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

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 and application of α-dicarbonyl compounds

technical field

The invention relates to the field of biochemical analysis, in particular to a method for detecting an α-dicarbonyl compound and its application.

Background technique

α-Dicarbonyl compounds are a class of active intermediates and reactive metabolites mainly from food thermal processing and glucose metabolism in the body. Such substances can chemically modify proteins, DNA and other important life substances in the body, resulting in structural changes and functional damages, thereby causing various diseases such as aging or diabetes, which are increasingly attracting attention in the fields of food safety and biomedicine. Therefore, how to detect the types, contents and changes of such potential hazards in food and biological samples is of great significance for healthy diet and disease prevention.

α-dicarbonyl compounds have strong hydrophilicity, low stability, lack of ultraviolet absorbing groups and fluorescent chromophores, and the actual sample matrix composition containing α-dicarbonyl compounds is often complicated, which ultimately makes it impossible to analyze them. Accurate detection. Therefore, α-dicarbonyl compounds need to be pretreated before they can be analyzed. By derivatizing α-dicarbonyl compounds with some compounds, the chromatographic retention of such substances can be improved, and optical groups can be added to achieve their separation, Qualitative and quantitative.

Traditional α-dicarbonyl compound derivatives are mostly o-diamine compounds, of which o-phenylenediamine is the most common. O-phenylenediamine has the advantages of fast derivatization, chromophore generation, cheap and easy availability, and is widely used in pre-column derivatization of chromatographic methods. However, it is difficult to store, unstable at room temperature, easily oxidized to phenazine, and has certain biological toxicity, which can cause corrosion to skin, mucous membranes, etc.

Arginine containing a guanidine reactive group is also a traditional α-dicarbonyl compound derivatizing agent, which has the advantages of low biological toxicity and good selectivity, and is suitable for the determination of samples with complex matrices. However, arginine is more polar and has poor chromatographic retention.

There are also traditional α-dicarbonyl compound derivatizing agents that derivatize α-dicarbonyl compounds and then apply them to chromatographic mass spectrometry analysis, which will have the problem of low sensitivity. This is because α-dicarbonyl compounds are different substances. The difference in molecular weight is large, the long-chain α-dicarbonyl compound retains the structure of hexose C6, but the short-chain α-dicarbonyl compound has a simple structure and small molecular weight, resulting in a big difference in volatility between the two.

SUMMARY OF THE INVENTION

Based on this, the present invention provides a detection method for an α-dicarbonyl compound, which adopts the reagents and derivatives with stable properties, high sensitivity, wide detection range, and good repeatability and specificity.

The present invention is realized through the following technical solutions.

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

Mix 2-guanidinobenzimidazole with solvent to prepare derivatization reagent;

Mixing the sample to be tested with the derivatization reagent to carry out a derivatization reaction to prepare a derivatized product;

performing liquid chromatography tandem mass spectrometry analysis on the derivatized product;

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

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

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

The derivatization reaction is carried out in an environment of pH 7-8.

In one embodiment, the conditions used in the liquid chromatography further include: using a gradient elution program, the gradient elution program includes: 0 min to 6 min, the volume percentage of mobile phase B is 10%; 6 min to 10 min, The volume percentage of mobile phase B changes from 10% to 20%; 10min～16min, the volume percentage of mobile phase B changes from 20% to 90%; 16min～24min, the volume percentage of mobile phase B remains at 90%; 24min～25min , the volume percentage of mobile phase B was varied from 90% to 10%.

In one embodiment, the conditions used in the liquid chromatography further include: the flow rate is 0.8mL/min～1.2mL/min; the column temperature is 20℃～25℃; the chromatographic column is octadecylsilane bonded silica gel column; and/or

The size of the chromatographic column used in the liquid chromatography includes: the column length is 120 mm-130 mm, the inner diameter is 4.4 mm-4.8 mm, and the particle size of the filler is 4 μm-6 μm.

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

In one embodiment, the conditions used in the mass spectrometry include: the ionization method is electrospray ionization; the flow rate of the atomizing gas is 2L/min-4L/min; the flow rate of the drying gas is 8L/min-12L/min; The tube temperature is 220°C to 280°C; the heating module temperature is 350°C to 450°C; the ion source interface voltage is 3.5kV to 4.5kV; the scanning mode is multiple reaction monitoring scanning.

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 glyoxal, methylglyoxal, 2,3-butanedione, D-glucurone, 2-chloro-2-phenyl One or more of acetaldehyde and 1-phenyl-1,2-propanedione.

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

The present invention also provides an application of the above-mentioned method for detecting α-dicarbonyl compounds in detecting α-dicarbonyl compounds in Chinese medicinal materials, biological tissues and foods.

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

The inventors selected 2-guanidinobenzimidazole as a derivatizing reagent for α-dicarbonyl compounds, and analyzed the derivatized products by liquid chromatography tandem mass spectrometry, and established a new method using 2-guanidinobenzimidazole as a mass spectrometer Derivatization of α-dicarbonyl compounds of sensitive probes. As an electron donor, 2-guanidinobenzimidazole can easily attack the α-dicarbonyl structure, thereby forming a five-membered ring with a relatively stable structure, and finally achieve the purpose of derivatization, improve the ionization efficiency and mass spectrometry response, and can obtain better sensitivity , which is embodied in that the detection sensitivity of short-chain α-dicarbonyl compounds is increased by 15-65 times. At the same time, the detection method for α-dicarbonyl compounds of the present invention has a wide detection range, can meet the detection of long-chain and short-chain α-dicarbonyl compounds at the same time, and has good repeatability, high accuracy, and relatively high specificity and selectivity. it is good.

Description of drawings

Fig. 1 is the different derivatization temperature effect investigation contrast diagram provided by the present invention;

Fig. 2 is not dehydration and dehydration product contrast diagram under different derivatization temperatures provided by the present invention;

Fig. 3 is the different derivatization time effect investigation contrast diagram provided by the present invention;

Fig. 4 is a comparison diagram of different derivatization pH effects provided by the present invention;

Fig. 5 is a comparison diagram of different chromatographic column effects provided by the present invention;

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

Fig. 7 is each derivatization product product ion spectrogram provided by the present invention;

Fig. 8 is a graph showing the stability of each derivatized product provided by the present invention.

Detailed ways

In order to facilitate understanding of the present invention, the present invention will be described more fully hereinafter with reference to the related drawings. The preferred embodiments of the invention are shown in the accompanying drawings. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough and complete understanding of the present disclosure is provided.

Unless otherwise defined, 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 terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present 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 α-dicarbonyl compound, comprising the following steps:

Mix 2-guanidinobenzimidazole with solvent to prepare derivatization reagent;

Mix the sample to be tested with the derivatization reagent, carry out a derivatization reaction, and prepare a derivatized product;

The derivatized product is analyzed by liquid chromatography tandem mass spectrometry;

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

In a specific example, the temperature of the derivatization reaction is 30°C to 50°C. Preferably, the temperature of the derivatization reaction is 40°C.

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

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

In a specific example, the conditions used in the liquid chromatography further include: using a gradient elution program, the gradient elution program includes: 0min-6min, the volume percentage of the mobile phase B is 10%; 6min-10min, the mobile phase B Volume percentage changed from 10% to 20%; 10min～16min, the volume percentage of mobile phase B changed from 20% to 90%; 16min～24min, the volume percentage of mobile phase B remained at 90%; 24min～25min, mobile phase B The volume percentage of 90% to 10%.

In a specific example, the conditions used in the liquid chromatography further include: the flow rate is 0.8mL/min-1.2mL/min; the chromatographic column is an octadecylsilane-bonded silica gel column; and the column temperature is 20°C to 25°C.

In a specific example, the size of the chromatographic column used in liquid chromatography includes: the column length is 120 mm-130 mm, the inner diameter is 4.4 mm-4.8 mm, and the particle size of the filler is 4 μm-6 μm.

More specifically, the column used for liquid chromatography was MLtimate XB-C18 (125 mm×4.6 mm, particle size 5 μM).

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

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

In a specific example, the conditions used in mass spectrometry include: the ionization method is electrospray ionization; the flow rate of the atomizing gas is 2L/min-4L/min; the flow rate of the drying gas is 8L/min-12L/min; the temperature of the desolvation tube The temperature is 220℃～280℃; the temperature of the heating module is 350℃～450℃; the ion source interface voltage is 3.5kV～4.5kV; the scanning mode is multiple reaction monitoring scanning.

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 the group consisting of glyoxal, methylglyoxal, 2,3-butanedione, D-glucurone, 2-chloro-2-phenylacetaldehyde One or more of 1-phenyl-1,2-propanedione.

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

In a specific example, the Chinese herbal medicine is selected from the group consisting of black medicine, Magnolia officinalis, Polygonatum japonica, A. chinensis, A. chinensis, Psoraleae, Bupleurum, Schisandra japonica, Evodia Fructus, Hentai Radix, Turmeric, Epimedium One or more of , Chuanxiong, Xiaoji, Inula, Rhubarb, Forsythia, Rehmannia glutinosa, Radix Isatidis, Dangshen and Codonopsis.

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

The present invention also provides an application of the above-mentioned detection method for α-dicarbonyl compounds in detecting α-dicarbonyl compounds in Chinese medicinal materials, biological tissues and foods.

The cleavage of each derivatized product obtained by the detection method of α-dicarbonyl compound provided by the present invention has a similar rule. This rule can not only accurately judge whether the derivatization reaction really occurs, but also facilitate the subsequent spectrum analysis and data analysis. And the common product ions m/z 159.0 and m/z 134.0 are formed. According to this characteristic, the precursor ion scanning mode can be used to screen the non-target reactive metabolite α-dicarbonyl compounds to expand the metabolic spectrum.

The detection method and application of the α-dicarbonyl compound of the present invention will be further described in detail below with reference to specific examples. The raw materials used in the following examples, unless otherwise specified, are all commercially available products.

Example 1

The present embodiment provides a method for detecting an α-dicarbonyl compound, which is specifically as follows:

1. Reagents

2-guanidinobenzimidazole (2-GBI), glyoxal (GO), methylglyoxal (MGO), 2,3-butanedione (DA), o-phenylenediamine, arginine were all purchased from Liaoning Cook Biotechnology Co., Ltd.; D-Glucurone (GS) and 2-guanidinobenzimidazole were purchased from Sigma Company (Shanghai, China); 2-oxo-2-phenylacetaldehyde (2 -OPT), 1-phenyl-1,2-propanedione (1-PPD) were purchased from Beijing Inoke Technology Co., Ltd.; disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium hydroxide were purchased from Aladdin Biochemical Technology Co., Ltd.; methanol was purchased from Inokai Technology Co., Ltd.; formic acid was purchased from Tianjin Kemeiou Chemical Reagent Co., Ltd.

Second, the experimental process

1. Derivatization reaction

Preparation of guanidinobenzimidazole solution: Accurately weigh 2-GBI standard product, dissolve and dilute with methanol (chromatographic grade) or ddH2O to obtain 10mM mother solution of each standard product, and store at -20°C in the dark.

The reaction was carried out in a 1.5 mL centrifuge tube, and the total volume of the reaction solution was 1 mL. Add 2-guanidinobenzimidazole solution, PBS buffer solution and α-dicarbonyl compound in sequence according to Table 1, and vortex for 2min. After reacting for 6 h under the condition of 40°C water bath heating and pH 7.4, it was placed at -20°C for 20 min to terminate the reaction. Passed through a 0.22 μm filter for LC-MS/MS detection and analysis. Ensure at least three parallel samples.

Table 1

NOTE: Except for PBS, the concentration is 10 μM.

2. Liquid chromatography-mass spectrometry detection and analysis

(1) Liquid chromatography conditions are as follows:

Analytical instrument: triple quadrupole liquid mass spectrometer (Shimadzu, LC-MS 8050);

Chromatographic column: MLtimate XB-C18 (125mm×4.6mm, particle size 5μM);

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

Mobile phase B: methanol;

Flow rate: 1mL/min;

Injection volume: 10 μL;

Elution mode: gradient elution (the elution program is shown in Table 2);

Column temperature: 22°C.

Table 2

(2) The mass spectrometry conditions are shown in Table 3, and the positive ion mode is used to analyze:

table 3

3. Change the conditions

(1) Other conditions remain unchanged, only the derivative temperature is changed: 40°C is changed to 24°C, 37°C, 60°C and 80°C respectively;

(2) Other conditions remain unchanged, only the derivation time is changed: 6h is changed to 2h, 4h, 12h and 24h respectively.

(3) Other conditions remain unchanged, only the derivatization pH is changed: adjust the pH in the range of 3-10 by PBS buffer solution.

(4) Other conditions remain unchanged, only the type of chromatographic column is changed: MLtimate XB-C18 column (125mm×4.6mm, particle size 5μM) is changed to Diamonsil C18 column (150×2.1mm, particle size 3μM).

3. Experimental results

1. Conditional filter

(1) 24, 37, 40, 60, and 80 °C were selected to investigate the derivatization effects of different α-dicarbonyl compounds. The experimental results are shown in Figure 1. Under the condition of ensuring a single variable, it is found that MGO has the best derivatization strength among the six α-dicarbonyl compounds, followed by short-chain dicarbonyl compounds such as GO and DA, and the derivatization strength of 1-PPD, 2-OPT, and GS. weakened sequentially. This is because in theory glyoxal has the highest reactivity, but in practice glyoxal has a higher degree of polymerization in aqueous solution, so methylglyoxal has the best reactivity. In addition, the optimum derivatization temperatures of glyoxal and methylglyoxal were both 40°C, and the other four α-dicarbonyl compounds were all 24°C. But except for 2,3-butanedione, the response changes from 24°C to 40°C for the remaining three α-dicarbonyl compounds were all within the acceptable range. Therefore, 40°C was chosen as the derivatization reaction temperature.

During the experiment, it was found that the higher the derivatization temperature, the darker the color of the reaction solution. The color of the solution was yellow at 60°C and brown at 80°C. At the same time, the derivatization products (not dehydrated) and by-products (dehydration) were monitored. Taking 2-GBI-GO as an example, it was found that only a small part of the derivatized products were not dehydrated further as the temperature increased, as shown in Figure 2. Combined with this experimental phenomenon, it is speculated that when the derivatization temperature exceeds a certain limit, the derivatization is likely to be denatured, resulting in a significant decrease in the derivatization effect.

(2) To ensure that the variables are single, the reaction is continuous for 24 hours in units of hours, and five time points (2, 4, 6, 12, and 24 hours) are selected for investigation. It can be concluded from Figure 3 that GO, MGO, 2-OPT and 1-PPD obtained the best derivatization effect at 6h. GS reached the optimal derivatization time at 12h and the derivatization effect of the subsequent 12h fluctuated less, but the derivatization effects of the other five compounds decreased to varying degrees, and MGO was the most significant. Therefore, 6h was chosen as the optimal derivatization time.

(3) In view of the complexity of the actual sample matrix containing α-dicarbonyl compounds, its pH may vary with external factors. The detection and analysis of it needs to be in a relatively stable acid-base environment, and the addition of buffer solution can greatly alleviate this problem. Choose a PBS buffer to adjust the pH, optimizing in the pH 3-10 range. It can be concluded from Figure 4 that MGO reaches the peak response at pH 7.4, and GO, DA, 2-OPT, 1-PPD, and GS have no major fluctuations in the pH range of 4-10, and are less affected by pH. At the same time, pH 7.4 is mild, and it is also the acid-base condition for the cultivation and growth of most living organisms. Therefore, pH 7.4 is selected as the optimal derivatization pH.

(4) In liquid and mass analysis, the chromatographic column is the core component of sample separation. The chromatographic column needs to meet the requirements of high separation efficiency, good selectivity, and good column efficiency. When the analysis target properties and structures are different, the separation effect of the chromatographic column will also be different. In order to achieve better separation and analysis purposes, the present invention selects Diamonsil C18 column (150 × 2.1 mm, particle diameter 3 μM) and MLtimate XB C18 column (125 mm × 4.6 mm, particle diameter 5 μM) to the above-mentioned six α-dicarbonyl compounds. Separate for comparison. It can be drawn in Figure 5 that the above six compounds were not completely separated by the Diamonsil C18 column; while the MLtimate XB C18 column had better separation effect, higher response, and less time-consuming. Based on the above, XB C18 was selected as the chromatographic column.

2. Results of mass spectrometry fragmentation law

The detection was carried out under the above optimal conditions, ^and the cracking rule of the derivatized product of α ^- dicarbonyl compound was analyzed. After a molecule of water is lost, a carbon-nitrogen double bond is formed, and a product ion of m/z 216.0 is obtained; it is cut off from the two carbon-nitrogen single bonds of the imidazole ring to obtain a product ion of m/z 159.1, which has the highest response, so The m/z 159.1 was selected as the quantitative ion; it was broken at the carbon-nitrogen double bond to form the product ion of m/z 134.1, as shown in Figure 6.

In the secondary mass spectra of six derivatized products of 2-GBI and GO, MGO, DA, 2-OPT, 1-PPD, and GS (as shown in Figure 7), it was found that the derivatized products had similar cracking rules. This rule can not only accurately judge whether the derivatization reaction really occurs, but also facilitate the subsequent spectrum analysis and data analysis, and form common product ions m/z 159.0 and m/z 134.0, which can be used according to this characteristic. The ion scan mode screens non-target reactive metabolites α-dicarbonyl compounds to expand the metabolic spectrum.

3. Stability investigation of derivatives

Derivatives were placed at 24°C in a dark environment for stability investigation. Samples were taken for detection at 0, 2, 4, 8, 16, and 24 h, respectively, and the peak area changes were observed, as shown in Figure 8. It was found that under this storage condition, the content of six reactive metabolites changed little within 24h, which were all within the acceptable range.

2. Liquid quality parameter optimization

A mass spectrometry multiple reaction monitoring method was established for the above six reactive metabolites, and the mass spectrometry parameters were optimized, such as precursor ion, product ion and collision voltage. The optimized parameters are shown in Table 4.

Table 4

Example 2

The present embodiment provides a method for detecting α-dicarbonyl compounds in Chinese medicinal materials, the details are as follows:

1. Reagents and reagents

Black medicine, Magnolia officinalis, Polygonatum japonica, Zuiyucao leaves, Coleus vine, Psoraleae, Bupleurum, Schisandra chinensis, Evodia, White snake grass, Turmeric, Epimedium, Chuanxiong, thistle, Inula flower, Rhubarb , Forsythia, Rehmannia glutinosa, Radix Isatidis, Twenty-one Chinese herbal medicines are purchased from local pharmacies in Changsha. Other reagents are the same as in Example 1.

If the above reagents do not make special instructions, the purity is greater than 98%. All water used in the experiment was ddH2O.

2. Derivatization reaction conditions and instrument conditions

1. Derivatization reaction

The reaction was carried out in a 1.5 mL centrifuge tube, and the total volume of the reaction solution was 1 mL. Add 2-guanidinobenzimidazole solution, PBS buffer solution and α-dicarbonyl compound in sequence according to Table 1, and vortex for 2min. After reacting for 6 h under the condition of 40°C water bath heating and pH 7.4, it was placed at -20°C for 20 min to terminate the reaction. Passed through a 0.22 μm filter for LC-MS/MS detection and analysis. Ensure at least three parallel samples.

2. Liquid chromatography-mass spectrometry detection and analysis

(1) Liquid chromatography conditions are as follows:

Analytical instrument: triple quadrupole liquid mass spectrometer (Shimadzu, LC-MS 8050);

Chromatographic column: MLtimate XB-C18 (125mm×4.6mm, particle size 5μM);

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

Mobile phase B: methanol;

Flow rate: 1mL/min;

Injection volume: 10 μL;

Elution mode: gradient elution (the elution program is shown in Table 5);

Column temperature: 22°C.

table 5

(2) Mass spectrometry conditions are as shown in 3 in Example 1, and are analyzed in positive ion mode:

3. The experimental process

1. Solution preparation

Accurately weigh (measure) each standard (GO, MGO, DA, GS, 2-OPT, 1-PPD and 2-GBI), dissolve and dilute with methanol (chromatographic grade) or ddH2O to obtain 10mM mother solution of each standard , store at -20°C away from light.

2. Establishment of standard curve

The six standard curves consist of six standard solution points of a concentration gradient, respectively. The concentrations of GO, MGO and DA are in the range of 2.25pmol/L-2.25μmol/L, while the concentrations of 2-OPT and 1-PPD are in the range of 90nmol/L-2.25μMol/L, and the concentration of GS is in the range of 225nmol/L-2.25 μmol/L. At least three parallel samples are guaranteed.

After the derivatization was completed, it was placed at -20 °C for 20 min to terminate the reaction. The samples were then processed through a membrane (0.22 [mu]M) and analyzed by mass spectrometry. Finally, taking the concentration of the above six reactive metabolites as the abscissa and the peak area of the response as the ordinate, the software IBM SPSS Statistics 26 was used to analyze and process the data, and six standard curves were obtained.

3. Treatment of Chinese herbal medicines

Accurately weigh 6.00 mg of Chinese herbal medicine into a 10 mL centrifuge tube, add 4 mL of ddH2O to infiltrate for 5 min, and then ultrasonicate for 20 min. Then add 2 mL of methanol, vortex for 5 min, mix well, centrifuge at 15,000 r/min for 15 min to precipitate protein, take 4 mL of supernatant, dilute it by one time, and dilute to 8 mL. Store at -20°C for later use.

4. Chinese herbal medicine derivatization

Take 450 μL of 2-GBI solution into a 1.5 mL centrifuge tube, add 100 μL of PBS buffer solution (pH 7.4), and then add 450 μL of the treated Chinese herbal medicine solution, water bath at 40 °C for 6 hours, and after derivatization, place it at -20 °C 20min to stop the reaction. After passing through the membrane (0.22 μm), it was transferred to a 1.5 mL brown injection bottle and entered into the mass spectrometer for detection.

4. Methodological investigation

1. Linear correlation and sensitivity investigation

A standard curve was drawn for GO, MGO, DA, 2-OPT, 1-PPD and GS, with the reciprocal concentration (1/c) as the weight, and the software IBM SPSS Statistics 26 was used to fit a linear regression equation. The linear correlations (R) were 0.934, 0.985, 0.996, 0.991, 0.997, and 0.990, respectively, showing a good linear relationship, as shown in Table 6. The limit of quantification and detection limit reached pmol/L level, indicating that the method has high sensitivity and can be used for the analysis of α-dicarbonyl compounds in Chinese herbal medicines.

Table 6

2. Accuracy inspection

Six standard solutions with low, medium and high concentrations of α-dicarbonyl compounds were prepared and added to black medicine and Hedera alba, and the standard addition recovery rate of the method was investigated to reflect the accuracy of the method. The experimental results are shown in Tables 7 and 8. The recovery rates of standard addition of black medicine were in the range of 91.4-105.8%, and the recovery of standard addition of Hedera alba were in the range of 85.3-113.0%. It shows that the method has good reproducibility and stability, and also shows that the detection results of this method are accurate and reliable.

Table 7 Spike recovery rate of black medicine

Table 8 The recovery rate of standard addition of Hedera japonica

3. Precision inspection

In order to evaluate the precision of the method, six target α-dicarbonyl compounds in black medicine and white snake grass were detected in parallel for three consecutive days for three consecutive days. The relative standard deviation of three parallel tests was used to represent the intra-day precision, and the relative standard deviation of three consecutive three-day testing samples reflected the inter-day precision of the reaction, as shown in Table 9. The intra-day precision was in the range of 1.1-10.1%, while the inter-day precision was in the range of 4.3-14.3%. The above data show that the method has good precision and can be applied to the determination of α-dicarbonyl compounds in black medicine and Hedera chinensis.

Table 9

4. Analysis of actual samples

After completing the verification of the mass spectrometry multiple reaction monitoring method for α-dicarbonyl compounds, quantitative analysis of black medicine and Hedera alba was carried out, and the detection results are shown in Table 10.

It was found that the content of α-dicarbonyl compounds in black medicine and white snake grass has a big difference, the MGO content in black medicine is the highest, about 0.715μg/g; the white flower snake grass has the highest DA content, about 0.523μg/g. The RSDs are all within 2.51-10.14%, and the detection errors are within the acceptable range.

Table 10

Example 3

The present embodiment provides a method for detecting α-dicarbonyl compounds in cells, the details are as follows:

1. Reagents and reagents

Hydrogen peroxide (H2O2, 30% v/v) and perchloric acid (PCA) were purchased from Tianjin Xinyuan Co., Ltd.; methanol, acetonitrile, and quercetin were purchased from Enokay Technology Co., Ltd.; L-butthionine Sulfoxide was purchased from Shanghai Xian Ding Biotechnology Co., Ltd.; formic acid, alcohol (75%, v/v), and glucose (Glu) were purchased from Tianjin Kemeiou Chemical Reagent Co., Ltd.; MEM medium and fetal bovine serum were Purchased from Thermo Fisher Scientific; trypsin and antibiotics were purchased from Shanghai Dingsang Biotechnology Co., Ltd.; Annexin V-FITC/PI apoptosis detection kit and double-stained apoptosis detection kit were purchased in Anhui Noron Biotechnology Co., Ltd.; physiological saline was purchased from Hunan Kelun Pharmaceutical Co., Ltd.; MTT kit was purchased from Shanghai Sangon Bioengineering Co., Ltd.; HuMan GLO ELISA kit was purchased from Shanghai Jining Industrial Co., Ltd. Other reagents are the same as 2.2.1.

If the above reagents do not make special instructions, the purity is greater than 98%. All water used in the experiment was ddH ₂ O.

2. Derivatization reaction conditions and instrument conditions

The derivatization reaction conditions and instrument conditions are the same as those in Example 2.

3. The experimental process

1. Establishment of apoptosis model

(1) HEK 293 cell recovery

First, pre-adjust the temperature of ddH2O to 37°C, then put on protective measures such as gloves, masks and hats. Adjusted to 37°C), shake gently to dissolve completely within 60s. After the dissolution is complete, disinfect the outer wall of the cryovial with alcohol and place it on a clean bench. In the ultra-clean bench, prepare a sterilized centrifuge tube in advance, and transfer 10 mL of the newly prepared culture base into the centrifuge tube, then add the cell suspension in the cryopreservation tube, and centrifuge at room temperature (1000r/min) 3min, discard the supernatant medium. Add 1 mL of medium to the HEK 293 cell pellet, resuspend it, transfer it to a T25 cell culture flask, add medium to a volume of 5 mL, and place it in a 37°C, 5% CO2 incubator for incubation. The culture medium was changed after two days, and the cells were passaged when about 80% confluent.

(2) HEK 293 cell culture

HEK 293 cells were cultured in MEM medium containing 10% fetal bovine serum in an incubator at 37°C, saturated humidity, and 5% CO2.

(3) HEK 293 cell plating

Cell counts were performed after well-grown HEK 293 cells were digested with 0.25% trypsin. 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 μM H2O2, respectively, and incubated for 6 h for MTT detection and analyzed by graphpad prism7.0 software.

(4) Detection of MTT in HEK 293 cells

After HEK 293 cells were incubated for 6 h, 1/10 (v/v) MTT solution (5 mg/mL) was added to each well, and then cultured in a 37°C, 5% CO2 incubator for 4 h. Carefully aspirate the culture supernatant from the wells to avoid removing the purple crystals. 100 μL of DMSO was added to each well and shaken for 10 min to fully dissolve the purple crystals. Select 490 nm as the detection wavelength, measure the light absorption value of each well, and record the results. The cell growth curve was plotted with the concentration as the abscissa and the relative viability as the ordinate. The survival rate curve was calculated and drawn by Gradpad software, and the cell morphology was recorded by taking pictures.

2. HEK 293 cell administration experiment

(1) HEK 293 cell administration group

Divided into three experimental groups according to the purpose of the experiment:

Blank control group: 5mM glucose and 50mM H ₂ O ₂ were added to the culture medium for cell modeling;

Experimental group 1: 5mM glucose and 12μM quercetin were added to the medium, and after incubation for 24 hours, 50μM H ₂ O ₂ was added to conduct cell modeling;

Experimental group 2: 5 mM glucose and 150 μM butthionine sulfoximine were added to the medium, and after incubation for 24 h, 50 μM H ₂ O ₂ was added to conduct cell modeling.

The above three groups were sampled at 0, 6, 16, 24, and 36 h after adding 50 mM H2O2, and the apoptosis rate, GLO enzyme activity and α-dicarbonyl compound content were detected.

HEK 293 cell recovery and culture operations are the same as above.

(2) Detection of apoptosis rate

The cells in the above groups were collected by centrifugation, washed twice with 4°C normal saline, and then added 500 μL of PBS buffer solution to resuspend the cells to control the concentration at 10 ⁷ /mL. Then, 100 μL of cell suspension was placed in a 5 mL flow tube, 5 μL of Annexin V-FITC was added, and after thorough mixing, 5 μL of PropidiuM Iodide was added and mixed, and incubated for 15 min at room temperature in a dark environment. Finally, cell apoptosis was detected by flow cytometry.

(3) Detection of GLO enzyme content

Refer to the kit instructions for specific operations, as follows:

Set standard wells, sample wells to be tested and blank wells (blank control wells do not add sample and enzyme labeling reagents, and the rest of the steps are the same), and add 50 μL of standards of different concentrations to each standard well. First add 40 μL of HEK 293 cell supernatant dilution to the sample well of the enzyme-labeled coating plate, and then add 10 μL of HEK 293 cell supernatant (the final dilution of the sample is 5 times). When adding the sample, pay attention to adding the sample to the bottom of the well of the microtiter plate, and try not to touch the wall of the well. Shake gently and mix well. Add 100 μL of enzyme labeling reagent to each well, except for blank wells. After sealing the ELISA-coated plate with a sealing film, incubate at 37°C for 60 min. After the incubation, open the sealing membrane, remove the supernatant, spin dry, fill each well with washing solution (dilute the concentrated washing solution 20 times with ddH ₂ O), and remove it after standing for 30 s. Repeat this operation 5 times, and then spin again Dry. First add 50 μL of chromogenic reagent A to each well, then add 50 μL of chromogenic reagent B, shake gently, mix well, develop color at 37°C for 15 min in a dark environment, and then add 50 μL of stop solution to stop the reaction (at this time, the blue turns yellow). The blank well was set to zero, the color of the sample was positively correlated with its GLO enzyme content, the OD450 value was measured by a microplate reader, the GLO standard curve was drawn, and then the GLO enzyme content was calculated. Note that the measurement time should be controlled within 15 minutes after adding the stop solution.

3. Detection of α-dicarbonyl compounds in HEK 293 cells

(1) Pretreatment of HEK 293 cells

Each sample (containing 10 ⁶ HEK 293 cells) was taken, 2 mL of PBS buffer solution was added, cells were resuspended, cells were lysed by sonication on ice for 15 min (10 s, 1 w), and then 500 μL of 1 M PCA was added. Centrifuge at 12000 r/min for 15 min, and take the supernatant for later use.

(2) Methodological verification

Derivatization of alpha-dicarbonyl compounds in HEK 293 cell supernatant ensures at least three parallel samples. The method validation mainly examined the intra-day precision, inter-day precision, recovery rate of standard addition, LOQ and detection limit LOD, and evaluated the linearity, sensitivity, specificity, repeatability and sensitivity of the method.

4. Experimental results

1. Draw a standard curve for GO, MGO, DA, 2-OPT, 1-PPD and GS, and use the software IBM SPSS Statistics 26 to fit a linear regression equation with the reciprocal concentration (1/c) as the weight. The linear correlation (R) was 0.934, 0.985, 0.996, 0.991, 0.997, 0.990, respectively, showing a good linear relationship. The limit of quantification and detection limit reached pmol/L level, indicating that the method has high sensitivity and can be used for the analysis of α-dicarbonyl compounds in HEK 293 cells. See Table 11.

Table 11

2. The precision was examined by three consecutive tests, three times a day. The intra-day precision RSDs of the three groups of cell samples were all in the range of 0.5-14.6%, and the inter-day precisions were all in the range of 1.4-13.2%. The test results are shown in Table 12, Table 13 and Table 14.

Table 12

Table 13

Table 14

3. The standard solutions of 10, 100 and 1000nM were prepared and added to different samples, and the standard addition recovery rates of the blank control group (Glu) were measured to be between 90.1-114.5%. The experimental group (Que) and the experimental group ( The recoveries of spiked BSO) were in the range of 88.2-112.4% and 92.5-110.4%. In conclusion, the method has good accuracy, reproducibility and stability. It also shows that the method has high reliability.

The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present invention, which are convenient for a specific and detailed understanding of the technical solutions of the present invention, but should not be construed as a limitation on the protection scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the patent of the present invention should be based on the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims (10)

1. a detection method of α-dicarbonyl compound, is characterized in that, comprises the steps: Mix 2-guanidinobenzimidazole with solvent to prepare derivatization reagent; Mixing the sample to be tested with the derivatization reagent to carry out a derivatization reaction to prepare a derivatized product; performing liquid chromatography tandem mass spectrometry analysis on the derivatized product; Wherein, the conditions adopted by the liquid chromatography include: mobile phase A is formic acid aqueous solution; mobile phase B is methanol. 2. the detection method of α-dicarbonyl compound according to claim 1, is characterized in that, the temperature of derivatization reaction is 35 ℃～45 ℃; and/or The time of the derivatization reaction is 6h～12h; and/or The derivatization reaction is carried out in an environment of pH 7-8. 3. the detection method of α-dicarbonyl compound according to claim 1, is characterized in that, the condition that described liquid chromatography adopts also comprises: adopts gradient elution procedure, and described gradient elution procedure comprises: 0min～6min , the volume percentage of mobile phase B is 10%; 6min～10min, the volume percentage of mobile phase B changes from 10% to 20%; 10min～16min, the volume percentage of mobile phase B changes from 20% to 90%; 16min～24min , the volume percentage of mobile phase B remained at 90%; 24min ~ 25min, the volume percentage of mobile phase B changed from 90% to 10%. 4. the detection method of α-dicarbonyl compound according to claim 1, is characterized in that, the condition that described liquid chromatography adopts also comprises: flow velocity is 0.8mL/min～1.2mL/min; Column temperature is 20 ℃ ~25°C; the chromatographic column is an octadecylsilane-bonded silica gel column; and/or The size of the chromatographic column used in the liquid chromatography includes: the column length is 120 mm-130 mm, the inner diameter is 4.4 mm-4.8 mm, and the particle size of the filler is 4 μm-6 μm. 5 . The method for detecting α-dicarbonyl compounds according to claim 1 , wherein, in the aqueous formic acid solution, the mass fraction of formic acid is 0.08% to 0.12%. 6 . 6 . The method for detecting α-dicarbonyl compounds according to claim 1 , wherein the conditions used in the mass spectrometry include: ionization mode is electrospray ionization; atomization gas flow rate is 2L/min～4L/min ; Drying gas flow rate is 8L/min～12L/min; Desolvation tube temperature is 220℃～280℃; Heating module temperature is 350℃～450℃; Ion source interface voltage is 3.5kV～4.5kV; Scanning mode is multiple reaction Monitoring scans. 7 . The method for detecting α-dicarbonyl compounds according to claim 1 , wherein the solvent is selected from one or both of methanol and water. 8 . 8 . The method for detecting an α-dicarbonyl compound according to claim 1 , wherein the α-dicarbonyl compound is selected from the group consisting of glyoxal, methylglyoxal, 2,3- One or more of butanedione, D-glucurone, 2-chloro-2-phenylacetaldehyde and 1-phenyl-1,2-propanedione. 9. The method for detecting α-dicarbonyl compounds according to any one of claims 1 to 6, wherein the source of the sample to be tested is selected from one or more of Chinese medicinal materials, biological tissues and food . 10. Application of the method for detecting α-dicarbonyl compounds according to any one of claims 1 to 9 in detecting α-dicarbonyl compounds in Chinese medicinal materials, biological tissues and foods.

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