Optimized 3-nitrophenylhydrazine derivatization method and application

Abstract

The invention discloses an optimized 3-nitrophenylhydrazine derivatization method and an application of the optimized 3-nitrophenylhydrazine derivatization method. According to the optimized method disclosed by the invention, the alpha-keto acid metabolite is subjected to direct derivatization reaction by adopting 3-nitrophenylhydrazine; the method comprises the following steps: carrying out a 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl) carbodiimide / pyridine derivatization reaction on a metabolite only containing carboxyl; a 3-nitrophenylhydrazine derivatization method or a 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl) carbodiimide / pyridine derivatization method or a non-derivatization method is adopted for metabolites only containing carbonyl; a non-derivatization method is adopted for other metabolites. According to the method disclosed by the invention, a specific reaction with carbonyl and carboxyl can be carried out, so that non-targeted metabonomics analysis is enhanced, and metabolites in a biological sample can be more comprehensively analyzed.

Description

Technical Field

[0001] The present invention belongs to the field of metabolite analysis technology, and specifically relates to an optimized 3-nitrophenylhydrazine derivatization method and application. Specifically, the optimized method of the present invention can detect more compounds with higher detection sensitivity, especially compounds containing carbonyl and carboxyl groups. Background Art

[0002] There are many metabolites containing carbonyl and carboxyl groups in organisms, which play important roles in multiple key metabolic pathways, such as the glycolysis pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle. Most carbonyl-containing metabolites are highly reactive and participate in a wide range of biochemical pathways in the body. Their disorders are associated with many disease states, such as cardiovascular disease, cancer, and Alzheimer's disease. However, carbonyl-containing metabolites have poor ionization ability and unstable chemical properties, making them difficult to analyze accurately. On the other hand, carboxyl-containing metabolites are widely distributed in organisms, have important effects on physiological activities, and are closely related to cancer, type 2 diabetes, intestinal diseases, etc. However, as polar compounds, carboxyl metabolites have poor retention on non-polar chromatographic columns and are difficult to separate effectively. In addition, due to poor separation and low sensitivity, higher concentrations of biological samples are required for analysis. Although the use of ion pair reagents can improve peak shape and resolution, it will contaminate the ion source of the mass spectrometer and shorten the service life of the chromatographic column. Therefore, the development of more stable and sensitive analytical methods is crucial for studying such metabolites.

[0003] Chemical derivatization technology is an effective strategy to improve the sensitivity and selectivity of metabolite detection. It can change the physical and chemical properties of target metabolites by modifying their structures and thus enhancing the analytical effect. Phenylhydrazine derivatization can modify both carbonyl and carboxyl groups at the same time, and has the advantages of simple operation and high efficiency. However, there are some limitations in the practical application of phenylhydrazine derivatization: some metabolites containing carbonyl and carboxyl groups may have low reactivity to derivatization reagents or not react with derivatization reagents; not all metabolites will show increased mass spectrometry response after derivatization; the C=N double bond formed after derivatization may introduce isomers, complicating the detection and identification process; the derivatization process of metabolites containing multiple derivatizable groups is complicated, and may produce multiple derivatization products, increasing the difficulty of identification; the stability of the derivatization products also varies significantly depending on the chemical properties of the metabolites, etc.

[0004] Therefore, it is necessary to develop an optimized phenylhydrazine derivatization method in order to overcome the limitations of this method in practical applications. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide an optimized 3-nitrophenylhydrazine derivatization method and application to address the deficiencies of the prior art, and to provide a reference for the study of carbonyl and carboxyl metabolites.

[0006] The present invention detects carbonyl and carboxyl metabolites based on an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS) method of phenylhydrazine derivatization, and screens out a suitable phenylhydrazine derivatization reagent by comparing the derivatization efficiency of different phenylhydrazine reagents and the mass spectrometry detection sensitivity of the derivatized products.

[0007] The present invention selects five different phenylhydrazine reagents to derivatize a mixture of 10 standard products containing carboxyl, carbonyl and α-keto acid, and determines that 3-nitrophenylhydrazine has the best derivatization effect. On this basis, the present invention optimizes the derivatization conditions of 3-nitrophenylhydrazine, thereby obtaining an optimized 3-nitrophenylhydrazine derivatization method.

[0008] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0009] The present invention provides an optimized 3-nitrophenylhydrazine derivatization method, comprising the following steps:

[0010] (1) Direct derivatization of α-keto acid metabolites using 3-nitrophenylhydrazine;

[0011] (2) For metabolites containing only carboxyl groups, a 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl) carbodiimide / pyridine derivatization reaction is used;

[0012] (3) For metabolites containing only carbonyl groups, 3-nitrophenylhydrazine derivatization or 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine derivatization or no derivatization is used;

[0013] (4) For other metabolites, a non-derivatized method was adopted.

[0014] The present invention detects carbonyl and carboxyl metabolites based on the ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS) method of phenylhydrazine derivatization. According to the structural characteristics of carbonyl and carboxyl metabolites, the derivatization method is optimized and classified and analyzed, thereby improving the detection sensitivity and detection range of metabolites. The optimized 3-nitrophenylhydrazine derivatization method of the present invention can detect metabolites more comprehensively.

[0015] Preferably, the reaction temperature of the 3-nitrophenylhydrazine direct derivatization reaction is 37°C.

[0016] Preferably, the reaction temperature of the 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine derivatization reaction is 4°C.

[0017] Preferably, in the derivatization reaction, the concentration of 3-nitrophenylhydrazine is 10-15 mM.

[0018] The present invention detects the limit of detection (LOD) of 3-nitrophenylhydrazine derivatization, takes four standard products of indoleacetic acid, indolepyruvic acid, indole-3-pyruvic acid and pyruvic acid as research objects, and the LOD of the derivatization product obtained by the optimized 3-nitrophenylhydrazine derivatization method reaches the pM level.

[0019] The present invention optimizes the stability of the 3-nitrophenylhydrazine derivatization product and determines that the 3-nitrophenylhydrazine derivatization product is stable within 8 hours after derivatization, and mass spectrometry detection should be performed within this period of time.

[0020] The present invention detects the linearity of 3-nitrophenylhydrazine derivatization products, taking indolepyruvic acid and indoleacetic acid as research objects, and the results show that indolepyruvic acid has a linear range of 20-1000nM (R 2 =0.9968), indoleacetic acid was in the range of 2-500 nM (R 2 =0.9997), a good linear relationship was shown.

[0021] The present invention also provides an application of the above-mentioned optimized 3-nitrophenylhydrazine derivatization method in non-targeted metabolomics analysis.

[0022] In the embodiment of the present invention, the samples for the non-targeted metabolomics analysis are Bacteroides Thetaiotaomicron (BT bacteria) and mouse feces samples.

[0023] The present invention also provides a method for detecting metabolites in a biological sample using the above-mentioned optimized 3-nitrophenylhydrazine derivatization method, comprising the following steps:

[0024] (1) preparing a lysate of the test substance by conventional methods;

[0025] (2) Take a portion of the lysate, add water, 55% methanol aqueous solution of 3-nitrophenylhydrazine·HCl, 55% methanol aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide·HCl and 55% methanol aqueous solution of pyridine, react at 4°C for 30 minutes, and evaporate to dryness;

[0026] (3) Take an equal amount of the second lysate, add water, 55% methanol aqueous solution of 3-nitrophenylhydrazine·HCl and 55% methanol aqueous solution, react at 37°C for 30 min, and evaporate to dryness;

[0027] (4) Take an equal amount of the third lysate, add water and 55% methanol aqueous solution, mix well, and evaporate to dryness;

[0028] (5) The samples prepared in step (2), step (3), and step (4) were respectively re-dissolved in 25% methanol water, injected within 8 hours, and metabolites were detected.

[0029] In the present invention, the BT bacteria lysate, mouse feces sample lysate or other lysate of the test object can be prepared by conventional methods known in the art.

[0030] A specific preferred embodiment of the present invention is that the preparation method of BT bacterial lysate is as follows: 200 μL BT bacterial culture solution is centrifuged at 4°C and 18000 rpm for 10 min, and the supernatant is discarded. 500 μL pre-cooled MeOH is added and homogenized 5 times (60 Hz, 60 s). The mixture is then centrifuged at 4°C and 18000 rpm for 10 min, and the supernatant is transferred for further analysis.

[0031] A specific preferred embodiment of the present invention is that the preparation method of mouse feces sample lysate is as follows: each mouse feces is collected separately, freeze-dried, and homogenized beads are added to grinder to grind to powder. Weigh 15 mg of feces freeze-dried powder, add 200 μL ultrapure water and 2 homogenized beads, and wet and homogenize in a grinder 3 times (60 Hz, 60 s). Add 800 μL of pre-cooled methanol containing 25 μM internal standard (4-chloro-L-phenylalanine), shake for 10 minutes, place at -20°C for 1 hour, 18000 rpm, 4°C centrifuge for 10 minutes, and take the supernatant as mouse feces lysate.

[0032] Preferably, the metabolites are detected by UPLC-MS, and the results of UPLC-MS are imported into Progenesis QI for metabolite identification.

[0033] Beneficial effects:

[0034] (1) The present invention selects 5 different phenylhydrazine reagents to derivatize a mixture of standard products containing carbonyl and carboxyl groups, and finds that 3-nitrophenylhydrazine has the highest derivatization efficiency. On this basis, the present invention divides the derivatization of 3-nitrophenylhydrazine into three specific cases based on the differences in the chemical structures of metabolites, namely, direct derivatization of 3-nitrophenylhydrazine, derivatization of 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl) carbodiimide / pyridine, and no derivatization. Dividing the samples into three groups for analysis can detect metabolites more comprehensively and improve the detection coverage of metabolites.

[0035] (2) The present invention verified through 25 kinds of standard products and found that in Progenesis QI software, by setting the corresponding adduct ions, including setting the adduct ion [M+(3-NPH)-H] in the negative ion mode - ,[M+2(3-NPH)-H] - ,[M+3(3-NPH)-H]- ; In positive ion mode, add ion setting [M+(3-NPH)+H] + ,[M+2(3-NPH)+H] + ,[M+3(3-NPH)+H] + ; Non-targeted identification based on 3-nitrophenylhydrazine derivatization can be established to accurately identify more metabolites without standards, making it possible to discover more unknown metabolites.

[0036] (3) The present invention applies the optimized 3-nitrophenylhydrazine derivatization method to the analysis of BT bacteria and mouse fecal samples, bringing possibilities for biological sample analysis and new biomarker discovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 AC is the mass spectrometric response of the standard mixture derivatized with five selected phenylhydrazine reagents: Figure 1 A is the product of direct derivatization of phenylhydrazine; Figure 1 B is a primary derivatization reaction product obtained by derivatization of phenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine; Figure 1 C is a secondary derivatization reaction product obtained by derivatization of phenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine; Figure 1 DF is the comparison of mass spectrometric responses of metabolites before and after derivatization: Figure 1 D is the product of direct derivatization of 3-nitrophenylhydrazine; Figure 1 E is a primary derivatization reaction product obtained by derivatization with 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine; Figure 1 F is a secondary derivatization reaction product obtained by derivatization with 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine.

[0038] Figure 2 This is the optimized 3-nitrophenylhydrazine derivatization strategy.

[0039] Figure 3 To optimize the derivatization conditions; Figure 3 A is the optimization of reaction temperature; Figure 3 B is the stability study of the derivatized product.

[0040] Figure 4 The figure is a linear relationship diagram of 3-nitrophenylhydrazine derivatization products represented by indolepyruvic acid and indoleacetic acid.

[0041] Figure 5 is the LOD detection of 3-nitrophenylhydrazine derivatization; wherein Figure 5 A is indoleacetic acid, LOD is 2nM, Figure 5B is indolepyruvic acid, LOD is 20nM, Figure 5 C is indole-3-pyruvic acid, LOD is 1nM, Figure 5 D is pyruvate, and LOD is 2 pM.

[0042] Figure 6 The concentration optimization of BT bacterial lysate derivatization and the XIC of representative metabolites in BT bacterial lysate; Figure 6 A is the concentration optimization of 3-nitrophenylhydrazine derivatization, Figure 6 B is the concentration optimization of 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine derivatization; Figure 6 C is the XIC of the representative metabolites in BT bacterial lysate without derivatization, Figure 6 D is the XIC of the representative metabolites in BT bacterial lysate after optimized 3-nitrophenylhydrazine derivatization method.

[0043] Figure 7 The concentration of mouse fecal lysate derivatization was optimized with XICs of representative compounds in mouse fecal samples; Figure 7 A is the concentration optimization of 3-nitrophenylhydrazine derivatization, Figure 7 B is the concentration optimization of 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine derivatization; Figure 7 C is the XIC of the representative metabolites in mouse fecal samples without derivatization, Figure 7 D is the XIC of the optimized 3-nitrophenylhydrazine derivatization method for representative metabolites in mouse fecal samples. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0045] The experimental methods in the examples of the present invention that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by raw material or product manufacturers. Reagents that do not specify specific sources are conventional reagents purchased from the market.

[0046] The present invention uses a Waters ACQUITY I-Class UPLC liquid phase system (Milford, MA, USA) combined with a Waters Xevo G2-XS Q-Tof mass spectrometer (Milford, MA, USA).

[0047] Chromatographic separation was performed using a Waters ACQUITY UPLC HSS T3 column (1.8 μm, 100 mm×2.1 mm), column temperature 40° C., autosampler 8° C., flow rate 0.3 mL / min, mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile.

[0048] Gradient elution conditions: 0-1.0 min, 15% B; 1.0-12.0 min, 15%-70% B; 12.0-14.0 min, 70% B; 14.0-15.0 min, 70%-15% B; 15.0-23.0 min, 15% B. DDA detection and data acquisition were performed in positive and negative ion modes, and the parameters were set as follows: m / z range 50-1200 Da; capillary voltage +3.0 kV / -2.5 kV for positive / negative ion modes, respectively; sampling cone voltage 40 V; ion source temperature 100 °C; desolvation temperature 400 °C; cone gas flow rate 50 L / h; desolvation gas flow rate 600 L / h. MassLynx (version 4.1) was used for data acquisition.

[0049] MassLynx (version 4.1) was used for peak extraction and peak area integration; GraphPad Prism 9.0.0 was used to draw line graphs and bar graphs; metabolites were identified using Progenesis QI (version 2.3.6275.47962). In Progenesis QI software, the corresponding adduct ions were set, including the adduct ion setting [M+(3-NPH)-H] in negative ion mode. - ,[M+2(3-NPH)-H] - ,[M+3(3-NPH)-H] - ; In positive ion mode, add ion setting [M+(3-NPH)+H] + ,[M+2(3-NPH)+H] + ,[M+3(3-NPH)+H] + .

[0050] Example 1 Confirmation of 3-nitrophenylhydrazine derivatization

[0051] To select the best derivatization reagent, 10 standards containing carboxyl (indoleacetic acid, indole-3-propionic acid, 4-hydroxybenzoic acid, L-glutamine), carbonyl (indole-3-carboxaldehyde, 3-acetylindole) and α-keto acid (pyruvic acid, 2-ketobutyric acid, indolepyruvic acid, 3-indoleglyoxylic acid) were dissolved in 55% methanol water to prepare a 100μM mixed standard. Take 200μL of the mixed standard, add 100μL of 60mM different phenylhydrazine derivatization reagents (solvent is 55% methanol aqueous solution), add 100μL of 36mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 2.0% pyridine in 55% methanol aqueous solution or 100μL of 55% methanol aqueous solution. React at 4℃ for 30min and evaporate to dryness. Redissolve in 1mL 25% methanol aqueous solution for injection. Different phenylhydrazine derivatization reagents including 2-bromophenylhydrazine, 3-bromophenylhydrazine, 4-bromophenylhydrazine, 4-chlorophenylhydrazine and 3-nitrophenylhydrazine were compared in their derivatization reaction efficiency.

[0052] The results showed that without the addition of 1-ethyl-(3-dimethylaminopropyl) carbodiimide / pyridine, the carboxyl metabolites basically did not react with the five phenylhydrazines, and the mass spectrometric response of the products obtained after the carbonyl-containing metabolites were derivatized with 3-nitrophenylhydrazine was better than that of the derivatized products obtained with other phenylhydrazine reagents ( Figure 1 A). In order to improve the derivatization efficiency of carboxyl groups, 1-ethyl-(3-dimethylaminopropyl) carbodiimide / pyridine was added, and it was found that compared with other reagents, 3-nitrophenylhydrazine still had the best derivatization effect ( Figure 1 B). Theoretically, in the presence of 1-ethyl-(3-dimethylaminopropyl) carbodiimide / pyridine, α-keto acids containing both carbonyl and carboxyl groups will undergo secondary derivatization. During the test, it was found that after adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide / pyridine, α-keto acids could be extracted into primary and secondary derivatization products, but their secondary derivatization reaction was not complete ( Figure 1 B and Figure 1 C), resulting in the coexistence of two derivatization products.

[0053] To compare the mass spectrometric responses of the standards before and after derivatization with 3-nitrophenylhydrazine, 10 standards containing carboxyl groups (indoleacetic acid, indole-3-propionic acid, 4-hydroxybenzoic acid, L-glutamine), carbonyl groups (indole-3-carboxaldehyde, 3-acetylindole) and α-keto acids (pyruvic acid, 2-ketobutyric acid, indolepyruvic acid, 3-indoleglyoxylic acid) were dissolved in 55% methanol water to prepare a 100 μM mixed standard. 200 μL of the mixed standard was taken and added with 100 μL of 60 mM 3-nitrophenylhydrazine·HCl in 55% methanol water, 100 μL of 36 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide·HCl and 2.0% pyridine in 55% methanol water or 100 μL of 55% methanol water. The reaction was allowed to react at 4°C for 30 min and evaporated to dryness. Take another 200 μL of the mixed standard, add 200 μL of 55% methanol-water solution, mix well, evaporate to dryness, and reconstitute with 1 mL of 25% methanol-water. Further compare the mass spectrometric response of the standard before and after 3-nitrophenylhydrazine derivatization.

[0054] For carbonyl compounds, the mass spectrometric response of 1H-indole-3-carboxaldehyde was enhanced more significantly after direct derivatization with 3-nitrophenylhydrazine. For 3-acetylindole, the mass spectrometric response decreased after direct derivatization with 3-nitrophenylhydrazine, while the mass spectrometric response after derivatization with 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine was similar to that before derivatization.

[0055] For carboxyl compounds, after adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide / pyridine, the mass spectrometric responses of some metabolites before and after 3-nitrophenylhydrazine derivatization were similar, such as 4-hydroxybenzoic acid and L-glutamine; the mass spectrometric responses of some metabolites were significantly enhanced, such as the mass spectrometric response of indoleacetic acid after derivatization increased by 191 times compared with that before derivatization, and the mass spectrometric response of indole-3-propionic acid after derivatization increased by 32 times compared with that before derivatization ( Figure 1 E).

[0056] For α-keto acids, 3-nitrophenylhydrazine derivatization can significantly improve the mass spectrometric response of these compounds compared to underivatized compounds. However, the addition of 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine can confound the derivatization process. For example, for pyruvic acid and 2-ketobutyric acid, the mass spectrometric intensity of their secondary derivatization products ( Figure 1 F) and the mass spectrum intensity of the primary derivatization product ( Figure 1 In addition, for indolepyruvic acid, after adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine, the mass spectrometric response of its secondary derivatization product ( Figure 1 F) is not as good as the mass spectrometric response of the primary derivatization product ( Figure 1 The above data further confirmed that α-keto acids are suitable for direct derivatization of 3-nitrophenylhydrazine.

[0057] In summary, according to the structural characteristics of the compounds, the present invention proposes three derivatization strategies based on the 3-nitrophenylhydrazine derivatization method (see Figure 2 ), that is, for α-keto acids, 3-nitrophenylhydrazine is used for direct derivatization; for carboxyl-containing compounds, 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine is used for derivatization; for carbonyl-containing compounds, 3-nitrophenylhydrazine direct derivatization, 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine derivatization or no derivatization is used; for other compounds, no derivatization method is used.

[0058] Example 2 Temperature Optimization of 3-Nitrophenylhydrazine Derivatization

[0059] Dissolve the 10 standards in Example 1 in 55% methanol water to prepare a 100 μM mixed standard. Take 200 μL of the mixed standard, add 100 μL of 60 mM 3-nitrophenylhydrazine hydrochloride in 55% methanol water, add 100 μL of 36 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (containing 2.0% pyridine) in 55% methanol water or 100 μL of 55% methanol water. React at 4°C, 25°C and 37°C for 30 min, evaporate to dryness. Redissolve with 1 mL of 25% methanol water.

[0060] The results showed that for α-keto acids, especially indolepyruvic acid and 3-indoleglyoxylic acid, the mass spectrometric response of the products obtained by derivatization at 37°C was higher than that of the products obtained by derivatization at the other two temperatures ( Figure 3 A). For compounds containing only carboxyl groups, such as 4-hydroxybenzoic acid and L-glutamine, derivatization at 4°C is more favorable ( Figure 3 A).

[0061] Therefore, in order to obtain the best derivatization effect, based on Example 1, the optimal reaction temperature for direct derivatization of 3-nitrophenylhydrazine was determined to be 37°C, while the optimal temperature for the derivatization reaction of 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine was determined to be 4°C.

[0062] Example 3 Stability, Linearity and LOD of the Optimized 3-Nitrophenylhydrazine Derivatization Method

[0063] The 10 standards in Example 1 were subjected to 3-nitrophenylhydrazine derivatization reaction using the optimized method (refer to the method in Example 2). The standards of indole-3-carboxaldehyde, 3-acetylindole, sodium indoleacetate, indole-3-propionic acid, 4-hydroxybenzoic acid, L-glutamine, sodium pyruvate, 2-ketobutyric acid, indolepyruvic acid and 3-indoleglyoxylic acid were dissolved in 55% methanol water and gradiently diluted to 5000nM, 2500nM, 1000nM, 500nM, 250nM, 100nM, 50nM, 25nM, 10nM, 5nM, 1nM, 0.1nM, 0.01nM mixed standard solutions. Divide into 2 groups, one group takes 100μL of each gradient mixed standard, adds 100μL of 25μM 4-chloro-L-phenylalanine in 55% methanol aqueous solution, 100μL of 60mM 3-NPH·HCl in 55% methanol aqueous solution, 100μL of 36mM EDC·HCl and 2.0% pyridine in 55% methanol aqueous solution, reacts at 4℃ for 30min, and evaporates. The other group takes 100μL of each gradient mixed standard, adds 100μL of 125μM 4-chloro-L-phenylalanine in 55% methanol aqueous solution, adds 100μL of 60mM 3-NPH·HCl in 55% methanol aqueous solution, 100μL of 55% methanol aqueous solution, reacts at 37℃ for 30min, and evaporates. Redissolve in 500μL of 25% methanol water.

[0064] Dissolve in 55% methanol water, prepare a 100μM mixed standard of the above 10 standards, divide into 2 groups according to the above method, perform 3-NPH derivatization reaction, evaporate, and re-dissolve in 500μL 25% methanol water. At the same time, prepare a 25% methanol aqueous solution of 100μM mixed standard of sodium pyruvate, 2-butyric acid, indolepyruvic acid and 3-indole glyoxylic acid, take 100μL, add 100μL 125μM 4-chloro-L-phenylalanine in 25% methanol aqueous solution, and add 300μL 25% methanol water. Place the above samples in a sample tray at 8℃ for 72h and measure the stability.

[0065] The results showed that under the conditions of 3-nitrophenylhydrazine and 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine derivatization, the mass spectrometry intensity of most of the derivatized products of the standards did not decrease significantly in the first 8 hours, but began to fluctuate at 16 hours, especially for 1H-indole-3-carboxaldehyde, the mass spectrometry signal decreased significantly at 72 hours compared with the starting time point ( Figure 3 B) Therefore, the 3-nitrophenylhydrazine derivatization product is stable within 8 hours after reconstitution.

[0066] The linearity of the derivatized products was investigated using 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine-derivatized indoleacetic acid and 3-nitrophenylhydrazine-derivatized indolepyruvic acid as representatives. Figure 4 As shown, Figure 4 A is the linear relationship diagram of the 3-nitrophenylhydrazine derivatization products of indoleacetic acid, Figure 4 B is the linear relationship diagram of the 3-nitrophenylhydrazine derivatization products of indolepyruvic acid. 2 =0.9997), indolepyruvic acid was in the range of 20 to 1000 nM (R 2 =0.9968), a good linear relationship was shown.

[0067] LOD was tested using four standards: indoleacetic acid, indolepyruvic acid, indole-3-propionic acid and pyruvic acid. Figure 5 As shown, the LOD of indoleacetic acid is 2nM, the LOD of indolepyruvic acid is 20nM, the LOD of indole-3-pyruvic acid is 1nM, and the LOD of pyruvic acid is 2pM. At the same time, the derivatized products all have a high signal-to-noise ratio. The above results confirm that derivatization significantly improves the mass spectrometry detection sensitivity of the corresponding compounds.

[0068] Example 4 Optimization of 3-nitrophenylhydrazine derivatization concentration of BT bacteria

[0069] To optimize the derivatization conditions, 200 μL of BT bacterial culture was centrifuged at 4°C, 18,000 rpm for 10 min, and the supernatant was discarded. 500 μL of pre-cooled MeOH was added and homogenized 5 times (60 Hz, 60 s). The mixture was then centrifuged at 4°C, 18,000 rpm for 10 min, and the supernatant was transferred for further analysis.

[0070] Take 110 μL of BT bacteria methanol lysis solution with the medium discarded, add 90 μL of water, 25 μL of 10mM, 25mM, 50mM, 100mM, 150mM 3-nitrophenylhydrazine·HCl in 55% methanol aqueous solution, 25 μL of 6mM, 15mM, 30mM, 60mM, 90mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide·HCl and 5.0% pyridine in 55% methanol aqueous solution, react at 4°C for 30min, and evaporate to dryness. Take 110 μL of BT bacteria methanol lysis solution with the medium discarded, add 90 μL of water, 25 μL of 10mM, 25mM, 50mM, 100mM, 150mM 3-nitrophenylhydrazine·HCl in 55% methanol aqueous solution, 25 μL of 55% methanol aqueous solution, react at 37°C for 30min, and evaporate to dryness. Reconstitute with 250 μL of 25% methanol in water for injection.

[0071] The results of UPLC-MS were imported into Progenesis QI for identification. From the identification results of QI, 11 representative metabolites were selected to evaluate the efficiency of derivatization. When the concentration of 3-nitrophenylhydrazine was in the range of 1 to 5 mM, the MS signal had significant fluctuations and poor reproducibility. When the concentration of 3-nitrophenylhydrazine was increased to 10 to 15 mM, the MS response of the detected derivatized products showed better stability and reproducibility, such as Figure 6 A and Figure 6 As shown in B. Therefore, we chose 10 mM for subsequent analysis.

[0072] Example 5 Analysis of 3-nitrophenylhydrazine derivatization of BT bacterial lysate

[0073] On the basis of Example 4, the present invention further compares the advantages of the optimized 3-nitrophenylhydrazine derivatization method for detecting metabolites with reference to the existing 25 standards. The 25 standards include: sodium indoleacetate, indole lactic acid, indole-3-acetaldehyde, indole-3-carboxaldehyde, indolepyruvic acid, L-tryptophan, 5-hydroxyindoleacetic acid, indole-3-acrylic acid, 5-hydroxytryptophan, indole-3-propionic acid, DL-kynurenine, sodium pyruvate, phenylpyruvic acid, 4-hydroxyphenylpyruvic acid, 4-hydroxyphenyllactic acid, 4-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, quinolinic acid, L-glutamic acid, L-glutamine, L-phenylalanine, L-lysine, L-histidine, ornithine and L-tyrosine.

[0074] To further analyze the 3-nitrophenylhydrazine derivatization effect of BT bacterial lysate, 110 μL of BT bacterial methanol lysate was added with 90 μL of water, 25 μL of 100 mM 3-nitrophenylhydrazine·HCl in 55% methanol aqueous solution, 25 μL of 60 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide·HCl and 5.0% pyridine in 55% methanol aqueous solution, reacted at 4°C for 30 min, and evaporated; 110 μL of BT bacterial methanol lysate was added with 90 μL of water, 25 μL of 100 mM 3-nitrophenylhydrazine·HCl in 55% methanol aqueous solution, and 25 μL of 55% methanol aqueous solution, reacted at 37°C for 30 min, and evaporated; 110 μL of BT bacterial methanol lysate was added with 90 μL of water and 50 μL of 55% methanol aqueous solution, mixed well, and evaporated; all were reconstituted with 250 μL of 25% methanol water. Concentrated BT bacteria methanol lysis buffer: 400 μL BT bacteria methanol lysis buffer, evaporate to dryness, and reconstitute with 200 μL 25% methanol water.

[0075] Using existing standards as reference, the advantages of 3-nitrophenylhydrazine derivatization of BT bacterial lysate in detecting metabolites were compared.

[0076] In the BT bacterial lysate without 3-nitrophenylhydrazine derivatization, only 10 metabolites could be detected, and the mass spectrometry response was relatively low (mass spectrometry response range was 1e 3 ~3e 5 ), and were all eluted within the first 2 minutes, with poor separation efficiency ( Figure 6 C). In contrast, the optimized 3-nitrophenylhydrazine derivatization method allowed the detection of 18 metabolites while greatly improving the mass spectrometry response (MS response range of 1e 3 ~3e 7 ) and improved the separation efficiency, with elution time evenly distributed from 1min to 12min ( Figure 6 D).

[0077] We further combined the optimized 3-nitrophenylhydrazine derivatization method with QI identification and applied it to the non-targeted metabolomics analysis of BT bacteria. The results of the non-targeted metabolomics analysis without derivatization showed that only 45 metabolites were detected and identified within the range of all metabolites. The number of detected metabolites was increased to 113 after the lysate was concentrated 10 times.

[0078] After optimized 3-nitrophenylhydrazine derivatization, 75 metabolites were detected directly from unconcentrated BT bacterial lysate, of which 9 were identified by 3-nitrophenylhydrazine derivatization and 71 were identified by 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine derivatization (5 of which were duplicates). Only 17 metabolites overlapped with the results obtained without derivatization, and 25 metabolites overlapped with the results obtained by concentrated bacterial lysate.

[0079] The above results indicate that the optimized 3-nitrophenylhydrazine derivatization method can provide more comprehensive metabolite detection results. Of the 75 metabolites detected based on derivatization, 66 were detected by 3-nitrobenzenehydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine derivatization (accounting for 88%), 4 were detected by 3-nitrophenylhydrazine derivatization, and 5 metabolites could be detected by both derivatization methods.

[0080] These results indicate that the three sample processing methods can comprehensively consider the structural characteristics of different metabolites, thereby identifying more possible metabolites.

[0081] Example 6 Optimization of 3-nitrophenylhydrazine derivatization concentration of mouse fecal samples

[0082] Six-week-old C57BL / 6 male mice purchased from Vital River Laboratory Animal Co., Ltd were selected. The feces of each mouse were collected separately, freeze-dried, and ground into powder in a grinder with homogenate beads. Weigh 15 mg of fecal freeze-dried powder, add 200 μL of ultrapure water and 2 homogenate beads, and wet and homogenize in a grinder 3 times (60 Hz, 60 s). Add 800 μL of pre-cooled methanol containing 25 μM internal standard (4-chloro-L-phenylalanine), shake for 10 min, place at -20 ° C for 1 h, 18000 rpm, 4 ° C for 10 min, and take the supernatant as the mouse fecal lysate.

[0083] To 137.5 μL of mouse fecal lysate, add 62.5 μL of water, 25 μL of 10 mM, 25 mM, 50 mM, 100 mM, 150 mM 3-nitrophenylhydrazine·HCl in 55% methanol aqueous solution, 25 μL of 6 mM, 15 mM, 30 mM, 60 mM, 90 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide·HCl and 5.0% pyridine in 55% methanol aqueous solution, react at 4°C for 30 min, and evaporate to dryness. To 137.5 μL of mouse fecal lysate, add 62.5 μL of water, 25 μL of 10 mM, 25 mM, 50 mM, 100 mM, 150 mM 3-nitrophenylhydrazine·HCl in 55% methanol aqueous solution and 25 μL of 55% methanol aqueous solution, react at 37°C for 30 min, and evaporate to dryness. Reconstitute with 250 μL of 25% methanol in water for injection.

[0084] The UPLC-MS results were imported into Progenesis QI software, and the results obtained were similar to those in Example 4, that is, within the concentration range of 1 to 5 mM, the derivatized products all had large mass spectrometry signal fluctuations and poor repeatability. When the 3-nitrophenylhydrazine concentration was increased to 10 to 15 mM, the mass spectrometry response of the derivatized products detected became more stable and repeatable, as shown in FIG. Figure 7 A and Figure 7 B. Therefore, 10 mM was selected for subsequent analysis of mouse fecal samples.

[0085] Example 7 Comparison of mouse fecal samples before and after 3-nitrophenylhydrazine derivatization

[0086] Take 137.5 μL of mouse feces lysate in Example 6, add 62.5 μL of water, 25 μL of 100 mM 3-nitrophenylhydrazine·HCl in 55% methanol solution, 25 μL of 60 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide·HCl and 5.0% pyridine in 55% methanol solution, react at 4°C for 30 min, and evaporate. Take 137.5 μL of mouse feces lysate, add 62.5 μL of water, 25 μL of 100 mM 3-nitrophenylhydrazine·HCl in 55% methanol solution and 25 μL of 55% methanol solution, react at 37°C for 30 min, and evaporate. Take 137.5 μL of mouse feces lysate, add 62.5 μL of water, 50 μL of 55% methanol solution, mix well, and evaporate. Redissolve with 250 μL of 25% methanol solution for injection.

[0087] In the mouse fecal samples, when 3-nitrophenylhydrazine derivatization was not used, only 15 metabolites could be detected with reference to the metabolites of the aforementioned 25 standards, and the mass spectrometry response was relatively low. At the same time, the metabolites were concentrated in the first 6.5 minutes, and the separation effect was poor ( Figure 7 C). After 3-nitrophenylhydrazine derivatization, 20 metabolites were sensitively detected, the mass spectrometry response was improved, and the metabolites were separated within 1-11 minutes, and the separation efficiency was improved ( Figure 7 D). The above data once again demonstrate that the optimized 3-nitrophenylhydrazine derivatization of the present invention can improve the detection sensitivity and chromatographic separation ability of metabolites.

[0088] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. An optimized 3-nitrophenylhydrazine derivatization method, characterized in that: The following steps are involved: (1) Direct derivatization of α-keto acid metabolites using 3-nitrophenylhydrazine; (2) For metabolites containing only carboxyl groups, a 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl) carbodiimide / pyridine derivatization reaction is used; (3) For metabolites containing only carbonyl groups, 3-nitrophenylhydrazine derivatization or 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine derivatization or no derivatization is used; (4) For other metabolites, a non-derivatized method was adopted.

2. The method according to claim 1, characterized in that The reaction temperature of the 3-nitrophenylhydrazine direct derivatization reaction is 37°C.

3. The method according to claim 1, characterized in that The reaction temperature of the 3-nitrophenylhydrazine / 1-ethyl-(3-dimethylaminopropyl)carbodiimide / pyridine derivatization reaction is 4°C.

4. The method according to claim 1, characterized in that In the derivatization reaction, the concentration of 3-nitrophenylhydrazine is 10-15 mM.

5. The method according to claim 1, characterized in that Four standards, namely indoleacetic acid, indolepyruvic acid, indole-3-propionic acid and pyruvic acid, were selected for LOD detection, and the LOD of the derivatization product obtained by the optimized 3-nitrophenylhydrazine derivatization method reached the pM level.

6. The method according to claim 1, characterized in that The derivatization product obtained by the optimized 3-nitrophenylhydrazine derivatization method is stable within 8 hours after re-dissolution.

7. Use of the optimized 3-nitrophenylhydrazine derivatization method according to any one of claims 1 to 6 in non-targeted metabolomics analysis, characterized in that: The samples for the non-targeted metabolomics analysis were Bacteroides Thetaiotaomicron and mouse feces samples.

8. A method for detecting metabolites in a biological sample using the optimized 3-nitrophenylhydrazine derivatization method according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) preparing a lysate of the test substance by conventional methods; (2) Take a portion of the lysate, add water, 55% methanol aqueous solution of 3-nitrophenylhydrazine·HCl, 55% methanol aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide·HCl and 55% methanol aqueous solution of pyridine, react at 4°C for 30 minutes, and evaporate to dryness; (3) Take an equal amount of the second lysate, add water, 55% methanol aqueous solution of 3-nitrophenylhydrazine·HCl and 55% methanol aqueous solution, react at 37°C for 30 min, and evaporate to dryness; (4) Take an equal amount of the third lysate, add water and 55% methanol aqueous solution, mix well, and evaporate to dryness; (5) The samples prepared in step (2), step (3), and step (4) were respectively re-dissolved in 25% methanol water, injected within 8 hours, and metabolites were detected.

9. The method according to claim 8, characterized in that The metabolites were detected by UPLC-MS method, and the results of UPLC-MS were imported into Progenesis QI for metabolite identification.

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