Normal-pressure mass spectrum determination method of chiral amino acid

Abstract

The invention provides a normal-pressure mass spectrum determination method of chiral amino acid, and belongs to the field of detection. The specific detection method comprises the following steps: selecting central ions and chiral ligands to prepare a dynamic test solution, mixing the dynamic test solution with a to-be-detected sample solution, constructing and setting a normal pressure ionization source, performing mass spectrometric detection, performing data processing and the like. The method is high in analysis speed, high in universality, simple to operate and low in organic reagent consumption. The chiral amino acid is measured by adopting a mass spectrometry kinetics method, and real-time and accurate analysis can be achieved. The kinetic sample solution is introduced into the normal-pressure mass spectrum, so that (simple) sample pretreatment is not needed, and rapid, simple and low-cost operation of chiral detection is realized. The method has wide application prospects in the fields of life medicine analysis, food safety, quality control and the like.

Description

Technical Field

[0001] The invention belongs to the detection field and relates to a normal pressure mass spectrometry method for chiral amino acids. Technical Background

[0002] Amino acids, as important chiral active molecules, play a vital role in life systems. Amino acids with different chirality exhibit different pharmacological and toxicological characteristics. Therefore, the chiral analysis of amino acids has received attention in the fields of life and medical analysis, food safety and quality control. A variety of chiral identification techniques have been developed, including circular dichroism, liquid chromatography, gas chromatography, capillary electrophoresis, nuclear magnetic resonance and chiral sensors. In these techniques, circular dichroism and sensors are usually interfered by the matrix. Chromatographic techniques, especially chromatography with chiral stationary phases, have traditionally been used for high-precision and repeatable chiral identification. However, these chromatographic techniques usually require hours of analysis time and high consumption of organic reagents.

[0003] Mass spectrometry has the advantages of accurate and simultaneous detection of multiple target substances. Kinetic chiral analysis is a method for analyzing based on the rate difference of different chiral isomers or diastereomers in chemical reactions. Combining kinetic methods with mass spectrometry (KM-MS) can realize real-time detection of enantiomers. The current typical KM-MS method is reported in the literature (WATao, D.Zhang, ENNikolaev, and RG Cooks. J.Am.Chem.Soc., 2000, 122, 10598-10609 and WATao and RG Cooks. Angew. Chem. Int. Ed., 2001, 40, 757-760). However, the existing KM-MS method is generally used to identify pure chiral compounds on a typical electrospray ionization source (ESI). Due to the low matrix tolerance of ESI and the chiral ligands that may have endogenous interference, it is still difficult to analyze complex unknown samples.

[0004] Atmospheric pressure mass spectrometry is a mass spectrometry analysis technology performed under atmospheric pressure. It has the advantages of fast test speed, no (simple) sample pretreatment, and can perform in-situ real-time analysis of the target. Combining atmospheric pressure mass spectrometry and kinetic chiral analysis methods is expected to combine the advantages of high matrix tolerance and real-time speed to achieve online detection of enantiomers in complex actual samples. Summary of the invention

[0005] Based on the technical defects of current chiral analysis methods such as long analysis time, high reagent consumption, and low matrix tolerance, the technical purpose of the present invention is to combine the kinetic method with atmospheric pressure mass spectrometry to provide a chiral amino acid enantiomeric excess (ee) determination method with high matrix tolerance and fast analysis speed, which can realize the chiral analysis of amino acids in actual samples such as food and biological samples.

[0006] The specific implementation steps of the method for chiral amino acid configuration analysis provided by the present invention include the following steps:

[0007] Step 1, preparing a kinetic test solution containing a central ion and a chiral ligand; the concentration of the chiral ligand is 1 μM-1 mM; the molar ratio of the central ion to the chiral ligand is 1:1-1:5; the central ion is a transition metal ion;

[0008] The chiral ligand structure is:

[0009]

[0010] Wherein L is unsubstituted naphthalene, anthracene, phenanthrene, pyrene or benzopyrene; or naphthalene, anthracene, phenanthrene, pyrene or benzopyrene substituted with one or more methoxy groups, hydroxyl groups, amino groups or methyl groups; n is an integer of 0-2;

[0011] Step 2, mixing the kinetic test solution in step 1 with the amino acid standard solution and the sample solution to be tested respectively;

[0012] Step 3, loading the mixed solution in step 2 onto the surface of a normal pressure ionization source, wherein the normal pressure ion source is an electrospray mode normal pressure ion source, and the mass spectrometer adopts a tandem mass spectrometer, setting the normal pressure ionization source parameters and the mass spectrum acquisition method, and performing sample detection;

[0013] Step 4, according to the mass spectrometry results of step 3, calculate the "R value", use the test results of amino acid standards with different ee to obtain a linear equation, and calculate the enantiomeric excess in the sample to be tested.

[0014] In one embodiment, the chiral ligand comprises

[0015] The selected chiral ligand is an exogenous compound that will not interfere with the chiral amino acids in the sample. It should have a similar structural size to the amino acid and contain a rigid side chain structure.

[0016] During the ionization process, the central ion (M 2+ ), chiral ligand (ref) and analyte (A) form a ternary complex ion [M 2+ (ref)2A-H] +, and dissociated in the tandem mass spectrometer to produce the target product fragment ion PI1: [M 2+ (ref)AH] + and PI2: [M 2+ (ref)2-H] + .

[0017] In one embodiment, in step 1, the central ion includes Mn 2+ 、Ni 2+ 、Co 2+ , Cu 2+ or Mg 2+ ; The central ion concentration is 1 μM-1 mM; the solvent used in the kinetic test solution includes one or more of methanol, acetonitrile, water, isopropanol or acetone.

[0018] In one embodiment, in step 2, the concentrations of the amino acid standard solution and the sample to be tested are 1 μM-1 mM, and the ee is -1 to 1; three kinetic chiral analysis samples are prepared for each sample; and the solvent of the amino acid standard solution includes one or more of methanol, acetonitrile, water, isopropanol or acetone.

[0019] The amino acids that can be analyzed are alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or a mixture of one or more of histidine.

[0020] In one embodiment, in step 2, the volume ratio of the kinetic test solution and the amino acid standard solution is 5:1 to 1:5, and the total volume after mixing is 1 μL-100 μL;

[0021] The volume ratio of the kinetic test solution to the sample solution to be tested is 5:1 to 1:5, and the total volume after mixing is 1 μL-100 μL.

[0022] In one embodiment, step 3 specifically includes: setting the position parameters and ionization parameters of the atmospheric pressure ion source, setting the mass spectrometry acquisition method, dropping the mixed solution on the surface of the atmospheric pressure ion source, turning on the mass spectrometry acquisition, and recording the mass spectrometry data.

[0023] In one embodiment, in step 3, the electrospray mode atmospheric pressure ion source includes a paper spray ion source, a chip spray ion source, a desorption electrospray ion source, a probe electrospray ion source, or an extraction electrospray ion source;

[0024] The tandem mass spectrometer includes a triple quadrupole mass analyzer, a quadrupole-time of flight mass analyzer, a quadrupole-ion trap mass analyzer or a quadrupole-electrostatic field orbital trap mass analyzer;

[0025] Before sample detection, the ion source position parameters and voltage parameters were optimized and adjusted according to the mass spectrometry signal intensity.

[0026] In one embodiment, in step 3, the ion source heating temperature is 30°C-300°C.

[0027] In one embodiment, in step 3, the mass spectrometry acquisition method includes positive ion mode, negative ion mode, selected ion monitoring mode, and selected reaction monitoring mode;

[0028] The sample volume is 1μL-50μL, which is added dropwise by a pipette or automatically by a syringe pump;

[0029] The detection order is: the standard solution is detected in the order of ee from low to high, and then the sample to be tested is detected.

[0030] Data processing uses the signal peak area intensity of PI1 and PI2 ions in the mass spectrum from the start of spray detection to the disappearance of the signal to calculate the signal peak area ratio R of PI1 and PI2 for quantitative analysis.

[0031] In one embodiment, step 4 is specifically:

[0032] Step 4-1, extracting an ion current chromatogram according to the mass-to-charge ratio of the target product fragments in the obtained mass spectrum, integrating the peak areas, and calculating the R values ​​of the amino acid standard solution and the unknown sample to be tested;

[0033] Step 4-2, plotting a graph with ee as the horizontal coordinate and the lnR value of the amino acid standard solution as the vertical coordinate, performing a linear fit on the data points in the graph to obtain a linear equation;

[0034] Step 4-3, the sample to be tested lnR 样 Substitute into the linear equation and calculate the enantiomeric excess of the chiral amino acid in the sample to be tested.

[0035] Beneficial effects:

[0036] (1) The kinetic method can achieve real-time detection without the need for amino acid derivatization and can rapidly determine the amino acid ee in the sample;

[0037] (2) Compared with chromatography and other methods, it consumes less organic solvents and is environmentally friendly;

[0038] (3) Using atmospheric pressure mass spectrometry detection, the analysis and testing process is easy to operate, the analysis is fast, and the throughput is high. The samples do not require or only require simple pretreatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a diagram of the thermal assisted paper spray ion source (side view and top view)

[0040] Figure 2 This is the secondary mass spectrum of the ternary complex ion of phenylalanine (taking the D configuration as an example)

[0041] Figure 3 is the standard curve of ee of tyrosine and phenylalanine

[0042] Figure 4 It is the total ion current chromatogram of a single sample test process

[0043] Figure 5 This is the secondary mass spectrum of the ternary complex ion of tyrosine (taking the D configuration as an example). DETAILED DESCRIPTION

[0044] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0045] Example 1: Determination of the ee value of phenylalanine in amino acid capsules

[0046] (1) Prepare the kinetic test solution with Cu 2+ as the central ion at a concentration of 600 μM, 3-(2-naphthyl)-L-alanine as the chiral ligand at a concentration of 1200 μM, and the solvent was 50% methanol in water;

[0047] (2) Prepare a series of phenylalanine (Phe) standard solutions, in which the total concentration of L-Phe and D-Phe is 100 μM, the ee is -1.0, -0.9, -0.8, -0.4, 0, 0.4, 0.8, 0.9 and 1.0, respectively, and the solvent is 50% methanol in water. Prepare the sample solution, pour out the contents of the amino acid capsule and grind it appropriately. Weigh the powder and prepare a 200 μM sample solution using pure water as the solvent. Then dilute it with pure methanol in equal times to obtain a solution with a final concentration of 100 μM. Finally, mix 50 μL of the kinetic test solution and 50 μL of the sample solution to be tested in a volume ratio of 1:1 to obtain a kinetic chiral analysis sample.

[0048] (3) Build a thermally assisted paper spray ion source. See the device for details. Figure 1 , install the ion source in front of the entrance of the quadrupole-ion trap mass spectrometer. The selected paper spray paper is in the shape of an isosceles triangle, with the vertex as the spray tip, an angle of 30°, and a base length of 6 mm. Set the height between the paper base tip and the mass spectrometer inlet to 4 mm and the distance to 5 mm, set the heating ring voltage to 7 V (146 ° C), and the spray voltage to 4 kV. Set the mass spectrometer acquisition mode to positive ion mode and selected reaction monitoring mode. The monitored ion pairs are parent ion m / z 657.2 and daughter ions m / z 442.1, 492.1, and the fragmentation energy is 10 V.

[0049] (4) Use a pipette to take 7 μL of Phe standard kinetic chiral analysis samples and capsule kinetic chiral analysis samples with ee of -1.0, -0.9, -0.8, -0.4, 0, 0.4, 0.8, 0.9 and 1.0, and drop them onto the paper spray blank paper sheet at one time, start the mass spectrometry test, and record the mass spectrometry data. Replace the paper spray paper sheet before each sample analysis. The secondary mass spectrum of the ternary complex formed by Phe is shown in Figure 2 .

[0050] (5) Extract the ion current chromatograms of the ion pairs m / z 442.1 and m / z 492.1 in the mass spectrum, integrate the peak areas of the two ion pairs of each sample, and calculate the R values ​​of the Phe standard solution and the amino acid capsule sample with ee of -1.0, -0.9, -0.8, -0.4, 0, 0.4, 0.8, 0.9 and 1.0.

[0051] (6) Plot the ee value as the horizontal axis and the lnR value of the Phe standard solution with different ee values ​​as the vertical axis, and fit the data points to obtain the standard curve. Figure 3 , the linear equations are y=1.08882x+0.38106, and the correlation coefficient is R 2 =0.9996.

[0052] (7) The R value of the capsule sample was substituted into the standard curve, and the ee of Phe in the amino acid capsule was calculated to be -0.99, with an RSD of 1.6%. The authenticity of the sample was confirmed. The test process for a single sample was completed within 30 seconds, and the total ion chromatogram was shown in Figure 4 .

[0053] Example 2: Determination of the ee value of tyrosine in amino acid food additives

[0054] (1) The types, concentrations and solvents of the central ions and chiral ligands used in preparing the kinetic solution are the same as those in Example 1;

[0055] (2) Prepare a series of tyrosine (Tyr) mixed standard solutions, wherein the total concentration of L-Tyr and D-Tyr is 100 μM, the ee is -1.0, -0.9, -0.8, -0.4, 0, 0.4, 0.8, 0.9 and 1.0 respectively, and the solvent is 50% methanol water solution. Prepare sample solutions, prepare food additives into 100 μM sample solutions with 50% methanol water. Mix them evenly according to the same volume ratio as in Example 1.

[0056] (3) The construction of the thermally assisted paper spray ion source was the same as in Example 1. The monitored ion pairs were parent ion m / z 673.2 and daughter ions m / z 458.1 and 492.1, and the fragmentation energy was 10V.

[0057] (4) Use a pipette to take 7 μL of Tyr standard kinetic chiral analysis samples and food additive kinetic chiral analysis samples with ee of -1.0, -0.9, -0.8, -0.4, 0, 0.4, 0.8, 0.9 and 1.0 in turn, and drop them onto the paper spray blank paper at one time, start the mass spectrometry test, and record the mass spectrometry data. Replace the paper spray paper before each sample analysis. The secondary mass spectrum of the ternary complex formed by Tyr is shown in Figure 5 ;

[0058] (5) Extract the ion current chromatograms of the ion pairs m / z 458.1 and m / z 492.1 in the mass spectrum, integrate the peak areas of the two ion pairs for each sample, and calculate the R values ​​of the Tyr standard solution and food additive samples with ee values ​​of -1.0, -0.9, -0.8, -0.4, 0, 0.4, 0.8, 0.9, and 1.0.

[0059] (6) Plot the ee value as the horizontal axis and the lnR value of the Tyr standard solution with different ee as the vertical axis, and fit the data points to obtain the standard curve. Figure 3 , the linear equations are y=1.12863x+0.88027, and the correlation coefficient is R 2 =0.9995.

[0060] (7) The R value of the food additive sample was substituted into the standard curve, and the ee of Tyr in the food additive was calculated to be -0.99, with an RSD of 1.2%, and the authenticity of the sample was confirmed.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for determining chiral amino acids by atmospheric pressure mass spectrometry, characterized in that: The method comprises the following steps: Step 1, preparing a kinetic test solution containing a central ion and a chiral ligand; the concentration of the chiral ligand is 1 μM-1 mM; the molar ratio of the central ion to the chiral ligand is 1:1-1:5; the central ion is a transition metal ion; The chiral ligand structure is Wherein L is unsubstituted naphthalene, anthracene, phenanthrene, pyrene or benzopyrene; or naphthalene, anthracene, phenanthrene, pyrene or benzopyrene substituted with one or more methoxy groups, hydroxyl groups, amino groups or methyl groups; n is an integer of 0-2; Step 2, mixing the kinetic test solution in step 1 with the amino acid standard solution and the sample solution to be tested respectively; Step 3, loading the mixed solution in step 2 onto the surface of a normal-pressure ionization source, wherein the normal-pressure ion source is an electrospray mode normal-pressure ion source, and the mass spectrometer adopts a tandem mass spectrometer, setting the normal-pressure ionization source parameters and the mass spectrum acquisition method to perform sample detection; Step 4, according to the mass spectrometry results of step 3, calculate the "R value", use the test results of amino acid standards with different ee to obtain a linear equation, and calculate the enantiomeric excess in the sample to be tested.

2. A method for determining chiral amino acids by mass spectrometry at normal pressure as described in claim 1, characterized in that: The chiral ligand comprises 3. A method for determining chiral amino acids by mass spectrometry at normal pressure as described in claim 1, characterized in that: In step 1, the central ion includes Mn 2+ 、Ni 2+ 、Co 2+ , Cu 2+ or Mg 2+ ; The central ion concentration is 1 μM-1 mM; the solvent used in the kinetic test solution includes one or more of methanol, acetonitrile, water, isopropanol or acetone.

4. A method for determining chiral amino acids by atmospheric pressure mass spectrometry according to claim 1, characterized in that: In step 2, the concentrations of the amino acid standard solution and the sample to be tested are 1 μM-1 mM, and the ee is -1 to 1; three kinetic chiral analysis samples are prepared for each sample; the solvent of the amino acid standard solution includes one or more of methanol, acetonitrile, water, isopropanol or acetone.

5. A method for determining chiral amino acids by mass spectrometry at normal pressure as described in claim 1, characterized in that: In step 2, the volume ratio of the kinetic test solution and the amino acid standard solution is 5:1 to 1:5, and the total volume after mixing is 1 μL-100 μL; The volume ratio of the kinetic test solution to the sample solution to be tested is 5:1 to 1:5, and the total volume after mixing is 1 μL-100 μL.

6. A method for determining chiral amino acids by mass spectrometry at normal pressure as described in claim 1, characterized in that: Step 3 is specifically as follows: setting the position parameters and ionization parameters of the atmospheric pressure ion source, setting the mass spectrometry acquisition method, dropping the mixed solution onto the surface of the atmospheric pressure ion source, turning on the mass spectrometry acquisition, and recording the mass spectrometry data.

7. A method for determining chiral amino acids by mass spectrometry at normal pressure as described in claim 1, characterized in that: In step 3, the electrospray mode atmospheric pressure ion source includes a paper spray ion source, a chip spray ion source, a desorption electrospray ion source, a probe electrospray ion source or an extraction electrospray ion source; The tandem mass spectrometer includes a triple quadrupole mass analyzer, a quadrupole-time of flight mass analyzer, a quadrupole-ion trap mass analyzer or a quadrupole-electrostatic field orbital trap mass analyzer; Before sample detection, the ion source position parameters and voltage parameters were optimized and adjusted according to the mass spectrometry signal intensity.

8. The method for determining chiral amino acids by mass spectrometry at normal pressure according to claim 1, characterized in that: In step 3, the ion source is heated to a temperature of 30°C-300°C.

9. The method for determining chiral amino acids by mass spectrometry at normal pressure according to claim 1, characterized in that: In step 3, the mass spectrometry acquisition method includes positive ion mode, negative ion mode, selected ion monitoring mode, and selected reaction monitoring mode; The sample volume is 1μL-50μL, which is added dropwise by a pipette or automatically by a syringe pump; The detection order is: the standard solution is detected in the order of ee from low to high, and then the sample to be tested is detected.

10. A method for determining chiral amino acids by atmospheric pressure mass spectrometry according to claim 1, characterized in that: Step 4 is as follows: Step 4-1, extracting an ion current chromatogram according to the mass-to-charge ratio of the target product fragments in the obtained mass spectrum, integrating the peak areas, and calculating the R values ​​of the amino acid standard solution and the unknown sample to be tested; Step 4-2, plotting a graph with ee as the horizontal coordinate and the lnR value of the amino acid standard solution as the vertical coordinate, performing a linear fit on the data points in the graph to obtain a linear equation; Step 4-3, the sample to be tested lnR 样 Substitute the linear equation into the equation and calculate the enantiomeric excess of the chiral amino acid in the sample to be tested.

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