode: Full MS, MS 2 Resolution: 35000 (Full Scan) and 17500 (MS 2 ) Perform mass spectrometry analysis on the injected solution and record the molecular weight information and mass spectrum. The experimental results are as follows: Figure 2 and Figure 3 shown.

Claims

1. A method for identifying the structure of drug carboxylic acid metabolites based on chemical derivatization and high-resolution mass spectrometry, characterized in that: The following steps are involved: Step 1: Sample pretreatment: taking a biological sample, adding an extraction solvent, vortexing, centrifuging, and collecting the supernatant; the biological sample includes a plasma sample; the extraction solvent is acetonitrile, and the volume of the extraction solvent is 3 times the volume of the biological sample; Step 2, drying with nitrogen and re-dissolving: drying the supernatant with nitrogen, adding a re-dissolving solvent for re-dissolving to obtain a re-solution solution; the re-dissolving solvent is acetonitrile; Step 3, carboxylic acid derivatization reaction: the reconstituted solution is mixed with a derivatization reagent, trimethylsilyldiazomethane, and reacted at 30-60° C. for 1-60 minutes to form a carboxylic acid methylation product; the concentration of the trimethylsilyldiazomethane is 0.1-1 mol / L; Step 4: High-resolution mass spectrometry detection: The solution after the reaction in step 3 is injected into a high-resolution mass spectrometer for detection to analyze the change in molecular weight. If a molecular weight change of +14 Da occurs, it is identified as a carboxylic acid metabolite.

2. The method according to claim 1, characterized in that When the biological sample is a plasma sample, the extraction solvent is acetonitrile, the added volume is 3 times the volume of the plasma sample, vortexing is performed for 1 minute, and the centrifugation condition is 15000 rpm for 10 minutes.

3. The method according to claim 1, characterized in that The temperature of the nitrogen drying is 30-40°C.

4. The method according to claim 1, wherein In step 3, the mixing ratio of the reconstituted solution and trimethylsilyldiazomethane is 1:

1.

5. The method according to claim 1, wherein In the high-resolution mass spectrometry detection, the ion source is ESI(+), the ion source spray voltage is 3.8 kV, the capillary temperature is 320°C, the sheath gas is 40 units, the auxiliary gas is 10 units, the purge gas is 0 units, the S-lens is 55 units, the collision energy NCE is 25, 45, and 60, the scan mode is Full MS and MS2, and the resolution is Full Scan 35000 and MS2 17500.

6. Use of the method according to any one of claims 1 to 5 in identifying carboxylic acid metabolites.

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