WO2022120752A1 - Method for quantitative analysis of free amino acids in biological sample by liquid chromatography-tandem mass spectrometry - Google Patents

Method for quantitative analysis of free amino acids in biological sample by liquid chromatography-tandem mass spectrometry Download PDF

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WO2022120752A1
WO2022120752A1 PCT/CN2020/135390 CN2020135390W WO2022120752A1 WO 2022120752 A1 WO2022120752 A1 WO 2022120752A1 CN 2020135390 W CN2020135390 W CN 2020135390W WO 2022120752 A1 WO2022120752 A1 WO 2022120752A1
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sample
amino acids
liquid chromatography
mass spectrometry
tandem mass
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Chinese (zh)
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罗茜
陈志宇
傅磊
李芳�
李文波
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中国科学院深圳先进技术研究院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers

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  • the invention belongs to the field of analysis and detection, and in particular relates to a method for quantitatively analyzing free amino acids in biological samples by using liquid chromatography-tandem mass spectrometry.
  • Amino acids are one of the most important metabolites in the body and have a wide range of biological functions. It is not only the basic building block of various proteins, enzymes and other biological macromolecular active substances in the body and an important precursor of nitrogen-containing biological small molecules such as polypeptides, neurotransmitters and polyamines, but also participates in the metabolism and synthesis of carbohydrates and lipids. , purine and pyrimidine synthesis.
  • the imbalance of amino acid metabolism can lead to abnormal physiological functions, affect the normal progress of the body's metabolism, and then lead to diseases, which has become one of the inducements or manifestations of many diseases. Therefore, amino acids are important targets in the metabolic process, and monitoring the content of amino acids in biological samples is of great significance for assisting clinical diagnosis and elucidating the effects on the body's physiological functions.
  • the existing amino acid analysis methods are mainly direct analysis method and derivatization indirect analysis method.
  • There are many analytical methods for amino acids including amino acid analyzers, liquid chromatography, gas chromatography, capillary electrophoresis, and mass spectrometry.
  • Amino acid analyzers have good reproducibility and reliable results, but they all have complex hardware configurations, high maintenance costs, poor flexibility, low resolution, and long analysis cycles.
  • Both liquid chromatography and gas chromatography require derivatization, but this method leads to complex sample processing steps, long analysis times, difficulty in achieving high-throughput detection, and distorted results for low-level amino acids.
  • Mass spectrometry technology has the advantages of high accuracy, high sensitivity, good stability and repeatability.
  • Liquid chromatography-mass spectrometry mainly improves amino acids on the chromatographic column by derivatizing amino acids with phenyl isothiocyanate, o-phthalaldehyde, 6-aminoquinoline-N-hydroxysuccinimidyl carbamate, etc.
  • the retention behavior of amino acids or the addition of volatile ion pair reagents such as sodium pentanesulfonate, sodium n-hexanesulfonate and other alkyl sulfonate solutions are used to analyze amino acids, but both methods will pollute the system.
  • Amino acids play an important role in the development of biotechnology, protein research, and the food and pharmaceutical industries. Therefore, it is particularly important to develop an amino acid analysis and detection method that is easy to operate, has high sensitivity, good selectivity, high detection throughput, and is suitable for a variety of biological samples. important.
  • the present invention aims to solve the technical problems mentioned in the background art, and provides a liquid chromatography-mass spectrometry analysis method suitable for amino acid content in various biological samples such as serum and brain tissue.
  • this method has a simple sample pretreatment method, does not require derivatization, does not add buffer salt reagents to the mobile phase, has high sensitivity, high selectivity, and is suitable for a variety of biological samples, such as serum, brain tissue and other biological samples.
  • the analysis of amino acid content provides a new reference method and provides important technical support for the evaluation of amino acid content in common clinical and biological samples and related biological and medical research.
  • a method for quantitative analysis of free amino acids in biological samples by liquid chromatography-tandem mass spectrometry characterized in that the method comprises the following steps:
  • Step 1) pre-processing the biological sample to obtain the processed biological sample
  • Step 2) placing the biological sample processed in step 1) in a refrigerated centrifuge for centrifugation, collecting the supernatant and concentrating to near dryness under a nitrogen blower;
  • Step 3 mix the sample obtained in step 2) with a solvent, filter it, and store it at 4°C for testing;
  • step 4 the biological sample obtained in step 3) is analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to obtain the type and content of free amino acids in the biological sample.
  • UPLC-MS/MS ultra-high performance liquid chromatography-tandem mass spectrometry
  • the biological samples include serum samples, plasma samples, brain tissue samples, and cell samples.
  • step 1) when the biological sample is a serum sample or a plasma sample, the step of preprocessing the serum sample or the plasma sample is: placing the serum sample or plasma sample at 4°C for 12000- Centrifuge at 15000rpm for 10-20min, take the upper serum or plasma for use, add the upper serum or plasma sample into methanol solution equivalent to 3-5 times the sample volume, shake and mix, and let stand for 10-15min to precipitate the protein in the sample .
  • step 1) when the biological sample is a brain tissue sample, the step of preprocessing the brain tissue sample is as follows: adding 500 ⁇ L of methanol/water mixed solvent per 50 mg of brain tissue sample, the brain tissue After adding the methanol/water mixed solvent, the samples were placed in a tissue grinder at 4°C and ground at 120 Hz for 10 min. The volume ratio of methanol and water in the methanol/water mixed solvent was 1:1.
  • step 2) the condition of refrigerated centrifugation is centrifugation at 12000-15000rpm for 10-20min at 4°C.
  • step 3 when the biological sample is a serum sample, add 150-250 ⁇ L of solvent per 50 ⁇ L of serum sample and mix well; when the biological sample is a brain tissue sample, per 50 mg of brain tissue sample, add 500 ⁇ L of solvent and mix well;
  • a 0.22 ⁇ m cellulose acetate filter membrane is used for filtration;
  • the solvent is a methanol/water mixed solvent, or an acetonitrile/water mixed solvent, and the volume ratio of methanol to water in the methanol/water mixed solvent is 1:1 , the volume ratio of acetonitrile and water in the acetonitrile/water mixed solvent is 1:1.
  • the analytical column used for the analysis by ultra-high performance liquid chromatography-tandem mass spectrometry is ACQUITY UPLC HSS T3, size: 2.1 ⁇ 100 mm, 1.8 ⁇ m.
  • step 4 the mobile phase in the analysis by ultra-high performance liquid chromatography-tandem mass spectrometry: phase A is 0.1% formic acid-water, and phase B is 0.1% formic acid-acetonitrile; the gradient used is The elution program is as follows: the initial ratio is 100% for phase A, and 0% for phase B for 0.5 min; 0.5-5 min, phase A drops to 40%, and phase B rises to 60%; at 5.1 min, it returns to the initial gradient Equilibrate the system to 7.0 min; the flow rate of the mobile phase is always 0.2 mL/min during the entire elution process; the temperature of the column oven is 40 °C; the injection volume is 5-10 ⁇ L; the injection needle cleaning solution is methanol/water mixed solvent, methanol The volume ratio of methanol and water in the /water mixed solvent was 1:1.
  • step 4 retention time locking and characteristic ion pair locking are used to quantify amino acids present in qualitatively screened biological samples and their content by external standard method, ionization of tandem mass spectrometry
  • the mode is mixed electrospray ionization of positive and negative ion modes;
  • the detection method is multiple reaction detection (MRM);
  • the ion source temperature is 200 °C
  • the desolvation temperature is 400 °C
  • the desolvation gas flow rate is 10L/min
  • the capillary voltage is 4.0kV
  • the cone voltage is 30kV
  • the collision gas is argon
  • the pressure is 270kPa
  • the collision energy is 20V.
  • Liquid chromatography-tandem mass spectrometry conditions are as follows:
  • the present invention has the following beneficial effects:
  • the biological sample analyte to be detected in the present invention does not need derivatization, and the biological sample pretreatment method is simple.
  • the present invention uses the ACQUITY UPLC HSS T3 chromatographic column with a special stationary phase, which has good retention and separation effects on small molecular organic compounds with water solubility and high polarity, and does not need to add buffer salt solution in the mobile phase. Different kinds of amino acids can also be retained and separated on the column.
  • the present invention can analyze the amino acid content in different types of biological samples such as serum, plasma and tissue.
  • the present invention develops an analytical method with high sensitivity and high selectivity without derivatization and addition of ion pair reagents.
  • Figure 1 (A) shows the total ion chromatogram of 18 free amino acid standards separated by HSS T3 column
  • Figure 1 (B) shows the total ion chromatogram of 18 free amino acids in serum samples separated by HSS T3 column.
  • the retention time of the amino acid in Figure 1(B) and Figure 1(A) are the same, and the chromatographic peak corresponding to the retention time is the same amino acid.
  • Figure 2 shows the qualitative ion chromatograms for the detection of three free amino acid standards, in which (a) the extracted ion chromatogram of histidine based on HSS T3 chromatographic column; (b) the picture of alanine based on HSS T3 chromatographic column separation The extracted ion chromatogram of ; (c) is the extracted ion chromatogram of asparagine based on HSS T3 chromatographic column separation.
  • Figure 3 shows the qualitative ion chromatograms for the detection of three free amino acid standards, in which (a) is the extracted ion chromatogram of aspartic acid based on HSS T3 chromatographic column separation; (b) is the chromatogram of isoleucine based on HSS T3 column The extracted ion chromatogram of column separation; (c) is the extracted ion chromatogram of leucine based on HSS T3 column separation.
  • Figure 4 shows the qualitative ion chromatograms for the detection of three free amino acid standards, in which (a) is the extracted ion chromatogram of lysine based on HSS T3 chromatographic column separation; (b) the picture shows methionine based on HSS T3 chromatographic column The extracted ion chromatogram of the separation; (c) is the extracted ion chromatogram of serine based on HSS T3 column separation.
  • Figure 5 shows the qualitative ion chromatograms for the detection of three free amino acid standards, in which (a) is the extracted ion chromatogram of threonine based on HSS T3 chromatographic column; (b) is the separation of valine based on HSS T3 chromatographic column The extracted ion chromatogram of ; (c) is the extracted ion chromatogram of proline based on HSS T3 chromatographic column separation.
  • Figure 6 is a qualitative ion chromatogram for the detection of three free amino acid standards, glutamic acid, glutamine, and tryptophan.
  • Figure 7 shows the qualitative ion chromatograms for the detection of three free amino acid standards, glycine, phenylalanine, and tyrosine.
  • Figure 8 is the standard curve of six free amino acids, wherein, A: histidine; B: alanine; C: asparagine; D: aspartic acid; E: isoleucine; F: leucine .
  • Figure 9 is a standard curve of six free amino acids, wherein G: lysine; H: methionine; I: serine; J: threonine; K: valine; L: proline.
  • Figure 10 is a standard curve of six free amino acids, wherein, M: glutamic acid; N: glutamine; O: tryptophan; P: glycine; Q: phenylalanine; R: tyrosine.
  • the standard substance was determined for amino acids, the UPLC-MS/MS used for the instrumental analysis of amino acid content was Shimadzu's UPLC-MS8060, and the analytical column used was ACQUITY UPLC HSS T3 (2.1 ⁇ 100mm, 1.8 ⁇ m);
  • the phases consisted of phase A at 0.1% formic acid-water and phase B at 0.1% formic acid-acetonitrile.
  • Gradient elution program the initial ratio is 100% for phase A, 0% for phase B, and lasts for 0.5min; 0.5-5min, phase A drops to 40%, phase B rises to 60%; return to the initial gradient at 5.1min Equilibrate the system to 8.0 min.
  • the flow rate of the mobile phase was always 0.2 mL/min throughout the elution process; the temperature of the column oven was 40 °C; the injection volume was 5 ⁇ L; the syringe cleaning solution was methanol/water (v/v, 1:1).
  • the ionization mode of the tandem mass spectrometer is electrospray ionization in positive ion mode; the detection method is multiple reaction detection (MRM); the ion source temperature is 200 °C, the desolvation temperature is 400 °C, the desolvation gas flow rate is 10 L/min, and the capillary voltage is 4.0kV, the cone voltage is 30kV, the collision gas is argon, and the pressure is 270kPa.
  • MRM multiple reaction detection
  • FIG. 1(A) The total ion current chromatogram of 18 kinds of free amino acid standards obtained by the above analysis method based on HSS T3 chromatographic column separation is shown in Figure 1(A).
  • Figures 2-7 are extracted ion chromatograms of 18 free amino acid standards separated by HSS T3 column.
  • Step 1) Obtaining serum samples: centrifuge the blood samples obtained from mice at 4°C and 10,000 ⁇ g for 10 min, and take the upper serum for use; pipette 50 ⁇ L of serum samples and place them in a 1.5 mL centrifuge tube , add methanol solution equivalent to 3 times the sample volume, shake and mix, and let stand for 10 to 15 minutes to precipitate the protein in the sample;
  • Step 2) centrifuge the sample obtained in step 1) at 12000rpm for 10min at 4°C, collect the supernatant sample and concentrate it to near dryness under a nitrogen blower; use methanol/water mixed solvent (v/v, 1:1) to determine Adjust the sample volume to 0.5-1.0mL, vortex and mix, filter the sample with a 0.22 ⁇ m cellulose acetate filter, and store at 4°C for testing;
  • Step 3 the UPLC-MS/MS used in the instrumental analysis of the amino acid content in the sample obtained in step 2) is Shimadzu's UPLC-MS 8060, and the analytical column used is ACQUITY UPLC HSS T3 (2.1 ⁇ 100mm, 1.8 ⁇ m);
  • the mobile phases used consisted of 0.1% formic acid-water phase A and 0.1% formic acid-acetonitrile phase B.
  • Gradient elution procedure the initial ratio is 100% for phase A, 0% for phase B, and lasts for 0.5min; 0.5-5min, phase A drops to 40%, phase B rises to 60%; return to the initial gradient at 5.1min Equilibrate the system to 8.0 min.
  • the flow rate of the mobile phase was always 0.2 mL/min throughout the elution process; the temperature of the column oven was 40 °C; the injection volume was 5 ⁇ L; the syringe cleaning solution was methanol/water (v/v, 1:1).
  • the ionization mode of the tandem mass spectrometer is electrospray ionization in positive ion mode; the detection method is multiple reaction detection (MRM); the ion source temperature is 200 °C, the desolvation temperature is 400 °C, the desolvation gas flow rate is 10 L/min, and the capillary voltage is 4.0kV, the cone voltage is 30kV, the collision gas is argon, and the pressure is 270kPa.
  • MRM multiple reaction detection
  • the amino acid content in the sample was detected according to the UPLC-MS/MS conditions in the above table 1, and mixed standard solutions of different mass concentrations were taken for UPLCMS/MS determination. Coordinates draw standard curve for analyzing the content of amino acids in the sample: obtain their standard curve, and obtain the corresponding linear regression equation and correlation coefficient.
  • Figures 8-10 are the standard curves of 18 free amino acids, as follows:
  • the amino acid content in the serum sample is calculated by substituting its response value on the instrument into the standard curve constructed by the standard sample. It can be seen from Table 2 that the amino acid content in the actual serum sample, as well as the detection limit of the instrument for 18 amino acids and the linear range of the 18 amino acid standard sample curve.
  • This embodiment is the same as the detection method in Embodiment 1, except that the brain tissue sample is used as the biological sample to be detected:
  • the brain tissue samples were obtained by placing the mouse brain tissue samples in a clean 1.5 mL centrifuge tube, adding 500 ⁇ L of methanol-water (v/v, 1:1) solution, and placing them in a tissue grinder at 4°C. Grind for 10 min at 120 Hz. Other detection steps are the same as in Example 1.

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Abstract

A method for quantitative analysis of free amino acids in a biological sample by liquid chromatography-tandem mass spectrometry. The method comprises the following steps: step 1), preprocessing a biological sample to obtain a processed biological sample; step 2), placing the biological sample processed in step 1) in a freeze centrifuge for centrifugation, collecting a supernatant, and concentrating the supernatant to a near-dry state under a nitrogen blowing instrument; step 3), uniformly mixing the sample obtained in step 2) with a solvent, then filtering, and storing the sample at 4°C for later testing; and step 4) performing ultra-high performance liquid chromatography-tandem mass spectrometry analysis on the biological sample obtained in step 3) to obtain the types and contents of free amino acids in the biological sample. According to the method, the contents of amino acids in different types of biological samples such as serum, plasma, and brain tissue can be analyzed, derivatization does not need to be performed on biological sample analytes to be tested, and the preprocessing method is simple.

Description

利用液相色谱-串联质谱定量分析生物样本中游离氨基酸的方法Method for quantitative analysis of free amino acids in biological samples by liquid chromatography-tandem mass spectrometry 技术领域technical field
本发明属于分析检测领域，具体的，涉及利用液相色谱-串联质谱定量分析生物样本中游离氨基酸的方法。The invention belongs to the field of analysis and detection, and in particular relates to a method for quantitatively analyzing free amino acids in biological samples by using liquid chromatography-tandem mass spectrometry.
背景技术Background technique
氨基酸是机体重要的代谢物之一，具有广泛的生物学功能。不仅是生物体内各种蛋白质、酶等生物大分子活性物质的基本组成单元和多肽、神经递质和多胺等含氮生物小分子物质的重要前体物质，还参与糖类和脂质代谢合成、嘌呤和嘧啶合成等。氨基酸代谢失衡会导致生理功能异常，影响机体代谢的正常进行，进而导致疾病，已成为众多疾病的诱因或表现形式之一。因此，氨基酸作为代谢过程中的重要靶标，监测生物样本中氨基酸的含量对于辅助临床诊断和阐明对机体生理机能的影响具有重要意义。Amino acids are one of the most important metabolites in the body and have a wide range of biological functions. It is not only the basic building block of various proteins, enzymes and other biological macromolecular active substances in the body and an important precursor of nitrogen-containing biological small molecules such as polypeptides, neurotransmitters and polyamines, but also participates in the metabolism and synthesis of carbohydrates and lipids. , purine and pyrimidine synthesis. The imbalance of amino acid metabolism can lead to abnormal physiological functions, affect the normal progress of the body's metabolism, and then lead to diseases, which has become one of the inducements or manifestations of many diseases. Therefore, amino acids are important targets in the metabolic process, and monitoring the content of amino acids in biological samples is of great significance for assisting clinical diagnosis and elucidating the effects on the body's physiological functions.
现有的氨基酸分析方法主要为直接分析法和衍生化间接分析法。对于氨基酸的分析方法众多，包括氨基酸分析仪、液相色谱法、气相色谱法、毛细管电泳和质谱法。氨基酸分析仪测定法重现性好，结果可靠，但是均在硬件配置复杂，维护成本高，灵活性差，分辨率低，分析周期长。液相色谱法和气相色谱法均需进行衍生化，但该方法导致样品处理步骤复杂分析时间长、难以实现高通量检测、且低含量氨基酸会出现结果失真。质谱技术具有精确度高、灵敏度高、稳定性和重复性好的优点，结合分离能力高的液相色谱已成为目前氨基酸分析最常用的方法。液相色谱-质谱法主要通过将氨基酸与异硫氰酸苯酯、邻苯二甲醛、6-氨基喹啉-N-羟基琥珀酰亚胺基氨基甲酸酯等衍生化改善氨基酸在色谱柱上的保留行为或添加挥发性离子对试剂如戊烷磺酸钠、正己磺酸钠等烷基磺酸盐溶液对氨基酸进行分析，但是这两种方法均会对系统有污染。The existing amino acid analysis methods are mainly direct analysis method and derivatization indirect analysis method. There are many analytical methods for amino acids, including amino acid analyzers, liquid chromatography, gas chromatography, capillary electrophoresis, and mass spectrometry. Amino acid analyzers have good reproducibility and reliable results, but they all have complex hardware configurations, high maintenance costs, poor flexibility, low resolution, and long analysis cycles. Both liquid chromatography and gas chromatography require derivatization, but this method leads to complex sample processing steps, long analysis times, difficulty in achieving high-throughput detection, and distorted results for low-level amino acids. Mass spectrometry technology has the advantages of high accuracy, high sensitivity, good stability and repeatability. Combined with liquid chromatography with high separation ability, it has become the most commonly used method for amino acid analysis. Liquid chromatography-mass spectrometry mainly improves amino acids on the chromatographic column by derivatizing amino acids with phenyl isothiocyanate, o-phthalaldehyde, 6-aminoquinoline-N-hydroxysuccinimidyl carbamate, etc. The retention behavior of amino acids or the addition of volatile ion pair reagents such as sodium pentanesulfonate, sodium n-hexanesulfonate and other alkyl sulfonate solutions are used to analyze amino acids, but both methods will pollute the system.
近年来，研究者们开始尝试采用硅胶、氨基、氰基等正相色谱填料或表面含极性基团的亲水性互作用(HILIC)高效液相色谱柱对氨基酸进行分离，虽然基于HILIC色谱柱的液相色谱-质谱法可实现对氨基酸无需衍生化分析，但由于HILIC色谱柱对待分析物质的保留能力会受如有机改良剂的含量、流速、流动相缓冲体系的pH值、缓冲盐种类和浓度等多种因素的综合影响。因此，在实际应用中需要严格控制色谱参数，应用条件较为严格。而且，在使用HILIC色谱柱对氨基酸含量进行分析时，需要在流动相中添加甲酸铵缓冲盐溶液。缓冲盐容易析出、残留，对色谱系统和色谱柱造成污染和堵塞，影响色谱的保留能力。而通过对氨基酸进行衍生化的间接分析法，会增加整个分析检测过程的实验步骤，且结果易受衍生化试剂、衍生化步骤、所生成衍生物等因素的影响，难以直接、快速地对样本中氨基酸含量进行分析检测。In recent years, researchers have begun to use silica gel, amino, cyano and other normal phase chromatography packings or hydrophilic interaction (HILIC) high performance liquid chromatography columns containing polar groups on the surface to separate amino acids, although based on HILIC chromatography Column-based liquid chromatography-mass spectrometry can realize the analysis of amino acids without derivatization, but the retention capacity of the analyte in the HILIC column will be affected by, for example, the content of organic modifiers, flow rate, pH value of the mobile phase buffer system, and types of buffer salts. The combined effect of various factors such as concentration and concentration. Therefore, it is necessary to strictly control the chromatographic parameters in practical applications, and the application conditions are relatively strict. Furthermore, when using HILIC column to analyze amino acid content, it is necessary to add ammonium formate buffered saline solution to the mobile phase. Buffer salts are easy to precipitate and remain, causing pollution and blockage to chromatographic systems and chromatographic columns, and affecting the retention capacity of chromatography. However, the indirect analysis method of derivatization of amino acids will increase the experimental steps of the entire analysis and detection process, and the results are easily affected by factors such as derivatization reagents, derivatization steps, and derivatives generated. It is difficult to directly and quickly analyze the sample. The amino acid content was analyzed and detected.
氨基酸在生物技术发展、蛋白质研究以及食品、医药行业领域的重要作用，因此，开发一种操作简便、灵敏度高、选择性好、检测通量高、适用于多种生物样本的氨基酸分析检测方法尤为重要。Amino acids play an important role in the development of biotechnology, protein research, and the food and pharmaceutical industries. Therefore, it is particularly important to develop an amino acid analysis and detection method that is easy to operate, has high sensitivity, good selectivity, high detection throughput, and is suitable for a variety of biological samples. important.
发明内容SUMMARY OF THE INVENTION
本发明旨在解决背景技术中提到的技术问题，提供一种适用于血清、脑组织等多种生物样本中氨基酸含量的液相色谱-质谱分析方法。与已有的方法比较，该方法样本前处理方法简单、无需衍生化、流动相不添加缓冲盐试剂、高灵敏度、高选择性以及适用多种生物样本，为如血清、脑组织等生物样本中氨基酸含量的分析提供一种新的参考方法，为常见临床、生物样本中氨基酸含量的评估及其相关生物学、医学研究提供重要的技术支撑。The present invention aims to solve the technical problems mentioned in the background art, and provides a liquid chromatography-mass spectrometry analysis method suitable for amino acid content in various biological samples such as serum and brain tissue. Compared with the existing methods, this method has a simple sample pretreatment method, does not require derivatization, does not add buffer salt reagents to the mobile phase, has high sensitivity, high selectivity, and is suitable for a variety of biological samples, such as serum, brain tissue and other biological samples. The analysis of amino acid content provides a new reference method and provides important technical support for the evaluation of amino acid content in common clinical and biological samples and related biological and medical research.
为了达到上述目的，本发明采用了如下技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:
利用液相色谱-串联质谱定量分析生物样本中游离氨基酸的方法，其特征在于，所述方法包括以下步骤：A method for quantitative analysis of free amino acids in biological samples by liquid chromatography-tandem mass spectrometry, characterized in that the method comprises the following steps:
步骤1)、将生物样品进行预处理，得到处理后的生物样品；Step 1), pre-processing the biological sample to obtain the processed biological sample;
步骤2)、将步骤1)中处理后的生物样品置于冷冻离心机中离心，收集上层清液在氮吹仪下浓缩至近干；Step 2), placing the biological sample processed in step 1) in a refrigerated centrifuge for centrifugation, collecting the supernatant and concentrating to near dryness under a nitrogen blower;
步骤3)、将步骤2)中所得的样品用溶剂混匀后用过滤，4℃储存待测；Step 3), mix the sample obtained in step 2) with a solvent, filter it, and store it at 4°C for testing;
步骤4)、将步骤3)中所得的生物样品进行超高效液相色谱-串联质谱联用(UPLC-MS/MS)分析，得到生物样品中游离氨基酸的种类和含量。In step 4), the biological sample obtained in step 3) is analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to obtain the type and content of free amino acids in the biological sample.
在本发明的技术方案中，所述生物样品包括血清样品、血浆样品、脑组织样品、细胞样品。In the technical solution of the present invention, the biological samples include serum samples, plasma samples, brain tissue samples, and cell samples.
在本发明的技术方案中，步骤1)中，当生物样品为血清样品或血浆样品时，对血清样品或血浆样品进行预处理的步骤为：将血清样品或血浆样品置于4℃下12000-15000rpm的条件下离心10-20min，取上层血清或血浆备用，将上层血清或血浆样品，加入相当于3-5倍样品体积的甲醇溶液后震荡混匀，静置10～15min沉淀样品中的蛋白质。In the technical solution of the present invention, in step 1), when the biological sample is a serum sample or a plasma sample, the step of preprocessing the serum sample or the plasma sample is: placing the serum sample or plasma sample at 4°C for 12000- Centrifuge at 15000rpm for 10-20min, take the upper serum or plasma for use, add the upper serum or plasma sample into methanol solution equivalent to 3-5 times the sample volume, shake and mix, and let stand for 10-15min to precipitate the protein in the sample .
在本发明的技术方案中，步骤1)中，当生物样品为脑组织样品时，对脑组织样品进行预处理的步骤为：按照每50mg脑组织样品加入甲醇/水混合溶剂500μL，将脑组织样品加入甲醇/水混合溶剂后，置于组织研磨器中4℃，120Hz下研磨10min，甲醇/水混合溶剂中甲醇和水的体积比为1:1。In the technical solution of the present invention, in step 1), when the biological sample is a brain tissue sample, the step of preprocessing the brain tissue sample is as follows: adding 500 μL of methanol/water mixed solvent per 50 mg of brain tissue sample, the brain tissue After adding the methanol/water mixed solvent, the samples were placed in a tissue grinder at 4°C and ground at 120 Hz for 10 min. The volume ratio of methanol and water in the methanol/water mixed solvent was 1:1.
在本发明的技术方案中，步骤2)中，冷冻离心的条件为4℃下12000-15000rpm离心10-20min。In the technical solution of the present invention, in step 2), the condition of refrigerated centrifugation is centrifugation at 12000-15000rpm for 10-20min at 4°C.
在本发明的技术方案中，步骤3)中，当生物样品为血清样品时，按照每50μL的血清样品，加入150-250μL的溶剂混匀；当生物样品脑组织样品时，按照每50mg脑组织样品，加入500μL的溶剂混匀；In the technical solution of the present invention, in step 3), when the biological sample is a serum sample, add 150-250 μL of solvent per 50 μL of serum sample and mix well; when the biological sample is a brain tissue sample, per 50 mg of brain tissue sample, add 500 μL of solvent and mix well;
优选的，步骤3)中，采用0.22μm醋酸纤维素滤膜进行过滤；溶剂为甲醇/水混合溶剂，或乙腈/水混合溶剂，甲醇/水混合溶剂中甲醇和水的体积比为 1:1，乙腈/水混合溶剂中乙腈和水的体积比为1:1。Preferably, in step 3), a 0.22 μm cellulose acetate filter membrane is used for filtration; the solvent is a methanol/water mixed solvent, or an acetonitrile/water mixed solvent, and the volume ratio of methanol to water in the methanol/water mixed solvent is 1:1 , the volume ratio of acetonitrile and water in the acetonitrile/water mixed solvent is 1:1.
在本发明的技术方案中，超高效液相色谱-串联质谱联用分析所用的分析柱为ACQUITY UPLC HSS T3，规格：2.1×100mm,1.8μm。In the technical solution of the present invention, the analytical column used for the analysis by ultra-high performance liquid chromatography-tandem mass spectrometry is ACQUITY UPLC HSS T3, size: 2.1×100 mm, 1.8 μm.
在本发明的技术方案中，步骤4)中，超高效液相色谱-串联质谱联用分析时的流动相：A相为0.1％甲酸-水，B相为0.1％甲酸-乙腈；采用的梯度洗脱程序为：起始比例为A相100％，B相为0％，持续0.5min；0.5～5min，A相降至40％，B相升至60％；5.1min时恢复至起始梯度平衡系统至7.0min；整个洗脱过程流动相的流速始终为0.2mL/min；柱温箱温度为40℃；进样体积为5～10μL；进样针清洗液为甲醇/水混合溶剂，甲醇/水混合溶剂中甲醇和水的体积比为1:1。In the technical solution of the present invention, in step 4), the mobile phase in the analysis by ultra-high performance liquid chromatography-tandem mass spectrometry: phase A is 0.1% formic acid-water, and phase B is 0.1% formic acid-acetonitrile; the gradient used is The elution program is as follows: the initial ratio is 100% for phase A, and 0% for phase B for 0.5 min; 0.5-5 min, phase A drops to 40%, and phase B rises to 60%; at 5.1 min, it returns to the initial gradient Equilibrate the system to 7.0 min; the flow rate of the mobile phase is always 0.2 mL/min during the entire elution process; the temperature of the column oven is 40 °C; the injection volume is 5-10 μL; the injection needle cleaning solution is methanol/water mixed solvent, methanol The volume ratio of methanol and water in the /water mixed solvent was 1:1.
在本发明的技术方案中，步骤4)中，利用保留时间锁定和特征离子对锁定的方式对定性筛查的生物样品中存在的氨基酸及通过外标法对其含量进行定量，串联质谱的电离模式为正、负离子模式混合的电喷雾电离；检测方式为多重反应检测(MRM)；离子源温度为200℃，去溶剂温度为400℃，去溶剂气流速为10L/min，毛细管电压为4.0kV，锥孔电压为30kV，碰撞气为氩气，压力为270kPa，碰撞能为20V。In the technical solution of the present invention, in step 4), retention time locking and characteristic ion pair locking are used to quantify amino acids present in qualitatively screened biological samples and their content by external standard method, ionization of tandem mass spectrometry The mode is mixed electrospray ionization of positive and negative ion modes; the detection method is multiple reaction detection (MRM); the ion source temperature is 200 °C, the desolvation temperature is 400 °C, the desolvation gas flow rate is 10L/min, and the capillary voltage is 4.0kV , the cone voltage is 30kV, the collision gas is argon, the pressure is 270kPa, and the collision energy is 20V.
液相色谱-串联质谱条件如下：Liquid chromatography-tandem mass spectrometry conditions are as follows:
Figure PCTCN2020135390-appb-000001
Figure PCTCN2020135390-appb-000001
Figure PCTCN2020135390-appb-000002
Figure PCTCN2020135390-appb-000002
与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
1、本发明待检测的生物样品分析物不需要衍生化，生物样品进行预处理方法简单。1. The biological sample analyte to be detected in the present invention does not need derivatization, and the biological sample pretreatment method is simple.
2、本发明使用了特殊固定相的ACQUITY UPLC HSS T3色谱柱，该色谱柱对水溶性、极性大的小分子有机化合物有较好的保留和分离效果，无需在流动相中添加缓冲盐溶液也可使不同种类的氨基酸在色谱柱上得到保留和分离。2. The present invention uses the ACQUITY UPLC HSS T3 chromatographic column with a special stationary phase, which has good retention and separation effects on small molecular organic compounds with water solubility and high polarity, and does not need to add buffer salt solution in the mobile phase. Different kinds of amino acids can also be retained and separated on the column.
3、本发明能够对血清、血浆、组织等不同类型的生物样本中氨基酸含量的分析。3. The present invention can analyze the amino acid content in different types of biological samples such as serum, plasma and tissue.
4、本发明为实现高通量测定血清、血浆、体液、组织等生物样本中常见游离氨基酸的含量，开发了一种无需衍生化和添加离子对试剂、高灵敏度、高选择性的分析方法。4. In order to achieve high-throughput determination of the content of common free amino acids in biological samples such as serum, plasma, body fluids, and tissues, the present invention develops an analytical method with high sensitivity and high selectivity without derivatization and addition of ion pair reagents.
附图说明Description of drawings
图1中(A)图为18种游离氨基酸标准品基于HSS T3色谱柱分离总离子流色谱图；图1中(B)图血清样本中18种游离氨基酸基于HSS T3色谱柱分离总离子流色谱图，其中，图1(B)中和图1(A)中的氨基酸保留时间是相同的，对应保留时间的色谱峰为同一个氨基酸。Figure 1 (A) shows the total ion chromatogram of 18 free amino acid standards separated by HSS T3 column; Figure 1 (B) shows the total ion chromatogram of 18 free amino acids in serum samples separated by HSS T3 column. In the figure, the retention time of the amino acid in Figure 1(B) and Figure 1(A) are the same, and the chromatographic peak corresponding to the retention time is the same amino acid.
图2为三种游离氨基酸标准品检测定性离子色谱图，其中(a)图为组氨酸基于HSS T3色谱柱分离的提取离子色谱图；(b)图为丙氨酸基于HSS T3色谱柱分离的提取离子色谱图；(c)图为天冬酰胺基于HSS T3色谱柱分离的提取离子色谱图。Figure 2 shows the qualitative ion chromatograms for the detection of three free amino acid standards, in which (a) the extracted ion chromatogram of histidine based on HSS T3 chromatographic column; (b) the picture of alanine based on HSS T3 chromatographic column separation The extracted ion chromatogram of ; (c) is the extracted ion chromatogram of asparagine based on HSS T3 chromatographic column separation.
图3为三种游离氨基酸标准品检测定性离子色谱图，其中(a)图为天冬氨酸基于HSS T3色谱柱分离的提取离子色谱图；(b)图为异亮氨酸基于HSS T3色谱柱分离的提取离子色谱图；(c)图为亮氨酸基于HSS T3色谱柱分离的提取离子色谱图。Figure 3 shows the qualitative ion chromatograms for the detection of three free amino acid standards, in which (a) is the extracted ion chromatogram of aspartic acid based on HSS T3 chromatographic column separation; (b) is the chromatogram of isoleucine based on HSS T3 column The extracted ion chromatogram of column separation; (c) is the extracted ion chromatogram of leucine based on HSS T3 column separation.
图4为三种游离氨基酸标准品检测定性离子色谱图，其中(a)图为赖氨酸基于HSS T3色谱柱分离的提取离子色谱图；(b)图为甲硫氨酸基于HSS T3色谱柱分离的提取离子色谱图；(c)图为丝氨酸基于HSS T3色谱柱分离的提取离子色谱图。Figure 4 shows the qualitative ion chromatograms for the detection of three free amino acid standards, in which (a) is the extracted ion chromatogram of lysine based on HSS T3 chromatographic column separation; (b) the picture shows methionine based on HSS T3 chromatographic column The extracted ion chromatogram of the separation; (c) is the extracted ion chromatogram of serine based on HSS T3 column separation.
图5为三种游离氨基酸标准品检测定性离子色谱图，其中(a)图为苏氨酸基于HSS T3色谱柱分离的提取离子色谱图；(b)图为缬氨酸基于HSS T3色谱柱分离的提取离子色谱图；(c)图为脯氨酸基于HSS T3色谱柱分离的提取离子色谱图。Figure 5 shows the qualitative ion chromatograms for the detection of three free amino acid standards, in which (a) is the extracted ion chromatogram of threonine based on HSS T3 chromatographic column; (b) is the separation of valine based on HSS T3 chromatographic column The extracted ion chromatogram of ; (c) is the extracted ion chromatogram of proline based on HSS T3 chromatographic column separation.
图6为三种游离氨基酸标准品检测定性离子色谱图，谷氨酸、谷氨酰胺、色氨酸。Figure 6 is a qualitative ion chromatogram for the detection of three free amino acid standards, glutamic acid, glutamine, and tryptophan.
图7为三种游离氨基酸标准品检测定性离子色谱图，甘氨酸、苯丙氨酸、酪氨酸。Figure 7 shows the qualitative ion chromatograms for the detection of three free amino acid standards, glycine, phenylalanine, and tyrosine.
图8为六种游离氨基酸的标准曲线，其中，A：组氨酸；B：丙氨酸；C：天冬酰胺；D：天冬氨酸；E：异亮氨酸；F：亮氨酸。Figure 8 is the standard curve of six free amino acids, wherein, A: histidine; B: alanine; C: asparagine; D: aspartic acid; E: isoleucine; F: leucine .
图9为六种游离氨基酸的标准曲线，其中，G：赖氨酸；H：甲硫氨酸；I：丝氨酸；J：苏氨酸；K：缬氨酸；L：脯氨酸。Figure 9 is a standard curve of six free amino acids, wherein G: lysine; H: methionine; I: serine; J: threonine; K: valine; L: proline.
图10为六种游离氨基酸的标准曲线，其中，M：谷氨酸；N：谷氨酰胺；O：色氨酸；P：甘氨酸；Q：苯丙氨酸；R：酪氨酸。Figure 10 is a standard curve of six free amino acids, wherein, M: glutamic acid; N: glutamine; O: tryptophan; P: glycine; Q: phenylalanine; R: tyrosine.
具体实施方式Detailed ways
下面结合实施例以及对比例，对本发明作进一步地详细说明，但本发明的实施方式不限于此。The present invention will be further described in detail below with reference to the examples and comparative examples, but the embodiments of the present invention are not limited thereto.
实施例1Example 1
标准品组氨酸、丙氨酸、天冬酰胺、天冬氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、丝氨酸、苏氨酸、缬氨酸、脯氨酸、谷氨酸、谷氨酰胺、色氨酸、甘氨酸、苯丙氨酸、酪氨酸。Standard histidine, alanine, asparagine, aspartic acid, isoleucine, leucine, lysine, methionine, serine, threonine, valine, proline , Glutamate, Glutamine, Tryptophan, Glycine, Phenylalanine, Tyrosine.
标准品的配置：Standard configuration:
称取18种氨基酸的标准品各100mg，溶解于甲醇-水混合溶液(v/v,1:1)，逐级稀释并将每种标准品混合，得到终浓度为1ppm的18种氨基酸混合标准样品，逐级稀释至浓度为500、200、100、50、10和5pbb用于标准样品曲线的建立。Weigh 100 mg of each standard of 18 amino acids, dissolve in methanol-water mixed solution (v/v, 1:1), dilute step by step and mix each standard to obtain a final concentration of 1 ppm of 18 amino acids mixed standard Samples, serially diluted to concentrations of 500, 200, 100, 50, 10 and 5 pbb were used for standard sample curve establishment.
将标准品进行氨基酸的测定，氨基酸含量的仪器分析采用的UPLC-MS/MS为Shimadzu的UPLC-MS8060，所用的分析柱为ACQUITY UPLC HSS T3(2.1×100mm,1.8μm)；所使用的的流动相由0.1％甲酸-水的A相和0.1％甲酸-乙腈的B相组成。梯度洗脱程序：起始比例为A相100％，B相为0％，持续0.5min；0.5～5min，A相降至40％，B相升至60％；5.1min时恢复至起始梯度平衡系统至8.0min。整个洗脱过程流动相的流速始终为0.2 mL/min；柱温箱温度为40℃；进样体积为5μL；进样针清洗液为甲醇/水(v/v，1:1)。串联质谱的电离模式为正离子模式下电喷雾电离；检测方式为多重反应检测(MRM)；离子源温度为200℃，去溶剂温度为400℃，去溶剂气流速为10L/min，毛细管电压为4.0kV，锥孔电压为30kV，碰撞气为氩气，压力为270kPa。The standard substance was determined for amino acids, the UPLC-MS/MS used for the instrumental analysis of amino acid content was Shimadzu's UPLC-MS8060, and the analytical column used was ACQUITY UPLC HSS T3 (2.1×100mm, 1.8μm); The phases consisted of phase A at 0.1% formic acid-water and phase B at 0.1% formic acid-acetonitrile. Gradient elution program: the initial ratio is 100% for phase A, 0% for phase B, and lasts for 0.5min; 0.5-5min, phase A drops to 40%, phase B rises to 60%; return to the initial gradient at 5.1min Equilibrate the system to 8.0 min. The flow rate of the mobile phase was always 0.2 mL/min throughout the elution process; the temperature of the column oven was 40 °C; the injection volume was 5 μL; the syringe cleaning solution was methanol/water (v/v, 1:1). The ionization mode of the tandem mass spectrometer is electrospray ionization in positive ion mode; the detection method is multiple reaction detection (MRM); the ion source temperature is 200 °C, the desolvation temperature is 400 °C, the desolvation gas flow rate is 10 L/min, and the capillary voltage is 4.0kV, the cone voltage is 30kV, the collision gas is argon, and the pressure is 270kPa.
18种游离氨基酸标准品氨基酸含量分析检测方法中的监测离子对和相应的碰撞能参数详见下表1。The monitoring transitions and the corresponding collision energy parameters in the amino acid content analysis and detection methods of the 18 free amino acid standards are shown in Table 1 below.
通过上述分析方法得到18种游离氨基酸标准品基于HSS T3色谱柱分离总离子流色谱图，如图1(A)所示。图2-图7为18种游离氨基酸标准品基于HSS T3色谱柱分离的提取离子色谱图。The total ion current chromatogram of 18 kinds of free amino acid standards obtained by the above analysis method based on HSS T3 chromatographic column separation is shown in Figure 1(A). Figures 2-7 are extracted ion chromatograms of 18 free amino acid standards separated by HSS T3 column.
将血清样本作为生物样本利用液相色谱-串联质谱定量分析血清样本中游离氨基酸的方法：A method for quantitative analysis of free amino acids in serum samples by liquid chromatography-tandem mass spectrometry using serum samples as biological samples:
步骤1)、血清样本的获得：将从小鼠体内获取的血液样本置于4℃，10,000×g的条件下离心10min，取上层血清备用；移取50μL的血清样品置于1.5mL的离心管中，加入相当于3倍样本体积的甲醇溶液后震荡混匀，静置10～15min沉淀样本中的蛋白质；Step 1) Obtaining serum samples: centrifuge the blood samples obtained from mice at 4°C and 10,000 × g for 10 min, and take the upper serum for use; pipette 50 μL of serum samples and place them in a 1.5 mL centrifuge tube , add methanol solution equivalent to 3 times the sample volume, shake and mix, and let stand for 10 to 15 minutes to precipitate the protein in the sample;
步骤2)、将步骤1)所得的样本置于4℃下12000rpm离心10min，收集上层样本清液在氮吹仪下浓缩至近干；用甲醇/水混合溶剂(v/v，1：1)定容样本体积至0.5～1.0mL，涡旋振荡混匀后用0.22μm醋酸纤维素滤膜对样本进行过滤，4℃储存待测；Step 2), centrifuge the sample obtained in step 1) at 12000rpm for 10min at 4°C, collect the supernatant sample and concentrate it to near dryness under a nitrogen blower; use methanol/water mixed solvent (v/v, 1:1) to determine Adjust the sample volume to 0.5-1.0mL, vortex and mix, filter the sample with a 0.22μm cellulose acetate filter, and store at 4°C for testing;
步骤3)、对步骤2)所得的样本中氨基酸含量的仪器分析采用的UPLC-MS/MS为Shimadzu的UPLC-MS 8060，所用的分析柱为ACQUITY UPLC HSS T3(2.1×100mm,1.8μm)；所使用的的流动相由0.1％甲酸-水的A相和0.1％甲酸-乙腈的B相组成。梯度洗脱程序：起始比例为A相100％，B相为0％，持续0.5min；0.5～5min，A相降至40％，B相升至60％；5.1min 时恢复至起始梯度平衡系统至8.0min。整个洗脱过程流动相的流速始终为0.2mL/min；柱温箱温度为40℃；进样体积为5μL；进样针清洗液为甲醇/水(v/v，1:1)。串联质谱的电离模式为正离子模式下电喷雾电离；检测方式为多重反应检测(MRM)；离子源温度为200℃，去溶剂温度为400℃，去溶剂气流速为10L/min，毛细管电压为4.0kV，锥孔电压为30kV，碰撞气为氩气，压力为270kPa。Step 3), the UPLC-MS/MS used in the instrumental analysis of the amino acid content in the sample obtained in step 2) is Shimadzu's UPLC-MS 8060, and the analytical column used is ACQUITY UPLC HSS T3 (2.1 × 100mm, 1.8 μm); The mobile phases used consisted of 0.1% formic acid-water phase A and 0.1% formic acid-acetonitrile phase B. Gradient elution procedure: the initial ratio is 100% for phase A, 0% for phase B, and lasts for 0.5min; 0.5-5min, phase A drops to 40%, phase B rises to 60%; return to the initial gradient at 5.1min Equilibrate the system to 8.0 min. The flow rate of the mobile phase was always 0.2 mL/min throughout the elution process; the temperature of the column oven was 40 °C; the injection volume was 5 μL; the syringe cleaning solution was methanol/water (v/v, 1:1). The ionization mode of the tandem mass spectrometer is electrospray ionization in positive ion mode; the detection method is multiple reaction detection (MRM); the ion source temperature is 200 °C, the desolvation temperature is 400 °C, the desolvation gas flow rate is 10 L/min, and the capillary voltage is 4.0kV, the cone voltage is 30kV, the collision gas is argon, and the pressure is 270kPa.
通过上述分析方法得到血清样本中18种游离氨基酸基于HSS T3色谱柱分离总离子流色谱图，如图1(B)所示。The total ion chromatogram of 18 free amino acids in serum samples separated by HSS T3 chromatographic column was obtained by the above analysis method, as shown in Figure 1(B).
样本中氨基酸含量分析检测方法中的监测离子对和相应的碰撞能参数详见下表1。The monitoring transitions and the corresponding collision energy parameters in the analysis and detection method of amino acid content in the sample are shown in Table 1 below.
表1.氨基酸监测离子对和碰撞能参数Table 1. Amino acid monitoring transition and collision energy parameters
Figure PCTCN2020135390-appb-000003
Figure PCTCN2020135390-appb-000003
Figure PCTCN2020135390-appb-000004
Figure PCTCN2020135390-appb-000004
按上述表1中UPLC-MS/MS条件对样本中的氨基酸含量进行检测，取不同质量浓度的混合标准品溶液进行UPLCMS/MS测定，以各氨基酸浓度为横坐标，定量离子质谱峰面积为纵坐标绘制标准曲线，用于分析样本中中氨基酸的含量：获得它们的标准曲线，并求出相应的线性回归方程及相关系数。The amino acid content in the sample was detected according to the UPLC-MS/MS conditions in the above table 1, and mixed standard solutions of different mass concentrations were taken for UPLCMS/MS determination. Coordinates draw standard curve for analyzing the content of amino acids in the sample: obtain their standard curve, and obtain the corresponding linear regression equation and correlation coefficient.
图8-图10为18种游离氨基酸的标准曲线，具体如下：Figures 8-10 are the standard curves of 18 free amino acids, as follows:
组氨酸标准曲线：根据定量离子对(如表1所示)，以组氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3A所示)，得到标准曲线函数关系为：Y＝(30077.4)X+(0)，相关系数R 2＝0.9981244。 Histidine standard curve: According to the quantitative ion pair (as shown in Table 1), with the histidine concentration as the abscissa, and the quantitative ion peak area as the ordinate, the standard curve (as shown in Figure 3A), the standard curve function relationship is obtained is: Y=(30077.4)X+(0), and the correlation coefficient R 2 =0.9981244.
丙氨酸标准曲线：根据定量离子对(如表1所示)，以丙氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3B所示)，得到标准曲线函数关系为：Y＝(11003.6)X+(0)，相关系数R 2＝0.9957335。 Alanine standard curve: According to the quantitative ion pair (as shown in Table 1), take the alanine concentration as the abscissa, and the quantitative ion peak area as the ordinate. is: Y=(11003.6)X+(0), and the correlation coefficient R 2 =0.9957335.
天冬酰胺标准曲线：根据定量离子对(如表1所示)，以天冬酰胺浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3C所示)，得到标准曲线函数关系为：Y＝(4647.43)X+(0)，相关系数R 2＝0.9970109。 Asparagine standard curve: According to the quantitative ion pair (as shown in Table 1), with the asparagine concentration as the abscissa, and the quantitative ion peak area as the ordinate, the standard curve (as shown in Figure 3C), the standard curve function relationship is obtained is: Y=(4647.43)X+(0), and the correlation coefficient R 2 =0.9970109.
天冬氨酸标准曲线：根据定量离子对(如表1所示)，以天冬氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3D所示)，得到标准曲线函数关系为：Y＝(10943.0)X+(0)，相关系数R 2＝0.9960386。 Aspartic acid standard curve: According to the quantitative ion pair (as shown in Table 1), take the aspartic acid concentration as the abscissa, and the quantitative ion peak area as the ordinate. The functional relationship is: Y=(10943.0)X+(0), and the correlation coefficient R 2 =0.9960386.
异亮氨酸标准曲线：根据定量离子对(如表1所示)，以异亮氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3E所示)，得到标准曲线函数关系为：Y＝(8714.00)X+(0)，相关系数R 2＝0.9998885。 Isoleucine standard curve: According to the quantitative ion pair (as shown in Table 1), take the isoleucine concentration as the abscissa, and the quantitative ion peak area as the ordinate. The functional relationship is: Y=(8714.00)X+(0), and the correlation coefficient R 2 =0.9998885.
亮氨酸标准曲线：根据定量离子对(如表1所示)，以亮氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3F所示)，得到标准曲线函数关系为：Y＝(43756.6)X+(0)，相关系数R 2＝0.9999542。 Leucine standard curve: According to the quantitative ion pair (as shown in Table 1), take the leucine concentration as the abscissa, and the quantitative ion peak area as the ordinate. is: Y=(43756.6)X+(0), and the correlation coefficient R 2 =0.9999542.
赖氨酸标准曲线：根据定量离子对(如表1所示)，以赖氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3G所示)，得到标准曲线函数关系为：Y＝(30098.6)X+(0)，相关系数R 2＝0.9984927。 Lysine standard curve: According to the quantitative ion pair (as shown in Table 1), take the lysine concentration as the abscissa, and the quantitative ion peak area as the ordinate. is: Y=(30098.6)X+(0), and the correlation coefficient R 2 =0.9984927.
甲硫氨酸标准曲线：根据定量离子对(如表1所示)，以甲硫氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3H所示)，得到标准曲线函数关系为：Y＝(18482.5)X+(0)，相关系数R 2＝0.9995012。 Methionine standard curve: According to the quantitative ion pair (as shown in Table 1), take the methionine concentration as the abscissa, and the quantitative ion peak area as the ordinate. The functional relationship is: Y=(18482.5)X+(0), and the correlation coefficient R 2 =0.9995012.
丝氨酸标准曲线：根据定量离子对(如表1所示)，以丝氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3I所示)，得到标准曲线函数关系为：Y＝(9933.69)X+(0)，相关系数R 2＝0.9975547。 Serine standard curve: According to the quantitative ion pair (as shown in Table 1), with the concentration of serine as the abscissa, and the peak area of the quantitative ion as the ordinate, the standard curve (as shown in Figure 3I), the standard curve function relationship obtained is: Y= (9933.69)X+(0), the correlation coefficient R 2 =0.9975547.
苏氨酸标准曲线：根据定量离子对(如表1所示)，以苏氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3J所示)，得到标准曲线函数关系为：Y＝(16270.7)X+(0)，相关系数R 2＝0.9993402。 Threonine standard curve: According to the quantitative ion pair (as shown in Table 1), take the threonine concentration as the abscissa and the quantitative ion peak area as the ordinate. is: Y=(16270.7)X+(0), and the correlation coefficient R 2 =0.9993402.
缬氨酸标准曲线：根据定量离子对(如表1所示)，以缬氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3K所示)，得到标准曲线函数关系为：Y＝(64463.1)X+(0)，相关系数R 2＝0.9982837。 Valine standard curve: According to the quantitative ion pair (as shown in Table 1), take the concentration of valine as the abscissa, and the peak area of the quantitative ion as the ordinate. is: Y=(64463.1)X+(0), and the correlation coefficient R 2 =0.9982837.
脯氨酸标准曲线：根据定量离子对(如表1所示)，以脯氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3L所示)，得到标准曲线函数关系为：Y＝(71507.9)X+(0)，相关系数R 2＝0.9973854。 Proline standard curve: According to the quantitative ion pair (as shown in Table 1), take the proline concentration as the abscissa, and the quantitative ion peak area as the ordinate. is: Y=(71507.9)X+(0), and the correlation coefficient R 2 =0.9973854.
谷氨酸标准曲线：根据定量离子对(如表1所示)，以谷氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3M所示)，得到标准曲线函数关系为：Y＝(8460.51)X+(0)，相关系数R 2＝0.9999456。 Glutamate standard curve: According to the quantitative ion pair (as shown in Table 1), take the glutamic acid concentration as the abscissa, and the quantitative ion peak area as the ordinate. is: Y=(8460.51)X+(0), and the correlation coefficient R 2 =0.9999456.
谷氨酰胺标准曲线：根据定量离子对(如表1所示)，以谷氨酰胺浓度为 横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3N所示)，得到标准曲线函数关系为：Y＝(11079.7)X+(0)，相关系数R 2＝0.9999321。 Glutamine standard curve: According to the quantitative ion pair (as shown in Table 1), with the glutamine concentration as the abscissa, and the quantitative ion peak area as the ordinate, the standard curve (as shown in Figure 3N), the standard curve function relationship is obtained is: Y=(11079.7)X+(0), and the correlation coefficient R 2 =0.9999321.
色氨酸标准曲线：根据定量离子对(如表1所示)，以色氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3O所示)，得到标准曲线函数关系为：Y＝(15734.1)X+(0)，相关系数R 2＝0.9999879。 Tryptophan standard curve: According to the quantitative ion pair (as shown in Table 1), take the tryptophan concentration as the abscissa, and the quantitative ion peak area as the ordinate. is: Y=(15734.1)X+(0), and the correlation coefficient R 2 =0.9999879.
甘氨酸标准曲线：根据定量离子对(如表1所示)，以甘氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3P所示)，得到标准曲线函数关系为：Y＝(950.571)X+(0)，相关系数R 2＝0.9985857。 Glycine standard curve: According to the quantitative ion pair (as shown in Table 1), with the concentration of glycine as the abscissa, and the peak area of the quantitative ion as the ordinate, the standard curve (as shown in Figure 3P), the standard curve function relationship is: Y= (950.571)X+(0), the correlation coefficient R 2 =0.9985857.
苯丙氨酸标准曲线：根据定量离子对(如表1所示)，以苯丙氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3Q所示)，得到标准曲线函数关系为：Y＝(26210.0)X+(0)，相关系数R 2＝0.9999878。 Phenylalanine standard curve: According to the quantitative ion pair (as shown in Table 1), with the phenylalanine concentration as the abscissa, and the quantitative ion peak area as the ordinate, the standard curve (as shown in Figure 3Q), the standard curve is obtained The functional relationship is: Y=(26210.0)X+(0), and the correlation coefficient R 2 =0.9999878.
酪氨酸标准曲线：根据定量离子对(如表1所示)，以酪氨酸浓度为横坐标，定量离子峰面积为纵坐标回执标准曲线(如图3R所示)，得到标准曲线函数关系为：Y＝(8763.37)X+(0)，相关系数R 2＝0.9999597。 Tyrosine standard curve: According to the quantitative ion pair (as shown in Table 1), take the tyrosine concentration as the abscissa, and the quantitative ion peak area as the ordinate. is: Y=(8763.37)X+(0), and the correlation coefficient R 2 =0.9999597.
结果表明：18种氨基酸在较宽的质量浓度范围内具有良好的线性关系，相关系数R 2均在0.995以上。 The results showed that the 18 amino acids had a good linear relationship in a wide range of mass concentration, and the correlation coefficients R 2 were all above 0.995.
根据18种游离氨基酸的标准曲线计算血清样本中氨基酸的含量，以及标准溶液在信噪比S/N＝3下的检出限和S/N＝10下的定量限：Calculate the content of amino acids in serum samples according to the standard curve of 18 free amino acids, and the detection limit of the standard solution under the signal-to-noise ratio S/N=3 and the quantification limit under S/N=10:
应用表1中的参数方法对血清样本中的游离氨基酸的测定，利用保留时间锁定和特征离子对锁定的方式对定性筛查的生物样品中存在的氨基酸及通过外标法对其含量进行定量，UPLC-MS分析血清样本中氨基酸含量范围及分析方法检出限、定量限如下表2所示：Determination of free amino acids in serum samples by applying the parameter method in Table 1, using retention time locking and characteristic ion pair locking methods to quantify the amino acids present in qualitatively screened biological samples and their content by external standard method, The range of amino acid content in serum samples analyzed by UPLC-MS and the detection limit and quantification limit of the analytical method are shown in Table 2 below:
表2 UPLC-MS分析血清样本中氨基酸含量范围及分析方法检出限、定量限Table 2 Content range of amino acids in serum samples analyzed by UPLC-MS and the detection limit and quantification limit of the analytical method
Figure PCTCN2020135390-appb-000005
Figure PCTCN2020135390-appb-000005
Figure PCTCN2020135390-appb-000006
Figure PCTCN2020135390-appb-000006
其中，血清样本中的氨基酸含量通过将其在仪器上的响应值代入至标准样品构建的标准曲线中计算得到。从表2可以看出实际血清样品中的氨基酸含量，以及仪器对18种氨基酸的检出限和18种氨基酸标准样品曲线的线性范围。Wherein, the amino acid content in the serum sample is calculated by substituting its response value on the instrument into the standard curve constructed by the standard sample. It can be seen from Table 2 that the amino acid content in the actual serum sample, as well as the detection limit of the instrument for 18 amino acids and the linear range of the 18 amino acid standard sample curve.
实施例2Example 2
本实施例和实施例1的检测方法一样，不同的是以脑组织样本作为待检测的生物样品：This embodiment is the same as the detection method in Embodiment 1, except that the brain tissue sample is used as the biological sample to be detected:
脑组织样本的获取是将小鼠脑组织样本置于干净的1.5mL的离心管中，加入500μL的甲醇-水(v/v，1:1)溶液后，置于组织研磨器中4℃，120Hz 下研磨10min。其他检测步骤同实施例1。The brain tissue samples were obtained by placing the mouse brain tissue samples in a clean 1.5 mL centrifuge tube, adding 500 μL of methanol-water (v/v, 1:1) solution, and placing them in a tissue grinder at 4°C. Grind for 10 min at 120 Hz. Other detection steps are the same as in Example 1.
为了使本领域的技术人员更清楚明了地理解本发明，现结合具体实施方式和附图，对本发明进行详细介绍。以下实施例仅是为了对该方法的实际操作进行说明举例，而非在实施方式上进行限制。除下述操作实例外，还可在细节处加以优化或改动，这里无需也无法对所有的实施方式予以穷举。只要是在本发明实施例基础上做出的常识性的改动方案，都处于本发明的保护范围。In order to make those skilled in the art understand the present invention more clearly, the present invention will now be described in detail with reference to the specific embodiments and accompanying drawings. The following examples are only for illustrating the actual operation of the method, rather than limiting the implementation. In addition to the following operational examples, details may be optimized or modified, and all implementations need not and cannot be exhaustive here. As long as it is a common-sense modification scheme made on the basis of the embodiments of the present invention, it falls within the protection scope of the present invention.
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受所述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the described embodiments, and any other changes, modifications, substitutions, and combinations made without departing from the spirit and principle of the present invention , simplification, all should be equivalent replacement modes, and are all included in the protection scope of the present invention.
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention. Thus, provided that these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims (10)

  1. 利用液相色谱-串联质谱定量分析生物样本中游离氨基酸的方法，其特征在于，所述方法包括以下步骤：A method for quantitative analysis of free amino acids in biological samples by liquid chromatography-tandem mass spectrometry, characterized in that the method comprises the following steps:
    步骤1)、将生物样品进行预处理，得到处理后的生物样品；Step 1), pre-processing the biological sample to obtain the processed biological sample;
    步骤2)、将步骤1)中处理后的生物样品置于冷冻离心机中离心，收集上层清液在氮吹仪下浓缩至近干；Step 2), placing the biological sample processed in step 1) in a refrigerated centrifuge for centrifugation, collecting the supernatant and concentrating to near dryness under a nitrogen blower;
    步骤3)、将步骤2)中所得的样品用溶剂混匀后用过滤，4℃储存待测；Step 3), mix the sample obtained in step 2) with a solvent, filter it, and store it at 4°C for testing;
    步骤4)、将步骤3)中所得的生物样品进行超高效液相色谱-串联质谱联用分析，得到生物样品中游离氨基酸的种类和含量。In step 4), the biological sample obtained in step 3) is analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry to obtain the type and content of free amino acids in the biological sample.
  2. 根据权利要求1所述的利用液相色谱-串联质谱定量分析生物样本中游离氨基酸的方法，其特征在于，所述生物样品包括血清样品、血浆样品、脑组织样品、细胞样品。The method for quantitative analysis of free amino acids in biological samples by liquid chromatography-tandem mass spectrometry according to claim 1, wherein the biological samples include serum samples, plasma samples, brain tissue samples, and cell samples.
  3. 根据权利要求2所述的利用液相色谱-串联质谱定量分析生物样本中游离氨基酸的方法，其特征在于，步骤1)中，当生物样品为血清样品或血浆样品时，对血清样品或血浆样品进行预处理的步骤为：将血清样品或血浆样品置于4℃下12000-15000rpm的条件下离心10-20min，取上层血清或血浆备用，将上层血清或血浆样品，加入相当于3-5倍样品体积的甲醇溶液后震荡混匀，静置10～15min沉淀样品中的蛋白质。The method for quantitative analysis of free amino acids in biological samples by liquid chromatography-tandem mass spectrometry according to claim 2, wherein in step 1), when the biological sample is a serum sample or a plasma sample, the serum sample or plasma sample is The steps of pretreatment are: centrifuge the serum or plasma samples at 12000-15000rpm at 4°C for 10-20min, take the upper serum or plasma for later use, and add 3-5 times of the upper serum or plasma to the samples. The methanol solution of the sample volume was shaken and mixed, and the protein in the sample was precipitated by standing for 10-15 min.
  4. 根据权利要求2所述的利用液相色谱-串联质谱定量分析生物样本中游离氨基酸的方法，其特征在于，步骤1)中，当生物样品为脑组织样品时，对脑组织样品进行预处理的步骤为：按照每50mg脑组织样品加入甲醇/水混合溶剂500μL，将脑组织样品加入甲醇/水混合溶剂后，置于组织研磨器中4℃，120Hz下研磨10min，甲醇/水混合溶剂中甲醇和水的体积比为1:1。The method for quantitative analysis of free amino acids in biological samples by liquid chromatography-tandem mass spectrometry according to claim 2, wherein in step 1), when the biological sample is a brain tissue sample, the brain tissue sample is pretreated The steps are: add 500 μL of methanol/water mixed solvent per 50 mg of brain tissue sample, add the brain tissue sample to methanol/water mixed solvent, place it in a tissue grinder at 4 °C, grind at 120 Hz for 10 min, and add methanol in methanol/water mixed solvent for 10 min. The volume ratio to water is 1:1.
  5. 根据权利要求1所述的利用液相色谱-串联质谱定量分析生物样本中游离氨基酸的方法，其特征在于，步骤2)中，冷冻离心的条件为4℃下12000-15000rpm离心10-20min。The method for quantitative analysis of free amino acids in biological samples by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, in step 2), the conditions of refrigerated centrifugation are centrifugation at 12000-15000rpm for 10-20min at 4°C.
  6. 根据权利要求1所述的利用液相色谱-串联质谱定量分析生物样本中游离氨基酸的方法，其特征在于，步骤3)中，当生物样品为血清样品时，按照每50μL的血清样品，加入150-250μL的溶剂混匀；当生物样品脑组织样品时，按照每50mg脑组织样品，加入500μL的溶剂混匀；The method for quantitative analysis of free amino acids in a biological sample by liquid chromatography-tandem mass spectrometry according to claim 1, wherein in step 3), when the biological sample is a serum sample, add 150 μL of serum sample per 50 μL. -250μL of solvent to mix well; for biological sample brain tissue samples, add 500μL of solvent for every 50mg of brain tissue sample and mix well;
    优选的，步骤3)中，采用0.22μm醋酸纤维素滤膜进行过滤；溶剂为甲醇/水混合溶剂，或乙腈/水混合溶剂，甲醇/水混合溶剂中甲醇和水的体积比为1:1，乙腈/水混合溶剂中乙腈和水的体积比为1:1。Preferably, in step 3), a 0.22 μm cellulose acetate filter membrane is used for filtration; the solvent is a methanol/water mixed solvent, or an acetonitrile/water mixed solvent, and the volume ratio of methanol to water in the methanol/water mixed solvent is 1:1 , the volume ratio of acetonitrile and water in the acetonitrile/water mixed solvent is 1:1.
  7. 根据权利要求1所述的利用液相色谱-串联质谱定量分析生物样本中游离氨基酸的方法，其特征在于，超高效液相色谱-串联质谱联用分析所用的分析柱为ACQUITY UPLC HSS T3，规格：2.1×100mm,1.8μm。The method for quantitatively analyzing free amino acids in biological samples using liquid chromatography-tandem mass spectrometry according to claim 1, wherein the analytical column used in the analysis by ultra-high performance liquid chromatography-tandem mass spectrometry is ACQUITY UPLC HSS T3, and the specification : 2.1×100mm, 1.8μm.
  8. 根据权利要求1所述的利用液相色谱-串联质谱定量分析生物样本中游离氨基酸的方法，其特征在于，步骤4)中，超高效液相色谱-串联质谱联用分析时的流动相：A相为0.1％甲酸-水，B相为0.1％甲酸-乙腈；采用的梯度洗脱程序为：起始比例为A相100％，B相为0％，持续0.5min；0.5～5min，A相降至40％，B相升至60％；5.1min时恢复至起始梯度平衡系统至7.0min；整个洗脱过程流动相的流速始终为0.2mL/min；柱温箱温度为40℃；进样体积为5～10μL；进样针清洗液为甲醇/水混合溶剂，甲醇/水混合溶剂中甲醇和水的体积比为1:1。The method for quantitatively analyzing free amino acids in biological samples using liquid chromatography-tandem mass spectrometry according to claim 1, wherein in step 4), the mobile phase in the analysis by ultra-high performance liquid chromatography-tandem mass spectrometry: A The phase is 0.1% formic acid-water, and the phase B is 0.1% formic acid-acetonitrile; the gradient elution program used is: the initial ratio is 100% for phase A, and 0% for phase B, for 0.5min; 0.5-5min, phase A decreased to 40%, phase B increased to 60%; at 5.1 min, the system was restored to the initial gradient equilibrium system to 7.0 min; the flow rate of the mobile phase was always 0.2 mL/min during the entire elution process; the temperature of the column oven was 40 °C; The sample volume is 5-10 μL; the syringe cleaning solution is methanol/water mixed solvent, and the volume ratio of methanol and water in the methanol/water mixed solvent is 1:1.
  9. 根据权利要求1所述的利用液相色谱-串联质谱定量分析生物样本中游离氨基酸的方法，其特征在于，步骤4)中，串联质谱的电离模式为正、负离子模式混合的电喷雾电离；检测方式为多重反应检测(MRM)；离子源温度 为200℃，去溶剂温度为400℃，去溶剂气流速为10L/min，毛细管电压为4.0kV，锥孔电压为30kV，碰撞气为氩气，压力为270kPa，碰撞能为20V。The method for quantitatively analyzing free amino acids in biological samples by liquid chromatography-tandem mass spectrometry according to claim 1, wherein in step 4), the ionization mode of the tandem mass spectrometer is electrospray ionization in which positive and negative ion modes are mixed; The method is multiple reaction detection (MRM); the ion source temperature is 200 °C, the solvent removal temperature is 400 °C, the solvent removal gas flow rate is 10 L/min, the capillary voltage is 4.0 kV, the cone voltage is 30 kV, and the collision gas is argon. The pressure is 270kPa and the collision energy is 20V.
  10. 根据权利要求1所述的利用液相色谱-串联质谱定量分析生物样本中游离氨基酸的方法，其特征在于，液相色谱-串联质谱条件如下：The method for quantitatively analyzing free amino acids in biological samples using liquid chromatography-tandem mass spectrometry according to claim 1, wherein the liquid chromatography-tandem mass spectrometry conditions are as follows:
    Figure PCTCN2020135390-appb-100001
    Figure PCTCN2020135390-appb-100001
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