CN112834656A - UPLC-MS/MS-based plasma cardiovascular disease related biomarker targeted metabonomics quantification method - Google Patents
UPLC-MS/MS-based plasma cardiovascular disease related biomarker targeted metabonomics quantification method Download PDFInfo
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Abstract
The invention belongs to the field of bioanalysis, and relates to a UPLC-MS/MS-based sensitive and reliable plasma cardiovascular disease related biomarker targeted metabonomics quantification method. By optimizing mass spectrum parameters and a liquid chromatography method, one-time sample injection within 16min is realized, and high-flux, high-sensitivity and high-resolution detection is carried out on 20 biomarkers metabolized by pathways such as aromatic amino acid catabolism, trimethylamine oxide biosynthesis and histidine metabolism. The method has good linearity, the linear correlation coefficients are both more than 0.99, the in-day precision and the in-day precision are respectively 1.12-14.12% and 0.30-13.74%, and the recovery rate and the stability can also meet the analysis requirements of biological samples. The targeted metabonomics method has proved to have strong capability of accurately analyzing metabolic markers, provides valuable information for large-scale biomarker verification, is a material basis for clarifying potential cardiovascular diseases, and provides powerful support for clinical diagnosis or early warning.
Description
Technical Field
The invention belongs to the field of bioanalysis, and relates to a UPLC-MS/MS-based plasma cardiovascular disease related biomarker targeted metabonomics quantification method.
Background
Cardiovascular disease (CVD) has become a leading cause of death and a serious public health challenge worldwide. According to the data of the world health organization, the number of deaths caused by cardiovascular diseases in 2016 is up to 1790 ten thousand. Without early prevention and intervention strategies, the prevalence of cardiovascular disease will continue to increase. Atherosclerosis, which results from the deposition and progressive accumulation of oxidized low density lipoprotein cholesterol (LDL-C), is a major cause of CVD. However, its underlying pathogenesis has not been fully elucidated, which prevents early warning and effective risk assessment of CVD. Exploring the pathophysiological basis of this complex disease is an urgent need for diagnosing atherosclerotic cerebrovascular disease, and exploring disease-related sensitive, specific and reliable biomarkers is a research approach.
Metabonomics, as an emerging "omics" strategy, is used to describe changes of small molecule metabolites in biological systems under diseases or abnormal conditions, and has been widely applied to the fields of disease candidate biomarker identification, pathological mechanism research, clinical drug discovery and evaluation, and the like. In recent years, a number of metabolomics-based biomarker identification and pathophysiological mechanism exploration have been increasingly applied to cardiovascular diseases. Several metabonomic studies have shown that plasma TMAO is a new marker of cardiovascular risk, has an atherogenic effect, and its biosynthetic precursors such as carnitine, choline, betaine, trimethyllysine and γ -butyrolactone can also be independent risk factors for CVD. In addition, aromatic amino acids (phenylalanine, tyrosine, tryptophan) and their microbial metabolites have also been shown to be closely related to cardiovascular disease, corresponding to their key role in immune regulation.
Non-targeted metabolome technology and targeted metabolome technology are the primary methods of metabolomic research. The non-target metabolome can provide more comprehensive and richer metabolome component information of a biological sample, and has the defects of peak alignment, peak identification error, high false positive rate identification, difficult reproduction of results among different instruments, difficult detection of low-abundance trace substances and the like. The targeted metabonomics are also called quantitative metabonomics, can accurately quantify related metabolites of specific metabolic pathways or components in a complex biological sample, and have higher sensitivity and accuracy compared with a non-targeted metabonomic group. Therefore, the establishment of a high-sensitivity high-coverage targeted metabonomics analysis method has important significance for accurately evaluating endogenous metabolites, especially key trace metabolites. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with Multiple Reaction Monitoring (MRM) can provide higher sensitivity and selectivity for the validation and quantification of potential biomarkers. LC-MS/MS techniques have been used to demonstrate the association between cardiovascular disease and certain metabolites (e.g. valine, isoleucine and leucine) but no report has been made to verify the accuracy and reproducibility of the methods used, but the content of these methodologies is clearly crucial to obtaining undoubted results, and in addition the relevant metabolic markers are not comprehensive, and there is no reported high-throughput quantitative method that covers all relevant markers.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention establishes a sensitive and reliable liquid chromatogram-tandem mass spectrometry (UPLC-MS/MS) for simultaneously quantifying cardiovascular disease biomarkers, including 20 biomarkers of TMAO, carnitine, succinic acid, phenylalanine, tryptophan, tyrosine and the like, and verifies the accuracy, precision, linearity, stability, extraction recovery rate and other performances of the method, thereby ensuring the accuracy and reliability of the quantification method. The method is expected to provide accurate quantitative results of the biomarkers, so that the pathological mechanism of cardiovascular diseases is more deeply understood.
The invention provides a UPLC-MS/MS-based plasma cardiovascular disease related biomarker targeted metabonomics quantification method, which comprises the following steps:
(1) preparation of standards
(1.1) respectively dissolving a standard product and an Internal Standard (IS) in a first solvent to respectively obtain a standard stock solution and an IS stock solution;
(1.2) diluting the standard stock solution in a gradient way, wherein the gradient dilution ratio is 1-1/400;
(1.3) selecting standard quality control products: selecting standard stock solution, and defining the standard stock solution as a high-concentration quality control product HQC, a medium-concentration quality control product MQC and a low-concentration quality control product LQC after non-dilution, 1/20 dilution and 1/100 dilution;
(1.4) all the prepared standard solutions were stored in a 4 ℃ refrigerator.
In the step (1.1), the first solvent is methanol and/or water; preferably, methanol/water (v/v-1/1).
In step (1.1), the concentration of the standard stock solution is 1 mg/mL.
In the step (1.1), the standard substance is L-histidine, L-lysine, succinic acid, L-threonine, L-tryptophan, L-tyrosine, trimethylamine-nitrogen oxide (TMAO), betaine, carnitine, choline chloride, 2-hydroxybutyric acid, 3-hydroxybutyric acid, indole-3-acetic acid (IAA), Indoleacrylic Acid (IA), indole-3-propionic acid (IPA), pantothenic acid, phenylacetylglutamine (PAGLn), phenylalanine, phenyllactic acid, or phenylpyruvic acid.
In step (1.1), the IS stock solution has a concentration of 1 mg/mL.
In the step (1.1), the Internal Standard (IS) IS a stable isotope labeled internal standard substance (IS) and comprises trimethylamine-D9-oxide (TMAO-D9), 3-hydroxybutyric acid-D4 sodium salt (3HB-D4), L-lysine-4, 4, 5, 5-D4 hydrochloride (lysine-D4), D-phenylalanine-D7 (phenylalanine-D7), indole-2, 4, 5, 6, 7-D5-3-acetate (IAA-D5), L-tyrosine-13C 9, 15N (tyrosine-13C 9, 15N), L-tryptophan-D5 (tryptophan-D5), phenylacetyl-D5-L-glutamine (PAGLn-D5), choline chloride-D9 (choline-D9), Succinic acid-2, 2, 3, 3-d4 (succinic acid-d 4).
In step (1.2), the gradient dilution ratio is 1, 1/2, 1/4, 1/10, 1/20, 1/40, 1/100, 1/200 and 1/400.
(2) Sample preparation
Plasma sample preparation:
(a) taking a plasma sample in an EP tube, and adding an Internal Standard (IS) and a methanol/water solution; then, adding a protein precipitator to precipitate protein; then, after low-speed vortex, high-speed low-temperature centrifugation is carried out;
(b) transferring the centrifuged supernatant of the step (a) and centrifuging and concentrating;
(c) redissolving the concentrated residue of step (b) in a second solvent; then evenly mixing the mixture by low-speed vortex, and centrifuging the mixture at high speed and low temperature; then separating the supernatant and filtering with a pore plate filter;
preparing a low-concentration quality control product, a medium-concentration quality control product, a high-concentration quality control product and a standard working calibration sample: and (c) preparing the low-concentration quality control product, the medium-concentration quality control product, the high-concentration quality control product and the standard working calibration sample according to the steps (a) - (c).
Blank control sample preparation: the blank control sample was prepared by replacing plasma with ultrapure water (blank substrate) and methanol/water solution with IS standard stock solution, and then following steps (a) - (c).
Wherein, the standard working calibration sample specifically refers to a QC quality control sample after all plasma samples are mixed.
In step (a), the plasma sample is taken in an amount of 50-150. mu.L, preferably 100. mu.L.
In step (a), the internal standard is taken at 5-15. mu.L, preferably 10. mu.L.
In the step (a), the internal standard IS IS a stable isotope labeled internal standard substance (IS) and comprises trimethylamine-D9-oxide (TMAO-D9), 3-hydroxybutyric acid-D4 sodium salt (3HB-D4), L-lysine-4, 4, 5, 5-D4 hydrochloride (lysine-D4), D-phenylalanine-D7 (phenylalanine-D7), indole-2, 4, 5, 6, 7-D5-3-acetate (IAA-D5), L-tyrosine-13C 9, 15N (tyrosine-13C 9, 15N), L-tryptophan-D5 (tryptophan-D5), phenylacetyl-D5-L-glutamine (PAGLn-D5), choline chloride-D9 (choline-D9), Succinic acid-2, 2, 3, 3-d4 (succinic acid-d 4).
In step (a), the volume of the methanol/water solution is 10 to 30. mu.L, preferably 20. mu.L.
In step (a), the protein precipitating agent is acetonitrile and/or methanol, preferably acetonitrile.
In step (a), the volume of the protein precipitant is 300-500. mu.L, preferably 300. mu.L.
In step (a), the low speed is 2000-3000rpm, preferably 2500 rpm.
In step (a), the vortex time is 3-10min, preferably 5 min.
In step (a), the high speed is 10000-12000rpm, preferably 10000 rpm.
In step (a), the low temperature is 4-10 ℃, preferably 4 ℃.
In step (b), the concentration time is 1-3h, preferably 2 h.
In step (c), the second solvent is acetonitrile/water, methanol/water, preferably acetonitrile/water.
In the step (c), the volume of the second solvent is 100-300. mu.L, preferably 100. mu.L.
In step (c), the high speed is 10000-12000rpm, preferably 10000 rpm.
In step (c), the low temperature is 4-10 ℃, preferably 4 ℃.
In step (c), the low speed is 2000-3000rpm, preferably 2500 rpm.
In step (c), the well plate is a 96 well plate.
(3) UPLC-MS/MS analysis
The LC-MS system adopts a Waters ACQUITY UPLC I-Class system and an AB SCIEX 5500Q-Trap system in series.
A Waters HSS T3 column (1.8 μm,100 mm. times.2.1 mm) was fitted using a positive and negative ion scheduled MRM mode, the column temperature was set at 35 ℃, mobile phase A was 0.1% FA-water, mobile phase B was acetonitrile, the flow rate was 0.25mL/min, and the analytical gradient was as follows: 0-10min, 2% B-100% B; 10-11min, 100% B; 11.1-16min, 2% B.
The mass spectrum parameters were as follows: the declustering voltage DP and the collision energy CE are optimized according to the specificity of each compound, the source parameters gas 1: 60 psi; gas 2: 50 psi; air curtain air: 20 psi; source temperature: at 450 ℃; spraying voltage: 5500/-4500V.
In a specific embodiment, the method specifically comprises the steps of:
preparation of standards
Standard substance was dissolved or diluted in methanol/water (50/50, v/v) to prepare standard stock solutions and IS stock solutions, respectively, at a final concentration of 1.0 mg/mL. Based on the detected concentrations of the corresponding metabolites in plasma (metabolite concentration range in plasma: 1/20-1/200, unit is mg/mL), mixed standards of the corresponding concentration gradients were prepared. Standard working calibration solutions were obtained after serial dilution of the combined standard stock solutions at dilution ratios of 1, 1/2, 1/4, 1/10, 1/20, 1/40, 1/100, 1/200 and 1/400. To make the detection point in the middle of the linear range, the concentration at the 1/20 dilution point was approximately equal to five times the concentration detected in plasma. No dilution, 1/20 dilution, and 1/100 dilution were defined as high, medium, and low concentration quality controls (HQC, MQC, LQC), respectively. All standard solutions were stored in a 4 ℃ freezer prior to on-machine analysis.
Sample preparation
Plasma sample preparation: 100 μ L of plasma samples were taken in 1.5mL EP tubes and 10 μ L of internal standard and 20 μ L of MeOH/water (50:50, v/v) solution were added. In addition, 300. mu.L of acetonitrile was added to precipitate the protein, vortexed at 2500rpm for 5min, and centrifuged at 10000rpm (4 ℃, 5 min). The supernatant was transferred and concentrated by centrifugation for 2 hours. The residue was redissolved in 100. mu.L acetonitrile/H2O (2:98, v/v), vortexed for 5min, and then centrifuged at 10000rpm (4 ℃, 5 min). Prior to liquid chromatography-mass spectrometry, the supernatant was separated and filtered with a 96-well plate filter (Captiva, agilent technologies, usa).
Low, medium, and high concentration Quality Control (QC) and standard working calibration samples were prepared according to the plasma sample preparation method described.
A blank sample was prepared by replacing 100. mu.L of plasma with only 100. mu.L of ultrapure water (blank substrate) and 20. mu.L of MeOH/water (50:50, v/v) solution with 20. mu.L of internal standard IS stock solution, and then following the plasma sample preparation procedure described.
LC-MS/MS analysis
The LC-MS system adopts a Waters ACQUITY UPLC I-Class system and an AB SCIEX 5500Q-Trap system in series. The method adopts a positive and negative ion scheduled MRM mode, is provided with a Waters HSS T3 chromatographic column (1.8 mu m,100mm multiplied by 2.1mm), the column temperature is set to be 35 ℃, the mobile phase A is 0.1 percent FA-water, the mobile phase B is acetonitrile, the flow rate is 0.25mL/min, and the analytical gradient is as follows: 0-10min, 2% B-100% B; 10-11min, 100% B; 11.1-16min, 2% B. The mass spectrum parameters were as follows: the declustering voltage DP and the collision energy CE are optimized according to the specificity of each compound, the source parameters gas 1: 60 psi; gas 2: 50 psi; air curtain air: 20 psi; source temperature: at 450 ℃; spraying voltage: 5500/-4500V.
The method has high flux, high sensitivity and high separation degree, realizes one-needle sample injection within 15min, and simultaneously carries out high-flux absolute quantitative analysis on the cardiovascular disease biomarkers.
The invention also provides the use of the method for simultaneous quantification of cardiovascular disease biomarkers.
The cardiovascular disease biomarkers include TMAO, choline, carnitine, betaine, phenylalanine, phenylpyruvic acid, PAGLn, phenyllactic acid, threonine, tyrosine, tryptophan, IAA, IA, IPA, L-histidine, lysine, pantothenic acid, 2-hydroxybutyrate, 3-hydroxybutyrate, succinate.
The invention also provides the application of the method in qualitative/quantitative/absolute quantitative/simultaneous quantitative analysis of biomarkers.
The biomarkers include 20 biomarkers of pathway metabolism such as aromatic amino acid catabolism (such as phenylalanine and tryptophan tyrosine), trimethylamine oxide biosynthesis (such as trimethylamine oxide, choline, carnitine and betaine) and histidine metabolism (such as histidine), specifically TMAO, choline, carnitine, betaine, phenylalanine, phenylpyruvic acid, PAGLn, phenyllactic acid, threonine, tyrosine, tryptophan, IAA, IA, IPA, L-histidine, lysine, pantothenic acid, 2-hydroxybutyrate, 3-hydroxybutyrate and succinate.
The invention has the beneficial effects that: the invention adopts ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to develop and verify a sensitive and reliable targeted metabonomics method for quantifying the biomarkers related to the blood plasma cardiovascular diseases. By optimizing mass spectrum parameters and a liquid chromatography method, the detection of 20 biomarkers comprising pathway metabolism of aromatic amino acid catabolism (such as phenylalanine and tryptophan tyrosine), trimethylamine oxide biosynthesis (such as trimethylamine oxide, choline, carnitine and betaine) and histidine metabolism (such as histidine) and the like in high flux, high sensitivity and high resolution is realized by one sample injection. The result shows that the method has good linearity, the linear correlation coefficients are both larger than 0.99, the RSD of MQC and HQC is smaller than 15%, the RSD of LQC is smaller than 20%, the intra-day precision and the inter-day precision are respectively 1.12-14.12% and 0.30-13.74%, and the recovery rate and the stability can also meet the analysis requirements of biological samples. The targeted metabonomics method has proved to have strong capability of accurately analyzing metabolic markers, provides valuable information for large-scale biomarker verification, is a material basis for clarifying potential cardiovascular diseases, and provides powerful support for clinical diagnosis or early warning.
Drawings
FIG. 1 typical XIC obtained by the MRM method of the LC-MS/MS project. (A-B) XIC (without IS peak) obtained by the double UPLC method in positive (A) and negative (B) ion mode. (C-D) XIC, obtained by one-dimensional (1D) UPLC method in positive (C) and negative (D) ion modes (including IS peaks). (E) XICs of the 20 quantified metabolites in table 1 showed good peak shapes.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
Example 1
Experiments and methods
Reagents and chemicals
All reagents were mass spectral grade, methanol (MeOH) and Acetonitrile (ACN) were purchased from fisher technologies ltd (Fair lann, NJ, USA), Formic Acid (FA) was purchased from merck (Darmstadt, Germany), and ultrapure water was obtained from wa haha (hangzhou, china). All chemicals purchased from commercial sources are analytical reagents, standards including L-histidine, L-lysine, succinic acid, L-threonine, L-tryptophan, L-tyrosine, trimethylamine-nitric oxide (TMAO), betaine, carnitine, choline chloride, 2-hydroxybutyric acid, 3-hydroxybutyric acid, indole-3-acetic acid (IAA), Indoleacrylic Acid (IA), indole-3-propionic acid (IPA), pantothenic acid, phenylacetylglutamine (PAGLn), phenylalanine, phenyllactic acid, phenylpyruvic acid, and stable isotope labeled Internal Standards (IS) were purchased from Yuanyi Biotech Limited (Shanghai, China), Bailingwei science and technology Limited (Beijing, China) and Toronto research chemical company (Toronto, Canada), including trimethylamine-d 9-oxide (TMAO-d9), 3-hydroxybutyric acid-D4 sodium salt (3HB-D4), L-lysine-4, 4, 5, 5-D4 hydrochloride (lysine-D4), D-phenylalanine-D7 (phenylalanine-D7), indole-2, 4, 5, 6, 7-D5-3-acetate (IAA-D5), L-tyrosine-13C 9, 15N (tyrosine-13C 9, 15N), L-tryptophan-D5 (tryptophan-D5), phenylacetyl-D5-L-glutamine (PAGLn-D5), choline chloride-D9 (choline-D9), succinic acid-2, 2, 3, 3-D4 (succinic acid-D4).
Preparation of standards
Standard and IS stock solutions were prepared separately by dissolving or diluting the standard substance in methanol/water (50/50, v/v) to a final concentration of 1.0 mg/mL. And preparing a mixed standard substance with corresponding concentration gradient according to the detected concentration of the corresponding metabolite in the blood plasma. Standard working calibration solutions were obtained after serial dilution of the combined standard stock solutions at dilution ratios of 1, 1/2, 1/4, 1/10, 1/20, 1/40, 1/100, 1/200 and 1/400. To make the detection point in the middle of the linear range, the concentration at the 1/20 dilution point was approximately equal to five times the concentration detected in plasma. No dilution, 1/20 dilution, and 1/100 dilution were defined as high, medium, and low concentration quality controls (HQC, MQC, LQC), respectively. All standard solutions were stored in a 4 ℃ freezer prior to on-machine analysis.
Sample preparation
100 μ L of plasma samples were taken in 1.5mL EP tubes and 10 μ L of internal standard and 20 μ L of MeOH/water (50:50, v/v) solution were added. In addition, 300. mu.L of acetonitrile was added to precipitate the protein, vortexed at 2500rpm for 5min and centrifuged at 10000rpm (4 ℃, 5 min. the supernatant was transferred and centrifugally concentrated for 2 hours. the residue was redissolved in 100. mu.L of acetonitrile/H2O (2:98, v/v), vortexed for 5min and then centrifuged at 10000rpm (4 ℃, 5 min.) Prior to the liquid chromatography-mass spectrometry, the supernatant was separated and filtered with a 96-well plate filter (Captiva, Agilent technologies, USA.) Low, Medium, high concentration Quality Control (QC) and standard working calibration samples were prepared according to the plasma sample preparation method described, replacing 100. mu.L of plasma with 100. mu.L of ultrapure water (blank matrix) only and 20. mu.L of IS working solution for 20. mu.L of MeOH/water (50:50, v/v).
LC-MS/MS analysis
The LC-MS system adopts a Waters ACQUITY UPLC I-Class system and an AB SCIEX 5500Q-Trap system in series. The method adopts a positive and negative ion scheduled MRM mode, is provided with a Waters HSS T3 chromatographic column (1.8 mu m,100mm multiplied by 2.1mm), the column temperature is set to be 35 ℃, the mobile phase A is 0.1 percent FA-water, the mobile phase B is acetonitrile, the flow rate is 0.25mL/min, and the analytical gradient is as follows: 0-10min, 2% B-100% B; 10-11min, 100% B; 11.1-16min, 2% B. The mass spectrum parameters were as follows: the declustering voltage DP and the collision energy CE are optimized according to the specificity of each compound, the source parameters gas 1: 60 psi; gas 2: 50 psi; air curtain air: 20 psi; source temperature: at 450 ℃; spraying voltage: 5500/-4500V.
Methodology validation
The linearity, selectivity, accuracy, precision, stability and recovery of the method were evaluated as follows according to the united states Food and Drug Administration (FDA) guidelines "validation of analytical methods and" validation of bioanalytical methods ":
(1) linearity
A calibration curve for the calibration standard sample was established for 9 calibration points using a weighted (1/x) linear regression analysis method. R2 values are determinant coefficients, R2>0.99 are considered acceptable. LOD and LOQ are calculated as 3 and 10 times the signal-to-noise ratio (S/N), respectively.
(2) Accuracy and precision
Each concentration was replicated 3 times by analyzing the QC samples for accuracy and precision within and between days at 3 concentration levels (LQC, MQC and HQC). The blended samples QCs are also used to assess accuracy.
(3) Recovery rate
Calculated by comparing the peak area ratios of the respective standards at 3 concentration levels before and after pretreatment.
(4) Stability of
Carrying out three times of freeze-thaw cycles from-80 ℃ to room temperature on the plasma QC sample, and evaluating freeze-thaw stability; plasma QC samples were stored in an autosampler at 4 ℃ for 12h, 24h, 48h and 72h and their stability was studied.
(5) Data analysis
Data processing quantitative analysis and method validation analysis were performed using MultiQuant (version 3.0.3, AB SCIEX, Concord, ON, Canada). The standard curve IS drawn by the internal standard method according to the area ratio of the peak area of the standard substance to the corresponding IS. Statistical analysis SPSS statistical software (version 22.0, IBM, Armonk, NY, USA)
Results and discussion
Development of LC-MS/MS analysis method
A UPLC-MS/MS quantitative analysis method was established to identify 20 metabolites in plasma (see Table 1). These metabolites are biomarkers of CVD screened by experimental findings and literature mining, and involved pathways include aromatic amino acid catabolism, TMAO biosynthesis, lysine metabolism, histidine metabolism, and the like, and are partially confirmed to be associated with intestinal flora disorders.
Optimal MRM ion pairs are a prerequisite for tandem mass spectrometry to obtain efficient and reliable quantitative data. The parent and product ion pairs with high sensitivity and selectivity are selected as the final MRM ion pair for quantification. The DP and CE of a single MRM ion pair are further optimized to increase ionization efficiency to maximum sensitivity. The MRM ion pairs, DP and CE of the 20 metabolites are listed in table 1. In addition, the advantages of the schedulemm mode over the traditional MRM mode are that each MRM ion pair can be scanned over an expected retention time (tR) window, the selectivity and sensitivity can be improved, and a better signal-to-noise ratio (S/N) can be obtained leading to a more accurate quantitative result, especially simultaneous quantification of a large number of metabolites. Therefore, the present invention establishes a schedulemm method (table 1) comprising MRM ion pairs, DP, CE, and associated tR, with an MRM detection window of 150s and a target scan time of 0.8 s.
By optimizing analysis conditions such as column type, mobile phase gradient and flow rate of the UPLC, a better analysis effect is obtained. The HSS T3 chromatographic column has high column efficiency and retention capacity, and the separation effect on hydrophilic high-polarity compounds is superior to that of other chromatographic columns. The present invention compares several different mobile phase conditions, including one-dimensional UPLC methods and two-dimensional UPLC methods of different gradients. The two-dimensional UPLC method allows for the efficient separation of both strongly polar and weakly polar metabolites, as has been demonstrated in previous reports. However, the use of the dual column extended the analysis time and did not significantly improve the metabolite separation for the same retention behavior (FIGS. 1A-1B). In contrast, a 16 minute one-dimensional chromatographic mobile phase gradient (including a 5 minute column re-equilibration) was chosen because it shortens run time while ensuring better resolution. Typical Total Ion Chromatograms (TICs) from UPLC-MS/MS positive and negative ion mode analysis showed a uniform distribution of metabolite retention times (fig. 1C-D). The ion chromatograms (XICs) of the extracted 20 metabolites exhibited good peak shapes in positive and negative ion mode (fig. 1E). The result shows that the established method can better separate the plasma sample and has high sensitivity.
TABLE 1 LC-MS/MS schedulEMRM parameters for biomarkers for quantitative CVD
M represents a metabolite associated with cardiovascular disease; "IS" stands for internal standard. Some internal standards were collected in positive and negative ion modes for calibration.
Abbreviations: trimethylamine-N-oxide (TMAO); phenylacetylglutamine (PAGln); indole-3-acetic acid (IAA), Indoleacrylic Acid (IA), indole 3-propionic acid (IPA).
Standard curve
Table 2 shows the linear correlation equation, linear correlation coefficient (R2), linear range, limit of quantitation (LOQ) and limit of detection (LOD) for all analytes. All calibration curves in Table 2 are acceptable, R2 ≧ 0.99. It is noted that overfitting concentration points for certain analytes, such as tyrosine, indoleacrylic acid (IAA) and phenyllactic acid, are eliminated. The linear range of the calibration standard is 0.0025-1.0 μ g/mL to 0.4-160 μ g/mL. The lowest limit of quantitation of the obtained analyte is lower than the lowest concentration of the sample, the range is 0.09-139.86 ng/mL, and the lowest limit of quantitation of the analyte is 0.022-41.96 ng/mL.
These show that the method has a high sensitivity.
TABLE 2.20 Linear correlation equations for metabolites, R2, Linear Range, LOQ and LOD
Abbreviations: trimethylamine-N-oxide (TMAO); phenylacetylglutamine (PAGln); indole-3-acetic acid (IAA), Indoleacrylic Acid (IA), indole 3-propionic acid (IPA).
Accuracy and precision
Accuracy is used to assess the proximity between measured and true values, which is determined by comparing the calculated concentration to the actual concentration using a calibration curve. Precision, including intra-day and inter-day precision, was assessed by the Relative Standard Deviation (RSD) of daily measurements and three-day results in a single test run. Table 3 summarizes the daytime and daytime accuracy and precision results for the 20 analytes in the quality control samples. The accuracy rates of MQC and HQC are 90.84-104.17% and 90.37-106.82% respectively, the absolute error is less than 15%, the accuracy rate of LQC is 80.86-110.87, and the absolute error is less than 20%. The daily and daytime precision of the LQC, MQC, HQC and plasma quality control is respectively 1.12-14.12% and 0.30-13.74%, and the daily and daytime precision is less than 15%. In conclusion, both accuracy and precision tests meet the FDA requirements.
TABLE 3.20 accuracy and precision of analytes
Abbreviations: trimethylamine-N-oxide (TMAO); phenylacetylglutamine (PAGln); indole-3-acetic acid (IAA), Indoleacrylic Acid (IA), indole 3-propionic acid (IPA).
Extraction recovery rate
Extraction recovery is also an important parameter for process validation. In the experiment of the invention, the recovery rate is evaluated by calculating the peak area ratio of the quality control product before and after the pretreatment of three repeated samples of LQC, MQC and HQC. As shown in Table 4, the recovery rates of all analytes were 80.05-107.33% for LQC, 94.98-118.09% for MQC, and 90.05-113.56% for HQC, with relative standard deviations of less than 15%. The above recovery results indicate that the recovery rate of the process is acceptable.
TABLE 4.20 recovery of extraction of the Compounds
Abbreviations: trimethylamine-N-oxide (TMAO); phenylacetylglutamine (PAGln); indole-3-acetic acid (IAA), Indoleacrylic Acid (IA), indole 3-propionic acid (IPA).
Stability of
To evaluate the effect of repeated freeze-thaw cycles and long-term storage in the 4 ℃ autosampler on plasma metabolite content, 3 freeze-thaw cycle stabilities and autosampler stabilities were evaluated. The metabolite concentrations were calculated using the standard curve and compared to the initial state to obtain the stability study results. As can be seen from tables 5 and 6, the accuracy of all samples is 85.13-114.86% of that of the fresh samples, the RSD is less than 15%, and the stability is better under all test conditions. Therefore, the stability of the method can meet the analysis requirement of the biological sample.
TABLE 5 results for stability of three freeze-thaw cycles
Abbreviations: trimethylamine-N-oxide (TMAO); phenylacetylglutamine (PAGln); indole-3-acetic acid (IAA), Indoleacrylic Acid (IA), indole 3-propionic acid (IPA)
TABLE 6 autosampler stability results after 12h, 24h, 48h, 72h storage
Abbreviations: trimethylamine-N-oxide (TMAO); phenylacetylglutamine (PAGln); indole-3-acetic acid (IAA), Indoleacrylic Acid (IA), indole 3-propionic acid (IPA)
Conclusion
The invention develops a sensitive and reliable UPLC-MS/MS schedulEMRM method which is used for simultaneously quantifying all biomarkers related to cardiovascular diseases in cardiovascular disease experiments or documents. The method has the advantages of good accuracy, precision, sensitivity, acceptable stability and extraction recovery rate. Although the nature of the analytes varies widely, all analytes show good resolution and satisfactory peak shape. One-needle sample injection is realized within 16min, and the efficiency of target quantitative analysis in large-batch analysis and detection is greatly improved. Furthermore, more metabolites can be quantified simultaneously by only modifying the corresponding MRM ion pairs, DPs and CEs. The method not only helps to find reliable cardiovascular disease biomarkers, but also provides valuable information for clarifying potential material bases of the cardiovascular disease biomarkers, and further provides corresponding support for early diagnosis and prognosis of the cardiovascular disease.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.
Claims (11)
1. A UPLC-MS/MS-based plasma cardiovascular disease-associated biomarker targeted metabolomics quantification method, characterized in that the method comprises the following steps:
(1) preparation of standards
(1.1) respectively dissolving a standard product and an internal standard IS in a first solvent to respectively obtain a standard stock solution and an IS stock solution;
(1.2) diluting the standard stock solution in a gradient, wherein the gradient dilution ratio is 1-1/400;
(1.3) selecting standard quality control products: selecting standard stock solution, and defining the stock solution as a high-concentration quality control product, a medium-concentration quality control product and a low-concentration quality control product after non-dilution, 1/20 dilution and 1/100 dilution;
(2) sample preparation
Plasma sample preparation:
(a) taking a plasma sample in an EP tube, and adding an internal standard IS and a methanol/water solution; then, adding a protein precipitator to precipitate protein; then, after low-speed vortex, high-speed low-temperature centrifugation is carried out;
(b) transferring the supernatant after centrifugation in the step (a) and performing centrifugal concentration;
(c) redissolving the concentrated residue of step (b) in a second solvent; then evenly mixing the mixture by low-speed vortex, and centrifuging the mixture at high speed and low temperature; then separating the supernatant and filtering with a pore plate filter;
preparing a high-concentration quality control product, a medium-concentration quality control product, a low-concentration quality control product and a standard working calibration sample: prepared according to the steps (a) - (c);
blank control sample preparation: preparing said blank control sample by replacing said plasma with ultrapure water only and replacing said methanol/water solution with said IS stock solution according to said steps (a) - (c);
(3) UPLC-MS/MS analysis.
2. The method according to claim 1, wherein in step (1.1), the first solvent is methanol and/or water; the concentration of the standard stock solution is 1 mg/mL; the standard substance is L-histidine, L-lysine, succinic acid, L-threonine, L-tryptophan, L-tyrosine, trimethylamine-nitrogen oxide, betaine, carnitine, choline chloride, 2-hydroxybutyric acid, 3-hydroxybutyric acid, indole-3-acetic acid, indoleacrylic acid, indole-3-propionic acid, pantothenic acid, phenylacetylglutamine, phenylalanine, phenyllactic acid and phenylpyruvic acid; the concentration of the IS stock solution IS 1 mg/mL; the internal standard IS IS an internal standard substance marked by stable isotopes and comprises trimethylamine-D9-oxide, 3-hydroxybutyric acid-D4 sodium salt, L-lysine-4, 4, 5, 5-D4 hydrochloride, D-phenylalanine-D7, indole-2, 4, 5, 6, 7-D5-3-acetate, L-tyrosine-13C 9, 15N, L-tryptophan-D5, phenylacetyl-D5-L-glutamine, choline chloride-D9 and succinic acid-2, 2, 3, 3-D4.
3. The method of claim 1, wherein in step (1.2), the gradient dilution is at a ratio of 1, 1/2, 1/4, 1/10, 1/20, 1/40, 1/100, 1/200, and 1/400.
4. The method of claim 1, wherein in step (a), the plasma sample is taken in an amount of 50-150 μ L; the internal standard quantity is 5-15 mu L; the internal standard IS IS an internal standard substance marked by stable isotopes and comprises trimethylamine-D9-oxide, 3-hydroxybutyric acid-D4 sodium salt, L-lysine-4, 4, 5, 5-D4 hydrochloride, D-phenylalanine-D7, indole-2, 4, 5, 6, 7-D5-3-acetate, L-tyrosine-13C 9, 15N, L-tryptophan-D5, phenylacetyl-D5-L-glutamine, choline chloride-D9 and succinic acid-2, 2, 3, 3-D4.
5. The method of claim 1, wherein in step (a), the volume of the methanol/water solution is 10-30 μ L; the protein precipitant is acetonitrile and/or methanol; the volume of the protein precipitant is 300-; the low speed is 2000-3000 rpm; the vortex time is 3-10 min; the high speed is 10000-12000 rpm; the low temperature is 4-10 ℃.
6. The method of claim 1, wherein in step (b), the concentration is carried out for a period of 1-3 hours.
7. The method of claim 1, wherein in step (c), the second solvent is acetonitrile/water, methanol/water; the volume of the second solvent is 100-; the high speed is 10000-12000 rpm; the low temperature is 4-10 ℃; the low speed is 2000-3000 rpm.
8. The method of claim 1, wherein the LC-MS/MS analysis method of step (3) is performed by using a Waters ACQUITY UPLC I-Class system in series with an AB SCIEX 5500Q-Trap system;
a Waters HSS T3 column (1.8 μm,100 mm. times.2.1 mm) was fitted using a positive and negative ion scheduled MRM mode, the column temperature was set at 35 ℃, mobile phase A was 0.1% FA-water, mobile phase B was acetonitrile, the flow rate was 0.25mL/min, and the analytical gradient was as follows: 0-10min, 2% B-100% B; 10-11min, 100% B; 11.1-16min, 2% B;
the mass spectrum parameters were as follows: the declustering voltage DP and the collision energy CE are optimized according to the specificity of each compound, the source parameters gas 1: 60 psi; gas 2: 50 psi; air curtain air: 20 psi; source temperature: at 450 ℃; spraying voltage: 5500/-4500V.
9. The method of claim 1, wherein the method combines high throughput, high sensitivity and high resolution, and allows for one needle injection within 16min while performing high throughput absolute quantitative analysis of cardiovascular disease biomarkers.
10. Use of the method of claim 1 for simultaneously quantifying cardiovascular disease biomarkers comprising TMAO, choline, carnitine, betaine, phenylalanine, phenylpyruvic acid, PAGln, phenyllactic acid, threonine, tyrosine, tryptophan, IAA, IA, IPA, L-histidine, lysine, pantothenic acid, 2-hydroxybutyrate, 3-hydroxybutyrate, succinate.
11. Use of the method of claim 1 for qualitative/quantitative/absolute quantitative/simultaneous quantitative analysis of biomarkers comprising TMAO, choline, carnitine, betaine, phenylalanine, phenylpyruvic acid, PAGln, phenyllactic acid, threonine, tyrosine, tryptophan, IAA, IA, IPA, L-histidine, lysine, pantothenic acid, 2-hydroxybutyrate, 3-hydroxybutyrate, succinate.
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