CN115184478A - A Quantitative Analysis Method of 70 Amino Substances in Plasma Based on "Intrafibrous Derivatization" Strategy and Its Use - Google Patents

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a quantitative analysis method of 70 amino substances in blood plasma based on a fiber internal derivatization strategy and application thereof. The analysis method comprises the steps of an 'intra-fiber derivatization' strategy, liquid phase separation, mass spectrometry and data processing. The amino-containing substance refers to a collection of metabolites containing primary amine and secondary amine groups in blood plasma, is an endogenous substance with important physiological effects, and can be used as a cancer marker for diagnosis and treatment of cancers. Due to the characteristics of high polarity of the amino-containing substance and the like, the defects of long sample pretreatment time, complex steps, high consumption of organic solvent, low sensitivity and the like exist when the derivative analysis is carried out by adopting an LC-MS technology. The invention provides a simplified high-sensitivity and high-accuracy analysis method for amino-containing substances, and provides a basis for clinical cancer diagnosis and treatment.

Description

Determination of 70 amino acids in plasma based on an "intrafibrillar derivatization" strategy Quantitative analysis methods and their uses

technical field

The invention belongs to the technical field of medicine, and in particular relates to a quantitative analysis method for 70 kinds of amino substances in plasma based on an "intrafiber derivatization" strategy and use thereof.

Background technique

Amino-containing substances refer to the collection of all metabolites containing primary and secondary amine groups in plasma, including biogenic amines, nucleosides and their metabolites amino acids, purines and pyrimidines. It exists widely in living organisms and participates in important life activities such as cell proliferation, synthesis of biological macromolecules such as proteins and nucleic acids, and is closely related to the occurrence and development of cancer.

Due to the high polarity of amino-containing substances, pre-column derivatization is still the most commonly used method in order to improve the separation ability, sensitivity and quantitative accuracy when using LC-MS analysis. However, long sample preparation times and large consumption of organic solvents resulting from complex sample preparation steps are major problems for most current derivatization methods. In recent years, in situ derivatization methods that react directly in the sample matrix and omit the extraction step have received extensive attention, but in order to reduce matrix effects, post-derivatization still needs to be combined with extraction techniques. And limited metabolite detection coverage, poor stability and robustness are still issues that need to be addressed.

In recent years, microextraction technology represented by solid-phase microextraction and liquid-phase microextraction technology has been widely used in the analysis of in vivo samples without or with less use of organic solvents. Carbon nanofibers (CNFs/CFs) grown on carbon fibers were used as the extraction substrate, and the nanopores of carbon nanofibers were limited to the solvent to extract the analytes according to the principle of "similar compatibility". The solvent consumption is low, the extraction and derivatization reactions are rapid due to the huge specific surface area of CNFs/CFs, and the material is durable.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problem of poor analysis effect of amino-containing substances by LC-MS, the present invention adopts the strategy of "intrafiber derivatization" to establish a simultaneous quantitative analysis method for 70 kinds of amino-containing substances in plasma. The analytical method described above measured the concentrations of 70 amino-containing substances in the plasma of healthy people and lung cancer patients, and screened out novel lung cancer diagnostic biomarkers by independent sample t-test, PLS-DA, univariate and multivariate ROC analysis.

Specifically, the present invention is achieved through the technical solutions of the following aspects:

In a first aspect, the present invention provides a method for quantitative analysis of 70 amino-containing substances in plasma based on an "intrafibrous derivatization" strategy, the analysis method comprising the following steps:

(1) "Intrafiber derivatization" strategy:

a. Take a certain amount of plasma, dilute it with a certain amount of distilled water, add a certain amount of internal standard solution, mix methanol and distilled water in a certain proportion to prepare a mixed solution, and immerse the CNFs/CFs in a certain amount of the mixed solution for 20s , take out CNFs/CFs, and then immerse the treated CNFs/CFs into the above-mentioned diluted plasma and internal standard mixed solution, and vortex for a certain period of time;

b. Take out the CNFs/CFs after the above extraction, add a certain amount of Na ₂ CO ₃ solution dropwise to the CNFs/CFs, and then add a certain amount of benzoyl chloride acetonitrile solution dropwise, react for a certain period of time, take out, and put in a certain amount After ultrasonic desorption in the desorption solvent for a certain period of time, the supernatant was dried and redissolved for LC-MS analysis;

(2) Liquid phase separation:

a. Using C18 as stationary phase,

b. Mobile phase A: formic acid water, mobile phase B: formic acid methanol,

Gradient elution and flow rate control;

(3) MS determination:

Electrospray ion source, positive ion scan, multiple reaction ion monitoring mode.

As an optional method, in the above quantitative analysis method, in step (1)a operation, the amount of distilled water is 100 μL-400 μL, the mixing ratio of methanol and water is 1:1-3, and the amount of methanol and water mixed solvent is 50 μL-200 μL.

As an optional method, in the above quantitative analysis method, in the operation of step (1)b, the concentration of the benzoyl chloride acetonitrile solution is 2%-6%, the concentration of the Na ₂ CO ₃ solution is 200mM-600 mM, and the amount of the benzoyl chloride acetonitrile solution is used. For 50 μL-200 μL, the amount of Na ₂ CO ₃ solution is 50 μL-200 μL.

As an optional method, in the above quantitative analysis method, in the operation of step (1)b, the derivatization reaction time is 2min-10min, and the ultrasonic desorption solvent is 300μL-1000μL.

As an optional method, in the above quantitative analysis method, the gradient elution in step (2) is that the methanol volume concentration increases from 50% to 95% within 60 minutes, and the injection volume is 5 μL-15 μL.

As an optional way, in the above-mentioned quantitative analysis method, in step (3),

MS measurement conditions are triple quadrupole tandem mass spectrometry system, electrospray ionization source (ESI source), positive ion detection; source temperature 300°C; capillary voltage 4000V; vacuum (10e-5) 2.5 Torr; nitrogen as atomizing gas and drying gas, atomizing gas pressure 15psi; drying gas flow rate 11L/min; scanning mode is multiple reaction ion monitoring mode (MRM),

Amino-containing substances in plasma identified by MS are selected from one or more of the following: ethanolamine, glycine, sarcosine, L-alanine, gamma aminobutyric acid, L-serine, hypotaurine, cytosine , histamine, L-proline, L-valine, L-homoserine, taurine, L-threonine, pipecolic acid, acetyl putrescine, agmatine, aminolevulinic acid, L -Leucine, L-Isoleucine, L-Asparagine, Aspartic Acid, Adenine, L-Glutamine, L-Glutamic Acid, L-Methionine, Guanine, Xanthine, Tryptamine , L-phenylalanine, homogentisic acid, L-arginine, L-citrulline, 1,3-propanediamine, L-homocysteine, quinolinic acid, putrescine, vanilla almond acid, vanillic acid, cadaverine, 5-hydroxyindole-3-acetic acid, guanidinoacetic acid, D-cysteine, L-carnosine, N ¹ ,N ⁸ -diacylated spermamidine, 4-hydroxyproline acid, L-ornithine, L-homocysteine, tyramine, uridine, L-lysine, octopamine, 3-hydroxyanthranilic acid, L-histidine, adenosine, 3- Methoxytyramine, 3,4-dihydroxyphenylacetic acid, serotonin, L-tyrosine, N8-acylated ^spermamidine , levodopa, metanephrine, L-tryptophan, L-canine Uridine, L-cystine, ^spermamidine , norepinephrine, N1, ^N12 -diacylated spermine, epinephrine or spermine.

As an optional method, in the above quantitative analysis method, the quantitative analysis method further includes (4) a data processing step, and according to different uses of the quantitative analysis method, an appropriate data processing method is selected for analysis.

As an optional method, in the above quantitative analysis method, the quantitative analysis method further includes (4) a data processing step, the quantitative analysis method is used for screening cancer diagnostic biomarkers, and the step (4) is specifically: first, through an independent Sample T test and PLS-DA were used to screen diagnostic biomarkers of cancer, and their diagnostic ability was evaluated by multivariate ROC curve.

In a second aspect, the present invention provides the use of the quantitative analysis method described in the first aspect above in screening cancer diagnostic biomarkers.

Preferably, the cancer is lung cancer.

In a third aspect, the present invention provides a cancer biomarker composition consisting of sarcosine, alanine, gamma-aminobutyric acid, proline, Isoleucine, glutamic acid, glutamine, phenylalanine and citrulline, the cancer biomarker composition is obtained by screening using the quantitative analysis method described in the first aspect.

Preferably, the cancer is lung cancer.

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

The invention solves the problems in the prior art that LC-MS is used to analyze the ubiquitous pretreatment steps of amino-containing substances, which leads to long sample pretreatment time and consumption of a large amount of organic solvents, and develops a simplified high sensitivity, A high-accuracy method based on the "intrafibrous derivatization" strategy can quantitatively analyze 70 amino-type substances in plasma, and a lung cancer biomarker composition was successfully screened by the quantitative analysis method, which is a clinical cancer biomarker composition. Diagnosis and treatment provide more options.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the specification, and are used to explain the present invention together with the embodiments of the present invention, and do not constitute a limitation to the present invention. In the attached image:

Fig. 1 is a total ion current chromatogram obtained by the method of Example 1.

FIG. 2 is a graph of the PLS-DA score of lung cancer biomarkers screened according to the method described in Example 1. FIG.

Figure 3 is a ROC curve diagram for judging the combined diagnostic ability of the screened lung cancer markers.

Figure 4 is a total ion current chromatogram obtained according to the method of Example 2.

FIG. 5 is a total ion current chromatogram obtained by implementing the method in Example 3. FIG.

FIG. 6 is a total ion current chromatogram obtained by implementing the method in Example 4. FIG.

FIG. 7 is a total ion current chromatogram obtained by implementing the method in Example 5. FIG.

FIG. 8 is a total ion current chromatogram obtained by implementing the method in Example 6. FIG.

Detailed ways

The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate the present invention, but not to limit the scope of the present invention.

If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased through formal channels.

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

Example 1:

(1) "Intrafiber derivatization" strategy:

a. Take 100 μL of human plasma into an EP tube, dilute with 200 μL of distilled water, and add 10 μL of mixed internal standard solution (see Table 2 for specific types of internal standards). Mix methanol and distilled water at 2:1, v/v to prepare a mixed solution, immerse the CNFs/CFs into a certain amount of the mixed solution for 20s, take out the CNFs/CFs, and then immerse the treated CNFs/CFs into the above-mentioned mixture. In the diluted plasma and internal standard mixed solution, vortex extraction for 3 min;

b. Take out the CNFs/CFs after the above extraction, add 50 μL 400mM Na ₂ CO ₃ solution dropwise to the CNFs/CFs, and then add 50 μL 4% (v/v) benzoyl chloride acetonitrile solution dropwise, after 5min reaction, take out, After being put into 300 μL methanol for ultrasonic desorption for 5 min, the supernatant was dried and redissolved for LC-MS analysis;

(2) Liquid phase separation:

Column: Kinetex XB-C ₁₈ (4.6×100mm, 2.6μm); Mobile phase: A phase: 0.1% (v/v) formic acid water, B phase: 0.1% (v/v) formic acid methanol; flow rate: 0.4mL ·min ⁻¹ ; injection volume: 5 μL; column temperature: 25° C.; see Table 1 for the gradient elution procedure.

Table 1: Gradient elution procedure

(3) MS determination:

Agilent 6420 triple quadrupole tandem mass spectrometer system, electrospray ionization source (ESI source), positive ion detection; source temperature 300°C; capillary voltage 4000V; vacuum degree (10e-5) 2.5 Torr; nitrogen as atomizing gas and drying Gas, atomizing gas pressure 15psi; drying gas flow rate 11L/min; scanning mode is multiple reaction ion monitoring mode (MRM), other parameters and analyte ion channels are shown in Table 2.

Table 2: Mass Spectrometry Parameters of Amino-Containing Substances

(4) Data processing:

The accuracy and precision results of the method described in Example 1 are shown in Table 3 and Table 4. Meet the relevant requirements for quantitative determination of biological samples. The accuracy and precision results of the methods described in Examples 2 to 6 below are similar to those of the method described in Example 1, and all meet the relevant requirements for quantitative determination of biological samples. Due to the limited length of the description, no specific details are listed here. result. The total ion current chromatogram of 70 amino-containing substances in the plasma determined in Example 1 is shown in Figure 1 .

Table 3: Method Precision Results

Table 4: Accuracy results of the method

The method described in Example 1 of the present invention can be used to screen biomarkers of lung cancer by comparing the detection results of the plasma of lung cancer patients with that of healthy people.

Blood samples were collected from healthy subjects and lung cancer patients, including 34 healthy adults; 20 lung cancer patients, lung cancer patients and healthy adults collected venous blood on an empty stomach in the morning, and the collected blood samples were placed in pre-labeled heparinized test tubes , centrifuged at 3500 rpm for 10 min, separated plasma, and stored frozen in an ultra-low temperature refrigerator. This study complies with the Declaration of Helsinki and was approved by the Ethics Committee of the Northern Theater General Hospital with ethics number K(2016)38. All subjects understood the purpose of the study and signed a written informed consent.

Using the measured concentrations of amino-containing metabolites in plasma as independent variables, multivariate statistical analysis was used to reduce the dimensionality of the data to find biomarkers in plasma that distinguish healthy patients from lung cancer patients. First, the markers were preliminarily screened using the P value (P<0.05) of the independent sample T test and the VIP value (VIP>1.5) of the orthogonal partial least squares discriminant analysis (OPLS-DA); Regression-ROC curve method was used to further screen and evaluate the combined diagnostic ability of biomarkers. The score chart is shown in Figure 2. It can be seen that the healthy group and lung cancer patients are clearly distinguished, and the biomarkers of lung cancer are finally determined as sarcosine, alanine, γ-aminobutyric acid, proline, isoleucine , glutamic acid, glutamine, phenylalanine and citrulline. The ROC curve for judging the combined diagnostic ability of the screened lung cancer biomarkers is shown in Figure 3. The area under the curve (AUC) value of 1 indicates that the selected lung cancer biomarkers have a strong combined diagnostic ability for lung cancer.

The present invention takes Example 1 as an example to illustrate the use of the quantitative detection method of the present invention for screening cancer diagnostic biomarkers. Similar results were obtained using the quantitative detection methods described in Examples 2-6.

Example 2:

The difference between this embodiment and Example 1 is the amount of distilled water in the step (1) a and the gradient elution program in the step (2), and the specific process is:

(1) "Intrafiber derivatization" strategy:

a. Take 100 μl of human plasma into an EP tube, dilute with 100 μL of distilled water, and add 10 μL of mixed internal standard solution (see Table 2 for specific types of internal standards). Mix methanol and distilled water at 2:1, v/v to prepare a mixed solution, immerse the CNFs/CFs into 100 μL of the mixed solution for 20 s, take out the CNFs/CFs, and immerse the treated CNFs/CFs into the above diluted solution. In the mixed solution of plasma and internal standard, vortex extraction for 3 min;

b. Take out the CNFs/CFs after the above extraction, add 50 μL 400mM Na ₂ CO ₃ solution dropwise to the CNFs/CFs, and then add 50 μL 4% (v/v) benzoyl chloride acetonitrile solution dropwise, after 5min reaction, take out, After being put into 300 μL methanol for ultrasonic desorption for 5 min, the supernatant was dried and redissolved for LC-MS analysis;

(2) Liquid phase separation:

Column: Kinetex XB-C ₁₈ (4.6×100mm, 2.6μm); Mobile phase: A phase: 0.1% (v/v) formic acid water, B phase: 0.1% (v/v) formic acid methanol; flow rate: 0.4mL ·min ⁻¹ ; injection volume: 5 μL; column temperature: 25° C.; see Table 3 for the gradient elution procedure.

Table 3: Gradient elution procedure

(3) MS determination:

The MS measurement conditions are the same as those in Example 1, and other parameters and ion channels of the analyte are shown in Table 2.

(4) Data processing:

The total ion current chromatogram of 70 amino-containing substances in the plasma determined in Example 2 is shown in FIG. 4 . The biomarkers of lung cancer were screened by independent sample T test and PLS-DA, and their diagnostic ability was evaluated by multi-factor ROC curve. amino acid, isoleucine, glutamic acid, glutamine, phenylalanine and citrulline.

Example 3:

The difference between this embodiment and embodiment 1 is the mixing ratio and consumption of methanol and water in the step (1) a and the vortex extraction time, and the specific process is:

(1) "Intrafiber derivatization" strategy:

a. Take 100 μL of human plasma into an EP tube, dilute with 200 μL of distilled water, and add 10 μL of mixed internal standard solution (see Table 2 for specific types of internal standards). Mix methanol and distilled water at 3:1, v/v to prepare a mixed solution, immerse the CNFs/CFs into 50 μL of the mixed solution for 20 s, remove the CNFs/CFs, and immerse the treated CNFs/CFs into the above-diluted plasma and the internal standard mixed solution, vortex extraction for 2min;

b. Take out the above-extracted CNFs/CFs, drop 50 μL of 400 mM Na ₂ CO ₃ solution onto the CNFs/CFs, and then dropwise add 50 μL of 4% (v/v) benzoyl chloride acetonitrile solution. After 5 minutes of reaction, take it out, put it into 300 μL methanol for ultrasonic desorption for 5 minutes, dry and redissolve the supernatant for LC-MS analysis;

(2) Liquid phase separation:

Column: Kinetex XB-C ₁₈ (4.6×100mm, 2.6μm); Mobile phase: A phase: 0.1% (v/v) formic acid water, B phase: 0.1% (v/v) formic acid methanol; flow rate: 0.4mL ·min ^-1 ; Injection volume: 5 μL; Column temperature: 25°C; Gradient elution procedure is shown in Table 1;

(3) MS determination:

The MS measurement conditions are the same as those in Example 1, and other parameters and ion channels of the analyte are shown in Table 2.

(4) Data processing:

The total ion current chromatogram of 70 amino-containing substances in the plasma determined in Example 3 is shown in Figure 5 . The biomarkers of lung cancer were screened by independent sample T test and PLS-DA, and their diagnostic ability was evaluated by multi-factor ROC curve. amino acid, isoleucine, glutamic acid, glutamine, phenylalanine and citrulline.

Example 4:

The difference between this embodiment and embodiment 1 is the concentration of Na ₂ CO ₃ solution and the concentration of benzoyl chloride acetonitrile solution in the step (1)b, and the specific process is:

(1) "Intrafiber derivatization" strategy:

a. Take 100 μL of human plasma into an EP tube, dilute with 200 μL of distilled water, and add 10 μL of mixed internal standard solution (see Table 2 for specific types of internal standards). Mix methanol and distilled water at 2:1, v/v to prepare a mixed solution. Immerse the CNFs/CFs in 100 μL of the mixed solution for 20s, take out the CNFs/CFs, immerse the treated CNFs/CFs into the above diluted plasma and internal standard mixed solution, and extract by vortexing for 3 min;

b. Take out the CNFs/CFs after the above extraction, add 50 μL 600mM Na ₂ CO ₃ solution dropwise to the CNFs/CFs, and then add dropwise 50 μL 6% (v/v) benzoyl chloride acetonitrile solution, after 5 minutes of reaction, take out, After being put into 300 μL methanol for ultrasonic desorption for 5 min, the supernatant was dried and redissolved for LC-MS analysis;

(2) Liquid phase separation:

Column: Kinetex XB-C ₁₈ (4.6×100mm, 2.6μm); Mobile phase: A phase: 0.1% (v/v) formic acid water, B phase: 0.1% (v/v) formic acid methanol; flow rate: 0.4mL ·min ⁻¹ ; injection volume: 5 μL; column temperature: 25° C.; see Table 1 for the gradient elution procedure.

(3) MS determination:

The MS measurement conditions are the same as those in Example 1, and other parameters and ion channels of the analyte are shown in Table 2.

(4) Data processing:

The total ion current chromatogram of the 70 amino-containing substances in the plasma determined in Example 4 is shown in FIG. 6 . The biomarkers of lung cancer were screened by independent sample T test and PLS-DA, and their diagnostic ability was evaluated by multi-factor ROC curve. amino acid, isoleucine, glutamic acid, glutamine, phenylalanine and citrulline.

Example 5:

The difference between this embodiment and Example 1 is the Na ₂ CO ₃ solution consumption and the benzoyl chloride acetonitrile solution consumption in the step (1) b, and the specific process is:

(1) "Intrafiber derivatization" strategy:

a. Take 100 μL of plasma into an EP tube, dilute with 200 μL of distilled water, and add 10 μL of mixed internal standard solution (see Table 2 for specific types of internal standards). Mix methanol and distilled water at 2:1, v/v to prepare a mixed solution. Immerse CNFs/CFs in 100 μL mixed solution for 20s, take out CNFs/CFs, immerse the treated CNFs/CFs in the above diluted plasma and internal standard mixed solution, and extract by vortex for 3 min;

b. Take out the CNFs/CFs after the above extraction, add 20 μL 400mM Na ₂ CO ₃ solution dropwise to the CNFs/CFs, and then add 20 μL 4% (v/v) benzoyl chloride acetonitrile solution dropwise, after 5 minutes of reaction, take out, Put it into 300 μL methanol for ultrasonic desorption for a certain period of time, then dry and redissolve the supernatant for LC-MS analysis;

(2) Liquid phase separation:

Column: Kinetex XB-C ₁₈ (4.6×100mm, 2.6μm); Mobile phase: A phase: 0.1% (v/v) formic acid water, B phase: 0.1% (v/v) formic acid methanol; flow rate: 0.4mL ·min ⁻¹ ; injection volume: 5 μL; column temperature: 25° C.; see Table 1 for the gradient elution procedure.

(3) MS determination:

The MS measurement conditions are the same as those in Example 1, and other parameters and ion channels of the analyte are shown in Table 2.

(4) Data processing:

The total ion current chromatogram of 70 amino-containing substances in the plasma determined in Example 5 is shown in Figure 7 . The biomarkers of lung cancer were screened by independent sample T test and PLS-DA, and their diagnostic ability was evaluated by multi-factor ROC curve. amino acid, isoleucine, glutamic acid, glutamine, phenylalanine and citrulline.

Example 6:

The difference between this embodiment and Embodiment 1 is the derivatization reaction time and the desorption solvent consumption in the step (1) b, and the specific process is:

(1) "Intrafiber derivatization" strategy:

a. Take 100 μL of human plasma into an EP tube, dilute with 200 μL of distilled water, and add 10 μL of mixed internal standard solution (see Table 2 for specific types of internal standards). Mix methanol and distilled water at a ratio of 2:1, v/v to prepare a mixed solution. Immerse the CNFs/CFs in 100 μL of the mixed solution for 20s, take out the CNFs/CFs, immerse the treated CNFs/CFs into the above diluted plasma and internal standard mixed solution, and extract by vortexing for 3 min;

b. Take out the CNFs/CFs after the above extraction, add 50 μL 400mM Na ₂ CO ₃ solution dropwise to the CNFs/CFs, and then add 50 μL 4% (v/v) benzoyl chloride acetonitrile solution dropwise, react for 2 min, take out, Put it into 500 μL methanol for ultrasonic desorption for 5 min, then dry and redissolve the supernatant for LC-MS analysis;

(2) Liquid phase separation:

Column: Kinetex XB-C ₁₈ (4.6×100mm, 2.6μm); Mobile phase: A phase: 0.1% (v/v) formic acid water, B phase: 0.1% (v/v) formic acid methanol; flow rate: 0.4mL ·min ⁻¹ ; injection volume: 5 μL; column temperature: 25° C.; see Table 1 for the gradient elution procedure.

(3) MS determination:

The MS measurement conditions are the same as those in Example 1, and other parameters and ion channels of the analyte are shown in Table 2.

(4) Data processing:

The total ion current chromatogram of 70 amino-containing substances in plasma determined in Example 6 is shown in FIG. 8 . The biomarkers of lung cancer were screened by independent sample T test and PLS-DA, and their diagnostic ability was evaluated by multi-factor ROC curve. amino acid, isoleucine, glutamic acid, glutamine, phenylalanine and citrulline.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention. Thus, provided that these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims (10)

1. A quantitative analysis method of 70 amino-containing substances in blood plasma based on a strategy of 'derivatization in fiber', which is characterized by comprising the following steps: the analysis method comprises the following steps:

(1) "derivatization within fibers" strategy:

a. diluting a certain amount of plasma with a certain amount of distilled water, adding a certain amount of internal standard solution, mixing methanol and distilled water according to a certain proportion to prepare a mixed solution, immersing the CNFs/CFs into the certain amount of mixed solution for 20s, taking out the CNFs/CFs, immersing the treated CNFs/CFs into the diluted plasma and internal standard mixed solution, and performing vortex extraction for a certain time;

b. taking out the extracted CNFs/CFs, and adding a certain amount of Na ₂ CO ₃ Dripping the solution on CNFs/CFs, dripping a certain amount of benzoyl chloride acetonitrile solution, reacting for a certain time, taking out, putting into a certain amount of desorption solvent for ultrasonic desorption for a certain time, blow-drying, redissolving, and taking supernatant for LC-MS analysis;

(2) Liquid phase separation:

a. c18 is adopted as a stationary phase,

b. mobile phase A: formic acid water, mobile phase B: the formic acid methanol is used as a raw material,

gradient elution and flow rate control;

(3) MS measurement:

electrospray ion source, positive ion scanning, and multiple reactive ion monitoring mode.

2. The quantitative analysis method according to claim 1, characterized in that: in the operation of the step (1) a, the dosage of distilled water is 100-400 mu L, the mixing ratio of methanol to water is 1-3, the dosage of the mixed solvent of methanol and water is 50-200 mu L, and the vortex extraction time is 1-5min.

3. The quantitative analysis method according to claim 1 or claim 2, characterized in that: in the operation of the step (1) b, the concentration of benzoyl chloride acetonitrile solution is 2-6 percent, v/v, na ₂ CO ₃ The concentration of the solution is 200mM-600mM, the dosage of the benzoyl chloroacetonitrile solution is 50 mu L-200 mu L, na ₂ CO ₃ The dosage of the solution is 50-200. Mu.L.

4. The quantitative analysis method according to any one of claims 1 to 3, characterized in that: in the operation of the step (1) b, the derivatization reaction time is 2min-10min, and the ultrasonic desorption solvent is 300 mu L-1000 mu L.

5. The quantitative analysis method according to any one of claims 1 to 4, characterized in that: in the step (2), the gradient elution is that the volume concentration of methanol is increased from 50 percent to 95 percent within 60 minutes, and the injection volume is 5-15 muL.

6. The quantitative analysis method according to any one of claims 1 to 5, characterized in that: in the step (3), the amino-containing substance identified by MS in the plasma is selected from one or more of the following: ethanolamine, glycine, sarcosine, L-alanine, gamma-aminobutyric acid, L-serine, hypotaurine, cytosine, histamine, L-proline, L-valine, L-homoserine, taurine, L-threonine, pipecolic acid, acetylputrescine, agmatine, aminolevulinic acid, L-leucine, L-isoleucine, L-asparagine, aspartic acid, adenine, L-glutamine, L-glutamic acid, L-methionine, guanine, xanthine, tryptamine, L-phenylalanine, homogentisic acid, L-arginine, L-citrulline, 1, 3-propanediamine, L-homocysteine, uroquinolinic acid, putrescine, vanilla mandelic acid, vanillic acid, cadaverine, 5-hydroxyindole-3-acetic acid, guanidinoacetic acid, D-citrulline-cysteine, L-carnosine, N ¹ ,N ⁸ Diacylated spermidine, 4-hydroxyproline, L-ornithine, L-homocysteine, tyramine, uridine, L-lysine, octopamine, 3-hydroxyanthranilic acid, L-histidine, adenosine, 3-methoxytyramine, 3, 4-dihydroxyphenylacetic acid, 5-hydroxytryptamine, L-tyrosine, N-dihydroxyphenylacetic acid ⁸ Acylated spermidine, levodopa, metaadrenaline, L-tryptophan, L-kynurenine, L-cystine, spermidine, noradrenaline, N ¹ ,N ¹² -diacylated spermine, epinephrine or spermine.

7. The quantitative analysis method according to any one of claims 1 to 6, characterized in that: the quantitative analysis method further comprises (4) a data processing step of selecting an appropriate data processing method for analysis according to the use of the quantitative analysis method.

8. The quantitative analysis method according to any one of claims 1 to 6, characterized in that: the quantitative analysis method also comprises (4) a data processing step, the quantitative analysis method is used for screening the cancer diagnosis biomarkers, and the step (4) is specifically as follows: the diagnostic biomarkers of cancer were first screened by independent sample T-test, PLS-DA and their diagnostic ability was evaluated by a multifactorial ROC curve.

9. Use of the quantitative analysis method of any one of claims 1 to 8 for screening cancer diagnostic biomarkers.

10. A cancer biomarker composition, characterized by: the cancer biomarker composition consists of: sarcosine, alanine, gamma-aminobutyric acid, proline, isoleucine, glutamic acid, glutamine, phenylalanine and citrulline, which is screened using the quantitative analysis method described in any one of claims 1 to 8.

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