Sensitization probe kit and method for detecting multiple aldehyde ketone compounds by using sensitization probe kit

Abstract

The invention relates to a sensitization probe kit and a method for detecting various aldehyde ketone compounds by using the sensitization probe kit, the kit comprises N, N-diethyl-1, 4-phenylenediamine and / or an isotope compound of the N, N-diethyl-1, 4-phenylenediamine as a sensitization probe, and particularly comprises any one of the N, N-diethyl-1, 4-phenylenediamine and the isotope compound of the N, N-diethyl-1, 4-phenylenediamine as a pair of sensitization probes, and the N, N-diethyl-1, 4-phenylenediamine and the isotope compound of the N, N-diethyl-1, 4-phenylenediamine as a pair of sensitization probes. The method is used for quantitatively detecting aldehyde ketone compounds by adopting an internal standard method. According to the method, N, N-diethyl-1, 4-phenylenediamine and / or an isotope compound of N, N-diethyl-1, 4-phenylenediamine are / is taken as a sensitizing probe, the aldehyde ketone compounds in a sample to be detected are subjected to derivatization labeling and then are subjected to quantitative detection, and particularly, the aldehyde ketone compounds represented by carbohydrate metabolites in a biological sample are quantitatively detected by adopting an internal standard method. Quantitative detection of 14 kinds of saccharide metabolites can be completed within 11 minutes, wherein four kinds of oligosaccharides (lactose, maltose, maltotriose and maltotetraose) which are difficult to detect together with monosaccharide at the same time are included, and the detection sensitivity can reach 0.007-0.458 fmol.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and more particularly to a sensitized probe detection kit and a method for detecting various aldehyde and ketone compounds using the same. Background Art

[0002] Aldehyde and ketone compounds represented by carbohydrate metabolites are basic substances for energy metabolism, involved in multiple metabolic pathways, and also play important roles in cell communication and signal transduction. The changes in their content and types in organisms are closely related to physiological and pathological changes. Therefore, synchronous quantitative analysis of such metabolites helps to reveal the association between their functions, change rules and diseases. However, there are problems in the quantitative detection of carbohydrate metabolites based on liquid chromatography-mass spectrometry, such as poor chromatographic retention behavior, low ionization efficiency, low sensitivity, and difficulty in obtaining stable isotope internal standards. Sensitized probes for aldehyde and ketone compounds are a class of reagents that can specifically interact with aldehydes and ketones, which can improve their chromatographic retention behavior and ionization efficiency, and thus can achieve improved sensitivity for the detection of aldehyde and ketone compounds.

[0003] Currently, the probes commonly used for labeling carbohydrate metabolites such as aldose and ketose can be classified into amine-based, hydrazine-based and polycyclic-based. Amine-based probes include 2-aminobenzamide (2-AB), 2-aminopyridine (2-AP) and 2-aminobenzoic acid (2-AA). Hydrazine-based probes include 2,4-dinitrophenylhydrazine (2,4-DNPH) and its isotope compound d3-DNPH, 3-nitrophenylhydrazine (3-NPH). Polycyclic-based probes include 1-phenyl-3-methyl-5-pyrazolone (PMP) and its isotope compound d5-PMP, 3-amino-9-ethylcarbazole (AEC).

[0004] Among them, amines are mainly represented by 2-aminobenzamide (2-AB). For example, Fang et al. used 2-AB to label 9 monosaccharides and then used a reversed-phase C18 column to achieve good chromatographic separation of the analytes. After labeling, the products carry protonatable groups, effectively improving the ionization efficiency and detection sensitivity (Reference 1); hydrazine-based compounds are mainly represented by 2,4-dinitrophenylhydrazine (2,4-DNPH). For example, Karamanos et al. used 2,4-DNPH to label neutral monosaccharides at 65 °C and also achieved reversed-phase chromatographic separation (Reference 2); polycyclic compounds are mainly represented by 1-phenyl-3-methyl-5-pyrazolone (PMP). For example, Xu et al. used PMP to label 10 neutral monosaccharides, 2 N-acetylated glycosamines, and 2 acidic monosaccharides, and then used a reversed-phase chromatographic column. Detection and analysis can be completed within 10 minutes in the positive ion mode, with a sensitivity up to the femtomole level, and it is used to detect the carbohydrate metabolites in infant feces (Reference 3). However, 2-AB lacks a stable isotope probe that can be used as an internal standard. The labeling reaction of 2,4-DNPH uses a relatively high temperature, which easily increases the probability of side reactions, and 2,4-DNPH is highly flammable. For safety reasons, it has been less used; PMP can react with both aldehyde-ketone metabolites and fatty acid metabolites, with relatively low selectivity, and the formation of double-derivative products has a large steric hindrance in the structure, which risks affecting the labeling reaction efficiency with oligosaccharide metabolites. Although a stable isotope probe d 5 -PMP of PMP has been synthesized by a team in the past three years, its use cost is relatively high.

[0005] At present, certain progress has been made in the quantitative technology of carbohydrate metabolites taking aldose and ketose as examples based on probe derivatization assistance. Although it has improved the chromatographic retention of carbohydrate metabolites, there are problems such as a very limited number of carbohydrate metabolites that can be quantitatively detected (such as it is difficult to detect lactose, maltose, maltotriose, and maltotetraose in oligosaccharides), a long detection time, or a low detection sensitivity. Quantitative detection based on the internal standard method with a pair of probes can avoid the influence of interference factors such as detection instruments and matrices. Compared with the external standard method for quantitative detection, the results of quantitative detection using the internal standard method are more accurate. However, the commercially available amine sensitization probe detection kit for the detection of aldehyde-ketone compounds lacks a suitable stable isotope probe. Therefore, it is still necessary to develop probes that can improve chromatographic retention to distinguish isomers, can cover more oligosaccharide metabolites, and have stable isotopes, which is conducive to further improving the detection sensitivity and coverage of aldehyde-ketone compounds, especially carbohydrate metabolites.

[0006] Literature source:

[0007] Reference 1: FANG J, QIN G, MA J, et al. Quantification of plant cell wall monosaccharides by reversed-phase liquid chromatography with 2-aminobenzamide pre-column derivatization and a non-toxic reducing reagent 2-picoline borane[J]. Journal of Chromatography A, 2015, 1414: 122-8.

[0008] Reference 2: KARAMANOS N, TSEGENIDIS T, ANTONOPOULOS C. Analysis of neutral sugars as dinitrophenyl-hydrazones by high-performance liquid chromatography[J]. Journal of Chromatography A, 1987, 405: 221-8.

[0009] Reference 3: XU G, AMICUCCI M J, CHENG Z, et al. Revisiting monosaccharide analysis - quantitation of a comprehensive set of monosaccharides using dynamic multiple reaction monitoring[J]. Analyst, 2017, 143(1): 200-7. SUMMARY OF THE INVENTION

[0010] In view of the above defects or improvement requirements of the prior art, the present invention provides a sensitizing probe kit and a method for detecting various aldehyde and ketone compounds. The purpose is to synthesize an N,N-diethyl-1,4-phenylenediamine and its isotope series compounds, and it is found that when used as a sensitizing probe for aldehyde and ketone compounds, based on LC-ESI-MS / MS technology and using the internal standard method, the quantitative detection of 14 aldehyde and ketone compounds represented by carbohydrate metabolites can be completed within 11 minutes, including 4 oligosaccharides (lactose, maltose, maltotriose, and maltotetraose) that are difficult to detect simultaneously with monosaccharides, and the detection sensitivity can reach 0.007 - 0.458 fmol. Thus, the technical problems of limited types of carbohydrate metabolites detected by existing sensitizing probes and low detection sensitivity are solved.

[0011] To achieve the above object, according to one aspect of the present invention, there is provided an application of a compound in the preparation of a sensitizing probe detection kit, wherein the detection kit is used for quantitatively detecting aldehyde and ketone compounds, and the compound serves as a sensitizing probe, including N,N-diethyl-1,4-phenylenediamine and / or its isotope compound, and the structural formula of N,N-diethyl-1,4-phenylenediamine is shown as follows:

[0012]

[0013] The isotope compound of N,N-diethyl-1,4-phenylenediamine includes any one or more of the compounds shown by the following structural formulas.

[0014]

[0015] Preferably, in the above application, the compound includes any one of N,N-diethyl-1,4-phenylenediamine and its isotope compound.

[0016] Preferably, in the above application, the isotope compound of N,N-diethyl-1,4-phenylenediamine is d 2 -DEPPD.

[0017] In addition, according to another aspect of the present invention, there is also provided a detection kit for aldehyde and ketone compounds, which includes N,N-diethyl-1,4-phenylenediamine and / or its isotope compound as a sensitizing probe.

[0018] Preferably, the kit is used for quantitatively detecting aldehyde and ketone compounds by internal standard method, and includes any one of N,N-diethyl-1,4-phenylenediamine and its isotope compound, wherein N,N-diethyl-1,4-phenylenediamine is the first sensitizing probe, and the isotope compound of N,N-diethyl-1,4-phenylenediamine is the second sensitizing probe.

[0019] Preferably, the isotope compound of N,N-diethyl-1,4-phenylenediamine in the kit is selected from any one of the following:

[0020]

[0021] Preferably, the kit further includes a reducing agent, an organic acid and / or a standard product of aldehyde and ketone compounds; the organic acid includes acetic acid, formic acid, oxalic acid, or citric acid; the reducing agent includes 2-picBH 3 、NaBH 3 CN、or NaBH(OAc) 3 .

[0022] Preferably, the kit is used for quantitatively detecting aldehyde and ketone compounds represented by carbohydrate metabolites, and the standard products include one or more standard products of L-arabinose, D-xylose, D-ribose, D-xylulose, L-fucose, galactose, D-glucose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, lactose, maltose, maltotriose, and maltotetraose; preferably including L-arabinose, D-xylose, D-ribose, D-xylulose, L-fucose, galactose, D-glucose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, lactose, maltose, maltotriose, and maltotetraose.

[0023] In addition, according to another aspect of the present invention, a method for quantitatively detecting aldehyde and ketone compounds based on a sensitizing probe is provided, which includes the step of derivatizing and labeling the aldehyde and ketone compounds in the sample to be tested with N,N-diethyl-1,4-phenylenediamine and / or its isotope compound as a sensitizing probe, and performing quantitative detection after labeling.

[0024] Preferably, the method quantitatively detects aldehyde and ketone compounds by an internal standard method, which includes the steps of labeling the aldehyde and ketone compounds in the sample to be tested with N,N-diethyl-1,4-phenylenediamine as the first sensitizing probe, and labeling the corresponding standard product with an isotope compound of N,N-diethyl-1,4-phenylenediamine as the second sensitizing probe. After labeling, the two are mixed as the sample for analysis, and LC-ESI-MS / MS is used for quantitative detection.

[0025] Preferably, the method quantitatively detects aldehyde and ketone compounds represented by carbohydrate metabolites in a biological sample. The reaction conditions for derivatizing the sample to be tested are to add an excessive amount of reducing agent and a derivatizing reagent as a sensitizing probe to the biological sample extract for mixing, and add 5% - 20% organic acid by volume ratio and react at 25 - 40 °C for 2 - 4 h to complete derivatization labeling.

[0026] Generally speaking, compared with the prior art through the above technical solutions conceived by the present invention, since it is found that the synthesized N,N-diethyl-1,4-phenylenediamine and / or its isotope compound can be used as a sensitizing probe for quantitative detection of aldehyde and ketone compounds, the following beneficial effects can be obtained:

[0027] Since it is found that the synthesized N,N - diethyl - 1,4 - phenylenediamine and / or its isotope compounds can be used as a sensitizing probe for the detection of aldehyde - ketone compounds, they can be applied to the preparation of a sensitizing probe detection kit for the quantitative detection of aldehyde - ketone compounds. In particular, taking N,N - diethyl - 1,4 - phenylenediamine and its isotope compounds as a pair of sensitizing probes and using the internal standard method for the quantitative detection of aldehyde - ketone compounds represented by carbohydrate metabolites, the quantitative detection of 14 carbohydrate metabolites can be completed in 11 minutes, including 4 oligosaccharides (lactose, maltose, maltotriose, and maltotetraose) that are difficult to detect simultaneously with monosaccharides, and the detection sensitivity can reach 0.007 - 0.458 fmol. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the synthetic route of the sensitizing probe and its isotope probe of the present invention;

[0029] Figure 2 is the structural schematic diagram of;

[0030] Figure 3 is the structural schematic diagram of the isotope substitute;

[0031] Figure 4 is the optimization of the derivatization conditions of representative carbohydrate metabolites; Figure 4 In, a is the reducing agent 2 - picBH 3 with different molar ratios of the carbonyl group to be measured, b is the different molar ratios of the derivatizing agent DEPPD to the carbonyl group to be measured; c is the acetic acid content in the reaction system; d is the reaction temperature; e is the reaction time;

[0032] Figure 5 is the optimization of the derivatization conditions of the plasma sample extract; keeping the addition amount of 10% (V / V) acetic acid, reaction temperature and time, and keeping the concentration ratio of the reducing agent to the derivatizing agent, optimize the amount of the required derivatizing reagent;

[0033] Figure 6 is the optimization of the derivatization conditions of the urine sample extract; keeping the addition amount of 10% (V / V) acetic acid, reaction temperature and time, and keeping the concentration ratio of the reducing agent to the derivatizing agent, optimize the amount of the required derivatizing reagent;

[0034] Figure 7It is the UPLC-MS / MS chromatogram of the labeled products of 14 carbohydrate metabolites; in the figure, 1 is the labeled product of D-xylulose; 2 is the labeled product of D-xylose; 3 is the labeled product of L-arabinose; 4 is the labeled product of D-ribose; 5 is the labeled product of L-fucose; 6 is the labeled product of D-galactose; 7 is the labeled product of D-glucose; 8 is the labeled product of D-mannose; 9 is the labeled product of N-acetyl-D-glucosamine; 10 is the labeled product of N-acetyl-D-galactosamine; 11 is the labeled product of lactose; 12 is the labeled product of maltose; 13 is the labeled product of maltotriose; 14 is the labeled product of maltotetraose. Detailed implementation mode

[0035] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following will, in conjunction with the accompanying drawings and preferred embodiments, detail the specific implementation mode, structure, features and their effects of the present invention as follows.

[0036] Term: N-Boc-1,4-phenylenediamine is N-(tert-butoxycarbonyl)-1,4-phenylenediamine, and its molecular formula is C 11 H 16 N 2 O 2 ;

[0037] 2-picBH 3 is 2-methylpyridine borane, and its molecular formula is C 6 H 10 BN; Oligosaccharides are generally low-degree polymers formed by connecting 2 to 10 monosaccharide molecules through glycosidic bonds. For example, the oligosaccharides in the present invention are lactose, maltose, maltotriose, maltotetraose, etc.

[0038] Although there are more than a hundred existing aldehyde and ketone compound sensitizing probes, there is no obvious pattern among the various sensitizing probes, and there are still many difficulties in the research of aldehyde and ketone compound sensitizing probes, such as the selectivity problem: the actual detection system is complex. In addition to aldehyde and ketone compounds, there are also various other organic substances, metal ions, etc. For example, in biological samples, amino acids, proteins, etc. may interfere with the specific reaction between the probe and aldehyde and ketone; it is difficult to distinguish due to similar structures and difficult to improve the sensitivity; the detection lower limit requirement is high, and the sensitivity of some current probes is difficult to meet the requirements; the response speed is slow and it is difficult to achieve rapid real-time detection, etc.

[0039] Surprisingly, the present invention discovers that N,N-diethyl-1,4-phenylenediamine or its isotopic compound d synthesized by using methanol as a solvent, adding N-Boc-1,4-phenylenediamine and acetaldehyde in a molar ratio of 1:50, and under the action of the reducing agent 2-picBH 3 (or its isotopic compound) and hydrochloric acid methanol solution 2-DEPPD can be used as a sensitizing probe for aldehyde and ketone compounds. Taking N,N-diethyl-1,4-phenylenediamine and its isotope compounds as a pair of sensitizing probes, 14 kinds of carbohydrate metabolites can be quantitatively detected within 11 minutes based on the LC-ESI-MS / MS technique using the internal standard method. Among them, there are 4 kinds of oligosaccharides (lactose, maltose, maltotriose, and maltotetraose) that are difficult to detect simultaneously with monosaccharides. This method not only has a wider detection coverage but also a lower detection limit, with a sensitivity as high as 0.007 - 0.458 fmol and a linear range up to 4 - 6 orders of magnitude.

[0040] Based on this, the present invention provides an application of a compound in the preparation of a detection kit for sensitizing probes. The compound is used as a sensitizing probe for aldehyde and ketone compounds, including N,N-diethyl-1,4-phenylenediamine and / or its isotope compounds. The structural formula of N,N-diethyl-1,4-phenylenediamine is shown as follows:

[0041]

[0042] In the present invention, the isotope compounds of N,N-diethyl-1,4-phenylenediamine include those in which at least one carbon atom and / or hydrogen atom in the two ethyl groups of the N,N-diethyl-1,4-phenylenediamine structure is replaced by its isotope, specifically including any one or more of the isotope compounds shown in the following structural formulas:

[0043]

[0044] The isotope compound is preferably d 2 -DEPPD, and its structural formula is as shown in (1) above.

[0045] In addition, the present invention also provides a detection kit for aldehyde and ketone compounds, which includes N,N-diethyl-1,4-phenylenediamine and / or its isotope compounds as sensitizing probes; preferably, it includes any one of N,N-diethyl-1,4-phenylenediamine and its isotope compounds as a sensitizing probe for quantitatively detecting aldehyde and ketone compounds by the internal standard method.

[0046] The structural formula of the N,N-diethyl-1,4-phenylenediamine is shown as follows:

[0047]

[0048] The isotope compound of N,N-diethyl-1,4-phenylenediamine is one in which at least one carbon atom and / or hydrogen atom in the two ethyl groups of the N,N-diethyl-1,4-phenylenediamine structure is replaced by its isotope, specifically including any one or more of the isotope markers shown in the following structural formulas:

[0049]

[0050] The kit further includes standards of reducing agents, organic acids, and / or aldehyde-ketone compounds.

[0051] In some embodiments, the standard of the aldehyde-ketone compound is the standard of a sugar metabolite, including standards of one or more of L-arabinose, D-xylose, D-ribose, D-xylulose, L-fucose, galactose, D-glucose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, lactose, maltose, maltotriose, and maltotetraose; preferably including L-arabinose, D-xylose, D-ribose, D-xylulose, L-fucose, galactose, D-glucose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, lactose, maltose, maltotriose, and maltotetraose.

[0052] The organic acids include acetic acid, formic acid, oxalic acid, or citric acid, etc., which are used to convert cyclic sugar metabolites into linear forms to expose the carbonyl terminus, promote the derivatization reaction of sugar metabolites, and improve the labeling efficiency.

[0053] The reducing agent includes 2-picBH 3 , sodium cyanoborohydride (NaBH 3 CN), or sodium triacetoxyborohydride (NaBH(OAc) 3 ), etc.

[0054] In some embodiments, for the kit, quantitative detection of aldehyde-ketone compounds is performed by an internal standard method based on LC-ESI-MS / MS technology, which includes any one of N,N-diethyl-1,4-phenylenediamine and its isotopic compound as a pair of sensitizing probes, where one probe is used to label the aldehyde-ketone compound to be detected, and the other probe is used to label the standard corresponding to the aldehyde-ketone compound and then used as an internal standard. For example, when using the kit to detect aldehyde-ketone compounds represented by sugar metabolites in a biological sample, N,N-diethyl-1,4-phenylenediamine can be used as the first sensitizing probe to label the sugar metabolites in the biological sample to be detected, and the isotopic compound of N,N-diethyl-1,4-phenylenediamine can be used as the second sensitizing probe to label the standard corresponding to the sugar metabolite and then used as an internal standard; the sugar metabolites include one or more of L-arabinose, D-xylose, D-ribose, D-xylulose, L-fucose, galactose, D-glucose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, lactose, maltose, maltotriose, and maltotetraose.

[0055] In some embodiments, the isotopic compound of N,N-diethyl-1,4-phenylenediamine in the kit is preferably d 2 -DEPPD.

[0056] In the present invention, the N,N-diethyl-1,4-phenylenediamine or d 2 -DEPPD is prepared according to the following method:

[0057] Using methanol as a solvent, an excessive amount of reducing agent is added to N-Boc-1,4-phenylenediamine and mixed to dissolve. An excessive amount of acetaldehyde is added to carry out a reductive amination reaction with N-Boc-1,4-phenylenediamine. The organic solvent and the remaining acetaldehyde are removed to obtain a pale yellow viscous oily substance, which is the intermediate N-Boc-diethyl-1,4-phenylenediamine;

[0058] Then, a hydrochloric acid-methanol solution is added to cause the intermediate to remove the tert-butoxycarbonyl group. The unreacted organic reagent is removed, and freeze-dried to obtain a pale yellow viscous powder, which is the synthesized N,N-diethyl-1,4-phenylenediamine (DEPPD).

[0059] For example, using methanol as a solvent, N-Boc-1,4-phenylenediamine and 2-picBH 3 are added to the solvent in a molar ratio of 1:4 and mixed to dissolve. An excessive amount (20 - 50 times, preferably 50 times) of acetaldehyde is added to carry out a reductive amination reaction with N-Boc-1,4-phenylenediamine. The reaction is stirred at room temperature for more than 2 hours. The organic solvent and the remaining acetaldehyde are removed by reduced pressure evaporation to obtain a pale yellow viscous oily substance, which is the intermediate N-Boc-diethyl-1,4-phenylenediamine; then, a hydrochloric acid-methanol solution is added to cause the intermediate to remove the tert-butoxycarbonyl group. The reaction is stirred at room temperature for more than half an hour. The unreacted organic reagent is removed by rotary evaporation, and freeze-dried to obtain a pale yellow viscous powder, which is the synthesized N,N-diethyl-1,4-phenylenediamine (DEPPD).

[0060] For the synthesis method of the isotope compound of the N,N-diethyl-1,4-phenylenediamine, referring to the synthesis of DEPPD, the above 2-picBH 3 is replaced with 2-picBD 3 (the commercial substance is stored in dichloromethane), 2-picBD 3 , CD 3 CHO, CD 3 CDO, CH 3 13 CHO or 13 CH 3 13 CHO, and other steps are the same as the synthesis of DEPPD to obtain the corresponding isotope compound of N,N-diethyl-1,4-phenylenediamine.

[0061] In some embodiments, the reaction is carried out by adding acetaldehyde according to a molar ratio of acetaldehyde to N-Boc-1,4-phenylenediamine of 50:1.

[0062] In addition, the present invention also provides a method for quantitatively detecting aldehyde and ketone compounds based on a sensitizing probe, which includes a step of derivatizing and labeling the aldehyde and ketone compounds in a sample to be tested with a derivatizing reagent using N,N-diethyl-1,4-phenylenediamine and / or its isotope compound as a sensitizing probe, and performing quantitative detection after labeling. In some embodiments, LC-ESI-MS / MS is used for quantitative detection after derivatization and labeling with the sensitizing probe.

[0063] Among them, for quantitative detection of aldehyde and ketone compounds represented by carbohydrate metabolites in biological samples by external standard method, the reaction conditions for derivatization are preferably carried out according to the molar ratio of carbohydrate metabolite standard: reducing agent 2-picBH 3 : derivatizing reagent such as DEPPD is 1:7:100; for the plasma sample extract (extracted with five volumes of pre-cooled methanol), add 2-picBH solution with the same volume and a concentration of 56 mmol / L and DEPPD solution with a concentration of 800 mmol / L; 3 for the urine sample extract (extracted with five volumes of pre-cooled methanol), add 2-picBH solution with the same volume and a concentration of 42 mmol / L and DEPPD solution with a concentration of 600 mmol / L; 3

[0064] Add 10% (V / V) acetic acid to the above reaction system, react at 30 °C for 3 hours, and then add 2% (V / V) formic acid to the reaction system to terminate the reaction.

[0065] More preferably, the internal standard method is used for quantitative detection of aldehyde and ketone compounds, which includes a step of labeling the aldehyde and ketone compounds to be tested with N,N-diethyl-1,4-phenylenediamine as the first sensitizing probe and labeling the corresponding standard with its isotope compound as the second sensitizing probe. After derivatization and labeling, the two are mixed as the sample for analysis and quantitative detection is carried out. For example, in some embodiments, the aldehyde and ketone compounds are carbohydrate metabolites in biological samples, and the method specifically includes the following steps:

[0066] (1) Pretreatment of biological samples: Protein precipitation treatment is carried out on the collected plasma or urine with pre-cooled methanol, and the supernatant is collected by centrifugation, which is the sample to be tested;

[0067] (2) Labeling of carbohydrate metabolites and their standards: Use DEPPD as the first sensitizing probe to label the target carbohydrate metabolites in the sample to be tested, and use the isotope compound of DEPPD as the second sensitizing probe to label the corresponding carbohydrate metabolite standards. Specifically:

[0068] An excessive amount of a reducing agent and a derivatizing agent as a sensitizing probe are added to a biological sample extract and mixed. An organic acid is added at a volume ratio of 5-20% and reacted at 25-40 °C for 2-4 h to derivatize the target carbohydrate metabolites in the sample to be tested, obtaining the target derivatives labeled with DEPPD; preferably, the reducing agent, the derivatizing agent as a sensitizing probe, and the carbonyl in the biological sample extract are mixed at a molar ratio of 6-8:75-125:1, more preferably at a molar ratio of 7:100:1. By volume ratio, 10% of the organic acid is added and reacted at 30 °C for 3 h. The organic acid includes acetic acid, formic acid, oxalic acid, citric acid, etc.; the reducing agent includes 2-picBH 3 , sodium cyanoborohydride (NaBH 3 CN), or sodium triacetoxyborohydride (NaBH(OAc) 3 ), etc.

[0069] And the standard is labeled with an isotope compound of DEPPD according to the same ratio and reaction conditions. After labeling, the two are mixed as the detection sample; the target carbohydrate metabolites include one or more of L-arabinose, D-xylose, D-ribose, D-xylulose, L-fucose, galactose, D-glucose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, lactose, maltose, maltotriose, and maltotetraose.

[0070] (3) Quantitative detection by internal standard method: The detection sample is quantitatively detected and analyzed by LC-ESI-MS / MS using the internal standard method.

[0071] In some embodiments, the detection conditions of LC-ESI-MS / MS are specifically as follows:

[0072] In an ultra-high performance liquid system, water containing 0.1% (V / V) formic acid is used as mobile phase A, and acetonitrile containing 0.1% (V / V) formic acid is used as mobile phase B. The chromatographic column used is a Waters Acquity BEH C 18 chromatographic column, with a specification of 2.1x100 mm and a particle size of 1.7 μm. The column oven is set at 35 °C, the flow rate is 0.4 mL / min, and the injection volume is 1 μL. The gradient change of phase B for each run is as follows: 2% B is maintained from 0 to 1 minute; from 1 to 5 minutes, 2 → 6% B; from 5.1 to 8 minutes, 90% B is maintained; from 8.1 to 11 minutes, 2% B is maintained.

[0073] The ion source temperature of the mass spectrometry system is 600 °C, the ionization voltage is 5500 volts, the curtain gas pressure value is set to 35 psi, and the nebulizing gas pressure and auxiliary heater pressure are set to 55 psi. The sampling mode is the multi-reaction monitoring positive mode with time windows, where the parent ion, daughter ion, retention time, declustering voltage value, and collision energy are set according to the target analyte to be measured.

[0074] The following are examples

[0075] Example 1 N,N-Diethyl-1,4-phenylenediamine and its isotope probe d 2 - Preparation of N,N-Diethyl-1,4-phenylenediamine

[0076] Experimental materials: N-Boc-1,4-phenylenediamine (>95%) was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.; 2-picoline borane complex (2-picBH 3 , 95%), analytical grade acetaldehyde (CH 3 CHO, 99%), analytical grade methanol (MeOH, ≥99.9%) were purchased from Sigma-Aldrich Co., LLC (USA); deuterated methanol (CD 3 OD, 99.8%) containing 0.03% (V / V) tetramethylsilane (Tetramethylsilane, TMS) was purchased from Cambridge Isotope Laboratories, Inc. (USA); deuterated 2-picoline borane complex (2-picBD 3 , 95%; deuterium abundance >99%) was purchased from Yunnan Suri Biomedical Technology Co., Ltd.; analytical grade concentrated hydrochloric acid (HCl, 36.0% - 38.0%) was purchased from Sinopharm Chemical Reagent Co., Ltd.; ultrapure water was prepared by the Milli-Q system (Millipore, USA) in the laboratory.

[0077] (1) Preparation of N,N-Diethyl-1,4-phenylenediamine (N,N-diethyl-1,4-phenylenediamine, DEPPD). The synthesis route of DEPPD is as Figure 1 shown as follows:

[0078] Using an analytical balance, weigh 208 mg (about 1 mmol) of N-Boc-1,4-phenylenediamine powder and 2-picBH 3428 mg (about 4 mmol) of the powder was placed in a 50 mL round-bottom flask. It was transferred to a fume hood, 5 mL of methanol solution was added to the round-bottom flask, and it was stirred well to dissolve using a magnetic stir bar. 3 mL of acetaldehyde solution (about 50 mmol) was added, and a soft rubber stopper was inserted to prevent the loss of acetaldehyde by volatilization. The reaction mixture was stirred at room temperature for 2 hours, and then rotary evaporation under reduced pressure was carried out using a rotary evaporator to obtain a pale yellow viscous oily substance. 12 mL of 6 mmol / L hydrochloric acid methanol solution was continuously added to the round-bottom flask, a soft rubber stopper was inserted to prevent the volatilization of concentrated hydrochloric acid, the reaction mixture was stirred at room temperature for 1 hour, the organic reagent was removed by rotary evaporation, and it was freeze-dried to obtain a pale yellow viscous powder. The product was collected to obtain N,N-diethyl-1,4-phenylenediamine (DEPPD), and the structure was as shown Figure 2 shown in the upper left of

[0079] (2)d 2 -N,N-diethyl-1,4-phenylenediamine (d 2 -N,N-diethyl-1,4-phenylenediamine, d 2 -DEPPD) was prepared, and the synthesis route of d 2 -DEPPD was as shown Figure 1 shown below:

[0080] Replace 2-picBH 3 powder with 4.8 mL of 2-picBD 3 solution (about 4 mmol, with dichloromethane as the solvent), and the remaining steps were the same as those for the synthesis of DEPPD, then d 2 -N,N-diethyl-1,4-phenylenediamine (d 2 -DEPPD) was obtained, and the structure was as shown Figure 2 shown in the upper right of

[0081] N,N-diethyl-1,4-phenylenediamine and d 2 -N,N-diethyl-1,4-phenylenediamine were characterized by one-dimensional proton nuclear magnetic resonance spectroscopy on a Bruker 600 MHz superconducting nuclear magnetic resonance spectrometer, and the data acquisition and processing software was Bruker TopSpin 3.6.0. N,N-diethyl-1,4-phenylenediamine and d 2 -N,N-diethyl-1,4-phenylenediamine were characterized by high-resolution mass spectrometry on a Shimadzu LC-30AD UHPLC liquid phase system combined with a Sciex X500R mass spectrometry system.

[0082] The structures of N,N-diethyl-1,4-phenylenediamine were identified using one-dimensional proton nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry, and the results were as follows:

[0083] 1H chemical shift δ 1.138 (t, 6H), 3.5565 (m, 4H), 6.984 (d, 2H), 7.241 (d, 2H); HRMS-ESI (+), [M+H] + , m / z, calculated: 165.1386, measured: 165.1366.

[0084] Using one-dimensional proton nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry, d 2 -N,N-diethyl-1,4-phenylenediamine was used for structural identification, and the results are as follows:

[0085] 1 H chemical shift δ 1.156 (d, 6H), 3.589 (m, 2H), 7.096 (d, 2H), 7.344 (d, 2H); HRMS-ESI (+), [M+H] + , m / z, calculated: 167.1512, measured: 167.1497.

[0086] According to this synthetic route, CD 3 CHO、CD 3 CDO、CH 3 13 CHO and 13 CH 3 13 One or more of CHO and 2-picBH 3 Corresponding replacements are made to obtain isotopic compounds in which at least one carbon atom and / or hydrogen atom in the diethyl group of the N,N-diethyl-1,4-phenylenediamine structure is replaced by its isotope, such as Figure 3 As shown, it is used as a sensitized probe for aldehydes and ketones.

[0087] Example 2: Using N,N-diethyl-1,4-phenylenediamine as a sensitized probe to detect carbohydrate metabolites in biological samples

[0088] Experimental materials: Analytical grade methanol (MeOH, ≥99.9%), L-arabinose (>98%), D-xylose (>98%), D-ribose (>98%), L-fucose (>97%), D-galactose (>97%) and D-mannose (>98%) were purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.; D-xylulose (>98%) was purchased from Shanghai Aladdin Chemical Reagent Co., Ltd.; D-glucose (>98%) was purchased from Sinopharm Group; N-acetyl-D-glucosamine (≥97%) and N-acetyl-D-galactosamine (≥97%) were purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd.; lactose (≥97%) was purchased from Sigma-Aldrich, USA; maltose (≥95%), maltotriose (≥96%) and maltotetraose (≥98%) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0089] Based on the approval of the Ethics Committee of Fudan University (PE21087), human plasma and urine were obtained from healthy Chinese adult volunteers recruited in the Human Phenome Project who had signed informed consent forms. Once the samples were obtained by the clinical standard sampling method, they were immediately frozen with liquid nitrogen and stored at -80 °C for further analysis.

[0090] Preparation of standard stock solutions and working solutions: The standards of 14 carbohydrate metabolites were separately dissolved in 50% methanol aqueous solution. After preparing the single-standard stock solutions using volumetric flasks, they were diluted to single-standard solutions of 2.0 mmol / L. The same volume was taken from each single-standard solution and mixed to obtain a mixed carbohydrate standard solution. The 50% methanol aqueous solution was used to perform stepwise serial dilutions to obtain 14 linear samples with different concentrations. The concentrations from high to low were named L1 - L14, and the concentrations (μmol / L) were 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.9063, 1.9531, 0.9766, 0.4883, and 0.2441 μmol / L, respectively. All the prepared single-standard solutions, mixed carbohydrate standard solutions, and linear samples were stored in a -80 °C refrigerator.

[0091] Pretreatment of biological samples: Carbohydrate metabolites have higher solubility in methanol solution and can cause the proteins in biological samples (such as blood samples and urine samples) to aggregate and precipitate, which can be removed by high-speed centrifugation to prevent protein precipitates from causing interference or blockage in subsequent steps. Specifically, 20 μL of human plasma or urine was mixed with 100 μL of pre-cooled methanol (-20 °C), sonicated in an ice bath for 5 min, and then centrifuged at 4 °C and 14000 rpm for 10 min in a low-temperature centrifuge to obtain the supernatant.

[0092] Optimization of the derivatization (diethylation) conditions for 8 representative carbohydrate metabolites. Specifically, first, the ratio of the reducing agent and derivatizing reagent to the analyte, the amount of acid, reaction temperature, and reaction time were systematically optimized using representative carbohydrate metabolites to determine the optimal derivatization conditions as follows:

[0093] Let the reducing agent be 2-picBH 3Three parallel samples were prepared under the conditions that the molar ratio of the reducing agent to the carbonyl group to be measured was 3, 4, 5, 6, 7, and 8 times. The peak areas of the derivatives of each carbohydrate metabolite were collected. The maximum average peak area was normalized as the ordinate, and the molar ratio of the reducing agent to the carbonyl group to be measured was used as the abscissa. The optimal dosage of the reducing agent was found to be 7 times. Subsequently, with the dosage of the reducing agent fixed, three parallel samples were prepared under the conditions that the molar ratio of DEPPD to the carbonyl group to be derivatized was 50, 100, 150, and 200 times. The peak areas of the derivatives of each carbohydrate metabolite were collected. The maximum average peak area was normalized as the ordinate, and the molar ratio of DEPPD to the carbonyl group to be measured was used as the abscissa. The optimal dosage of the derivatization reagent was found to be 100 times; after confirming the dosages of the reducing agent and the derivatization reagent, three parallel samples were prepared under the conditions that the acetic acid content in the reaction system was 5%, 10%, 20%, and 30%. The peak areas of the derivatives of each carbohydrate metabolite were collected. The maximum average peak area was normalized as the ordinate, and the acetic acid content was used as the abscissa. The most suitable acetic acid content in the reaction system was found to be 10%; using the above optimal dosages of the reducing agent, DEPPD, and acetic acid content, three parallel samples were incubated at reaction temperatures of 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, and 70 °C respectively. The peak areas of the derivatives of each carbohydrate metabolite were collected. The maximum average peak area was normalized as the ordinate, and the heating temperature was used as the abscissa. The optimal reaction temperature was found to be 30 °C; using the above optimal dosages of the reducing agent, DEPPD, acetic acid content, and reaction temperature, the reaction time was investigated for 1, 2, 3, 4, 5, and 6 hours for three parallel samples each. The peak areas of the derivatives of each carbohydrate metabolite were collected. The maximum average peak area was normalized as the ordinate, and the reaction time was used as the abscissa. The most suitable reaction duration was found to be 3 hours.

[0094] Figure 4 where a and b are the different molar ratios of the reducing agent 2-picBH 3 and the derivatizing agent DEPPD to the carbonyl group to be measured; c is the acetic acid content in the reaction system; d is the reaction temperature; e is the reaction time. Among them, C 5 H 10 O 5 -ketose is D-xylulose; C 5 H 10 O 5 -aldose is D-ribose; C 6 H 12 O 5 is L-fucose; C 6 H 12 O 6 is D-galactose; C 8 H 15 NO 6 is N-acetyl-D-glucosamine; C 12 H 22 O11 is maltose; C 18 H 32 O 16 is maltotriose; C 24 H 42 O 21 is maltotetraose.

[0095] Taking 8 representative carbohydrate metabolites as the detection objects, the optimized derivatization conditions are as follows: Accurately pipette 10 μL of the working solution of standards L1-L14 into a 1.5 mL EP tube, add 10 μL of the reducing agent 2-picBH 3 solution with a concentration of 14 mmol / L, 10 μL of the derivatizing agent DEPPD with a concentration of 200 mmol / L, and 3.3 μL of acetic acid. After vortex mixing, incubate at 30 °C for 3 hours (900 rpm), and add 1 μL of formic acid to the above mixture to quench the reaction.

[0096] For real biological samples, the total amount of carbohydrate analytes in the samples is unknown, and the amount of derivatization reagent in the biological samples needs to be determined. Using the optimized results of the acetic acid addition amount, reaction temperature, and reaction time in the previous text, while keeping the ratio of the reducing agent to the derivatizing reagent at 7:100 unchanged, the derivatization conditions for plasma samples and urine samples were optimized as follows:

[0097] Take 10 μL each of the plasma sample extract after extraction with pre-cooled methanol and the urine sample extract after extraction with pre-cooled methanol. Select a reaction system with 10% acetic acid content, and add 10 μL of reducing agents with concentrations of 14 mmol / L, 28 mmol / L, 42 mmol / L, 56 mmol / L, and 70 mmol / L respectively, and then correspondingly add 10 μL of derivatizing reagents with concentrations of 200 mmol / L, 400 mmol / L, 600 mmol / L, 800 mmol / L, and 1000 mmol / L. Prepare three parallel samples for each concentration. After the derivatization is completed by incubating at 30 °C for 3 hours, sample and collect the peak areas of the derivatization products of each carbohydrate metabolite, and normalize them with the maximum average peak area of the derivatization products of the carbohydrate metabolites.

[0098] Figure 5 and Figure 6 are the optimized results of the derivatization conditions for the plasma sample extract and the urine sample extract respectively; while keeping the acetic acid addition amount of 10% (V / V), reaction temperature, and time, and keeping the concentration ratio of the reducing agent to the derivatizing reagent, optimize the amount of derivatizing reagent required. Select the condition with a derivatizing reagent concentration of 800 mmol / L for derivatization labeling of the plasma sample after extraction, and select the condition with a derivatizing reagent concentration of 600 mmol / L for derivatization labeling of the urine sample after extraction.

[0099] For plasma as the biological sample, the preferred derivatization conditions are as follows: 10 μL of the supernatant plasma after extraction with pre-cooled methanol and centrifugation is added with 10 μL of 2-picBH 3 solution with a concentration of 56 mmol / L and 10 μL of the working solution of DEPPD solution with a concentration of 800 mmol / L.

[0100] For urine as the biological sample, the preferred derivatization conditions are as follows: 10 μL of the urine in the supernatant layer after extraction with pre-cooled methanol and centrifugation is added with 10 μL of 2-picBH 3 solution with a concentration of 42 mmol / L and 10 μL of the working solution of DEPPD solution with a concentration of 600 mmol / L, and other reaction conditions remain unchanged.

[0101] Data acquisition: High-resolution mass spectrometry detection of the labeled product is carried out on a Shimadzu LC-30AD UHPLC liquid phase system combined with a Sciex X500R mass spectrometry system. The detection conditions are as follows: In the ultra-high performance liquid phase system, water containing 0.1% (V / V) formic acid is used as mobile phase A, and acetonitrile containing 0.1% (V / V) formic acid is used as mobile phase B. The chromatographic column used is a Waters Acquity HSS T3 chromatographic column, with a specification of 2.1 x 100 mm and a particle size of 1.8 μm. The column oven is set at 40 °C, the flow rate is 0.4 mL / min, and the injection volume is 1 μL. The gradient change of phase B for each run is as follows: 0 - 1.5 minutes, hold 1% B; 1.5 - 3 minutes, 1 → 10% B; 3.1 - 4 minutes, 40% → 51% B; 4.1 - 7 minutes, hold 80% B; 7.1 - 9 minutes, hold 1% B.

[0102] The mass spectrometry system is collected in both positive and negative ion modes. The ion source temperature is set at 450 °C, the ionization voltage is (±) 5500 V, the collision gas is selected with the Medium option, the curtain gas pressure is set at 35 psi, and the nebulizing gas and auxiliary heating gas are set at 50 psi.

[0103] Investigation of labeling efficiency: After obtaining the optimal derivatization reaction conditions, the derivatization efficiency is evaluated by collecting the peak areas of the under-labeled substrate and the total substrate of each sugar metabolite to be measured. The results are shown in Table 1.

[0104] Table 1 Derivatization efficiency of representative sugar metabolites

[0105]

[0106] As can be seen from the results in Table 1, the derivatization efficiencies of 8 metabolic sugar metabolites are all above 94%, and the derivatization efficiency of L-fucose can reach 99.5%.

[0107] Example 3 N,N - Diethyl - 1,4 - phenylenediamine and d 2 -N,N - Diethyl - 1,4 - phenylenediamine as a Sensitizing Probe for the Simultaneous Quantitative Detection of Multiple Carbohydrate Metabolites

[0108] The biological samples were pretreated and labeled according to the optimized derivatization conditions for real biological samples in Example 2. Meanwhile, d 2 -DEPPD was used as an internal standard by labeling a working solution with a known concentration under the same reaction conditions. To achieve accurate quantification, the DEPPD - labeled biological samples and d 2 -DEPPD - labeled internal standard working solutions were mixed and diluted 10 - fold with acetonitrile - water (volume ratio 2:98), and then subjected to LC - ESI - MS / MS detection and analysis.

[0109] Quantitative analysis was performed using a Sciex QTRAP 6500+ mass spectrometer equipped with an electrospray ionization (ESI) source and coupled with a Shimadzu LC - 30AD UPLC system. Subsequently, the liquid - phase parameters were optimized. The optimization process of the liquid phase was as follows: Water containing 0.1% (V / V) formic acid and acetonitrile, which are the most common in reverse - phase chromatography, were selected as mobile phase A and B respectively. The flow rate was set at 0.4 mL / min, the initial column temperature was set at 40°C, the recommended value by the column manufacturer. The mobile - phase gradient was that phase B changed from 2% to 15% within 2 to 15 minutes. Three types of chromatographic columns, namely Agilent Zorbax Eclipse Plus RRHD C18 column (2.1×100 mm, particle size 1.8 μm), Waters Acquity HSS T3 column (2.1×100 mm, particle size 1.8 μm) and Waters Acquity BEH C18 column (2.1×100 mm, particle size 1.7 μm), were tested respectively, and the chromatograms of the L1 - derived samples were collected. By comparing the peak shapes of each analyte and the separation trend between peaks, the Waters Acquity BEH C18 column was selected as the most suitable chromatographic column. Subsequently, the chromatographic peak separation effects of two elution gradients were investigated, where mobile phase B changed from 1% to 6% within 1 to 6 minutes and from 2% to 6% within 1 to 5 minutes. Also, the chromatographic peak separation effects of this chromatographic column under three working conditions with column oven temperatures of 30°C, 35°C, and 40°C were investigated respectively.

[0110] The optimized liquid - phase analysis parameters are as follows:

[0111] Using Waters Acquity BEH C18 chromatographic column (2.1×100 mm, particle size 1.7 μm), column temperature was set at 35 °C, mobile phases A and B were water and acetonitrile containing 0.1% (V / V) formic acid respectively, flow rate was 0.4 mL / min, injection volume was 1 μL; chromatographic elution gradient, expressed as the percentage of mobile phase B: 0 - 1 minute, 0 → 2%; 1 - 5 minutes, 2% → 6%; 5.1 - 8 minutes, 6% → 90%; 8.1 - 11 minutes, 90% → 2%, where the total volume of mobile phase A and mobile phase B was 100%.

[0112] All labeled carbohydrate metabolites were quantitatively analyzed in the positive ion mode using the multiple reaction monitoring mode (MRM). The important acquisition parameters of the mass spectrometry system were optimized. The optimization process was as follows: First, the capillary voltage value of the ion source was optimized. A range of 3500 to 5500 volts that the instrument could tolerate was selected. The peak area data of each component were collected three times respectively for 5 capillary voltage values at intervals of 500 volts. The maximum average peak area of the derivative product of each carbohydrate metabolite was used for normalization, and the optimal capillary voltage value was obtained as 5500 volts. Then, the obtained optimal capillary voltage value was taken to optimize the temperature of the ion source. An ion source temperature range of 300 to 600 °C was selected. The peak areas of each component were collected three times respectively for 7 ion source temperatures at intervals of 50 °C. The maximum average peak area of the derivative product of each carbohydrate metabolite was used for normalization, and the optimal ion source temperature was obtained as 600 °C. After selecting the optimal ion source parameters, for each carbohydrate metabolite to be measured, its corresponding two compound-dependent parameters - declustering potential (DP) and collision energy (CE) were optimized. A range of DP values from 20 to 80 V was selected, and the peak areas of the derivative products of each carbohydrate metabolite were collected three times respectively for 13 DP values at intervals of 5 V. The maximum average peak area of the derivative product of each carbohydrate metabolite was used for normalization; the optimization method for the CE value was similar. A range of 20 to 50 V was selected, and the peak areas of the derivative products of each carbohydrate metabolite were collected three times respectively for 16 CE values at intervals of 2 V. The maximum average peak area of the derivative product of each carbohydrate metabolite was used for normalization.

[0113] The ion source temperature was 600 °C, the ionization voltage was 5500 volts, and the nebulizing gas and auxiliary heating gas were set at 35 and 50 psi respectively. The ion pair, retention time, DP value, and CE value detection parameters corresponding to each analyte are shown in Table 2. Sciex Analyst and OS (v1.7) software were used for data acquisition and processing respectively.

[0114] Table 2 MRM detection parameter settings for quantitative detection of carbohydrate labeled products

[0115]

[0116]

[0117] In the table, such as "D-xylulose 1-d 2 " In "d 2 " indicates the product labeled with the second labeling reagent (i.e., deuterated reagent).

[0118] The UPLC-MS / MS chromatograms of the labeled products of 14 carbohydrate metabolites are as Figure 7 shown Figure 7 In it, 1 is the labeled product of D-xylulose; 2 is the labeled product of D-xylose; 3 is the labeled product of L-arabinose; 4 is the labeled product of D-ribose; 5 is the labeled product of L-fucose; 6 is the labeled product of D-galactose; 7 is the labeled product of D-glucose; 8 is the labeled product of D-mannose; 9 is the labeled product of N-acetyl-D-glucosamine; 10 is the labeled product of N-acetyl-D-galactosamine; 11 is the labeled product of lactose; 12 is the labeled product of maltose; 13 is the labeled product of maltotriose; 14 is the labeled product of maltotetraose.

[0119] After labeling the carbohydrate metabolites, 14 carbohydrate metabolites can be detected by UPLC-MS / MS, including 8 monosaccharides (including 1 ketose and 7 aldoses), 2 N-acetylated glycosamines and 4 oligosaccharides (2 disaccharides, 1 trisaccharide and 1 tetrasaccharide), and the total amount of L-arabinose and D-xylose is quantified.

[0120] Example 4 verifies the sensitivity and accuracy of this method

[0121] This example verifies this quantitative method in terms of sensitivity, linear range, precision and accuracy, as follows:

[0122] Use d 2 -DEPPD-labeled working solution with known concentration as the internal standard, and use DEPPD to label the above-mentioned linearly diluted L1-L14 samples. Collect the spectra according to the liquid chromatography-mass spectrometry conditions in Example 3, and obtain the standard curve, linear range and correlation coefficient (R 2)。The steps of data processing are as follows: First, import the collected raw data in the Analytics module of Sciex OS 2.1 software, select "New" to create a new result table. At this time, a pop-up window will display the method for creating a new data processing method. Use the linear sample data (it is recommended to use L1) to create a data processing method, set appropriate integration parameters, and save the data processing method after confirming that the metabolite component names and retention times are correct. Then, use this data processing method to let the OS software automatically batch process the raw data. After generating the preliminary results, manually check the peak attribution and integration of each metabolite and its corresponding chromatographic peak in each sample. Subsequently, use the peak area ratio and concentration ratio of metabolites and their internal standards in the linear samples (L1-L14) to construct a linear equation, obtain the linear range, and obtain the detection limit (LOD) and quantification limit (LOQ) of each carbohydrate metabolite at signal-to-noise ratios of 3 and 10 respectively. The results are shown in Table 3.

[0123] The precision was evaluated by using carbohydrate metabolites labeled with DEPPD at three concentration levels of low (L7), medium (L4), and high (L2), and the intra-day difference and inter-day difference of collecting three times within a day and collecting continuously for three days. The results are shown in Table 4.

[0124] The isotope effect was evaluated by calculating the difference in retention time and the ratio of ionization efficiency between DEPPD-labeled and d 2 -DEPPD-labeled carbohydrate metabolites (n = 5, data of 5 repeated sample collections). The results are shown in Table 5. At the same time, add the same volume of d 2 -DEPPD-labeled carbohydrates (n = 3) to classical biological fluids (human plasma and urine) to evaluate the matrix effect. The results are shown in Table 6.

[0125] Add linear samples at three concentration levels of low (L7), medium (L4), and high (L2) of the mixed carbohydrate standard products to the methanol extracts of human plasma samples and urine samples in a volume ratio of 1:1, and calculate the ratio of the difference to the actually added metabolite concentration to obtain the spiked recovery rates at the three concentration levels. The results are shown in Table 7.

[0126] Table 3 Linear equations, R 2 , linear ranges, and LOD and LOQ after sensitization of aldose and ketose metabolites

[0127]

[0128]

[0129] Table 3 results show that the quantitative linear ranges of the 14 carbohydrate metabolite labeled products can cover 4-6 orders of magnitude and have good linearity (R 2(>0.99), the lowest values of the limit of detection (LOD, signal-to-noise ratio of 3:1) and the limit of quantification (LOQ, signal-to-noise ratio of 10:1) can reach 7.2*10 -18 and 1.43*10 -17 mol, respectively.

[0130] Table 4 Evaluation of precision (n = 3)

[0131]

[0132] The results of the methodological validation showed that at three concentration levels of low (L7), medium (L4), and high (L2), the relative standard deviation (RSD) values of the within-day and between-day differences in the peak areas of all analytes were lower than 20%.

[0133] Table 5 Evaluation of isotope effect of the aldose-ketose quantification method (n = 5)

[0134]

[0135] In the table, "d0 sensitized product" is the "product labeled with DEPPD", and "d 2 sensitized product" is the "product labeled with d 2 -DEPPD".

[0136] The results of the isotope effect validation showed that the difference in the retention times of the chromatographic peaks of the d0 and d2 derivatives of each detected substance was within 1.13 s, and the signal response ratio of the d0 / d2 products was 0.95 - 1.02, indicating that the chromatographic behaviors and ionization efficiencies of the derivatives of each substance were consistent, and the isotope effect could be ignored.

[0137] Table 6 Investigation of matrix effect of plasma samples and urine samples in the aldose-ketose quantification method (n = 3)

[0138]

[0139]

[0140] The results of the matrix effect validation for two biological matrices of plasma samples and urine samples showed that the matrix effect of plasma samples was 78% - 116%, and that of urine samples was 81% - 100%, and the matrix effect was not obvious.

[0141] Table 7 Spike recovery of biological samples (n = 3)

[0142]

[0143] The results of the spike recovery showed that the spike recoveries at three concentration levels of low, medium, and high in human urine samples and plasma samples were all within 80% - 120%, indicating good quantitative accuracy of this method.

[0144] The quantitative analysis method established by this method is simple and rapid, and can complete the quantitative analysis of 14 carbohydrate metabolites (aldehyde and ketone metabolites) within 11 minutes of detection time, which is suitable for high-throughput analysis of large samples.

[0145] Example 5 uses this method to quantitatively detect various carbohydrate metabolites in biological samples

[0146] Quantitatively detect and analyze carbohydrate metabolites in complex biological samples of classical biological fluids (human plasma and urine) as follows:

[0147] After taking out the human plasma sample or urine sample from the -80 °C refrigerator, it is placed in ice water to thaw for 1 hour, and both biological samples are extracted by the protein precipitation method. Use a pipette to transfer 40 μL of human plasma or urine into a 1.5 mL centrifuge tube in a biological safety cabinet, add 200 μL of methanol pre-cooled at -20 °C for more than 4 hours, vortex and mix evenly, ultrasonically bath in ice water for 5 minutes, then place it in a high-speed centrifuge, set the centrifuge temperature to 4 °C, the rotation speed to 14000 rpm, centrifuge for 10 minutes, collect the supernatant into a new 1.5 mL centrifuge tube, and place it in the -80 °C refrigerator for later use.

[0148] Subsequently, the extracts of the standard products and biological samples are derivatized. The derivatization steps of the internal standard are as follows: Take out 10 μL of the linear sample L1 into a 1.5 mL centrifuge tube, and sequentially add 3.3 μL of acetic acid solution, 10 μL of 2-picBH 3 solution with a concentration of 14 mmol / L and 10 μL of d 2 -DEPPD solution with a concentration of 200 mmol / L. Note that the 2-picBH 3 solution needs to be prepared and used immediately to ensure its reducibility. Subsequently, the mixed solution is vortexed thoroughly and then placed in a thermostatic mixer at 30 °C for reaction for 3 hours. After the incubation time is reached, add 1 μL of formic acid to quench the reaction, and seal and store it in the -20 °C refrigerator for later use.

[0149] The derivatization steps of the linear sample are as follows: Take 10 μL of each of the linear samples L1 to L14 and transfer them to a 1.5 mL centrifuge tube, and sequentially add 3.3 μL of acetic acid solution, 10 μL of 2-picBH 3 solution with a concentration of 14 mmol / L and 10 μL of DEPPD solution with a concentration of 200 mmol / L. Note that the 2-picBH 3 solution is also prepared and used immediately to ensure its reducibility. The mixed solution is vortexed thoroughly and placed in a thermostatic mixer for reaction for 3 hours. After the incubation time is reached, add 1 μL of formic acid to quench the reaction. Mix the DEPPD-derivatized linear sample and the d 2 -DEPPD-derivatized internal standard working solution evenly at a volume ratio of 10:1, and use ACN / H2 The O(2:98, V / V) solution was diluted 10-fold and transferred to a 2-mL liquid-phase vial with a liner for UHPLC-ESI-MS / MS detection and analysis.

[0150] The derivatization step for biological samples was as follows: 10 μL of the extracted plasma sample was taken into a 1.5-mL centrifuge tube, and 3.3 μL of acetic acid solution, 10 μL of 2-picBH solution with a concentration of 56 mmol / L, and 10 μL of DEPPD solution with a concentration of 800 mmol / L were added successively. 3 10 μL of the urine sample extracted with methanol was taken into a 1.5-mL centrifuge tube, and 3.3 μL of acetic acid solution, 10 μL of 2-picBH solution with a concentration of 42 mmol / L, and 10 μL of DEPPD solution with a concentration of 600 mmol / L were added successively. 3 Note that the 2-picBH solution was also prepared and used immediately to ensure its reducing property. Subsequently, the mixtures of plasma samples and urine samples were vortexed thoroughly and placed in a thermostatic mixer at 30 °C for 3 hours. After the incubation time reached, 1 μL of formic acid was added to quench the reaction. 3 The two DEPPD-derivatized biological samples and the d-DEPPD-derivatized internal standard working solution were mixed evenly at a volume ratio of 10:1, diluted 10-fold with ACN / H2O (2:98, V / V) solution, and transferred to a 2-mL liquid-phase vial with a liner for UHPLC-ESI-MS / MS detection and analysis. Generally, 3 - 5 samples were prepared in parallel for each detection. If the prepared samples could not be detected in time, they were temporarily stored in a -20 °C refrigerator during the waiting period. 2 The quantitative detection results are shown in Table 8, where n is the number of samples prepared in parallel.

[0151] Table 8 Quantitative results of carbohydrate metabolites in biological samples (n = 5)

[0152] In the table, "-" indicates that the metabolite was not detected in healthy adults.

[0153]

[0154] This method can rapidly and highly-throughput detect 14 carbohydrate metabolites. However, due to using biological samples from healthy adults, some individual metabolites were not detected. Therefore, 10 carbohydrate metabolites were detected in the plasma samples of healthy adults, and 13 carbohydrate metabolites were detected in the urine samples of healthy adults. When the body is in a special state, such as in patients with chronic kidney disease undergoing peritoneal dialysis, maltotriose and maltotetraose can be detected in the plasma and urine of the patients.

[0155]

[0156] ​In summary, the quantitative analysis method established based on the sensitizing probe in the present invention is simple and rapid, and can complete the quantitative analysis of 14 carbohydrate metabolites within a detection time of 11 minutes, which is suitable for large-sample high-throughput analysis. Compared with the UPLC-MS / MS detection method based on probe sensitization in the existing literature, the results are shown in Table 9.

[0157] Table 9 Comparison of this probe with existing probe-assisted methods for quantitative analysis of carbohydrate metabolites

[0158]

[0159] Reference 1 only covers phosphorylated monosaccharides, and the types of sugars it covers are less than those of this application. The carboxylic acid coverage types of References 2 and 3 are slightly improved compared with Reference 1, but they lack isotope internal standard substances, and the analysis time is 2 - 12 times that of this application. References 4 and 5 are both hydrazine-based probes, and their coverage and sensitivity are lower than those of this application. Compared with probe PMP, which has better performance in all aspects and a sensitivity reaching the fmol level among the currently reported ones, Reference 6 has a comparable analysis time, and the linear range is 4 to 6 orders of magnitude for both. While the sensitivity of the probe-assisted method for quantitative analysis of carbohydrate metabolites in this paper has increased by 8 to 34 times. Due to its large steric hindrance, Reference 7 fails to cover oligosaccharide types. Considering the three aspects of detection coverage, sensitivity, and efficiency, this application is significantly superior to the existing probe quantitative methods.

[0160] The specific references in Table 9 are as follows:

[0161] 1. LI S, LIU F-L, ZHANG Z, et al. Ultrasensitive Determination of Sugar Phosphates in Trace Samples by Stable Isotope Chemical Labeling Combined with RPLC–MS[J]. Analytical Chemistry, 2022, 94(11): 4866 - 73.

[0162] 2. TAKEMOTO H, HASE S, IKENAKA T. Microquantitative analysis of neutral and amino sugars as fluorescent pyridylamino derivatives by high-performance liquid chromatography[J]. Analytical biochemistry, 1985, 145(2): 245 - 50.

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[0168] As described above, it is only the preferred embodiment of the present invention, and it does not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments by using the disclosed technical content within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any brief modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. Use of a compound in the preparation of a sensitization probe detection kit, characterized in that: The detection kit is used for quantitative detection of aldehyde and ketone compounds, wherein the compounds are used as sensitization probes, including N,N-diethyl-1,4-phenylenediamine and / or its isotope compounds, wherein the structural formula of N,N-diethyl-1,4-phenylenediamine is as follows: The isotopic compound of N,N-diethyl-1,4-phenylenediamine includes the compound shown in the following structural formula: Or any one or more of the above.

2. The use according to claim 1, characterized in that The compound includes any one of N,N-diethyl-1,4-phenylenediamine and its isotope compound, wherein the isotope compound of N,N-diethyl-1,4-phenylenediamine is preferably d2-DEPPD.

3. A detection kit for aldehyde and ketone compounds, characterized in that: It includes N,N-diethyl-1,4-phenylenediamine and / or its isotope compounds as a sensitization probe.

4. The kit according to claim 3, characterized in that Used for the quantitative detection of aldehyde and ketone compounds by internal standard method, it includes N,N-diethyl-1,4-phenylenediamine and any one of its isotope compounds, wherein N,N-diethyl-1,4-phenylenediamine is the first sensitization probe and the isotope compound of N,N-diethyl-1,4-phenylenediamine is the second sensitization probe.

5. The kit according to claim 4, characterized in that The isotope compound of N,N-diethyl-1,4-phenylenediamine is selected from any one of the following:

6. The kit according to any one of claims 3 to 5, characterized in that It also includes reducing agents, organic acids and / or standard substances of aldehyde and ketone compounds, wherein the organic acids include acetic acid, formic acid, oxalic acid, or citric acid; and the reducing agents include 2-picBH3, NaBH3CN, or NaBH(OAc)3.

7. The kit according to claim 6, characterized in that For quantitative detection of aldehyde and ketone compounds represented by carbohydrate metabolites, the standard substance includes one or more standard substances of L-arabinose, D-xylose, D-ribose, D-xylulose, L-fucose, galactose, D-glucose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, lactose, maltose, maltotriose, and maltotetraose; Preferred include L-arabinose, D-xylose, D-ribose, D-xylulose, L-fucose, galactose, D-glucose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, lactose, maltose, maltotriose and maltotetraose.

8. A method for quantitatively detecting aldehyde and ketone compounds based on a sensitized probe, characterized in that: The method comprises the steps of using N,N-diethyl-1,4-phenylenediamine and / or its isotope compounds as sensitization probes, carrying out derivatization labeling on aldehyde and ketone compounds in the sample to be tested, and carrying out quantitative detection after labeling.

9. The method according to claim 8, characterized in that The internal standard method is used to quantitatively detect aldehyde and ketone compounds, which includes the steps of using N,N-diethyl-1,4-phenylenediamine as a first sensitization probe to label the aldehyde and ketone compounds in the sample to be tested, and using an isotope compound of N,N-diethyl-1,4-phenylenediamine as a second sensitization probe to label the corresponding standard. After labeling, the two are mixed as a sample for quantitative detection using LC-ESI-MS / MS.

10. The method according to claim 8 or 9, characterized in that The quantitative detection of aldehyde and ketone compounds represented by carbohydrate metabolites in biological samples, the reaction conditions for the derivatization of the sample to be tested are to add an excess of a reducing agent and a derivatization reagent as a sensitization probe to the biological sample extract and mix them, and then add 5% to 20% of an organic acid by volume and react at 25 to 40°C for 2 to 4 hours to complete the derivatization labeling.

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