Method for detecting content of compound in fermentation liquid or reaction liquid through ultrahigh flux

Abstract

The invention provides an ultrahigh-flux detection method for multiple small molecule compounds in fermentation liquor or reaction liquor, and relates to the technical field of analytical chemistry and the field of synthetic biology. Fermentation liquid or reaction liquid is excited to a capillary tube in a droplet form through a sound wave exciter, is transmitted to an ESI source, enters a mass spectrum after being ionized, and then is subjected to quantitative analysis by adopting a standard curve method. According to the method, liquid chromatography and the like are not used for separation, derivatization is not needed, samples are directly transmitted to mass spectrometry for detection after being injected, the analysis time is short, 50 small molecule compounds can be rapidly detected at ultrahigh flux, and the mass spectrometry adopts an electrospray ionization and positive and negative ion simultaneous scanning detection mode and a multi-reaction detection scanning mode. According to the detection method provided by the invention, the detection speed is remarkably increased, ultrahigh-flux detection can be realized, and the method is suitable for detection of synthetic biological high-flux samples.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical detection, and specifically provides a mass spectrometry method for ultra-high throughput detection of 50 small molecule compounds in fermentation broth or reaction solution. Background Art

[0002] Small molecule compounds such as amino acids, organic acids, and sugars are important biomanufacturing products and have a wide range of applications in fields such as medicine, food, cosmetics, and agriculture. During the biosynthesis process, when conducting research on industrial strain screening, enzyme directed evolution, fermentation process optimization, etc., it is necessary to perform high-throughput detection of target products for a large number of strains and mutants, and screen out test bodies with potential high yields of target products such as amino acids, organic acids, and sugars. In addition, the content information of their by-products, intermediate products, etc. is also helpful for further research.

[0003] High-throughput detection techniques for small molecule compounds such as amino acids, organic acids, and sugars include 1) optical means (ultraviolet, fluorescence, chemiluminescence, etc.), which have the advantages of easy use, low cost, and high throughput. However, their pretreatment is cumbersome, and in most cases, the detection target is one compound, and there is no optical method that can simultaneously detect amino acids, organic acids, and sugars. 2) Chromatography techniques (including high performance liquid chromatography, gas chromatography, etc.) can simultaneously detect some compounds, but these techniques require long chromatographic separation and a large amount of organic reagents. For gas chromatography, derivatization and other treatments are also required, and the operation is cumbersome. In addition, different types of compounds require different chromatographic columns, and there is still no chromatographic method that can simultaneously detect amino acids, organic acids, and sugars. 3) Mass spectrometry techniques can simultaneously detect multiple compounds, but current mass spectrometry techniques need to be combined with liquid chromatography, with long analysis time and high cost, and cannot meet the requirements of high-throughput detection.

[0004] To solve the above problems, based on acoustic wave excited mass spectrometry, the present invention has developed an ultra-high throughput detection method that can simultaneously qualitatively and quantitatively detect 50 small molecule substances such as amino acids, organic acids, and sugars without derivatization and separation. Summary of the Invention

[0005] The present invention provides an ultra-high throughput mass spectrometry method for simultaneous detection of multiple small molecule compounds in fermentation broth or reaction solution in view of the above existing problems.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides an ultra-high throughput mass spectrometry detection method for multiple small molecule compounds in fermentation broth or reaction solution, comprising the following steps:

[0008] 1) Solid-liquid separation of the fermentation broth or reaction solution to obtain a sample liquid;

[0009] 2) Prepare a series of standard solutions of the mixed compounds to be measured with different concentrations, using water as the solvent, and conduct mass spectrometry detection;

[0010] Preferably, the mass spectrometry detection conditions are as follows:

[0011] Carrier liquid: by volume ratio, methanol: acetonitrile = 4 - 6: 6 - 4, which also contains one or two or three of water with a volume concentration of 0 - 10%, ammonium fluoride with a concentration of 0 - 2 mM, and formic acid with a volume concentration of 0 - 0.1%;

[0012] Injection interval time: 0 - 2 s;

[0013] Mass spectrometry conditions: GAS1 70 - 90 psi, GAS2 70 - 90 psi, curtain gas 20 - 30 psi, CAD gas 6 - 15 psi, temperature 300 - 400 °C, spray voltage is 5 - 6 kV in positive ion ionization mode and 3 - 5 kV in negative ion ionization mode, and multi - reaction monitoring mode MRM scanning is used;

[0014] 3) Plot the standard curve of the concentration of the compound to be measured and the peak area of the daughter ion of the MRM ion pair;

[0015] 4) Inject the sample liquid obtained in step 1) for mass spectrometry detection under the mass spectrometry analysis conditions in step 2), obtain the corresponding parent ion and daughter ion mass - to - charge ratio information. By finding the corresponding matching parent ion and daughter ion mass - to - charge ratio, the corresponding compound in the sample liquid can be qualitatively confirmed. At the same time, substituting the peak area of the daughter ion of its MRM ion pair into the standard curve of the corresponding compound in step 3), the content information of the compound in the fermentation broth or reaction solution can be obtained.

[0016] Specifically, the mass spectrometry detection mode, the corresponding parent ion and daughter ion mass - to - charge ratio information, that is, the MRM ion pair, of the target compound to be measured are determined in advance.

[0017] 1) Detect the standard mother liquor of each compound using positive and negative ion modes respectively, and determine the parent ion m / z and its ion response intensity under positive and negative ions;

[0018] 2) In the positive ion mode, input the parent ion m / z determined in 1), use the production function, preset the declustering voltage to 60 V, set the collision energies to 80, 60, 40, 20, 10 V respectively, observe the secondary fragment situation, and find all the secondary fragment m / z of the compound in the positive ion mode, so as to determine all the MRM ion pairs in the positive ion mode;

[0019] 3) In the positive ion mode, using the MRM function, input all the MRM ion pairs determined in 2), and optimize the declustering voltage and collision energy respectively to obtain the ion response intensity of each MRM ion pair at the optimal declustering voltage and collision energy;

[0020] 4) In the negative ion mode, input the precursor ion m / z determined in 1), use the production mode, preset the declustering voltage to -60 V, set the collision energies to -80, -60, -40, -20, -10 V respectively, observe the secondary fragment situation, and find all the secondary fragment m / z of the compound in the negative ion mode, so as to determine all the MRM ion pairs in the negative ion mode;

[0021] 5) In the negative ion mode, using the MRM function, input all the MRM ion pairs determined in 4), and optimize the declustering voltage and collision energy respectively to obtain the ion response intensity of each MRM ion pair at the optimal declustering voltage and collision energy; Summarize the MRM ion pairs of the compound and the ion response intensities at the optimal declustering voltage and collision energy, select the interference-free characteristic ion pairs for a specific compound, and when there are multiple characteristic ion pairs, select the one with a high ion response intensity as the optimal characteristic ion pair.

[0022] Preferably, the fermentation broth or reaction solution in step 1) includes at least one or two or more of microbial fermentation broth or metabolites, enzyme reaction solution, and chemical reaction solution;

[0023] Preferably, it is a microbial fermentation broth or metabolite; the microbial fermentation broth is a fermentation broth obtained by culturing and fermenting microorganisms;

[0024] The content of the target analyte compound in the liquid fermentation broth is 0.001 - 10 mg / mL.

[0025] Specifically, the analyte compound in step 2) is a metabolite produced by at least one or two or more of animal and plant cells and microorganisms;

[0026] The concentration of the analyte compound standard solution is 0.001 - 5 mg / mL.

[0027] More specifically, the compound is selected from at least one or two or more of amino acids, organic acids, and sugars; preferably, the amino acid compounds are selected from at least one or two or more of glutamic acid, aspartic acid, and isoleucine.

[0028] Preferably, the solid-liquid separation in step 1) can be carried out by centrifugation or filtration; the specific parameters of the centrifugation are a speed of 3000 - 6000 rpm and a time of 5 - 30 minutes; the parameters of filtration are membrane filtration with a pore size of 0.22 μm - 0.45 μm.

[0029] Specifically, the specific operation of step 2) is as follows: Weigh the reference substance of the compound to be tested, dissolve it in a solvent, and prepare a standard stock solution with a concentration of 1 mg / mL. Take the standard stock solution and dilute it with a solvent to obtain a standard solution of the target compound to be tested with a concentration range of 0.001 - 1 mg / mL.

[0030] More specifically, the specific operation of drawing the standard curve in step 3) is to select 3 - 4 different mass concentrations within the range of 0.001 - 5 mg / mL to draw the standard curve.

[0031] In the specific embodiment, draw the standard curve of the concentration of the compound to be tested and the height of the parent ion peak, and calculate the corresponding linear regression equation, correlation coefficient, limit of quantification, precision, and recovery rate.

[0032] In a specific embodiment, the target compound to be tested is one or more of the following. The corresponding mass spectrometry detection mode, the mass-to-charge ratios of the corresponding parent ion and daughter ion, that is, the MRM ion pairs are as follows:

[0033]

[0034]

[0035]

[0036] Preferably, after determining the injection interval time and MRM ion pairs of the target compound to be tested, the detection time for every 4 kinds of compound MRM ion pairs is 1.5 s, and the total detection time for 50 kinds of compounds is 19 s.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention can perform high-throughput detection of multiple compounds, without the need for liquid or gas phase separation, with a short detection time, can achieve the detection of high-throughput samples, and can adjust the mass spectrometry conditions as needed to increase the number of target compounds to be tested. In the examples, taking 50 common compounds in fermentation broth or reaction solution as an example, the detection time is less than 20 seconds. First, the present invention effectively avoids the interference problem of direct mass spectrometry detection without a separation system by optimizing the MRM ion pairs, and improves the accuracy of qualitative detection of compounds. At the same time, the present invention effectively improves the poor peak shape phenomenon of compounds in mass spectrometry quantitative analysis by optimizing conditions such as the type and proportion of the carrier liquid, effectively improves the detection sensitivity of compounds, and increases the accuracy of compound content determination.

[0039] Optimization steps for the carrier solution: 1) First, optimize the type of carrier solution. Methanol, acetonitrile, ultrapure water, and their binary and ternary mixtures are used as carrier solutions to detect the target compounds to be measured. Observe the peak shape, calculate the half-peak width, extract the peak-to-peak signal-to-noise ratio, and integrate the peak area. Select the carrier solution with a good peak shape, narrow half-peak width, high signal-to-noise ratio, and large peak area for the next optimization. 2) Optimize the carrier solution ratio. Based on the type of carrier solution selected in 1), further optimize the carrier solution ratio, such as methanol:acetonitrile 7:2, methanol:acetonitrile 5:5, methanol:acetonitrile 2:7, etc. Observe the peak shape, calculate the half-peak width, extract the peak-to-peak signal-to-noise ratio, and integrate the peak area. Select the carrier solution with a good peak shape, narrow half-peak width, high signal-to-noise ratio, and large peak area for the next optimization. 3) Optimize the carrier solution additives. Under the optimized carrier solution ratio in 2), add different contents of formic acid solution (0.001%, 0.005%, 0.01%, 0.05%, 0.1% v / v), different concentrations of ammonium fluoride solution (0.5 mM, 1 mM, 2 mM), and add formic acid and ammonium fluoride simultaneously, and compare with no additives. Observe the peak shape, calculate the half-peak width, extract the peak-to-peak signal-to-noise ratio, and integrate the peak area. Select the type and amount of carrier solution additives with a good peak shape, narrow half-peak width, high signal-to-noise ratio, and large peak area. Finally, obtain the optimal type, ratio, and additive conditions of the carrier solution. In addition, due to the large differences in the properties of 50 compounds, the applicable carrier solution additives are not completely the same. After optimization according to this step, the peak emergence situation of each compound under each additive can be obtained. When the target compound to be measured is part of the 50 compounds, the carrier solution conditions can be appropriately adjusted.

[0040] The present invention is fast and convenient, without the need for complex pretreatment and derivatization of samples, and also greatly reduces the volatilization, decomposition, or oxidation of amino acids and organic acids during the pretreatment process, avoiding the distortion of compound content. Therefore, the method of the present invention is very reliable, with high accuracy and precision, and the RSD range for the 50 compounds in the examples is 2.3 - 14.9%. Description of the Drawings

[0041] Figure 1 Extracted ion chromatogram of 50 compounds detected by LC-MS.

[0042] Figure 2 Detection of glutamic acid (low concentration: 0.1 mM; medium concentration: 1 mM; high concentration: 10 mM) using different carrier solutions.

[0043] Figure 3 Interference of asparagine when detecting aspartic acid using different ion pairs.

[0044] Figure 4 Interference of glutamine and lysine when detecting glutamic acid using different ion pairs. Detailed Description of the Invention

[0045] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the present invention will be further illustrated below in conjunction with specific embodiments. However, the following embodiments are only the preferred embodiments of the present invention, not all of them. Based on the embodiments in the implementation manners, other embodiments obtained by those skilled in the art without creative efforts all belong to the protection scope of the present invention. The technical and scientific terms used in the embodiments have the same meanings as those commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0046] Example 1: Investigation of standard curve, linear range, limit of quantification and precision

[0047] 1. Materials and methods

[0048] (1) Test materials

[0049] Aspartic acid, glutamic acid, isoleucine, hydroxyproline, 3-hydroxypipecolic acid, asparagine, glutamine, glycine, cysteine, arginine, methionine, tyrosine, proline, tryptophan, serine, threonine, valine, histidine, leucine, lysine, phenylalanine, alanine, 5-aminolevulinic acid, pipecolic acid, 5-hydroxypipecolic acid, glyoxylic acid, citric acid, malic acid, succinic acid, fumaric acid, lactic acid, α-ketoglutaric acid, pyruvic acid, oxaloacetic acid, protocatechuic acid, gallic acid, β-hydroxy-β-methylbutyric acid, acrylic acid, dihydroxyacetone, glucose, xylose, trehalose, erythrulose, maltotriose, erythritol, arabitol, mannitol, inositol, sorbitol, penicillin G standard were all purchased from sigma company.

[0050] (2) Instrument equipment

[0051] Eppendorf centrifuge

[0052] SCIEX EchoMS sonic excitation coupled mass spectrometer

[0053] Preparation of standard solution

[0054] The standards in step (1) were respectively prepared into a 1 mg / mL standard stock solution, added with aqueous solution, and diluted into a series of mixed standard solutions of test compounds with different concentrations (0.001, 0.005, 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 1 mg / mL) for standby.

[0055] (3) Mass spectrometry conditions

[0056] Carrier solution (V / V): 50% methanol, 50% acetonitrile, 0% water, 0 mM ammonium fluoride

[0057] Mass spectrometry conditions: GAS1 90 psi, GAS2 70 psi, curtain gas 25 psi, CAD gas 9 psi, temperature 300 °C, spray voltage 5.5 kV in positive ion ionization mode and 4.5 kV in negative ion ionization mode. Optimize the MRM ion pairs and mass spectrometry parameters for 50 compounds respectively according to the following steps:

[0058] 1) Detect the 1 mg / mL standard stock solution of each compound using positive and negative ion modes respectively to determine the precursor ion m / z and its ion response intensity under positive and negative ions;

[0059] 2) In positive ion mode, input the precursor ion m / z determined in 1), use the product ion function, preset the declustering voltage to 60 V, set the collision energies to 80, 60, 40, 20, 10 V respectively, observe the secondary fragment situation, and find all the secondary fragment m / z of this compound in positive ion mode, so as to determine all the MRM ion pairs in positive ion mode;

[0060] 3) In positive ion mode, use the MRM function, input all the MRM ion pairs determined in 2), optimize the declustering voltage and collision energy respectively, and obtain the ion response intensity of each MRM ion pair under the optimal declustering voltage and collision energy;

[0061] 4) In negative ion mode, input the precursor ion m / z determined in 1), use the product ion mode, preset the declustering voltage to -60 V, set the collision energies to -80, -60, -40, -20, -10 V respectively, observe the secondary fragment situation, and find all the secondary fragment m / z of this compound in negative ion mode, so as to determine all the MRM ion pairs in negative ion mode;

[0062] 5) In negative ion mode, use the MRM function, input all the MRM ion pairs determined in 4), optimize the declustering voltage and collision energy respectively, and obtain the ion response intensity of each MRM ion pair under the optimal declustering voltage and collision energy;

[0063] 6) Summarize all the MRM ion pairs of 50 compounds and the ion response intensity under the optimal declustering voltage and collision energy, select the interference-free characteristic ion pairs for each compound, and when there are multiple characteristic ion pairs for a compound, select the one with higher ion response intensity as the optimal characteristic ion pair;

[0064] 7) According to the above optimization results, determine the MRM ion pairs, declustering voltage and collision energy parameters for the mass spectrometry detection of 50 compounds as shown in Table 1.

[0065] Table 1. MRM ion pairs, declustering voltage and collision energy parameters for 50 compounds

[0066]

[0067]

[0068]

[0069] Finally, the injection interval time was optimized. Using the MRM ion pairs and mass spectrometry parameters determined in Table 1, the injection interval time was adjusted to 0 s, 0.5 s, 1 s, 1.5 s, and 2 s respectively, and the peak emergence of 50 compounds was detected. The half-peak width and the separation between two injections were calculated, and the injection interval time that could achieve complete separation without residue was selected. In this experiment, it was 0.5 s.

[0070] 2. Experimental Results

[0071] After test analysis, the linear equations (with the compound concentration as the abscissa and the daughter ion peak area of the MRM ion pair as the ordinate), linear ranges, correlation coefficients, and quantification limits of the 50-compound standard solution are shown in Table 2.

[0072] Table 2. Standard curves, linear ranges, quantification limits, and precisions of 50 compounds

[0073]

[0074]

[0075]

[0076] It can be seen from the above results that the 50 compounds meet the quantification requirements, show a good linear relationship within the linear range, and can be used to detect the compound content in the fermentation broth or reaction solution with high sensitivity and low quantification limit.

[0077] Example 2: Determination of the Content of 50 Compounds in Corynebacterium glutamicum Fermentation Broth

[0078] The difference between this example and Example 1 is that the sample for injection analysis in this example is Corynebacterium glutamicum fermentation broth, and the preparation method is as follows:

[0079] Take the logarithmic-phase bacterial liquid of Corynebacterium glutamicum and centrifuge it at 6000 rpm for 30 minutes at 4°C. Take 25 μL of the supernatant and transfer it to a dedicated 384-well plate for EchoMS (Sciex, EchoMS-384 plate). After centrifuging the EchoMS plate containing the supernatant at 1500 rpm for 5 minutes, shake it horizontally for 2 minutes, and then put it into the EchoMS acoustic excitation coupled mass spectrometer for content determination according to the mass spectrometry conditions in Example 1.

[0080] After determination, the contents of 50 compounds in the fermentation broth of Corynebacterium glutamicum are shown in Table 3, where "N.D." indicates that the concentration is below the quantification limit.

[0081] Table 3. Contents of 50 Compounds in the Fermentation Broth of Corynebacterium glutamicum

[0082]

[0083]

[0084] Comparative Example 1: Determination of 50 Compounds under the Conditions of the Existing Technology Method

[0085] The difference between this comparative example and Example 2 is that in this comparative example, LC-MS (ultra-high performance liquid chromatography-triple quadrupole mass spectrometer) was used to detect 50 compounds in the fermentation broth described in Example 2. The conditions are as follows:

[0086] Liquid phase conditions:

[0087] Mobile phase: A: 25 mM ammonium acetate, 10 mM ammonia, aqueous solution; B: 100% acetonitrile

[0088] Chromatographic column: waters Xbridge BEH amide column (100 mm × 2.1 mm, 1.7 μm)

[0089] Flow rate: 0.3 mL / min

[0090] Gradient: 0 - 2 min, 95% B; 2 - 18 min, 95 - 65% B; 18 - 20 min, 65 - 40% B: 20 - 22 min, 40% B; 20 - 20.1 min, 40 - 95% B; and 22.1 - 30 min, 95% B.

[0091] Mass spectrometry conditions:

[0092] Ion source parameters: GAS1 55 psi, GAS2 55 psi, curtain gas 35 psi, CAD gas 9 psi, temperature 550 °C, spray voltage 5.5 kV in positive ion ionization mode, spray voltage 4.5 kV in negative ion ionization mode, and multi-reaction monitoring mode MRM scanning; the ion pairs of each target compound to be measured and their corresponding fragmentation voltages and collision energy parameters are the same as in Table 1.

[0093] Using the above conditions to detect 50 compounds, the extracted ion chromatogram is as Figure 1 , and the time required for each sample is 30 min, which is much longer than 20 s in Example 2.

[0094] Comparative Example 2: Detecting glutamic acid under conditions outside the scope of the claims of the present technology

[0095] The difference between this comparative example and Example 1 is that in this comparative example, the types and proportions of the carrier liquids are as follows:

[0096] Carrier liquid 1: 100% methanol, 0% acetonitrile, 0% water

[0097] Carrier liquid 2: 22% methanol, 78% acetonitrile, 0% water

[0098] Carrier liquid 3: 0% methanol, 70% acetonitrile, 30% water.

[0099] From Figure 2 it can be seen that when analyzing glutamic acid at high (10 mM), medium (1 mM), and low concentrations (0.1 mM) using carrier liquids 1, 2, and 3, there are problems such as peak tailing, many burrs, and poor sensitivity. When detecting under the conditions of Example 1, these situations can be effectively improved, and better quantitative results can be obtained.

[0100] When the analyte is part of 50 compounds, on the basis of the carrier liquid in Example 1, additives can be appropriately increased, and the types and proportions of the additives are determined according to the analyte. For example, when the analyte is glutamic acid and aspartic acid, the carrier liquid can be adjusted to 50% methanol, 50% acetonitrile, 0% water, 2 mM ammonium fluoride, 0% formic acid, and higher sensitivity can be obtained.

[0101] Comparative Example 3:

[0102] The difference between this comparative example and Example 1 is that when detecting aspartic acid and glutamic acid, the carrier liquid is: 50% methanol, 50% acetonitrile, 0% water, 2 mM ammonium fluoride, 0% formic acid; any one of the MRM ion pairs in Table 4 is used.

[0103] Table 4. Aspartic acid and glutamic acid ion pairs in Comparative Example 1

[0104]

[0105]

[0106] In addition to the MRM ion pair m / z132→88 (negative ion) in Example 1 for aspartic acid, it can also generate ion pairs such as m / z134→116 (positive ion), m / z134→88 (positive ion), m / z134→74 (positive ion), m / z134→70 (positive ion), m / z132→115 (negative ion), m / z132→71 (negative ion) in Table 4. When detecting aspartic acid, using any of the above MRM ion pairs will also generate signals in the asparagine reference solution, such as Figure 3This is because the molecular weight of aspartic acid, 133.037 Da, is extremely close to the molecular weight of the M+1 isotope peak of asparagine, 133.056 Da, with a difference of only 0.019 Da, which is much smaller than the resolution of triple quadrupole mass spectrometry, 1 Da. Therefore, when the sample contains both aspartic acid and asparagine, the M+1 isotope peak of asparagine will also enter the parent ion channel of MRM. In addition, due to the very similar structures of aspartic acid and asparagine, the M+1 isotope peak of asparagine will also produce daughter ions m / z116, m / z88, m / z74, m / z70, m / z115, m / z71 that are consistent with those of aspartic acid, and the ion response intensity is relatively high, thus interfering with the detection of aspartic acid. Therefore, the MRM ion pairs m / z134→116 (positive ion), m / z134→88 (positive ion), m / z134→74 (positive ion), m / z134→70 (positive ion), m / z132→115 (negative ion), m / z132→71 (negative ion) do not have specificity. In Example 1, the MRM ion pair m / z132→88 (negative ion) is generated by the C3H7O2N- structure of aspartic acid, and the M+1 isotope of asparagine does not have this structure. Therefore, m / z132→88 (negative ion) has specificity and is not easily interfered with, resulting in accurate qualitative and quantitative analysis.

[0107] Similarly, in addition to the MRM ion pair m / z146→102 (negative ion) in Example 1, aspartic acid can also generate ion pairs such as m / z148→130 (positive ion), m / z148→102 (positive ion), m / z148→84 (positive ion), m / z146→128 (negative ion) in Table 4. When detecting glutamic acid, when using any of the above MRM ion pairs, signals will also be generated in the glutamine and lysine reference substances, such as Figure 4This is because the molecular weight of glutamic acid, 147.053 Da, only differs by 0.02 Da from the molecular weight of the M+1 isotope peak of glutamine, 147.073 Da, and by 0.05 Da from the molecular weight of the M+1 isotope peak of lysine, 147.109 Da. Both are less than the resolution of triple quadrupole mass spectrometry, 1 Da. Therefore, when the sample contains glutamine and lysine, the M+1 isotope peaks of glutamine and lysine will also enter the parent ion channel of MRM. Due to the very similar structures of glutamic acid, glutamine, and lysine, the M+1 isotope peak of glutamine will also produce daughter ions m / z130, m / z102 (positive ion), m / z84, and m / z128 that are consistent with aspartic acid. The M+1 isotope peak of lysine will also produce daughter ions m / z130, m / z102, and m / z84 that are consistent with aspartic acid, and the ion response intensity is relatively high, thus interfering with the detection of glutamic acid. Therefore, the MRM ion pairs m / z148→130 (positive ion), m / z148→102 (positive ion), m / z148→84 (positive ion), and m / z146→128 (negative ion) do not have specificity. The MRM ion pair m / z146→102 (negative ion) in Example 1 is generated by the C4H9O2N- structure of glutamic acid. The M+1 isotope of lysine cannot produce C4H9O2N- fragments in the negative ion mode. The M+1 isotope of asparagine can theoretically produce a very small amount of C4H9O2N- fragments under certain mass spectrometry parameters. However, it cannot be detected under the optimized mass spectrometry parameters (declustering voltage -20 V, collision energy -20 V) in Example 1. Therefore, the glutamic acid MRM ion pair m / z132→88 (negative ion) and the mass spectrometry parameters (declustering voltage -20 V, collision energy -20 V) have specificity, are not easily interfered with, and are accurate in qualitative and quantitative analysis.

Claims

1. A method for detecting the content of small molecule compounds in fermentation broth or reaction solution with ultra-high throughput, characterized in that, It includes the following steps: 1) Solid-liquid separation of the fermentation broth or reaction solution to obtain a sample liquid; 2) Prepare a series of standard solutions of the mixed compounds to be measured with different concentrations, using water as the solvent, and perform mass spectrometry detection; Preferably, the mass spectrometry detection conditions are: Carrier liquid: by volume ratio, methanol: acetonitrile = 4-6:6-4, which also contains one or two or three of water with a volume concentration of 0-10%, ammonium fluoride with a concentration of 0-2 mM, and formic acid with a volume concentration of 0-0.1%; Injection interval time: 0-2 s; Mass spectrometry conditions: GAS1 70-90 psi, GAS2 70-90 psi, curtain gas 20-30 psi, CAD gas 6-15 psi, temperature 300-400 °C, spray voltage is 5-6 kV in positive ion ionization mode and 3-5 kV in negative ion ionization mode, and multi-reaction monitoring mode MRM scanning is used; 3) Plot the standard curve of the concentration of the compound to be measured and the peak area of the daughter ion of the MRM ion pair; 4) Inject the sample liquid obtained in step 1) for mass spectrometry detection under the mass spectrometry analysis conditions in step 2), obtain the corresponding parent ion and daughter ion mass-to-charge ratio information, qualitatively confirm the corresponding compound in the sample liquid by finding the corresponding matching parent ion and daughter ion mass-to-charge ratio, and at the same time substitute the peak area of the daughter ion of its MRM ion pair into the standard curve of the corresponding compound in step 3), and the content information of the compound in the fermentation broth or reaction solution can be obtained.

2. The method according to claim 1, wherein Among them, the mass spectrometry detection mode, the corresponding parent ion and daughter ion mass-to-charge ratio information, that is, the MRM ion pair, of the target compound to be measured are determined in advance; Preferably, the specific method is: 1) Detect the standard mother liquor of each compound using positive and negative ion modes respectively, and determine the parent ion m / z and its ion response intensity under positive and negative ions; 2) In the positive ion mode, input the parent ion m / z determined in 1), use the production function, preset the declustering voltage to 60 V, set the collision energies to 80, 60, 40, 20, 10 V respectively, observe the secondary fragment situation, and find all the secondary fragment m / z of the compound in the positive ion mode, so as to determine all the MRM ion pairs in the positive ion mode; 3) In the positive ion mode, use the MRM function, input all the MRM ion pairs determined in 2), optimize the declustering voltage and collision energy respectively, and obtain the ion response intensity of each MRM ion pair under the optimal declustering voltage and collision energy; 4) In the negative ion mode, input the parent ion m / z determined in 1), use the production mode, preset the declustering voltage to -60 V, set the collision energies to -80, -60, -40, -20, -10 V respectively, observe the secondary fragment situation, and find all the secondary fragment m / z of the compound in the negative ion mode, so as to determine all the MRM ion pairs in the negative ion mode; 5) In the negative ion mode, using the MRM function, input all the MRM ion pairs determined in 4), optimize the declustering voltage and collision energy respectively, and obtain the ion response intensity of each MRM ion pair at the optimal declustering voltage and collision energy; summarize the MRM ion pairs of the compound and the ion response intensity at the optimal declustering voltage and collision energy, select the interference-free characteristic ion pairs for a specific compound, and when there are multiple characteristic ion pairs, select the one with a high ion response intensity as the optimal characteristic ion pair.

3. The method according to claim 1, wherein the fermentation broth or reaction solution in step 1) comprises at least one or two or more of microbial fermentation broth or metabolites, enzyme reaction solution, and chemical reaction solution; Preferably, it is microbial fermentation broth or metabolites; the microbial fermentation broth is a fermentation broth obtained by culturing and fermenting microorganisms; the content of the target compound to be measured in the liquid fermentation broth is 0.001 - 10 mg / mL.

4. The method according to claim 3, characterized in that, The compound to be measured in step 2) is metabolites produced by at least one or two or more of animal and plant cells and microorganisms; the concentration of the standard solution of the compound to be measured is 0.001 - 5 mg / mL.

5. The method according to claim 3 or 4, characterized in that, The compound is selected from at least one or two or more of amino acids, organic acids, and sugars; preferably, the amino acid compounds are selected from at least one or two or more of glutamic acid, aspartic acid, and isoleucine.

6. The method according to claim 3 or 4, wherein solid-liquid separation in step 1) can be carried out by centrifugation or filtration; the specific parameters of the centrifugation are a speed of 3000 - 6000 rpm and a time of 5 - 30 minutes; the parameters of filtration are membrane filtration with a pore size of 0.22 μm - 0.45 μm.

7. The detection method according to claim 1, wherein The specific operation of step 2) is: weigh the reference substance of the compound to be measured, dissolve it with a solvent to prepare a standard stock solution of 1 mg / mL; take the standard stock solution and dilute it with a solvent to prepare a standard solution of the target compound to be measured with a concentration of 0.001 - 1 mg / mL.

8. The method according to claim 1, wherein The specific operation of drawing the standard curve in step 3) is to select 3 - 4 different mass concentrations within the mass concentration range of 0.001 - 5 mg / mL to draw the standard curve.

9. The method according to claim 8, wherein Draw the standard curve of the concentration of the compound to be measured and the height of the parent ion peak, and calculate the corresponding linear regression equation, correlation coefficient, quantification limit, precision, and recovery rate.

10. The detection method according to claim 1, characterized in that, The target compound to be measured is one or more of the following, and the corresponding mass spectrometry detection mode, the mass-to-charge ratio information of the corresponding parent ion and daughter ion, that is, the MRM ion pair, is as follows: Preferably, after determining the injection interval time and MRM ion pairs of the target compound to be measured, the detection time for every 4 MRM ion pairs of compounds is 1.5 s.

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