WO2022120752A1 - Méthode d'analyse quantitative d'acides aminés libres en échantillon biologique par chromatographie liquide/spectrométrie de masse en tandem - Google Patents

Méthode d'analyse quantitative d'acides aminés libres en échantillon biologique par chromatographie liquide/spectrométrie de masse en tandem 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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PCT/CN2020/135390
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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

Méthode d'analyse quantitative d'acides aminés libres dans un échantillon biologique par chromatographie liquide/spectrométrie de masse en tandem. La méthode comprend les étapes consistant : 1) à prétraiter un échantillon biologique pour obtenir un échantillon biologique traité ; 2) à mettre en place l'échantillon biologique traité à l'étape 1) dans une centrifugeuse de congélation pour centrifugation, à collecter un surnageant et à concentrer le surnageant à un état presque sec sous un instrument de soufflage d'azote ; 3) à mélanger de manière homogène l'échantillon obtenu à l'étape 2) avec un solvant puis à filtrer et à stocker l'échantillon à 4 °C pour un test ultérieur ; et 4) à effectuer une analyse par chromatographie liquide à très hautes performances/spectrométrie de masse en tandem sur l'échantillon biologique obtenu à l'étape 3) pour obtenir les types et les teneurs en acides aminés libres dans l'échantillon biologique. Selon la méthode, les teneurs en acides aminés dans différents types d'échantillons biologiques tels que le sérum, le plasma et le tissu cérébral peuvent être analysées, la dérivation est inutile sur des analytes d'échantillons biologiques à tester et la méthode de prétraitement est simple.
PCT/CN2020/135390 2020-12-10 2020-12-10 Méthode d'analyse quantitative d'acides aminés libres en échantillon biologique par chromatographie liquide/spectrométrie de masse en tandem WO2022120752A1 (fr)

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CN115201360B (zh) * 2022-07-04 2024-03-26 南京市公安局刑事侦查局 血、尿中氟乙酸的离子色谱串联三重四极杆质谱检测方法
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CN115201367B (zh) * 2022-07-11 2024-01-26 中国计量科学研究院 一种污染源中含硫有机化合物的检测方法
CN115201367A (zh) * 2022-07-11 2022-10-18 中国计量科学研究院 一种污染源中含硫有机化合物的检测方法
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