High coverage analysis method for carbonyl compounds in Baijiu and application of high coverage analysis method

Abstract

The invention belongs to the technical field of Baijiu detection and analysis, and relates to a high coverage analysis method for carbonyl compounds in Baijiu and application, the analysis method comprises the following steps: concentrating and pretreating a Baijiu sample, adding a derivatization reagent to obtain a to-be-detected Baijiu sample, and then obtaining non-targeted mass spectrum data of the to-be-detected Baijiu sample; the method comprises the following steps: preprocessing obtained non-targeted mass spectrometric data of a white spirit sample to be detected, screening out carbonyl compounds conforming to specific characteristics, annotating the screened carbonyl compounds, and defining corresponding qualitative grades according to different annotation scores. According to the method, carbonyl compounds in a white spirit sample are derivatized by synthesizing a derivatization reagent 3-bromoquinoline ester hydrazide, potential carbonyl compounds are screened and annotated on the basis of mass spectrum characteristics of derivatization products, and the method has remarkable advantages in analysis of high-boiling-point trace carbonyl compounds in Maotai-flavor white spirit and has the advantages of high coverage, high sensitivity, high sensitivity and the like. And simplicity and rapidness are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of liquor detection and analysis, and relates to a high coverage analysis method for carbonyl compounds in liquor and application thereof. Background Art

[0002] Maotai-flavor liquor is famous for its mellow taste and elegant and delicate aroma. It is a unique alcoholic beverage in China. Its unique flavor comes from a series of complex production processes, including 8 grain-based microbial fermentations, 7 high-temperature distillations, and the base liquor is blended into the final finished liquor in precise proportions after long-term aging. During the liquor brewing process, this powerful and reproducible metabolism of sorghum components (such as carbohydrates, proteins, etc.) by microorganisms forms a complex compound system.

[0003] There are more than 3,000 flavor compounds in liquor, most of which are volatile substances, including esters, aldehydes, ketones, sulfur-containing compounds, etc. Esters contribute fruity or other specific flavors, while non-volatile substances tend to contribute to the taste and overall flavor of liquor, forming the structural framework of the liquor's taste and affecting consumers' direct perception of liquor.

[0004] The Maillard reaction is a series of non-enzymatic reactions of carbonyl compounds (aldehydes, ketones) and amino compounds (amino acids, proteins), and is the main source of flavor substances in food and beverages. Carbonyl compounds are the main intermediates or end products of the Maillard reaction. In the primary stage of the Maillard reaction, carbonyl compounds and amino groups condense to form imine derivatives, which are further cyclized. In the intermediate stage, reduced ketones and dehydrogenated reduced ketones are produced through different reaction pathways. In the final stage, aldehydes and amines polymerize at low temperatures to form polymers. Carbonyl compounds have an important influence on the flavor and function of liquor, such as providing caramel flavor and changing color. Dicarbonyl compounds and active carbonyl compounds are highly active and react with biological macromolecules such as proteins and nucleic acids, affecting the nutritional value and safety of liquor. Therefore, high-coverage analysis of carbonyl compounds in liquor helps to explain the source of flavor and provide guidance for improving brewing technology.

[0005] Chemical derivatization combined with high-resolution mass spectrometry is a common method for characterizing low-abundance, low-sensitivity metabolites by introducing special groups to improve the chromatographic retention and ionization efficiency of metabolites. Currently, a variety of reagents for carbonyl compound derivatization have been developed, such as GP, HIQB, DnsHZ, etc., and further introduction of carbon or hydrogen light and heavy isotopes can achieve accurate quantification of metabolites based on the same retention time and peak area.

[0006] However, the molecular weight of traditional derivatization reagents is too small to allow metabolites to cross the conventional metabolite detection range. In addition, the synthesis of light and heavy isotope reagents is expensive. In terms of metabolite annotation, the traditional search method based on the secondary mass spectrometry database is not suitable for the annotation of derivatized products because it is impossible to obtain a large number of standard samples to establish a standard mass spectrometry library, and the derivatization group affects the mass spectrometry fragmentation of the metabolites, resulting in a large deviation in the mass spectrometry prediction method. Summary of the invention

[0007] The present invention aims to provide a high coverage analysis method for carbonyl compounds in liquor and its application. A new derivatization reagent 3-bromoquinoline ester hydrazide BqaHz based on natural bromine isotopes is synthesized, and after derivatization of carbonyl compounds in liquor samples, an ultra-high performance liquid chromatography-high resolution mass spectrometry platform is used to detect the derivatized carbonyl compounds, and potential carbonyl compounds are screened based on the mass spectrum characteristics of the derivatized products. The derivatized carbonyl compounds are annotated in combination with the primary m / z, tR, MS / MS spectra and molecular similarity, and the corresponding qualitative grades are defined according to different annotation scores. Not only the chromatographic retention coefficient of the carbonyl compounds is improved, but also the carbonyl compounds are made into a form that can be identified by mass spectrometry MS and MS / MS. The present invention has significant advantages in the analysis of high boiling point and trace carbonyl compounds in Maotai-flavor liquor, and has the characteristics of high coverage, high sensitivity, simplicity and rapidity.

[0008] In a first aspect, the present invention provides a high coverage analysis method for carbonyl compounds in liquor, comprising the following steps:

[0009] Step 1, data collection of carbonyl compounds in liquor samples: after the liquor samples are concentrated and pretreated, a derivatization reagent is added to obtain a liquor sample to be tested, and then non-targeted mass spectrometry data of the liquor sample to be tested is obtained; the derivatization reagent is 3-bromoquinoline ester hydrazide;

[0010] Step 2, screening and annotation of carbonyl compounds: pre-process the non-targeted mass spectrometry data of the liquor sample to be tested, screen out carbonyl compounds that meet specific characteristics, compare with the database of liquor carbonyl compounds, predict retention time tR, annotate the screened carbonyl compounds according to retention time tR, m / z, MS / MS similarity, and molecular similarity, and define corresponding qualitative levels according to different annotation scores.

[0011] In some embodiments, the method for collecting data on carbonyl compounds in liquor samples in step 1 comprises the following steps:

[0012] (1.1) Pretreatment: Concentrate the liquor sample and blank sample by 20-25 times to obtain the liquor sample to be tested and the blank sample;

[0013] (1.2) Derivatization: Add the derivatization reagent 3-bromoquinoline ester hydrazide to derivatize the liquor sample to be tested and the blank sample respectively;

[0014] (1.3) Obtaining non-targeted mass spectrometry data of the liquor sample to be tested: using ultra-high performance liquid chromatography-high resolution mass spectrometry to detect the derivatized liquor sample to be tested, and obtaining non-targeted mass spectrometry data of the liquor sample to be tested;

[0015] Preferably, the blank sample consists of 53% by volume of ethanol water;

[0016] Preferably, the concentration method is rotary evaporation, the rotation speed of the rotary evaporation is 100-200 rpm / min; the temperature of the rotary evaporation is 40-60° C.; the pressure of the rotary evaporation is -0.1±0.01 MPa;

[0017] Preferably, the rotation speed of the rotary evaporation is 150-180 rpm / min; the temperature of the rotary evaporation is 45-55° C.; the pressure of the rotary evaporation is -0.1-0.09 MPa; the liquor sample and the blank sample are concentrated 22-23 times;

[0018] Preferably, the rotation speed of the rotary evaporation is 200 rpm / min; the temperature of the rotary evaporation is 60° C.; the pressure of the rotary evaporation is -0.1 MPa, and the liquor sample and the blank sample are concentrated 25 times.

[0019] In some embodiments, the derivatization reagent has an equivalent of 1-20 eq, an acidity of 0.1%-5%, a reaction temperature of 4-80°C, and a reaction time of 5-120 min;

[0020] Preferably, the derivatization reagent has an equivalent of 10-20 eq, an acidity of 1%-4%, a reaction temperature of 4-80°C, and a reaction time of 20-100 min;

[0021] Preferably, the derivatization method is: after adding an acid-base reagent to the concentrated liquor sample to adjust the acidity to 0.1%-5%, add 1-20eq 3-bromoquinoline ester hydrazide solution, make up to volume, mix thoroughly, and react at 4-80°C for 5-120min.

[0022] In some embodiments, in step 2, the data preprocessing method is: peak alignment and peak extraction are performed on the non-targeted mass spectrometry data of the liquor sample to be tested obtained in step 1, and compounds in the blank sample whose area is greater than 5 times the peak area of ​​the liquor sample to be tested are removed;

[0023] Preferably, the parameters of the peak extraction are as follows: MS1 deviation, 0.005Da; MS / MS deviation, 0.01Da; retention time deviation, 0.2min, minimum peak width, 3Scan; minimum peak height, 1000; other parameters use default parameters;

[0024] Preferably, the annotation method is: using molecular network SGMNS software to annotate carbonyl compounds in the measured sample;

[0025] Preferably, the specific feature of step 2 is that in the MS spectrum, the [M] of the derivatized carbonyl compound + and [M+2] + The peak area ratio is 0.95-1.05. In the MS / MS spectrum, the mass-to-charge ratio m / z of characteristic fragment 1 is 154.0863±10ppm, the mass-to-charge ratio m / z of characteristic fragment 2 differs from that of the parent ion by 0±10ppm, and characteristic fragment 3 is the fragment lost when characteristic fragment 2 is stripped of bromoquinoline, and the mass-to-charge ratio m / z difference between it and characteristic fragment 2 is 206.9684±10ppm.

[0026] In some embodiments, the data information in the database of liquor carbonyl compounds includes the CAS number, CID, SMILES, InChI, and Mass information of the carbonyl compounds.

[0027] In some embodiments, the retention time tR is predicted by: establishing a QSRR model to predict the retention time of carbonyl compounds in a self-built library;

[0028] Preferably, the retention time tR is predicted by: arranging the real tR of the standard, then importing the SMILES and tR of the standard into the QSRR_Automator software, converting the SMILES into molecular descriptors, and then fitting the relationship between the molecular descriptors and the real tR by the software to establish a QSRR model;

[0029] Preferably, the mean absolute error of the QSRR model is 6s, and the linear correlation coefficient R is greater than 0.999.

[0030] In some embodiments, the derivatization reagent 3-bromoquinoline ester hydrazide has the structural formula:

[0031] In some embodiments, the preparation method of the derivatization reagent 3-bromoquinoline ester hydrazide is: dissolving tert-butyloxycarbonyl protected 3-bromoquinoline ester hydrazide in an organic solvent, adding 1-2 mL of trifluoroacetic acid to react, removing the organic solvent after the reaction, separating and purifying to obtain 3-bromoquinoline ester hydrazide;

[0032] Preferably, the preparation method of the tert-butoxycarbonyl protected 3-bromoquinoline ester hydrazide is: dissolving 3-bromoquinoline propylamine and tert-butoxycarbonyl protected glutaric acid hydrazide succinamide ester in an organic solvent at a molar equivalent of 1.2-1.5, adding 1-2 mL of triethanolamine, reacting at 100-180° C. for 5-10 h, removing the organic solvent after the reaction, separating and purifying to obtain tert-butoxycarbonyl protected 3-bromoquinoline ester hydrazide;

[0033] Preferably, the synthesis method of the tert-butoxycarbonyl protected glutaric acid hydrazide succinamide ester is as follows: 1.2-1.5 molar equivalents of tert-butoxycarbonyl protected glutaric acid hydrazide and N-hydroxysuccinimide are dissolved in an organic solvent, 1.2-1.5 molar equivalents of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is used as a condensing agent, and the mixture is reacted at 100-180° C. for 5-10 hours. After the reaction is completed, the organic solvent is removed, and the mixture is separated and purified to obtain tert-butoxycarbonyl protected glutaric acid hydrazide succinamide ester;

[0034] Preferably, the synthesis method of the 3-bromoquinoline propylamine is: 3-bromoquinoline is dissolved in an organic solvent, and then 1-1.5 molar equivalents of 1-bromopropylamine are added, and the mixture is reacted at 100-180° C. for 5-10 hours. After the reaction is completed, the solvent is removed, and the mixture is separated and purified to obtain 3-bromoquinoline propylamine.

[0035] In some embodiments, the qualitative grade of the carbonyl compound is as follows:

[0036] Level 4: Know the tR and molecular weight of the ions in the sample;

[0037] Level 3: Knowing the elemental C, H, O, N, S composition of the compound in the sample, the m / z search of the candidate structure has a corresponding candidate structure in the database with an error of 10-20ppm; the absolute deviation between the predicted retention time tR of the candidate structure and the actual retention time tR in the sample is greater than 60s;

[0038] Level 2: Knowing the elemental composition of the compound in the sample, the m / z search of the candidate structure has a corresponding candidate structure in the database, with a difference of 10-20ppm; the absolute deviation between the predicted retention time tR of the candidate structure and the actual retention time tR in the sample is less than 60s, and the SGMNS score is greater than 0.7;

[0039] Level 1: Knowing the elemental composition of the compound in the sample, the m / z search of the candidate structure has a corresponding candidate structure in the database, with a difference of 10-20ppm; the absolute deviation between the predicted retention time tR of the candidate structure and the actual retention time tR in the sample is less than 60s, and the SGMNS score is greater than 0.7; the absolute deviation between the predicted retention time tR of the candidate structure and the actual retention time tR of the standard is less than 30s.

[0040] In a second aspect, the present invention provides an application of any one of the above-mentioned analytical methods in detecting carbonyl compounds in liquor.

[0041] In summary, the present application includes at least one of the following beneficial technical effects:

[0042] By synthesizing a new derivatization reagent 3-bromoquinoline ester hydrazide BqaHz based on the natural isotope of bromine, the carbonyl compounds in the liquor samples were derivatized, and the derivatized carbonyl compounds were detected using an ultra-high performance liquid chromatography-high-resolution mass spectrometry platform. Potential carbonyl compounds were screened based on the mass spectrometric characteristics of the derivatized products, and the derivatized carbonyl compounds were annotated in combination with the primary m / z, tR, MS / MS spectra and molecular similarity, and the corresponding qualitative grades were defined according to different annotation scores. Not only the chromatographic retention coefficient of the carbonyl compounds was improved, but also the carbonyl compounds were made identifiable by mass spectrometry MS and MS / MS, which has significant advantages in the analysis of high-boiling point and trace carbonyl compounds in Maotai-flavor liquors, and has the characteristics of high coverage, high sensitivity, simplicity and rapidity. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The synthetic route of 3-bromoquinoline ester hydrazide (BqaHz) is shown below;

[0044] Figure 2 The figure is the optimization result of the derivatization conditions, where (A) is the reagent equivalent, (B) is the system acidity, (C) is the reaction temperature, and (D) is the reaction time;

[0045] Figure 3 The mass spectrometry characteristics of carbonyl compounds after derivatization, wherein (A) is the MS and MS / MS spectrum characteristics of carbonyl compounds after derivatization, and (B) is the deviation distribution of the mass spectrometry characteristics of 100 standards;

[0046] Figure 4 This is the overall analysis flow chart of carbonyl compounds in Maotai-flavor liquor;

[0047] Figure 5 Annotate the flow chart for carbonyl compounds using 3-phenylpropanal as an example;

[0048] Figure 6 This is an example of the background network of the SGMNS molecular network. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0050] The analysis of hydroxyl compounds mainly involves derivatization followed by detection, annotation and grading. Existing derivatization reagents have problems such as small molecular weight, inability to make metabolites cross the conventional metabolite detection range, and high cost for the synthesis of light and heavy isotope reagents.

[0051] Example 1

[0052] A derivatization reagent 3-bromoquinoline ester hydrazide (BqaHz) has a chemical structural formula of:

[0053]

[0054] Example 2

[0055] A method for synthesizing a derivatization reagent 3-bromoquinoline ester hydrazide (BqaHz), such as Figure 1 As shown, the following steps are included:

[0056] (1) Synthesis of 3-bromoquinoline propylamine (Compound 1):

[0057] Dissolve 3-bromoquinoline in a solvent, then add 1-1.5 molar equivalents of 1-bromopropylamine, and react at 100-180° C. for 5-10 hours. After the reaction, remove the solvent, and separate and purify the crude product by column chromatography to obtain 3-bromoquinoline propylamine (Compound 1);

[0058] (2) Synthesis of tert-butyloxycarbonyl protected glutaric acid hydrazide succinamide ester (Compound 2):

[0059] The tert-butoxycarbonyl protected glutaric acid hydrazide and N-hydroxysuccinimide are dissolved in dimethyl sulfoxide (DMSO) solvent in a molar equivalent of 1.2-1.5, and 1.2-1.5 molar equivalents (eq) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is used as a condensation agent. The reaction is carried out at 100-180° C. for 5-10 hours. After the reaction is completed, the DMSO solvent is removed, and the crude product is separated and purified to obtain tert-butoxycarbonyl protected glutaric acid hydrazide succinamide ester (compound 2);

[0060] (3) Synthesis of tert-butyloxycarbonyl protected BqaHz (Compound 3):

[0061] Dissolve 3-bromoquinoline propylamine and tert-butoxycarbonyl protected glutaric acid hydrazide succinamide ester in dimethyl sulfoxide (DMSO) solvent at a molar equivalent of 1.2-1.5, add 1-2 mL of triethanolamine (TEA), react at 100-180° C. for 5-10 h, remove the dimethyl sulfoxide (DMSO) solvent after the reaction, separate and purify to obtain tert-butoxycarbonyl protected BqaHz (compound 3);

[0062] (4) Synthesis of 3-bromoquinoline ester hydrazide BqaHz (Compound 4):

[0063] The tert-butyloxycarbonyl-protected BqaHz (compound 3) is dissolved in dimethyl sulfoxide (DMSO) solvent, and 1-2 mL of trifluoroacetic acid (TFA) is added to react. After the reaction is completed, the DMSO solvent is removed, and the mixture is separated and purified to obtain 3-bromoquinoline ester hydrazide BqaHz (compound 4).

[0064] Specifically, in this embodiment, in step (1), the reactant 3-bromoquinoline (1.03 g, 5 mmol) is dissolved in 50 mL of acetonitrile solvent, 1.5 molar equivalents of 1-bromopropylamine (1.03 g, 7.5 mmol) is added, and the reaction is stirred for 10 h under 130° C. oil bath heating conditions. After the reaction is completed, the acetonitrile solvent is removed by rotary evaporation. The crude product is separated and purified by column chromatography, the separation filler is 200-400 mesh silica gel, the mobile phase is a methanol-chloroform mixture, and the volume ratio of methanol to chloroform is 1:3. The organic phase is removed by rotary evaporation under reduced pressure to obtain 1.13 g of a light yellow oily product 3-bromoquinoline propylamine, with a yield of 85.2%. 1 H NMR (400MHz, DMSO-d6, ppm) δ10.07(s,1H), 9.72(s,1H), 8.73(d,1H), 8.46(d,1H), 8. 33(dd,1H), 8.12(dd,1H), 8.00(m,2H,-NH2), 5.22(t,2H), 3.08(tt,2H), 2.36(t,2H); 13 C NMR (400 MHz, DMSO-d6, ppm) δ 151.5, 144.3, 138.4, 136.2, 130.5, 129.4, 127.3, 126.4, 118.7, 56.8, 36.2, 27.5; HR-MS (C12H14BrN2+): theoretical value: 265.0335, found value [M]+: 265.0336.

[0065] In some embodiments, the solvent in step (1) is acetonitrile, dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF), etc., and the above solvents are not prone to produce side reactions.

[0066] In some embodiments, the amount of 1-bromopropylamine added in step (1) is 1.1, 1.2, 1.3, 1.4, 1.5, etc. molar equivalents. When the amount of 1-bromopropylamine added is less than 1 molar equivalent, the amount of 1-bromopropylamine added is too small, and the yield of 3-bromoquinoline propylamine (compound 1) obtained is low. Similarly, when the amount of 1-bromopropylamine added is greater than 1 molar equivalent, the reactant 3-bromoquinoline is too small, and the reaction cannot be fully carried out, resulting in a low yield of 3-bromoquinoline propylamine (compound 1).

[0067] In some embodiments, the reaction temperature in step (1) is 100°C, 110°C, 120°C, 140°C, 150°C, 160°C, 170°C or 180°C, etc. When the temperature is too low, the reaction is incomplete and the product yield is low; when the temperature is too high, the generation of by-products increases and the product purity decreases.

[0068] In some embodiments, the reaction time in step (1) is 5 h, 6 h, 7 h, 7.5 h, 8 h, 9 h, etc. If the reaction time is too short, it is not conducive to the complete reaction and the product yield is low. If the reaction time is too long, the product will no longer be generated after the reaction is completed, which reduces the synthesis efficiency.

[0069] Specifically, in this embodiment, in step (2), t-butyloxycarbonyl-protected glutaric acid hydrazide (1.48 g, 6 mmol) and N-hydroxysuccinimide (0.69 g, 6 mmol) were dissolved in 50 mL of dimethyl sulfoxide (DMSO) solvent at a molar equivalent of 1:1.2, 1.2 molar equivalents of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (1.38 g, 7.2 mmol) was used as a condensing agent, and the reaction was stirred at 120° C. for 10 h. After the reaction was completed, the DMSO solvent was removed by rotary evaporation. The crude product was separated and purified by column chromatography, the separation filler was 200-400 mesh silica gel, the mobile phase was a methanol-chloroform mixture, and the volume ratio of methanol to chloroform was 1:3. The organic phase was removed by rotary evaporation under reduced pressure to obtain 1.656 g of the product with a yield of 80.2%. 1 H NMR (400MHz, DMSO-d6, ppm) δ11.27(m,1H), 10.08(m,1H), 2.64(t,4H), 2.30(tt,2H), 2.19(t,2H), 2.20(tt,2H), 1.42(s,9H); 13 C NMR (400 MHz, DMSO-d6, ppm) δ 176.7, 169.1, 167.5, 156.6, 79.3, 37.7, 31.0, 28.5, 25.6, 20.6; HR-MS (C14H21N3O7): theoretical value: 343.1380, found value [M+H] + :344.1450.

[0070] In some embodiments, the solvent in step (2) is acetonitrile, dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF), etc.

[0071] In some embodiments, in step (2), the molar equivalents of tert-butyloxycarbonyl (Boc) protected glutaric acid hydrazide and N-hydroxysuccinimide are 1.2, 1.3, 1.4 or 1.5, etc.; tert-butyloxycarbonyl protected glutaric acid hydrazide and N-hydroxysuccinimide are dissolved in dimethyl sulfoxide (DMSO) solvent at a molar equivalent of 1.2-1.5, the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide condensation agent is 1.3, 1.4 or 1.5 molar equivalents, the reaction temperature is 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, 160°C, 170°C or 180°C, etc., the reaction time is 5h, 6h, 7h, 8h, 9h or 10h, etc., which can achieve maximum yield of tert-butyloxycarbonyl protected glutaric acid hydrazide succinamide ester (Compound 2).

[0072] Specifically, in this embodiment, in step (3), 3-bromoquinoline propylamine (compound 1) and tert-butoxycarbonyl protected glutaric acid hydrazide succinamide ester (compound 2) are dissolved in 50 mL of dimethyl sulfoxide (DMSO) solvent at a molar equivalent of 1:1.5, 1 mL of triethanolamine (TEA) is added, and the reaction is stirred for 10 hours under 130° C. oil bath heating conditions. After the reaction is completed, the DMSO solvent is removed by rotary evaporation, and semi-preparative liquid phase separation and purification are performed. The mobile phases are water (containing 0.1% by volume of TFA) and acetonitrile (containing 0.1% by volume of TFA), respectively. A linear gradient is used: 5% of the aqueous phase increases to 50% of the aqueous phase, and the time is 40 minutes. The effluent from 27-29 minutes is collected and vacuum freeze-dried to obtain 1.48 g of a light yellow oily product tert-butoxycarbonyl protected BqaHz (compound 3), with a yield of 60.8%. 1 H NMR (400MHz, DMSO-d6, ppm) δ11.27(m,1H), 10.08(m,1H), 10.00(s,1H), 9.64(s,1H), 8.65(d,1H), 8.46(d,1H), 8.28(dd,1H) , 8.06(dd,1H), 7.72(s,1H), 5.20(t,2H), 3.03(t,2H), 2.36(tt,2H), 2.30(tt,2H), 2.19(t,2H), 2.04(tt,2H), 1.44(s,9H); 13 C NMR (400 MHz, DMSO-d6, ppm) δ 176.7, 172.5, 156.4, 151.2, 144.6, 138.5, 136.3, 130.4, 129.4, 127.3, 126.4, 118.5, 79.3, 56.8, 38.7, 37.2, 36.5, 28.5, 28.3, 22.5; HR-MS (C22H31Br2N4O4+): theoretical value: 573.0707, found value [M] +:573.0708.

[0073] In some embodiments, the solvent in step (3) is acetonitrile, dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF), etc.

[0074] In some embodiments, in step (3), the molar equivalents of 3-bromoquinoline propylamine and tert-butyloxycarbonyl protected glutaric acid hydrazide succinamide ester are 1.2, 1.3, 1.4 or 1.5, etc.; the amount of triethanolamine (TEA) is 1.1, 1.5 or 2 mL, the reaction temperature is 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, 160°C, 170°C or 180°C, etc., and the reaction time is 5h, 6h, 7h, 8h, 9h or 10h, etc., which can maximize the yield of tert-butyloxycarbonyl protected BqaHz (compound 3).

[0075] In this embodiment, in step (4), the tert-butyloxycarbonyl-protected BqaHz (compound 3) is completely dissolved in 20 mL of dimethyl sulfoxide (DMSO) solvent, 1 mL of trifluoroacetic acid (TFA) is added and stirred for 5 h. After the reaction is completed, the dimethyl sulfoxide (DMSO) solvent is removed by rotary evaporation, and semi-preparative liquid phase separation and purification are performed. The mobile phases are water (containing 0.1% by volume of TFA) and acetonitrile (containing 0.1% by volume of TFA), respectively. A linear gradient is used: 5% of the aqueous phase increases to 50% of the aqueous phase in 40 min. The effluent from 20-23 min is collected and vacuum-dried to obtain 0.94 g of a light yellow oily product BqaHz (compound 4) with a yield of 92.2%. 1 H NMR (400MHz, DMSO-d6, ppm) δ10.02(s,1H), 9.70(s,1H), 8.76(s,1H), 8.48(d,1H), 8.38(d,1H), 8.18(dd,1H), 8.02(dd, 1H), 8.00(m,2H,-NH2), 7.68(s,1H), 5.20(t,2H), 3.03(t,2H), 2.32(tt,2H), 2.28(tt,2H), 2.18(t,2H), 2.02(tt,2H); 13 CNMR (400MHz, DMSO-d6, ppm) δ 176.7, 172.6, 151.4, 144.6, 138.6, 136.1, 130.5, 129.4, 127.2, 126.4, 118.6, 56.8, 38.6, 37.2, 36.4, 28.5, 22.5,; HR-MS (C17H22BrN4O2+): theoretical value: 393.0921, measured value [M] + :393.0920. The chemical structure of BqaHz is:

[0076] In some embodiments, the solvent in step (4) is acetonitrile, dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF), etc.

[0077] In some embodiments, the amount of trifluoroacetic acid (TFA) used in step (4) is 1.1, 1.8 or 2 mL, which can maximize the yield of 3-bromoquinoline ester hydrazide BqaHz (compound 4).

[0078] Example 3 Optimization of derivatization reaction conditions

[0079] Since BqaHz was synthesized for the first time and used for derivatization of carbonyl compounds, in order to detect as many carbonyl compounds in liquor as possible, this example systematically optimized the reagent equivalent, reaction time, reaction temperature and acidity of the derivatization reaction, and investigated the reagent equivalent of 1, 1.5, 2, 5, 10, 20 eq, acidity of 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, reaction time of 5, 15, 30, 60, 90, 120 min, reaction temperature of 4, 20, 40, 50, 60, 80 ° C, and evaluated by comparing the peak areas of UPLC-MS detected by 7 standards after derivatization under different reaction conditions, the method is as follows:

[0080] Add 4 μL of dilute hydrochloric acid (10 mM), 20 μL of BqaHz solution to 20 μL of standard solution, and then add 156 μL of acetonitrile to 200 μL. Mix thoroughly and react at 60°C and 1000 rpm for 1 hour. Centrifuge and dry the reaction solution at 30°C and 1500 rpm. Redissolve with 200 μL of 20% acetonitrile solution (volume ratio) before analysis, centrifuge at 14000g, 4°C for 10 minutes, and place 180 μL of supernatant in a sample injection bottle for UPLC-MS analysis.

[0081] The results are as follows Figure 2 As shown, an excess of derivatization reagent makes the reaction more complete, and the presence of hydrogen ions is conducive to the activation of the carbonyl functional group, that is, the nucleophilic reaction, but too many hydrogen ions will weaken the reactivity of the nucleophilic reagent. The reaction temperature and reaction time are 60°C and 1h respectively, which can make the reaction proceed fully. Therefore, the optimal reaction conditions finally determined are: reagent equivalent 20eq, acidity 0.1%, reaction time 1h, reaction temperature 60°C.

[0082] Example 4 Exploring the Characteristics of Carbonyl Compound Derivatization Mass Spectrometry

[0083] To summarize the MS and MS / MS spectrometric characteristics of carbonyl compounds after derivatization, a mixed standard consisting of at least 80 different carbonyl compound standards (mixed by equal volumes of more than 80 single standard stock solutions) was derivatized and then non-targetedly analyzed by ultra-performance liquid chromatography-high-resolution mass spectrometry.

[0084] In this embodiment, the specific method for exploring the characteristic law of mass spectrometry of carbonyl compound derivatization is as follows: 100 carbonyl compound standards are prepared into a mixed standard, and the derivatization reagent BqaHz prepared in Example 2 is added to perform derivatization according to the optimal reaction conditions preferably obtained in Example 3. The 100 carbonyl compound standards are shown in the following table:

[0085] Standard product information table for establishing QSRR model

[0086]

[0087]

[0088]

[0089] In other embodiments, the number of carbonyl compound standards is 90, 110, 120, or other numbers greater than 80, and the specific derivatization reaction conditions are as follows: the amount of the derivatization reagent BqaHz is 20 equivalents (eq), the acidity is 0.1%, the reaction time is 1 h, and the reaction temperature is 60°C.

[0090] After derivatization, the mixed standard was analyzed non-targeted by ultra-high performance liquid chromatography-high resolution mass spectrometry, wherein the ultra-high performance liquid chromatography conditions were: phase A and phase B were 0.1% formic acid / water (volume ratio) and 0.1% formic acid / acetonitrile (volume ratio), respectively. The flow rate was 0.30 mL / min, the chromatographic column was Waters ACQUITY UPLC@BEH C18 (2.1 mm×100 mm, 1.7 μm), the column temperature was 30°C, the sample injection chamber temperature was set to 6°C, and the sample injection volume was 3 μL. The elution gradient started with 5% B, maintained for 5 min, then linearly increased to 18% B within 8 min, linearly increased to 95% B within 2 min, and maintained for 3 min, then linearly returned to the initial ratio within 0.1 min, and maintained for 1.9 min.

[0091] The mass spectrometry conditions are:

[0092] The scanning mode was full scan plus secondary mass spectrometry acquisition mode in positive ion mode (full MS / dd-MS 2). The spray voltage was 3.5 kV, the temperature of the ion transfer tube was 300 °C, and the temperature of the auxiliary gas heater was 350 °C. The sheath gas and auxiliary gas flow rates were 45 and 10 (in arbiRtary units), respectively. The S-lens was set to 50.0 (in arbiRtary units). In the full scan mass spectrometry setting, the scan range was m / z 300-1200, and the resolution was 60000. The maximum injection time (maximum IT) and automatic gain control target (AGC target) were set to 50 ms and 1×10 6 In the secondary mass spectrometry setting, the scan range was m / z 50-1500, and the resolution was 30000. The maximum injection time (maximum IT) and automatic gain control target (AGC target) were 50 ms and 1×10 5 Ion capacity. The collision energy was a mixture of 15%, 30% and 45% normalized energies. Secondary acquisition was triggered by the top 10 ions with the highest response intensity in each scan cycle.

[0093] Summarize the UPLC-MS spectrum and find that ( Figure 3 ), the mass spectrometry characteristics of carbonyl compounds after derivatization are as follows: In the MS spectrum, the [M] of all single-derivatized carbonyl compound standards + and [M+2] + The peak area ratio is 0.95-1.05. For the MS / MS spectrum, all standards contain three characteristic fragments. Fragment 1 is the characteristic fragment of BqaHz, with a mass-to-charge ratio of m / z of 154.0863±10ppm. Fragment 2 is the unfragmented parent ion, and the difference between its mass-to-charge ratio m / z and the parent ion is 0±10ppm. Fragment 3 is the fragment after the parent ion is debrominated and lost, and the difference between its mass-to-charge ratio m / z and fragment 2 is 206.9684±10ppm. Based on this, the compounds with the above characteristic spectra in the sample are marked as carbonyl compounds derived by BqaHz. The molecular weight of the carbonyl compound can be calculated according to fragment 3, M=m / z(fragment 3)-m / z(fragment 1)+m / z(H2O)-m / z(N2H4).

[0094] Example 5 Construction of a database of liquor carbonyl compounds

[0095] The database includes CAS, CID, SMILES, InChI, Mass and other information of carbonyl compounds.

[0096] Specifically, in this embodiment, the compounds found in liquor were mined from open source literature by text mining method, the compounds reported in literature related to liquor (keywords: Baijiu, Chinese liquor) were mined by ChemDataExtRactor software, the literature abstracts were obtained from the Web of Science website, the full text of the literature and supplementary materials were obtained from the Pubmedcentral website, and after downloading the literature, the compound information was mined from the abstract, the main text and the supplementary materials respectively to obtain the literature library;

[0097] An extended compound library was established by predicting transformation reactions, and then the transformation reactions were predicted using BiotRansformer3.0 software to obtain the extended library. The reaction types included: Phase I (CYO450) TRansformation, EC-Based TRansformation, Phase ll TRansformation, Human Gut Microbial TRansformation, Environmental Microbial TRansformation, Human and Human Gut Microbial TRansformation (AllHuman), Human and Human Gut Microbial TRansformation (SuperBio), Custom Human Multi-Step TRansformation (MultiBio) and Abiotic TRansformation (AbioticBio).

[0098] The basic information of the compounds was supplemented through open source websites, and the basic information of the compounds was obtained from the Pubchem website, including CAS number, CID, SMILES, InChI, Mass, etc. The carbonyl compounds in the database were screened out separately, and the derivatized SMILES were obtained and organized in an excel file, and finally a self-built carbonyl compound database containing 737 reported compounds and 3451 predicted compounds was constructed.

[0099] Example 6 Retention time prediction

[0100] The retention time tR and SMILES of the derivatized standards collected in Example 4 were collected and imported into the QSRR_Automator software. The contribution of 300-400 molecular descriptors to tR was calculated by the machine learning algorithm of the software. Finally, a quantitative structure chromatography retention correlation model was established with an average absolute error of less than 30s and a linear correlation coefficient R greater than 0.99.

[0101] Retention time is an important parameter of compound annotation. In order to improve the accuracy and qualitative grade of carbonyl compound annotation, the present invention establishes QSRR model to predict the retention time of carbonyl compounds in self-built library. Specifically, in the present embodiment, the real tR of 100 standard products used in the summary of carbonyl compound derivatization mass spectrum characteristic regularity in the above-mentioned embodiment 4 is sorted out, and then the SMILES and tR of 100 standard products are imported into QSRR_Automator software, and the software converts SMILES into 380 molecular descriptors under default parameters, and then the software uses linear regression, support vector machine regression and random forest regression algorithm to fit the relationship between molecular descriptors and real tR, and then 5 internal cross validations are carried out, and finally the QSRR model is established. The mean absolute error of the model is 6s, and the linear correlation coefficient R is greater than 0.999. It is found that the model performs well in combination with the final score and the mean absolute error, and the tR of 4188 carbonyl compounds in the self-built library is predicted using the model.

[0102] Example 7

[0103] A high coverage analysis method for carbonyl compounds in liquor comprises the following steps:

[0104] Step 1, data collection of carbonyl compounds in liquor samples: after the liquor samples are concentrated and pretreated, they are derivatized to obtain liquor samples to be tested, and then non-targeted mass spectrometry data of the liquor samples to be tested are obtained;

[0105] Step 2, screening and annotation of carbonyl compounds: pre-process the non-targeted mass spectrometry data of the liquor sample to be tested, screen out carbonyl compounds that meet specific characteristics, compare with the database of liquor carbonyl compounds, predict retention time tR, annotate the screened carbonyl compounds according to retention time tR, m / z, MS / MS similarity, and molecular similarity, and define corresponding qualitative levels according to different annotation scores.

[0106] In this embodiment, the method for collecting data on carbonyl compounds in liquor samples in step 1 comprises the following steps:

[0107] (1.1) Pretreatment: Concentrate the liquor sample and blank sample by 20-25 times to obtain the liquor sample to be tested and the blank sample;

[0108] (1.2) Derivatization: adding the derivatization reagent BqaHz prepared in Example 2 to derivatize the liquor sample to be tested and the blank sample respectively;

[0109] (1.3) Obtaining non-targeted mass spectrometry data of the liquor sample to be tested: Using ultra-high performance liquid chromatography-high resolution mass spectrometry to detect the derivatized liquor sample to be tested, and obtaining non-targeted mass spectrometry data of the liquor sample to be tested.

[0110] In some embodiments, in step (1.1), the specific method for concentrating the liquor sample and the blank sample is rotary evaporation, and the rotation speed of the rotary evaporation is 100-200rpm / min, such as 120rpm / min, 130rpm / min, 140rpm / min, 150rpm / min, 160rpm / min, 170rpm / min, 180rpm / min, 190rpm / min, 200rpm / min, etc. The temperature of the rotary evaporation is 40-60°C, such as 40°C, 45°C, 50°C, 55°C, 60°C, etc. The pressure of the rotary evaporation is -0.1±0.01MPa, such as -0.11MPa, -0.1MPa, -0.09MPa, etc. The concentration multiple is 20-25 times, such as 20 times, 21 times, 23 times, 24 times, 25 times, etc. Too small a concentration multiple will result in low derivatization efficiency and reduced product detection limit.

[0111] In this embodiment, in step (1.1), the samples of the six kinds of sauce-flavor liquors SFB1-SFB6 are all from Guizhou Xijiu Co., Ltd., China, and the blank sample is composed of 53% ethanol water (volume ratio). The specific method of concentrating the liquor samples and the blank sample is: 50 mL of the six kinds of sauce-flavor liquor samples SFB1-SFB6 and the blank sample are rotary evaporated at 200 rpm / min, 60°C, -0.1 MPa and fixed to 2 mL.

[0112] In this embodiment, in step (1.2), the specific derivatization method is: add 4 μL of dilute hydrochloric acid (10 mM), 20 μL of BqaHz solution, and then add 156 μL of acetonitrile to 200 μL to 20 μL of concentrated wine sample or blank sample, mix thoroughly, react at 60°C and 1000 rpm for 1 h for derivatization, and centrifuge and dry the reaction solution at 30°C and 1500 rpm.

[0113] In this embodiment, in step (1.3), the reaction solution obtained in step (5.2) was re-dissolved with 200 μL of 20% acetonitrile solution (volume ratio) before analysis, and centrifuged at 14000g, 4°C for 10 min, and 180 μL of the supernatant was placed in an injection bottle for UPLC-MS analysis to obtain non-targeted mass spectrometry data of the liquor sample to be tested.

[0114] The data were collected using an analytical instrument combining an ACQUITY UPLC ultra-high performance liquid chromatography system (UPLC, Waters, Milford, MA, USA) and a Q Exactive HF high-resolution mass spectrometer (Thermo Fisher Scientific, Rockford, IL, USA).

[0115] The liquid chromatography conditions were as follows: phase A and phase B were 0.1% formic acid / water (volume ratio) and 0.1% formic acid / acetonitrile (volume ratio), respectively. The flow rate was 0.30 mL / min, the chromatographic column was Waters ACQUITY UPLC@BEH C18 (2.1 mm×100 mm, 1.7 μm), the column temperature was 30°C, the sample injection chamber temperature was set to 6°C, and the sample injection volume was 3 μL. The elution gradient started with 5% B, maintained for 5 min, then linearly increased to 18% B within 8 min, linearly increased to 95% B within 2 min, and maintained for 3 min, then linearly returned to the initial ratio within 0.1 min, and maintained for 1.9 min.

[0116] The mass spectrometry analysis conditions were: full scan in positive ion mode plus secondary mass spectrometry acquisition mode (fullMS / dd-MS 2 ). The spray voltage was 3.5 kV, the temperature of the ion transfer tube was 300 °C, and the temperature of the auxiliary gas heater was 350 °C. The sheath gas and auxiliary gas flow rates were 45 and 10 (in arbiRtary units), respectively. The S-lens was set to 50.0 (in arbiRtary units). In the full scan mass spectrometry setting, the scan range was m / z 300-1200, and the resolution was 60000. The maximum injection time (maximum IT) and automatic gain control target (AGC target) were set to 50 ms and 1×10 6 In the secondary mass spectrometry setting, the scan range was m / z 50-1500, and the resolution was 30000. The maximum injection time (maximum IT) and automatic gain control target (AGC target) were 50 ms and 1×10 5 Ion capacity. The collision energy was a mixture of 15%, 30% and 45% normalized energies. Secondary acquisition was triggered by the top 10 ions with the highest response intensity in each scan cycle.

[0117] In this embodiment, the method of step 2, screening and annotation of carbonyl compounds is as follows:

[0118] Screening of carbonyl compounds: The non-targeted mass spectrometry data of the liquor sample to be tested obtained in step (1.3) was converted into mgf files using MSconvert software, and the peak area was extracted from the raw mass spectrometry data using MS-DIAL (v 5.1) software, with the following parameters: MS1 deviation, 0.005Da; MS / MS deviation, 0.01Da; retention time deviation, 0.2min, minimum peak width, 3Scan; minimum peak height, 1000; other parameters were the default parameters. Compounds whose areas in the blank sample were more than 5 times the peak areas of the liquor sample to be tested were removed to obtain a compound list of the liquor sample to be tested (the list included tR, m / z, MS / MS, etc. of the compounds). Python code was then used to screen out the carbonyl compounds corresponding to the mass spectrometry features summarized in Example 4 in the obtained compound list, i.e., potential carbonyl compounds, for structural annotation.

[0119] Notes on carbonyl compounds:

[0120] The molecular network SGMNS (StRucture-Guided Molecular Network) software was used to annotate the carbonyl compounds in the measured samples, and a background molecular network was established based on the standard. A comparison search was performed with the database of white wine carbonyl compounds established in Example 5. The retention time tR of the white wine sample to be tested was predicted using the method in Example 6. The screened carbonyl compounds were annotated according to the retention time tR, m / z, MS / MS similarity, and molecular similarity, and different annotation scores were defined as corresponding qualitative grades.

[0121] The principle is as follows: first, you need to prepare the seed table file, the peak table file to be annotated, the secondary mass spectrum mgf file to be annotated, and the database table file. The seed table file includes the real tR, m / z, and derivatized SMILES of 100 carbonyl compound standards, the peak table file to be annotated contains the experimental tR and m / z of the screened carbonyl compounds, the secondary mass spectrum file to be annotated includes the MS / MS spectrum information of the screened carbonyl compounds and 100 standards, and the database file includes the tR, m / z, and derivatized SMILES of 4188 carbonyl compounds and 100 standards.

[0122] Then, SGMNS first calculated the similarity of SMILES molecular descriptors of 4188 carbonyl compounds in the database and 100 standards according to the algorithm to establish a molecular network. Subsequently, the ions in the peak table to be annotated were matched to the network according to tR and m / z. The software assigned the MS / MS spectrum of the seed to the compound in the database of that m / z and calculated the similarity between the MS / MS spectrum and the experimental MS / MS spectrum. Finally, the software calculated the weighted score score according to tR, m / z, molecular similarity sim1, and MS / MS similarity sim2. The parameters used in this embodiment are as follows: tR deviation is less than 30%, m / z deviation is less than 10ppm, sim1 is greater than 0.6, and sim2 is greater than 0.5.

[0123] Qualitative grades of carbonyl compounds are as follows Figure 4 As shown, the molecular formula, i.e., the elemental composition (C, H, O, N, S), was first calculated according to the molecular weight using Xcalibur (v 2.1) software. The ions for which the theoretical molecular formula was not calculated were defined as level 4. The ions for which the theoretical molecular formula was calculated were matched to the database, with ΔM less than 10 ppm, and the qualitative level was defined as level 3. For the ions with molecular formulas, candidate compounds with a score greater than 0.8 were screened according to SMGNS, and the qualitative level was defined as level 2. For ions with candidate structures, the tR of the standard was collected by derivatization method for comparison, and compounds with a tR deviation less than 0.3 min were screened, and the qualitative level was defined as level 1.

[0124] Take 3-phenylpropanaldehyde as an example to explain the specific annotation process, such as Figure 5 As shown:

[0125] The tR of the screening ion is 13.03min and the m / z is 509.1550. The molecular weight is first calculated to be 134.0735, and then the molecular formula is calculated to be C9H 10 O, matched 9 candidate structures in the database, and annotated using SMGNS. The score of 3-phenylpropanaldehyde was 0.91, and the predicted tR was closest to the true tR. The mass spectrum information of 3-phenylpropanaldehyde was collected and compared with the experimental information. Finally, the screened ion was annotated as 3-phenylpropanaldehyde, and the annotation level was level 1.

[0126] Using the above screening and annotation process, the present invention screened out 773, 803, 919, 937, 960 and 951 carbonyl compounds in the six Maotai-flavor liquors SFB1-SFB6, respectively, as shown in the following table:

[0127] Table of levels of detected carbonyl compounds

[0128]

[0129]

[0130] After removing the features of repeated detection, the number of non-redundant carbonyl compounds was 1080, achieving high coverage detection of carbonyl compounds in Maotai-flavor liquor, among which the numbers of annotation levels 1, 2, 3 and 4 were 35, 108, 673 and 265, respectively. Level 4 means knowing the tR and molecular weight of the ions in the sample, Level 3 means knowing the elemental (C, H, O, N, S) composition of the compounds in the sample, and the m / z search of the candidate structure has the corresponding candidate structure in the database with an error of 10-20ppm; the absolute deviation between the predicted retention time of the candidate structure and the actual retention time in the sample is greater than 60s, Level 2 means knowing the elemental composition of the compounds in the sample, and the m / z search of the candidate structure has the corresponding candidate structure in the database with a difference of 10-20ppm; the absolute deviation between the predicted tR of the candidate structure and the actual tR in the sample is less than 60s, and the SGMNS score is greater than 0.7; Level 1 means knowing the elemental composition of the compounds in the sample, and the m / z search of the candidate structure has the corresponding candidate structure in the database with a difference of 10-20ppm; the absolute deviation between the predicted tR of the candidate structure and the actual tR in the sample is less than 60s, and the SGMNS score is greater than 0.7; the absolute deviation between the predicted tR of the candidate structure and the actual tR of the standard is less than 30s. The qualitative grades of the reported flavor substances, such as furfural, 5-methyl-2-furfural, benzaldehyde, etc., were all grade 1.

[0131] like Figure 6 As shown, based on the specific content of the background network at qualitative level 1 and level 2, each compound is connected to the seed compound with the highest molecular similarity, and the MS / MS spectrum similarity is calculated to obtain the final weighted score.

[0132] The present invention establishes a new comprehensive analysis method for carbonyl compounds in Maotai-flavor liquor, synthesizes a new derivatization reagent 3-bromoquinoline ester hydrazide BqaHz based on natural bromine isotopes, derivatizes carbonyl compounds in samples, uses ultra-high performance liquid chromatography-high resolution mass spectrometry platform to detect derivatized carbonyl compounds, screens potential carbonyl compounds based on mass spectrometry characteristics of derivatized products, and annotates derivatized carbonyl compounds in combination with primary m / z, tR, MS / MS spectra and molecular similarity. The derivatization reagent not only improves the chromatographic retention coefficient of carbonyl compounds, but also makes carbonyl compounds identifiable in mass spectrometry MS and MS / MS. The present invention has significant advantages in detecting high-boiling point and trace carbonyl compounds in Maotai-flavor liquor, and has the characteristics of high coverage, high sensitivity, simplicity and rapidity.

[0133] Traditional spectral search methods are used to annotate derivative products and face the following problems:

[0134] First, there are not enough public standard MS / MS spectra, and the derivatization groups affect the fragmentation behavior of molecules, which reduces the accuracy of MS / MS spectra predicted by software; second, establish a personalized derivatization database, such as the database MyCompundID established by Li Liang et al., which contains more than 400 standardized compounds, but this method is time-consuming and labor-intensive, and only targets specific derivatization reagents. Xie Xiaoyu et al. established a derivatization method to detect carbonyl compounds in Baijiu Daqu, and a total of 487 carbonyl compounds were detected. When using the traditional spectrum search method, the molecular information of 4 public compound libraries was first obtained, and then the MS / MS spectrum prediction was performed using open source software to calculate the similarity with the experimental spectrum. The final number of qualitative level 1 and level 2 was 48.

[0135] Compared with the study, the present invention has significant improvements in derivatization efficiency and qualitative accuracy. First, the derivatization reagent developed by the present invention detected 1081 carbonyl compounds in Maotai-flavor liquor, which is more than twice the number. In addition, the molecular network SGMNS used in the present invention does not need to predict MS / MS spectra, but assigns MS / MS spectra of compounds in the database based on molecular similarity, which reduces false positive identification. The total number of qualitative level 1 and level 2 in the present invention is 143, which is more than 3 times the number, and the identification accuracy is significantly improved. In summary, the present invention has high performance for high coverage analysis of carbonyl compounds and can be extended to the characterization of other specific functional group compounds.

[0136] It is to be understood that the present invention is described by some embodiments, and it is known to those skilled in the art that various changes or equivalent substitutions may be made to these features and embodiments without departing from the spirit and scope of the present invention. In addition, under the teachings of the present invention, these features and embodiments may be modified to adapt to specific circumstances and materials without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the scope of protection of the present invention.

Claims

1. A high coverage analysis method for carbonyl compounds in liquor, characterized in that: The following steps are involved: Step 1, data collection of carbonyl compounds in liquor samples: after the liquor samples are concentrated and pretreated, a derivatization reagent is added to obtain a liquor sample to be tested, and then non-targeted mass spectrometry data of the liquor sample to be tested is obtained; the derivatization reagent is 3-bromoquinoline ester hydrazide; Step 2: Screening and annotation of carbonyl compounds: Preprocess the non-targeted mass spectrometry data of the liquor sample to be tested, screen out carbonyl compounds that meet specific characteristics, compare them with the database of liquor carbonyl compounds, and predict the retention time t R , according to the retention time t R , m / z, MS / MS similarity, and molecular similarity were used to annotate the screened carbonyl compounds, and the corresponding qualitative levels were defined according to different annotation scores.

2. The analysis method according to claim 1, characterized in that The method for collecting data on carbonyl compounds in liquor samples in step 1 comprises the following steps: (1.1) Pretreatment: Concentrate the liquor sample and blank sample by 20-25 times to obtain the liquor sample to be tested and the blank sample; (1.2) Derivatization: Add the derivatization reagent 3-bromoquinoline ester hydrazide to derivatize the liquor sample to be tested and the blank sample respectively; (1.3) Obtaining non-targeted mass spectrometry data of the liquor sample to be tested: using ultra-high performance liquid chromatography-high resolution mass spectrometry to detect the derivatized liquor sample to be tested, and obtaining non-targeted mass spectrometry data of the liquor sample to be tested; Preferably, the blank sample consists of 53% by volume of ethanol water; Preferably, the concentration method is rotary evaporation, the rotation speed of the rotary evaporation is 100-200 rpm / min; the temperature of the rotary evaporation is 40-60° C.; the pressure of the rotary evaporation is -0.1±0.01 MPa; Preferably, the rotation speed of the rotary evaporation is 150-180 rpm / min; the temperature of the rotary evaporation is 45-55° C.; the pressure of the rotary evaporation is -0.1-0.09 MPa; the liquor sample and the blank sample are concentrated 22-23 times; Preferably, the rotation speed of the rotary evaporation is 200 rpm / min; the temperature of the rotary evaporation is 60° C.; the pressure of the rotary evaporation is -0.1 MPa, and the liquor sample and the blank sample are concentrated 25 times.

3. The analysis method according to claim 1, characterized in that The derivatization reagent has an equivalent of 1-20 eq, an acidity of 0.1%-5%, a reaction temperature of 4-80°C, and a reaction time of 5-120 min; Preferably, the derivatization reagent has an equivalent of 10-20 eq, an acidity of 1%-4%, a reaction temperature of 4-80°C, and a reaction time of 20-100 min; Preferably, the derivatization method is: after adding an acid-base reagent to the concentrated liquor sample to adjust the acidity to 0.1%-5%, add 1-20eq 3-bromoquinoline ester hydrazide solution, make up to volume, mix thoroughly, and react at 4-80°C for 5-120min.

4. The analysis method according to claim 1, characterized in that In step 2, the data preprocessing method is: peak alignment and peak extraction are performed on the non-targeted mass spectrometry data of the liquor sample to be tested obtained in step 1, and compounds whose areas in the blank sample are greater than 5 times the peak areas of the liquor sample to be tested are removed; Preferably, the parameters of the peak extraction are as follows: MS1 deviation, 0.005Da; MS / MS deviation, 0.01Da; retention time deviation, 0.2min, minimum peak width, 3Scan; minimum peak height, 1000; other parameters use default parameters; Preferably, the annotation method is: using molecular network SGMNS software to annotate carbonyl compounds in the measured sample; Preferably, the specific feature of step 2 is that in the MS spectrum, the [M] of the derivatized carbonyl compound + and [M+2] + The peak area ratio is 0.95-1.

05. In the MS / MS spectrum, the mass-to-charge ratio m / z of characteristic fragment 1 is 154.0863±10ppm, the mass-to-charge ratio m / z of characteristic fragment 2 differs from that of the parent ion by 0±10ppm, and characteristic fragment 3 is the fragment lost when characteristic fragment 2 is stripped of bromoquinoline, and the mass-to-charge ratio m / z difference between it and characteristic fragment 2 is 206.9684±10ppm.

5. The analysis method according to claim 1, characterized in that The data information in the database of white wine carbonyl compounds includes the CAS number, CID, SMILES, InChI, and Mass information of the carbonyl compounds.

6. The analysis method according to claim 1, characterized in that The retention time tR is predicted by: establishing a QSRR model to predict the retention time of carbonyl compounds in a self-built library; Preferably, the retention time tR is predicted by: arranging the real tR of the standard, then importing the SMILES and tR of the standard into the QSRR_Automator software, converting the SMILES into molecular descriptors, and then fitting the relationship between the molecular descriptors and the real tR by the software to establish a QSRR model; Preferably, the mean absolute error of the QSRR model is 6s, and the linear correlation coefficient R is greater than 0.

999.

7. The analysis method according to any one of claims 1 to 6, characterized in that The structural formula of the derivatization reagent 3-bromoquinoline ester hydrazide is:

8. The analysis method according to any one of claims 1 to 6, characterized in that: The preparation method of the derivatization reagent 3-bromoquinoline ester hydrazide is: Dissolve tert-butyloxycarbonyl protected 3-bromoquinoline ester hydrazide in an organic solvent, add 1-2 mL of trifluoroacetic acid to react, remove the organic solvent after the reaction is completed, separate and purify to obtain 3-bromoquinoline ester hydrazide; Preferably, the preparation method of the tert-butyloxycarbonyl protected 3-bromoquinoline ester hydrazide is: Dissolve 3-bromoquinoline propylamine and tert-butoxycarbonyl protected glutaric acid hydrazide succinamide ester in an organic solvent at a molar equivalent of 1.2-1.5, add 1-2 mL of triethanolamine, react at 100-180° C. for 5-10 hours, remove the organic solvent after the reaction, separate and purify to obtain tert-butoxycarbonyl protected 3-bromoquinoline ester hydrazide; Preferably, the synthesis method of the tert-butyloxycarbonyl protected glutaric acid hydrazide succinamide ester is: The tert-butoxycarbonyl protected glutaric acid hydrazide and N-hydroxysuccinimide are dissolved in an organic solvent in a molar equivalent of 1.2-1.5, 1.2-1.5 molar equivalent of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is used as a condensation agent, and the reaction is carried out at 100-180° C. for 5-10 hours. After the reaction is completed, the organic solvent is removed, and the mixture is separated and purified to obtain tert-butoxycarbonyl protected glutaric acid hydrazide succinamide ester; Preferably, the synthesis method of the 3-bromoquinoline propylamine is: 3-bromoquinoline is dissolved in an organic solvent, and then 1-1.5 molar equivalents of 1-bromopropylamine are added, and the mixture is reacted at 100-180° C. for 5-10 hours. After the reaction is completed, the solvent is removed, and the mixture is separated and purified to obtain 3-bromoquinoline propylamine.

9. The analysis method according to claim 1, characterized in that The qualitative grades of the carbonyl compounds are as follows: Level 4: Know the tR and molecular weight of the ions in the sample; Level 3: Knowing the elemental C, H, O, N, S composition of the compound in the sample, the m / z search of the candidate structure has a corresponding candidate structure in the database with an error of 10-20ppm; The absolute deviation between the predicted retention time tR of the candidate structure and the actual retention time tR in the sample is greater than 60s; Level 2: Knowing the elemental composition of the compound in the sample, the m / z search of the candidate structure has a corresponding candidate structure in the database with a difference of 10-20ppm; The absolute deviation between the predicted retention time tR of the candidate structure and the actual retention time tR in the sample is less than 60s, and the SGMNS score is greater than 0.7; Level 1: Knowing the elemental composition of the compound in the sample, the m / z search of the candidate structure has a corresponding candidate structure in the database with a difference of 10-20ppm; The absolute deviation between the predicted retention time tR of the candidate structure and the actual retention time tR in the sample is less than 60s, and the SGMNS score is greater than 0.7; the absolute deviation between the predicted retention time tR of the candidate structure and the actual retention time tR of the standard is less than 30s.

10. Use of the analytical method according to any one of claims 1 to 9 in detecting carbonyl compounds in liquor.

Images