CN114858947A

CN114858947A - Detection method of alpha-dicarbonyl compound and application thereof

Info

Publication number: CN114858947A
Application number: CN202210628793.6A
Authority: CN
Inventors: 郭宾; 刘雅璇; 丑芳; 黄礼斌; 方静
Original assignee: Hunan Normal University
Current assignee: Hunan Normal University
Priority date: 2022-06-06
Filing date: 2022-06-06
Publication date: 2022-08-05
Anticipated expiration: 2042-06-06
Also published as: CN114858947B

Abstract

The invention provides a method for detecting an alpha-dicarbonyl compound, which comprises the following steps: mixing 2-guanidinobenzimidazole with a solvent to prepare a derivatization reagent; mixing a sample to be tested with the derivatization reagent, and carrying out derivatization reaction to prepare a derivatization product; subjecting the derivatized product to liquid chromatography tandem mass spectrometry; wherein the conditions used for the liquid chromatography include: the mobile phase A is formic acid aqueous solution; the mobile phase B is methanol. The reagent and the derivative adopted by the detection method have stable properties, high sensitivity, wide detection range and better repeatability and specificity.

Description

Detection method of alpha-dicarbonyl compound and application thereof

Technical Field

The invention relates to the field of biochemical analysis, in particular to a detection method of an alpha-dicarbonyl compound and application thereof.

Background

Alpha-dicarbonyl compounds are a class of reactive intermediates and reactive metabolites that arise primarily from the thermal processing of foods and from processes of the body's sugar metabolism. The substances can chemically modify important living substances in vivo such as proteins, DNA and the like, so that the structural change and the functional damage of the substances are caused, and various diseases such as aging or diabetes and the like are caused, thereby attracting increasing attention in the fields of food safety and biomedicine. Therefore, how to detect the types, contents and changes of the potential hazards in food and biological samples has great significance for healthy diet and disease prevention and treatment.

The alpha-dicarbonyl compound has strong hydrophilicity and low stability, is lack of an ultraviolet absorbing group and a fluorescent chromophoric group, and simultaneously the matrix components of an actual sample containing the alpha-dicarbonyl compound are often complex, so that the alpha-dicarbonyl compound can not be accurately detected. Therefore, the alpha-dicarbonyl compound needs to be pretreated for analysis, and the alpha-dicarbonyl compound is subjected to derivatization by virtue of a plurality of compounds, so that the chromatographic retention of the substances is improved, optical groups are added, and the separation, the qualification and the quantification of the substances are realized.

The conventional alpha-dicarbonyl compound derivatizing agents are mostly o-diamine compounds, of which o-phenylenediamine is the most common. The o-phenylenediamine has the advantages of fast derivation, chromophore generation, low price, easy obtainment and the like, and has wide application in the pre-column derivation of a chromatographic method. However, it is difficult to store itself, is unstable at room temperature, is easily oxidized to phenazine, and also has a certain biotoxicity, causing corrosion of the skin, mucous membranes, and the like.

Arginine containing a guanidino reactive group is also a traditional alpha-dicarbonyl compound derivative, has the advantages of low biological toxicity and good selectivity, and is suitable for determination of samples with complex matrixes. However, arginine is more polar and the chromatographic retention is poor.

The problem of low sensitivity occurs when the conventional alpha-dicarbonyl compound derivatization agent is applied to chromatography-mass spectrometry combined analysis after the alpha-dicarbonyl compound is subjected to derivatization reaction, because the molecular weight difference of different substances of the alpha-dicarbonyl compound is large, the structure of hexose C6 is reserved in the long-chain alpha-dicarbonyl compound, but the structure of the short-chain alpha-dicarbonyl compound is simple and the molecular weight is small, so that the volatility of the short-chain alpha-dicarbonyl compound is greatly different.

Disclosure of Invention

Based on the method, the adopted reagent and the derivative have stable properties, high sensitivity, wide detection range and better repeatability and specificity.

The invention is realized by the following technical scheme.

A method for detecting an alpha-dicarbonyl compound, comprising the steps of:

mixing 2-guanidinobenzimidazole with a solvent to prepare a derivatization reagent;

mixing a sample to be tested with the derivatization reagent, and carrying out derivatization reaction to prepare a derivatization product;

subjecting the derivatized product to liquid chromatography tandem mass spectrometry;

wherein the conditions used for the liquid chromatography include: the mobile phase A is aqueous solution of formic acid; the mobile phase B is methanol.

In one embodiment, the temperature of the derivatization reaction is 30 ℃ to 50 ℃; and/or

The time of derivatization reaction is 6-12 h; and/or

And performing derivatization reaction in an environment with the pH value of 7-8.

In one embodiment, the conditions employed for liquid chromatography further comprise: employing a gradient elution procedure comprising: 0 min-6 min, the volume percentage of the mobile phase B is 10 percent; 6-10 min, the volume percentage of the mobile phase B is changed from 10% to 20%; 10-16 min, and the volume percentage of the mobile phase B is changed from 20% to 90%; 16-24 min, and keeping the volume percentage of the mobile phase B at 90%; the volume percentage of the mobile phase B is changed from 90 percent to 10 percent from 24min to 25 min.

In one embodiment, the conditions employed for liquid chromatography further comprise: the flow rate is 0.8mL/min to 1.2 mL/min; the column temperature is 20-25 ℃; the chromatographic column is an octadecyl silane bonded silica gel column; and/or

The liquid chromatography employs chromatographic columns of sizes including: the column length is 120 mm-130 mm, the inner diameter is 4.4 mm-4.8 mm, and the grain diameter of the filler is 4 μm-6 μm.

In one embodiment, the mass fraction of formic acid in the formic acid aqueous solution is 0.08% -0.12%.

In one embodiment, the mass spectrum employs conditions comprising: the ionization mode is electrospray ionization; the atomizing airflow speed is 2L/min-4L/min; the drying airflow speed is 8L/min-12L/min; the temperature of the desolventizing tube is 220-280 ℃; the temperature of the heating module is 350-450 ℃; the voltage of the ion source interface is 3.5 kV-4.5 kV; the scanning mode is a multiple response monitoring scan.

In one embodiment, the solvent is selected from one or both of methanol and water.

In one embodiment, the α -dicarbonyl compound is selected from one or more of glyoxal, methylglyoxal, 2, 3-butanedione, D-glucosone, 2-chloro-2-phenylacetaldehyde and 1-phenyl-1, 2-propanedione.

In one embodiment, the source of the sample to be tested is selected from one or more of traditional Chinese medicinal materials, biological tissues and food.

The invention also provides an application of the alpha-dicarbonyl compound detection method in detection of alpha-dicarbonyl compounds in traditional Chinese medicinal materials, biological tissues and foods.

Compared with the prior art, the detection method of the alpha-dicarbonyl compound has the following beneficial effects:

the inventor selects 2-guanidinobenzimidazole as a derivative reagent of an alpha-dicarbonyl compound, and performs liquid chromatography tandem mass spectrometry on the derivative product to establish an alpha-dicarbonyl compound derivative method using the 2-guanidinobenzimidazole as a mass spectrometry sensitization probe. The 2-guanidinobenzimidazole is used as an electron donor and is easy to attack an alpha-dicarbonyl structure, so that a five-membered ring with a stable structure is formed, the purpose of derivatization is finally achieved, the ionization efficiency and mass spectrum response are improved, good sensitivity can be obtained, and the detection sensitivity of the short-chain alpha-dicarbonyl compound is improved by 15-65 times. Meanwhile, the detection method of the alpha-dicarbonyl compound has wide detection range, can simultaneously meet the detection requirements of long-chain and short-chain alpha-dicarbonyl compounds, and has good repeatability, high accuracy and better specificity and selectivity.

Drawings

FIG. 1 is a comparison chart of different derivatization temperature effect investigation provided by the invention;

FIG. 2 is a graph comparing the undehydrated and dehydrated products at different derivatization temperatures provided by the present invention;

FIG. 3 is a comparison chart of effect investigation of different derivatization times provided by the present invention;

FIG. 4 is a comparison graph for examining the effect of different derivatization pH values provided by the invention;

FIG. 5 is a comparison chart of different chromatographic column effect investigation provided by the present invention;

FIG. 6 is a secondary mass fragmentation diagram of 2-GBI-GO provided by the present invention;

FIG. 7 is a proton ion spectrum of each of the derivatized products provided herein;

FIG. 8 is a graph showing the stability of each of the derivatized products provided by the present invention.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention provides a method for detecting an alpha-dicarbonyl compound, which comprises the following steps:

mixing a sample to be detected with a derivatization reagent, and carrying out derivatization reaction to prepare a derivatization product;

performing liquid chromatography tandem mass spectrometry on the derivatization product;

wherein, the conditions adopted by the liquid chromatography comprise: the mobile phase A is aqueous solution of formic acid; the mobile phase B is methanol.

In one specific example, the temperature of the derivatization reaction is 30 ℃ to 50 ℃. Preferably, the temperature of the derivatization reaction is 40 ℃.

In a specific example, the time of the derivatization reaction is 6h to 12 h. Preferably, the time of the derivatization reaction is 6 h.

In a specific example, the derivatization reaction is carried out in an environment with a pH of 7-8. Preferably, the derivatization reaction is carried out in an environment having a pH of 7.4. More specifically, pH adjustment was achieved by addition of PBS buffer solution.

In one particular example, the conditions employed for liquid chromatography further include: a gradient elution procedure is employed, the gradient elution procedure comprising: 0 min-6 min, the volume percentage of the mobile phase B is 10 percent; 6-10 min, the volume percentage of the mobile phase B is changed from 10% to 20%; 10-16 min, and the volume percentage of the mobile phase B is changed from 20% to 90%; 16 min-24 min, the volume percentage of the mobile phase B is kept to be 90 percent; the volume percentage of the mobile phase B is changed from 90 percent to 10 percent from 24min to 25 min.

In one specific example, the conditions employed for liquid chromatography further include: the flow rate is 0.8mL/min to 1.2 mL/min; the chromatographic column is an octadecylsilane chemically bonded silica gel column; the column temperature is 20-25 ℃.

In one particular example, liquid chromatography employs column sizes including: the column length is 120 mm-130 mm, the inner diameter is 4.4 mm-4.8 mm, and the grain diameter of the filler is 4 μm-6 μm.

More specifically, the liquid chromatography is performed on a column of μ M Ltimate XB-C18(125 mm. times.4.6 mm, particle size 5 μ M).

In a specific example, the mass fraction of formic acid in the formic acid aqueous solution is 0.08% -0.12%.

More specifically, the mass fraction of formic acid in the aqueous formic acid solution was 0.1%.

In one specific example, mass spectrometry employs conditions that include: the ionization mode is electrospray ionization; the atomizing airflow speed is 2L/min-4L/min; the drying airflow speed is 8L/min-12L/min; the temperature of the desolventizing tube is 220-280 ℃; the temperature of the heating module is 350-450 ℃; the voltage of the ion source interface is 3.5 kV-4.5 kV; the scanning mode is a multiple response monitoring scan.

In a specific example, the solvent is selected from one or both of methanol and water.

In a specific example, the α -dicarbonyl compound is selected from one or more of glyoxal, methylglyoxal, 2, 3-butanedione, D-glucosone, 2-chloro-2-phenylacetaldehyde and 1-phenyl-1, 2-propanedione.

In a specific example, the source of the sample to be tested is selected from one or more of traditional Chinese medicinal materials, biological tissues and food.

In a specific example, the Chinese medicinal material is selected from one or more of radix Linderae, cortex Magnoliae officinalis, rhizoma Polygonati Odorati, Buddleja officinalis leaf, caulis Trachelospermi, fructus Psoraleae, radix bupleuri, fructus Schisandrae chinensis, fructus evodiae, herba Hedyotidis Diffusae, radix Curcumae, herba Epimedii, rhizoma Chuanxiong, herba Cephalanoploris, Inulae flos, radix Et rhizoma Rhei, fructus forsythiae, radix rehmanniae, radix Isatidis, radix Dipsaci, and radix Codonopsis.

In one particular example, the biological tissue is a cell. More specifically, the cells are selected from HEK 293 cells.

The cracking of each derivative product obtained by the detection method of the alpha-dicarbonyl compound provided by the invention has similar rules. The rule can not only accurately judge whether the derivatization reaction really occurs, but also facilitate subsequent spectrum resolution and data analysis. And common daughter ions m/z 159.0 and m/z 134.0 are formed, and the non-target reactive metabolite alpha-dicarbonyl compound can be screened by utilizing a precursor ion scanning mode according to the characteristic, so that the metabolic spectrum is expanded.

The method for detecting an α -dicarbonyl compound and the use thereof according to the present invention will be described in further detail with reference to specific examples. The starting materials used in the following examples are all commercially available products unless otherwise specified.

Example 1

This example provides a method for detecting an α -dicarbonyl compound, which includes:

reagent

2-guanidinobenzimidazole (2-GBI), Glyoxal (GO), Methylglyoxal (MGO), 2, 3-butanedione (DA), o-phenylenediamine, and arginine were purchased from Liaoning Cuke Biotechnology, Inc.; D-Glucosone (GS), 2-guanidinobenzimidazole were purchased from Sigma, Shanghai, China; 2-oxo-2-phenylacetaldehyde (2-OPT), 1-phenyl-1, 2-propanedione (1-PPD) were purchased from Beijing YinoKai science, Inc.; disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium hydroxide were all purchased from alatin Biotechnology, Inc.; methanol was purchased from enokay technologies ltd; formic acid was purchased from chemical reagents ltd, miuiou, department of tianjin.

Second, the experimental procedure

1. Derivatization reaction

Preparation of guanidinobenzimidazole solution: accurately weighing 2-GBI standard substance, dissolving with methanol (chromatographic grade) or ddH2O, diluting to obtain 10mM mother liquor, and storing at-20 deg.C in dark place.

The reaction was carried out in a 1.5mL centrifuge tube, and the total volume of the reaction solution was 1 mL. 2-guanidinobenzimidazole solution, PBS buffer solution, alpha-dicarbonyl compound were added in sequence as in Table 1 and vortexed for 2 min. Reacting for 6h under the conditions of water bath heating at 40 ℃ and pH of 7.4, and then placing at-20 ℃ for 20min to terminate the reaction. The mixture was filtered through a 0.22 μm filter for LC-MS/MS detection analysis. At least three replicates were guaranteed.

TABLE 1

Note: the concentrations were 10. mu.M except PBS.

2. Liquid chromatography-mass spectrometry detection analysis

(1) The liquid chromatography conditions were as follows:

an analytical instrument: triple quadrupole LC-MS 8050;

a chromatographic column: μ M Ltimate XB-C18(125mm × 4.6mm, particle size 5 μ M);

mobile phase A: 0.1% formic acid in water (v/v);

mobile phase B: methanol;

flow rate: 1 mL/min;

sample introduction volume: 10 mu L of the solution;

and (3) an elution mode: gradient elution (elution procedure as shown in table 2);

column temperature: at 22 ℃.

TABLE 2

(2) The mass spectrometry conditions are shown in table 3, using positive ion mode analysis:

TABLE 3

3. Changing conditions

(1) Other conditions were unchanged, only the temperature of derivation was changed: changing the temperature of 40 ℃ to 24 ℃, 37 ℃, 60 ℃ and 80 ℃ respectively;

(2) other conditions were unchanged, only the derivation time was changed: the 6h is changed into 2h, 4h, 12h and 24h respectively.

(3) Other conditions were unchanged, only the derivatization pH was changed: the pH value is adjusted within the range of 3-10 by PBS buffer solution.

(4) Other conditions were unchanged, only the type of column was changed: a μ M Ltimate XB-C18 column (125 mm. times.4.6 mm, particle size 5 μ M) was changed to a Diamonsil C18 column (150. times.2.1 mm, particle size 3 μ M).

Third, experimental results

1. Conditional screening

(1) The derivatization effects of different alpha-dicarbonyl compounds are examined by selecting 24 ℃, 37 ℃, 40 ℃, 60 and 80 ℃ respectively, and the experimental results are shown in figure 1. Under the condition of single guarantee variable, the derivation strength of MGO in six alpha-dicarbonyl compounds is the best, and the derivation strength of short-chain dicarbonyl compounds such as GO, DA and the like, 1-PPD, 2-OPT and GS are weakened in sequence. This is because the reactivity of glyoxal is theoretically the highest, but in practice, glyoxal is polymerized in an aqueous solution to a high degree, and thus, methylglyoxal is the most reactive. In addition, the optimal derivatization temperature of glyoxal and methylglyoxal is 40 ℃, and the optimal derivatization temperature of other four alpha-dicarbonyl compounds is 24 ℃. However, the response of the remaining three α -dicarbonyl compounds, except 2, 3-butanedione, from 24 ℃ to 40 ℃ varied within acceptable ranges. Therefore, 40 ℃ was selected as the derivatization reaction temperature.

In the experimental process, the higher the derivatization temperature is, the darker the reaction solution is, the yellow solution is at 60 ℃ and the brown solution is at 80 ℃. At the same time, the derivatized product (not dehydrated) and by-product (dehydrated) were monitored, and in the case of 2-GBI-GO, the temperature was found to be elevated, with only a small portion of the derivatized product being further dehydrated, as shown in FIG. 2. In combination with the experimental phenomenon, it is speculated that when the derivatization temperature exceeds a limit, the derivatization substance is easy to denature, thereby causing great reduction of derivatization effect.

(2) Ensuring that the variable is single, continuously reacting for 24 hours by taking the hour as a unit, and selecting five time points (2, 4, 6, 12 and 24 hours) for examination. It can be derived from FIG. 3 that GO, MGO, 2-OPT, 1-PPD gave the best derivatization effect at 6 h. GS reaches the optimal derivatization time in 12h, the subsequent derivatization effect fluctuation in 12h is small, but the derivatization effects of other five compounds are reduced in different degrees, and MGO is the most remarkable. Therefore, 6h was chosen as the optimal derivatization time.

(3) Since the actual sample matrix containing the α -dicarbonyl compound is complex, its pH may vary depending on external factors. The detection and analysis of the compound are carried out in a relatively stable acid-base environment, and the problem can be greatly relieved by adding the buffer solution. PBS buffer was selected to adjust pH and optimized within the pH range of 3-10. As can be seen from FIG. 4, MGO reached a peak response at pH7.4, and GO, DA, 2-OPT, 1-PPD, and GS did not fluctuate greatly in the pH range of 4-10 and were less disturbed by pH. Meanwhile, the pH7.4 condition is mild, and is also an acid-base condition for culturing and growing most of organisms, so that the pH7.4 is selected as the optimal derivatization pH.

(4) In liquid chromatography, a chromatography column is a central element of a sample. The chromatographic column needs to meet the requirements of high separation efficiency, good selectivity, good column efficiency and the like. When the analysis target properties and structures are different, the chromatographic columns also have different separation effects. For better separation analysis purposes, the invention compared the separation of the six alpha-dicarbonyl compounds described above using a Diamonsil C18 column (150X 2.1mm, particle size 3. mu.M) and a μ M Ltimate XB C18 column (125mm X4.6 mm, particle size 5. mu.M). As can be seen in fig. 5, the Diamonsil C18 column did not completely separate the six compounds described above; while the Μ ltate XB C18 column has better separation, higher response and takes less time. And combining the above steps, selecting XB 18 as a chromatographic column.

2. Results of mass spectrum fragmentation law

And (3) detecting under the optimal conditions, and analyzing the cracking rule of the derivative product of the alpha-dicarbonyl compound: the product is generated into [ M ] under the positive ion mode of an ESI ion source ⁺ H ⁺ ]The parent ion (m/z 234.1) loses one molecule of water to form a carbon-nitrogen double bond, and a daughter ion of m/z 216.0 is obtained; and then the carbon-nitrogen single bond at two positions of the imidazole ring is broken to obtain the daughter ion of m/z 159.1, and the ion response is highest, so that m/z 159.1 is selected as quantitative ion; and is broken at the carbon-nitrogen double bond to form m/z 134.1 daughter ionAs shown in fig. 6.

In the secondary mass spectrum (shown in figure 7) of six derivative products of 2-GBI, GO, MGO, DA, 2-OPT, 1-PPD and GS, each derivative product has a similar cracking rule. The rule can accurately judge whether the derivatization reaction really occurs or not, is convenient for subsequent spectrum resolution and data analysis, forms common daughter ions m/z 159.0 and m/z 134.0, and can screen a non-target reactive metabolite alpha-dicarbonyl compound by using a precursor ion scanning mode according to the characteristic to expand a metabolic spectrum.

3. Stability study of derivatives

And (3) placing the derivative in a dark environment at 24 ℃ for stability investigation. Samples were taken at 0, 2, 4, 8, 16, and 24 hours and their peak area changes were observed, as shown in FIG. 8. Under the storage condition, the content of the six reactive metabolites is kept for 24 hours, and the content variation range is small and is within an acceptable range.

2. Optimization of liquid quality parameters

And establishing a mass spectrum multi-reaction monitoring method for the six reactive metabolites, and optimizing mass spectrum parameters of the mass spectrum multi-reaction monitoring method, such as parent ions, daughter ions and collision voltage. The optimization parameters are shown in table 4.

TABLE 4

Example 2

The embodiment provides a method for detecting an alpha-dicarbonyl compound in a traditional Chinese medicinal material, which comprises the following specific steps:

reagent and reagent

Radix linderae, mangnolia officinalis, radix polygonati officinalis, buddleia, Chinese starjasmine stem, fructus psoraleae, radix bupleuri, schisandra chinensis, fructus evodiae, spreading hedyotis herb, radix curcumae, herba epimedii, ligusticum wallichii, field thistle, inula flower, rheum officinale, fructus forsythiae, radix rehmanniae recen, radix isatidis, teasel root and radix codonopsis are purchased from local drug stores in Changsha. The other reagents were the same as in example 1.

If the reagents are not specially stated, the purity is more than 98%. The water used in the experiment was ddH 2O.

Secondly, derivatization reaction conditions and instrument conditions

1. Derivatization reaction

2. Liquid chromatography-mass spectrometry detection analysis

(1) The liquid chromatography conditions were as follows:

an analytical instrument: triple quadrupole LC-MS 8050;

mobile phase A: an aqueous solution (v/v) containing 0.1% formic acid;

mobile phase B: methanol;

flow rate: 1 mL/min;

sample introduction volume: 10 mu L of the solution;

and (3) an elution mode: gradient elution (elution procedure shown in table 5);

column temperature: at 22 ℃.

TABLE 5

(2) Mass spectrometry conditions were as shown in example 1, 3, using positive ion mode analysis:

third, the experimental process

1. Solution preparation

Accurately weighing (measuring) each standard (GO, MGO, DA, GS, 2-OPT, 1-PPD and 2-GBI), dissolving with methanol (chromatographic grade) or ddH2O, diluting to obtain 10mM each standard mother liquor, and storing at-20 deg.C in dark place.

2. Establishment of a Standard Curve

The six standard curves consist of six standard solution points with concentration gradients. The concentrations of GO, MGO and DA are in the range of 2.25pmol/L to 2.25. mu. Mol/L, while the concentrations of 2-OPT and 1-PPD are in the range of 90nmol/L to 2.25. mu. Mol/L and the concentration of GS is in the range of 225nmol/L to 2.25. mu. Mol/L. At least three replicates were guaranteed.

After derivatization, the mixture is placed at the temperature of minus 20 ℃ for 20min, and the reaction is stopped. The samples were then subjected to membrane treatment (0.22. mu.M) for mass spectrometry. Finally, the concentrations of the six reactive metabolites are used as abscissa, the peak area of response is used as ordinate, and the data is analyzed and processed by using software IBM SPSS staticisics 26, so that six standard curves are obtained.

3. Treatment of Chinese medicinal materials

Accurately weighing 6.00mg of Chinese herbal medicines into a 10mL centrifuge tube, adding 4mL ddH2O, soaking for 5min, and performing ultrasonic treatment for 20 min. Then adding 2mL of methanol, vortexing for 5min, fully and uniformly mixing, centrifuging for 15min at 15000r/min, precipitating protein, taking 4mL of supernatant, diluting by one time, and fixing the volume to 8 mL. Storing at-20 deg.C for use.

4. Derivatization of Chinese herbs

Putting 450 μ L of 2-GBI solution into a 1.5mL centrifuge tube, adding 100 μ L of PBS buffer solution (pH7.4), adding 450 μ L of the treated Chinese herbal medicine solution, performing water bath at 40 deg.C for 6h, and after derivatization, placing at-20 deg.C for 20min to terminate the reaction. After passing through a membrane (0.22 μm), the membrane was transferred to a 1.5mL brown sample vial and examined by a mass spectrometer.

Fourth, methodology investigation

1. Linear correlation and sensitivity investigation

Standard curves were plotted for GO, MGO, DA, 2-OPT, 1-PPD and GS, weighted by the reciprocal concentration (1/c), and fitted with a linear regression equation using the software IBM SPSS statics 26. The linear correlation degrees (R) are respectively 0.934, 0.985, 0.996, 0.991, 0.997 and 0.990, and a better linear relation is shown in a table 6. The limit of quantitation and the limit of detection both reach pmol/L level, which shows that the method has higher sensitivity and can be used for the analysis of the alpha-dicarbonyl compound of the Chinese herbal medicine.

TABLE 6

2. Accuracy survey

Six standard solutions with low, medium and high concentrations of alpha-dicarbonyl compounds are prepared and added into the lindera aggregate and the oldenlandia diffusa, and the standard recovery rate of the method is inspected, so that the accuracy of the method is reflected. The experimental results are shown in tables 7 and 8. The standard recovery rate of radix Linderae is 91.4-105.8%, and the standard recovery rate of herba Hedyotidis Diffusae is 85.3-113.0%. The method has good reproducibility and stability, and simultaneously, the method also has accurate and reliable detection results.

TABLE 7 recovery rate of lindera strychnifolia by adding standard

TABLE 8 recovery of hop by adding standard

3. Precision survey

In order to evaluate the precision of the method, six target alpha-dicarbonyl compounds in the combined spicebush root and the oldenlandia diffusa are detected in parallel three times and three consecutive days. The intra-day precision was represented by the relative standard deviation of the triplicate measurements, and the inter-day precision of the reaction was reflected by the relative standard deviation of the three consecutive days of the measurement, as shown in Table 9. The daily precision is in the range of 1.1-10.1%, and the daytime precision is in the range of 4.3-14.3%. The data show that the method has better precision and can be applied to the content determination of the alpha-dicarbonyl compounds of the combined spicebush root and the oldenlandia diffusa.

TABLE 9

4. Analysis of actual samples

After the mass spectrometry multiple reaction monitoring method for the alpha-dicarbonyl compound was verified, quantitative analysis was performed on lindera strychnifolia and oldenlandia diffusa, and the detection results are shown in table 10.

The content of alpha-dicarbonyl compounds in the combined spicebush root and the oldenlandia diffusa is greatly different, and the MGO content in the combined spicebush root is the highest and is about 0.715 mu g/g; the maximum DA content in the hedyotis diffusa is about 0.523 mu g/g. The RSD of the detection probe is within 2.51-10.14%, and the detection error is within an acceptable range.

TABLE 10

Example 3

This example provides a method for detecting α -dicarbonyl compounds in cells, which comprises the following steps:

reagent and reagent

Hydrogen peroxide (H2O2, 30% v/v), perchloric acid (PCA) available from Tianjin Xin Yuan GmbH; methanol, acetonitrile, quercetin were all purchased from enokay technologies ltd; l-buthionine sulfoximine is purchased from Shanghai Xiandong Biotechnology GmbH; formic acid, ethanol (75%, v/v), glucose (Glu) were purchased from Mimi European Chemicals, Inc., Tianjin; MEM medium and fetal bovine serum were purchased from Saimer Feishel scientific Co; trypsin and antibiotics are purchased from Shanghai Dingsang Biotechnology GmbH; the Annexin V-FITC/PI apoptosis detection kit and the double-staining apoptosis detection kit are purchased from Anlun Biotechnology GmbH; normal saline was purchased from Kolen pharmaceutical Co., Ltd, Hunan; the MTT kit is purchased from Shanghai biological engineering Co., Ltd; the HuMan GLO ELISA kit was purchased from Shanghai Jinning industries, Inc. The other reagents are the same as 2.2.1.

If the reagents are not specially stated, the purity is more than 98%. All water used in the experimental process is ddH ₂ O。

Secondly, derivatization reaction conditions and instrument conditions

The derivatization reaction conditions and the apparatus conditions were the same as in example 2.

Third, the experimental process

1. Establishment of apoptosis model

(1) HEK 293 cell resuscitation

Firstly, the temperature of ddH2O is preset to 37 ℃, then, the HEK 293 cryopreservation tube (containing 1mL of cell mixed liquid) is taken out from a liquid nitrogen tank by wearing protective measures such as gloves, a mask and a hat, and immediately placed in ddH2O (preset to 37 ℃) and shaken gently to be completely dissolved in 60 s. After the dissolution is completed, the outer wall of the cryopreservation tube is disinfected by alcohol and placed on a super clean bench. In a super clean bench, a sterile centrifuge tube is prepared in advance, 10mL of freshly prepared culture medium is transferred into the centrifuge tube, the cell suspension in the frozen tube is added, centrifugation (1000r/min) is carried out for 3min at normal temperature, and the upper culture medium is discarded. Then 1mL of culture medium was added to the HEK 293 cell pellet, the mixture was resuspended and mixed, and then transferred to a T25 cell culture flask, and the volume of the culture medium was adjusted to 5mL, and the flask was incubated at 37 ℃ in a 5% CO2 incubator. After two days, the culture medium is replaced, and the cells can be subcultured when the cells are fused to about 80%.

(2) HEK 293 cell culture

HEK 293 cells were cultured in MEM medium containing 10% fetal bovine serum at 37 ℃ in a saturated humidity incubator containing 5% CO 2.

(3) HEK 293 cell plating

After the HEK 293 cells in good growth state were digested with 0.25% pancreatic enzyme, the cells were counted. HEK 293 cells were then seeded into 96-well plates at a density of 8000 cells/well. After the HEK 293 cells were stabilized, they were treated with 0, 50, 100, 200, 400, 800, 1600, 2000, 2500, 3000 μ M H2O2, incubated for 6h and then assayed for MTT by graphpad prism 7.0 software.

(4) MTT assay for HEK 293 cells

HEK 293 cells were incubated for 6h, then 1/10(v/v) MTT solution (5mg/mL) was added per well, followed by incubation for 4h at 37 ℃ in a 5% CO2 incubator. The culture supernatant in the wells was carefully aspirated away to avoid aspiration of the purple crystals. Then, 100. mu.L of DMSO was added to each well, and the mixture was shaken for 10min to dissolve the purple crystals sufficiently. 490nm was selected as the detection wavelength, the absorbance of each well was measured, and the results were recorded. The cell growth curve is plotted with concentration as abscissa and relative survival rate as ordinate. Survival rate curves were calculated and plotted by grapdad software and cell morphology was recorded by photography.

2. Experiment of HEK 293 cell administration

(1) HEK 293 cell dosing group

Three experimental groups were divided according to experimental purpose:

blank control group: 5mM glucose and 50mM H were added to the medium ₂ O ₂ Performing cell modeling;

experimental group 1: adding 5mM glucose and 12 μ M quercetin into the culture medium, incubating for 24 hr, and adding 50 μ M H ₂ O ₂ Performing cell modeling;

experimental group 2: 5mM glucose and 150. mu.M thionine sulfoxide was added to the medium, and after incubation for 24h, 50. mu. M H was added ₂ O ₂ And performing cell modeling.

The three groups are sampled at 0, 6, 16, 24 and 36 hours after 50mM H2O2 is added, and the apoptosis rate, the GLO enzyme activity and the content of alpha-dicarbonyl compound are detected.

The HEK 293 cells were recovered and cultured as above.

(2) Apoptosis rate detection

Centrifuging and collecting the above cells, washing the cells with 4 deg.C physiological saline for 2 times, adding 500 μ LPBS buffer solution, and resuspending the cells to a concentration of 10 ⁷ and/mL. Then take 100mu.L of the cell suspension is placed in a 5mL flow tube, 5 mu.L of Annexin V-FITC is added, after the mixture is fully mixed, 5 mu.L of Propidium Iodid is added, the mixture is mixed, and the mixture is incubated for 15min at room temperature in a dark environment. And finally detecting the apoptosis condition of the cells by using a flow cytometer.

(3) GLO enzyme content detection

The specific operation refers to the kit specification, as follows:

and setting a standard substance hole, a sample hole to be detected and a blank hole (the blank reference hole is not added with a sample and an enzyme-labeled reagent, and the rest steps are the same), and adding 50 mu L of standard substances with different concentrations into the standard substance hole. 40 μ L of HEK 293 cell supernatant was added to the wells of the enzyme-labeled coated plate, followed by 10 μ L of HEK 293 cell supernatant (final dilution of the sample was 5-fold). During sample adding, the sample is added to the bottom of the hole of the enzyme label plate, and the hole wall is not touched as much as possible, and the sample is shaken and mixed evenly. Add enzyme labeling reagent 100. mu.L to each well except for blank wells. The plate was sealed with a sealing plate and incubated at 37 ℃ for 60 min. Opening the membrane after incubation, removing supernatant, spin-drying, and filling each well with washing solution (using ddH to concentrate washing solution) ₂ Diluting with O20 times), standing for 30s, removing, repeating the operation for 5 times, and spin-drying. Adding 50 μ L of color-developing agent A into each well, adding 50 μ L of color-developing agent B, shaking gently, mixing, developing at 37 deg.C in dark environment for 15min, adding 50 μ L of stop solution, and stopping reaction (at this time, blue color turns yellow immediately). And (3) adjusting to zero by using a blank hole, enabling the color of the sample to be in positive correlation with the GLO enzyme content, measuring an OD450 value by using an enzyme-labeling instrument, drawing a GLO standard curve, and calculating to obtain the GLO enzyme content. Note that the measurement time should be controlled within 15min after the addition of the stop solution.

3. Content detection of alpha-dicarbonyl compound in HEK 293 cell

(1) HEK 293 cell pretreatment

Samples (containing 10) ⁶ Individual HEK 293 cells) were added 2mL PBS buffer, the cells were resuspended, lysed by sonication on ice for 15min (10s, 1w), and then 500 μ L1M PCA was added. Then, the mixture is centrifuged at 12000r/min for 15min, and the supernatant is taken for later use.

(2) Methodology validation

Derivatization of α -dicarbonyl compounds in the supernatants of HEK 293 cells ensured at least three replicates. The methodological verification mainly considers the daily precision, the daytime precision, the adding standard recovery rate, the LOQ and the detection limit LOD, and evaluates the linearity, the sensitivity, the specificity, the repeatability and the sensitivity of the method.

Fourth, experimental results

1. Standard curves were plotted for GO, MGO, DA, 2-OPT, 1-PPD and GS, weighted by the reciprocal concentration (1/c), and fitted with a linear regression equation using the software IBM SPSS statics 26. The linear correlation degrees (R) are respectively 0.934, 0.985, 0.996, 0.991, 0.997 and 0.990, and have better linear relation. The limit of quantitation and the limit of detection both reach pmol/L level, which shows that the method has higher sensitivity and can be used for analyzing alpha-dicarbonyl compounds in HEK 293 cells. See table 11.

TABLE 11

2. Precision was examined by three consecutive tests, three times a day. The daily precision RSD of the three groups of cell samples is between 0.5 and 14.6 percent, and the daily precision RSD is between 1.4 and 13.2 percent. The results are shown in Table 12, Table 13 and Table 14.

TABLE 12

Watch 13

TABLE 14

3. Standard solutions of 10 nM, 100 nM and 1000nM are prepared and added to different samples, and the standard recovery rates of the blank control group (Glu) and the experimental group (Que) and the experimental group (BSO) are respectively in the range of 88.2-112.4% and 92.5-110.4%. In conclusion, the method has better accuracy, reproducibility and stability. Meanwhile, the method is shown to have higher reliability.

All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments, are within the scope of the appended claims. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.

Claims

1. A method for detecting an alpha-dicarbonyl compound, comprising the steps of:

2. The method for detecting an α -dicarbonyl compound according to claim 1, wherein a temperature of the derivatization reaction is 35 ℃ to 45 ℃; and/or

The time of derivatization reaction is 6-12 h; and/or

3. The method for detecting an α -dicarbonyl compound according to claim 1, wherein the liquid chromatography further comprises: employing a gradient elution procedure comprising: 0 min-6 min, the volume percentage of the mobile phase B is 10 percent; 6-10 min, the volume percentage of the mobile phase B is changed from 10% to 20%; 10-16 min, and the volume percentage of the mobile phase B is changed from 20% to 90%; 16 min-24 min, the volume percentage of the mobile phase B is kept to be 90 percent; the volume percentage of the mobile phase B is changed from 90% to 10% within 24-25 min.

4. The method for detecting an α -dicarbonyl compound according to claim 1, wherein the liquid chromatography further comprises: the flow rate is 0.8mL/min to 1.2 mL/min; the column temperature is 20-25 ℃; the chromatographic column is an octadecylsilane chemically bonded silica gel column; and/or

5. The method for detecting an α -dicarbonyl compound according to claim 1, wherein a mass fraction of formic acid in the aqueous formic acid solution is 0.08% to 0.12%.

6. The method for detecting an α -dicarbonyl compound of claim 1, wherein the mass spectrometry employs conditions comprising: the ionization mode is electrospray ionization; the atomizing airflow speed is 2L/min-4L/min; the drying airflow speed is 8L/min-12L/min; the temperature of the desolventizing tube is 220-280 ℃; the temperature of the heating module is 350-450 ℃; the ion source interface voltage is 3.5 kV-4.5 kV; the scanning mode is a multiple response monitoring scan.

7. The method for detecting an α -dicarbonyl compound according to any one of claims 1 to 6, wherein the solvent is one or two selected from methanol and water.

8. The method for detecting an α -dicarbonyl compound according to any one of claims 1 to 6, wherein the α -dicarbonyl compound is one or more selected from the group consisting of glyoxal, methylglyoxal, 2, 3-butanedione, D-glucosone, 2-chloro-2-phenylacetaldehyde and 1-phenyl-1, 2-propanedione.

9. The method for detecting an α -dicarbonyl compound according to any one of claims 1 to 6, wherein the source of the sample to be detected is one or more selected from the group consisting of a Chinese medicinal material, a biological tissue and a food.

10. The use of the method for detecting an α -dicarbonyl compound according to any one of claims 1 to 9 for detecting an α -dicarbonyl compound in a Chinese medicinal material, a biological tissue and a food.