CN113933421A

CN113933421A - LC-MSMS technology-based method for quantitatively analyzing theanine and metabolites thereof

Info

Publication number: CN113933421A
Application number: CN202111166734.3A
Authority: CN
Inventors: 谢永明; 王朝阳; 王俊霞
Original assignee: Shanghai Applied Protein Technology Co Ltd
Current assignee: Shanghai Applied Protein Technology Co Ltd
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2022-01-14

Abstract

The invention provides a method for quantitatively analyzing theanine and metabolites thereof based on an LC-MSMS (liquid chromatography-mass spectrometry) technology, belonging to the technical field of detection of theanine and metabolites thereof, wherein a sample to be detected is subjected to pretreatment of liquid chromatography tandem mass spectrometry, and each pretreated sample is respectively subjected to liquid chromatography tandem mass spectrometry so as to accurately and quantitatively analyze theanine and metabolites thereof; the standard sample uses 1x phosphate buffer salt solution as a substitute matrix in the preparation process, is simultaneously suitable for plasma and urine, and has the advantages of improving the sample pretreatment efficiency and saving the cost.

Description

LC-MSMS technology-based method for quantitatively analyzing theanine and metabolites thereof

Technical Field

The invention belongs to the technical field of detection of theanine and metabolites thereof, and particularly relates to a method for quantitatively analyzing the theanine and the metabolites thereof, which is based on an LC-MSMS (liquid chromatography-mass spectrometry) technology and is simultaneously suitable for blood and urine.

Background

Catecholamines (CA) are a class of neurohormones synthesized and secreted by adrenergic neurons and containing catechol and amine groups, including Dopamine (DA), Norepinephrine (NE) and epinephrine (E), the major metabolites of which are 3-methoxytyramine (3-MT), methoxynorepinephrine (NMN), and Methoxyepinephrine (MN). Clinically, the method has important clinical significance for diagnosing and treating pheochromocytoma, paraganglioma, neuroblastoma, hypertension, myocardial infarction, adrenal medullary hyperplasia and other diseases. The consensus of the diagnosis and treatment experts of pheochromocytoma and paraganglioma is published in 2020 of China, and the consensus is clear: MN and NMN in plasma or urine are used as the first-choice indexes for diagnosing pheochromocytoma and paraganglioma, and the indexes of NE, E, DA, 3-MT and the like of the plasma or urine are simultaneously suggested to be measured. The method for measuring CA level also suggests LC-MSMS method or high performance liquid chromatography.

The analysis and detection of catecholamine substances at home and abroad mainly comprise a high performance liquid chromatography, a fluorescence photometry, a capillary electrophoresis method, a chemiluminescence method, an electrochemical analysis and a liquid chromatography-mass spectrometry method. Although the methods such as fluorescence, capillary electrophoresis, chemiluminescence, electrochemical analysis and the like are simple, the methods have obvious defects in the aspects of sensitivity, interference resistance, joint detection of various compounds and the like. The high performance liquid chromatography can only realize the detection of DA \ E \ NE 3 prototypes due to the properties of the compounds, and can not accurately determine indexes such as intermediate metabolites and the like.

For the LC-MSMS method, many test methods are available on the market: the invention of 'a method for detecting plasma catecholamine by using liquid chromatography tandem mass spectrometry' applied by Hangzhou medical inspection institute Limited company in 2016 at present, adopts dansyl chloride to perform derivatization extraction and then performs detection, and has the advantages of complex sample treatment, difficult popularization, poor specificity and capability of detecting only 3 compounds. The invention of 'liquid quality analysis method for detecting content of catecholamine metabolites in 24H urine' applied in 2019 by Beijing and Hei medical diagnostic technology GmbH only detects 3 catecholamine metabolites. Similarly, the invention of the high performance liquid chromatography-tandem mass spectrometry detection method for free catecholamine and metabolites thereof in human urine, which is applied in 2018 by Hangzhou Kaili spectral accurate medical detection technology Limited, is also only applicable to 5 catecholamine compounds. Generally, the problems of complex derivatization pretreatment, incapability of reaching sensitivity, capability of detecting only partial compounds and the like mainly exist in the process of detecting catecholamine substances in blood plasma or urine by using a liquid chromatography-mass spectrometry technology. There were also no derivatization methods tested, but the 6 compounds all peaked at the 1min peak position with poor resolution.

Disclosure of Invention

Based on the problems in the prior art, the invention provides a method for quantitatively analyzing the theanine and the metabolite thereof based on the LC-MSMS technology, which is simultaneously suitable for plasma and urine and has the advantages of improving the sample pretreatment efficiency and saving the cost.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a method for quantitatively analyzing theanine and metabolites thereof based on LC-MSMS technology comprises the steps of carrying out pretreatment of liquid chromatography tandem mass spectrometry on samples to be detected, respectively carrying out liquid chromatography tandem mass spectrometry on each pretreated sample, and accurately and quantitatively analyzing the theanine and the metabolites thereof; the standard samples used 1x phosphate buffered saline as a surrogate matrix during the formulation process.

According to the above scheme, the said theanine is Dopamine (DA), Noradrenaline (NE) and adrenaline (E); the metabolites are 3-methoxytyramine (3-MT), methoxynorepinephrine (NMN) and Methoxyepinephrine (MN).

According to the scheme, the method for quantitatively analyzing the theanine and the metabolites thereof based on the LC-MSMS technology comprises the following detailed steps:

step S1, preparing a calibrator solution, a quality control solution and an internal standard working solution with series concentrations;

step S2, adding an internal standard working solution into a sample to be detected, then adding an ammonium acetate buffer solution, shaking and mixing, and then centrifuging to obtain a supernatant;

step S3, adding methanol into the SPE solid phase extraction column for activation, and adding ammonium acetate to balance the SPE solid phase extraction column;

and step S4, adding the supernatant obtained in the step S2 into the SPE solid phase extraction column processed in the step S3, drying the SPE solid phase extraction column by positive pressure, adding water, methanol and isopropanol into the SPE solid phase extraction column in sequence for leaching, eluting the SPE solid phase extraction column by using an acetonitrile solution, drying the eluent by using nitrogen after collecting the eluent, and re-dissolving the eluent by using 100 mu L of pure water.

And step S5, performing liquid chromatography-tandem mass spectrometry on the redissolution finally obtained in the step S4.

According to the above scheme, the conditions of the liquid chromatography are as follows:

and (3) analyzing the column: ACE EXCEL 1.7C18-PFP 100 × 2.1 mm;

elution gradient:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0.00	98	2
1.00	98	2
			2.50	90	10
3.50	5	95
			4.50	5	95
5.00	98	2
			5.50	98	2

Mobile phase: phase A: an aqueous solution containing 0.1% acetonitrile; phase B: methanol solution containing 0.1% acetonitrile; flow rate: 0.4 mL/min.

According to the above scheme, the mass spectrum conditions are as follows:

an ionization mode: spot spray ionization positive ion mode (ESI +); the detection mode is as follows: multiple Reaction Monitoring (MRM); ion source Temperature (TEM): 550 ℃; ion source gas 1(GS 1): 55 psi; ion source gas 2(GS 2): 55 psi; air curtain gas (CUR): 40 psi; spray voltage (IS): 5500V; collision gas (CAD): 8.

according to the above scheme, the MRM parameters of the compounds in the mass spectrometric detection are as follows:

compound (I)	Q1	Q3	Dwell(ms)	DP	CE
						Dopamine	154.1	137.2	10	3	15
Dopamine	154.1	91	10	3	32
						dopamine-D4	158.1	95	10	3	32
Adrenalin	166.1	107.1	10	34	25
						Adrenalin	166.1	135.2	10	34	21
epinephrine-D6	172.1	112.1	10	34	25
						Norepinephrine	152	107.1	10	35	24
Norepinephrine	152	135.1	10	35	17
						norepinephrine-D6	158	111	10	35	24
Methoxy norepinephrine	166.1	134.2	10	25	22
						Methoxy norepinephrine	166.1	121.2	10	25	23
Methoxy norepinephrine-D3	169.1	137.2	10	25	22
						Methoxy adrenalin	180.1	165.1	10	42	23
Methoxyproterenol	180.1	148.2	10	42	24
						Methoxyproterenol-D3	183.1	151.2	10	42	24
3-methoxytyramine	151	91	10	38	26
						3-methoxytyramine	151	119.1	10	38	19
3-methoxytyramine-D4	155	123.1	10	38	19

Denotes the quantitative ion pair of the compound.

The invention has the beneficial effects that:

1. the method can be used for detecting catecholamine in two different types of samples, namely the plasma sample and the urine sample, saves cost, improves efficiency and is convenient for clinical popularization.

2. The method adopts non-derivatization pretreatment, saves fussy derivatization time and steps, and has good reproducibility.

Drawings

FIG. 1 is a liquid chromatography-tandem mass spectrometry analysis result graph of dopamine in example two.

FIG. 2 is a liquid chromatography-tandem mass spectrometry analysis result spectrum of isotopically labeled dopamine in example two.

FIG. 3 is a liquid chromatography-tandem mass spectrometry analysis of norepinephrine in example two.

FIG. 4 is a liquid chromatography-tandem mass spectrometry analysis of norepinephrine internal isotope label of example two.

FIG. 5 is a liquid chromatography-tandem mass spectrometry analysis of norepinephrine in example two.

FIG. 6 is a liquid chromatography-tandem mass spectrometry analysis of norepinephrine internal isotope label of example two.

FIG. 7 is a liquid chromatography-tandem mass spectrometry analysis of noradrenaline in example two.

FIG. 8 is a liquid chromatography-tandem mass spectrometry analysis of noradrenaline with isotopic internal standard according to example two.

FIG. 9 is a diagram of the results of liquid chromatography-tandem mass spectrometry of metanephrin in example two.

FIG. 10 is a liquid chromatography-tandem mass spectrometry analysis of the isotopically labeled epinephrine of example two.

FIG. 11 is a graph showing the results of liquid chromatography-tandem mass spectrometry analysis of 3-methyltyramine in example two.

FIG. 12 is a liquid chromatography-tandem mass spectrometry analysis result spectrum of 3-methyltyramine as an isotope internal standard in example two.

FIG. 13 is a linear profile of dopamine in example two.

FIG. 14 is a linear profile of dopamine in example one.

Figure 15 is a linear profile of epinephrine in example two.

Figure 16 is a linear profile of epinephrine in example one.

Figure 17 is a linear profile of norepinephrine in example two.

FIG. 18 is a linear profile of norepinephrine in example one.

FIG. 19 is a linear profile of metanephrin in example two.

FIG. 20 is a linear profile of metanephrin in the first example.

FIG. 21 is a linear map of noradrenaline in example two.

FIG. 22 is a linear map of noradrenaline in example one.

FIG. 23 is a linear map of 3-methoxytyramine in example two.

FIG. 24 is a linear map of 3-methoxytyramine in example one.

The figures are LC-MS/MS detection analysis result graphs which are result displays in the embodiment, characters in the graphs are result displays, and the results change according to the result of each detection analysis, namely the characters in the graphs are irrelevant to whether the detection method provided by the invention can be repeatedly implemented, and the characters in the graphs are unclear, so that a person skilled in the art can repeatedly implement the detection method provided by the invention.

Detailed Description

The technical solution of the present invention will be described below with reference to the specific embodiments and the accompanying drawings.

The first embodiment is as follows: quantitative analysis of theanine and its metabolites in plasma.

A method for quantitatively analyzing theanine and metabolites thereof based on LC-MSMS technology comprises the steps of carrying out pretreatment of liquid chromatography tandem mass spectrometry on samples to be detected, respectively carrying out liquid chromatography tandem mass spectrometry on each pretreated sample, and accurately and quantitatively analyzing the theanine and the metabolites thereof; the standard sample uses 1x phosphate buffer salt solution as a substitute matrix in the preparation process; the theanine is Dopamine (DA), Norepinephrine (NE) and epinephrine (E); the metabolites are 3-methoxytyramine (3-MT), methoxynorepinephrine (NMN) and Methoxyepinephrine (MN), and the whole method comprises the following detailed steps:

step S1, preparing a calibrator (standard) solution, a quality control solution and an internal standard working solution with series concentrations;

preparation of stock solution in step S11: dopamine (DA), Noradrenaline (NE), adrenaline (E), 3-methoxytyramine (3-MT), methoxynoradrenaline (NMN) and Methoxyadrenaline (MN) were accurately weighed using an analytical balance, and isotopically labeled dopamine-d 4(DA-d4), adrenaline-d 6(E-d6), noradrenaline-d 6(NE-d6), methoxyadrenaline-d 3(MN-d3), methoxynoradrenaline-d 3(NMN-d3), 3-methoxytyramine-d 4(3-MT-d4) were dissolved in 5% HCl (1M) + 95% MeOH (50% original concentration) to prepare standard stock solutions and stock solutions as follows:

step S12, preparation of standard curve working solution and quality control working solution:

for the standard curve working solution, the stock solution of the standard prepared in step S11 was diluted with 50% MeOH solution containing 0.2% FA and mixed to obtain a mixed standard working solution. Calculating the working solution of the mixed standard product according to proper concentration, diluting according to different times, and finally obtaining the working solution of a calibration curve and the quality control working solution which are suitable for respective matrix concentration ranges, wherein the detailed concentration points are as follows:

for the quality control working solution, the stock solution of the standard product prepared in step S11 is diluted with 50% MeOH solution containing 0.2% FA, and mixed to obtain a mixed quality control working solution, wherein the detailed concentration points are as follows:

preparation of step S13 internal standard working solution

Diluting the internal standard stock solution prepared in the step S11 by using 50% MeOH solution containing 0.2% FA and mixing to obtain mixed internal standard working solution, wherein the concentration of the noradrenaline isotope internal standard working solution is 4ng/mL, the concentration of the adrenaline isotope internal standard working solution is 2ng/mL, the concentration of the dopamine isotope internal standard working solution is 2ng/mL, the concentration of the methoxy noradrenaline isotope internal standard working solution is 2ng/mL, the concentration of the methoxy adrenaline isotope internal standard working solution is 2ng/mL, and the concentration of the 3-methoxytyramine isotope internal standard working solution is 2 ng/mL.

Step S14, preparation of calibration curve and quality control product:

a) preparation of Standard Curve samples

For the calibrator, the standard curve sample was obtained by diluting the standard working solution 20-fold using 1xPBS as the surrogate substrate.

The linear range of plasma is: the linear range of dopamine, adrenaline, methoxyadrenaline and 3-methoxytyramine is as follows: 50-5000pmol/L, linear range of noradrenaline and noroxyarenine: 100-10000 pmol/L.

b) Preparation of quality control product

For the quality control product, dividing the mixed human plasma (n is more than or equal to 6) into three parts, diluting the quality control working solution by 20 times to obtain the quality control product, and finally forming:

DA，E，MN，3-MT：X，X+150pM，X+2000pM,X+4000pM；

NE，NMN：X，X+300pM，X+4000pM,X+8000pM；

wherein X is mixed plasma.

Step S2, transferring 400 mu L of plasma sample to be detected into a 96-well plate by using a pipette, adding 30 mu L of internal standard solution, then adding 300 mu L of 50mM/L ammonium acetate buffer solution, shaking for 5min, centrifuging for 10min at 4000rpm, and taking supernatant;

step S3, adding 300 μ L of methanol into the SPE solid phase extraction column for activation, and then adding 300 μ L of 50mM ammonium acetate to balance the SPE solid phase extraction column;

and step S4, adding the supernatant obtained in the step S2, a calibrator (standard) solution with a series of concentrations and a quality control solution into the SPE solid-phase extraction column processed in the step S3, drying the SPE solid-phase extraction column under positive pressure, sequentially adding 400 mu L of water, 400 mu L of methanol and isopropanol into the SPE solid-phase extraction column for leaching, wherein the volume usage of the isopropanol is equal to that of the supernatant obtained in the step S2, finally eluting the mixture twice by using 200 mu L of 5% acetonitrile solution, merging the eluents, blowing the eluents by using nitrogen gas for drying, and finally redissolving the eluates by using 100 mu L of pure water.

And step S5, detecting the re-dissolved solution finally obtained in the step S4 by using high performance liquid chromatography triple quadrupole mass spectrometry, obtaining a standard curve equation by fitting the peak area ratio of the standard substance and the internal standard substance, wherein the standard curve equation is shown in the attached figures 14, 16, 18, 20, 22 and 24, and calculating the concentration of the standard substance to be detected.

The UPLC-MSMS is adopted, and the instrument model is as follows: shimadzu LC-30A ultra high performance liquid tandem AB Sciex 6500plus mass spectrometer, the conditions of the liquid chromatogram are as follows:

and (3) analyzing the column: ACE EXCEL 1.7C18-PFP 100 × 2.1 mm;

elution gradient:

The mass spectrum conditions were as follows:

an ionization mode: spot spray ionization positive ion mode (ESI +); ion source Temperature (TEM): 550 ℃; ion source gas 1(GS 1): 55 psi; ion source gas 2(GS 2): 55 psi; air curtain gas (CUR): 40 psi; spray voltage (IS): 5500V; collision gas (CAD): 8; mass spectrum acquisition time: 5.5min, detection mode: multiple Reaction Monitoring (MRM); the multiple reaction monitoring parameters were as follows:

Denotes the quantitative ion pair of the compound.

Example two: and (3) quantitatively analyzing the theanine and the metabolites thereof in the urine.

The difference between this embodiment and the first embodiment lies in the steps S12-S14, and the detailed differences are as follows:

preparation of step S13 internal standard working solution

Diluting the internal standard stock solution prepared in the step S11 by using 50% MeOH solution containing 0.2% FA and mixing to obtain mixed internal standard working solution, wherein the concentration of the noradrenaline isotope internal standard working solution is 500ng/mL, the concentration of the adrenaline isotope internal standard working solution is 1200ng/mL, the concentration of the dopamine isotope internal standard working solution is 5000ng/mL, the concentration of the methoxy noradrenaline isotope internal standard working solution is 1000ng/mL, the concentration of the methoxy adrenaline isotope internal standard working solution is 500ng/mL, and the concentration of the 3-methoxytyramine isotope internal standard working solution is 2500 ng/mL.

Step S14, preparation of calibration curve and quality control product:

a) preparation of Standard Curve samples

The linear reference range for urine is: the linear range of dopamine and norepinephrine is 1.00-200 mu M/L; the linear range of adrenaline and methoxy adrenaline is 0.05-10.0 mu M/L; the linear range of the methoxy norepinephrine and the 3-methoxytyramine is 0.1 to 20 mu M/L.

b) Preparation of quality control product

For the quality control product, dividing the mixed and acidified 24h urine (n is more than or equal to 6) of a person into three parts, diluting the quality control working solution by 20 times to obtain the quality control product, and finally forming:

E，MN：Y，Y+7.5，Y+250,Y+375nM；

NMN，3-MT：Y，Y+15，Y+500,Y+750nM；

DA，NE：Y，Y+150，Y+5000,Y+7500nM；

wherein Y is the background value of the mixed urine.

The results of the detection analysis and the linear profiles in this example are shown in FIGS. 1-13, 15, 17, 19, 21 and 23.

The three-day verification of the performance index of the method is carried out on the analysis method provided by the invention, and the results are as follows:

the 6 target compounds of the plasma matrix and the urine matrix are verified to meet the following performances.

The present invention is provided by the above embodiments only for illustrating and not limiting the technical solutions of the present invention, and although the above embodiments describe the present invention in detail, those skilled in the art should understand that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and any modifications and equivalents may fall within the scope of the claims.

Claims

1. A method for quantitatively analyzing theanine and metabolites thereof based on LC-MSMS technology is characterized in that a sample to be detected is subjected to pretreatment of liquid chromatography tandem mass spectrometry, each pretreated sample is respectively subjected to liquid chromatography tandem mass spectrometry, and the theanine and the metabolites thereof are accurately and quantitatively analyzed; the standard samples used 1x phosphate buffered saline as a surrogate matrix during the formulation process.

2. The method for the quantitative analysis of the theanine and its metabolites based on the LC-MSMS technology as claimed in claim 1, wherein the theanine is dopamine, norepinephrine and epinephrine; the metabolites are 3-methoxytyramine, methoxynorepinephrine and methoxyepinephrine.

3. The method for the quantitative analysis of theanine and its metabolites based on LC-MSMS technology as claimed in claim 2, which comprises the following detailed steps:

4. The method for the quantitative analysis of theanine and its metabolites based on LC-MSMS technology as claimed in claim 3, wherein the conditions of the liquid chromatography are as follows:

and (3) analyzing the column: ACE EXCEL 1.7C18-PFP 100 × 2.1 mm;

elution gradient:

5. The method for the quantitative analysis of theanine and its metabolites based on LC-MSMS technology as claimed in claim 4, wherein the mass spectrometric conditions are as follows:

an ionization mode: point spray ionization positive ion mode; the detection mode is as follows: monitoring multiple reactions; ion source temperature: 550 ℃; ion source gas 1: 55 psi; ion source gas 2: 55 psi; air curtain air: 40 psi; spraying voltage: 5500V; collision gas: 8.

6. the method for the quantitative analysis of theanine and its metabolites based on LC-MSMS technology as claimed in claim 5, wherein the MRM parameters of the compound in the mass spectrometric detection are as follows:

Denotes the quantitative ion pair of the compound.