Method for rapidly detecting 20 amino acids in plasma based on LC-MS / MS platform

Abstract

The invention discloses a method for rapidly detecting 20 amino acid prototypes in plasma based on an LC-MS / MS technology. The method comprises the following steps: (1) sample pretreatment: diluting a sample by 20 times with normal saline; (2) mixing the diluted liquid obtained in the step A1 with acetonitrile containing an internal standard, transferring the mixture into a centrifugal tube, and carrying out vortex centrifugal treatment; and (3) taking out the centrifugal tube, transferring the centrifuged extracting solution on the upper layer into a pore plate, loading the sample into LC-MS / MS for detection, and obtaining the amino acid component content of the plasma sample according to the standard curve. The invention provides a novel method for detecting the concentration of amino acids in human plasma, which can be used for detecting 20 amino acid prototypes in plasma through verification of parallelism, specificity, linearity, quantitation limit, accuracy, instrument precision, extraction recovery rate, matrix effect and stability of a substitute matrix. The method is simple in pretreatment, short in mass spectrum acquisition time, low in cost and convenient for large-scale clinical sample detection in a laboratory.

Description

Technical Field

[0001] The invention belongs to the technical field of amino acid detection, and specifically relates to a method for rapidly detecting 20 kinds of amino acids in plasma based on an LC-MS / MS platform. Background Art

[0001] Amino acids are a class of amphipathic organic compounds and are the basic building blocks of biological functional macromolecule proteins. According to the ability of amino acids to be synthesized in the body, amino acids can be divided into essential amino acids and non-essential amino acids. Essential amino acids refer to those that cannot be synthesized by the human body and must be ingested through food (such as lysine, tryptophan, etc.); while non-essential amino acids can be synthesized from other amino acids or compounds in the body (such as glutamic acid, alanine, etc.). A series of diseases including malnutrition, metabolic diseases, mental illness and cancer can affect the metabolic transport of amino acids, leading to changes in amino acids in human plasma. In clinical practice, the determination of amino acid profiles in plasma can be used as a diagnostic indicator for metabolic diseases and mental illnesses. However, at present, there are relatively few reports on the use of plasma amino acid detection for amino acid metabolism-related diseases at home and abroad. The main reason is that amino acids are substances with extremely high endogenous concentrations, and without complex pre-treatment methods such as derivatization, the sensitivity and specificity of conventional detection methods cannot meet the detection of amino acid profiles in plasma.

[0001] At present, common methods for amino acid detection include ultraviolet spectrophotometry and high performance liquid chromatography. These methods have problems such as complex pre-treatment, inaccurate qualitative and quantitative analysis, high cost, long analysis time, or high technical requirements for experimental personnel. Liquid chromatography tandem mass spectrometry (LC-MS / MS) is increasingly widely used in various fields such as medicine, food, environment, forensics, and clinical medicine. LC-MS / MS has the characteristics of high sensitivity, strong selectivity, and good accuracy. Its application range in clinical testing far exceeds that of radioimmunoassay and chemical testing, which is unmatched by other methods. Summary of the invention

[0001] In order to solve the above technical problems, the purpose of the present invention is to provide a method for detecting the concentrations of 20 amino acid prototypes in human plasma based on LC-MS / MS technology. The method has simple pretreatment, short mass spectrometry acquisition time, low cost, and is convenient for laboratories to carry out large-scale clinical sample testing.

[0001] To achieve the above object, the present invention is implemented through the following technical solutions: (1) Sample pretreatment: dilute the sample 20 times with physiological saline; (2) mixing the diluted liquid obtained in step (1) with acetonitrile containing an internal standard, transferring the mixture into a centrifuge tube, and subjecting the mixture to vortex centrifugation; (3) Take out the centrifuge tube, transfer the extract from the upper layer into the well plate, load it into LC-MS / MS for detection, and obtain the amino acid content of the plasma sample according to the standard curve.

[0001] In step (2), the method for preparing the acetonitrile solution containing the internal standard comprises: first preparing a high concentration of tranexamic acid aqueous solution, and then using the acetonitrile solution to prepare a 100 ng / mL internal standard solution.

[0001] In step (2), the centrifugal speed is 10000-20000 r / min, and the time is 5-15 minutes.

[0001] Step (3) uses high performance liquid phase tandem mass spectrometry to detect the pretreated plasma sample, and uses the internal standard method for quantification, with the concentration of various amino acids in the plasma sample of the standard curve as the horizontal coordinate, and the ratio of the peak area of ​​various amino acids to the peak area of ​​the internal standard as the vertical coordinate, to establish a standard curve equation, substitute the ratio of the peak area of ​​various amino acids in the plasma sample to the internal standard into the standard curve equation, and calculate the concentration of 20 amino acid prototypes in the plasma to be tested.

[0001] In step (3), 0.1% formic acid, 5 mM ammonium formate and 5 mM ammonium acetate are added to separate a group of isomers; the liquid chromatography conditions of the high performance liquid phase tandem mass spectrometry are: Chromatographic column: Agilent AdvanceBio Hilic-Z (2.1 mm × 100 mm, 2.7 μm); Flow rate: 0.25 mL / min; Mobile phase A: 0.1% formic acid, 5 mM ammonium formate, and 5 mM ammonium acetate in ultrapure water; Mobile phase B: 0.1% formic acid, 5 mM ammonium formate, and 5 mM ammonium acetate in acetonitrile water (90:10 / v:v); Needle washing solution: methanol: water (50:50); Column temperature: 35°C; Autosampler temperature: 4°C; The mobile phase gradient is shown in Table 1 below:

[0001] Table 1

[0001] Preferably, the mass spectrometry parameters of 20 amino acids by LC / MS / MS are shown in Table 2 below:

[0001] Table 2

[0001] Preferably, in step (3), the method for preparing the calibration curve comprises: sample pretreatment: diluting the sample 20 times with physiological saline; mixing the obtained diluted liquid with acetonitrile containing the internal standard, and transferring it to a centrifuge tube for vortex centrifugation; transferring the extract obtained from the upper layer of centrifugation to a well plate, loading the sample on LC-MS / MS for detection, and obtaining a standard curve.

[0001] Preferably, the calibrator comprises 8 level calibrators, and the calibrator background matrix is ​​physiological saline.

[0001] Preferably, the amount of the calibrator is 50-200 μL, the concentration of the acetonitrile solution containing the internal standard is 0.1 μg / mL-0.2 μg / mL, and the amount added is 250-1000 μL.

[0001] In some embodiments, the amount of the calibrator is 50 μL, the concentration of the internal standard acetonitrile solution is 0.1 mg / L, and the amount added is 250 μL.

[0001] Preferably, the centrifugal speed is 10000-20000 r / min, and the time is 5-15 minutes.

[0001] In some embodiments, the centrifugation speed is 14000 r / min and the time is 10 minutes.

[0001] Preferably, the calibration curve is obtained by an internal standard quantitative method, specifically comprising: establishing a calibration curve with the concentration ratio of the standard as the x-axis and the peak area ratio of the standard to the internal standard as the y-axis.

[0001] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention verifies the feasibility of using physiological saline as a substitute matrix, eliminating the problem that amino acids are extremely endogenous in plasma and cannot be quantified by mass spectrometry. Compared with other methods such as liquid phase, mass spectrometry has higher precision, accuracy and sensitivity. (2) The sample does not require complex pre-treatment methods such as derivatization, and the method is simple to operate, the sample preparation time is short, and the method has good reproducibility. (3) The present invention uses LC-MS / MS technology to establish a method for simultaneously determining the concentrations of 20 amino acid prototypes in human serum, which is simple to operate, has stable and reliable results, and is rapid. After verification of the parallelism, specificity, linearity, quantitative limit, accuracy, instrument precision, extraction recovery rate, matrix effect and stability of the substitute matrix, it can be used for clinical detection of serum amino acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0001] Figure 1 The following is a typical chromatogram showing the selectivity of 20 amino acids.

[0001] Figure 2 The standard curve was determined by internal standard method for 20 kinds of amino acids. DETAILED DESCRIPTION

[0001] In order to more clearly illustrate the present invention, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. By reading the detailed description of the non-limiting embodiments with reference to the following drawings, other features, purposes and advantages of the present invention will become more obvious. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0001] Example 1

[0001] 1. Detection method 1.1 Chromatographic conditions Chromatographic column: Agilent AdvanceBio Hilic-Z (2.1 mm × 100 mm, 2.7 μm); Flow rate: 0.25 mL / min; Mobile phase A: 0.1% formic acid, 5 mM ammonium formate, and 5 mM ammonium acetate in ultrapure water; Mobile phase B: 0.1% formic acid, 5 mM ammonium formate, and 5 mM ammonium acetate in acetonitrile water (90:10 (v:v)); Needle washing solution: methanol: water (50:50) Column temperature: 35°C; Autosampler temperature: 4°C; The mobile phase gradient is shown in Table 3 below:

[0001] Table 3 time Mobile phase A% Mobile phase B% Flow rate (mL / min) 0.0 0 100 0.25 4.0 10 90 0.25 8.0 40 60 0.25 10.0 50 50 0.25 10.5 0 100 0.25 15.0 0 100 0.25

[0001] 1.2 Mass spectrometry conditions Electrospray ionization (ESI) was used to scan the samples in positive ion MRM mode. Curtain gas, 35psi; collsion gas, 5psi; ion source gas1, 50psi; ion source gas2, 55psi; ion spray voltage, 5500V; and temperature, 600℃. Other parameters are shown in Table 4 below:

[0001] Table 4

[0001] 2. Methods

[0001] 2.1 Solution preparation Accurately weigh 20 kinds of amino acids, and prepare them into 1 mg / mL standard stock solutions with Watsons distilled water respectively. The stock solutions are stored in a -80°C refrigerator. Before use, mix them well and prepare them into a mixed solution of 4 μg / mL 3-Mel; 80 μg / mL Ser, Gly; 16 μg / mL Asp; 160 μg / mL Ala; 40 μg / mL Arg, Cit, Glu, His, Ile, Leu, Lys, Orn, Met, Phe, Pro, Thr, Trp, Tyr, and Val with a diluent (acetonitrile). Then, 3-Mel was diluted with acetonitrile to a concentration of 200, 400, 1000, 1600, 2000, 2400, 3200, and 4000 ng / mL; Ser and Gly were diluted to a concentration of 4000, 8000, 20000, 32000, 40000, 48000, 64000, and 80000 ng / mL; Asp was diluted to a concentration of 800, 1600, 4000, 6400, 9600, 12800, and 16000 ng / mL; Ala was diluted to a concentration of 1. The concentrations of Arg, Cit, Glu, His, Ile, Leu, Lys, Orn, Met, Phe, Pro, Thr, Trp, Tyr and Val were diluted into a mixed standard series working solution with concentrations of 2000, 4000, 10000, 16000, 20000, 24000, 32000 and 40000 ng / mL.

[0001] Accurately weigh tranexamic acid, prepare it into a 1 mg / mL standard stock solution with distilled water, and store it in a -80°C refrigerator. Dilute it with diluent (acetonitrile) to a concentration of 100 ng / mL internal standard working solution before use.

[0001] 2.2 Preparation of standard curve and serum sample pretreatment Take 5 μL of the mixed stock solution of each concentration and place it in a 1.5 mL centrifuge tube, add 95 μL of normal saline and then add 500 μL of tranexamic acid internal standard solution, place it on a vortex instrument and vortex for 3 min, centrifuge it at 4°C, 13000 rpm for 10 min, prepare the standard curve, take 100 μL of the supernatant and place it in an injection bottle, prepare the standard curve solution, and perform LC-MS / MS analysis according to the conditions of steps "1.1" and "1.2".

[0001] Accurately pipette 5 μL of serum sample into a 1.5 mL centrifuge tube, dilute 20 times with normal saline, then accurately pipette 5 μL of diluted serum sample, add 95 μL of normal saline and then 500 μL of tranexamic acid internal standard solution, vortex on a vortexer for 3 min, centrifuge at 4°C, 13,000 rpm for 10 min, pipette 100 μL of supernatant into a sample injection bottle, and perform LC-MS / MS analysis according to the conditions of steps "1.1" and "1.2".

[0001] 3. Methodological investigation With reference to the FDA and ICH M10 guidelines for validation of quantitative analysis methods for biological samples, the parallelism, specificity, linearity, detection limit, quantification limit, accuracy, instrument precision, extraction recovery, matrix effect and stability of the established analytical method were verified using quality control products of different concentrations of a single amino acid.

[0001] 3.1 Parallelism of surrogate matrix Since the endogenous concentration of amino acids in human blank plasma is extremely high and cannot be completely removed, human blank plasma diluted with normal saline (so that the endogenous concentration of amino acids is between LLOQ and LQC) was used as the real matrix, and the parallelism of normal saline as a surrogate matrix was investigated at the LQC and HQC levels (n=6). The results showed that the slope ranged from 86.2% to 107.3%, indicating that normal saline can be used as a surrogate matrix in this experiment.

[0001] 3.2 Exclusivity Investigation Take 6 portions of blank saline from different sources, prepare amino acid substitution matrix samples containing LLOQ concentration according to step "2.2", and perform LC-MS / MS analysis according to the conditions of steps "1.1" and "1.2". The results are shown in Figure 1 , showing that in blank saline solutions from different sources, the interference of endogenous components within the retention time of the chromatographic peaks of the amino acids to be analyzed was lower than 20% LLOQ concentration response, indicating that the method had good specificity.

[0001] 3.3 Investigation of linearity and limit of quantitation Use blank saline as the matrix and prepare the standard saline sample of amino acids according to the method in step "2.2". After processing, inject and analyze. The ratio of the peak area of ​​the analyte to the corresponding internal standard (Y) is the ordinate, the concentration of the analyte (X) is the abscissa, and the weight is W = 1 / X 2 Regression analysis was performed to obtain the standard curve of each amino acid. Figure 2 , showing that the 20 amino acids were linear within the concentration range, and the linearity was good, indicating that the linearity of each analyte in the serum was good within the corresponding concentration range.

[0001] 3.4 Precision and accuracy test Using blank saline as the matrix, quality control samples at four levels of LLOQ, QL, QM, and QH were prepared according to the method in step "2.2". Six samples were prepared for each concentration level as an analysis batch. After processing according to the method in step "2.2", the samples were analyzed and the intra-batch accuracy and instrument precision of the quality control samples were calculated based on the accompanying standard curve. Three analysis batches were prepared and measured on different days, and the inter-batch accuracy and instrument precision were calculated. The results showed that the precision accuracy errors were within 15% of the labeled value, indicating that the accuracy and instrument precision of this determination method meet the requirements for quantitative analysis of biological samples.

[0001] 3.5 Sample extraction recovery and matrix effect Using the diluted human blank serum as the matrix, the quality control samples at three levels of QL, QM, and QH were prepared according to the method of step "2.2", with 6 copies at each concentration level. After processing according to the method of step "2.2", the peak area A1 of the analyte was obtained. In addition, 95 μL of the diluted human blank serum was taken, 500 μL of acetonitrile was added, vortexed for 3 minutes, centrifuged at 13000 rpm for 10 minutes, all the supernatants were aspirated, and 5 μL of the quality control working solutions at three levels of QL, QM, and QH were added, with 6 copies at each concentration level, mixed and injected for analysis to obtain the peak area A2 of the analyte. The sample extraction recovery rate was calculated based on A1 / A2, and the results showed that the extraction recovery rates of 20 amino acids were between 85.9% ± 6.3% and 101.6% ± 12.3%, indicating that the sample pretreatment method is suitable for the extraction and analysis of the analytes.

[0001] Blank samples were prepared from at least 6 different sources of diluted blank human plasma, and a certain amount of analyte and internal standard were added after pretreatment, so that their final concentrations were consistent with the injection concentrations of LQC and HQC, respectively. Pure solutions containing a certain amount of analyte and internal standard were prepared at the same time, and their final concentrations were consistent with the injection concentrations of LQC and HQC, respectively. Each concentration was measured in parallel for at least 6 copies. The results showed that the RSD values ​​of each amino acid were within 15%, indicating that the method of the present invention had no obvious matrix effect.

[0001] 3.6 Stability investigation Using diluted human blank serum as the matrix, quality control samples at two levels, QL and QH, were prepared according to step "2.2", with three copies of different concentration levels under each condition. The results showed that the average deviation between the measured values ​​and the theoretical values ​​of each amino acid before and after treatment, under the conditions of automatic sample injection and long-term freeze-thaw storage was within ±15%, indicating that each amino acid was stable under the conditions of this experiment.

[0001] 3.7 Dilution reliability study Since the concentration of amino acids in human plasma samples is much higher than the quantitative upper limit of this experiment, and considering that high concentrations of amino acids may contaminate the mass spectrometry detection environment and thus affect the detection results, and because there is no blank matrix without amino acids, physiological saline is used as a blank matrix to dilute and inject plasma samples. The results show that the precision and accuracy of each amino acid after dilution are within ±15%, indicating that dilution of the analyte by 5-200 times is reliable.

[0001] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for rapid detection of 20 amino acid prototypes in plasma based on LC-MS / MS technology, characterized in that: The method is implemented by the following steps: (1) Sample pretreatment: dilute the sample 20 times with physiological saline; (2) mixing the diluted liquid obtained in step A1 with acetonitrile containing an internal standard, transferring the mixture into a centrifuge tube, and subjecting the tube to vortex centrifugation; (3) Take out the centrifuge tube, transfer the extract from the upper layer into the well plate, load it into LC-MS / MS for detection, and obtain the amino acid content of the plasma sample according to the standard curve.

2. The method according to claim 1, characterized in that The 20 amino acids are: 3-methylhistidine, alanine, arginine, aspartic acid, citrulline, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

3. The method according to claim 1, characterized in that In step (2), the method for preparing the acetonitrile solution containing the internal standard comprises: first preparing a high concentration of tranexamic acid aqueous solution, and then using the acetonitrile solution to prepare a 100 ng / mL internal standard solution.

4. The method according to claim 1, characterized in that: In step (2), the centrifugal speed is 10000-20000 r / min, and the time is 5-15 minutes.

5. The method according to claim 1, characterized in that In step (3), the mass spectrometry parameters of 20 amino acids by LC-MS / MS are shown in the following table:

6. The method according to claim 1, characterized in that In step (3), the liquid chromatography conditions of LC-MS / MS are as follows: 0.1% formic acid, 5 mM ammonium formate and 5 mM ammonium acetate are added to separate a group of isomers; wherein, the chromatographic column is Agilent Advance Bio Hilic-Z (2.1 mm×100 mm, 2.7 μm); the flow rate is 0.25 mL / min; the mobile phase A is an ultrapure water phase of 0.1% formic acid, 5 mM ammonium formate and 5 mM ammonium acetate; the mobile phase B is an acetonitrile water phase of 0.1% formic acid, 5 mM ammonium formate and 5 mM ammonium acetate at a ratio of 90:10 / v:v; the mobile phase gradient is shown in the following table:

7. The method according to claim 1, characterized in that In step (3), the method for preparing the calibration curve is: dilute the calibration sample 20 times, add the internal standard acetonitrile solution, centrifuge, take the upper layer of the extract into a 96-well plate, load LC-MS / MS for detection, and obtain the calibration curve.

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