Method for separating amino acid L / D type enantiomer based on LC-MS (liquid chromatography-mass spectrometry) and quantitative detection method

Abstract

The invention discloses an amino acid L / D type enantiomer separation and quantitative detection method based on LC-MS, and belongs to the technical field of biological analysis. Aiming at the problems of difficulty in separation of amino acid L / D type enantiomers in human serum, low detection sensitivity, complex analysis process and the like, the method disclosed by the invention is simple in pretreatment, short in instrument collection time, high in chiral enantiomer separation degree, small in matrix effect, high in sensitivity, low in cost and convenient for large-scale clinical sample detection in a laboratory; the method not only can make up the limitation that only the L type is detected at present, but also can provide a key analysis method for early warning of diseases, drug effect and toxicokinetics research and precise medical evaluation.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical technology, specifically relating to a method for separating and quantitatively detecting L / D enantiomers of amino acids based on LC-MS. Background Technology

[0002] Most natural amino acids have a chiral center on the α-carbon, and the same amino acid can be divided into two enantiomers: L-type and D-type. Although L / D enantiomers are chemically identical, they may exhibit significant differences in recognition, transport, metabolism, and biological effects in vivo. Therefore, distinguishing and quantifying the L / D enantiomers is more sensitive in capturing abnormalities in the amino acid metabolic network than simply measuring their total amounts, and has potential value for early disease warning and stratified assessment.

[0003] Current conventional methods for amino acid detection mostly focus on the detection of L-form or achiral "total" amino acids. The few studies on D-form detection also primarily target individual amino acids, lacking standardized, accurate, and rapid methods for the simultaneous separation and quantification of L / D amino acids within the same analytical process. This results in two main problems: firstly, it is difficult to accurately assess the true abundance and biological significance of D-amino acids; secondly, it is difficult to utilize the L / D ratio or enantiomeric excess (ee) as a more sensitive indicator of metabolic pathway changes. Serum, due to its convenient collection, high representativeness, and repeatable follow-up, has become an important matrix for amino acid profiling in clinical and translational research. However, the content of D-form amino acids in serum is usually much lower than their corresponding L-form forms. Furthermore, to accurately reflect metabolic changes and simultaneously cover the L / D enantiomers of various common amino acids in serum analysis within a complex matrix, the following key challenges are still commonly encountered: 1. Insufficient synergy between separation and detection: Many methods only achieve one of the two: enantiomer separation or quantitative detection. It is difficult to achieve both baseline separation and high-sensitivity mass spectrometry response within a reasonable gradient time, which limits high-throughput clinical applications. 2. Difficulty in resolving enantiomers: The L-type and D-type amino acids differ only in mirror image structure. They are highly polar and structurally similar. Chiral stationary phases or derivatization systems require strict baseline separation for multiple targets simultaneously, which can easily lead to peak overlap and inaccurate quantitative results. 3. Challenges in quantifying low-abundance D-type amino acids: D-configurations are mostly at trace levels and coexist with L-type amino acids. Insufficient method sensitivity can easily lead to peak overlap and crosstalk between L / D enantiomers, raising the lower limit of quantification. 4. Handling the risk of induced racemization: High temperature, strong alkali or metal ion pretreatment conditions may cause the sample to racemize, weakening the authenticity of enantiomeric information; 5. Limited coverage and transferability: Existing studies often focus on L-type or a few D-type amino acids, making it difficult to stably cover multiple amino acids under uniform conditions. The reproducibility and standardization of detection also need to be improved. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for separating and quantitatively detecting L / D enantiomers of amino acids based on LC-MS, so as to solve the technical problems of inaccurate quantification of low-abundance D-type amino acids and difficulty in separating chiral enantiomers in existing methods.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: to provide a method for separating and quantitatively detecting L / D enantiomers of amino acids based on LC-MS, comprising the following steps: S1. Mix the sample to be tested with a methanol / acetonitrile solution containing formic acid at a volume ratio of 0.5-1.5:5-7, then sonicate for 10-20 min, then precipitate at -25~-15℃ for 0.5-1.5 h, and finally centrifuge at 3-5℃ for 8-12 min to obtain the supernatant. S2. The supernatant was analyzed by mass spectrometry using high performance liquid chromatography, and the composition and content of chiral amino acids in the sample were obtained according to the standard curve of amino acids.

[0006] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, the concentration of formic acid in the methanol / acetonitrile solution is 0.05-0.15 wt%.

[0007] Furthermore, the centrifugation speed is 9000-11000 r / min.

[0008] Furthermore, the liquid chromatography conditions were as follows: column temperature 20-30℃, flow rate 0.1-0.2mL / min, pressure 200-400bar, and injection volume 3-7μL.

[0009] Furthermore, the chiral selector used in liquid chromatography is a chiral crown ether.

[0010] Furthermore, mass spectrometry employed multiple reaction monitoring (MRM) analysis under the following conditions: electrospray ionization source, carrier gas N2, ionization mode positive ion mode, mass spectrometry scanning mode MRM, dryer temperature 280-320℃, dryer flow rate 8-12 L / min, nebulizer pressure 30-40 psi, capillary voltage 3500-4500 V, sheath gas temperature 200-300℃, sheath gas flow rate 10-13 L / min, and nozzle voltage 1200-1700 V.

[0011] Furthermore, the chiral amino acids are phenylalanine, alanine, theanine, methionine, citrulline, glutamic acid, glutamine, lysine, tyrosine, tryptophan, serine, threonine, aspartic acid, asparagine, or valine.

[0012] The beneficial effects of this invention are as follows: 1. Broad Amino Acid Coverage: The method of this invention can successfully achieve efficient simultaneous separation and detection of L / D enantiomers of 15 amino acids in serum without any derivatization steps, including phenylalanine, alanine, theanine, methionine, citrulline, glutamic acid, glutamine, lysine, tyrosine, tryptophan, serine, threonine, aspartic acid, asparagine, and valine. Compared with traditional methods for determining single amino acids or total amounts, the method of this invention can comprehensively cover the L and D enantiomers of these amino acids, supporting more accurate metabolic data analysis.

[0013] 2. High sensitivity and low detection limit: The detection limit of the method of this invention can reach the ng / L level, which can effectively detect low abundance D-amino acids in serum, avoiding the problem of inaccurate quantification of low abundance D-amino acids in existing methods, and has significantly better sensitivity than traditional methods.

[0014] 3. Highly efficient and stable separation and quantitative detection of L / D enantiomers: Through simple and efficient isocratic elution, the L / D enantiomers of 15 amino acids are ensured to achieve efficient separation and stable peak elution in a short time, without the need for complex operations such as derivatization and multiple analyses, which significantly improves the analytical throughput and detection efficiency of D-type amino acid detection in complex serum samples.

[0015] 4. Simple pretreatment operation: Compared with traditional methods, serum samples can be purified and amino acids extracted through short-time ultrasonic extraction and centrifugation, avoiding the influence of protein and other matrix effects on sample detection.

[0016] 5. Good Recovery Rate: The spiked recovery rate of this method is good and stable. Introducing 0.1% formic acid into the methanol / acetonitrile extraction system, by controlling the acidic environment during the extraction process, can achieve efficient protein precipitation, stable enantiomeric configuration, and improved detection sensitivity for low-abundance D-amino acids. This ensures high accuracy and small error in the detection results of L / D enantiomers.

[0017] 6. Wide applicability and strong transferability: The method of this invention is not only applicable to the detection of serum samples, but also has strong transferability, and can be widely applied to the analysis of other biological samples such as plasma, urine, and tissues. The detection equipment is compatible with conventional laboratory equipment, and has good accessibility and economic feasibility.

[0018] 7. Applicable to clinical testing: Compared with methods that only detect the total amount of amino acids or a single configuration, this invention can more sensitively capture abnormal changes in the amino acid metabolic network by simultaneously detecting L / D enantiomers, providing more accurate physiological and pathological information. It is helpful for early disease warning, drug metabolism research and the development of biomarkers, and has good practical value and promotion prospects. Attached Figure Description

[0019] Figure 1 Total ion current (TIC) plot for 15 chiral amino acids; Figure 2 This is a diagram of multiple reaction monitoring (MRM) patterns for 15 chiral amino acids. Figure 3 The optimized collision energy diagram for aspartate ion pairs; Figure 4 The optimized collision energy diagram for glutamine ion pairs; Figure 5 Optimized collision energy diagram for theanine ion pairs; Figure 6 Optimized collision energy diagram for tryptophan ion pairs; Figure 7 Optimized collision energy diagram for methionine ion pairs; Figure 8 Optimized collision energy diagram for tyrosine ion pairs; Figure 9 A comparison chart showing the separation effect of phenylalanine; Figure 10 A comparison chart showing the separation effect of alanine; Figure 11 A comparison chart showing the separation effect of methionine; Figure 12 A comparison chart showing the separation effect of glutamic acid; Figure 13 A comparison chart showing the separation effect of glutamine; Figure 14 A comparison chart showing the separation effect of tyrosine; Figure 15 A comparison chart showing the separation effect of serine; Figure 16 A comparison of the separation effects of threonine; Figure 17 A comparison chart showing the separation effect of valine; Figure 18 This is a graph showing the detection of D-alanine in serum samples. Figure 19 This is a graph showing the detection of D-aspartic acid in serum samples. Figure 20 The image shows the detection of D-citrulline in serum samples. Figure 21 This is a graph showing the detection of D-tyrosine in serum samples. Figure 22 The image shows the detection of D-serine in serum samples. Figure 23 This is a graph showing the detection of D-threonine in serum samples. Figure 24 This is a graph showing the detection of D-tryptophan in serum samples. Detailed Implementation

[0020] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0021] Liquid chromatography-triple quadrupole mass spectrometry system (1290 Infinity II-G6475A, Agilent Technologies, USA); low-temperature high-speed centrifuge (MicroCL 21R, Thermo Fisher Scientific); vortex mixer (UMV-2, Yusheng); CNC ultrasonic cleaner (KQ-500DE, Kunshan Shumei); chromatographic column (CROWNPAK® CR-I+, 3×150mm; 5μm, Daicel Chemical Industry Co., Ltd., Tokyo, Japan); acetonitrile (HPLC, Aladdin); formic acid (HPLC, Aladdin); methanol (HPLC, Aladdin); 1.5mL polypropylene centrifuge tubes (White Shark).

[0022] L-type amino acid compound standards (100mg, Alta): including L-phenylalanine, L-alanine, L-theanine, L-methionine, L-citrulline, L-glutamic acid, L-glutamine, L-lysine, L-tyrosine, L-tryptophan, L-serine, L-threonine, L-aspartic acid, L-asparagine, and L-valine; D-type amino acid compound standards (100mg, Alta): including D-phenylalanine, D-alanine, D-theanine, D-methionine, D-citrulline, D-glutamic acid, D-glutamine, D-lysine, D-tyrosine, D-tryptophan, D-serine, D-threonine, D-aspartic acid, D-asparagine, and D-valine.

[0023] Example 1: Determination of L / D type amino acid standard curve A working standard curve containing 15 L / D amino acids was prepared using pure water: 30 amino acids were accurately weighed and quantitatively dissolved in ultrapure water to prepare standard stock solutions of 125 μmol / mL. These stock solutions were then serially diluted with ultrapure water to 0.0625 μmol / L, 0.125 μmol / L, 0.25 μmol / L, 0.625 μmol / L, 1.25 μmol / L, 2.5 μmol / L, 6.25 μmol / L, 12.5 μmol / L, and 25 μmol / L, yielding mixed standard solutions of chiral amino acids at different dilution concentrations. These solutions were then detected using liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS / MS) under the following conditions: Liquid chromatography was performed using an Agilent 1290 Infinity II system, and mass spectrometry was performed using an Agilent G6475A electrospray triple quadrupole mass spectrometer. The chromatographic column used was a Daicel crown ether chiral column (CROWNPAK® CR-I+), 150 mm in length, 3 mm in inner diameter, and with 5 μm particle diameter packing material. Elution was performed isocratically with 5 wt% formic acid in acetonitrile / ethanol / water (80:15:5). The column temperature was 25 °C, the flow rate was 0.15 mL / min, the upper limit of pressure was 300 bar, and the injection volume was 5 μL.

[0024] The tandem mass spectrometry (MS / MS) conditions were as follows: positive ion mode, ESI electrospray ionization source, and the ion source parameters were: drying gas temperature 300℃, drying gas flow rate 10 L / min, nebulizing gas pressure 35 psi, capillary voltage 4000 V, sheath gas temperature 250℃, sheath gas flow rate 11 L / min, and nozzle voltage 1500 V. The instrument acquisition parameters for liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS / MS) are shown in Table 1.

[0025] Table 1.15 Instrument data for chiral amino acids

[0026] Based on the results in Table 1, the following was established: Figure 1 The total ion current chromatogram (TIC) of the 15 chiral amino acids shown is as follows: Figure 2 The multiple reaction monitoring (MRM) chromatogram shown indicates that, based on the properties of the CROWNPAK CR-I(+) chiral column, the first chromatographic peak on the left side of the MRM chromatogram represents the D-enantiomer of the amino acid, and the second chromatographic peak represents the L-enantiomer. The collision energies of novel ion pairs for six amino acids (tryptophan, theanine, aspartic acid, glutamine, methionine, and tyrosine) were optimized to improve the response of amino acid enantiomers in mass spectrometry. The optimized ion pairs for these six amino acids exhibited good response performance. Figures 3-8As shown, these new ion pairs, at a concentration of 125 μmol / L, achieved an optimized mass spectrometry response intensity of up to 1.4 × 10⁻⁶. 5 This invention ensures highly sensitive detection and accurate quantification of low-abundance D-type amino acids in serum, especially in complex matrices. The peak overlap and low response issues that may exist in existing traditional methods are effectively improved in this invention. Through optimized collision energy, this invention enables efficient separation and quantification of L / D enantiomers of 15 amino acids in the same analytical procedure, providing strong support for high-throughput detection and clinical applications.

[0027] Fifteen chiral amino acids were quantified based on standard curves fitted at nine concentration levels for each chiral amino acid. The linearity results of the L and D enantiomers of the 15 chiral amino acids are shown in Table 2.

[0028] Table 2.15 Linearity results of the standard curve for chiral amino acids

[0029] Table 2 shows that the L and D enantiomers of the 15 chiral amino acids exhibited good linearity in the concentration range of 0.0625-25 μmol / L, with a correlation coefficient (R0). 2 The value is generally greater than 0.99, which can be used to calculate the content of L and D enantiomers of 15 chiral amino acids in actual sample detection.

[0030] Example 2 Spiked Recovery Test To verify the separation and detection efficiency of the L and D enantiomers of chiral amino acids in this invention, a spiked recovery experimental group was set up for recovery testing. The spiked recovery test group consisted of 15 chiral amino acid mixed standards added to standard fetal bovine serum at a spike concentration of 10 μmol / L. Each test group contained three parallel samples, and a blank control group without spikes was also included. The formula for calculating the spiked recovery rate is as follows: ; Where a is the spiked recovery rate (%), c0 is the spiked concentration of the blank control group (μmol / L), c1 is the spiked concentration of the test group (μmol / L), and c2 is the actual spiked concentration (μmol / L).

[0031] The specific detection steps are as follows: Add 300 μL of a methanol / acetonitrile (1:1; v / v) solution containing 0.1 wt% formic acid to 50 μL of spiked fetal bovine serum / standard fetal bovine serum sample in a 1.5 mL polypropylene centrifuge tube; vortex for 1 min to mix and then sonicate for 15 min to extract amino acids; incubate the extracted sample at -20℃ for 1 h to precipitate proteins, and then centrifuge at 4℃ and 10000 r / min for 10 min; transfer the obtained supernatant to a high-performance liquid chromatography (HPLC) sample vial for analysis, and set the detection conditions of a liquid chromatography-tandem triple quadrupole mass spectrometer (LC-MS / MS) for sample detection. The detection method disclosed in Example 1 was used, and the limits of quantitation and spiked recovery results are shown in Table 3.

[0032] Table 3.15 Spiking recoveries of chiral amino acids

[0033] As shown in Table 3, after removing the background values ​​of chiral amino acids in the blank control, the spiked recoveries at a spiking concentration of 10 μmol / L were between 71% and 120%, with a relative deviation of less than 20%, which meets the generally accepted good recovery standard (70-120%). This confirms that the method of the present invention can accurately achieve the simultaneous extraction, separation, and qualitative and quantitative detection of L and D enantiomers of 15 chiral amino acids.

[0034] Example 3: Comparison of experimental results between traditional detection methods and the method of the present invention for serum samples. Using the method of Example 1, 15 chiral amino acid standards were analyzed under the same chromatographic instrument conditions. The method of this invention was compared with existing techniques, with the chromatographic method disclosed in the literature Sugimoto H, Kakehi M, and Jinno F. Bioanalytical method for the simultaneous determination of D- and L-serine in human plasma[J]. Analytical Biochemistry, 2015, 487: 38–44 serving as a control. The specific chromatographic conditions of this existing method were: a mobile phase of 10% acetonitrile aqueous solution containing 0.3% (v / v) trifluoroacetic acid, a flow rate of 0.15 mL / min, and isocratic elution mode for separation.

[0035] The chromatographic separation results obtained by the two methods under the same detection conditions are as follows: Figures 9-17 As shown, the blue spectrum represents the separation result obtained using existing methods, and the red spectrum represents the separation result obtained using the method of this invention. Figures 9-17It is evident that, using existing techniques, the enantiomers of various chiral amino acids exhibit broad peak shapes, with some compounds showing peak overlap, insufficient resolution, or incomplete baseline separation. This is particularly evident in the short retention time region, where chiral recognition is limited, making effective separation and accurate quantification of each enantiomer difficult. In contrast, the method of this invention demonstrates significantly improved chromatographic behavior for many chiral amino acids, with sharp, symmetrical enantiomer peaks, reasonable retention time distribution, and baseline separation or near-baseline separation achieved for the L / D enantiomers of most chiral amino acids, resulting in a significantly improved overall resolution. Furthermore, the method of this invention exhibits stable responses and good reproducibility for each amino acid, which is beneficial for subsequent qualitative and high-throughput quantitative analysis.

[0036] In summary, a direct comparison with existing technologies confirms that the method of this invention is significantly superior to existing technologies in terms of separation efficiency, resolution, and overall chromatographic performance of chiral amino acids. It is particularly suitable for the simultaneous high-throughput detection of multiple chiral amino acids and has greater practical value and promotional significance.

[0037] Example 4: Detection of chiral amino acids in actual human serum samples To further verify the applicability and stability of the method of the present invention in real biological samples, the LC-MS / MS detection method of Example 1 was used to detect and analyze chiral amino acids in 50 actual human serum samples. All serum samples were processed according to the pretreatment procedure established in this invention, and detection and quantification were performed under the same chromatographic and mass spectrometric conditions.

[0038] Based on the established amino acid standard curve, the L-type and D-type enantiomers of detectable amino acids in human serum samples were quantitatively calculated, and their detection in 50 human serum samples is shown in Table 4.

[0039] Table 4. Detection of chiral amino acids in 50 human serum samples

[0040] The results show that the method of this invention can stably detect multiple D-type amino acids in complex serum matrices, with low detection limits and high detection rates. Table 4 shows that D-alanine, D-citrulline, D-tyrosine, D-tryptophan, D-serine, D-threonine, and D-aspartic acid can be detected in all or most human serum samples, with a detection rate of 100%, indicating that these D-type amino acids have a stable baseline level in human serum. Among them, the average concentration of D-alanine was 3.2106±2.4083 μmol / L, significantly higher than other D-type amino acids; followed by D-serine, D-aspartic acid, and D-threonine, with average concentrations of 1.9786±0.6728 μmol / L, 1.6163±0.5412 μmol / L, and 1.2760±0.2353 μmol / L, respectively; while the concentrations of D-tyrosine, D-citrulline, and D-tryptophan were relatively lower. The concentrations were low, at 0.4716±0.1141 μmol / L, 0.3335±0.0815 μmol / L, and 0.1812±0.1008 μmol / L, respectively, indicating that these amino acids are at even lower abundance levels in human serum. However, the method of this invention can still achieve reliable detection under low detection limits (e.g., the detection limit for tryptophan is 0.0027 μmol / L), demonstrating the high sensitivity of the method of this invention in the analysis of trace D-type amino acids.

[0041] Compared to their corresponding D-forms, the L-enantiomers of each amino acid were present in significantly higher concentrations in human serum, and were detected in 100% of the 50 samples. For example, the average concentrations of L-alanine, L-serine, and L-threonine were 366.1017±75.8134 μmol / L, 179.6505±29.1570 μmol / L, and 152.3915±33.5804 μmol / L, respectively, which were significantly higher than their D-enantiomers, consistent with the physiological characteristic that the vast majority of amino acids in the human body are predominantly L-forms.

[0042] Figures 18-24 This study presents the MRM detection results of some representative D-type amino acids in actual human serum samples, including D-alanine, D-aspartic acid, D-citrulline, D-tyrosine, D-serine, D-threonine, and D-tryptophan. Figures 18-24 As can be seen, each D-type amino acid exhibited good peak shape and clear chromatographic response in the serum matrix, achieving effective separation from its corresponding L-type enantiomer without obvious interference peaks or baseline drift, further demonstrating the good separation ability and quantitative reliability of the method of the present invention for D-type amino acids in actual human samples.

[0043] In summary, the LC-MS / MS-based detection method established in this invention enables the stable and accurate detection of multiple low-abundance D-amino acids in complex serum matrices without derivatization, and achieves efficient separation and accurate quantification of these amino acids with their corresponding L-enantiomers. Compared to methods that only detect total amino acid levels or single configurations, this method provides more refined enantiomer-level information, offering an important technical means for reflecting human amino acid metabolism status based on changes in D-amino acid levels and L / D ratios, screening for potential metabolic abnormalities, and conducting auxiliary assessments and research on related diseases. Furthermore, the method is simple to operate and convenient for large-scale clinical sample testing in laboratories.

Claims

1. A method for separating and quantitatively detecting L / D enantiomers of amino acids based on LC-MS, characterized in that, Includes the following steps: S1. Mix the sample to be tested with a methanol / acetonitrile solution containing formic acid at a volume ratio of 0.5-1.5:5-7, then sonicate for 10-20 min, then precipitate at -25~-15℃ for 0.5-1.5 h, and finally centrifuge at 3-5℃ for 8-12 min to obtain the supernatant. S2. The supernatant was analyzed by mass spectrometry using high performance liquid chromatography, and the composition and content of chiral amino acids in the sample were obtained according to the standard curve of amino acids.

2. In the method for separating and quantitatively detecting L / D enantiomers of amino acids based on LC-MS according to claim 1, the concentration of formic acid in the methanol / acetonitrile solution is 0.05-0.15 wt%.

3. The method for separating and quantitatively detecting L / D enantiomers of amino acids based on LC-MS according to claim 1, wherein the centrifugation speed is 9000-11000 r / min.

4. The method for separating and quantitatively detecting L / D enantiomers of amino acids based on LC-MS according to claim 1, characterized in that, The liquid chromatography conditions were as follows: column temperature 20-30℃, flow rate 0.1-0.2 mL / min, pressure 200-400 bar, and injection volume 3-7 μL.

5. The method for separating and quantitatively detecting L / D enantiomers of amino acids based on LC-MS according to claim 1, characterized in that, The chiral selector used in liquid chromatography is a chiral crown ether.

6. The method for separating and quantitatively detecting L / D enantiomers of amino acids based on LC-MS according to claim 1, characterized in that, Mass spectrometry was performed using multiple reaction monitoring (MRM). The analytical conditions were as follows: electrospray ionization source, carrier gas N2, ionization mode positive ion mode, mass spectrometry scanning mode multiple reaction monitoring, dryer temperature 280-320℃, dryer flow rate 8-12 L / min, nebulizer pressure 30-40 psi, capillary voltage 3500-4500 V, sheath gas temperature 200-300℃, sheath gas flow rate 10-13 L / min, and nozzle voltage 1200-1700 V.

7. The method for separating and quantitatively detecting L / D enantiomers of amino acids based on LC-MS according to claim 1, characterized in that, The chiral amino acid is phenylalanine, alanine, theanine, methionine, citrulline, glutamic acid, glutamine, lysine, tyrosine, tryptophan, serine, threonine, aspartic acid, asparagine, or valine.

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