阅读说明：本技术 一种同时测定动物组织中氨基酸和核苷酸的lc-ms/ms的检测方法 (LC-MS/MS detection method for simultaneously determining amino acids and nucleotides in animal tissues ) 是由 韦晓群 刘鸿婷 邹轶 郭成贤 赵晋楷 欧纪锋 孙远明 雷红涛 于 2019-08-30 设计创作，主要内容包括：本发明涉及食品及生物检测领域,具体涉及一种同时测定动物组织中氨基酸和核苷酸的LC-MS/MS的检测方法。本方法根据酸性洗脱和碱性洗脱双重保留机理,采用超高效液相色谱-静电离子轨道肼质谱系统,使用XBridge BEH Amide色谱柱,以乙腈-甲酸水溶液-氨水为流动相进行三元梯度洗脱,无需添加离子对和衍生化试剂,建立了氨基酸和核苷酸液相色谱-质谱联用直接分析方法。本发明简单易行,大大降低前处理时间和成本,实现以氨基酸为典型的样品中极性物质的分离,以氨基酸和核苷酸为典型例子,有效解决同时分离带正电荷离子和带负电荷离子的问题。(The invention relates to the field of food and biological detection, in particular to an LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) detection method for simultaneously determining amino acids and nucleotides in animal tissues. According to the method, an ultra-high performance liquid chromatography-electrostatic ion orbital hydrazine mass spectrometry system is adopted according to a dual retention mechanism of acidic elution and alkaline elution, an XBridge BEH Amide chromatographic column is used, acetonitrile-formic acid aqueous solution-ammonia water is used as a mobile phase for carrying out ternary gradient elution, ion pairs and derivatization reagents are not required to be added, and a direct analysis method of amino acid and nucleotide liquid chromatography-mass spectrometry is established. The method is simple and easy to implement, greatly reduces the pretreatment time and cost, realizes the separation of polar substances in a sample by taking amino acid as a typical example, and effectively solves the problem of simultaneously separating positively charged ions and negatively charged ions by taking amino acid and nucleotide as typical examples.)

1. An LC-MS/MS detection method for simultaneously determining amino acids and nucleotides in animal tissues, characterized by comprising the steps of:

(1) extraction and removal of macromolecular substance interferences: mixing the minced sample to be detected with water, and uniformly performing vortex oscillation; adding methanol, and uniformly vortexing and oscillating; centrifuging, and collecting supernatant to obtain extract 1;

(2) removal of lipid interference on results: adding an organic reagent n-hexane into the extract 1 prepared in the step (1), and uniformly performing vortex oscillation; centrifuging, and collecting supernatant to obtain extract 2;

(3) preparing a liquid to be tested before loading on a machine: diluting the extract 2 prepared in the step (2) with water to a constant volume, and filtering to obtain a pre-machine test solution;

(4) and (3) determination: detecting the pre-machine liquid to be detected prepared in the step (3) by adopting an ultra-high performance liquid chromatography-electrostatic ion orbital hydrazine mass spectrometry system, and calculating to obtain the content of corresponding amino acid and nucleotide in a sample to be detected according to a standard curve linear regression equation of the amino acid and the nucleotide;

the chromatographic conditions for detection in the step (4) are as follows:

ternary gradient elution was performed using an XBridge BEH Amide (4.6mm × 150mm,5 μm) column with a-B-C as the mobile phase, where a: acetonitrile, B: 0.09-0.1% V/V formic acid aqueous solution, C: 0.09-0.1% of ammonia water with V/V;

the mass spectrum conditions of the detection in the step (4) are as follows:

an ionization mode: electrospray ionization;

an ionization mode: scanning positive ions and negative ions simultaneously;

scanning mode: full Scan/ddMS²；

Resolution ratio: 14000 FWHM;

ejection voltage: positive spectrum condition/first grade 3.0KV, negative spectrum condition/first grade 2.8 KV; positive spectrum condition/second level 3.0KV, negative spectrum condition/second level 2.8 KV;

capillary temperature: at 320 ℃.

2. The LC-MS/MS detection method for simultaneous determination of amino acids and nucleotides in animal tissues according to claim 1, characterized in that:

the volume ratio of the methanol to the water in the step (1) is (5-7.5): (2.5-5).

3. The LC-MS/MS detection method for simultaneous determination of amino acids and nucleotides in animal tissues according to claim 1, characterized in that:

the mass ratio of the total volume of the methanol and the water to the sample in the step (1) is 10: 0.5.

4. the LC-MS/MS detection method for simultaneous determination of amino acids and nucleotides in animal tissues according to claim 1, characterized in that:

the volume ratio of the total volume of the methanol and the water in the step (1) to the volume of the n-hexane in the step (2) is 1: 1.

5. the LC-MS/MS detection method for simultaneous determination of amino acids and nucleotides in animal tissues according to claim 1, characterized in that:

the standard curve linear regression equation of the amino acids and the nucleotides in the step (4) is obtained by the following method:

preparing amino acid and nucleotide standard substance working solutions with a series of concentration gradients, and detecting the amino acid and nucleotide standard substance working solutions by adopting an ultra-high performance liquid chromatography-electrostatic ion orbital hydrazine mass spectrometry system to obtain chromatograms corresponding to the standard substance working solutions; and (3) drawing a standard curve by taking the concentration of each standard substance working solution as a horizontal coordinate and the peak area as a vertical coordinate to obtain a standard curve linear regression equation of the amino acid and the nucleotide.

6. The LC-MS/MS detection method for simultaneous determination of amino acids and nucleotides in animal tissues according to claim 1, characterized in that:

the concentration gradient of the standard working solution is as follows:

cystine standard working solutions 0.125, 0.25, 0.5, 0.75, 1.0 and 1.25 μmol/L, other amino acid standard working solutions 0.25, 0.5, 1.0, 1.5, 2.0 and 2.5 μmol/L;

the nucleotide standard working solution was 10ppb, 20ppb, 40ppb, 60ppb, 80ppb, 100 ppb.

7. The LC-MS/MS detection method for simultaneous determination of amino acids and nucleotides in animal tissues according to claim 1, characterized in that:

the chromatographic conditions for detection in the step (4) are as follows:

the column temperature is 30 ℃, the sample injection amount is 2-10 mu L, and the flow rate is 100 mu L/min; elution conditions: equilibrate for 5min and elute with gradient for 20 min.

8. The LC-MS/MS detection method for simultaneous determination of amino acids and nucleotides in animal tissues according to claim 1, characterized in that:

the procedure of the ternary gradient elution is as follows:

9. the LC-MS/MS detection method for simultaneous determination of amino acids and nucleotides in animal tissues according to claim 1, characterized in that:

the amino acid is at least one of glycine, L-alanine, L-serine, L-proline, L-valine, L-threonine, L-leucine, L-isoleucine, L-aspartic acid, L-lysine, L-glutamic acid, L-methionine, L-histidine, L-phenylalanine, L-arginine, L-tyrosine and L-cystine.

10. The LC-MS/MS detection method for simultaneous determination of amino acids and nucleotides in animal tissues according to claim 1, characterized in that:

the nucleotide is at least one of cytosine nucleotide, uracil nucleotide, adenine nucleotide, hypoxanthine nucleotide and guanine nucleotide.

Technical Field

The invention relates to the field of food and biological detection, in particular to an LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) detection method for simultaneously determining amino acids and nucleotides in animal tissues.

Background

Amino acid is a general name of a class of organic compounds containing amino and carboxyl, and the amino acid is a basis for forming protein and is an indispensable class of small molecular substances in life activities; amino acids are essential nutrients in organisms, and play an important role in the activity of biomacromolecules and physiological functions thereof. The amino acid analysis research in organisms is helpful for understanding the life process, and has important effects on the diagnosis of diseases and the research of medicines.

At present, nucleotide is one of the hottest food nutrition enhancers and is added into a formula product by a large number of food manufacturers for functional popularization. The reason for this is that nucleotides have common biological metabolic functions, and not only are precursor structural units of DNA and RNA, but also are components of coenzymes (e.g., NAD, CoA) and active intermediates (e.g., intermediates of glycogen and glycoprotein), and are involved in multiple metabolic reactions in energy metabolism and cell signaling as physiological regulators, allosteric effectors and cell agonists. From another perspective, it is also important in the field of biological functions, improves immunity, promotes iron absorption, participates in lipoprotein metabolism, regulates intestinal flora, and helps tissue growth and differentiation.

A detection method of nucleotide is not established in China, and methods for determining nucleotide at home and abroad are various, including a fluorescence method, a high-efficiency capillary electrophoresis method, a high-efficiency liquid chromatography, an ion exchange chromatography, a liquid chromatography-mass spectrometry method and other mass spectrometry methods, and the existing detection method has the problems of easy interference, complex operation, poor reproducibility and the like. In the liquid chromatography-mass spectrometry, the research form is mainly used, the high-resolution mass spectrometry is used for analyzing and quantifying the nucleotide content, the requirements on the aspects of instrument maintenance and the like are high, meanwhile, the nucleotide isotope internal standard is difficult to obtain, and the accuracy of a detection result is difficult to guarantee due to the influence of a matrix effect on the ionization efficiency.

In recent years, the rapid development of mass spectrometry technology meets the requirement of highly sensitive detection of amino acid to some extent. The amino acid is easy to ionize, good response can be obtained under an electrospray ionization (ESI) positive ion mode, and meanwhile, due to the fact that mass spectrum detection signals are mass-to-charge ratios (m/z), even if baseline separation cannot be achieved, quantitative analysis can be conducted through different mass-to-charge ratios, and therefore in liquid chromatography-mass spectrometry (LC/MS) analysis of the amino acid, retention of the amino acid on a chromatographic column becomes a key. At present, the LC/MS analysis method of amino acid improves the retention behavior of the amino acid mostly by derivatization or adding an ion pair reagent, but the addition of the derivatization reagent can influence the ionization of the mass spectrum ion source to a certain extent.

The separation and detection technology of amino acid is always one of the concerns of researchers, and although the amino acid analysis method is various, the improvement of the existing method to make the method have better specificity and sensitivity is always a hotspot and difficulty problem of research. In the liquid phase separation of amino acid, because amino acid substances contain two functional groups of amino and carboxyl, on one hand, the polarity of the amino acid substances is very strong, and the amino acid substances are difficult to be well reserved on a reversed phase C18 chromatographic column directly, on the other hand, the ultraviolet absorption is weak, and in 20 common amino acids in a living body, except tyrosine, tryptophan and phenylalanine, high sensitive response is difficult to be obtained on an ultraviolet detector (UV). In order to improve the problems of amino acid retention and detection, a derivatization reagent is usually used for derivatization treatment of amino acid before or after a column, and then the amino acid is detected on the machine, but for biological sample analysis, the introduction of the derivatization reagent further complicates an originally complex system, and increases the difficulty of analysis.

The nucleotide belongs to a substance with negative charges, can be separated and quantified by applying anion exchange chromatography, but the ion chromatographic column has long balance time, the capillary electrophoresis method with low detection efficiency easily causes unstable detection results because the pH value of a buffer solution has great influence on the separation of the nucleotide. The nucleotide is detected by adopting the high performance liquid chromatography, and in the actual detection process, the general C18 chromatographic column is found to be difficult to well reserve and separate 5 nucleotides, a target compound is easily interfered by a foreign peak, an ion pair reagent is contained in a mobile phase, the chromatographic column has longer equilibrium time, the condition of retention time drift is easily caused, the impurity removal effect of sample pretreatment is not ideal, and the influence on the service life of the chromatographic column is larger.

Disclosure of Invention

In order to overcome the defects and shortcomings of the existing amino acid and nucleotide separation technology, the invention aims to provide the LC-MS/MS detection method for simultaneously determining the amino acid and the nucleotide in the animal tissue, the pretreatment operation of the method is more convenient, the derivatization treatment of a sample is not needed, and the better separation of at least 17 amino acids and 5 nucleotides can be realized based on the structural characteristics of the amino acids and the nucleotides.

The purpose of the invention is realized by the following technical scheme:

an LC-MS/MS detection method for simultaneously determining amino acids and nucleotides in animal tissues, comprising the following steps:

(1) extraction and removal of macromolecular substance interferences: mixing the minced sample to be detected with water, and uniformly performing vortex oscillation; adding methanol, and uniformly vortexing and oscillating; centrifuging, and collecting supernatant to obtain extract 1;

(2) removal of lipid interference on results: adding an organic reagent n-hexane into the extract 1 prepared in the step (1), and uniformly performing vortex oscillation; centrifuging, and collecting supernatant to obtain extract 2;

(3) preparing a liquid to be tested before loading on a machine: diluting the extract 2 prepared in the step (2) with water to a constant volume, and filtering to obtain a pre-machine test solution;

(4) and (3) determination: detecting the pre-machine liquid to be detected prepared in the step (3) by adopting an ultra-high performance liquid chromatography-electrostatic ion orbital hydrazine Mass Spectrometer (UHPLC-Thermo Fisher QOxctive Mass Spectrometer), and calculating to obtain the content of corresponding amino acid and nucleotide in a sample to be detected according to a standard curve linear regression equation of the amino acid and the nucleotide;

the amount of the sample in the step (1) is preferably 0.5 g;

the volume ratio of the methanol to the water in the step (1) is preferably (5-7.5): (2.5-5), the two components form a precipitator to precipitate macromolecular substances such as protein and the like; wherein, water is added firstly and vortexed, so that the minced sample can be completely and uniformly dispersed, and the problems of local precipitation, incomplete extraction and incomplete removal of macromolecular substances caused by directly adding a methanol aqueous solution, and further the accuracy of a final result is influenced are avoided;

the volume ratio of methanol to water in step (1) is more preferably 3: 1;

the mass ratio of the total volume of methanol and water to the sample in step (1) is preferably 10:0.5 (mL: g);

the ratio of the total volume of methanol and water in the step (1) to the volume of n-hexane in the step (2) is preferably 1: 1;

the centrifugation condition in the steps (1) and (2) is preferably 800-4000 r/min for 10-15 min;

the aperture of the filter membrane for filtering in the step (3) is preferably 0.1-0.5 μm;

the standard curve linear regression equation of the amino acids and the nucleotides in the step (4) is obtained by the following method:

preparing amino acid and nucleotide standard substance working solutions with a series of concentration gradients, and detecting the amino acid and nucleotide standard substance working solutions by adopting an ultra-high performance liquid chromatography-electrostatic ion orbital hydrazine mass spectrometry system to obtain chromatograms corresponding to the standard substance working solutions; taking the concentration of each standard substance working solution as a horizontal coordinate and a peak area as a vertical coordinate, drawing a standard curve, and obtaining a standard curve linear regression equation of the amino acid and the nucleotide;

the concentration gradient of the standard working solution is preferably as follows:

cystine standard working solutions 0.125, 0.25, 0.5, 0.75, 1.0 and 1.25 μmol/L, other amino acid standard working solutions 0.25, 0.5, 1.0, 1.5, 2.0 and 2.5 μmol/L;

10ppb, 20ppb, 40ppb, 60ppb, 80ppb, 100ppb of the working solutions of the nucleotide standards;

the chromatographic conditions for detection in the step (4) are as follows:

ternary gradient elution was performed using an XBridge BEH Amide (4.6mm × 150mm,5 μm) column with a-B-C as the mobile phase, where a: acetonitrile, B: 0.09-0.1% V/V formic acid aqueous solution, C: 0.09-0.1% of ammonia water with V/V;

the chromatographic conditions for the detection in step (4) are more preferably:

the column temperature is 30 ℃, the sample injection amount is 2-10 mu L, the flow rate is 100 mu L/min, and the elution conditions are as follows: balancing for 5min, and gradient eluting for 20 min;

the three-way gradient elution procedure is preferably as follows:

the mass spectrum conditions of the detection in the step (4) are as follows:

an ionization mode: electrospray ionization;

an ionization mode: scanning positive ions and negative ions simultaneously;

scanning mode: full Scan/ddMS²；

Resolution ratio: 14000 FWHM;

ejection voltage: 3.0KV under the positive spectrum condition (first level) and 2.8KV under the negative spectrum condition (first level); 3.0KV under the positive spectrum condition (secondary level), and 2.8KV under the negative spectrum condition (secondary level);

capillary temperature: 320 ℃;

auxiliary gas heating temperature: 300 ℃;

the atomizing gas, the sheath gas, the auxiliary gas and the collision gas are all high-purity nitrogen;

the amino acid is at least one of glycine (Gly), L-alanine (Ala), L-serine (Ser), L-proline (Pro), L-valine (Val), L-threonine (Thr), L-leucine (Leu), L-isoleucine (Ile), L-aspartic acid (Asp), L-lysine (Lys), L-glutamic acid (Glu), L-methionine (Met), L-histidine (His), L-phenylalanine (Phe), L-arginine (Arg), L-tyrosine (Tyr) and L-cystine (Cys);

the nucleotide is at least one of cytosine nucleotide (CMP), uracil nucleotide (UMP), adenine nucleotide (AMP), hypoxanthine nucleotide (IMP) and guanine nucleotide (GMP);

the principle of the invention is as follows:

aiming at the defects and shortcomings that in the prior art, a derivative reagent influences ionization of a mass spectrum ion source, a target compound is easily interfered by a hybrid peak, a chromatographic column has long balance time and is easy to generate retention time drift, and the removal effect of sample pretreatment on impurities is not ideal, so that the influence on the service life of the chromatographic column is large, a plurality of projects cannot be detected simultaneously, and the like, the invention develops a method for well retaining polar amino acids and nucleotides by using an Xbridge BEH Amide (4.6mm multiplied by 150mm,5 mu m) chromatographic column on the basis of an ultra-high performance liquid chromatography-electrostatic ion orbital hydrazine mass Spectrometer (UHPLC-Thermo Fisher Q activated Mass Spectrometer) system, greatly improves the retention problem of the amino acids and the nucleotides on LC/MS, and the like.

Firstly, the invention utilizes the characteristics of high sensitivity, strong anti-interference capability and the like of an ultra-high performance liquid chromatography four-level rod-electrostatic field orbital trap high-resolution mass spectrometry technology, extracts the accurate mass number of a target compound through a high-resolution mass spectrometry full-scan mode, automatically triggers a full-scan secondary mass spectrum by using a threshold value, eliminates matrix interference to a certain extent, has a simple sample pretreatment mode, and can simultaneously and rapidly screen and confirm 17 amino acids (glycine (Gly), L-alanine (Ala), L-serine (Ser), L-proline (Pro), L-valine (Val), L-threonine (Thr), L-leucine (Leu), L-isoleucine (Ile), L-aspartic acid (Asp), L-lysine (Lys), L-glutamic acid (Glu) in a sample and a standard product, L-methionine (Met), L-histidine (His), L-phenylalanine (Phe), L-arginine (Arg), L-tyrosine (Tyr), L-cystine (Cys)) and 5 nucleotides (cytosine nucleotide (CMP), uracil nucleotide (UMP), adenine nucleotide (AMP), inosinic acid (IMP) and guanine nucleotide (GMP)).

Secondly, the matrix of the Xbridge BEH Amide amino column used by the invention is triple bond Amide based on ethylene bridge hybrid particles (BEH), and the triple bond Amide bond combination technology provides excellent column service life and method robustness; the pH range which can be endured is wider, the stability of the chemical character is higher under the conditions of high pH and high temperature, the compatibility with a high pH mobile phase is facilitated, the MS detection is facilitated without derivatization, the sample pretreatment work before LC-MS analysis is greatly simplified, and the analysis sensitivity is improved. The BEH Amide chromatographic column adopts ethylene bridge hybrid particles, has a relatively high tower plate number due to the particle size of 5 mu m, has higher separation efficiency, and can effectively separate 17 amino acids and 5 nucleotides from impurities.

Thirdly, the invention optimizes the sample pretreatment and the chromatographic conditions, so that the sample pretreatment can remove more impurities, and methanol is adopted in the pretreatment process of the sample to be detected: the water-mixed organic solvent can effectively reduce the interference of macromolecular compounds in a sample to be detected, and normal hexane is added to remove grease. The chromatographic condition with better separation effect is selected, so that the amino acid and 5 nucleotides in the 17 amino acids are effectively separated from the impurities, and the accuracy of the detection data is ensured. Good separation results are obtained.

Compared with the prior art, the invention has the following advantages and effects:

(1) compared with the prior art, the method adopts a dual retention mechanism of acid elution and alkaline elution, adopts an Xbridge BEH Amide chromatographic column, can achieve the aim of simultaneously separating ions with positive charges and ions with negative charges on the basis of a procedure of gradient elution from acid to alkali, does not need to add ion pairs and derivatization reagents, and provides a new detection direction for the retention problem of strong polar substances.

(2) The method is simple and easy to implement, greatly reduces the pretreatment time and cost, effectively solves the problem of separating positively charged ions and negatively charged ions simultaneously by taking 17 amino acids and 5 nucleotides as typical examples, shortens the detection time of the two substances, reduces the error of instruments, and is convenient for uniformly processing and analyzing data.

(3) The invention provides a detection method for simultaneously identifying an LC-MS/MS positive ion mode and a negative ion mode for measuring 17 amino acids and 5 nucleotides in animal tissues, and the method has excellent reproducibility, accuracy and sensitivity.

(4) The invention provides technical support for relevant departments to make corresponding laws and regulations and provides reference for future research in the field of food.

Drawings

FIG. 1 is an extracted ion chromatogram (Amide column) of 17 amino acid standards; wherein, 1-NL is 2.64E6, m/z is 76.03550-76.04310; 2-NL:1.38E7, m/z 90.05046-90.05946; 3-NL:1.50E7, m/z 106.04457-106.05517; 4-NL 8.10E7, m/z 116.06481-116.07641; 5-NL 6.00E7, m/z 118.08036-118.09216; 2.47E7 for 6-NL, 120.05952-120.07152 for m/z; 7-NL 1.11E8, m/z 132.09530-132.10852; 8-NL 7.94E6, m/z 134.03808-134.05148; 9-NL:1.30E7, m/z 147.11206-147.11354; 10-NL:2.41E7, m/z 148.05969-148.06117; 11-NL 5.86E7, m/z 150.05758-150.05908; 12-NL:2.18E7, m/z 156.07597-156.07753; 13-NL 5.02E7, m/z 166.08543-166.08709; 14-NL 5.85E7, m/z 175.11807-175.11983; 15-NL:4.06E7, m/z 182.08206-182.08208; 16-NL 4.20E7, m/z 241.02991-241.0323.

FIG. 2 is an extracted ion chromatogram (Amide column) of 5 nucleotide standards; wherein, 1-NL is 8.87E7, m/z is 322.04296-322.04618; 2-NL:1.20E7, m/z 323.02967-323.0302; 3-NL:7.03E6, m/z 346.05408-346.05754; 4-NL 6.83E6, m/z 347.03808-347.04158; 5-NL 2.98E6, m/z 362.04891-362.05253.

FIG. 3 is an extracted ion chromatogram (C18 column) of a 17 amino acid standard; wherein, 1-NL is 2.46E7, m/z is 76.03550-76.04310; 2-NL 5.72E7, m/z 90.05046-90.05946; 3-NL:2.17E7, m/z 106.04457-106.05517; 4-NL 8.15E7, m/z 116.06481-116.07641; 5-NL 1.98E8, m/z 118.08036-118.09216; 6-NL 4.33E7, m/z 120.05952-120.07152; 7-NL 1.81E8, m/z 132.09530-132.10852; 8-NL 1.05E7, m/z 134.03808-134.05148; 2.13E7 for 9-NL, 147.11206-147.11354 for m/z; 10-NL:7.14E7, m/z 148.05969-148.06117; 11-NL:3.17E7, m/z 150.05758-150.05908; 12-NL:2.86E8, m/z 156.07597-156.07753; 13-NL:1.00E8, m/z 166.08543-166.08709; 14-NL:7.90E7, m/z 175.11807-175.11983; 15-NL:3.29E7, m/z 182.08206-182.08208; 16-NL 4.01E5, m/z 241.02991-241.03233.

FIG. 4 is an extracted ion chromatogram (C18 column) of 5 nucleotide standards; wherein, 1-NL is 2.26E5, m/z is 322.04296-322.04618; 2-NL 8.48E5, m/z 323.02967-323.0302; 3-NL:6.73E6, m/z 346.05408-346.05754; 4-NL:3.19E7, m/z 347.03808-347.04158; 5-NL:1.07E6, m/z 362.04891-362.05253.

FIG. 5 is an extracted ion chromatogram (Amide column, two-phase elution) of 17 amino acid standards; wherein, 1-NL is 5.51E6, m/z is 76.03550-76.04310; 2-NL:1.56E7, m/z 90.05046-90.05946; 3-NL:2.04E7, m/z 106.04457-106.05517; 4-NL2.04E7, m/z 116.06481-116.07641; 9.27E7 for 5-NL, 118.08036-118.09216 for m/z; 3.38E7 for 6-NL, 120.05952-120.07152 for m/z; 7-NL 1.56E7, m/z 132.09530-132.10852; 8-NL 1.03E7, m/z 134.03808-134.05148; 9-NL:1.77E7, m/z 147.11206-147.11354; 10-NL:3.16E7, m/z 148.05969-148.06117; 11-NL:1.02E8, m/z 150.05758-150.05908; 12-NL 8.12E7, m/z 156.07597-156.07753; 13-NL:1.71E8, m/z 166.08543-166.08709; 14-NL 9.90E7, m/z 175.11807-175.11983; 15-NL:1.08E8, m/z 182.08206-182.08208; 16-NL 2.19E7, m/z 241.02991-241.03233.

FIG. 6 is an extracted ion chromatogram (Amide column, two-phase elution) of 5 nucleotide standards; wherein, 1-NL is 3.16E5, m/z is 322.04296-322.04618; 2-NL:2.92E6, m/z 323.02967-323.0302; 3-NL:1.20E7, m/z 346.05408-346.05754; 4-NL 8.00E7, m/z 347.03808-347.04158; 5-NL 1.30E6, m/z 362.04891-362.05253.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The following examples used instruments and equipment:

(1) liquid chromatogram-mass spectrum/mass spectrum combination appearance: an ultra-high performance liquid chromatography-electrostatic ion orbital hydrazine Mass spectrometry (UHPLC-Thermo Fisher Q active Mass Spectrometer) system;

(2) an electronic balance: the accuracy is 0.01mg, 0844cf0173-1 (national measurement and test center of south China, Guangdong province research institute of metrology science);

(3) a centrifuge: the maximum rotation speed is 4000r/min (Changshan Xiangzhi centrifuge instruments Co., Ltd.);

(4) microporous filter membrane: 0.22 μm (Shanghai Anpu scientific instruments Co.);

(5) a liquid transferring gun: 5mL, 1000. mu.L, 100. mu.L,s (Brand, Germany);

(6) heat-resisting centrifuging tube of taking spiral shell lid or other heat-resisting centrifuging tube that can seal: 50 mL;

(7) an injector: 1mL and 2 mL.

Reagents and solutions used in the following examples:

the water is deionized water; 17 amino acid standards and 5 nucleotide standards: purchased from national standard substance research center, with purity more than or equal to 99.99%; methanol: carrying out chromatographic purification; n-hexane: and (4) carrying out chromatographic purification.