Application of 3, 4-diaminobenzophenone in detection of alpha-keto acid and detection method of 3, 4-diaminobenzophenone

Abstract

The invention relates to the field of biochemical detection, in particular to application of 3, 4-diaminobenzophenone in detection of alpha-keto acid and a detection method of the 3, 4-diaminobenzophenone. The invention discloses a reagent for detecting alpha-keto acid. The preparation method comprises the following steps: adding 2, 3, 4-diaminobenzophenone and a solvent; the detection method comprises the following steps: oscillating and uniformly mixing a to-be-detected sample and a reagent, then carrying out derivatization reaction, and after the derivatization reaction is finished, carrying out analysis by adopting matrix-assisted laser desorption / ionization time-of-flight mass spectrometry equipment, the reactive substrate 3, 4-diaminobenzophenone used in the invention can be subjected to a rapid derivatization reaction with alpha-keto acid under a heating condition, the problem of low ionization efficiency of alpha-keto acid is improved, other conventional substrates are not needed, and the sample pretreatment process is simplified; in addition, the reactive matrix shows good crystallinity on the MALDI target plate, and quantitative analysis of alpha-keto acid is facilitated.

Description

Technical Field

[0001] The invention relates to the field of biochemical detection, and in particular to the application of 3,4-diaminobenzophenone in detecting alpha-keto acid and a detection method thereof. Background Art

[0002] α-keto acid compounds are intermediates in many metabolic pathways in organisms, such as glycolysis, Krebs cycle, and amino acid metabolism. The level of α-keto acid in organisms also plays a key role in the diagnosis of metabolic disorders. In addition, α-keto acid compounds can also be used as dietary supplements, food additives, and antioxidants to prevent and treat a variety of diseases, and are also widely used in the fields of food and medicine. Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful analytical tool with high salt tolerance, high throughput, and high sensitivity, and has been widely used in the field of biochemistry. However, α-keto acid is a "small molecule" with low ionization efficiency, and generally needs to be derivatized before detection; and the conventional MALDI matrix is ​​also a small molecule compound, and the signal in the low mass range will affect the detection of α-keto acid compounds.

[0003] Reactive matrices can be used as both derivatization agents and MALDI matrices for the detection of "small molecules". On the one hand, reactive matrices have the function of derivatization agents and can undergo derivatization reactions with analytes to improve their ionization efficiency; on the other hand, reactive matrices also have the performance of MALDI matrices, eliminating the need to use other matrices for detection, thus simplifying the sample pretreatment process. Summary of the invention

[0004] The purpose of the present invention is to provide an α-keto acid compound analysis method based on reactive matrix-assisted laser desorption / ionization time-of-flight mass spectrometry in view of the deficiencies in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention is to provide a reagent for detecting α-keto acid, comprising 3,4-diaminobenzophenone and a solvent.

[0007] Preferably, in the reagent, the concentration of the 3,4-diaminobenzophenone is 3-6 mg / mL.

[0008] Preferably, the solvent includes: methanol and water.

[0009] More preferably, the volume ratio of methanol to water is 1:(1-2).

[0010] The second aspect of the present invention is to provide a method for detecting α-keto acid, comprising the steps of: shaking and mixing the sample to be tested with the above reagent and then performing a derivatization reaction, and after the derivatization reaction is completed, using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer for analysis.

[0011] Preferably, the reaction temperature of the derivatization reaction is 55-60° C., and the reaction time is 0.5-1 h.

[0012] Preferably, the sample to be tested includes: α-keto acid, methanol and water.

[0013] Preferably, in the sample to be tested, the molar mass of the α-keto acid is 10 -4 M.

[0014] Preferably, the α-keto acid includes: pyruvic acid and α-ketoglutaric acid.

[0015] The third aspect of the present invention is to provide the use of 3,4-diaminobenzophenone in detecting α-keto acid.

[0016] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0017] The reactive matrix 3,4-diaminobenzophenone used in the present invention can not only rapidly undergo a derivatization reaction with an α-keto acid under heating conditions, thereby improving the problem of low ionization efficiency of the α-keto acid, but also eliminates the need to use other conventional matrices, thereby simplifying the sample pretreatment process; in addition, the reactive matrix exhibits good crystallinity on a MALDI target plate, thereby facilitating the quantitative analysis of α-keto acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the chemical structure of the reactive substrate 3,4-diaminobenzophenone (DABP);

[0019] Figure 2 Schematic diagram of the derivatization of pyruvate (PA) and α-ketoglutarate (AKG) with the reactive substrate DABP;

[0020] Figure 3 is the MALDI mass spectrum of pyruvic acid (PA); wherein, Figure 3 a: DABP as matrix; Figure 3 b 1,5-DAN as matrix; Figure 3 c is 9-AA as the matrix;

[0021] Figure 4 is the MALDI mass spectrum of α-ketoglutaric acid (AKG); wherein, Figure 4 a: DABP as matrix; Figure 4 b 1,5-DAN as matrix; Figure 4 c is 9-AA as the matrix;

[0022] Figure 5 This is the crystallization image of the reactive matrix DABP on the MALDI target plate;

[0023] Figure 6 "Spot-to-spot" reproducibility for the reactive matrix DABP. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0026] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0027] Example 1

[0028] This embodiment provides the use of 3,4-diaminobenzophenone (DABP) in the detection of pyruvic acid (PA).

[0029] Weigh pyruvic acid dissolved in a mixture of methanol and water (V 甲醇 :V 水 =1:1), configure 10 -4 M of the sample to be tested; weigh DABP dissolved in a mixed solution of methanol and water (V 甲醇 :V 水 =1:1), prepare 5 mg / mL DABP solution;

[0030] 50 μL of the sample to be tested and 50 μL of DABP solution were placed in a centrifuge tube and mixed evenly, and reacted in a 60°C water bath for 1 h. After the derivatization reaction was completed, 1 μL of the reaction solution was spotted on the MALDI target plate and dried at room temperature.

[0031] As a control, 1 μL of the sample to be tested and 1 μL of 1,5-naphthalenediamine (1,5-DAN, 10 mg / mL, the solvent used was a methanol-water mixture, V 甲醇 :V 水 =1:1) and 1 μL of 9-aminoacridine (9-AA, 10 mg / mL, the solvent used was methanol solution) were mixed, and then 1 μL of the mixture was spotted on the MALDI target plate and dried at room temperature.

[0032] The dried MALDI target plate was sent to MALDI-MS for analysis, and mass spectrometry analysis was performed on an Autoflex Max MALDI-TOF MS (Bruker Daltonics) equipped with a pulsed Nd:YAG laser (355 nm). The acceleration voltage was set to 20.55 kV in the reflectron negative ion mode, and 1000 laser shots were collected for each spectrum.

[0033] The MALDI mass spectra of pyruvic acid (PA) analyzed using DABP, 1,5-DAN and 9-AA as matrices are shown in Figure 2. Figure 3 As shown, it can be seen that when DABP is used as a matrix, an obvious mass spectrum peak of DABP-labeled pyruvate (PA) can be detected, while when 1,5-DAN and 9-AA are used as matrices, no signal response of pyruvate (PA) is detected.

[0034] Example 2

[0035] This embodiment provides the use of 3,4-diaminobenzophenone (DABP) in the detection of α-ketoglutaric acid (AKG).

[0036] Weigh α-ketoglutaric acid and dissolve it in a mixed solution of methanol and water (V 甲醇 :V 水 =1:1), configure 10 -4 M of the sample to be tested; weigh DABP dissolved in a mixed solution of methanol and water (V 甲醇 :V 水 =1:1), prepare 5 mg / mL DABP solution;

[0037] 50 μL of the sample to be tested and 50 μL of DABP solution were placed in a centrifuge tube and mixed evenly, and reacted in a 60°C water bath for 1 h. After the derivatization reaction was completed, 1 μL of the reaction solution was spotted on the MALDI target plate and dried at room temperature.

[0038] As a control, 1 μL of the sample to be tested and 1 μL of 1,5-naphthalenediamine (1,5-DAN, 10 mg / mL, the solvent used was a methanol-water mixture, V 甲醇 :V 水 =1:1) and 1 μL of 9-aminoacridine (9-AA, 10 mg / mL, the solvent used was methanol solution) were mixed, and then 1 μL of the mixture was spotted on the MALDI target plate and dried at room temperature.

[0039] The dried MALDI target plate was sent to MALDI-MS for analysis, and mass spectrometry analysis was performed on an Autoflex Max MALDI-TOF MS (Bruker Daltonics) equipped with a pulsed Nd:YAG laser (355 nm). The acceleration voltage was set to 20.55 kV in the reflectron negative ion mode, and 1000 laser shots were collected for each spectrum.

[0040] The MALDI mass spectra of α-ketoglutarate (AKG) analyzed using DABP, 1,5-DAN and 9-AA as matrices are shown in Figure 2. Figure 4 As shown, it can be seen that DABP as a matrix can detect the mass spectrum peak of DABP-labeled α-ketoglutarate (AKG), and a stronger signal is obtained than the commonly used alkaline matrix 1,5-DAN, while 9-AA as a matrix does not detect the signal response of α-ketoglutarate (AKG).

[0041] Example 3

[0042] DABP detected the reproducibility of the "spot-to-spot" mixed solution of pyruvate (PA) and α-ketoglutarate (AKG) on the MALDI target plate.

[0043] Weigh pyruvic acid and α-ketoglutaric acid and dissolve them in a mixed solution of methanol and water (V 甲醇 :V 水 =1:1) to prepare the sample to be tested; weigh DABP and dissolve it in a mixed solution of methanol and water (V 甲醇 :V 水 =1:1), prepare 5 mg / mL DABP solution;

[0044] 50 μL of the sample to be tested and 50 μL of DABP solution were placed in a centrifuge tube and mixed evenly, and reacted in a 60°C water bath for 1 h. After the derivatization reaction was completed, 1 μL of the reaction solution was spotted on the MALDI target plate and dried at room temperature.

[0045] The morphology of DABP was recorded under a MALDI-MS microscope. The signal-to-noise ratio of 20 sets of data collected at different locations of the sample point was used to evaluate the "point-to-point" reproducibility; the signal-to-noise ratio was obtained using Flex Analysis 3.3080 software (Bruker Daltonics).

[0046] The speckled crystal morphology of the reactive substrate DABP is shown in Figure 5 As shown in the figure, it can be seen that the reactive matrix DABP forms uniform crystals on the MALDI target plate; the signal-to-noise ratio change obtained by detecting the mixed solution of pyruvic acid (PA) and α-ketoglutaric acid (AKG) by MALDI MS is shown in the figure. Figure 6As shown, it can be seen that the reactive matrix DABP analysis of PA and AKG has good "point-to-point" reproducibility.

[0047] In summary, the reactive matrix 3,4-diaminobenzophenone used in the present invention can not only rapidly undergo a derivatization reaction with α-keto acid under heating conditions to improve the problem of low ionization efficiency of α-keto acid, but also eliminates the need to use other conventional matrices, thereby simplifying the sample pretreatment process; in addition, the reactive matrix exhibits good crystallinity on the MALDI target plate, which is helpful for the quantitative analysis of α-keto acid.

[0048] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A reagent for detecting α-keto acid, characterized in that Comprising; 3,4-diaminobenzophenone and a solvent.

2. The reagent according to claim 1, characterized in that In the reagent, the concentration of the 3,4-diaminobenzophenone is 3-6 mg / mL.

3. The reagent according to claim 1, characterized in that The solvent includes methanol and water.

4. The reagent according to claim 3, characterized in that The volume ratio of methanol to water is 1:(1-2).

5. A method for detecting α-keto acid, characterized in that: The steps include: The sample to be tested is shaken and mixed with the reagent according to any one of claims 1 to 4, and then subjected to a derivatization reaction. After the derivatization reaction is completed, a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer is used for analysis.

6. The detection method according to claim 5, characterized in that: The reaction temperature of the derivatization reaction is 55-60° C., and the reaction time is 0.5-1 h.

7. The detection method according to claim 5, characterized in that: The sample to be tested includes: α-keto acid, methanol and water.

8. The detection method according to claim 5, characterized in that: In the sample to be tested, the molar mass of the α-keto acid is 10 -4 M.

9. The detection method according to claim 5, characterized in that: The α-keto acids include: pyruvic acid and α-ketoglutaric acid.

10. Application of 3,4-diaminobenzophenone in the detection of α-keto acids.

Images