Method for rapidly analyzing amino acid in plasma based on sound wave excitation coupling mass spectrum system

Abstract

The invention discloses a method for rapidly analyzing multiple amino acids in plasma based on sound wave excitation coupled mass spectrometry, which is characterized by comprising the following steps: pretreating a plasma sample by using a protein precipitation method to obtain a loading solution for sample introduction; then, the sample loading solution is subjected to sound wave excitation sample introduction to triple quadrupole mass spectrometry for analysis, the characteristic ion pair peak area of the amino acid and the stable isotope internal standard substance is obtained, and then the multiple amino acids in the plasma sample are subjected to rapid quantitative analysis with a stable isotope internal standard method in combination with a standard curve. According to the invention, a non-contact sample introduction mode is adopted, so that no residue is left in the sample introduction process, the ultrafast collection speed of one sample can be analyzed per second, conventional liquid chromatography consumables such as a chromatographic column and a pre-column are not needed, and the method has the characteristics of simple pretreatment, small sample introduction volume, low matrix effect, high efficiency and rapidness in detection, good stability and reproducibility and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biological sample analysis, and in particular relates to a method for rapidly analyzing one or more amino acids in plasma based on an acoustic wave excitation coupled mass spectrometry system. Background Art

[0002] Amino acids are the basic building blocks of proteins. Their main functions in the human body include synthesizing peptides and proteins, regulating the activity of immune cells, energy supply, synthesizing neurotransmitters, etc. More and more evidence from metabolomics studies shows that disorders of amino acid metabolism in the human body are closely related to a series of cardiovascular diseases, diabetes, fetal metabolic diseases, etc. Establishing an efficient analytical method that can quickly detect the amino acid content in the blood will help to comprehensively and quickly evaluate the health status of the human body and guide clinical diagnosis and treatment.

[0003] At present, the commonly used amino acid detection instruments mainly include: amino acid analyzer determination, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, etc. However, the detection time of the above instruments is relatively long, at least more than 15 minutes, which cannot meet the requirements of clinical rapid detection, and there are limitations such as large sample dosage, large injection volume, and strong matrix effect. Therefore, it is very important to construct a method that is fast and efficient, requires less sample dosage, has a small injection volume, has a low matrix effect and has high accuracy, which can provide a favorable means for population health assessment and clinical diagnosis. Summary of the invention

[0004] In view of the technical problems existing in the above-mentioned existing methods, the present invention aims to provide a method for rapidly analyzing one or more amino acids in plasma based on acoustic excitation coupled mass spectrometry. The non-contact sampling method ensures that there is no residue in the sampling process, and can achieve an ultra-fast acquisition speed of analyzing one sample per second. There is no need to use conventional liquid chromatography consumables such as chromatographic columns and pre-columns. It has the characteristics of simple pretreatment, small injection volume, low matrix effect, efficient and rapid detection, good stability and reproducibility, etc.

[0005] The technical solution adopted by the present invention to solve the above-mentioned problems is:

[0006] A method for rapid analysis of multiple amino acids in plasma based on acoustic wave excitation coupled mass spectrometry, which uses a protein precipitation method to pre-treat a plasma sample to obtain a sample solution for injection; then the sample solution is injected into a triple quadrupole mass spectrometer using acoustic wave excitation for analysis to obtain characteristic ion pair peak areas of amino acids and stable isotope internal standards, and then the stable isotope internal standard method is used in combination with a standard curve to perform rapid quantitative analysis of multiple amino acids in the plasma sample.

[0007] According to the above scheme, the process of pretreating plasma samples using the protein precipitation method is as follows: Mix the plasma sample and the extraction solution at a volume ratio of 1:(3 - 5), vortex for 60 - 120 s, place at -15 to -25 °C for 10 - 20 min, then centrifuge at 1 - 7 °C at a speed of 9000 - 140000 rpm for more than 10 min, and take the supernatant; Use this supernatant directly as the sample loading solution for injection, or dilute this supernatant with ultrapure water and use it as the sample loading solution for injection. Among them, the extraction solution is composed of methanol and acetonitrile mixed at a volume ratio of (3 - 5):1, and is added with stable isotope internal standards with a concentration of 0.5 - 1.5 μg / mL; The stable isotope internal standards are arginine-D7 and tyrosine-D4. Preferably, the concentration of the stable isotope internal standard in the sample loading solution is in the range of 100 - 150 ng / mL. The isotope internal standard quantification method can reduce the errors caused by the sample pretreatment process and instrument instability, and improve the accuracy of the analysis results.

[0008] According to the above scheme, the non-contact injection method of acoustic wave excitation is adopted, and the specific conditions are as follows: The coupling fluid is ultrapure water, the carrier liquid is a methanol solution containing formic acid, the injection mode is the AQ mode (Aqueous), and the flow rate is 400 - 550 μL / min; The injection volume is 2.5 - 25 nL; Among them, the methanol solution containing formic acid uses methanol as the solvent, and the volume percentage of formic acid is 0.01 - 0.025%.

[0009] According to the above scheme, the triple quadrupole mass spectrometry is detected in the electrospray positive ion multiple reaction monitoring mode (Multiple Reaction Monitoring, MRM), and the specific conditions are as follows: The ion source temperature is set at 300 - 500 °C; The curtain gas is set at 20 - 40 psi, the nebulizing gas is set at 90 - 100 psi, the auxiliary heating gas is set at 60 - 80 psi, and 1 - 4 ion pair channels are used at the same time; The delay time is 1000 - 3000 ms, and the information of the characteristic ion pair peak area of amino acids and the mass spectrometry parameters are shown in Table 1-1.

[0010] According to the above scheme, the amino acids that can be rapidly analyzed by the method of the present invention include phenylalanine, arginine, methionine, tyrosine, ornithine, proline, tryptophan, serine, threonine and histidine, and one of these 10 amino acids can be rapidly analyzed, or 2 - 10 of these 10 amino acids can be rapidly analyzed.

[0011] According to the above-mentioned scheme, a series of standard solutions for drawing a standard curve are prepared by dissolving amino acid standards in water, with a concentration range between 0.02 and 20.0 μg / mL, and a stable isotope internal standard is added to each of them. Among them, the concentration of the stable isotope internal standard in each standard solution is 0.2 - 5 μg / mL. Generally, the number of standard solutions is not less than 5, and specific concentrations are set at intervals within the concentration range.

[0012] According to the above-mentioned scheme, the standard solution is injected into a triple quadrupole mass spectrometer for analysis by acoustic wave excitation to obtain the characteristic ion pair peak areas of amino acids and internal standards; then, with the concentration ratio of amino acids to internal standards in the standard solution as the abscissa and the ratio of the characteristic ion pair peak areas of amino acids to internal standards as the ordinate, a standard curve is established; then, according to the characteristic ion pair peak areas obtained from the sample solution, the peak area ratio of amino acids to internal standards in it is substituted into the standard curve to calculate the concentration ratio of each amino acid to internal standards in the sample solution, and then, in combination with the concentration of the internal standard in the sample solution and the volume relationship between the sample solution and the plasma sample, the content of each amino acid in the plasma sample is converted. Among them, the acoustic wave excitation injection conditions and triple quadrupole mass spectrometry analysis conditions for obtaining the characteristic ion pair peak areas of the sample solution are the same as those for obtaining the characteristic ion pair peak areas of the standard solution.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] 1. The present invention does not require solid-phase extraction or derivatization reactions, and does not need to use conventional liquid chromatography consumables such as chromatographic columns and pre-columns. After directly subjecting the plasma sample to protein precipitation pretreatment, it can be injected for analysis. The method is simple and easy to operate.

[0015] 2. Compared with previous amino acid detection methods, the present invention adopts acoustic wave excitation injection technology. This non-contact injection method ensures no residue during the injection process, and can achieve about one sample per second, with a greatly improved detection speed, an extremely short analysis time, and a small injection volume. For example, compared with the liquid chromatography-tandem mass spectrometry method which requires at least 15 minutes per sample, the present invention has increased by nearly 900 times.

[0016] 3. The present invention uses the stable isotope internal standard method for quantification, which has low matrix effects, high accuracy, and a wide linear range. The average spike recovery rate is between 98.3% and 126.0%, and the quantitative limit concentration range of the target amino acids is 0.02 - 0.05 ng / mL. The linear correlation coefficient R within this range up to 20 μg / mL 2 is greater than 0.998, showing good linear relationships and recovery rates. In addition, the quantitative stability and reproducibility are excellent, and the inter-day and intra-day precisions of the quality control samples are both less than 20.0%.

[0017] In summary, the method for rapid analysis of multiple amino acids in plasma based on acoustic wave excitation coupled mass spectrometry according to the present invention has simple pretreatment, small injection volume, small sample consumption, low matrix effect, high detection efficiency and speed, and has characteristics such as high accuracy, good stability and reproducibility, and can realize rapid and stable qualitative and quantitative analysis and detection of these 10 amino acids in clinical large cohort samples, especially precious samples. Description of the Drawings

[0018] Figures 1 to 3 It is the calibration curve graph of representative amino acids in Example 1. Among them, Figure 1 is phenylalanine; Figure 2 is arginine; Figure 3 is tyrosine.

[0019] Figures 4 to 6 It is the spectrum graph of representative amino acids in the actual sample measured in Example 1. Among them, Figure 4 is phenylalanine; Figure 5 is arginine; Figure 6 is tyrosine.

[0020] Figure 7 and Figure 8 are the peak areas of characteristic ion pairs of phenylalanine and tyrosine respectively under different carrier liquids in the comparative example.

[0021] Figure 9 and Figure 10 are respectively the peak areas of characteristic ion pairs and relative standard deviations of phenylalanine and tyrosine under different ion channels in Example 2.

[0022] Figure 11 and Figure 12 are respectively the peak areas of characteristic ion pairs of phenylalanine and tyrosine under different additional delay times in Example 3. Detailed Embodiments

[0023] The present invention will be further described below in conjunction with specific embodiments and drawings. These embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0024] The present invention uses an acoustic wave excitation coupled mass spectrometry system (Acoustic Ejection Mass Spectrometry, AEMS, SCIEX Echo TM ), which is a high-throughput system based on electrospray ionization mass spectrometry. By using acoustic wave excitation injection technology (ADE), it can accurately control the transfer of samples from the sample plate to the open-port probe sampling interface (Open-port probe sampling interface, OPI), and this open-port probe interface is directly connected to the mass spectrometry system (SCIEX Triple Quad TM 6500+).

[0025] In the following examples, the 10 amino acids include: phenylalanine, arginine, methionine, tyrosine, ornithine, proline, tryptophan, serine, threonine, and histidine.

[0026] Example 1

[0027] A method for rapidly analyzing 10 amino acids in plasma based on acoustic wave excitation coupled with mass spectrometry, the specific steps are as follows:

[0028] 1) Prepare a mixed standard solution containing 10 amino acids: The mixed standard solutions are all prepared with water as the solvent and adding 10 amino acid standards; the concentrations of the 10 amino acids in each mixed standard solution are the same, and the concentrations are successively 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 μg / mL. For example, in the mixed standard solution with an amino acid concentration of 0.02 μg / mL, the concentrations of the 10 amino acids are all 0.02 μg / mL.

[0029] Moreover, in the above mixed standard solutions, isotopically labeled standard products arginine-D7 and tyrosine-D4 are added, and the concentrations are both 1 μg / mL.

[0030] 2) Inject the above mixed standard solutions into a triple quadrupole mass spectrometer for analysis by acoustic wave excitation respectively to obtain the characteristic ion pair peak areas of the amino acids and internal standards; among them,

[0031] The specific conditions for acoustic wave excitation injection are: the coupling fluid uses ultrapure water (i.e., the solvent), the carrier liquid uses methanol containing 0.01% formic acid (i.e., the mobile phase), the flow rate is 440 μL / min, the injection mode is AQ (Aqueous) mode, and the injection volume is 2.5 nL; the triple quadrupole mass spectrometer uses multiple reaction ion monitoring (MRM) and positive electrospray ionization mode for detection. The specific analysis conditions are: the ion source temperature is set at 350 °C, the curtain gas is set at 25 psi, the nebulizing gas is set at 90 psi, the auxiliary heating gas is set at 70 psi, and 2 ion pair channels are used simultaneously; the delay time is 3000 ms. The ion pairs and mass spectrometry parameters of each standard product are shown in Table 1-1.

[0032] Table 1-1. Ion pair information and mass spectrometry parameters of 10 amino acids and 2 internal standards

[0033]

[0034] DP: Declustering Potential, the declustering voltage; EP: Entrance Potential, the entrance voltage; CE: Collision Energy, the collision voltage; CXP: Collision Cell Exit Potential, the collision cell exit voltage; the parent ion and the corresponding daughter ion are characteristic ion pairs.

[0035] 3) Using the mass spectrometry isotope internal standard quantification method, based on the peak areas of the characteristic ion pairs obtained in step 2), with the concentration ratio of each amino acid standard to the internal standard as the X-axis (i.e., the abscissa), and the peak area ratio of the characteristic ion pairs of the corresponding amino acid standard to the internal standard as the Y-axis (i.e., the ordinate), the standard curves of 10 amino acids were established as shown in Table 1-2.

[0036] Table 1-2 Standard curves of 10 amino acids

[0037]

[0038] Among them, the peak area diagrams of the characteristic ion pairs and the standard curves at different concentrations are as Figures 1 - 3 shown (taking phenylalanine, arginine, and tyrosine as examples);

[0039] 4) Taking 300 plasma samples of pregnant women in a certain area as actual plasma samples, the following method was used for pretreatment with protein precipitation:

[0040] Take 50 μL of the actual plasma sample, add 200 μL of the extraction solution of methanol: acetonitrile (V / V, 4:1) (containing 1 μg / mL of arginine-D7 and 1 μg / mL of tyrosine-D4), vortex for 90 s, place it at -20 °C for 15 min, then centrifuge at 15000 rpm for 15 min at 4 °C. Take the supernatant and dilute it 8 times with ultrapure water, and then take 65 μL and place it in a 384-well plate as the sample loading solution for injection.

[0041] 5) Inject the sample loading solution for injection obtained in step 4) into a triple quadrupole mass spectrometer for analysis by acoustic wave excitation to obtain the peak areas of the characteristic ion pairs of amino acids and the internal standard; among them, the specific conditions of acoustic wave excitation injection and the specific analysis conditions of the triple quadrupole mass spectrometer are the same as those in step 2).

[0042] According to the peak areas of the characteristic ion pairs obtained from the sample loading solution, substitute the peak area ratios of the amino acids to the internal standard into the standard curve, calculate the concentration ratios of the amino acids to the internal standard in the sample loading solution, and then, in combination with the concentration of the internal standard in the sample loading solution and the volume relationship between the sample loading solution and the plasma sample, convert the contents of the amino acids in the actual plasma sample. The content distributions of 10 amino acids in the plasma of 300 pregnant women are shown in Table 1-3 (Note: Since the data volume of the amino acid contents in the plasma of 300 pregnant women is large, the present invention specifically provides the distribution of these contents).

[0043] Specifically, as Figures 4 to 6 shown, the peak areas of the characteristic ion pairs of phenylalanine, arginine, and tyrosine in the sample loading solution are 22456, 3896, and 9384 respectively, and the peak areas of the internal standard are 348060, 5279, and 62480 respectively. By combining the corresponding standard curve and conversion, the contents of phenylalanine, arginine, and tyrosine in the actual plasma sample are calculated to be 277, 20, and 157 μg / mL respectively.

[0044] Table 1-3. Content distributions of 10 amino acids in the plasma of 300 pregnant women (μg / mL)

[0045]

[0046] 6) Method verification: including spiked recovery rate and precision within and between days.

[0047] Add 10 amino acids at low, medium, and high concentration levels to the actual plasma sample, and calculate the recovery rate of this method. Use the mixed sample as the quality control sample, insert the quality control sample once every 10 actual samples during the instrument analysis, insert it 7 times in total, inject 6 needles repeatedly each time, calculate the reproducibility of the detection within the day, inject 5 needles of the quality control sample at different times on two days respectively, calculate the RSD value of the quality control sample concentration. The RSD values of all compounds are less than 20%. The spiked recovery rate and precision within and between days of this detection method are shown in Table 1-4.

[0048] Table 1-4. Spiked recovery rate and precision within and between days

[0049]

[0050] Comparative example

[0051] To compare the effects of different carrier liquids on the analysis results, amino acid detections with different carrier liquids were carried out, and the effects of methanol solutions containing formic acid (with formic acid volume percentages of 0.01% and 0.025% respectively) on the analysis results were compared emphatically. In addition, the effects of ethanol solutions containing acetic acid (with acetic acid volume percentage of 0.025%) and methanol solutions with ammonium fluoride concentration of 2 mM on the analysis results were also compared. The experimental process was basically the same as steps 1) and 2) of Example 1. A 0.1 ug / mL mixed standard solution was injected into a triple quadrupole mass spectrometer for analysis by acoustic wave excitation, and the only difference was that the carrier liquids were different.

[0052] It can be seen from Figure 7 and Figure 8 that formic acid methanol is superior to other carrier liquids in terms of sensitivity improvement (signal-to-noise ratio increase), and 0.01% formic acid methanol performs the best. As an acidic additive, formic acid can improve the ionization efficiency, enhance the ionization degree of the target substance in mass spectrometry analysis, make amino acid molecules easier to be ionized, thereby increasing the signal-to-noise ratio and enhancing the detection sensitivity.

[0053] Example 2

[0054] To compare the effects of different numbers of ion pair channels on the analysis results, a comparative experiment with different numbers of ion pair channels was carried out. The experimental process was basically the same as steps 1) and 2) of Example 1. A 0.1 ug / mL mixed standard solution was injected into a triple quadrupole mass spectrometer for analysis by acoustic wave excitation, and the only difference was that the number of centrifugal channels was different.

[0055] It can be seen from Figure 9 and Figure 10 that 2 ion pair channels are superior to 3 and 4 ion pair channels in terms of reproducibility and peak shape. Using more ion pair channels will lead to an increase in signal complexity, thus affecting the signal stability and reproducibility. At the same time, more ion pair channels may introduce more interference and reduce the clarity of the peak shape. While 2 ion pair channels can provide sufficient sensitivity while maintaining good reproducibility and peak shape. Therefore, the present invention selects 2 ion pair channels for the detection of amino acids.

[0056] Example 3

[0057] To compare the effects of different additional delay times on the amino acid analysis results, a comparative experiment with different additional delay times was carried out. The experimental process was basically the same as steps 1) and 2) of Example 1. A 0.1 ug / mL mixed standard solution was injected into a triple quadrupole mass spectrometer for analysis by acoustic wave excitation, and the only difference was that the delay times were different.

[0058] It can be seen from Figure 11 and Figure 12It can be seen that a 3000 ms delay time has advantages in amino acid detection, such as reducing interference, obtaining a better peak shape, and accurate quantification. A longer delay time can effectively reduce signal interference, especially interference from the matrix or other ions. In addition, an appropriate delay time helps ions to enter the mass spectrometry analysis region stably, thereby improving the resolution of the peak shape and the selective detection of the target substance. Therefore, the present invention selects 3000 ms as the additional delay time to optimize the detection effect of amino acids.

[0059] The above embodiments are convenient for those skilled in the art to understand and apply the present invention. For those of ordinary skill in the art, without departing from the inventive concept of the present invention, several improvements and transformations can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A method for rapid analysis of amino acids in plasma based on acoustic wave excitation coupled mass spectrometry, characterized in that, The plasma sample was pretreated by protein precipitation method to obtain the sample loading solution for injection; then the sample loading solution was injected into a triple quadrupole mass spectrometer by acoustic wave excitation for analysis to obtain the characteristic ion pair peak areas of amino acids and stable isotope internal standards, and then one or more amino acids in the plasma sample were rapidly quantified by the stable isotope internal standard method in combination with the standard curve.

2. A method for rapidly analyzing amino acids in plasma based on acoustic wave excited coupling mass spectrometry according to claim 1, characterized in that This non-contact injection method of acoustic wave excitation was used, and the specific conditions were as follows: the coupling fluid was ultrapure water, the carrier liquid was a methanol solution containing formic acid, the injection mode was the solution mode, and the flow rate was 400 - 550 μL / min; the injection volume was 2.5 - 25 nL; among them, the methanol solution containing formic acid used methanol as the solvent, and the volume percentage of formic acid was 0.01 - 0.025%.

3. A method for rapid analysis of amino acids in plasma based on acoustic wave excitation coupled mass spectrometry according to claim 1, wherein The triple quadrupole mass spectrometer was detected in the electrospray positive ion multiple reaction monitoring mode, and the specific conditions were as follows: the ion source temperature was set at 300 - 500 °C; the curtain gas was set at 20 - 40 psi, the nebulizing gas was set at 90 - 100 psi, the auxiliary heating gas was set at 60 - 80 psi, and 1 - 4 ion pair channels were used simultaneously, and the delay time was 1000 - 3000 ms.

4. The method for rapid analysis of amino acids in plasma based on acoustic wave excitation coupled mass spectrometry according to claim 3, wherein Two ion pair channels were used, and the delay time was 3000 ms.

5. A method for rapid analysis of amino acids in plasma based on acoustic wave excitation coupled mass spectrometry according to claim 1, characterized in that, The standard solution was injected into the triple quadrupole mass spectrometer by acoustic wave excitation for analysis to obtain the characteristic ion pair peak areas of amino acids and internal standards; then, the concentration ratio of amino acids to internal standards in the standard solution was used as the abscissa, and the ratio of the characteristic ion pair peak areas of amino acids to internal standards was used as the ordinate to establish a standard curve; then, according to the characteristic ion pair peak areas obtained from the sample loading solution, the peak area ratio of amino acids to internal standards was substituted into the standard curve to calculate the concentration ratio of each amino acid to the internal standard in the sample loading solution, and then, in combination with the concentration of the internal standard in the sample loading solution and the volume relationship between the sample loading solution and the plasma sample, the content of each amino acid in the plasma sample was converted; among them, the acoustic wave excitation injection conditions and triple quadrupole mass spectrometry analysis conditions for obtaining the characteristic ion pair peak areas of the sample loading solution were the same as those for obtaining the characteristic ion pair peak areas of the standard solution.

6. The method for rapid analysis of amino acids in plasma based on acoustic wave excitation coupled mass spectrometry according to claim 4, wherein The series of standard solutions for drawing the standard curve were prepared by dissolving amino acid standards in water, and the concentration range was between 0.02 - 20.0 μg / mL, and all were added with stable isotope internal standards; among them, the concentration of the stable isotope internal standard in each standard solution was 0.2 - 5 μg / mL.

7. A method for rapid analysis of amino acids in plasma based on acoustic wave excitation coupled mass spectrometry according to claim 1, characterized in that, The process of pretreating the plasma sample by the protein precipitation method was as follows: the plasma sample and the extraction solution were mixed at a volume ratio of 1:(3 - 5), vortex - oscillated for 60 - 120 s, placed at - 15 - 25 °C for 10 - 20 min, and then centrifuged at 9000 - 140000 rpm for more than 10 min at 1 - 7 °C, and the supernatant was taken; the supernatant was directly used as the sample loading solution for injection, or the supernatant was diluted with ultrapure water and then used as the sample loading solution for injection.

8. A method for rapid analysis of amino acids in plasma based on acoustic wave excitation coupled mass spectrometry according to claim 7, characterized in that, The extraction solution is composed of methanol and acetonitrile mixed in a volume ratio of (3 - 5):1, and is added with stable isotope internal standards with a concentration of 0.5 - 1.5 μg / mL each; the stable isotope internal standards are arginine-D7 and tyrosine-D4.

9. A method for rapid analysis of amino acids in plasma based on acoustic wave excitation coupled mass spectrometry according to claim 8, characterized in that, The concentration of the stable isotope internal standard in the sample loading solution is in the range of 100 - 150 ng / mL.

10. A method for rapid analysis of amino acids in plasma based on acoustic wave excitation coupled mass spectrometry according to claim 1, characterized in that, The multiple amino acids include phenylalanine, arginine, methionine, tyrosine, ornithine, proline, tryptophan, serine, threonine, and histidine.

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