Mass spectrometric detection method for N heterocyclic ketone and / or N heterocyclic chiral amino compound and application of mass spectrometric detection method

Abstract

The invention provides a mass spectrometric detection method for N heterocyclic ketone and / or N heterocyclic chiral amino compounds, and in the mass spectrometric detection method, a declustering voltage value is 20-40V, preferably 30V. And the high-throughput screening method is applied to directional transformation of the functional enzyme, so that the screening efficiency of the functional enzyme mutant is improved.

Description

Technical Field

[0001] The present application relates to the field of detection technology, and in particular to a mass spectrometry detection method for N-heterocyclic ketones and / or N-heterocyclic chiral amino compounds and applications thereof. Background Art

[0002] N-heterocyclic amino compounds can be widely used in the synthesis of pesticides and medicines. Among them, N-heterocyclic chiral aminopyrrolidines, as important intermediates in the synthesis of the fifth-generation cephalosporin antibiotic ceftobiprole, have broad market application prospects. At present, enzymatic synthesis of N-heterocyclic chiral amino compounds is an important development direction. However, the current biocatalytic synthesis has problems such as low wild-type enzyme activity and poor stability, so it is necessary to modify the natural enzyme. Directed evolution is a commonly used protein modification method. It constructs a large number of mutant libraries through random mutations and combines high-throughput screening methods to obtain beneficial mutants. Therefore, establishing a high-throughput screening method for N-heterocyclic chiral amino compounds is a prerequisite for conducting directed evolution of functional enzymes.

[0003] At present, liquid chromatography or gas chromatography is mostly used for the detection of N-heterocyclic chiral amino compounds. The above methods have high detection sensitivity and good reproducibility. However, conventional liquid chromatography or gas chromatography still has shortcomings in trace systems and detection flux, and cannot achieve high-throughput screening. Therefore, the development of high-throughput analysis and detection methods for N-heterocyclic chiral amino compounds is of great significance for the directed evolution of functional enzymes and their application in the efficient synthesis of N-heterocyclic amino compounds. Summary of the invention

[0004] In view of the problems existing in the prior art, the present application provides a mass spectrometry detection method for N-heterocyclic ketones and N-heterocyclic chiral amino compounds. The high-throughput mass spectrometer can realize the high-throughput biological system screening of N-heterocyclic ketones and N-heterocyclic chiral amino compounds in biological experiments, and can quickly and accurately detect the content of N-heterocyclic ketones and N-heterocyclic chiral amino compounds in samples. Ultra-fast, large-scale and highly stable detection can be achieved. The detection method of the present application realizes high-throughput screening of N-heterocyclic ketones and N-heterocyclic chiral amino compounds, and is applied to the directed evolution screening of transaminases, which accelerates the screening efficiency of functional enzyme mutants.

[0005] The specific plan is as follows:

[0006] 1. A mass spectrometry detection method for N-heterocyclic ketones and / or N-heterocyclic chiral amino compounds,

[0007] Wherein, in the mass spectrometry detection method, the declustering voltage value is 20-40V, preferably 30V.

[0008] 2. The mass spectrometry detection method according to item 1, wherein, in the mass spectrometry detection method, the collision energy is 40-60V, preferably 50V.

[0009] 3. The mass spectrometry detection method according to item 1-2, wherein, in the mass spectrometry detection method, the ion spray voltage is: 4000-6000V, preferably 5000V;

[0010] 4. The mass spectrometry detection method according to item 1-3, wherein, in the mass spectrometry detection method, the ion source temperature is 200-400°C, preferably 300°C.

[0011] 5. The mass spectrometry detection method according to item 1-4, wherein, in the mass spectrometry detection method, the mobile phase used is methanol containing 1-5 mM ammonium fluoride; preferably methanol containing 2 mM ammonium fluoride;

[0012] Preferably, the mobile phase flow rate is 250-350 μL / min; preferably 300 μL / min.

[0013] 6. The mass spectrometry detection method according to item 1-5, wherein:

[0014] In the mass spectrometry detection method,

[0015] The ion source carrier gas 1 is: 80-100 psi nitrogen; preferably 90 psi nitrogen;

[0016] The ion source carrier gas 2 is: 40-50 psi nitrogen; preferably 45 psi nitrogen;

[0017] The curtain gas is: 10-30 psi nitrogen, preferably 20 psi nitrogen.

[0018] 7. The mass spectrometry detection method according to item 1, wherein:

[0019] The N-heterocyclic chiral amino compound is selected from one or more of (R)-N-tert-butyloxycarbonyl-3-aminopyrrole, (R)-N-benzyl-3-aminopiperidine, (R)-N-tert-butyloxycarbonyl-3-aminopiperidine, (R)-N-benzyloxycarbonyl-3-aminopiperidine and (R)-3-aminoazepane-1-carboxylic acid tert-butyl ester;

[0020] The N heterocyclic ketone is selected from one or more of N-tert-butyloxycarbonyl-3-pyrrolidone, N-benzyl-3-piperidone, N-tert-butyloxycarbonyl-3-piperidone, N-benzyloxycarbonyl-3-piperidone and N-tert-butyloxycarbonyl-3-azacycloheptanone.

[0021] 8. The mass spectrometry detection method according to item 7, wherein:

[0022] When the substance to be detected is (R)-N-tert-butyloxycarbonyl-3-aminopyrrole, the ion pair information of the mass spectrometry detection method is m / z 187 / 70;

[0023] When the substance to be detected is (R)-N-benzyl-3-aminopiperidine, the ion pair information of the mass spectrometry detection method is m / z 191 / 91;

[0024] When the substance to be detected is (R)-N-tert-butyloxycarbonyl-3-aminopiperidine, the ion pair information of the mass spectrometry detection method is m / z 201 / 84.1;

[0025] When the substance to be detected is (R)-N-benzyloxycarbonyl-3-aminopiperidine, the ion pair information of the mass spectrometry detection method is m / z 235 / 91;

[0026] When the substance to be detected is (R)-3-aminoazepane-1-carboxylic acid tert-butyl ester, the ion pair information of the mass spectrometry detection method is m / z 215 / 89.9;

[0027] When the substance to be detected is N-tert-butyloxycarbonyl-3-pyrrolidone, the ion pair information of the mass spectrometry detection method is m / z 184 / 109;

[0028] When the substance to be detected is N-benzyl-3-piperidone, the ion pair information of the mass spectrometry detection method is m / z190 / 90;

[0029] When the substance to be detected is N-tert-butyloxycarbonyl-3-piperidone, the ion pair information of the mass spectrometry detection method is m / z 200 / 89.9;

[0030] When the substance to be detected is N-benzyloxycarbonyl-3-piperidone, the ion pair information of the mass spectrometry detection method is m / z 234 / 91;

[0031] When the substance to be detected is N-tert-butyloxycarbonyl-3-azacycloheptanone, the ion pair information of the mass spectrometry detection method is m / z 214 / 89.9.

[0032] 9. A method for high-throughput screening of directed evolution of transaminases using the mass spectrometry detection method described in any one of items 1-8.

[0033] Technical Effects

[0034] The detection method of this application has a short detection time, 3s to detect one sample, which is 280 times faster than the traditional detection of 14min to detect one sample; this detection method can detect 384 / 1536 samples at a time to achieve rapid high-throughput detection; in high-throughput detection, the recovery rate is between 87%-98%, and the RSD is less than 5%, with high sensitivity and good reproducibility. This detection method was used for the first time to perform mass spectrometry quantitative analysis of N-heterocyclic ketones and N-heterocyclic chiral amino compounds, and was successfully applied to high-throughput rapid screening of transaminases, accelerating the screening speed of mutants with improved performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 shows The molecular structures of N-heterocyclic chiral amino compounds and N-heterocyclic ketones are given;

[0036] Figure 2 A standard curve of (R)-N-tert-butyloxycarbonyl-3-aminopyrrole compound content is shown;

[0037] Figure 3 The results of screening transaminase mutants using the detection method of the present application are shown. DETAILED DESCRIPTION

[0038] The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

[0039] Unless otherwise defined, the technical and scientific terms in this specification have the same meaning as those generally understood by those skilled in the art. Although similar or identical methods and materials as described herein may be used in experiments or actual uses, the materials and methods are described herein below. In the event of a conflict, the present specification including the definitions therein shall prevail. In addition, the materials, methods and examples are for illustration only and are not limiting. The present application is further described below in conjunction with specific examples, but is not intended to limit the scope of the present application.

[0040] The present application provides a mass spectrometry detection method for N-heterocyclic ketones and / or N-heterocyclic chiral amino compounds, in which the declustering voltage value is 20-40 V, preferably 30 V. For example, the declustering voltage can be 20 V, 22 V, 24 V, 26 V, 28 V, 30 V, 32 V, 34 V, 36 V, 38 V, 40 V or any range therebetween.

[0041] In the present application, when the substance to be detected is (R)-N-tert-butyloxycarbonyl-3-aminopyrrole, the ion pair information of the mass spectrometry detection method is m / z 187 / 70.

[0042] In the present application, when the substance to be detected is (R)-N-benzyl-3-aminopiperidine, the ion pair information of the mass spectrometry detection method is m / z 191 / 91.

[0043] In the present application, when the substance to be detected is (R)-N-tert-butyloxycarbonyl-3-aminopiperidine, the ion pair information of the mass spectrometry detection method is m / z 201 / 84.1.

[0044] In the present application, when the substance to be detected is (R)-N-benzyloxycarbonyl-3-aminopiperidine, the ion pair information of the mass spectrometry detection method is m / z 235 / 91.

[0045] In the present application, when the substance to be detected is (R)-3-aminoazepane-1-carboxylic acid tert-butyl ester, the ion pair information of the mass spectrometry detection method is m / z 215 / 89.9.

[0046] In the present application, when the substance to be detected is N-tert-butyloxycarbonyl-3-pyrrolidone, the ion pair information of the mass spectrometry detection method is m / z 184 / 109.

[0047] In the present application, when the substance to be detected is N-benzyl-3-piperidone, the ion pair information of the mass spectrometry detection method is m / z 190 / 90.

[0048] In the present application, when the substance to be detected is N-tert-butyloxycarbonyl-3-piperidone, the ion pair information of the mass spectrometry detection method is m / z 200 / 89.9.

[0049] In the present application, when the substance to be detected is N-benzyloxycarbonyl-3-piperidone, the ion pair information of the mass spectrometry detection method is m / z 234 / 91.

[0050] In the present application, when the substance to be detected is N-tert-butyloxycarbonyl-3-azacycloheptanone, the ion pair information of the mass spectrometry detection method is m / z 214 / 89.9.

[0051] In the present application, the detection method comprises the following steps: preparation of a test solution: dissolving an N-heterocyclic compound in ethanol, adding ten times the volume of a pure water solution, and vortexing to obtain a test solution of the N-heterocyclic compound; determination: performing high-throughput mass spectrometry detection on the test solution containing the N-heterocyclic compound to analyze the N-heterocyclic compound. The N-heterocyclic compound is an N-heterocyclic ketone or an N-heterocyclic chiral amino compound.

[0052] In some embodiments of the present application, in the mass spectrometry detection method, the declustering voltage value is 20-40 V, preferably 30 V. For example, the declustering voltage can be 20 V, 22 V, 24 V, 26 V, 28 V, 30 V, 32 V, 34 V, 36 V, 38 V, 40 V or any range therebetween.

[0053] In some embodiments of the present application, in the mass spectrometry detection method, the collision energy is 40-60 V, preferably 50 V. For example, the collision energy CE can be 40 V, 42 V, 44 V, 46 V, 48 V, 50 V, 52 V, 54 V, 56 V, 58 V, 60 V or any range therebetween.

[0054] In some embodiments of the present application, in the mass spectrometry detection method, the ion spray voltage is: 4000-6000 V, preferably 5000 V. For example, the ion spray voltage is: 4000 V, 4200 V, 4400 V, 4600 V, 4800 V, 5000 V, 5200 V, 5400 V, 5600 V, 5800 V, 6000 V or any range therebetween.

[0055] In some embodiments of the present application, in the mass spectrometry detection method, the temperature is 200-400° C., preferably 300° C. For example, the temperature can be 200° C., 220° C., 240° C., 260° C., 280° C., 300° C., 320° C., 340° C., 360° C., 380° C., 400° C. or any range therebetween.

[0056] In some embodiments of the present application, in the mass spectrometry detection method, the mobile phase is methanol containing 1-5 mM ammonium fluoride, preferably 2 mM ammonium fluoride. For example, the mobile phase can be 1 mM ammonium fluoride in methanol, 2 mM ammonium fluoride in methanol, 3 mM ammonium fluoride in methanol, 4 mM ammonium fluoride in methanol, 5 mM ammonium fluoride in methanol, or any range therebetween.

[0057] In some embodiments of the present application, in the mass spectrometry detection method, the mobile phase flow rate is 250-350 μL / min, preferably 300 μL / min. For example, the mobile phase flow rate can be 250 μL / min, 260 μL / min, 270 μL / min, 280 μL / min, 290 μL / min, 300 μL / min, 310 μL / min, 320 μL / min, 330 μL / min, 340 μL / min, 350 μL / min or any range therebetween.

[0058] In some embodiments of the present application, in the mass spectrometry detection method, the ion source carrier gas 1 is: 80-100 psi nitrogen, preferably 90 psi nitrogen; for example, the ion source carrier gas 1 can be 80 psi nitrogen, 82 psi nitrogen, 84 psi nitrogen, 86 psi nitrogen, 88 psi nitrogen, 90 psi nitrogen, 92 psi nitrogen, 94 psi nitrogen, 96 psi nitrogen, 98 psi nitrogen, 100 psi nitrogen or any range therebetween.

[0059] In some embodiments of the present application, in the mass spectrometry detection method, the ion source carrier gas 2 is: 40-50psi nitrogen, preferably 45psi nitrogen; for example, the ion source carrier gas 2 can be 40psi nitrogen, 41psi nitrogen, 42psi nitrogen, 43psi nitrogen, 44psi nitrogen, 45psi nitrogen, 46psi nitrogen, 47psi nitrogen, 48psi nitrogen, 49psi nitrogen, 50psi nitrogen or any range therebetween.

[0060] In some embodiments of the present application, in the mass spectrometry detection method, the curtain gas is: 10-30 psi nitrogen, preferably 20 psi nitrogen; for example, the curtain gas can be 10 psi nitrogen, 12 psi nitrogen, 14 psi nitrogen, 16 psi nitrogen, 18 psi nitrogen, 20 psi nitrogen, 22 psi nitrogen, 24 psi nitrogen, 26 psi nitrogen, 28 psi nitrogen, 30 psi nitrogen or any range therebetween.

[0061] In some embodiments of the present application, the mass spectrometry detection conditions are: ion spray voltage: 4000-6000V; ion source carrier gas 1: 80-100psi nitrogen; ion source carrier gas 2: 40-50psi nitrogen; curtain gas: 10-30psi nitrogen; ion source temperature: 200-400°C, declustering voltage: 20-40V, collision energy (CE): 40-60V, the mobile phase used is methanol containing 1-5mM ammonium fluoride, and the mobile phase flow rate is 250-350μL / min.

[0062] In some embodiments of the present application, the preparation of the standard solution is as follows: pure water is added to the N-heterocyclic ketone and N-heterocyclic chiral amino compound standards dissolved in ethanol to prepare at least seven concentrations of standard working solutions; the detection is performed, and the concentration is used as the horizontal coordinate and the peak area is used as the vertical coordinate to establish a standard curve. For example, the (R)-N-tert-butyloxycarbonyl-3-aminopyrrole standard is weighed, dissolved in ethanol, a standard stock solution is obtained, and ethanol is used to dilute to the required concentration in proportion to obtain at least seven concentrations of standard working solutions; after the pretreatment step, the high-throughput mass spectrometry is used for detection, and the detection conditions are ion pair information 187 / 70m / z; ion spray voltage is: 5000V; ion source carrier gas 1 is: 90psi nitrogen; ion source carrier gas 2 is: 45psi nitrogen; curtain gas is: 20psi nitrogen, the temperature is 300°C, the declustering voltage is: 30V, the collision energy CE is: 50V, and the mobile phase used is methanol containing 2mM ammonium fluoride. A standard curve y=6441.6x+1631.3 was established with concentration as the horizontal axis and peak area as the vertical axis.

[0063] In some embodiments of the present application, there is no limitation on the source of the N-heterocyclic chiral amino compound or N-heterocyclic ketone, for example, it can be obtained by mutation of fermentation broth.

[0064] In one embodiment of the present application, the fermentation broth of the transaminase mutant is added to an aqueous solution containing pyrrolidine and isopropylamine to carry out a catalytic reaction, and the test solution of (R)-1-Boc-3-aminopyrrolidine is obtained by centrifugation; determination: the test solution of (R)-1-Boc-3-aminopyrrolidine is subjected to mass spectrometry detection, and aminopyrrolidine is analyzed.

[0065] In the present application, there is no limitation on the source of the aminotransferase mutant, as long as the purpose of the present application is met. The aminotransferase can be derived from photosynthetic bacteria, Bacillus subtilis, Streptomyces cinnamomi, Streptomyces hygroscopicus or other bacteria.

[0066] In the present application, there is no limitation on the mutation method, as long as it is a mutation method known to those skilled in the art.

[0067] In the present application, the fermentation broth of the aminotransferase mutant is used as a catalyst to catalyze the reaction of an N-containing heterocyclic ketone or an N-containing heterocyclic chiral amino compound.

[0068] In the present application, the fermentation broth of the aminotransferase mutant is used as a catalyst to catalyze the reaction of pyrrolidine and isopropylamine to generate (R)-1-Boc-3-aminopyrrolidine.

[0069] In some embodiments of the present application, when the OD value of the fermentation broth is 1 to 3, the amount of the fermentation broth added is 0.075 g / 1 mL to 0.15 g / 1 mL. For example, the amount of the fermentation broth added can be 0.075 g / 1 mL, 0.08 g / 1 mL, 0.085 g / 1 mL, 0.09 g / 1 mL, 0.095 g / 1 mL, 0.105 g / 1 mL, 0.11 g / 1 mL, 0.115 g / 1 mL, 0.12 g / 1 mL, 0.125 g / 1 mL, 0.13 g / 1 mL, 0.135 g / 1 mL, 0.14 g / 1 mL, 0.145 g / 1 mL, 0.15 g / 1 mL or any range therebetween.

[0070] In some embodiments of the present application, the amount of isopropylamine added is 0.03 g / 1 mL to 0.05 g / 1 mL. For example, the amount of isopropylamine added is 0.03 g / 1 mL, 0.035 g / 1 mL, 0.04 g / 1 mL, 0.045 g / 1 mL, 0.05 g / 1 mL, or any range therebetween.

[0071] In some embodiments of the present application, the molar ratio of pyrrolidone to isopropylamine is 1:1.

[0072] The present application provides the above method to establish a high-throughput screening method for the directional modification of transaminase. Specifically, the mutant obtained by random mutation of transaminase is fermented in a 96-well plate to obtain a fermentation broth, and the OD value of the fermentation broth is about 2. Add fermentation broth to 1mL system, containing N-heterocyclic ketones and N-heterocyclic chiral amino compounds as reactants, water as solvent, and carry out catalytic reaction. The reaction is about 20min, and the reaction generates the target. After centrifugation, the supernatant is taken and transferred to a 384-well plate. Echo MS is used to detect and analyze the target N-heterocyclic ketones and N-heterocyclic chiral amino compounds. Each sample is tested 3 times to ensure that the results are accurately reproduced, and mutants with improved performance are screened out according to the content of the generated target.

[0073] Example

[0074] Example 1: Mass spectrometry detection method for (R)-N-tert-butyloxycarbonyl-3-aminopyrrole

[0075] Weigh (R)-N-tert-butyloxycarbonyl-3-aminopyrrole standard, dissolve it in ethanol to obtain a standard solution of the same concentration; add 7 times the reaction solution to the standard solution to prepare a standard solution, and use SCIEX Echo-MS high-throughput mass spectrometer to determine the setting of ion spray voltage: 5000V; ion source carrier gas 1: 90psi nitrogen; ion source carrier gas 2: 45psi nitrogen; curtain gas: 20psi nitrogen, ion source temperature: 300°C, declustering voltage: 30V, collision energy: 50V; the mobile phase used is methanol containing 2mM ammonium fluoride, and the flow rate is: 300μL / min. In Q1 mode, the scan range was set to 50-250Da for full scan, and the parent ion information was determined to be m / z 187; secondly, in Product Ion mode, the scan range was set to 10-200Da, and the product ion fragment information was determined to be m / z 70; finally, in MRM mode, the ion pair information of the compound was determined to be m / z 187 / 70. It was verified by experiments that the ion pair information determined was m / z 187 / 70 when the mobile phase contained different contents of ammonium fluoride in methanol and at different flow rates.

[0076] Under the above method, the ion pair was set to m / z 187, and the (R)-N-tert-butyloxycarbonyl-3-aminopyrrole standard was detected to have an obvious signal peak, confirming that this method meets the requirements for rapid detection of (R)-N-tert-butyloxycarbonyl-3-aminopyrrole by high-throughput mass spectrometry.

[0077] Example 2: Mass spectrometry standard curve for (R)-N-tert-butyloxycarbonyl-3-aminopyrrole

[0078] Weigh the (R)-N-tert-butyloxycarbonyl-3-aminopyrrole standard, dissolve it in ethanol to obtain a standard stock solution, dilute it with pure water to the required concentration in proportion, and obtain at least seven concentrations of standard working solutions; respectively transfer the same volume of the standard working solution to a 384-well plate, cover it with a sealing film, centrifuge it at 4000 rpm for 5 min, and use the high-throughput mass spectrometry detection method in Example 1 to draw a standard curve with concentration as the horizontal coordinate and peak area as the vertical coordinate: y=6441.6x+1631.3.

[0079] Example 3 Determination of Declustering Voltage in Mass Spectrometry Detection Conditions for (R)-N-tert-Butyloxycarbonyl-3-aminopyrrole

[0080] The (R)-N-tert-butyloxycarbonyl-3-aminopyrrole standard was weighed and dissolved in ethanol to obtain a standard solution of the same concentration; 7 times the reaction solution was added to the standard solution, and after the pretreatment step, the detection conditions described in Example 1 were referred to, except that the declustering voltage values ​​were set to 15, 20, 30, 40, and 45 for detection, as shown in Table 1. The relative error and reproducibility (CV) between the detection value and the theoretical value of the same concentration of (R)-N-tert-butyloxycarbonyl-3-aminopyrrole at different declustering voltage values ​​were calculated. When the declustering voltage value is 15, the relative error is 21.76%, the CV value is 2.31%, the result is poor in accuracy and good in reproducibility; when the declustering voltage value is 20, the relative error is 8.33%, the CV value is 1.05%, the result is poor in accuracy and good in reproducibility; when the declustering voltage value is 30, the relative error is 1.85%, the CV value is 1.04%, the result is the best in accuracy and reproducibility; when the declustering voltage value is 40, the relative error is 3.33%, the CV value is 1.09%, the result is poor in accuracy and good in reproducibility; when the declustering voltage value is 45, the relative error is 9.52%, the CV value is 1.43%, the result is poor in accuracy and good in reproducibility. The results show that when the declustering voltage is 20-40, the accuracy is high and the reproducibility is good. And when the declustering voltage is 30, it is the optimal condition, which meets the accuracy of the method and can accurately represent the content of (R)-N-tert-butyloxycarbonyl-3-aminopyrrole in the sample.

[0081] Table 1. Optimization of declustering voltage for (R)-N-tert-butyloxycarbonyl-3-aminopyrrole detection method

[0082]

[0083] The theoretical values ​​in Table 1 are the theoretical concentrations of (R)-N-tert-butoxycarbonyl-3-aminopyrrole, and the detected values ​​are the concentrations of (R)-N-tert-butoxycarbonyl-3-aminopyrrole detected under the detection conditions.

[0084] Example 4 Determination of Collision Energy in Mass Spectrometry Detection Conditions for (R)-N-tert-Butyloxycarbonyl-3-aminopyrrole

[0085] The detection conditions described in Example 1 were referred to, wherein the collision energies were set to 35, 40, 50, 60, and 65 for detection, and the differences were shown in Table 2. The relative error and reproducibility (CV) between the detection value and the theoretical value of the same concentration of (R)-N-tert-butyloxycarbonyl-3-aminopyrrole at different collision energies were calculated. When the collision energy is 35, the relative error is 72%, the CV value is 2.65%, the result accuracy is poor, and the reproducibility is good; when the collision energy is 40, the relative error is 3.92%, the CV value is 1.44%, the result accuracy is poor, and the reproducibility is good; when the collision energy is 50, the relative error is 1.85%, the CV value is 1.04%, the result accuracy is the best, and the reproducibility is the best; when the collision energy is 60, the relative error is 2.68%, the CV value is 2.05%, the result accuracy is poor, and the reproducibility is good; when the collision energy is 65, the relative error is 16.84%, the CV value is 2.33%, the result accuracy is poor, and the reproducibility is good; the results show that when the collision energy is 40-60, the accuracy is high and the reproducibility is good. And when the collision energy is 50, it is the optimal condition, which meets the accuracy of the method and can accurately represent the content of (R)-N-tert-butyloxycarbonyl-3-aminopyrrole in the sample.

[0086] Table 2 Collision energy optimization of (R)-N-tert-butyloxycarbonyl-3-aminopyrrole detection method

[0087]

[0088] Example 5 Determination of collision energy in mass spectrometry detection conditions for (R)-N-tert-butyloxycarbonyl-3-aminopyrrole Response value and reproducibility of detection method at different flow rates

[0089] Weigh (R)-1-Boc-3-aminopyrrolidine standard, dissolve in ethanol to obtain standard solution of the same concentration; add 7 times of reaction solution to the standard solution, and after pretreatment steps, refer to the detection conditions described in Example 1, wherein the flow rates are set to 200 μL / min, 250 μL / min, 300 μL / min, 350 μL / min, and 400 μL / min for detection, and the differences are shown in Table 3. Verify the response value and reproducibility (CV) of the same concentration of (R)-1-Boc-3-aminopyrrolidine at different flow rates, the concentration is 25 mM. When the flow rate is 200, the response value is 3.2e4, the CV value is 5.21%, the response value is low, and the reproducibility is poor; when the flow rate is 250, the response value is 3.0e5, the CV value is 3.55%, the response value is low, and the reproducibility is poor; when the flow rate is 300, the response value is 5.3e5, the CV value is 1.02%, the response value is high, and the reproducibility is good; when the flow rate is 350, the response value is 4.1e5, the CV value is 1.06%, the response value is good, and the reproducibility is good; when the flow rate is 400, the response value is 4.2e4, the CV value is 4.63%, the response value is low, and the reproducibility is poor; the results show that when the flow rate is between 250-350μL / min, the response value and reproducibility are good. And when the flow rate is set to 300μL / min, it is the optimal condition, with both a high response value and the best reproducibility.

[0090] Table 3 Optimization comparison of (R)-1-Boc-3-aminopyrrolidine detection method (flow rate)

[0091]

[0092]

[0093] Example 6: (R)-N-tert-butyloxycarbonyl-3-aminopyrrole high-throughput mass spectrometry detection method and feasibility verification

[0094] By optimizing the declustering voltage and collision energy, the ion spray voltage was determined to be 5000 V; ion source carrier gas 1 was 90 psi nitrogen; ion source carrier gas 2 was 45 psi nitrogen; curtain gas was 20 psi nitrogen, the ion source temperature was 300°C, the declustering voltage was 30 V, the collision energy was 50 V, the flow rate was 300 μL / min, the mobile phase used was methanol containing 2 mM ammonium fluoride, and the ion pair was set to 187 / 70.

[0095] The injection volume in the Echo injection system is 2.5 nL, and the detection rate is 3 seconds per sample.

[0096] For standard detection, the limit of detection (LOD), limit of quantification (LOQ), recovery and precision were calculated. By adding different concentrations of standard to the blank matrix, the measurement signal was compared with that of the blank sample, and the concentrations of the standard solution at the signal-to-noise ratio S / N=3 and S / N=10 were defined as the method LOD and LOQ, respectively. The experimental results showed that the LOD and LOQ of the target were close, which were 0.2mM and 0.71mM, respectively. Three standard solution concentrations of low (0.98mM), medium (1.9mM), and high (2.3mM) were set up, and 6 groups of parallels were set up for each. The recovery was determined and the relative standard deviation (RSD) was calculated to evaluate the accuracy and precision of the method. The results are shown in Table 4.

[0097] Table 4. Sensitivity, accuracy and precision of (R)-N-tert-butyloxycarbonyl-3-aminopyrrole detection method

[0098]

[0099] The initial concentration in Table 4 is the initial concentration of (R)-N-tert-butyloxycarbonyl-3-aminopyrrolidine, and the added concentration is the added concentration of (R)-N-tert-butyloxycarbonyl-3-aminopyrrolidine. The detection concentration is the concentration of (R)-N-tert-butyloxycarbonyl-3-aminopyrrolidine detected under the detection conditions. As shown in Table 4, the recovery rate in the sample is between 87% and 98%, and the RSD is less than 5%, indicating that the recovery rate and precision are good.

[0100] Example 7: (R)-N-benzyl-3-aminopiperidine high-throughput mass spectrometry detection method and feasibility verification

[0101] The ion spray voltage was set to 5000V; the ion source carrier gas 1 was set to 90psi nitrogen; the ion source carrier gas 2 was set to 45psi nitrogen; the curtain gas was set to 20psi nitrogen, the ion source temperature was 300℃, the declustering voltage was 30V, the collision energy was 50V, the flow rate was 300μL / min, and the mobile phase used was methanol containing 2mM ammonium fluoride. In Q1 mode, the scan range was set to 50-250Da for full scan, and the parent ion information was determined to be m / z 191; secondly, in Product Ion mode, the scan range was set to 10-200Da, and the daughter ion fragment information was determined to be m / z 91; finally, in MRM mode, the ion pair information of the compound was determined to be m / z 191 / 91. After experimental verification, the ion pair information determined was m / z 191 / 91 in methanol mobile phases with different contents of ammonium fluoride and different flow rates.

[0102] The injection volume in the Echo injection system is 2.5 nL, and the detection rate is 3 seconds per sample.

[0103] For standard detection, the limit of detection (LOD), limit of quantification (LOQ), recovery and precision were calculated. By adding different concentrations of standard to the blank matrix, the measurement signal was compared with that of the blank sample, and the concentrations of the standard solution at the signal-to-noise ratio S / N=3 and S / N=10 were defined as the method LOD and LOQ, respectively. The experimental results showed that the LOD and LOQ of the target were close, which were 0.45mM and 0.8mM, respectively. Three standard solution concentrations of low (1mM), medium (3mM), and high (5mM) were set up, and 6 groups of parallels were set up for each. The recovery was determined and the relative standard deviation (RSD) was calculated to evaluate the accuracy and precision of the method. The results are shown in Table 5.

[0104] Table 5. Sensitivity, accuracy and precision of (R)-N-benzyl-3-aminopiperidine detection method

[0105]

[0106] The initial concentration in Table 5 is the initial concentration of (R)-N-benzyl-3-aminopiperidine, and the added concentration is the added concentration of (R)-N-benzyl-3-aminopiperidine. The detection concentration is the concentration of (R)-N-benzyl-3-aminopiperidine detected under the detection conditions. As shown in Table 5, the recovery rate in the sample is between 85% and 91%, and the RSD is less than 5%, indicating that the recovery rate and precision are good.

[0107] Example 8: (R)-N-tert-butyloxycarbonyl-3-aminopiperidine high-throughput mass spectrometry detection method and feasibility verification

[0108] The ion spray voltage was set to 5000V; the ion source carrier gas 1 was set to 90psi nitrogen; the ion source carrier gas 2 was set to 45psi nitrogen; the curtain gas was set to 20psi nitrogen, the ion source temperature was 300℃, the declustering voltage was 30V, the collision energy was 50V, the flow rate was 300μL / min, and the mobile phase used was methanol containing 2mM ammonium fluoride. In Q1 mode, the scan range was set to 50-250Da for full scan, and the parent ion information was determined to be m / z 201; secondly, in Product Ion mode, the scan range was set to 10-200Da, and the daughter ion fragment information was determined to be m / z 84.1; finally, in MRM mode, the ion pair information of the compound was determined to be m / z 201 / 84.1. After experimental verification, the ion pair information determined was m / z 201 / 84.1 when the methanol mobile phase had different contents of ammonium fluoride and different flow rates.

[0109] The injection volume in the Echo injection system is 2.5 nL, and the detection rate is 3 seconds per sample.

[0110] For standard detection, the limit of detection (LOD), limit of quantification (LOQ), recovery and precision were calculated. By adding different concentrations of standard to the blank matrix, the measurement signal was compared with that of the blank sample, and the concentrations of the standard solution at the signal-to-noise ratio S / N=3 and S / N=10 were defined as the method LOD and LOQ, respectively. The experimental results showed that the target LOD and LOQ were close, which were 0.11mM and 0.94mM, respectively. Three standard solution concentrations of low (1mM), medium (3mM), and high (5mM) were set up, and 6 groups of parallels were set up for each. The recovery was determined and the relative standard deviation (RSD) was calculated to evaluate the accuracy and precision of the method. The results are shown in Table 6.

[0111] Table 6. Sensitivity, accuracy and precision of (R)-N-tert-butyloxycarbonyl-3-aminopiperidine detection method

[0112]

[0113] The initial concentration in Table 6 is the initial concentration of (R)-N-tert-butyloxycarbonyl-3-aminopiperidine, and the added concentration is the added concentration of (R)-N-tert-butyloxycarbonyl-3-aminopiperidine. The detection concentration is the concentration of (R)-N-tert-butyloxycarbonyl-3-aminopiperidine detected under the detection conditions. As shown in Table 6, the recovery rate in the sample is between 92% and 96%, and the RSD is less than 5%, indicating that the recovery rate and precision are good.

[0114] Example 9: (R)-N-Benzyloxycarbonyl-3-aminopiperidine high-throughput mass spectrometry detection method and feasibility verification

[0115] The ion spray voltage was set to 5000V; the ion source carrier gas 1 was set to 90psi nitrogen; the ion source carrier gas 2 was set to 45psi nitrogen; the curtain gas was set to 20psi nitrogen, the ion source temperature was 300℃, the declustering voltage was 30V, the collision energy was 50V, the flow rate was 300μL / min, and the mobile phase used was methanol containing 2mM ammonium fluoride. In Q1 mode, the scan range was set to 50-300Da for full scan, and the parent ion information was determined to be m / z 235; secondly, in Product Ion mode, the scan range was set to 10-200Da, and the daughter ion fragment information was determined to be m / z 91; finally, in MRM mode, the ion pair information of the compound was determined to be m / z 235 / 91. After experimental verification, the ion pair information determined was m / z 235 / 91 when the methanol mobile phase had different contents of ammonium fluoride and different flow rates.

[0116] The injection volume in the Echo injection system is 2.5 nL, and the detection rate is 3 seconds per sample.

[0117] For standard detection, the limit of detection (LOD), limit of quantification (LOQ), recovery and precision were calculated. By adding different concentrations of standard to the blank matrix, the measurement signal was compared with that of the blank sample, and the concentrations of the standard solution at the signal-to-noise ratio S / N=3 and S / N=10 were defined as the method LOD and LOQ, respectively. The experimental results showed that the LOD and LOQ of the target were close, which were 0.33mM and 1.01mM, respectively. Three standard solution concentrations of low (1mM), medium (3mM), and high (5mM) were set up, and 6 groups of parallels were set up for each. The recovery was determined and the relative standard deviation (RSD) was calculated to evaluate the accuracy and precision of the method. The results are shown in Table 7.

[0118] Table 7. Sensitivity, accuracy and precision of (R)-N-benzyloxycarbonyl-3-aminopiperidine detection method

[0119]

[0120] The initial concentration in Table 7 is the initial concentration of (R)-N-benzyloxycarbonyl-3-aminopiperidine, and the added concentration is the added concentration of (R)-N-benzyloxycarbonyl-3-aminopiperidine. The detection concentration is the concentration of (R)-N-benzyloxycarbonyl-3-aminopiperidine detected under the detection conditions. As shown in Table 7, the recovery rate in the sample is between 85% and 94%, and the RSD is less than 5%, indicating that the recovery rate and precision are good.

[0121] Example 10: High-throughput mass spectrometry detection method and feasibility verification of (R)-3-aminoazepane-1-carboxylic acid tert-butyl ester

[0122] The ion spray voltage was set to 5000V; ion source carrier gas 1 was set to 90psi nitrogen; ion source carrier gas 2 was set to 45psi nitrogen; curtain gas was set to 20psi nitrogen, ion source temperature was 300℃, declustering voltage was set to 30V, collision energy was set to 50V, flow rate was set to 300μL / min, and the mobile phase used was methanol containing 2mM ammonium fluoride. In Q1 mode, the scan range was set to 50-250Da for full scan, and the parent ion information was determined to be m / z 215; secondly, in Product Ion mode, the scan range was set to 10-200Da, and the daughter ion fragment information was determined to be m / z 89.9; finally, in MRM mode, the ion pair information of the compound was determined to be m / z 215 / 89.9. After experimental verification, the ion pair information determined was m / z 215 / 89.9 when the mobile phase contained different contents of ammonium fluoride and at different flow rates.

[0123] The injection volume in the Echo injection system is 2.5 nL, and the detection rate is 3 seconds per sample.

[0124] For standard detection, the limit of detection (LOD), limit of quantification (LOQ), recovery and precision were calculated. By adding different concentrations of standard to the blank matrix, the measurement signal was compared with that of the blank sample, and the concentrations of the standard solution at the signal-to-noise ratio S / N=3 and S / N=10 were defined as the method LOD and LOQ, respectively. The experimental results showed that the target LOD and LOQ were close, which were 0.64mM and 1.33mM, respectively. Three standard solution concentrations of low (1mM), medium (3mM), and high (5mM) were set up, and 6 groups of parallels were set up for each. The recovery was determined and the relative standard deviation (RSD) was calculated to evaluate the accuracy and precision of the method. The results are shown in Table 8.

[0125] Table 8. Sensitivity, accuracy and precision of the detection method for (R)-3-aminoazepane-1-carboxylic acid tert-butyl ester

[0126]

[0127] The initial concentration in Table 8 is the initial concentration of (R)-3-aminoazepane-1-carboxylic acid tert-butyl ester, and the added concentration is the added concentration of (R)-3-aminoazepane-1-carboxylic acid tert-butyl ester. The detection concentration is the concentration of (R)-3-aminoazepane-1-carboxylic acid tert-butyl ester detected under the detection conditions. As can be seen from Table 8, the recovery rate in the sample is between 86%-95%, and the RSD is less than 5%, indicating that the recovery rate and precision are good.

[0128] Example 11: High-throughput mass spectrometry detection method for N-tert-butyloxycarbonyl-3-pyrrolidone and feasibility verification

[0129] The ion spray voltage was set to 5000V; the ion source carrier gas 1 was set to 90psi nitrogen; the ion source carrier gas 2 was set to 45psi nitrogen; the curtain gas was set to 20psi nitrogen, the ion source temperature was 300℃, the declustering voltage was 30V, the collision energy was 50V, the flow rate was 300μL / min, and the mobile phase used was methanol containing 2mM ammonium fluoride. In Q1 mode, the scan range was set to 50-250Da for full scan, and the parent ion information was determined to be m / z 184; secondly, in Product Ion mode, the scan range was set to 10-200Da, and the daughter ion fragment information was determined to be m / z 109; finally, in MRM mode, the ion pair information of the compound was determined to be m / z 184 / 109. After experimental verification, the ion pair information determined was m / z 184 / 109 when the methanol mobile phase had different contents of ammonium fluoride and different flow rates.

[0130] The injection volume in the Echo injection system is 2.5 nL, and the detection rate is 3 seconds per sample.

[0131] For standard detection, the limit of detection (LOD), limit of quantification (LOQ), recovery and precision were calculated. By adding different concentrations of standard to the blank matrix, the measurement signal was compared with that of the blank sample, and the concentrations of the standard solution at the signal-to-noise ratio S / N=3 and S / N=10 were defined as the method LOD and LOQ, respectively. The experimental results showed that the target LOD and LOQ were close, which were 0.11mM and 0.65mM, respectively. Three standard solution concentrations of low (1mM), medium (3mM), and high (5mM) were set up, and 6 groups of parallels were set up for each. The recovery was determined and the relative standard deviation (RSD) was calculated to evaluate the accuracy and precision of the method. The results are shown in Table 9.

[0132] Table 9. Sensitivity, accuracy and precision of N-tert-butyloxycarbonyl-3-pyrrolidone detection method

[0133]

[0134] The initial concentration in Table 9 is the initial concentration of N-tert-butyloxycarbonyl-3-pyrrolidone, and the added concentration is the added concentration of N-tert-butyloxycarbonyl-3-pyrrolidone. The detected concentration is the concentration of N-tert-butyloxycarbonyl-3-pyrrolidone detected under the detection conditions. As shown in Table 9, the recovery rate in the sample is between 88% and 98%, and the RSD is less than 5%, indicating that the recovery rate and precision are good.

[0135] Example 12: High-throughput mass spectrometry detection method and feasibility verification of N-benzyl-3-piperidone

[0136] The ion spray voltage was set to 5000V; the ion source carrier gas 1 was set to 90psi nitrogen; the ion source carrier gas 2 was set to 45psi nitrogen; the curtain gas was set to 20psi nitrogen, the ion source temperature was 300℃, the declustering voltage was 30V, the collision energy was 50V, the flow rate was 300μL / min, and the mobile phase used was methanol containing 2mM ammonium fluoride. In Q1 mode, the scan range was set to 50-250Da for full scan, and the parent ion information was determined to be m / z 190; secondly, in Product Ion mode, the scan range was set to 10-200Da, and the daughter ion fragment information was determined to be m / z 91; finally, in MRM mode, the ion pair information of the compound was determined to be m / z 190 / 91. After experimental verification, the ion pair information determined was m / z 190 / 90 when the methanol mobile phase had different contents of ammonium fluoride and different flow rates.

[0137] The injection volume in the Echo injection system is 2.5 nL, and the detection rate is 3 seconds per sample.

[0138] For standard detection, the limit of detection (LOD), limit of quantification (LOQ), recovery and precision were calculated. By adding different concentrations of standard to the blank matrix, the measurement signal was compared with that of the blank sample, and the concentrations of the standard solution at the signal-to-noise ratio S / N=3 and S / N=10 were defined as the method LOD and LOQ, respectively. The experimental results showed that the target LOD and LOQ were close, which were 0.61mM and 1.04mM, respectively. Three standard solution concentrations of low (1mM), medium (3mM), and high (5mM) were set up, and 6 groups of parallels were set up for each. The recovery was determined and the relative standard deviation (RSD) was calculated to evaluate the accuracy and precision of the method. The results are shown in Table 10.

[0139] Table 10. Sensitivity, accuracy and precision of N-benzyl-3-piperidone detection method

[0140]

[0141] The initial concentration in Table 10 is the initial concentration of N-benzyl-3-piperidone, and the added concentration is the concentration of N-benzyl-3-piperidone added. The detection concentration is the concentration of N-benzyl-3-piperidone detected under the detection conditions. As shown in Table 10, the recovery rate in the sample is between 91% and 105%, and the RSD is less than 5%, indicating that the recovery rate and precision are good.

[0142] Example 13: High-throughput mass spectrometry detection method and feasibility verification of N-tert-butyloxycarbonyl-3-piperidone

[0143] The ion spray voltage was set to 5000V; the ion source carrier gas 1 was set to 90psi nitrogen; the ion source carrier gas 2 was set to 45psi nitrogen; the curtain gas was set to 20psi nitrogen, the ion source temperature was 300℃, the declustering voltage was 30V, the collision energy was 50V, the flow rate was 300μL / min, and the mobile phase used was methanol containing 2mM ammonium fluoride. In Q1 mode, the scan range was set to 50-250Da for full scan, and the parent ion information was determined to be m / z 200; secondly, in Product Ion mode, the scan range was set to 10-200Da, and the daughter ion fragment information was determined to be m / z 89.9; finally, in MRM mode, the ion pair information of the compound was determined to be m / z 200 / 89.9. After experimental verification, the ion pair information determined was m / z 200 / 89.9 when the methanol mobile phase had different contents of ammonium fluoride and different flow rates.

[0144] The injection volume in the Echo injection system is 2.5 nL, and the detection rate is 3 seconds per sample.

[0145] For standard detection, the limit of detection (LOD), limit of quantification (LOQ), recovery and precision were calculated. By adding different concentrations of standard to the blank matrix, the measurement signal was compared with that of the blank sample, and the concentrations of the standard solution at the signal-to-noise ratio S / N=3 and S / N=10 were defined as the method LOD and LOQ, respectively. The experimental results showed that the target LOD and LOQ were close, which were 0.33mM and 0.96mM, respectively. Three standard solution concentrations of low (1mM), medium (3mM), and high (5mM) were set up, and 6 groups of parallels were set up for each. The recovery was determined and the relative standard deviation (RSD) was calculated to evaluate the accuracy and precision of the method. The results are shown in Table 11.

[0146] Table 11. Sensitivity, accuracy and precision of N-tert-butyloxycarbonyl-3-piperidone detection method

[0147]

[0148] The initial concentration in Table 11 is the initial concentration of N-tert-butyloxycarbonyl-3-piperidone, and the added concentration is the added concentration of N-tert-butyloxycarbonyl-3-piperidone. The detection concentration is the concentration of N-tert-butyloxycarbonyl-3-piperidone detected under the detection conditions. As shown in Table 11, the recovery rate in the sample is between 93% and 96%, and the RSD is less than 5%, indicating that the recovery rate and precision are good.

[0149] Example 14: High-throughput mass spectrometry detection method and feasibility verification of N-benzyloxycarbonyl-3-piperidone

[0150] The ion spray voltage was set to 5000V; the ion source carrier gas 1 was set to 90psi nitrogen; the ion source carrier gas 2 was set to 45psi nitrogen; the curtain gas was set to 20psi nitrogen, the ion source temperature was 300℃, the declustering voltage was 30V, the collision energy was 50V, the flow rate was 300μL / min, and the mobile phase used was methanol containing 2mM ammonium fluoride. In Q1 mode, the scan range was set to 50-300Da for full scan, and the parent ion information was determined to be m / z 234; secondly, in Product Ion mode, the scan range was set to 10-200Da, and the daughter ion fragment information was determined to be m / z 91; finally, in MRM mode, the ion pair information of the compound was determined to be m / z 234 / 91. After experimental verification, the ion pair information determined was m / z 234 / 91 when the methanol mobile phase had different contents of ammonium fluoride and different flow rates.

[0151] The injection volume in the Echo injection system is 2.5 nL, and the detection rate is 3 seconds per sample.

[0152] For standard detection, the limit of detection (LOD), limit of quantification (LOQ), recovery and precision were calculated. By adding different concentrations of standard to the blank matrix, the measurement signal was compared with that of the blank sample, and the concentrations of the standard solution at the signal-to-noise ratio S / N=3 and S / N=10 were defined as the method LOD and LOQ, respectively. The experimental results showed that the target LOD and LOQ were close, which were 0.67mM and 1.38mM, respectively. Three standard solution concentrations of low (1mM), medium (3mM), and high (5mM) were set up, and 6 groups of parallels were set up for each. The recovery was determined and the relative standard deviation (RSD) was calculated to evaluate the accuracy and precision of the method. The results are shown in Table 12.

[0153] Table 12 Sensitivity, accuracy and precision of N-benzyloxycarbonyl-3-piperidone detection method

[0154]

[0155] The initial concentration in Table 12 is the initial concentration of N-benzyloxycarbonyl-3-piperidone, and the added concentration is the added concentration of N-benzyloxycarbonyl-3-piperidone. The detected concentration is the concentration of N-benzyloxycarbonyl-3-piperidone detected under the detection conditions. As shown in Table 12, the recovery rate in the sample is between 90% and 96%, and the RSD is less than 5%, indicating that the recovery rate and precision are good.

[0156] Example 15: High-throughput mass spectrometry detection method and feasibility verification of N-tert-butyloxycarbonyl-3-azacycloheptanone

[0157] The ion spray voltage was set to 5000V; ion source carrier gas 1 was set to 90psi nitrogen; ion source carrier gas 2 was set to 45psi nitrogen; curtain gas was set to 20psi nitrogen, ion source temperature was 300℃, declustering voltage was set to 30V, collision energy was set to 50V, flow rate was set to 300μL / min, and the mobile phase used was methanol containing 2mM ammonium fluoride. In Q1 mode, the scan range was set to 50-250Da for full scan, and the parent ion information was determined to be m / z 214; secondly, in Product Ion mode, the scan range was set to 10-200Da, and the daughter ion fragment information was determined to be m / z 89.9; finally, in MRM mode, the ion pair information of the compound was determined to be m / z 214 / 89.9. After experimental verification, the ion pair information determined was m / z 214 / 89.9 when the mobile phase contained different contents of ammonium fluoride and at different flow rates.

[0158] The injection volume in the Echo injection system is 2.5 nL, and the detection rate is 3 seconds per sample.

[0159] For standard detection, the limit of detection (LOD), limit of quantification (LOQ), recovery and precision were calculated. By adding different concentrations of standard to the blank matrix, the measurement signal was compared with that of the blank sample, and the concentrations of the standard solution at the signal-to-noise ratio S / N=3 and S / N=10 were defined as the method LOD and LOQ, respectively. The experimental results showed that the target LOD and LOQ were close, which were 0.28mM and 0.77mM, respectively. Three standard solution concentrations of low (1mM), medium (3mM), and high (5mM) were set up, and 6 groups of parallels were set up for each. The recovery was determined and the relative standard deviation (RSD) was calculated to evaluate the accuracy and precision of the method. The results are shown in Table 13.

[0160] Table 13. Sensitivity, accuracy and precision of N-tert-butyloxycarbonyl-3-azacycloheptanone detection method

[0161]

[0162] The initial concentration in Table 13 is the initial concentration of N-tert-butyloxycarbonyl-3-azacycloheptanone, and the added concentration is the added concentration of N-tert-butyloxycarbonyl-3-azacycloheptanone. The detection concentration is the concentration of N-tert-butyloxycarbonyl-3-azacycloheptanone detected under the detection conditions. As shown in Table 13, the recovery rate in the sample is between 92% and 96%, and the RSD is less than 5%, indicating that the recovery rate and precision are good.

[0163] Example 16: Application of (R)-N-tert-butyloxycarbonyl-3-aminopyrrole high-throughput mass spectrometry detection method to screening of transaminase mutant library

[0164] For the detection of (R)-N-tert-butyloxycarbonyl-3-aminopyrrole samples, a mutant library obtained by point saturation mutagenesis of transaminase was fermented in a 96-well plate to obtain a fermentation broth, and the OD value of the fermentation broth was about 2.

[0165] The mutation of transaminase is specifically described in the method disclosed in CN 112280761 B.

[0166] 10% fermentation broth, 1% N-tert-butoxycarbonyl-3-pyrrolidone, 4% isopropylamine as reactants and 85% water as solvent were added to 1 mL of the system for a catalytic reaction. The reaction lasted for about 20 minutes to generate the target product (R)-N-tert-butoxycarbonyl-3-aminopyrrole. After centrifugation at 4000 rpm for 5 minutes, the supernatant was transferred to a 384-well plate to obtain a sample containing compound 6.

[0167] The target compound (R)-N-tert-butyloxycarbonyl-3-aminopyrrole was detected and analyzed using Echo MS. Each sample was tested three times to ensure accurate reproducibility of the results. After 3 hours of testing 1,500 samples, mutants with improved performance were screened out based on the content of the generated target compound (R)-N-tert-butyloxycarbonyl-3-aminopyrrole. Figure 3 The results show that the peak height is proportional to the content of (R)-N-tert-butyloxycarbonyl-3-aminopyrrole. The content of (R)-N-tert-butyloxycarbonyl-3-aminopyrrole is calculated based on the peak area and compared with the content of (R)-N-tert-butyloxycarbonyl-3-aminopyrrole in the wild type (see patent document CN 112280761 B for the wild type). 23 mutants with a content increase of more than 1 times and 5 mutants with a content increase of more than 3 times were selected from 1500 samples. The specific results are shown in Table 14:

[0168] Table 14. Screening results of transaminase mutants by high-throughput detection method

[0169]

[0170] Although the present invention has been disclosed as above by way of embodiments, it is not intended to limit the present invention. Any person having ordinary knowledge in the technical field may make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the scope of the patent application attached hereto.

Claims

1. A mass spectrometry detection method for N-heterocyclic ketones and / or N-heterocyclic chiral amino compounds, in, In the mass spectrometry detection method, the declustering voltage value is 20-40V, preferably 30V.

2. The mass spectrometry detection method according to claim 1, wherein: In the mass spectrometry detection method, the collision energy is 40-60V, preferably 50V.

3. The mass spectrometry detection method according to claim 1-2, wherein: In the mass spectrometry detection method, the ion spray voltage is 4000-6000V, preferably 5000V.

4. The mass spectrometry detection method according to claims 1-3, wherein: In the mass spectrometry detection method, the ion source temperature is 200-400°C, preferably 300°C.

5. The mass spectrometry detection method according to claims 1-4, wherein: In the mass spectrometry detection method, the mobile phase used is methanol containing 1-5 mM ammonium fluoride; preferably methanol containing 2 mM ammonium fluoride; Preferably, the mobile phase flow rate is 250-350 μL / min; preferably 300 μL / min.

6. The mass spectrometry detection method according to claims 1-5, wherein: In the mass spectrometry detection method, The ion source carrier gas 1 is: 80-100 psi nitrogen; preferably 90 psi nitrogen; The ion source carrier gas 2 is: 40-50 psi nitrogen; preferably 45 psi nitrogen; The curtain gas is: 10-30 psi nitrogen, preferably 20 psi nitrogen.

7. The mass spectrometry detection method according to claim 1, wherein: The N-heterocyclic chiral amino compound is selected from one or more of (R)-N-tert-butyloxycarbonyl-3-aminopyrrole, (R)-N-benzyl-3-aminopiperidine, (R)-N-tert-butyloxycarbonyl-3-aminopiperidine, (R)-N-benzyloxycarbonyl-3-aminopiperidine and (R)-3-aminoazepane-1-carboxylic acid tert-butyl ester; The N heterocyclic ketone is selected from one or more of N-tert-butyloxycarbonyl-3-pyrrolidone, N-benzyl-3-piperidone, N-tert-butyloxycarbonyl-3-piperidone, N-benzyloxycarbonyl-3-piperidone and N-tert-butyloxycarbonyl-3-azacycloheptanone.

8. The mass spectrometry detection method according to claim 7, wherein: When the substance to be detected is (R)-N-tert-butyloxycarbonyl-3-aminopyrrole, the ion pair information of the mass spectrometry detection method is m / z 187 / 70; When the substance to be detected is (R)-N-benzyl-3-aminopiperidine, the ion pair information of the mass spectrometry detection method is m / z191 / 91; When the substance to be detected is (R)-N-tert-butyloxycarbonyl-3-aminopiperidine, the ion pair information of the mass spectrometry detection method is m / z 201 / 84.1; When the substance to be detected is (R)-N-benzyloxycarbonyl-3-aminopiperidine, the ion pair information of the mass spectrometry detection method is m / z 235 / 91; When the substance to be detected is (R)-3-aminoazepane-1-carboxylic acid tert-butyl ester, the ion pair information of the mass spectrometry detection method is m / z 215 / 89.9; When the substance to be detected is N-tert-butyloxycarbonyl-3-pyrrolidone, the ion pair information of the mass spectrometry detection method is m / z 184 / 109; When the substance to be detected is N-benzyl-3-piperidone, the ion pair information of the mass spectrometry detection method is m / z 190 / 90; When the substance to be detected is N-tert-butyloxycarbonyl-3-piperidone, the ion pair information of the mass spectrometry detection method is m / z200 / 89.9; When the substance to be detected is N-benzyloxycarbonyl-3-piperidone, the ion pair information of the mass spectrometry detection method is m / z234 / 91; When the substance to be detected is N-tert-butyloxycarbonyl-3-azacycloheptanone, the ion pair information of the mass spectrometry detection method is m / z 214 / 89.

9.

9. A method for high-throughput screening of transaminase directed evolution using the mass spectrometry detection method according to any one of claims 1 to 8.

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