Method for analyzing and detecting hydroxyl and amino substances in Baijiu and application

Abstract

The invention discloses a method for analyzing and detecting hydroxyl and amino substances in Baijiu and application, and relates to the technical field of Baijiu detection and analysis. The method comprises the following steps: carrying out derivatization reaction on bromine nicotinoyl chloride and hydroxyl and amino compounds in a white spirit sample to obtain a derivatized white spirit sample, carrying out data acquisition on the derivatized white spirit sample by utilizing a UPLC-HRMS platform, screening derivatized products by virtue of bromine isotope mass spectrum peaks, and determining the content of the bromine isotope mass spectrum peaks. And finally, performing high-coverage annotation on hydroxyl and amino compounds in the white spirit by combining multi-dimensional information including retention time, a primary mass spectrum and a secondary mass spectrum. The derivatization reaction does not need heating, the reaction time is short, a rapid and mild derivatization method is established, the loss of volatile compounds is avoided, and the analysis efficiency is improved; according to the invention, the natural isotope characteristics of bromine atoms are utilized to efficiently screen labeled compounds, and other heavy isotope labeling reagents are prevented from being additionally synthesized and used.

Description

Technical Field

[0001] The present application relates to the technical field of liquor detection and analysis, and specifically to a method for analyzing and detecting hydroxyl and amino substances in liquor and its application. Background Art

[0002] Hydroxyl and amino compounds significantly influence the flavor and quality of baijiu (Chinese liquor). High-coverage analysis and detection of these compounds in baijiu provides theoretical support for quality control during production. Liquid chromatography-mass spectrometry (LC-MS) has been increasingly used in baijiu analysis due to its stability and high coverage. However, the high polarity and low ionization efficiency of some hydroxyl and amino compounds reduce the sensitivity of LC-MS analysis.

[0003] Chemical derivatization, a common pretreatment method for LC-MS, can effectively improve overall analytical performance. Although a variety of derivatization methods have been developed for hydroxyl and amino compounds, most require high temperatures or long reaction times, potentially resulting in the loss of some volatile hydroxyl and amino groups. This also reduces analytical efficiency and limits their widespread application in practical applications. Therefore, developing a derivatization method with milder reaction conditions and faster reaction times is of great significance. Summary of the Invention

[0004] In order to solve at least one of the problems mentioned in the above background technology, the present application provides a method and application for the analysis and detection of hydroxyl and amino substances in white wine, wherein bromonicotinoyl chloride is used to undergo a derivatization reaction with hydroxyl and amino compounds in white wine. The derivatization reaction does not require heating and has a short reaction time, thereby establishing a rapid and mild derivatization method, avoiding the loss of volatile compounds and improving analysis efficiency. The UPLC-HRMS platform is then used to complete data acquisition, and the derivatization products are screened by bromine isotope mass spectrometry peak pairs without the need for heavy isotope labeling reagents. Finally, multidimensional information including retention time, primary mass spectra, and secondary mass spectra are combined to perform high-coverage annotation of hydroxyl and amino compounds in white wine.

[0005] The specific technical solutions provided in the embodiments of this application are as follows:

[0006] In a first aspect, a method for analyzing and detecting hydroxyl and amino substances in liquor is provided, comprising the following steps:

[0007] Step 1: Pretreating a liquor sample to obtain a liquor extract, adding a derivatization reagent and a catalyst to the liquor extract, causing the derivatization reagent to react with hydroxyl compounds and amino compounds in the liquor extract to obtain a derivatized liquor extract; wherein the derivatization reagent is bromonicotinoyl chloride;

[0008] Step 2: obtaining non-targeted mass spectrometry data of the derivatized liquor extract, and screening to obtain a list of unknown compounds corresponding to the hydroxyl compounds and amino compounds to be annotated and qualitatively identified in the derivatized liquor extract based on the non-targeted mass spectrometry data;

[0009] Step 3: Based on the list of unknown compounds, a search is performed from a preset database to obtain a list of candidate compounds for the hydroxyl compounds and amino compounds to be annotated and qualitatively characterized; and based on the non-targeted mass spectrometry data, the list of candidate compounds, and a preset quantitative structure-chromatographic retention relationship model, the hydroxyl compounds and amino compounds to be annotated and qualitatively characterized are annotated and qualitatively characterized.

[0010] In a specific embodiment, obtaining the unknown compound list in step 2 comprises the following steps:

[0011] The non-targeted mass spectrometry data are subjected to peak alignment, peak matching, and blank subtraction to obtain an original compound list; wherein the non-targeted mass spectrometry data include: precursor ion mass-to-charge ratio m / z, primary mass spectrum MS1, secondary mass spectrum MS2, and retention time t R ;

[0012] Based on the original compound list, precursor ions containing characteristic mass spectrum difference peak pairs of bromine atom isotopes are extracted to obtain a list of unknown compounds corresponding to the hydroxyl compounds and amino compounds to be annotated and qualitatively identified in the derivatized liquor extract.

[0013] In a specific embodiment, the quantitative structure-chromatographic retention relationship model in step 3 is configured to input the SMILES molecular structure of the compound and output the predicted retention time of the compound after derivatization with bromonicotinoyl chloride.

[0014] In a specific embodiment, the step three further includes the following steps:

[0015] Determining the actual retention time of the unknown compound to be annotated and the corresponding primary mass spectrum MS1 based on the unknown compound list and the non-targeted mass spectrometry data, and searching the database based on the actual retention time of the unknown compound and the corresponding primary mass spectrum MS1 to obtain multiple candidate compounds corresponding to the unknown compound;

[0016] comparing the secondary mass spectra MS2 of the plurality of candidate compounds with the secondary mass spectra MS2 of the unknown compound, and comparing the predicted retention time values ​​of the plurality of candidate compounds after derivatization with bromonicotinoyl chloride with the actual retention time of the unknown compound to obtain a comparison result;

[0017] The comparison result is matched with the annotation qualitative level to obtain a matching result, and the unknown compound is annotated and qualitatively evaluated based on the matching result.

[0018] In a specific embodiment, the annotation qualitative level includes:

[0019] Level 1: The actual retention time, primary mass spectrum MS1, and secondary mass spectrum MS2 of the unknown compound to be annotated and qualitatively identified match those of the corresponding standard;

[0020] Level 2: The actual retention time, primary mass spectrum MS1, and secondary mass spectrum MS2 of the unknown compound to be annotated and qualitatively identified match those of the corresponding compound in the database;

[0021] Level 3: The unknown compound to be annotated and qualitatively identified only has actual retention time and primary mass spectrum MS1 matching the corresponding compound in the database;

[0022] Level 4: The unknown compound to be annotated and qualitatively identified has only a primary mass spectrum MS1 that matches the corresponding compound in the database;

[0023] Level 5: The actual retention time, primary mass spectrum MS1 and secondary mass spectrum MS2 of the unknown compound to be annotated and qualitatively identified do not match those of the corresponding compound in the database, and only the molecular formula of the unknown compound to be annotated and qualitatively identified can be determined.

[0024] In a specific embodiment, the step 1 of adding a derivatization agent and a catalyst to the liquor extract comprises the following steps:

[0025] Add 5 to 15 μL of a derivatization reagent, bromonicotinoyl chloride acetonitrile solution, and 5 to 15 μL of a catalyst, 4-dimethylaminopyridine acetonitrile solution, to the liquor extract, and vortex for 20 to 40 seconds at room temperature to perform a derivatization reaction;

[0026] The concentration of the bromonicotinoyl chloride acetonitrile solution is 5 to 15 mg / mL, and the concentration of the 4-dimethylaminopyridine acetonitrile solution is 5 to 15 mg / mL.

[0027] In a specific embodiment, the step 1 of pre-treating the liquor sample to obtain the liquor extract comprises the following steps:

[0028] Concentrating the liquor sample to obtain a concentrated liquor sample;

[0029] The concentrated liquor sample is subjected to extraction, centrifugation, and freeze-drying operations in sequence to obtain a freeze-dried liquor extract;

[0030] 50 to 150 μL of ultra-dry acetonitrile is added to the freeze-dried liquor extract to dissolve it, thereby obtaining the liquor extract.

[0031] In a specific embodiment, the concentrated liquor sample is sequentially subjected to extraction, centrifugation, and freeze-drying operations, comprising the following steps:

[0032] Taking 20-40 μL of the concentrated liquor sample, adding 5-10 μL of a mixed internal standard solution, 200-400 μL of dichloromethane, and 100-200 μL of water to the concentrated liquor sample in sequence, and extracting by shaking for 4-6 minutes at a speed of 1500-2000 rpm and a temperature of 4-10°C;

[0033] After the extraction is completed, centrifuging at a speed of 14,000 to 15,000 rpm and a temperature of 4 to 10° C. for 10 to 15 minutes to obtain an organic layer and an aqueous layer, taking out 200 to 250 μL of the organic layer and 100 to 150 μL of the aqueous layer, and vacuum freeze-drying the organic layer and the aqueous layer to obtain the freeze-dried liquor extract;

[0034] Wherein, the mixed internal standard solution includes one or more of phenol-d5, benzyl alcohol-d7, glycerol-d5, sorbitol-13C6, n-hexanol-d13, phenylalanine-d5, and leucine-13C;

[0035] The concentrations of phenol-d5, benzyl alcohol-d7, glycerol-d5, sorbitol-13C6, n-hexanol-d13, phenylalanine-d5, and leucine-13C are 0.5-1.5 μg / mL.

[0036] In a specific embodiment, the concentration treatment adopts rotary evaporation, and the conditions of the rotary evaporation are a rotation speed of 100-200 rpm / min, a temperature of 50-60° C., and a pressure of -0.1-0.2 MPa. The concentration multiple of the concentration treatment is 20-30 times.

[0037] In a second aspect, a method for analyzing and detecting hydroxyl and amino substances in liquor as described above is provided for use in detecting hydroxyl and amino substances in liquor.

[0038] Beneficial effects:

[0039] The present invention targets hydroxyl and amino compounds in liquor, and utilizes bromonicotinoyl chloride to undergo a derivatization reaction with the hydroxyl and amino compounds in liquor. This derivatization reaction does not require heating (it can react at room temperature) and has a short reaction time, establishing a rapid and mild derivatization method that avoids the loss of volatile compounds and improves analysis efficiency. The UPLC-HRMS platform is then used to complete data acquisition, and derivatized products are screened by bromine isotope mass spectrometry peak pairs, without the need for heavy isotope labeling reagents. Finally, multidimensional information including retention time, primary mass spectrometry, and secondary mass spectrometry is combined to perform high-coverage annotation of hydroxyl and amino compounds in liquor. The method of the present invention has the characteristics of high coverage, high sensitivity, convenience, and economy, and is suitable for the detection of trace hydroxyl and amino compounds in liquor.

[0040] The derivatization reagent used in the present invention, bromonicotinoyl chloride, can label hydroxyl and amino compounds without the need for heating and prolonged reaction, generating labeled products with characteristic bromine isotope mass spectrometry peak pairs. The invention also introduces a pyridine structure that is easily ionized to significantly improve the sensitivity of mass spectrometry detection. Furthermore, the natural isotopic properties of bromine atoms are utilized to efficiently screen labeled compounds, avoiding the need for additional synthesis and use of other heavy isotope labeling reagents. Furthermore, the invention combines internal liquor databases, open source databases, and information based on retention time, primary mass spectrometry (MS1), and secondary mass spectrometry (MS2) to grade and annotate labeled compounds, thereby reducing the false positive rate of annotations and improving the accuracy and reliability of annotations. The present invention overcomes the limitations of complex matrix interference and provides an economical, efficient, and convenient technical means for the comprehensive analysis of hydroxyl and amino compounds in complex systems such as liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0042] Figure 1 This is the result diagram of external validation of the quantitative structure-chromatographic retention relationship model;

[0043] Figure 2 This is the chromatographic mass spectrometric characteristic diagram of the compound after derivatization with bromonicotinoyl chloride;

[0044] Figure 3 It is an annotated qualitative flow chart of hydroxyl and amino compounds in liquor;

[0045] Figure 4 This is an annotated qualitative diagram using p-hydroxyphenylethanol as an example;

[0046] Figure 5 This is a chart showing the quantities of five levels of hydroxyl and amino compounds detected in liquor samples. DETAILED DESCRIPTION

[0047] To make the purpose, technical solutions, and advantages of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are intended only to describe specific embodiments and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0049] Example 1

[0050] This embodiment provides a method for analyzing and detecting hydroxyl and amino substances in liquor, comprising the following steps:

[0051] Step 1: Pretreating a liquor sample to obtain a liquor extract, adding a derivatization reagent and a catalyst to the liquor extract, causing the derivatization reagent to react with hydroxyl compounds and amino compounds in the liquor extract to obtain a derivatized liquor extract; wherein the derivatization reagent is bromonicotinoyl chloride;

[0052] Step 2: Obtain non-targeted mass spectrometry data of the derivatized liquor extract, and screen to obtain a list of unknown compounds corresponding to the hydroxyl compounds and amino compounds to be annotated and qualitatively identified in the derivatized liquor extract based on the non-targeted mass spectrometry data;

[0053] Step 3: Based on the list of unknown compounds, a search is performed from a preset database to obtain a list of candidate compounds for the hydroxyl compounds and amino compounds to be annotated and qualitatively analyzed. Based on the non-targeted mass spectrometry data, the candidate compound list, and the preset quantitative structure-chromatographic retention relationship model, the hydroxyl compounds and amino compounds to be annotated and qualitatively analyzed are annotated and qualitatively analyzed.

[0054] The following details steps one to three:

[0055] In step 1, the liquor sample is pre-treated to obtain a liquor extract, including steps S11 to S13:

[0056] Step S11: Concentrate the liquor sample to obtain a concentrated liquor sample.

[0057] The concentration treatment in step S11 adopts rotary evaporation, and the conditions of the rotary evaporation are a rotation speed of 100-200 rpm / min, a temperature of 50-60° C., and a pressure of -0.1-0.2 MPa. The concentration multiple of the concentration treatment is 20-30 times.

[0058] The main purpose of concentration in this example is to enrich the test substance. If the concentration factor is too small, such as less than 20 times, the ideal enrichment effect will not be achieved, and the sensitivity of the analysis will be reduced. If the concentration factor is too large, such as greater than 30 times, some volatile substances will be lost, reducing the coverage of the analysis.

[0059] Step S12: extracting, centrifuging, and freeze-drying the concentrated liquor sample in sequence to obtain a freeze-dried liquor extract.

[0060] Take 20-40 μL of the concentrated liquor sample, add 5-10 μL of a mixed internal standard solution, 200-400 μL of dichloromethane and 100-200 μL of water to the concentrated liquor sample in sequence, and shake for 4-6 minutes at a speed of 1500-2000 rpm and a temperature of 4-10°C for extraction; after the extraction is completed, centrifuge for 10-15 minutes at a speed of 14000-15000 rpm and a temperature of 4-10°C to obtain an organic layer and an aqueous layer, take out 200-250 μL of the organic layer and 100-150 μL of the aqueous layer, and vacuum freeze-dry the organic layer and the aqueous layer to obtain a freeze-dried liquor extract.

[0061] In step 2, after the concentrated liquor sample is extracted and centrifuged, the organic and aqueous layers are removed and freeze-dried in a vacuum. The purpose of vacuum freeze-drying is to remove water from the system, reducing any interference from water during the subsequent derivatization process and preventing hydrolysis of the derivatization reagents.

[0062] The mixed internal standard solution includes one or more of phenol-d5, benzyl alcohol-d7, glycerol-d5, sorbitol-13C6, n-hexanol-d13, phenylalanine-d5, and leucine-13C. The concentrations of phenol-d5, benzyl alcohol-d7, glycerol-d5, sorbitol-13C6, n-hexanol-d13, phenylalanine-d5, and leucine-13C are 0.5 to 1.5 μg / mL. The purpose of adding the mixed internal standard solution in this example is to investigate the stability of the UPLC-HRMS platform operation; the stability of the UPLC-HRMS platform is determined by the relative standard deviation of the peak area of ​​the internal standard compound over multiple runs.

[0063] Step S13: adding 50-150 μL of ultra-dry acetonitrile to the freeze-dried liquor extract to dissolve it, thereby obtaining a liquor extract.

[0064] In step 1, a derivatization reagent and a catalyst are added to the liquor extract, and the derivatization reagent reacts with the hydroxyl compound and the amino compound in the liquor extract to obtain a reactant. The reactant is freeze-dried and redissolved in sequence to obtain a derivatized liquor extract; wherein the derivatization reagent is bromonicotinoyl chloride and the catalyst is 4-dimethylaminopyridine.

[0065] The above-mentioned process of adding a derivatization reagent and a catalyst to the liquor extract specifically comprises the following steps:

[0066] 5-15 μL of bromonicotinoyl chloride acetonitrile solution and 5-15 μL of 4-dimethylaminopyridine acetonitrile solution were added to the liquor extract, and the mixture was vortexed for 20-40 seconds at room temperature for a derivatization reaction; wherein the concentration of the bromonicotinoyl chloride acetonitrile solution was 5-15 mg / mL, and the concentration of the 4-dimethylaminopyridine acetonitrile solution was 5-15 mg / mL.

[0067] In this example, the volume of bromonicotinoyl chloride acetonitrile solution added was 5-15 μL, and the concentration was 5-15 mg / mL. If the volume of bromonicotinoyl chloride acetonitrile solution added is less than 5 μL, or the concentration is less than 5 mg / mL, the effective concentration of the derivatization reagent in the reaction system cannot be maintained, reducing the derivatization efficiency. If the volume of bromonicotinoyl chloride acetonitrile solution added is greater than 15 μL, or the concentration is greater than 15 mg / mL, the excess derivatization reagent will contaminate the mass spectrometer during subsequent LC-MS analysis, obscuring the signal of the target analyte and reducing the sensitivity of the analysis.

[0068] In this example, the catalyst solution of 4-dimethylaminopyridine in acetonitrile was added at a concentration of 5-15 mg / mL in a volume of 5-15 μL. Catalyst added in a volume less than 5 μL or a concentration less than 5 mg / mL will reduce derivatization efficiency. Catalyst added in a volume greater than 15 μL or a concentration greater than 15 mg / mL will mask the target analyte signal during subsequent LC-MS analysis, reducing analytical sensitivity.

[0069] In this embodiment, the vortex time after adding the derivatization reagent and catalyst is 20 to 40 seconds. If the vortex time is too short, the reaction will be incomplete and the derivatization efficiency will be reduced; if the vortex time is too long, the derivatization reagent will be hydrolyzed.

[0070] Wherein, the structural formula of bromonicotinoyl chloride is:

[0071]

[0072] The analysis targets in this example are hydroxyl groups and amino compounds (i.e., hydroxyl compounds and amino compounds) in liquor. The bromonicotinyl chloride used in this example can react efficiently with amino groups, phenolic hydroxyl groups, and alcoholic hydroxyl groups simultaneously. Hydroxyl compounds and amino compounds undergo nucleophilic substitution reactions with acyl chlorides under alkaline conditions, forming amides and esters, respectively. The reaction products produce distinct fragments in the secondary mass spectrometer. The reaction equations for bromonicotinyl chloride with hydroxyl compounds and amino compounds are:

[0073]

[0074] The chromatographic and mass spectrometric characteristics of the compounds after derivatization with bromonicotinoyl chloride, such as Figure 2 shown. Specifically, Figure 2 (A) is the EIC graph before derivatization, Figure 2 (B) is the EIC diagram after derivatization. Figure 2 (C) is the MS1 spectrum after 2-hydroxybutyric acid derivatization. Figure 2 (D) is the MS2 spectrum after 2-hydroxybutyric acid derivatization. Figure 2 (E) is the MS1 spectrum after benzylamine derivatization. Figure 2 (F) is the MS2 spectrum after benzylamine derivatization.

[0075] Compared to commonly used derivatization reagents, such as dansyl chloride, which react only with amino and phenolic hydroxyl groups and have poor reactivity with alcoholic hydroxyl groups, bromonicotinoyl chloride was used in this example for derivatization. The reaction is completed within 30 seconds, improving analytical efficiency. Furthermore, derivatization with bromonicotinoyl chloride does not require heating or prolonged reaction times, minimizing the loss of highly volatile amino or hydroxyl compounds during the derivatization process. The bromonicotinoyl chloride used in this example was purchased from a reagent company.

[0076] Step 2: Obtain non-targeted mass spectrometry data of the derivatized liquor extract. Based on the non-targeted mass spectrometry data, screen and obtain a list of unknown compounds corresponding to the hydroxyl compounds and amino compounds to be annotated and qualitatively identified in the derivatized liquor extract.

[0077] Obtaining the list of unknown compounds in step 2 includes the following steps:

[0078] Perform peak alignment, peak matching, and blank subtraction on the non-targeted mass spectrometry data to obtain the original compound list; the non-targeted mass spectrometry data includes: precursor ion mass-to-charge ratio m / z, primary mass spectrum MS1, secondary mass spectrum MS2, and retention time t R ;

[0079] Based on the original compound list, precursor ions containing characteristic mass spectral difference peak pairs of bromine atom isotopes were extracted to obtain a list of unknown compounds corresponding to the hydroxyl compounds and amino compounds to be annotated and qualitatively analyzed in the derivatized liquor extract.

[0080] The following details step two: After using UPLC-HRMS to obtain the non-targeted mass spectrometry data of the derivatized liquor extract, the non-targeted metabolomics data were peak aligned, peak matched, and blank subtracted using MS-DIAL software to obtain the original compound list. The original compound list contains the precursor ion mass-to-charge ratio, retention time, secondary mass spectrometry fragments, and chromatographic peak area. Then, bromonicotinoyl chloride-derivatized compounds were screened based on the natural isotope mass spectrometry peak pairs introduced by the bromine atom. Specifically, an internally written Python program was used to screen and extract precursor ions containing bromine atom isotope characteristic mass difference peak pairs, and the screened and extracted precursor ions were used to construct a list of unknown compounds. The fixed mass difference of the bromine atom isotope characteristic mass spectrometry peak pairs is 1.998Da±10ppm, and the fixed intensity ratio is 0.8~1.2.

[0081] The non-targeted mass spectrometry data were acquired using a liquid chromatography-mass spectrometry (LC-MS) platform. The LC analysis conditions and MS conditions were as follows:

[0082] (1) LC Analysis Conditions: An Agilent 1290 Series Ultra High Performance Liquid Chromatograph (Agilent, Santa Clara, CA, USA) was used, with a BEHC8 column (2.1 mm × 100 mm, 1.7 μm, Waters, Milford, MA, USA). The column temperature was 50°C, and the flow rate was 0.35 mL / min. Phase A consisted of an aqueous solution containing 0.1% by volume formic acid, and phase B consisted of an acetonitrile solution containing 0.1% by volume formic acid. The initial gradient was 5% by volume phase B, maintained for 1 min, then linearly increased to 100% by volume over 23 min, maintained for 4 min, then decreased to 5% by volume over 0.1 min, and equilibrated for 1.9 min.

[0083] (2) MS conditions: An Agilent 6546 triple quadrupole-time-of-flight (Q-TOF) mass spectrometer (Agilent, Santa Clara, CA, USA) was used with a spray voltage of 4.0 kV. The desolvation gas flow rate was 8 L / min and the temperature was 320°C. The sheath gas flow rate was 11 L / min and the temperature was 350°C. In the positive ion full-scan analysis mode, the mass-to-charge ratio range was 100-1100 Da. In the Auto-MS / MS analysis mode, the collision energy was 10 eV, 20 eV, and 40 eV, and the top five ions with the highest response intensity in each scan cycle were collected; the mass-to-charge ratio range was 50-1000 Da.

[0084] Step 3: Based on the list of unknown compounds, a search is performed from a preset database to obtain a list of candidate compounds for the hydroxyl compounds and amino compounds to be annotated and qualitatively analyzed. Based on the non-targeted mass spectrometry data, the candidate compound list, and the preset quantitative structure-chromatographic retention relationship model, the hydroxyl compounds and amino compounds to be annotated and qualitatively analyzed are annotated and qualitatively analyzed.

[0085] Among them, the preset databases include the internal liquor compound database, the yeast metabolism database (The Yeast Metabolome Database, YMDB) and the plant available active molecule database (Collective Molecular Activities of Useful Plants, CMAPU).

[0086] The preset quantitative structure-chromatographic retention relationship model (QSRR model) was set to input the SMILES molecular structure of the compound and output the corresponding retention time prediction value of the compound after derivatization with bromonicotinoyl chloride.

[0087] LC-MS is used to detect liquor samples to obtain mass spectrometry information for qualitative database search. Based on the database and a certain matching matrix, multiple candidate compounds corresponding to the unknown compound are given. The structures of the candidate compounds are known. How to determine whether the candidate compound accurately represents the unknown compound? The QSRR model is used to predict the t of the candidate compound. R , determine the predicted t R and the actual t of the unknown compound R Whether it matches.

[0088] The most standard approach is to determine t by comparing with a standard. R If all m / z information is consistent, then the unknown compound is basically considered to be the standard. However, in this example, there are many hydroxyl compounds and amino compounds to be detected, and all of them are compared with standards, which is slow. In addition, collecting standards requires a lot of effort and cost, and the annotation range is limited by the availability of standards. Therefore, a QSRR model is constructed. The QSRR model can be used to predict retention times based on the molecular formula of the compound in the absence of a standard, thereby expanding the range of annotated compounds.

[0089] And mass spectrometry measures t R The value will be different depending on different people and different experimental conditions. R values ​​(even for common substances), which may lead to the measurement of R The data are inaccurate, so a uniformly trained QSRR model is used to predict retention time to reduce measurement deviations caused by differences in experimental conditions.

[0090] Step three further includes the following steps:

[0091] Determine the actual retention time of the unknown compound to be annotated and the corresponding primary mass spectrum MS1 based on the unknown compound list and the non-targeted mass spectrometry data, and search the database based on the actual retention time of the unknown compound and the corresponding primary mass spectrum MS1 to obtain multiple candidate compounds corresponding to the unknown compound;

[0092] comparing the secondary mass spectra MS2 of the plurality of candidate compounds with the secondary mass spectra MS2 of the unknown compound, and comparing the predicted retention time values ​​of the plurality of candidate compounds after derivatization with bromonicotinoyl chloride with the actual retention time of the unknown compound to obtain a comparison result;

[0093] The comparison result is matched with the annotation qualitative level to obtain a matching result, and the unknown compound is annotated and qualitatively evaluated based on the matching result.

[0094] The steps for obtaining the predicted retention time values ​​of the above-mentioned multiple candidate compounds after derivatization with bromonicotinoyl chloride are as follows:

[0095] SMILES molecular structural formulas of multiple candidate compounds are obtained, and the SMILES molecular structural formulas of the multiple candidate compounds are input into a quantitative structure-chromatographic retention relationship model to obtain retention time prediction values ​​corresponding to the multiple candidate compounds after derivatization with bromonicotinoyl chloride.

[0096] The qualitative levels of annotation include:

[0097] Level 1: The actual retention time, primary mass spectrum MS1, and secondary mass spectrum MS2 of the unknown compound currently being annotated and qualitatively matched those of the corresponding standard. Level 1 matching conditions can be determined based on actual qualitative requirements. For ease of understanding, an example of a Level 1 matching condition is provided below: the MS1 difference between the unknown compound currently being annotated and qualitatively and the corresponding standard is less than 10 ppm, the MS2 similarity is greater than 0.5, and the actual retention time of the unknown compound currently being annotated and qualitatively matched with the predicted retention time of the standard is less than 0.5 minutes. If these matching conditions are met, the compound is considered Level 1.

[0098] Level 2: The actual retention time, primary mass spectrum MS1, and secondary mass spectrum MS2 of the unknown compound currently being annotated and qualitatively matched those of the corresponding compound in the database. Level 2 matching criteria can be determined based on actual qualitative requirements. For ease of understanding, an example of a Level 2 matching criteria is provided below: the MS1 difference between the unknown compound currently being annotated and qualitatively and the corresponding compound in the database is less than 10 ppm, the MS2 similarity is greater than 0.5, and the actual retention time of the unknown compound currently being annotated and qualitatively matched with the predicted retention time of the corresponding compound in the database is less than 0.5 minutes. If these matching criteria are met, the compound is considered Level 2.

[0099] Level 3: The unknown compound currently being annotated and qualitatively identified matches only its actual retention time and primary mass spectrum (MS1) with the corresponding compound in the database. Level 3 matching criteria can be determined based on actual qualitative requirements. For ease of understanding, here is an example of a Level 3 matching condition: the difference between the MS1 value of the unknown compound currently being annotated and qualitatively identified and the corresponding compound in the database is less than 10 ppm, and the difference between the actual retention time of the unknown compound currently being annotated and qualitatively identified and the predicted retention time of the corresponding compound in the database is less than 0.5 minutes. If these matching conditions are met, the compound is considered Level 3.

[0100] Level 4: The unknown compound currently being annotated and qualitatively identified only has a primary mass spectrum (MS1) that matches the corresponding compound in the database. Level 4 matching criteria can be determined based on actual qualitative needs. For ease of understanding, here is an example of a Level 4 matching condition: if the only difference between the MS1 spectra of the unknown compound currently being annotated and qualitatively identified and the corresponding compound in the database is less than 10 ppm, then the compound is considered Level 4.

[0101] Level 5: The actual retention time, primary mass spectrum MS1, and secondary mass spectrum MS2 of the unknown compound currently being annotated and qualitatively identified do not match the corresponding compound in the database. Only the molecular formula of the unknown compound currently being annotated and qualitatively identified can be determined. Level 5 matching criteria can be determined based on actual qualitative analysis needs. For ease of understanding, an example of Level 5 matching criteria is provided below: If the conditions of Levels 1 to 4 above are not met and only the molecular formula of the unknown compound currently being annotated and qualitatively identified can be determined, then the compound is considered Level 5.

[0102] The following uses parahydroxyphenylethanol and combines Figure 3 and Figure 4 Let's take this as an example to further explain step three:

[0103] In the non-targeted mass spectrometry data, a mass peak pair of m / z 322.0083 and m / z 324.0063 was found at a retention time of 9.67 min, and it was preliminarily determined that a compound labeled with bromonicotinoyl chloride existed. Through database search, the MS1 difference between the database data and the unknown compound was less than 10ppm, tR Three candidate compounds were identified with a difference of less than 0.5 min: p-hydroxyphenylethanol, 2-hydroxymethyl-6-methylphenol, and 2,6-dimethylhydroquinone. Further observations of the unknown compound's precursor ion MS2 revealed the presence of characteristic ions of the hydroxy compound bromonicotinyl chloride (183.9390, 201.9482, and 155.9440), as well as structural characteristic ions (107.0488 and 103.0535). Analysis revealed that the former corresponded to a methylphenol structural fragment, while the latter to a styrene structural fragment. Therefore, it was speculated that the unknown compound's corresponding structure was more likely to be p-hydroxyphenylethanol.

[0104] According to the above inference results, the unknown compound was first compared with the LC-MS analysis results of the p-hydroxyphenylethanol standard sample, and it was found that the t R The MS2 spectra were similar (similarity 0.71), so the compound was annotated as p-hydroxyphenylethanol, and the qualitative level was level one.

[0105] If the comparison result between the unknown compound and the standard sample of p-hydroxyphenylethanol does not meet the conditions of level 1, the next step of verification is to determine the t of the unknown compound and the p-hydroxyphenylethanol in the database. R , MS1, and MS2 meet the conditions of Level 2. If so, the compound is annotated as p-hydroxyphenylethanol and the qualitative level is Level 2. If not, the conditions of Levels 3, 4, and 5 are determined in sequence, and the unknown compound is annotated and qualitatively characterized based on the judgment results.

[0106] Figure 4 This is an annotated qualitative diagram taking p-hydroxyphenylethanol as an example. Figure 4 (A) is the MS2 spectrum generated at retention time 9.67 min and m / z 322.0083. Figure 4 (B) is the annotation of the secondary fragment structure generated by the parent ion 322.0083. Figure 4 (C) is the EIC comparison between the liquor sample and the p-hydroxyphenylethanol standard sample. Figure 4 (D) Comparison of MS2 between liquor sample and p-hydroxyphenylethanol standard sample.

[0107] Figure 5This chart shows the number of hydroxyl and amino compounds detected in five grades of liquor samples. SFB1 represents grade 1, SFB2 represents grade 2, SFB3 represents grade 3, SFB4 represents grade 4, and SFB5 represents grade 5. The petal chart in the upper right corner shows the number of unique and shared compounds across the five grades of liquor. The petals of different colors represent the number of unique compounds, while the petal core represents the number of shared compounds. The Upset chart provides a more detailed view and comparison of the relationship between the number of compounds detected in different grades of liquor. The bar chart on the left shows the number of compounds in a single liquor. The bar chart above shows the number of compounds in the intersection of the liquors.

[0108] Example 2

[0109] 1. Preparation of Liquor Samples

[0110] The liquor sample was concentrated 25 times by rotary evaporation under the conditions of 150 rpm / min, 55°C, and -0.1 MPa to obtain a concentrated liquor sample, which was then stored at 4°C. 30 μL of the concentrated liquor sample was taken, 5 μL of the mixed internal standard solution was added first, followed by 300 μL of dichloromethane and 150 μL of water, and the mixture was vortexed at 1800 rpm and 8°C for 5 minutes for extraction. After extraction, the mixture was centrifuged at 15,000 rpm and 4°C for 10 minutes, 220 μL of the organic layer and 100 μL of the aqueous layer were taken out, and the organic layer and the aqueous layer were freeze-dried (vacuum freeze-dried) to obtain a freeze-dried liquor extract.

[0111] 2. Derivatization of Liquor Samples

[0112] To the freeze-dried liquor extract, 100 μL of ultra-dry acetonitrile was added to dissolve the liquor extract to obtain a liquor extract. Subsequently, 10 μL of bromonicotinoyl chloride in acetonitrile (10 mg / mL) and 10 μL of 4-dimethylaminopyridine in acetonitrile (10 mg / mL) were added sequentially. After vortexing at room temperature for 30 seconds, the mixture was quenched by adding 20 μL of water to obtain the reaction product. The reaction product was freeze-dried and reconstituted in 50 μL of 50% acetonitrile to obtain the derivatized liquor extract.

[0113] 3. Qualitative annotation of liquor samples

[0114] After acquiring non-targeted mass spectrometric data from the derivatized liquor extract using UPLC-HRMS, MS-DIAL software was used to perform peak alignment, peak matching, and blank subtraction on the non-targeted metabolomics data to generate a raw compound list. An in-house Python program was used to screen and extract precursor ions containing characteristic bromine isotope mass difference peak pairs from the raw compound list. These screened precursor ions were then used to construct a list of unknown compounds.

[0115] Based on the list of unknown compounds, a search is performed from the preset database to obtain a list of candidate compounds for the hydroxyl compounds and amino compounds to be annotated and qualitatively analyzed. Based on the non-targeted mass spectrometry data, the candidate compound list and the preset quantitative structure-chromatographic retention relationship model, the hydroxyl compounds and amino compounds to be annotated and qualitatively analyzed are annotated and qualitatively analyzed.

[0116] Through the above-mentioned annotation qualitative process, 309 hydroxyl or amino compounds were detected in the liquor samples, including 14 at level one, 5 at level two, 51 at level three, 158 at level four, and 81 at level five.

[0117] Example 3

[0118] To evaluate the reliability of the established method, a mixed standard solution containing 10 hydroxyl and amino compounds was used to investigate the linear range, detection limit, quantification limit, intra-day and inter-day precision, and stability. As shown in Tables 1 and 2, the calibration curves of all 10 compounds showed good linearity within the range of 1-4 orders of magnitude (R 2 >0.99). Its limit of detection (LOD) and limit of quantification (LOQ) ranged from 0.001-0.159 nmol / mL and 0.002-0.317 nmol / mL, respectively. Good intra-day and inter-day precision was observed for hydroxyl and amino compounds at medium and high concentration levels, with relative standard deviations (RSDs) below 10% (1.20-8.69%). At low concentrations, the intra-day and inter-day RSD values ​​for precision of all hydroxyl and amino compounds were less than 20% (2.80-18.02%), confirming the high reliability of the method. In addition, reproducibility assessment showed that the RSD values ​​for all tested compounds ranged from 3.20-18.30%, all below 20%, validating the repeatability of the method. Regarding the stability of the bromonicotinoyl chloride-labeled compounds, the peak area RSD values ​​of all labeled compounds within 48 hours remained in the range of 2.90-11.53% (all below 15%), indicating that the bromonicotinoyl chloride-labeled compounds can be stably stored at 4°C for at least two days.

[0119] Table 1. Linear range, LOD, and LOQ of 10 hydroxyl and amino compounds

[0120]

[0121] Table 2. Intra-day and inter-day precision, repeatability, and stability of 10 hydroxyl and amino compounds

[0122]

[0123]

[0124] By comparing the changes in the chromatographic and mass spectrometric characteristics of a series of hydroxyl and amino compounds after labeling with bromonicotinoyl chloride, the following conclusions were drawn: Due to their high volatility and poor ionization efficiency, unlabeled hydroxyl and amino compounds are difficult to detect directly by LC-MS. However, labeling with bromonicotinoyl chloride significantly improves sensitivity, attributed to enhanced reverse-phase retention due to the introduction of the pyridine ring and enhanced ionization efficiency due to the tertiary amine. Furthermore, a precursor ion peak pair with a mass difference of 1.998 Da and an intensity ratio of approximately 1 was identified in the MS1 spectrum. This peak pair is derived from 78.9183Br and its isotope 80.9163Br. This demonstrates that bromonicotinoyl chloride-labeled compounds can be screened from a large number of precursor ions even in the absence of a heavy isotope labeling reagent. Furthermore, in the MS2 spectrum of the bromonicotinoyl chloride-labeled compound, a characteristic fragment at 183.9393, resulting from ester bond cleavage, was observed, along with a characteristic fragment at 201.9498 for hydroxyl compounds and a fragment at 200.9658 for amino compounds. These fragments help to more accurately screen and annotate hydroxyl and amino compounds. In short, bromonicotinoyl chloride helps to detect hydroxyl and amino compounds in complex systems with high coverage and high sensitivity.

[0125] Example 4

[0126] This embodiment provides a modeling method for the quantitative structure-chromatographic retention relationship model as in Example 1, comprising the following steps:

[0127] 86 hydroxyl compound and amino compound standards were selected for the establishment and evaluation of the quantitative structure-chromatographic retention relationship model (QSRR model). 66 of them were randomly selected as the training set, and the remaining 20 standards were used as the test set. The retention time (t R ).

[0128] The SMILES of all compounds in the training set and the t R The QSRR_Automator software was imported and the QSRR model was established using the random forest regression algorithm and internal cross-validation was performed. Subsequently, the QSRR model established above was used to predict the t R , and calculate the predicted t of the compounds in the test set R and actual t R The average absolute deviation (AAE) is used to evaluate the reliability of the established model. The average absolute deviation between the predicted and actual retention times of the test set is less than 1 minute.

[0129] The QSRR_Automator software converted the SMILES of the imported training set compounds into 371 molecular descriptors, and selected 11 of them for model construction. Random forest regression was selected for molecular descriptors and t R After 5 times of internal cross validation, the established model R 2 is 0.99. Figure 1 As shown, the test set was further used for external validation, and the t R With actual t R Perform linear fitting and obtain R 2 The calculated AAE is 34s, which proves that the established model can well predict the t of the compound labeled with bromonicotinoyl chloride. R .

[0130] The hydroxyl compound standards and amino compound standards used in the above model construction are shown in Table 3:

[0131] Table 3. Detailed list of hydroxyl compound standards and amino compound standards

[0132]

[0133]

[0134]

[0135]

[0136] Example 5

[0137] This embodiment provides an application of a method for analyzing and detecting hydroxyl and amino substances in liquor as in Example 1 in detecting hydroxyl and amino substances in liquor.

[0138] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they become aware of the basic creative concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present invention.

[0139] Obviously, those skilled in the art may make various changes and modifications to this application without departing from the spirit and scope of this application. Thus, if these modifications and variations of this application fall within the scope of the claims of this application and their equivalents, this application is intended to include these modifications and variations.

Claims

1. A method for analyzing and detecting hydroxyl and amino substances in liquor, characterized in that: The following steps are involved: Step 1: Pretreating a liquor sample to obtain a liquor extract, adding a derivatization reagent and a catalyst to the liquor extract, causing the derivatization reagent to react with hydroxyl compounds and amino compounds in the liquor extract to obtain a derivatized liquor extract; wherein the derivatization reagent is bromonicotinoyl chloride; Step 2: obtaining non-targeted mass spectrometry data of the derivatized liquor extract, and screening to obtain a list of unknown compounds corresponding to the hydroxyl compounds and amino compounds to be annotated and qualitatively identified in the derivatized liquor extract based on the non-targeted mass spectrometry data; Step 3: Based on the list of unknown compounds, a search is performed from a preset database to obtain a list of candidate compounds for the hydroxyl compounds and amino compounds to be annotated and qualitatively characterized; and based on the non-targeted mass spectrometry data, the list of candidate compounds, and a preset quantitative structure-chromatographic retention relationship model, the hydroxyl compounds and amino compounds to be annotated and qualitatively characterized are annotated and qualitatively characterized.

2. The method for analyzing and detecting hydroxyl and amino substances in liquor according to claim 1, wherein: The acquisition of the unknown compound list in step 2 includes the following steps: The non-targeted mass spectrometry data are subjected to peak alignment, peak matching, and blank subtraction to obtain an original compound list; wherein the non-targeted mass spectrometry data include: precursor ion mass-to-charge ratio m / z, primary mass spectrum MS1, secondary mass spectrum MS2, and retention time t R ; Based on the original compound list, precursor ions containing characteristic mass spectrum difference peak pairs of bromine atom isotopes are extracted to obtain a list of unknown compounds corresponding to the hydroxyl compounds and amino compounds to be annotated and qualitatively identified in the derivatized liquor extract.

3. The method for analyzing and detecting hydroxyl and amino substances in liquor according to claim 2, characterized in that: The quantitative structure-chromatographic retention relationship model in step 3 is set to input the SMILES molecular structure of the compound and output the predicted retention time of the compound after derivatization with bromonicotinoyl chloride.

4. The method for analyzing and detecting hydroxyl and amino substances in liquor according to claim 3, characterized in that: The step three further comprises the following steps: Determining the actual retention time of the unknown compound to be annotated and the corresponding primary mass spectrum MS1 based on the unknown compound list and the non-targeted mass spectrometry data, and searching the database based on the actual retention time of the unknown compound and the corresponding primary mass spectrum MS1 to obtain multiple candidate compounds corresponding to the unknown compound; comparing the secondary mass spectra MS2 of the plurality of candidate compounds with the secondary mass spectra MS2 of the unknown compound, and comparing the predicted retention time values ​​of the plurality of candidate compounds after derivatization with bromonicotinoyl chloride with the actual retention time of the unknown compound to obtain a comparison result; The comparison result is matched with the annotation qualitative level to obtain a matching result, and the unknown compound is annotated and qualitatively evaluated based on the matching result.

5. The method for analyzing and detecting hydroxyl and amino substances in liquor according to claim 4, characterized in that: The annotation qualitative levels include: Level 1: The actual retention time, primary mass spectrum MS1, and secondary mass spectrum MS2 of the unknown compound to be annotated and qualitatively identified match those of the corresponding standard; Level 2: The actual retention time, primary mass spectrum MS1, and secondary mass spectrum MS2 of the unknown compound to be annotated and qualitatively identified match those of the corresponding compound in the database; Level 3: The unknown compound to be annotated and qualitatively identified only has actual retention time and primary mass spectrum MS1 matching the corresponding compound in the database; Level 4: The unknown compound to be annotated and qualitatively identified has only a primary mass spectrum MS1 that matches the corresponding compound in the database; Level 5: The actual retention time, primary mass spectrum MS1 and secondary mass spectrum MS2 of the unknown compound to be annotated and qualitatively identified do not match those of the corresponding compound in the database, and only the molecular formula of the unknown compound to be annotated and qualitatively identified can be determined.

6. The method for analyzing and detecting hydroxyl and amino substances in liquor according to claim 1, characterized in that: The step 1 of adding a derivatization reagent and a catalyst to the liquor extract comprises the following steps: Add 5 to 15 μL of a derivatization reagent, bromonicotinoyl chloride acetonitrile solution, and 5 to 15 μL of a catalyst, 4-dimethylaminopyridine acetonitrile solution, to the liquor extract, and vortex for 20 to 40 seconds at room temperature to perform a derivatization reaction; The concentration of the bromonicotinoyl chloride acetonitrile solution is 5 to 15 mg / mL, and the concentration of the 4-dimethylaminopyridine acetonitrile solution is 5 to 15 mg / mL.

7. The method for analyzing and detecting hydroxyl and amino substances in liquor according to claim 1, characterized in that: The step 1 of pre-treating the liquor sample to obtain the liquor extract comprises the following steps: Concentrating the liquor sample to obtain a concentrated liquor sample; The concentrated liquor sample is subjected to extraction, centrifugation, and freeze-drying operations in sequence to obtain a freeze-dried liquor extract; 50 to 150 μL of ultra-dry acetonitrile is added to the freeze-dried liquor extract to dissolve it, thereby obtaining the liquor extract.

8. The method for analyzing and detecting hydroxyl and amino substances in liquor according to claim 7, characterized in that: The concentrated liquor sample is sequentially subjected to extraction, centrifugation, and freeze-drying operations, including the following steps: Taking 20-40 μL of the concentrated liquor sample, adding 5-10 μL of a mixed internal standard solution, 200-400 μL of dichloromethane, and 100-200 μL of water to the concentrated liquor sample in sequence, and extracting by shaking for 4-6 minutes at a speed of 1500-2000 rpm and a temperature of 4-10°C; After the extraction is completed, centrifuging at a speed of 14,000 to 15,000 rpm and a temperature of 4 to 10° C. for 10 to 15 minutes to obtain an organic layer and an aqueous layer, taking out 200 to 250 μL of the organic layer and 100 to 150 μL of the aqueous layer, and vacuum freeze-drying the organic layer and the aqueous layer to obtain the freeze-dried liquor extract; Wherein, the mixed internal standard solution includes one or more of phenol-d5, benzyl alcohol-d7, glycerol-d5, sorbitol-13C6, n-hexanol-d13, phenylalanine-d5, and leucine-13C; The concentrations of phenol-d5, benzyl alcohol-d7, glycerol-d5, sorbitol-13C6, n-hexanol-d13, phenylalanine-d5, and leucine-13C are 0.5-1.5 μg / mL.

9. The method for analyzing and detecting hydroxyl and amino substances in liquor according to claim 7, characterized in that: The concentration treatment adopts rotary evaporation, and the conditions of the rotary evaporation are a rotation speed of 100-200 rpm / min, a temperature of 50-60° C., and a pressure of -0.1-0.2 MPa. The concentration multiple of the concentration treatment is 20-30 times.

10. Use of the method for analyzing and detecting hydroxyl and amino substances in liquor according to any one of claims 1 to 9 in detecting hydroxyl and amino substances in liquor.

Images