A method for simultaneously detecting 18 kinds of amino acids in Daqu based on high-resolution liquid chromatography-mass spectrometry

Abstract

The application discloses a method for simultaneously detecting 18 kinds of amino acids in Daqu based on high-resolution liquid chromatography-mass spectrometry, and belongs to the technical field of liquor detection. Chromatographic conditions of the method include that an ethylene bridge hybrid particle with surface-bonded amide groups is used as a filler, ammonium acetate-formic acid aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B for gradient elution; mass spectrometry conditions include that an H-ESI ion source is used, the spray voltage in the positive ion mode is 3000-4000 V, and the spray voltage in the negative ion mode is 2000-3000 V; qualitative analysis is performed according to mass-to-charge ratios of the 18 kinds of amino acids, and quantitative analysis is performed by using a standard curve method. The method has the characteristics of simple pretreatment operation, easy-to-obtain reagents, high extraction efficiency and good stability, can realize synchronous and accurate qualitative and quantitative analysis of the 18 kinds of amino acids, and provides reliable data support for flavor substance research of Daqu.

Description

Technical Field

[0001] This invention belongs to the field of liquor detection technology, specifically relating to a method for simultaneously detecting 18 amino acids in Daqu (a type of Chinese liquor) based on a high-resolution liquid chromatography-mass spectrometry (LC-MS) instrument. Background Technology

[0002] Amino acids, as a crucial class of nitrogenous nutrients, are essential building blocks for microbial growth, reproduction, and metabolism. In the solid-state fermentation system of Baijiu (Chinese liquor), Daqu (a type of starter culture) plays a vital role in saccharification, fermentation, and aroma production. The composition and availability of its nitrogenous components, particularly the specific types, contents, and proportions of amino acids, directly influence the metabolic activity and direction of the microbial community during fermentation. Nitrogen source utilization efficiency is closely linked to protein synthesis, nucleotide metabolism, and the biosynthetic pathways of various flavor-active substances. Key components that determine the flavor profile of Baijiu, such as higher alcohols and pyrazines, primarily derive their precursors from the transformation and metabolism of amino acids. Therefore, the composition and proportion of amino acids in Daqu are crucial intrinsic factors in regulating the fermentation process, influencing microbial community function, and ultimately determining the quality and style of Baijiu. Especially in high-temperature Daqu, amino acids are not only important non-volatile flavor compounds but also a precursor library constituting complex flavor networks. Precise analysis of amino acids is of significant scientific value and guiding significance for understanding the causes of Baijiu flavor and optimizing production processes.

[0003] Currently, various techniques exist for the detection of amino acids, such as automated amino acid analyzers and pre- or post-column derivatization combined with high-performance liquid chromatography (HPLC). However, these traditional methods often face numerous challenges when applied to the complex Daqu (Chinese koji) matrix: cumbersome and time-consuming pretreatment steps; the derivatization process may introduce side reactions or lead to instability of the target analyte; and the ability to simultaneously separate and accurately quantify multiple amino acids in complex matrices is limited, often failing to balance high throughput and high sensitivity. These limitations restrict the ability to conduct comprehensive, rapid, and accurate analysis of the amino acid composition of Daqu, thereby affecting a deeper understanding of nitrogen metabolism mechanisms during fermentation and the fine-tuning of Baijiu (Chinese liquor) quality.

[0004] Therefore, providing a method that is simple to operate, has high extraction efficiency and good stability, and can simultaneously and efficiently separate and accurately identify and quantify multiple amino acids in Daqu (a type of Chinese liquor), with the characteristics of simple pretreatment, high analytical throughput and accurate and reliable results, is an urgent technical problem to be solved by those skilled in the art, which is to solve the problems of cumbersome pretreatment, unstable derivatization and insufficient ability to separate and identify complex matrices in the existing technology. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the simultaneous detection of 18 amino acids in Daqu (a type of Chinese liquor) based on high-resolution liquid chromatography-mass spectrometry (LC-MS). The method has simple pretreatment operation, readily available reagents, high extraction efficiency, and good stability. It can achieve simultaneous and accurate qualitative and quantitative analysis of 18 amino acids, providing reliable data support for the study of flavor substances in Daqu.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention discloses a method for the simultaneous detection of 18 amino acids in Daqu (a type of Chinese liquor) using high-resolution liquid chromatography-mass spectrometry (LC-MS). The chromatographic conditions include: using an ethylene-bridged hybrid column packed with amide groups as the packing material; using ammonium acetate-formic acid aqueous solution as mobile phase A; using acetonitrile as mobile phase B; and eluting according to the following gradient: 0 min, 4~6% A, 94~96% B; 0.5min, 4~6%A, 94~96%B; 4.0min, 28~32%A, 68~72%B; 5.0min, 48~52%A, 48~52%B; 7.0min, 48~52%A, 48~52%B; 9.0min, 4~6%A, 94~96%B; Mass spectrometry conditions include: using an H-ESI ion source, with a spray voltage of 3000-4000V in positive ion mode and 2000-3000V in negative ion mode; The mass-to-charge ratios of the 18 amino acids are as follows: L-aspartic acid: 132.03; L-glutamic acid: 146.05; L-serine: 104.04; glycine: 74.02; L-histidine: 154.06; L-arginine: 175.12; L-threonine: 118.05; L-alanine: 90.05; L-proline: 116.07; L-tryptophan: 205.10; L-valine: 118.09; L-methionine: 150.06; L-cysteine: 120.01; L-isoleucine: 132.10; L-leucine: 132.10; L-phenylalanine: 166.09; L-tyrosine: 182.08; L-lysine acetate: 147.11.

[0007] In some embodiments of the present invention, the concentration of ammonium acetate in the ammonium acetate-formic acid aqueous solution is 2 mmol / L, and the volume content of formic acid is 0.1%.

[0008] In some embodiments of the present invention, gradient elution is performed according to the following specifications: 0 min, 5% A, 95% B; 0.5 min, 5% A, 95% B; 4.0 min, 30% A, 70% B; 5.0 min, 50% A, 50% B; 7.0 min, 50% A, 50% B; 9.0 min, 5% A, 95% B.

[0009] In some embodiments of the present invention, the chromatographic column has the specifications of GT×Resolve Premier BEHAmide Columns 2.1×150mm; Preferably, the column temperature is 35~45℃; more preferably 40℃; the mobile phase flow rate is 0.3ml / min; In some embodiments of the present invention, the sheath gas flow rate is 0.5-2 Arb, the auxiliary gas flow rate is 5-15 Arb, the purge gas flow rate is 0.5-2 Arb, the ion transport tube temperature is 300-350℃, and the vaporization temperature is 300-400℃.

[0010] In some embodiments of the present invention, the mass spectrometry detection conditions are as follows: the spray voltage is 3500V in positive ion mode and 2500V in negative ion mode; the sheath gas flow rate is 1Arb, the auxiliary gas flow rate is 10Arb, and the purge gas flow rate is 1Arb; the ion transmission tube temperature is 325℃ and the vaporization temperature is 350℃.

[0011] In some embodiments of the present invention, when the method is used for qualitative analysis, it includes the following steps: Preparation of test solution: Take Daqu powder, weigh accurately, add solvent and extract by ultrasonication, let stand, centrifuge, collect the supernatant, dilute with solvent and filter through a filter membrane to obtain the test solution; Take the test solution, inject it into the liquid chromatography-mass spectrometry instrument, and perform the determination according to the above method. The target substance is identified by the mass-to-charge ratio.

[0012] In some embodiments of the present invention, when the method is used for quantification, it includes the following steps: Preparation of test solution: Take Daqu powder, weigh accurately, add solvent and extract by ultrasonication, let stand, centrifuge, collect the supernatant, dilute with solvent and filter through a filter membrane to obtain the test solution; Preparation of reference solutions: Accurately weigh appropriate amounts of each of the 18 amino acids, dissolve them separately in water and make up to volume to prepare single or mixed reference solutions with a concentration of 0.1~20 μmol / L; Take the reference solution and the test solution, respectively, and inject them into a liquid chromatography-mass spectrometry instrument. Determine the content of each amino acid in the test solution according to the method described in any one of claims 1-6. Calculate the content of each amino acid in the test solution according to the standard curve method.

[0013] In some embodiments of the present invention, when preparing the test solution, after ultrasonic extraction, the sample is allowed to stand at low temperature and then centrifuged at low temperature. Preferably, the mixture is left to stand at 2-6°C for at least 12 hours, more preferably 12-48 hours, and then centrifuged at 2-6°C.

[0014] In some embodiments of the present invention, the solvent used in preparing the test solution is an aqueous solution of acetonitrile, preferably, the volume content of acetonitrile is 40-60%, more preferably 50%.

[0015] Preferably, the concentration of Daqu in the test solution is 25~100 mg / ml, and more preferably 50 mg / ml.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention is scientifically designed and ingeniously conceived. Its pretreatment operation is simple and the reagents are readily available. It features high extraction efficiency and good stability, and can achieve simultaneous and accurate qualitative and quantitative analysis of 18 amino acids, providing reliable data support for the study of Daqu flavor substances.

[0017] This invention employs high-resolution liquid chromatography-mass spectrometry (LC-MS) to achieve rapid quantification of amino acids in Daqu (a type of Chinese medicinal herb), effectively solving the problems of cumbersome procedures and long processing times associated with traditional methods. Secondly, for complex matrices, this invention designs a three-step purification strategy of "acetonitrile extraction – low-temperature sedimentation – ultrasonic filtration and centrifugation," which effectively removes interfering components such as proteins, polysaccharides, and lipids, improving sample purity. Furthermore, by selecting a HILIC column and optimizing the mobile phase and gradient program, efficient baseline separation of various free amino acids is achieved, eliminating the derivatization step and simplifying the pretreatment process. Finally, based on the precise mass number obtained from high-resolution mass spectrometry and the MS / MS library, triple validation is performed using retention time, primary precise mass, and secondary fragmentation information, significantly improving the accuracy and reliability of qualitative and quantitative analysis and overcoming the limitations of low-resolution mass spectrometry in complex detection. Overall, this method improves upon each stage of extraction and purification, chromatographic separation, and mass spectrometry detection, synergistically enhancing analytical efficiency and result reliability. Attached Figure Description

[0018] Appendix Figure 1 The image shows the total ion chromatography separation of 18 target amino acids using liquid chromatography-mass spectrometry. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0020] The information on the instruments and equipment used in the embodiments of this invention is as follows: Thermo Fisher Vanquish ultra-high performance liquid chromatograph, Thermo Fisher OrbitrapExploris 120 mass spectrometer, Waters GT×Resolve Premier BEH Amide Columns 2.1×150mm, and Zhida autosampler.

[0021] Example 1 This embodiment discloses a method for qualitative analysis of 18 amino acids in Daqu (a type of Chinese liquor) based on a high-resolution liquid chromatography-mass spectrometry (LC-MS) instrument.

[0022] The chromatographic conditions are as follows: The chromatographic column was a GT×ResolvePremierBEHAmideColumns 2.1×150mm, with a column temperature of 40℃ and a mobile phase flow rate of 0.3 ml / min. Mobile phase A was ammonium acetate-formic acid aqueous solution, and mobile phase B was acetonitrile. The concentration of ammonium acetate in the ammonium acetate-formic acid aqueous solution was 2 mmol / L, and the volume content of formic acid was 0.1%. Gradient elution was performed according to the specifications in Table 1. Table 1 Gradient elution program

[0023] The mass spectrometry detection conditions were as follows: H-ESI ion source was used, spray voltage was 3500V in positive ion mode and 2500V in negative ion mode; sheath gas flow rate was 1Arb, auxiliary gas flow rate was 10Arb, purge gas flow rate was 1Arb; ion transmission tube temperature was 325℃ and vaporization temperature was 350℃.

[0024] The specific method is as follows: Preparation of the test solution: Weigh approximately 5g of Daqu powder accurately, place it in a container, add 25ml of 50v% acetonitrile aqueous solution, vortex mix for 6 minutes, then sonicate for 1 hour; then let stand at 4℃ for 24 hours; subsequently centrifuge at 12000g for 10 minutes at 4℃; collect the supernatant, dilute with 50v% acetonitrile aqueous solution at a volume ratio of 1:4, filter through a 0.22μm filter membrane, and use the resulting filtrate as the test solution.

[0025] Take 2 μL of the test solution and inject it into the liquid chromatography-mass spectrometry instrument. Perform the determination under the above conditions and qualitatively identify 18 amino acids using the mass-to-charge ratios described in Table 2.

[0026] Table 2. Retention times and corresponding precursor ions of 218 amino acids

[0027] Example 2 This embodiment discloses the methodological verification of the present invention for quantifying 18 amino acids in Daqu based on high-resolution liquid chromatography-mass spectrometry, and the chromatographic and mass spectrometry conditions are the same as those in Example 1.

[0028] 1. Examination of linear relationships A series of mixed standard solutions of varying concentrations were prepared for plotting a standard curve. At each concentration level, the concentration of each amino acid in the solution was identical. A total of eight concentration levels were set: 0.1 μmol / L, 0.2 μmol / L, 0.5 μmol / L, 1 μmol / L, 2 μmol / L, 5 μmol / L, 10 μmol / L, and 20 μmol / L.

[0029] Take 2 μL of each concentration of the mixed standard solution and inject it into the liquid chromatography-mass spectrometry (LC-MS) instrument. Measure the concentrations according to the conditions in Example 1, and record the mass spectrometric response values ​​(peak areas) of each amino acid. Plot a standard curve using linear regression with concentration as the x-axis and peak area as the y-axis to obtain the linear equation and correlation coefficient (R²). 2 The results are shown in Table 3.

[0030] The results showed that all 18 amino acids exhibited good linearity within the concentration range of 0.1–20 μmol / L. The correlation coefficients (R²) ranged from 0.9915 to 0.9993, with the vast majority (16 / 18) exceeding 0.992, meeting the stringent linearity requirements for quantitative analysis.

[0031] 2. Sensitivity verification (limit of detection and limit of quantitation) The signal-to-noise ratio (S / N) method was used for determination. The mixed standard solution was gradually diluted to the expected low concentration and analyzed by liquid chromatography-mass spectrometry. Six consecutive injections were performed near the finally determined limit of quantitation.

[0032] Determination of the limit of quantification (LOQ): Take the chromatographic peak of the target amino acid in the chromatogram and calculate the ratio of its peak height (H) to the baseline noise peak height (N) (S / N). S / N ≥ 10, and the relative standard deviation (RSD) of the peak area for six consecutive injections ≤ 20%. The lowest concentration that meets these conditions is the LOQ of the amino acid.

[0033] Determination of the limit of detection (LOD): Based on the LOQ, further dilute and take the concentration corresponding to S / N≥3 as the LOD of the amino acid.

[0034] The results are shown in Table 3: Limit of Quantification (LOQ): The LOQ of all amino acids was between 0.0015 and 0.18 μmol / L, none of which was higher than 0.2 μmol / L, indicating that the method has reliable quantitative ability for trace components.

[0035] Limit of Detection (LOD): The LOD of all amino acids is between 0.0005 and 0.06 μmol / L, demonstrating the excellent minimum detection capability of the method of this invention.

[0036] Table 3. Results of Methodological Investigation

[0037] Example 3 This embodiment discloses the method of the present invention for quantifying 18 amino acids in a sample based on a high-resolution liquid chromatography-mass spectrometry (LC-MS) instrument. The chromatographic and mass spectrometry conditions are the same as those in Example 1.

[0038] 1. Instruments and Conditions This embodiment uses the same high-resolution liquid chromatography-mass spectrometry system, chromatographic conditions, and mass spectrometry conditions as in Example 1 for detection.

[0039] 2. Test solution: prepared according to the method described in Example 1.

[0040] 3. Quantitative analysis: Take 2 μL of the test solution and inject it into the liquid chromatography-mass spectrometry instrument. Perform the determination according to the conditions in Example 1. The system automatically collects the chromatograms and high-resolution mass spectrometry data of the target amino acids in all test samples.

[0041] 4. Calculation: Calculate the content of each amino acid in the test solution using the following formula. X = C × M × ρ × 3 X—Amino acid content in Daqu (a type of Chinese liquor), expressed in micrograms per kilogram (μg / kg). C—The amino acid content in the test solution is calculated from the standard curve, in micromoles per liter (μmmol / L). M—The molecular weights of the 18 amino acids; ρ—the density of the solvent, here it is the density of water.

[0042] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the embodiments of the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for simultaneous detection of 18 amino acids in Daqu (a type of Chinese liquor) using high-resolution liquid chromatography-mass spectrometry (LC-MS), characterized in that, Chromatographic conditions included: a column packed with ethylene-bridged hybrid particles with surface-bonded amide groups; mobile phase A: ammonium acetate-formic acid aqueous solution; mobile phase B: acetonitrile; and gradient elution as specified below: 0 min, 4~6% A, 94~96% B; 0.5min, 4~6%A, 94~96%B; 4.0min, 28~32%A, 68~72%B; 5.0min, 48~52%A, 48~52%B; 7.0min, 48~52%A, 48~52%B; 9.0min, 4~6%A, 94~96%B; Mass spectrometry conditions include: using an H-ESI ion source, with a spray voltage of 3000-4000V in positive ion mode and 2000-3000V in negative ion mode; The mass-to-charge ratios of the 18 amino acids are as follows: L-aspartic acid: 132.03; L-glutamic acid: 146.05; L-serine: 104.04; glycine: 74.02; L-histidine: 154.06; L-arginine: 175.12; L-threonine: 118.05; L-alanine: 90.05; L-proline: 116.07; L-tryptophan: 205.10; L-valine: 118.09; L-methionine: 150.06; L-cysteine: 120.01; L-isoleucine: 132.10; L-leucine: 132.10; L-phenylalanine: 166.09; L-tyrosine: 182.08; L-lysine acetate: 147.

11.

2. The method for simultaneous detection of 18 amino acids in Daqu (a type of Chinese liquor) based on high-resolution liquid chromatography-mass spectrometry (LC-MS) according to claim 1, characterized in that, In the ammonium acetate-formic acid aqueous solution, the concentration of ammonium acetate is 2 mmol / L and the volume content of formic acid is 0.1%.

3. The method for simultaneous detection of 18 amino acids in Daqu (a type of Chinese liquor) based on high-resolution liquid chromatography-mass spectrometry (LC-MS) according to claim 1, characterized in that, Elute using the following gradient method: 0 min, 5% A, 95% B; 0.5 min, 5% A, 95% B; 4.0 min, 30% A, 70% B; 5.0 min, 50% A, 50% B; 7.0 min, 50% A, 50% B; 9.0 min, 5% A, 95% B.

4. The method for simultaneous detection of 18 amino acids in Daqu (a type of Chinese liquor) based on high-resolution liquid chromatography-mass spectrometry (LC-MS) according to claim 1, characterized in that, The chromatographic column specifications are GT×Resolve Premier BEH Amide Columns 2.1×150mm; Preferably, the column temperature is 35~45℃; more preferably 40℃; and the mobile phase flow rate is 0.3ml / min.

5. The method for simultaneous detection of 18 amino acids in Daqu (a type of Chinese liquor) based on high-resolution liquid chromatography-mass spectrometry (LC-MS) according to claim 1, characterized in that, The sheath gas flow rate is 0.5-2 Arb, the auxiliary gas flow rate is 5-15 Arb, and the purge gas flow rate is 0.5-2 Arb; the ion transport tube temperature is 300-350℃, and the vaporization temperature is 300-400℃.

6. The method for simultaneous detection of 18 amino acids in Daqu (a type of Chinese liquor) based on high-resolution liquid chromatography-mass spectrometry (LC-MS) according to claim 1, characterized in that, The specific mass spectrometry detection conditions are as follows: spray voltage of 3500V in positive ion mode and 2500V in negative ion mode; sheath gas flow rate of 1Arb, auxiliary gas flow rate of 10Arb, purge gas flow rate of 1Arb; ion transmission tube temperature of 325℃ and vaporization temperature of 350℃.

7. A method for simultaneous detection of 18 amino acids in Daqu (a type of Chinese liquor) based on a high-resolution liquid chromatography-mass spectrometry (LC-MS) instrument, as described in any one of claims 1-6, characterized in that... When used qualitatively, the method includes the following steps: Preparation of test solution: Take Daqu powder, weigh accurately, add solvent and extract by ultrasonication, let stand, centrifuge, collect the supernatant, dilute with solvent and filter through a filter membrane to obtain the test solution; Take the test solution, inject it into a liquid chromatography-mass spectrometry instrument, and determine it according to the method described in any one of claims 1-6. The target substance is identified by the mass-to-charge ratio.

8. A method for simultaneous detection of 18 amino acids in Daqu (a type of Chinese liquor) based on a high-resolution liquid chromatography-mass spectrometry (LC-MS) instrument, as described in any one of claims 1-6, characterized in that... When used for quantification, the method includes the following steps: Preparation of test solution: Take Daqu powder, weigh accurately, add solvent and extract by ultrasonication, let stand, centrifuge, collect the supernatant, dilute with solvent and filter through a filter membrane to obtain the test solution; Preparation of reference solutions: Accurately weigh appropriate amounts of each of the 18 amino acids, dissolve them separately in water and make up to volume to prepare single or mixed reference solutions with a concentration of 0.1~20 μmol / L; Take the reference solution and the test solution, respectively, and inject them into a liquid chromatography-mass spectrometry instrument. Perform the determination according to the method described in any one of claims 1-6. Calculate the content of each amino acid in the test solution based on the measured chromatographic peak area using the external standard method or the standard curve method.

9. A method for simultaneous detection of 18 amino acids in Daqu (a type of Chinese liquor) based on a high-resolution liquid chromatography-mass spectrometry (LC-MS) instrument, as described in claim 7 or 8, characterized in that... When preparing the test solution, after ultrasonic extraction, allow it to stand at low temperature, and then centrifuge at low temperature; Preferably, the mixture is left to stand at 2-6°C for at least 12 hours, more preferably 12-48 hours, and then centrifuged at 2-6°C.

10. A method for simultaneous detection of 18 amino acids in Daqu (a type of Chinese liquor) based on a high-resolution liquid chromatography-mass spectrometry (LC-MS) instrument, as described in claim 7 or 8, characterized in that... The solvent used in preparing the test solution is an aqueous solution of acetonitrile. Preferably, the volume content of acetonitrile is 40-60%, more preferably 50%. Preferably, the concentration of Daqu in the test solution is 25~100 mg / ml, and more preferably 50 mg / ml.

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