A method for identifying the structure of drug carboxylic acid metabolites based on derivatizing reagents and high resolution mass spectrometry

Abstract

The application relates to the field of biological medicine, and particularly relates to a method for identifying structures of drug carboxylic acid metabolites based on a derivatization reagent and high-resolution mass spectrometry. The method comprises the following steps: performing pretreatment on a biological sample (such as blood plasma, urine, feces and the like), precipitating proteins through an extraction solvent, and then centrifuging to collect supernatant; blowing nitrogen to dry the supernatant and redissolving; reacting with a derivatization reagent trimethylsilyl diazomethane at 30-60 DEG C for 1-60 minutes to make carboxylic acid metabolites generate methylation products; and identifying the carboxylic acid metabolites through molecular weight changes (+14 Da) by using high-resolution mass spectrometry. The method has high accuracy, is simple to operate, and is low in cost, is suitable for a variety of biological matrices and conventional small molecules, polypeptide and other carboxylic acid-containing metabolite structure identification, has important application value for 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] This invention belongs to the field of biomedicine and relates to a method for identifying the structure of drug carboxylic acid metabolites based on derivatization reagents and high-resolution mass spectrometry. Background Technology

[0002] The metabolic process of drugs in the body is closely related to their efficacy and safety. During drug development, it is necessary to detect and identify the main metabolites of the drug in the body and determine their specific structures. When a drug enters the body as an exogenous substance, the body often metabolizes it into molecules with higher polarity and better water solubility, such as carboxylic acids, glucuronic acids, and sulfate conjugates, which are then excreted, thus playing a detoxification role. Carboxylic acids are the metabolic forms of many different types of metabolites. For example, fatty alcohols are oxidized to aldehydes and then oxidized to form carboxylic acids; lactones undergo ring-opening hydrolysis to form carboxylic acids; and oxygen or nitrogen atoms are oxidized and dealkylated to form carboxylic acids—all common metabolic pathways.

[0003] However, determining the chemical structure of carboxylic acid metabolites is challenging in drug development. The pharmaceutical industry often uses high-resolution mass spectrometry (HMS) to identify the structures of drug metabolites. First-order mass spectrometry (MIMS) information is used to obtain the molecular weight of the metabolite. The exact change in molecular weight relative to the parent drug is used to infer changes in elemental composition. Finally, second-order mass spectrometry (MIMS) information provides the detailed structural information. For carboxylic acid metabolites, the molecular weight information from first-order HMS can be used to preliminarily determine the elemental composition. For example, the hydrolysis of lactones or lactams into carboxylic acids (+H₂O, molecular weight increases by 18 Da)... Figure 1 Case 1) Alcohol oxidation to form carboxylic acid (+O-2H, molecular weight increases by 14 Da) Figure 1 In scenario 2), the amide hydrolyzes into a carboxylic acid ( Figure 1 Case 3) Oxidation of nitrogen or oxygen heterocycles to form carboxylic acids (+2O, molecular weight increases by 32 Da) Figure 1 (Scenario 4) A preliminary judgment can be made that it is likely the formation of a carboxylic acid metabolite. However, there is also the possibility of other structural metabolites. For example, the oxidation of an alcohol to form a carboxylic acid with a molecular weight increase of 14 Da may be due to oxidative dehydrogenation; the oxidation of a nitrogen or oxygen heterocycle to form a carboxylic acid with a molecular weight increase of 32 Da may be due to dioxide oxidation; the hydrolysis of a lactone or lactam to form a carboxylic acid with a molecular weight increase of 18 Da may be due to oxidative ring-opening to an alcohol, etc. Further confirmation of the structure of carboxylic acid metabolites based on secondary data from high-resolution mass spectrometry also presents some difficulties. In some cases, the removal of 44 Da or 46 Da from mass spectrometric fragments can provide a preliminary indication that it is a carboxylic acid, but in many cases, such typical fragments cannot be generated, making precise confirmation of carboxylic acid metabolites extremely difficult.

[0004] Several other methods can confirm the results of carboxylic acid metabolites. One method is to separate more than 1 mg of the carboxylic acid metabolite into a standard using preparative liquid chromatography, and then confirm the structure using nuclear magnetic resonance (NMR) spectroscopy. However, in the early stages of new drug development, separating and synthesizing standards for the metabolite is difficult. This method in biological matrices requires a large number of samples to ensure the sensitivity of the NMR instrument, thus consuming a significant amount of time and money. Another method is to use derivatization to react the carboxylic acid group, thereby obtaining a specific mass spectrometry signal. David W. Johnson reported a derivatization method for the derivatization of carboxylic acid drugs. The specific procedure is as follows: First, alkyl dimethylaminoethyl ester iodide is prepared by quaternization of dimethylaminoethyl ester with alkyl iodine. Plasma sample processing involves mixing a labeled fatty acid mixture with plasma, acetonitrile, and hydrochloric acid, followed by heating, extraction with hexane, evaporation, reaction with oxalyl chloride, and then reaction with dimethylaminoethanol. Finally, quaternization is performed by adding iodide methyl to form a derivative. The reaction residue is then dissolved in a mixed solvent of acetonitrile / water / formic acid for electrospray tandem mass spectrometry analysis. However, this method involves a long process, requiring heating to a high temperature of 100°C and the use of highly reactive and toxic reagents such as iodomethane. In general, this method is time-consuming and unsafe.

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

[0006] The main objective of this invention is to provide an efficient, low-cost, rapid, and safe method for identifying the structure of carboxylic acid metabolites of drugs, in order to solve the problem of the difficulty in accurately identifying carboxylic acid metabolites in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for identifying the structure of drug carboxylic acid metabolites based on chemical derivatization and high-resolution mass spectrometry includes the following steps:

[0009] Step 1, Sample Pretreatment: Take biological samples, add extraction solvent, vortex and centrifuge, and collect the supernatant; the biological samples include 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;

[0010] Step 2, Nitrogen drying and redissolution: Dry the supernatant with nitrogen, add a redissolution solvent to redissolve, and obtain a redissolution solution; the redissolution solvent is one or a mixture of several of methanol, acetonitrile, water or dimethyl sulfoxide.

[0011] Step 3, Carboxylic acid derivatization reaction: The complex solution is mixed with the derivatization reagent trimethylsilyldiazomethane and reacted at 30-60℃ for 1-60 minutes to form a carboxylic acid methylation product; the concentration of the trimethylsilyldiazomethane is 0.1-1 mol / L;

[0012] Step 4: High-resolution mass spectrometry detection: Inject the solution after the reaction in step 3 into a high-resolution mass spectrometer for detection and analyze the change in molecular weight. If a molecular weight change of +14 Da is observed, it is identified as a carboxylic acid metabolite.

[0013] Furthermore, when the biological sample is a plasma sample, the extraction solvent is acetonitrile, the volume of which is added is 3 times the volume of the plasma sample, vortexing for 1 minute, and centrifugation conditions are 15000 rpm for 10 minutes.

[0014] Furthermore, the nitrogen drying temperature is 30-40°C.

[0015] Furthermore, in step two, the resolution solvent is acetonitrile, and the resolution volume is adjusted according to the sample concentration and subsequent experimental requirements.

[0016] Furthermore, in step three, the mixing ratio of the complex solution to trimethylsilyldiazomethane is 1:1.

[0017] 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 scanning modes are Full MS and MS2, and the resolutions are Full Scan 35000 and MS2 17500.

[0018] The above method is used to identify carboxylic acid metabolites.

[0019] The beneficial effects of this invention are:

[0020] 1. High accuracy: By reacting carboxylic acid metabolites with a specific derivatization reagent, trimethylsilyldiazomethane (TMSCHN2), characteristic methylated products (molecular weight increase of +14 Da) are generated. Combined with the precise molecular weight and fragment information of high-resolution mass spectrometry, the accuracy and sensitivity of carboxylic acid metabolite structure identification are significantly improved, avoiding misjudgments caused by the lack of typical fragments in traditional mass spectrometry.

[0021] 2. High efficiency: The pretreatment steps are simple, requiring only protein precipitation, centrifugation, drying and reconstitution, and derivatization reaction under mild conditions (30-60℃, 1-60 minutes). There is no need for complex separation or high-temperature operation. Moreover, the mass spectrometry detection speed is fast, and samples can be analyzed in batches, which greatly shortens the experimental cycle.

[0022] 3. Low cost: The reagent TMSCHN2 used is inexpensive and requires a small amount. The 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.

[0023] 4. The reaction conditions are mild, avoiding the use of high temperatures of 100°C and toxic reagents in existing derivatization methods, making the operation safer.

[0024] 5. Applicable to a variety of matrices: This invention is applicable to a variety of biological samples, including plasma, urine, and feces, and can meet the needs of different drug metabolism studies.

[0025] 6. Applicable to carboxylic acid metabolites of various drugs: This 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 polypeptides, and has good applicability. Attached Figure Description

[0026] Figure 1 This represents the carboxylic acid metabolites produced by different metabolic reactions and their corresponding molecular weight changes.

[0027] Figure 2 The images show the high-resolution mass spectra and corresponding chemical structures of plasma samples containing 10 μM diclofenac after treatment with and without TMSCHN2. A: without TMSCHN2 treatment; B: after TMSCHN2 treatment.

[0028] Figure 3 High-resolution mass spectra and corresponding chemical structures of 10 μM semaglutide plasma samples without TMSCHN2 treatment and with TMSCHN2 treatment. A: without TMSCHN2 treatment; B: with TMSCHN2 treatment.

[0029] Figure 4 A flowchart for identifying carboxylic acid metabolites using derivatization methods. Detailed Implementation

[0030] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected. Example 1

[0031] 1. Experimental preparation:

[0032] Two drugs containing carboxylic acid structures, diclofenac and smegglutide, were prepared into stock solutions with a concentration of 10 mM in dimethyl sulfoxide and water, respectively. The stock solutions were then diluted 1000 times to 10 μM in plasma to simulate actual in vivo plasma samples containing carboxylic acid metabolites.

[0033] Prepare the methylation reagent TMSCHN2, and prepare it in a 1 mol / L solution of methanol / acetonitrile / water (1:1:1). Also prepare the extraction solvent acetonitrile, the redissolving solvent acetonitrile, and experimental equipment and reagents such as a high-resolution mass spectrometer.

[0034] The high-resolution mass spectrometer is calibrated and adjusted to ensure its stable performance and accurate molecular weight measurement.

[0035] 2. Pre-processing steps:

[0036] The plasma sample was mixed with three times its volume of extraction solvent acetonitrile, shaken, and then centrifuged (15,000 rpm, 10 min) to remove the lower layer of plasma proteins and other biological matrix, and the upper layer containing potential carboxylic acid metabolites was collected.

[0037] 3. Nitrogen drying and reconstitution:

[0038] The extracted product was transferred to a nitrogen dryer, and the temperature was set to 30-40℃ and the nitrogen flow rate was adjusted for nitrogen drying. This ensured that any residual extraction solvent was completely removed while avoiding loss of potential carboxylic acid metabolites.

[0039] After drying, 200 μL of acetonitrile is added for reconstitution, depending on the properties of the sample and the requirements of subsequent experiments. The optimal reconstitution solvent and volume can be determined experimentally to ensure complete dissolution of potential carboxylic acid metabolites.

[0040] 4. Reaction with the derivatizing reagent TMSCHN2:

[0041] Mix the reconstituted solution with 1 mol / L TMSCHN2 at a predetermined ratio of 1:1. Accurate mixing can be performed using a micropipette or autosampler.

[0042] Place the mixed solution in a suitable reaction vessel, such as a polypropylene test tube or glass vial, and then place it in a constant temperature water bath to carry out the reaction. Control the reaction temperature (1 min to 60 min) and time to ensure that the derivatization reaction proceeds fully.

[0043] High-resolution mass spectrometry detection:

[0044] The reacted solution is injected into a high-resolution mass spectrometer using an autosampler. Appropriate mass spectrometry parameters are set, such as ion source type, scan range, and resolution, as follows.

[0045] Mass spectrometer: Q Exactive Plus (Thermo)

[0046] Ion source: ESI (+)

[0047] Ion source spray voltage: 3.8 kV

[0048] Capillary temperature: 320°C

[0049] Sheath gas: 40 units

[0050] Auxiliary gas: 10 units

[0051] Purge air: 0 units

[0052] S-lens: 55 units

[0053] Collision energy (NCE): 25, 45, 60

[0054] Scan modes: Full MS, MS 2

[0055] Resolution: 35000 (Full Scan) and 17500 (MS) 2 )

[0056] Mass spectrometry analysis was performed on the injected solution, and the molecular weight information and mass spectrum were recorded. The experimental results are as follows: Figure 2 and Figure 3 As 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, Includes the following steps: Step 1, Sample Pretreatment: Take a biological sample, add extraction solvent, vortex and centrifuge, and collect the supernatant; the biological sample includes 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, Nitrogen drying and redissolution: The supernatant is dried with nitrogen, and a redissolution solvent is added to redissolve the supernatant to obtain a redissolved solution; the redissolution solvent is acetonitrile. Step 3, Carboxylic acid derivatization reaction: The complex solution is mixed with the derivatization reagent trimethylsilyldiazomethane and reacted at 30-60℃ 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: Inject the solution after the reaction in step 3 into a high-resolution mass spectrometer for detection and analyze the change in molecular weight. If a molecular weight change of +14 Da is observed, 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, and the volume added is 3 times the volume of the plasma sample. The mixture is vortexed for 1 minute and centrifuged at 15,000 rpm for 10 minutes.

3. The method according to claim 1, characterized in that, The nitrogen drying temperature is 30-40℃.

4. The method according to claim 1, characterized in that, In step three, the mixing ratio of the complex solution to trimethylsilyldiazomethane is 1:

1.

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

6. The use of the method according to any one of claims 1-5 in the identification of carboxylic acid metabolites.

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