Design, synthesis and application of a class of N-alkanoylglycine homologues that can be used in retention index system

Abstract

The present invention discloses the design, synthesis and application of a class of N-alkanoylglycine homologues that can be used for a retention index system, and belongs to the technical field of organic chemistry. The present invention provides a class of N-alkanoylglycine homologues, which can not only cover a sufficiently wide chromatographic window on a reversed-phase liquid chromatography-mass spectrometry platform, but also have a sufficiently strong response in the positive and negative ion detection mode of the mass spectrometer, and are therefore an ideal set of retention index system calibration substances. The present invention provides a method for synthesizing N-alkanoylglycine homologues, and its preparation process is simple and convenient. The retention index system established by the present invention using N-alkanoylglycine homologues can calibrate the retention time of the compound, correct the offset of the retention time, allow better inter-laboratory comparability, and facilitate the identification of unknown compounds, so this type of substance is very suitable for application in the fields of organic chemistry and analytical chemistry.

Description

Technical Field

[0001] The invention relates to the technical field of organic chemistry, and in particular to the design, synthesis and application of a class of N-alkanoylglycine homologues that can be used in a retention index system. Background Art

[0002] The retention index system is a solution to the problem of poor reproducibility of compound retention times in the field of liquid chromatography-mass spectrometry analysis. By measuring the retention time of a compound relative to a set of reference calibrants, a method is provided to standardize retention times between different chromatographic methods and systems, which can correct retention time offsets, allow better inter-laboratory comparability, and facilitate the identification of unknown compounds. To establish a retention index system calibrant commonly used in the field of liquid chromatography-mass spectrometry, it is necessary not only to cover a sufficiently wide chromatographic window in liquid chromatography, but also to have a sufficiently strong response in both the positive and negative ion detection modes of the mass spectrometer. The design and synthesis of effective retention index system calibrants are of great significance for the standardization of compound retention times and assisting compound identification. Summary of the invention

[0003] In view of the above-mentioned defects of the prior art, in the first aspect of the present invention, a N-alkanoylglycine homologue is provided, and the structural formula of the N-alkanoylglycine homologue is as follows:

[0004]

[0005] Among them, R 1 C 1 ~C 22 The straight chain alkyl group, R 2 is hydrogen or deuterium.

[0006] In the second aspect of the present invention, there is provided a method for preparing the N-alkanoylglycine homologues according to the first aspect of the present invention, comprising the following steps:

[0007] dissolving normal saturated fatty acids in an organic solvent to obtain a fatty acid solution;

[0008] Dissolving glycine raw material and sodium hydroxide in water to obtain a pre-reaction solution;

[0009] An acid binding agent and a condensing agent are sequentially added to the fatty acid solution to complete activation, and then the pre-reaction solution is added to the obtained activated fatty acid solution to react, and after the reaction, the solution is purified to obtain an N-alkanoylglycine homologue.

[0010] Preferably, the structural formula of the normal saturated fatty acid is as follows:

[0011]

[0012] Among them, R1 C 1 ~C 22 of a straight chain alkyl group.

[0013] Preferably, the organic solvent is at least one of tetrahydrofuran, dichloromethane, dimethyl sulfoxide and methyl tert-butyl ether.

[0014] Preferably, the solid-liquid ratio of the normal saturated fatty acid and the organic solvent is 5 to 100 g / L.

[0015] Preferably, the structural formula of the glycine raw material is as follows:

[0016]

[0017] Among them, R 2 is hydrogen or deuterium, and the corresponding glycine raw material is glycine or glycine-2,2-D2, respectively.

[0018] Preferably, the molar ratio of the normal saturated fatty acid to the glycine raw material is 1:1.0-1.5.

[0019] Preferably, the material-liquid ratio of the glycine raw material to the pre-reaction solution is 0.5-70 g / L; the molar ratio of the glycine raw material to the sodium hydroxide is 1:0.7-1.3.

[0020] Preferably, the acid binding agent is triethylamine or diisopropylethylamine.

[0021] Preferably, the molar ratio of the normal saturated fatty acid to the acid binding agent is 1:1.0-1.5.

[0022] Preferably, the structural formula of the condensing agent is as follows:

[0023]

[0024] Among them, R 3 is methyl or propyl, and the corresponding condensing agent is methyl chloroformate or propyl chloroformate.

[0025] Preferably, the molar ratio of the normal saturated fatty acid to the condensing agent is 1:1.0-1.5.

[0026] Preferably, the reaction temperature is -10 to 10°C, and the reaction time is 0.5 to 2.0 hours.

[0027] The reaction process of the preparation method of the second aspect of the present invention is as follows:

[0028]

[0029] In the third aspect of the present invention, there is provided an application of the N-alkanoylglycine homologue of the first aspect of the present invention or the N-alkanoylglycine homologue prepared by the method of the second aspect of the present invention in liquid chromatography-mass spectrometry analysis, specifically the application of a standard containing the N-alkanoylglycine homologue as a calibrant in establishing a liquid chromatography retention index system.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0031] The invention synthesizes and prepares an N-alkanoylglycine homologue, which can cover a sufficiently wide chromatographic window on a reversed-phase liquid chromatography-mass spectrometry platform and has a sufficiently strong response in both positive and negative ion detection modes of the mass spectrometer, and is an ideal retention index system calibrant.

[0032] The invention provides N-alkanoylglycine homologues and a synthesis method. The method has a simple and convenient preparation process and can flexibly prepare a variety of different types of N-alkanoylglycine homologues.

[0033] The present invention provides an application of N-alkanoylglycine homologues in a liquid chromatography retention index system. Based on the characteristic that the compound is an ideal retention index system calibrant, after being used to establish a retention index system, the retention time of the compound can be converted into a more stable retention index, the retention time of the compound can be calibrated, the deviation of the retention time can be corrected, better inter-laboratory comparability can be achieved, and unknown compounds can be easily identified. This is of great significance for the standardization of compound retention time and the identification of auxiliary compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is the test result of liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis of standards containing N-alkanoylglycine homologues. DETAILED DESCRIPTION

[0035] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0036] Example 1

[0037] Preparation:

[0038] Weigh 500 mg of acetic acid standard (1.0 eq) into a 100 mL round-bottom flask, add 10 mL of tetrahydrofuran and stir to dissolve it, place the resulting solution in a 0°C low-temperature reaction cold trap to obtain a fatty acid solution; add triethylamine (1.5 eq) dropwise to the resulting fatty acid solution, then add methyl chloroformate (1.5 eq) dropwise, and stir at 0°C for activation for 30 min to obtain an activated fatty acid solution; add glycine (1.5 eq) and sodium hydroxide (1.5 eq) to 10 mL of water, vortex to dissolve the glycine and sodium hydroxide solids, and obtain a pre-reaction The obtained pre-reaction solution was added dropwise to the obtained activated fatty acid solution, and the reaction was stirred at 0°C for 0.5h. After the reaction was completed, the round-bottom flask was taken out, tetrahydrofuran was removed by rotary evaporation, and 10mL of 2mol / L hydrochloric acid was added dropwise to the round-bottom flask, and then 60mL of ethyl acetate was used to extract the obtained mixed solution three times, and the organic layers were collected and combined, and the organic layers were washed with saturated brine, and then dried with anhydrous sodium sulfate and filtered. The filtrate was subjected to rotary evaporation to remove ethyl acetate to obtain white powdery solid N-acetylglycine with a yield of 53.6%; 1 H NMR (400 MHz, Methanol-d 4 )δ3.89(s,2H),1.21(s,3H); HRMS:[M+H] + =118.0492, theoretical value [M+H] + =118.0499; [MH] - =116.0350, theoretical value [MH] - =116.0353.

[0039] Example 2

[0040] Preparation:

[0041] The preparation method of this example is basically the same as that of Example 1, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is white powdery solid deuterated N-acetylglycine with a yield of 57.2%; HRMS: [M+H] + =120.0613, theoretical value [M+H] + =120.0619; [MH] - =119.0552, theoretical value [MH] - =119.0551.

[0042] Example 3

[0043] Preparation:

[0044] Weigh 500 mg of propionic acid standard (1.0 eq) into a 100 mL round-bottom flask, add 10 mL of dichloromethane and stir to dissolve it, place the resulting solution in a 0°C low-temperature reaction cold trap to obtain a fatty acid solution; add triethylamine (1.5 eq) dropwise to the resulting fatty acid solution, then add methyl chloroformate (1.5 eq) dropwise, and stir at 0°C for activation for 30 min to obtain an activated fatty acid solution; add glycine (1.5 eq) and sodium hydroxide (1.5 eq) to 10 mL of water, vortex to dissolve the glycine and sodium hydroxide solids, and obtain a pre-reaction The obtained pre-reaction solution was added dropwise to the obtained activated fatty acid solution, and the reaction was stirred at 0°C for 0.5h. After the reaction was completed, the round-bottom flask was taken out, and dichloromethane was removed by rotary evaporation. Then 10mL of 2mol / L hydrochloric acid was added dropwise to the round-bottom flask, and then 60mL of ethyl acetate was used to extract the obtained mixed solution three times, and the organic layers were collected and combined. The organic layers were washed with saturated brine, and then dried with anhydrous sodium sulfate and filtered. The filtrate was subjected to rotary evaporation to remove ethyl acetate to obtain white powder solid N-propionylglycine with a yield of 61.2%; 1 H NMR (400 MHz, Methanol-d 4 )δ3.89(s,2H),2.27(q,2H),1.14(t,3H); HRMS:[M+H] + =132.0647, theoretical value [M+H] + =132.0655; [MH] - =130.0502, theoretical value [MH] - =130.0510.

[0045] Example 4

[0046] Preparation:

[0047] The preparation method of this example is basically the same as that of Example 3, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-propionylglycine with a yield of 59.4%; HRMS: [M+H] + =134.0779, theoretical value [M+H] + =134.0775; [MH] - =132.0642, theoretical value [MH] - =132.0635.

[0048] Example 5

[0049] Preparation:

[0050] Weigh 500 mg of n-butyric acid standard (1.0 eq) into a 100 mL round-bottom flask, add 10 mL of dimethyl sulfoxide and stir to dissolve it, place the resulting solution in a 0°C low-temperature reaction cold trap to obtain a fatty acid solution; add triethylamine (1.5 eq) dropwise to the resulting fatty acid solution, then add methyl chloroformate (1.5 eq) dropwise, and stir at 0°C for activation for 30 min to obtain an activated fatty acid solution; add glycine (1.5 eq) and sodium hydroxide (1.5 eq) to 10 mL of water, vortex to dissolve the glycine and sodium hydroxide solids, and obtain the pre- reaction solution; adding the obtained pre-reaction solution dropwise to the obtained activated fatty acid solution, and stirring the reaction at 0°C for 0.5h. After the reaction is completed, the round-bottom flask is taken out, dimethyl sulfoxide is removed by rotary evaporation, and then 10mL of 2mol / L hydrochloric acid is added dropwise to the round-bottom flask, and then 60mL of ethyl acetate is used to extract the obtained mixed solution three times, the organic layer is collected and combined, the organic layer is washed with saturated brine, and then anhydrous sodium sulfate is added to dry and filtered, and the filtrate is subjected to rotary evaporation to remove ethyl acetate to obtain white powder solid N-butyrylglycine with a yield of 63.9%; 1 H NMR (400 MHz, Methanol-d 4 )δ3.89(s,2H),2.22(t,2H),1.65(m,2H),0.96(t,3H); HRMS:[M+H] + =146.0824, theoretical value [M+H] + =146.0812; [MH] - =144.0668, theoretical value [MH] - =144.0666.

[0051] Example 6

[0052] Preparation:

[0053] The preparation method of this example is basically the same as that of Example 5, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-butyryl glycine with a yield of 60.4%; HRMS: [M+H] + =148.0939, theoretical value [M+H] + =148.0937; [MH] - =146.0800, theoretical value [MH] - =146.0792.

[0054] Example 7

[0055] Preparation:

[0056] Weigh 500 mg of n-valeric acid standard (1.0 eq) into a 100 mL round-bottom flask, add 10 mL of methyl tert-butyl ether and stir to dissolve it, place the resulting solution in a 0°C low-temperature reaction cold trap to obtain a fatty acid solution; add triethylamine (1.5 eq) dropwise to the resulting fatty acid solution, then add methyl chloroformate (1.5 eq) dropwise, and stir at 0°C for activation for 30 min to obtain an activated fatty acid solution; add glycine (1.5 eq) and sodium hydroxide (1.5 eq) to 10 mL of water, vortex to dissolve the glycine and sodium hydroxide solids, and obtain the pre- reaction solution; adding the obtained pre-reaction solution dropwise to the obtained activated fatty acid solution, and stirring the reaction at 0°C for 0.5h. After the reaction is completed, the round-bottom flask is taken out, and methyl tert-butyl ether is removed by rotary evaporation, and then 10mL of 2mol / L hydrochloric acid is added dropwise to the round-bottom flask, and then 60mL of ethyl acetate is used to extract the obtained mixed solution three times, and the organic layer is collected and combined, and the organic layer is washed with saturated brine, and then anhydrous sodium sulfate is added to dry and filtered. The filtrate is subjected to rotary evaporation to remove ethyl acetate to obtain white powder solid N-valerylglycine with a yield of 68.6%; 1 H NMR (400 MHz, Methanol-d 4 )δ3.89(s,2H),2.25(t,2H),1.60(tt,2H),1.37(dq,2H),0.93(t,3H); HRMS:[M+H] + =160.0980, theoretical value [M+H] + =160.0968; [MH] - =158.0824, theoretical value [MH] - =158.0823.

[0057] Example 8

[0058] Preparation:

[0059] The preparation method of this example is basically the same as that of Example 7, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-pentanoylglycine with a yield of 62.1%; HRMS: [M+H] + =162.1087, theoretical value [M+H] + =162.1094; [MH] - =160.0952, theoretical value [MH] - =160.0948.

[0060] Example 9

[0061] Preparation:

[0062] Weigh 500 mg of n-hexanoic acid standard (1.0 eq) into a 100 mL round-bottom flask, add 10 mL of tetrahydrofuran and stir to dissolve it, place the resulting solution in a 0°C low-temperature reaction cold trap to obtain a fatty acid solution; add diisopropylethylamine (1.5 eq) dropwise to the resulting fatty acid solution, then add methyl chloroformate (1.5 eq) dropwise, and stir at 0°C for activation for 30 min to obtain an activated fatty acid solution; add glycine (1.5 eq) and sodium hydroxide (1.5 eq) to 10 mL of water, vortex to dissolve the glycine and sodium hydroxide solids, and obtain to the pre-reaction solution; the obtained pre-reaction solution was added dropwise to the obtained activated fatty acid solution, and the reaction was stirred at 0°C for 0.5h. After the reaction was completed, the round-bottom flask was taken out, tetrahydrofuran was removed by rotary evaporation, and 10mL of 2mol / L hydrochloric acid was added dropwise to the round-bottom flask, and then 60mL of ethyl acetate was used to extract the obtained mixed solution three times, the organic layer was collected and combined, the organic layer was washed with saturated brine, and then anhydrous sodium sulfate was added to dry and filtered, and the filtrate was subjected to rotary evaporation to remove ethyl acetate to obtain white powder solid N-hexanoylglycine with a yield of 66.2%; 1 HNMR (400MHz, Methanol-d 4 )δ3.89(s,2H),2.39–2.06(t,2H),1.71–1.50(m,2H),1.47–1.15(m,4H),1.03–0.80(t,3H); HRMS:[M+H] + =174.1130, theoretical value [M+H] + =174.1125; [MH] - =172.0969, theoretical value [MH] - =172.0979.

[0063] Example 10

[0064] Preparation:

[0065] The preparation method of this example is basically the same as that of Example 9, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is white powdery solid N-hexanoylglycine with a yield of 69.3%; HRMS: [M+H] + =176.1252, theoretical value [M+H] + =176.1250; [MH] - =174.1102, theoretical value [MH] - =174.1105.

[0066] Embodiment 11

[0067] Preparation:

[0068] Weigh 500 mg of n-heptanoic acid standard (1.0 eq) into a 100 mL round-bottom flask, add 10 mL of tetrahydrofuran and stir to dissolve it, place the resulting solution in a 0°C low-temperature reaction cold trap to obtain a fatty acid solution; add triethylamine (1.2 eq) dropwise to the resulting fatty acid solution, then add methyl chloroformate (1.5 eq) dropwise, and stir at 0°C for activation for 30 min to obtain an activated fatty acid solution; add glycine (1.5 eq) and sodium hydroxide (1.5 eq) to 10 mL of water, vortex to dissolve the glycine and sodium hydroxide solids, and obtain the pre- Reaction solution; adding the obtained pre-reaction solution dropwise to the obtained activated fatty acid solution, and stirring the reaction at 0°C for 0.5h. After the reaction is completed, the round-bottom flask is taken out, tetrahydrofuran is removed by rotary evaporation, and then 10mL of 2mol / L hydrochloric acid is dropwise added to the round-bottom flask, and then 60mL of ethyl acetate is used to extract the obtained mixed solution three times, and the organic layer is collected and combined, and the organic layer is washed with saturated brine, and then anhydrous sodium sulfate is added to dry and filtered. The filtrate is subjected to rotary evaporation to remove ethyl acetate to obtain white powder solid N-heptanoylglycine with a yield of 70.2%; 1 H NMR (400 MHz, Methanol-d 4 )δ3.89(s,2H),2.24(t,2H),1.62(m,2H),1.42–1.24(m,6H),1.02–0.80(t,3H); HRMS:[M+H] + =188.1285, theoretical value [M+H] + =188.1281; [MH] - =186.1141, theoretical value [MH] - =186.1136.

[0069] Example 12

[0070] Preparation:

[0071] The preparation method of this example is basically the same as that of Example 11, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-heptanoylglycine with a yield of 73.3%; HRMS: [M+H] + =190.1422, theoretical value [M+H]+ =190.1407; [MH] - =188.1253, theoretical value [MH] - =188.1261.

[0072] Example 13

[0073] Preparation:

[0074] Weigh 500 mg of n-octanoic acid standard (1.0 eq) into a 100 mL round-bottom flask, add 10 mL of tetrahydrofuran and stir to dissolve it, place the resulting solution in a 0°C low-temperature reaction cold trap to obtain a fatty acid solution; add triethylamine (1.5 eq) dropwise to the resulting fatty acid solution, then add propyl chloroformate (1.5 eq) dropwise, and stir at 0°C for activation for 30 min to obtain an activated fatty acid solution; add glycine (1.5 eq) and sodium hydroxide (1.5 eq) to 1 0mL of water, vortex to dissolve glycine and sodium hydroxide solid to obtain a pre-reaction solution; add the obtained pre-reaction solution dropwise to the obtained activated fatty acid solution, and stir and react at 0°C for 0.5h. After the reaction is completed, take out the round-bottom flask, remove tetrahydrofuran by rotary evaporation, and then add 10mL of 2mol / L hydrochloric acid dropwise to the round-bottom flask, then filter and collect the precipitate, rinse the precipitate with 3mL of water and then drain to obtain white powder solid N-octanoylglycine, with a yield of 75.4%; 1 H NMR (400 MHz, DMSO-d 6 )δ8.10(t,1H),3.72(d,2H),2.11(t,2H),1.49(m,2H),1.25(m,8H),0.93–0.80(t,3H); HRMS:[M+H] + =202.1455, theoretical value [M+H] + =202.1438; [MH] - =200.1293, theoretical value [MH] - =200.1292.

[0075] Embodiment 14

[0076] Preparation:

[0077] The preparation method of this example is basically the same as that of Example 13, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-octanoylglycine with a yield of 72.3%; HRMS: [M+H] + =204.1567, theoretical value [M+H] + =204.1563; [MH] - =202.1422, theoretical value [MH] - =202.1418.

[0078] Embodiment 15

[0079] Preparation:

[0080] Weigh 500 mg of nonanoic acid standard (1.0 eq) into a 100 mL round-bottom flask, add 10 mL of tetrahydrofuran and stir to dissolve it, place the resulting solution in a 0°C low-temperature reaction cold trap to obtain a fatty acid solution; add triethylamine (1.2 eq) dropwise to the resulting fatty acid solution, then add propyl chloroformate (1.2 eq) dropwise, and stir at 0°C for activation for 30 min to obtain an activated fatty acid solution; add glycine (1.2 eq) and sodium hydroxide (1.2 eq) to 1 0mL of water, vortex to dissolve glycine and sodium hydroxide solid to obtain a pre-reaction solution; add the obtained pre-reaction solution dropwise to the obtained activated fatty acid solution, and stir and react at 0°C for 0.5h. After the reaction is completed, take out the round-bottom flask, remove tetrahydrofuran by rotary evaporation, and then add 10mL of 2mol / L hydrochloric acid dropwise to the round-bottom flask, then filter and collect the precipitate, rinse the precipitate with 3mL of water and then drain to obtain white powder solid N-nonanoylglycine, with a yield of 76.2%; 1 H NMR (400 MHz, DMSO-d 6 )δ12.45(s,1H),8.09(t,1H),3.71(d,2H),2.10(t,2H),1.47(m,2H),1.24(m,10H),0.90–0.81(t,3H); HRMS:[M+H] + =216.1613, theoretical value [M+H] + =216.1594; [MH] - =214.1450, theoretical value [MH] - =214.1449.

[0081] Example 16

[0082] Preparation:

[0083] The preparation method of this example is basically the same as that of Example 15, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-nonanoylglycine with a yield of 71.3%; HRMS: [M+H] + =218.1729, theoretical value [M+H] + =218.1720; [MH] - =216.1585, theoretical value [MH] - =216.1574.

[0084] Embodiment 17

[0085] Preparation:

[0086] Weigh 500 mg of n-decanoic acid standard (1.0 eq) into a 100 mL round-bottom flask, add 10 mL of tetrahydrofuran and stir to dissolve it, place the resulting solution in a 0°C low-temperature reaction cold trap to obtain a fatty acid solution; add triethylamine (1.2 eq) dropwise to the resulting fatty acid solution, then add propyl chloroformate (1.2 eq) dropwise, and stir at 0°C for activation for 30 min to obtain an activated fatty acid solution; add glycine (1.5 eq) and sodium hydroxide (1.5 eq) to 1 0mL of water, vortex to dissolve glycine and sodium hydroxide solid to obtain a pre-reaction solution; add the obtained pre-reaction solution dropwise to the obtained activated fatty acid solution, and stir and react at 0°C for 0.5h. After the reaction is completed, take out the round-bottom flask, remove tetrahydrofuran by rotary evaporation, and then add 10mL of 2mol / L hydrochloric acid dropwise to the round-bottom flask, then filter and collect the precipitate, rinse the precipitate with 3mL of water and then drain to obtain white powder solid N-decanoylglycine, with a yield of 80.2%; 1 H NMR (400 MHz, Methanol-d 4 )δ3.88(s,2H),2.24(t,2H),1.62(m,2H),1.45–1.14(m,12H),1.03–0.78(t,3H); HRMS:[M+H] + =230.1769, theoretical value [M+H] + =230.1751; [MH] - =228.1607, theoretical value [MH] - =228.1605.

[0087] Embodiment 18

[0088] Preparation:

[0089] The preparation method of this example is basically the same as that of Example 17, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-decanoylglycine with a yield of 78.7%; HRMS: [M+H] + =232.1882, theoretical value [M+H] + =232.1876; [MH] - =230.1362, theoretical value [MH] - =230.1731.

[0090] Embodiment 19

[0091] Preparation:

[0092] Weigh 500 mg of undecanoic acid standard (1.0 eq) into a 100 mL round-bottom flask, add 15 mL of tetrahydrofuran and stir to dissolve it, place the resulting solution in a 0°C low-temperature reaction cold trap to obtain a fatty acid solution; add triethylamine (1.5 eq) dropwise to the resulting fatty acid solution, then add propyl chloroformate (1.5 eq) dropwise, and stir at 0°C for activation for 30 min to obtain an activated fatty acid solution; add glycine (1.5 eq) and sodium hydroxide (1.5 eq) to 10 mL of water, vortex to dissolve glycine and sodium hydroxide solid to obtain a pre-reaction solution; add the obtained pre-reaction solution dropwise to the obtained activated fatty acid solution, and stir and react at -5°C for 0.5h. After the reaction is completed, take out the round-bottom flask, remove tetrahydrofuran by rotary evaporation, and then add 10mL of 2mol / L hydrochloric acid dropwise to the round-bottom flask, then filter and collect the precipitate, rinse the precipitate with 3mL of water and then drain to obtain white powder solid N-undecanoylglycine, with a yield of 85.1%; 1 H NMR (400 MHz, Methanol-d 4 )δ3.89(s,2H),2.24(t,2H),1.62(m,2H),1.40–1.19(m,14H),0.98–0.81(t,3H); HRMS:[M+H] + =244.1913, theoretical value [M+H] + =244.1907; [MH] - =242.1775, theoretical value [MH] - =242.1762.

[0093] Embodiment 20

[0094] Preparation:

[0095] The preparation method of this example is basically the same as that of Example 19, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-undecanoylglycine with a yield of 83.9%; HRMS: [M+H] + =246.2042, theoretical value [M+H] + =246.2033; [MH] - =244.1870, theoretical value [MH] - =244.1887.

[0096] Embodiment 21

[0097] Preparation:

[0098] Weigh 500 mg of dodecanoic acid standard (1.0 eq) into a 100 mL round-bottom flask, add 15 mL of tetrahydrofuran and stir to dissolve it, place the resulting solution in a 0°C low-temperature reaction cold trap to obtain a fatty acid solution; add triethylamine (1.5 eq) dropwise to the resulting fatty acid solution, then add propyl chloroformate (1.5 eq) dropwise, and stir at 0°C for activation for 30 min to obtain an activated fatty acid solution; add glycine (1.5 eq) and sodium hydroxide (1.5 eq) to 10 mL of water, vortex to dissolve glycine and sodium hydroxide solid to obtain a pre-reaction solution; add the obtained pre-reaction solution dropwise to the obtained activated fatty acid solution, and stir and react at 0°C for 0.5h. After the reaction is completed, take out the round-bottom flask, remove tetrahydrofuran by rotary evaporation, and then add 10mL of 2mol / L hydrochloric acid dropwise to the round-bottom flask, then filter and collect the precipitate, rinse the precipitate with 3mL of water and then drain to obtain white powder solid N-dodecanoylglycine with a yield of 87.4%; 1 H NMR (400 MHz, Methanol-d 4 )δ3.89(s,2H),2.24(t,2H),1.62(m,2H),1.30(m,16H),0.89(t,3H); HRMS:[M+H] + =258.2084, theoretical value [M+H] + =258.2064; [MH] - =256.1918, theoretical value [MH] - =256.1918.

[0099] Embodiment 22

[0100] Preparation:

[0101] The preparation method of this example is basically the same as that of Example 21, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-dodecanoylglycine with a yield of 89.9%; HRMS: [M+H] + =260.2196, theoretical value [M+H] + =260.2189; [MH] - =258.2054, theoretical value [MH] - =258.2044.

[0102] Embodiment 23

[0103] Preparation:

[0104] The preparation method of this example is basically the same as that of Example 21, except that the normal saturated fatty acid used in this example is a tridecanoic acid standard, and the obtained product is a white powdery solid N-tridecanoylglycine with a yield of 90.4%; 1 HNMR (400MHz, Methanol-d 4 )δ3.88(s,2H),2.42–2.15(t,2H),1.61(m,2H),1.29(m,18H),0.90(t,3H); HRMS:[M+H] + =272.2242, theoretical value [M+H] + =272.2220; [MH] - =270.2075, theoretical value [MH] - =270.2075.

[0105] Embodiment 24

[0106] Preparation:

[0107] The preparation method of this example is basically the same as that of Example 23, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-tridecanoylglycine with a yield of 86.9%; HRMS: [M+H] + =274.2358, theoretical value [M+H] + =274.2346; [MH] - =272.2210, theoretical value [MH] - =272.2200.

[0108] Embodiment 25

[0109] Preparation:

[0110] The preparation method of this example is basically the same as that of Example 21, except that the normal saturated fatty acid used in this example is a tetradecanoic acid standard product, and the obtained product is a white powdery solid N-tetradecanoylglycine with a yield of 92.1%; 1 HNMR (400MHz, Methanol-d 4 )δ3.89(s,2H),2.24(t,2H),1.61(m,2H),1.30(m,20H),1.05–0.76(t,3H); HRMS:[M+H] + =286.2399, theoretical value [M+H] +=286.2377; [MH] - =284.2230, theoretical value [MH] - =284.2231.

[0111] Embodiment 26

[0112] Preparation:

[0113] The preparation method of this example is basically the same as that of Example 25, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-tetradecanoylglycine with a yield of 93.3%; HRMS: [M+H] + =288.2522, theoretical value [M+H] + =288.2502; [MH] - =286.2355, theoretical value [MH] - =286.2357.

[0114] Embodiment 27

[0115] Preparation:

[0116] The preparation method of this example is basically the same as that of Example 21, except that the normal saturated fatty acid used in this example is a standard product of pentadecanoic acid, and the obtained product is a white powdery solid N-pentadecanoylglycine with a yield of 89.6%; 1 HNMR (400MHz, Methanol-d 4 )δ3.89(s,2H),2.24(t,2H),1.62(m,2H),1.29(m,22H),0.90(t,3H); HRMS:[M+H] + =300.2553, theoretical value [M+H] + =300.2533; [MH] - =298.2387, theoretical value [MH] - =298.2388.

[0117] Embodiment 28

[0118] Preparation:

[0119] The preparation method of this example is basically the same as that of Example 27, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-pentadecanoylglycine with a yield of 91.2%; HRMS: [M+H] +=302.2669, theoretical value [M+H] + =302.2658; [MH] - =300.2516, theoretical value [MH] - =300.2513.

[0120] Embodiment 29

[0121] Preparation:

[0122] The preparation method of this example is basically the same as that of Example 21, except that the normal saturated fatty acid used in this example is a standard hexadecanoic acid, and the obtained product is a white powdery solid N-hexadecanoylglycine with a yield of 93.6%; 1 HNMR (400MHz, Methanol-d 4 )δ3.88(s,2H),2.24(t,2H),1.62(m,2H),1.28(m,24H),1.03–0.74(t,3H); HRMS:[M+H] + =314.2715, theoretical value [M+H] + =314.2689; [MH] - =312.2541, theoretical value [MH] - =312.2544.

[0123] Embodiment 30

[0124] Preparation:

[0125] The preparation method of this example is basically the same as that of Example 29, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-hexadecanoylglycine with a yield of 92.1%; HRMS: [M+H] + =316.2828, theoretical value [M+H] + =316.2814; [MH] - =314.2671, theoretical value [MH] - =314.2669.

[0126] Embodiment 31

[0127] Preparation:

[0128] The preparation method of this example is basically the same as that of Example 21, except that the normal saturated fatty acid used in this example is a standard product of heptadecanoic acid, and the obtained product is a white powdery solid N-heptadecanoyl glycine with a yield of 91.9%;1 HNMR (400 MHz, DMSO-d 6 )δ8.08(t,1H),3.71(d,2H),2.10(t,2H),1.47(m,2H),1.24(m,26H),0.97–0.72(t,3H); HRMS:[M+H] + =328.2868, theoretical value [M+H] + =328.2845; [MH] - =326.2696, theoretical value [MH] - =326.2700.

[0129] Embodiment 32

[0130] Preparation:

[0131] The preparation method of this example is basically the same as that of Example 31, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-heptadecanoyl glycine with a yield of 94.3%; HRMS: [M+H] + =330.2993, theoretical value [M+H] + =330.2970; [MH] - =328.2830, theoretical value [MH] - =328.2825.

[0132] Embodiment 33

[0133] Preparation:

[0134] The preparation method of this example is basically the same as that of Example 21, except that the normal saturated fatty acid used in this example is a standard octadecanoic acid, and the obtained product is a white powdery solid N-octadecanoylglycine with a yield of 92.1%; 1 HNMR (400 MHz, DMSO-d 6 )δ8.07(t,1H),3.71(d,2H),2.09(t,2H),1.47(m,2H),1.23(m,28H),0.91–0.79(t,3H); HRMS:[M+H] + =342.3029, theoretical value [M+H] + =342.3001; [MH] - =340.2857, theoretical value [MH] - =340.2856.

[0135] Embodiment 34

[0136] Preparation:

[0137] The preparation method of this example is basically the same as that of Example 33, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-octadecanoylglycine with a yield of 91.6%; HRMS: [M+H] + =344.3146, theoretical value [M+H] + =344.3126; [MH] - =342.2986, theoretical value [MH] - =342.2981.

[0138] Embodiment 35

[0139] Preparation:

[0140] The preparation method of this example is basically the same as that of Example 21, except that the normal saturated fatty acid used in this example is a nonadecanoic acid standard product, and the obtained product is a white powdery solid N-nonadecanoylglycine with a yield of 94.3%; 1 HNMR (400 MHz, DMSO-d 6 )δ8.00(t,1H),3.67(d,2H),2.09(t,2H),1.47(t,2H),1.23(m,30H),0.98–0.72(t,3H); HRMS:[M+H] + =356.3188, theoretical value [M+H] + =356.3157; [MH] - =354.3011, theoretical value [MH] - =354.3012.

[0141] Embodiment 36

[0142] Preparation:

[0143] The preparation method of this example is basically the same as that of Example 35, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-nonadecanoyl glycine with a yield of 93.2%; HRMS: [M+H] + =358.3309, theoretical value [M+H] + =358.3282; [MH] - =356.3139, theoretical value [MH] - =356.3137.

[0144] Embodiment 37

[0145] Preparation:

[0146] The preparation method of this example is basically the same as that of Example 21, except that the normal saturated fatty acid used in this example is an eicosanoic acid standard product, and the obtained product is a white powdery solid N-eicosanoylglycine with a yield of 91.7%; 1 HNMR (400 MHz, DMSO-d 6 )δ8.08(t,1H),3.71(d,2H),2.09(t,2H),1.47(m,2H),1.23(m,32H),0.85(t,3H); HRMS:[M+H] + =370.3340, theoretical value [M+H] + =370.3313; [MH] - =368.3169, theoretical value [MH] - =368.3168.

[0147] Embodiment 38

[0148] Preparation:

[0149] The preparation method of this example is basically the same as that of Example 37, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-eicosanoylglycine with a yield of 92.2%; HRMS: [M+H] + =372.3468, theoretical value [M+H] + =372.3438; [MH] - =370.3297, theoretical value [MH] - =370.3293.

[0150] Embodiment 39

[0151] Preparation:

[0152] The preparation method of this example is basically the same as that of Example 21, except that the normal saturated fatty acid used in this example is a standard heneicosanoic acid, and the obtained product is a white powdery solid N-heneicosanoylglycine with a yield of 91.7%; 1 H NMR (400 MHz, DMSO-d 6)δ8.08(t,1H),3.71(d,2H),2.09(t,2H),1.48(m,2H),1.23(m,34H),0.97–0.72(t,3H); HRMS:[M+H] + =384.3497, theoretical value [M+H] + =384.3469; [MH] - =382.3327, theoretical value [MH] - =382.3324.

[0153] Embodiment 40

[0154] Preparation:

[0155] The preparation method of this example is basically the same as that of Example 39, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-heneicosanoylglycine with a yield of 89.8%; HRMS: [M+H] + =386.3612, theoretical value [M+H] + =386.3594; [MH] - =384.3454, theoretical value [MH] - =384.3449.

[0156] Embodiment 41

[0157] Preparation:

[0158] The preparation method of this example is basically the same as that of Example 21, except that the normal saturated fatty acid used in this example is a standard docosanoic acid, and the obtained product is a white powdery solid N-docosanoylglycine with a yield of 95.1%; 1 H NMR (400MHz, DMSO-d6) δ8.08(t,1H),3.71(d,2H),2.09(t,2H),1.48(m,2H),1.23(m,36H),0.93–0.74(t,3H); HRMS:[M+H] + =398.3654, theoretical value [M+H] + =398.3625; [MH] - =396.3485, theoretical value [MH] - =396.3480.

[0159] Embodiment 42

[0160] Preparation:

[0161] The preparation method of this example is basically the same as that of Example 41, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-docosanoylglycine with a yield of 94.8%; HRMS: [M+H] + =400.3776, theoretical value [M+H] + =400.3750; [MH] - =398.3611, theoretical value [MH] - =398.3605.

[0162] Embodiment 43

[0163] Preparation:

[0164] The preparation method of this example is basically the same as that of Example 21, except that the normal saturated fatty acid used in this example is a standard product of tricosanoic acid, and the obtained product is a white powdery solid N-tricosanoylglycine with a yield of 93.7%; 1 H NMR (400MHz, DMSO-d6) δ8.08(t,1H),3.71(d,2H),2.09(t,2H),1.48(m,2H),1.23(m,38H),0.93–0.74(t,3H); HRMS:[M+H] + =412.3800, theoretical value [M+H] + =412.3781; [MH] - =410.3642, theoretical value [MH] - =410.3636.

[0165] Embodiment 44

[0166] Preparation:

[0167] The preparation method of this example is basically the same as that of Example 43, except that the glycine raw material used in this example is glycine-2,2-D2, and the obtained product is a white powdery solid deuterated N-tricosanoylglycine with a yield of 92.1%; HRMS: [M+H] + =414.3939, theoretical value [M+H] + =414.3906; [MH] - =412.3768, theoretical value [MH] - =412.3761.

[0168] Embodiment 45

[0169] Application of the retention index system using a mixed standard containing N-alkanoylglycine homologues as a calibrant:

[0170] Use 200 μL acetonitrile (ACN): H 2 A mixed standard of N-acetylglycine to N-tricosanoylglycine with a concentration of 500 μg / L was prepared by using a 1:9 (v / v) solution, and the obtained mixed standard was analyzed by liquid phase high resolution mass spectrometry (LC-HRMS). The instrument used for the analysis was an Agilent 6546 LC / Q-TOF liquid chromatography-mass spectrometry instrument, which consisted of an Agilent 1290 Infinity II LC System and an Agilent 6546 QTOF mass analyzer equipped with an Agilent Jet Stream electrospray ionization source (Agilent Technologies Inc, California, USA). Water with 0.1% FA was used as mobile phase A, and ACN with 0.1% FA was used as mobile phase B. The chromatographic elution gradient was 0-1 min 2% B, 1-23 min 2% to 98% B, 23-25 ​​min maintained at 98% B, 25.10-30 min 2% B. The flow rate was set to 0.40 mL / min, the column temperature was set to 40 °C, the injection volume was 5 μL, and the chromatographic column model was ACQUITY UPLC BEH C18 1.7 μm 2.1*100 mm Column. The mass spectrometry analysis adopted the full scan mode, the mass-to-charge ratio scanning range was 50-1000, and the detection was performed in both positive and negative ion modes. The detection results are shown in Figure 1 .

[0171] like Figure 1 As shown in the figure, the results show that under a 25-min chromatographic gradient, the retention time of 22 N-alkanoylglycine homologues with different carbon numbers covers a time window from 0.8 to 24 min, and the difference between the RTs of each adjacent calibrant is about 1 min; in addition, the N-alkanoylglycine homologues have sufficiently strong responses in both the positive and negative ion modes of the mass spectrometer. It can be seen that the N-alkanoylglycine homologues are an ideal set of retention index system calibrants.

[0172] Embodiment 46

[0173] Application of using the N-alkanoylglycine homologue retention index system to calibrate compound retention times:

[0174] Use 200 μL acetonitrile (ACN): H 2O=1:9 (v / v) solution was prepared with a concentration of 500 μg / L of 73 kinds of compound standards (as shown in Table 1) and a mixed solution of N-acetylglycine to N-tricosanoylglycine at a concentration of 500 μg / L, and liquid phase high resolution mass spectrometry (LC-HRMS) analysis was performed. The analysis conditions were similar to those in Example 45, except that two different elution gradients were used for analysis twice, gradient 1: 0-1 min 2% B, 1-23 min 2% to 98% B, 23-25 ​​min maintained at 98% B, 25.10-30 min 2% B; gradient 2: 0-1 min 2% B, 1-13 min 2% to 98% B, 13-15 min maintained at 98% B, 15.10-20 min 2% B. The results are as follows. The retention time ranges of 73 compounds are 0.5-19.6 min (gradient 1) and 0.5-12.4 min (gradient 2). The retention time ranges of 22 N-alkanoylglycine calibrants are 0.8-24.0 min (gradient 1) and 0.8-15.0 min (gradient 2). The retention time of most compounds obtained by gradient 2 analysis is significantly earlier than that of gradient 1. The retention time of most compounds can be covered by N-alkanoylglycine homologues. The retention time of the compound can be corrected to the retention index using the following retention index calculation formula:

[0175]

[0176] In the formula, t represents the retention time of the compound standard, RI represents the retention index corresponding to the retention time of the compound standard, n and n+1 represent the carbon numbers of the normal fatty acid chains of the two calibration substances adjacent to the compound, and t Gly-Cn With t Gly-C(n+1) Represents the retention times of two calibrants adjacent to the compound.

[0177] Table 1: Compound Standard Information

[0178]

[0179]

[0180]

[0181] The N-alkanoylglycine retention index system can be used to convert the retention time of compounds with large variations into stable retention indices. For example, the retention time of dopamine detected in the positive ion mode of mass spectrometry is 5.3min (gradient 1) and 4.3min (gradient 2), and the retention time varies greatly. However, its retention time in the two analyses falls within the retention time range of 4.1-5.9min (gradient 1) and 3.6-4.6min (gradient 2) of N-pentanoylglycine and N-hexanoylglycine. Therefore, the retention index of this compound can be calculated to be 569 (gradient 1) and 572 (gradient 2); the retention time of 3-indolepropionic acid detected in the negative ion mode of mass spectrometry is 5.3min (gradient 1) and 4.3min (gradient 2). The retention time of N-heptanoylglycine (gradient 1) and N-octanoylglycine (gradient 2) is 8.2 min (gradient 1) and 5.9 min (gradient 2), which falls within the retention time range of 7.6-9.2 min (gradient 1) and 5.6-6.5 min (gradient 2) of N-heptanoylglycine and N-octanoylglycine, so the retention index of the compound is calculated to be 740 (gradient 1) and 741 (gradient 2). The above results show that the retention index system established based on N-alkanoylglycine homologues can calibrate the retention time of the compound.

[0182] Embodiment 47

[0183] Establishment of a retention index system using a mixed standard containing N-alkanoylglycine homologues as a calibrant and its application in LC-MS-based non-targeted metabolomics analysis:

[0184] Use 200 μL acetonitrile (ACN): HO 2 The mixed solutions of deuterated N-acetylglycine to deuterated N-tricosanoylglycine with a concentration of 500 μg / L were prepared by mixing deuterated N-acetylglycine with a ratio of 1:1:9 (v / v), and the obtained mixed solutions were added to actual human feces samples for liquid chromatography-mass spectrometry (LC-HRMS) analysis. The analysis conditions were referred to Example 45. The results are as follows: deuterated N-alkanoylglycine homologues (C 2 -C 23 ) can cover the entire gradient elution range (0.8-24 min). Using the formula of Example 46, the retention time of the detected metabolites can be converted into a more stable retention index, thereby facilitating the qualitative identification of the metabolites.

[0185] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. Application of an N-alkanoylglycine homologue in liquid chromatography-mass spectrometry analysis, Features: The standard containing N-alkanoylglycine homologues is used as a calibration substance in the establishment of a liquid chromatography retention index system. The retention time of the compound is calibrated to a retention index using the following retention index calculation formula: In the formula, t represents the retention time of the compound standard, RI represents the retention index corresponding to the retention time of the compound standard, n and n+1 represent the carbon numbers of the normal fatty acid chains of the two calibration substances adjacent to the compound, and t Gly-Cn With t Gly-C(n+1) represents the retention time of two calibrants adjacent to the compound; The structural formula of the N-alkanoylglycine homologue is as follows: ； Among them, R 1 C 1 ~C 22 The straight chain alkyl group, R 2 is hydrogen or deuterium.

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