Detection method for simultaneously detecting glutamic acid, citric acid and beta-citryl glutamic acid

Abstract

The invention relates to the technical field of chromatographic detection, in particular to a detection method for simultaneously detecting glutamic acid, citric acid and beta-citrylglutamic acid, and the detection method comprises the following steps: adding an internal standard solution into plasma to be detected, mixing, and carrying out pretreatment to obtain a solution of a product to be detected; carrying out series detection on the solution of the product to be detected by adopting an ultra-high performance liquid chromatograph-triple quadrupole tandem mass spectrometer to obtain the contents of glutamic acid, citric acid and beta-citryl glutamic acid in the solution of the product to be detected, wherein the internal standard solution comprises isotope markers of glutamic acid, citric acid and beta-citryl glutamic acid. According to the detection method, glutamic acid, citric acid and beta-citryl glutamic acid can be accurately measured at the same time, the detection efficiency is high, and the sensitivity and the specificity are high.

Description

Technical Field

[0001] The present application relates to the technical field of chromatographic detection, and particularly relates to a detection method for simultaneously detecting glutamic acid, citric acid, and β-citrylglutamic acid. Background Art

[0002] Glutamic acid is the most abundant free amino acid in the brain and is also the main excitatory neurotransmitter in the brain, widely participating in key functions such as learning, memory, and synaptic plasticity. Citric acid is the core metabolic intermediate of the tricarboxylic acid cycle (TCA cycle), participating in cellular energy generation, lipid synthesis, and redox balance. β-Citrylglutamic acid is a metabolite formed by the catalysis of glutamic acid and citric acid by the RIMKLB enzyme. RIMKLB is highly expressed in specific tissues (such as testis, brain, and placenta) and is closely related to the metabolic activities of these tissues.

[0003] Simultaneously detecting the contents of glutamic acid, citric acid, and β-citrylglutamic acid in a sample to be tested (such as cerebrospinal fluid, plasma, etc.) can reveal the complex pathological network of brain diseases from three dimensions: metabolic energy supply, neurotransmission regulation, and endogenous protection. Such multi-target analysis not only helps to break through the limitations of single-molecule research but also provides a new combination of biomarkers and therapeutic targets for precision medicine, promoting the leap from mechanism research to clinical application.

[0004] Traditional methods for detecting glutamic acid, citric acid, and β-citrylglutamic acid are mainly enzyme-linked methods and spectrophotometric methods. These methods are easily interfered by other components in the sample, resulting in low sensitivity and accuracy, and each detection can only measure one of glutamic acid, citric acid, and β-citrylglutamic acid, with low detection efficiency. Therefore, there is an urgent need to develop a determination method that can simultaneously detect the above three substances. Summary of the Invention

[0005] Based on this, one or more embodiments of the present application provide a detection method for simultaneously detecting glutamic acid, citric acid, and β-citrylglutamic acid. The detection method of the present application can accurately determine glutamic acid, citric acid, and β-citrylglutamic acid simultaneously, with high detection efficiency, high sensitivity, and high specificity.

[0006] The technical solution of the present application includes the following content:

[0007] A detection method for simultaneously detecting glutamic acid, citric acid, and β-citrylglutamic acid, comprising the following steps:

[0008] After adding an internal standard solution to the plasma to be tested and mixing, perform pretreatment to obtain a solution of the sample to be tested;

[0009] The solution of the product to be tested is subjected to tandem detection using an ultra-high performance liquid chromatograph-triple quadrupole tandem mass spectrometer to obtain the contents of glutamic acid, citric acid, and β-citrylglutamic acid in the solution of the product to be tested;

[0010] Among them, the internal standard solution contains isotope-labeled substances of glutamic acid, citric acid, and β-citrylglutamic acid.

[0011] In some embodiments, the conditions for detection by the ultra-high performance liquid chromatograph include: using a C18-bonded silica reverse-phase chromatographic column; using gradient elution; using a mobile phase including mobile phase A and mobile phase B, where mobile phase A is an aqueous solution containing ammonium acetate and ammonia water, and mobile phase B is an aqueous solution of acetonitrile containing ammonium acetate and ammonia water, and the volume concentration of acetonitrile in the aqueous solution of acetonitrile is 94% - 96%; the molar concentration of ammonium acetate in mobile phase A and mobile phase B is independently 8 mmol / L - 12 mmol / L, and the mass concentration of ammonia water is independently 0.03% - 0.05%.

[0012] In some embodiments, the sum of the volume concentrations of mobile phase A and mobile phase B is 100%; the program for gradient elution includes:

[0013] 0 - 2 min, the volume concentration of mobile phase A is maintained at 95%;

[0014] 2 - 2.5 min, the volume concentration of mobile phase A is reduced from 95% to 90%;

[0015] 2.5 - 3.5 min, the volume concentration of mobile phase A is reduced from 90% to 5%;

[0016] 3.5 - 4 min, the volume concentration of mobile phase A is increased from 5% to 95%.

[0017] In some embodiments, the conditions for detection by the ultra-high performance liquid chromatograph further include at least one of the following (1) - (3):

[0018] (1) The flow rate of the mobile phase is 0.3 mL / min - 0.4 mL / min;

[0019] (2) The injection volume is 0.5 μL - 1.5 μL;

[0020] (3) The length of the C18-bonded silica reverse-phase chromatographic column is 40 mm - 60 mm, the diameter is 2 mm - 2.2 mm, and the particle size of the packing is 1.6 μm - 1.8 μm.

[0021] In some of these embodiments, the conditions for detection by the triple quadrupole tandem mass spectrometer include: using multiple reaction monitoring scanning; using an electrospray ionization source; the spray voltage is 3000V to 3500V; the gas flow rate is 8L / min to 12L / min.

[0022] In some of these embodiments, the conditions for detection by the triple quadrupole tandem mass spectrometer further include at least one of the following (1) to (2):

[0023] (1) The nebulizing gas is 15 psi to 25 psi;

[0024] (2) The temperature is 380°C to 420°C.

[0025] In some of these embodiments, multiple reaction monitoring scanning mode is used to quantitatively and qualitatively detect the glutamic acid, the citric acid, and the β-citrylglutamic acid; the quantitative ion pair of the glutamic acid is 148.1→84, and the qualitative ion pair is 148.1→130.1; the quantitative ion pair of the citric acid is 191→87, and the qualitative ion pair is 191→111; the quantitative ion pair of the β-citrylglutamic acid is 320.1→284.1, and the qualitative ion pair is 322.1→276.1.

[0026] In some of these embodiments, the internal standard solution includes 13 C and 15 N-labeled glutamic acid, deuterium-labeled citric acid, and 13 C-labeled β-citrylglutamic acid.

[0027] In some of these embodiments, the steps of adding the internal standard solution to the plasma to be tested, mixing, and then performing pretreatment to obtain the solution to be tested include:

[0028] After adding the internal standard solution to the plasma to be tested and mixing, adding a first solvent and mixing, vortexing and ultrasonically vibrating for 10 min to 20 min, and then adding a second solvent and mixing to obtain a first mixture;

[0029] After centrifuging the first mixture at 12000 rpm to 13000 rpm for 8 min to 12 min, taking the lower layer liquid and mixing it with a third solvent to obtain a second mixture;

[0030] After incubating the second mixture at -25°C to -15°C for 0.5 h to 1.5 h, centrifuging it at 12000 rpm to 13000 rpm for 8 min to 12 min, drying the upper layer supernatant, and redissolving it with a solvent to obtain the solution to be tested;

[0031] The first solvent is a mixed solution of methyl tert-butyl ether and methanol, the second solvent is a mixed solution of water and methanol, and the third solvent is methanol.

[0032] In some of these embodiments, the detection method satisfies at least one of the following conditions:

[0033] (1) The volume ratio of methyl tert-butyl ether to methanol in the first solvent is (2 - 4):1;

[0034] (2) The volume ratio of water to methanol in the second solvent is (2 - 4):1;

[0035] (3) The volume ratio of the plasma to be tested to the first solvent is 1:(1 - 2);

[0036] (4) The volume ratio of the first mixture to the second solvent is 1:(0.1 - 0.3);

[0037] (5) The volume ratio of the second mixture to the third solvent is 1:(0.73 - 0.83).

[0038] The detection method of the present application adopts the internal standard method, uses the isotope-labeled substances of glutamic acid, citric acid and β-citrylglutamic acid as internal standards, and performs tandem detection on the sample to be tested using an ultra-high performance liquid chromatograph-triple quadrupole tandem mass spectrometer, realizing the simultaneous qualitative and quantitative detection of glutamic acid, citric acid and β-citrylglutamic acid, and greatly improving the detection efficiency.

[0039] All technical indicators of the detection method of the present application, such as the limit of quantification, precision and spiked recovery rate, meet the requirements, with good reproducibility, high spiked recovery rate and accurate detection results. Description of the Drawings

[0040] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0041] Figure 1 It is the chromatogram of glutamic acid in the standard curve of Example 1 of the present application; Figure 1 In it, A is the glutamic acid internal standard isotope; Figure 1 In it, B is the glutamic acid standard;

[0042] Figure 2 It is the chromatogram of β-citrylglutamic acid in the standard curve of Example 1 of the present application; Figure 2 In it, A is the β-citrylglutamic acid internal standard isotope; Figure 2 In it, B is the β-citrylglutamic acid standard;

[0043] Figure 3 Chromatogram of citric acid in the standard curve of Example 1 of this application; Figure 3 In it, A is the citric acid internal standard isotope; Figure 3 In it, B is the citric acid standard;

[0044] Figure 4 Chromatogram of glutamic acid in the sample to be tested in Example 1 of this application;

[0045] Figure 5 Chromatogram of β-citrylglutamic acid in the sample to be tested in Example 1 of this application;

[0046] Figure 6 Chromatogram of citric acid in the sample to be tested in Example 1 of this application. Detailed implementation manners

[0047] The present application will be further elaborated below in combination with the implementation manners and examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. In addition, it should be understood that after reading the content taught in the present application, those skilled in the art can make various changes or modifications to the present application, and these equivalent forms also fall within the protection scope of the appended claims of the present application.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the specification of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0049] Terms

[0050] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0051] The term "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when connecting at least three items with at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B.

[0052] In this application, terms such as "first", "second", "third", "fourth", etc. are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or quantity, nor can they be construed as implicitly specifying the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0053] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, as well as an open technical solution containing the listed features.

[0054] In this application, regarding numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical values are considered continuous within the above numerical intervals and include the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In addition, when providing multiple range descriptions of features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0055] In this application, the weight can be mass units well-known in the chemical industry such as μg, mg, g, kg, etc.

[0056] In this application, the correlation coefficient (R 2 ) represents the strength of the linear relationship of the standard curve and is a key indicator for evaluating the reliability of quantitative analysis. Generally, it is required that R 2 ≥0.99. The larger R 2 is, the more reliable it is; LOQ (limit of quantitation) refers to the lowest concentration or lowest amount at which the target substance can be reliably quantitatively determined under specific analysis conditions, reflecting the sensitivity of the analytical method. The lower the limit of quantitation, the higher the sensitivity of the analytical method.

[0057] In this application, when performing chromatographic peak detection, if the detection object is a reference substance but there are multiple non-target peaks, it may be due to inherent impurities in the reference substance or internal standard (different residues from different production processes). During actual detection, since the resolution between non-target peaks and target peaks (corresponding to the peaks to be measured) is large, it will not affect the qualitative or quantitative determination of the analyte. The detection method for simultaneously detecting glutamic acid, citric acid, and β-citrylglutamic acid in the embodiments of this application includes the following steps:

[0058] Add an internal standard solution to the plasma to be measured, mix them, and perform pretreatment to obtain a solution of the sample to be measured;

[0059] The solution of the sample to be tested is subjected to tandem detection using an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer to obtain the contents of glutamic acid, citric acid, and β-citrylglutamic acid in the solution of the sample to be tested;

[0060] Among them, the internal standard solution contains isotope-labeled substances of glutamic acid, citric acid, and β-citrylglutamic acid.

[0061] In the detection method of the embodiment of the present application, by using the internal standard method, with isotope-labeled substances of glutamic acid, citric acid, and β-citrylglutamic acid as internal standards, and subjecting the sample to be tested to tandem detection using an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer. Since the isotope-labeled substances and the substances to be tested have almost exactly the same molecular structure and chemical properties, they show similar behaviors in the processes of sample preparation, chromatographic separation, and mass spectrometry analysis, but have different mass numbers and can be effectively distinguished in mass spectrometry analysis. Therefore, simultaneous qualitative and quantitative detection of glutamic acid, citric acid, and β-citrylglutamic acid can be achieved, greatly improving the detection efficiency.

[0062] In the detection method of the embodiment of the present application, an ultra-high performance liquid chromatography is used in tandem detection, which has higher separation efficiency, sensitivity, and faster analysis speed compared with a high performance liquid chromatography.

[0063] All technical indicators such as the limit of quantification, precision, and spike recovery rate of the detection method of the embodiment of the present application meet the requirements, with good reproducibility, high spike recovery rate, and accurate detection results.

[0064] In some embodiments, based on the ratio of the peak area of the standard substance to the peak area of the corresponding internal standard isotope as the ordinate of the standard curve, and the concentration of the standard substance and the concentration of the corresponding internal standard isotope as the abscissa of the standard curve, a standard curve is plotted, and this standard curve can be used to calculate the concentration of the substance to be tested in the sample to be tested.

[0065] In this embodiment, since the properties of the isotope internal standard and the substance to be tested are highly similar, the influences such as matrix effect and ionization efficiency change during the analysis process are also similar. By comparing the signal ratio of the internal standard and the substance to be tested, errors can be effectively eliminated, and the accuracy and precision of quantitative analysis can be improved.

[0066] In some of these embodiments, the conditions for detection by ultra - performance liquid chromatography include: using a C18 - bonded silica reversed - phase chromatographic column; using gradient elution; using a mobile phase comprising mobile phase A and mobile phase B. Mobile phase A is an aqueous solution containing ammonium acetate and ammonia water, and mobile phase B is an aqueous solution of acetonitrile containing ammonium acetate and ammonia water. The volume concentration of acetonitrile in the aqueous solution of acetonitrile is 94% - 96%; the molar concentration of ammonium acetate in mobile phase A and mobile phase B is independently 8 mmol / L - 12 mmol / L, and the mass concentration of ammonia water is independently 0.03% - 0.05%.

[0067] Optionally, the volume concentration of the aqueous solution of acetonitrile in mobile phase B can be selected from 94% - 96%, for example, 94%, 95%, 96%, etc.

[0068] Glutamic acid, citric acid, and β - citroylglutamic acid have different chemical properties. Due to the presence of polar groups such as carboxyl, hydroxyl, or amino groups, they have strong polarity. In this embodiment, by using a chromatographic column with an appropriate polarity (C18 - bonded silica reversed - phase chromatographic column), mobile phase, and gradient elution, the above three components can be better separated.

[0069] In some of these embodiments, the sum of the volume concentrations of mobile phase A and mobile phase B is 100%; the gradient elution program includes:

[0070] From 0 to 2 min, the volume concentration of mobile phase A is maintained at 95%;

[0071] From 2 to 2.5 min, the volume concentration of mobile phase A decreases from 95% to 90%;

[0072] From 2.5 to 3.5 min, the volume concentration of mobile phase A decreases from 90% to 5%;

[0073] From 3.5 to 4 min, the volume concentration of mobile phase A increases from 5% to 95%.

[0074] In this embodiment, the polarity of glutamic acid, citric acid, and β - citroylglutamic acid has less influence, and the separation effect is better.

[0075] In some of these embodiments, the conditions for detection by ultra - performance liquid chromatography further include at least one of the following (1) - (3):

[0076] (1) The flow rate of the mobile phase is 0.3 mL / min - 0.4 mL / min;

[0077] (2) The injection volume is 0.5 μL - 1.5 μL;

[0078] (3) The length of the C18-bonded silica gel reversed-phase chromatographic column is 40 mm to 60 mm, the diameter is 2 mm to 2.2 mm, and the particle size of the packing material is 1.6 μm to 1.8 μm.

[0079] In this embodiment, the C18-bonded silica gel reversed-phase chromatographic column uses packing material with a particle size of 1.6 μm to 1.8 μm, which has higher separation efficiency and can also obtain narrower chromatographic peaks, thereby improving the resolution and sensitivity of detection. At the same time, it also improves the experimental throughput, effectively shortens the analysis time, and further improves the detection efficiency. The length of the C18-bonded silica gel reversed-phase chromatographic column is 40 mm to 60 mm, and the diameter is 2 mm to 2.2 mm. Combining with the small particle size of the packing material, the chromatographic peak can be made narrower, the peak height increases, and the response signal of the detector to the target compound is enhanced, thereby reducing the consumption of samples and solvents.

[0080] In some of these embodiments, the detection conditions of the triple quadrupole tandem mass spectrometer include: using multiple reaction monitoring scanning; using an electrospray ionization source; the spray voltage is 3000 V to 3500 V; the gas flow rate is 8 L / min to 12 L / min.

[0081] Glutamic acid, citric acid, and β-citrylglutamic acid have different ionization efficiencies in mass spectrometry analysis. In this application, by adjusting the ion source parameters of the spray voltage and gas flow rate, the ionization efficiencies of the above substances can be improved simultaneously, so that the signals of the three substances can be effectively detected under the same detection conditions.

[0082] In some of these embodiments, the detection conditions of the triple quadrupole tandem mass spectrometer further include at least one of the following (1) to (2):

[0083] (1) The nebulizing gas is 15 psi to 25 psi;

[0084] (2) The temperature is 380 °C to 420 °C.

[0085] In some of these embodiments, the multiple reaction monitoring scanning mode is used for quantitative and qualitative detection of glutamic acid, citric acid, and β-citrylglutamic acid; the quantitative ion pair of glutamic acid is 148.1 → 84, and the qualitative ion pair is 148.1 → 130.1; the quantitative ion pair of citric acid is 191 → 87, and the qualitative ion pair is 191 → 111; the quantitative ion pair of β-citrylglutamic acid is 320.1 → 284.1, and the qualitative ion pair is 322.1 → 276.1.

[0086] In this application, the qualitative ion pair is the ion pair used to confirm the identity of a compound, which is composed of characteristic daughter ions generated after the fragmentation of the parent ion, expressed as "parent ion → daughter ion". The daughter ion has the characteristics of strong selectivity and less interference. By matching the mass-to-charge ratio (m / z) and relative abundance ratio of the characteristic ion pair with a reference standard or a database, the qualitative confirmation of the compound can be achieved; the quantitative ion pair is the ion pair used to determine the concentration of a compound. Usually, the combination of the parent ion and daughter ion with the strongest response and the best stability is selected, expressed as "parent ion → daughter ion". The daughter ion has the characteristics of high response, good stability, and a wide linear range. By the peak area or peak height of the ion pair and combining with a standard curve, the content of the target compound in the sample can be calculated.

[0087] In some of these embodiments, the internal standard solution includes 13 C and 15 N-labeled glutamic acid, deuterium-labeled citric acid, and 13 C-labeled β-citrylglutamic acid.

[0088] In some of these embodiments, the steps of adding the internal standard solution to the plasma to be tested, mixing, and then performing pretreatment to obtain the solution to be tested include:

[0089] After adding the internal standard solution to the plasma to be tested and mixing, add the first solvent and mix, vortex and ultrasonicate for 10 min to 20 min, then add the second solvent and mix to obtain the first mixture;

[0090] After centrifuging the first mixture at 12000 rpm to 13000 rpm for 8 min to 12 min, take the lower layer liquid and mix it with the third solvent to obtain the second mixture;

[0091] After incubating the second mixture at -25 °C to -15 °C for 0.5 h to 1.5 h, centrifuge it at 12000 rpm to 13000 rpm for 8 min to 12 min, take the upper clear liquid and dry it, and re-dissolve it with a solvent to obtain the solution to be tested;

[0092] The first solvent is a mixed solution of methyl tert-butyl ether and methanol, the second solvent is a mixed solution of water and methanol, and the third solvent is methanol.

[0093] In this embodiment, the function of the first solvent is to dissolve lipids. After adding the first solvent and mixing, vortex oscillation and ultrasonic treatment for 10 - 20 min can fully react to break the cell wall; the function of the second solvent is to separate the lipid layer and the extract based on the principle of similar solubility. After centrifuging the first mixture at 12,000 - 13,000 rpm for 8 - 12 min, the substance to be measured can be concentrated in the lower layer; the function of the third solvent is to precipitate proteins. After taking the lower layer liquid and mixing it with the third solvent to obtain the second mixture, incubating at -25°C to -15°C for 0.5 - 1.5 h can fully precipitate proteins, and then centrifuging at 12,000 - 13,000 rpm for 8 - 12 min can separate the precipitated proteins to obtain the supernatant.

[0094] In this embodiment, the first solvent dissolves lipids using the principle of liquid - liquid extraction. Compared with solid - liquid extraction (a pretreatment method that requires multiple - step operations), the extraction process is simple, the cost is low, and no special equipment is needed. The first solvent is more environmentally friendly than traditional chloroform / methanol systems, MTBE / methanol systems, etc., reducing the health risks to operators; after the second solvent separates the lipid layer and the extract, the substance to be measured is concentrated in the lower layer, which is more conducive to separation and collection compared with the MTBE / methanol system (the organic phase formed by extraction is usually in the upper layer), reducing the errors caused by incomplete phase separation.

[0095] In some embodiments, the detection method satisfies at least one of the following conditions:

[0096] (1) The volume ratio of methyl tert - butyl ether to methanol in the first solvent is (2 - 4):1;

[0097] (2) The volume ratio of water to methanol in the second solvent is (2 - 4):1;

[0098] (3) The volume ratio of the plasma to be measured to the first solvent is 1:(1 - 2);

[0099] (4) The volume ratio of the first mixture to the second solvent is 1:(0.1 - 0.3);

[0100] (5) The volume ratio of the second mixture to the third solvent is 1:(0.73 - 0.83).

[0101] Optionally, the volume ratio of methyl tert - butyl ether to methanol in the first solvent can be selected from any ratio in (2 - 4):1, such as 2:1, 2.5:1, 3:1, 3.5:1, 4:1, etc.

[0102] Optionally, the volume ratio of water to methanol in the second solvent can be selected from any ratio in (2 - 4):1, such as 2:1, 2.5:1, 3:1, 3.5:1, 4:1, etc.

[0103] Optionally, the volume ratio of the plasma to be measured and the first solvent can be selected from any ratio in 1:(1-2), such as 1:1, 1:1.2, 1:1.4, 1:1.5, 1:1.7, 1:2, etc.

[0104] Optionally, the volume ratio of the first mixed solution and the second solvent can be selected from any ratio in 1:(0.1-0.3), such as 1:0.1, 1:0.2, 156:35, 1:0.3, etc.

[0105] Optionally, the volume ratio of the second mixed solution and the third solvent is any ratio in 1:(0.73-0.83), such as 1:0.73, 1:0.75, 1:0.78, 14:11, 1:0.8, 1:0.83, etc.

[0106] The following are some specific embodiments.

[0107] For the experimental parameters not specified in the following specific embodiments, preferably refer to the guidelines given in this application document, and it is also possible to refer to the experimental manuals in this field or other experimental methods known in this field, or refer to the experimental conditions recommended by the manufacturers.

[0108] The raw materials and reagents involved in the following specific embodiments can be obtained commercially, or those skilled in the art can prepare them according to known means.

[0109] Example 1

[0110] This example provides a method for detecting glutamic acid, citric acid and β-citrylglutamic acid in plasma, including the following steps:

[0111] 1. Prepare standard solutions

[0112] 1.1 Prepare standard curves

[0113] Accurately weigh 1.0 mg each of glutamic acid and citric acid standards with an analytical balance, dissolve them in 1 mL of methanol solution to make a 1 mg / mL solution, and dilute to a final concentration of 100 μg / mL as needed to obtain 100 μg / mL glutamic acid standard stock solution and 100 μg / mL citric acid standard stock solution. Dilute 1 mg / mL β-citrylglutamic acid to a final concentration of 100 μg / mL as needed to obtain 100 μg / mL β-citrylglutamic acid standard stock solution; subsequently, respectively pipette 400 μL of glutamic acid standard stock solution, 100 μL of β-citrylglutamic acid standard stock solution and 200 μL of citric acid standard stock solution into a 10 mL volumetric flask and make up the volume with a diluent (50:50 methanol aqueous solution) to obtain 4000 ng / mL glutamic acid standard stock solution, 1000 ng / mL β-citrylglutamic acid standard stock solution and 2000 ng / mL citric acid standard stock solution, as shown in STD6 in Table 1.

[0114] Prepare 5 volumetric flasks with a capacity of 5 mL, labeled as STD1-5 respectively. First, successively pipette 0.625, 1, 1.25, 2, and 2.5 mL of the glutamate standard stock solution, β-citrylglutamic acid standard stock solution, and citric acid standard stock solution into the STD1-5 volumetric flasks, dilute with methanol aqueous solution (v:v = 50:50) and make up to the mark to prepare the standard working solutions of each concentration for making the standard curve. The concentration range of glutamate is 500-2000 ng / mL, the concentration range of β-citrylglutamic acid is 125-500 ng / mL, and the concentration range of citric acid is 250-1000 ng / mL. The specific concentrations are shown in Table 1.

[0115] Table 1. Standard working solutions of standards

[0116]

[0117] 1.2 Preparation of standard internal standard solution

[0118] Accurately weigh 1 mg of isotope 13C-BCG and dissolve it in 1 mL of aqueous solution to prepare a 1 mg / mL stock solution. Dilute the β-citrylglutamic acid standard isotope internal standard solution (1 mg / mL) with methanol water (50:50) to 100 μg / mL for standby. Accurately weigh 1 mg of 13C,15N-Glu and 1 mg of Citric-d4 and dissolve them in methanol water (50:50) respectively to prepare 1 mg / mL stock solutions. Dilute the glutamate standard isotope internal standard solution (1 mg / mL) and the citric acid standard isotope internal standard solution (1 mg / mL) with methanol water (50:50) to 100 μg / mL for standby. Accurately pipette 10 μL of the β-citrylglutamic acid standard isotope internal standard solution (100 μg / mL), 10 μL of the glutamate standard isotope internal standard solution (100 μg / mL), and 10 μL of the citric acid standard isotope internal standard solution (100 μg / mL) into a 100 mL volumetric flask, dilute with methanol aqueous solution (v:v = 50:50) and make up to the mark to obtain the mixed isotope standard internal standard solution, as shown in Table 2.

[0119] Table 2 Mixed isotope standard internal standard solution

[0120]

[0121] 1.3 Establishment of standard curve

[0122] Use a pipette to separately aspirate 50 μL of standard working solutions of six different concentrations (Table 1), and add them to the corresponding centrifuge tubes. Then add 450 μL of phosphate buffered saline (PBS), and mix well. After adding 10 μL of mixed isotope standard internal standard solution,

[0123] add 700 μL of methyl tert-butyl ether / methanol (3:1), vortex and ultrasonically mix at room temperature for 15 min, add 350 μL of water / methanol (3:1), vortex for 10 s, and centrifuge at 12,500 rpm at 4 °C for 10 min. Connect a 1 mL pipette tip to a vacuum pump to remove the remaining lipid layer and the middle layer, transfer 300 μL of the lower layer to a new centrifuge tube, add 1.1 mL of pre-cooled methanol, vortex, and incubate in a -20 °C refrigerator for 1 h. After protein precipitation, vortex for 10 s, centrifuge at 12,700 rpm at 4 °C for 10 min, transfer 1 mL of the supernatant, and dry it under nitrogen. Re-dissolve with 200 μL of reconstitution solution (50:50 methanol / water), vortex for 30 s, and centrifuge at 12,500 rpm at 4 °C for 15 min for LC-MS / MS analysis.

[0124] Separate chromatograms of glutamic acid, β-citrylglutamic acid, and citric acid in the above three standard working solutions (STD1-6) and chromatograms of glutamic acid isotope, β-citrylglutamic acid isotope, and citric acid isotope in the corresponding mixed isotope standard internal standard solution are obtained ( Figures 1 - 3 ). Using the ratios of the peak areas of glutamic acid, β-citrylglutamic acid, and citric acid in the above three standard working solutions to the peak areas of glutamic acid isotope, β-citrylglutamic acid isotope, and citric acid isotope in the corresponding mixed isotope standard internal standard solution as the ordinates y1, y2, and y3 of the standard curve graph, and using the ratios of the concentrations of glutamic acid, β-citrylglutamic acid, and citric acid in the above standard working solutions to the concentrations of glutamic acid isotope, β-citrylglutamic acid isotope, and citric acid isotope in the corresponding mixed isotope standard internal standard solution as the abscissas x1, x2, and x3 of the standard curve graph, perform linear regression on the data obtained from the above detections respectively, fit to obtain the standard curve equations as Y1 = a×X1 + b, Y2 = c×X2 + d, and Y3 = e×X3 + f, and obtain the weighting coefficients a, b, c, d, e, and f. The obtained linear equations are shown in Table 3. That is, using the ratios of the concentrations of three standard working solutions to the concentrations of the corresponding mixed isotope standard internal standard solution as the abscissa, and the ratios of the quantitative chromatographic peak areas of three standard working solutions to the quantitative chromatographic peak areas of the corresponding mixed isotope standard internal standard solution as the ordinate to plot a graph to obtain the standard curve. The results show that the linear correlation coefficients (R 2 ) of the three analytes are all greater than 0.999, and the limits of quantification (LOQ) are shown in Table 3.

[0125] Table 3. Standard Curve and Correlation Coefficient

[0126]

[0127]

[0128] 2. Obtain the sample to be tested

[0129] 2.1 Collection of human plasma

[0130] Fast for at least 12 hours before plasma collection to ensure plasma quality. According to the requirements of "WS / T225-2022 Collection and Processing of Blood Specimens for Clinical Chemical Examination", use an EDTA anticoagulant tube to collect 1 mL of fasting venous blood. After collection, centrifuge at 4°C and 1600 g for 12 minutes, and collect the supernatant. If immediate detection is not carried out, store it at -80°C for later use.

[0131] 2.2 Pretreatment of plasma

[0132] Use a pipette to transfer 500 μL of the supernatant collected in the subsection "2.1 Collection of human plasma" into the corresponding centrifuge tube. After adding 10 μL of the mixed isotope standard internal standard solution, add 700 μL of methyl tert-butyl ether / methanol (3:1), vortex and ultrasonically mix at room temperature for 15 min, then add 350 μL of water / methanol (3:1), vortex for 10 s, and centrifuge at 4°C and 12500 rpm for 10 min; connect a 1 mL pipette tip to a vacuum pump to remove the remaining lipid layer and the middle layer, transfer 300 μL of the lower layer to a new centrifuge tube, add 1.1 mL of pre-cooled methanol, vortex and incubate in a -20°C refrigerator for 1 h; after protein precipitation, vortex for 10 s, centrifuge at 4°C and 12700 rpm for 10 min, transfer 1 mL of the supernatant, and dry it with nitrogen; re-dissolve with 200 μL of the reconstitution solution (50:50 methanol-water), vortex for 30 s, and centrifuge at 4°C and 12500 rpm for 15 min to obtain the sample to be tested.

[0133] 3. Qualitative and quantitative detection of glutamic acid, β-citrylglutamic acid and citric acid in the sample to be tested

[0134] Take the sample to be tested obtained above, and use a high performance liquid chromatograph-triple quadrupole tandem mass spectrometer for tandem detection (the detection conditions are shown in Table 4) to obtain the chromatograms of glutamic acid, β-citrylglutamic acid and citric acid in the above-mentioned sample to be tested ( Figures 4 - 6 ) and the chromatograms of glutamic acid isotope, β-citrylglutamic acid isotope and citric acid isotope in the corresponding mixed isotope standard internal standard solution (see Figures 1 - 3)。Substitute the peak area ratios Y1, Y2, and Y3 of glutamic acid, β-citrylglutamic acid, and citric acid in the above chromatogram and the glutamic acid isotope, β-citrylglutamic acid isotope, and citric acid isotope in the corresponding mixed isotope standard internal standard solution into the standard curve equation in step (c) (Table 3) above. By calculation, obtain the concentration ratios X1, X2, and X3 of glutamic acid, β-citrylglutamic acid, and citric acid in the sample to be detected and the glutamic acid isotope, β-citrylglutamic acid isotope, and citric acid isotope in the corresponding mixed isotope standard internal standard solution. The concentrations of the glutamic acid isotope, β-citrylglutamic acid isotope, and citric acid isotope in the mixed isotope standard internal standard solution are known. Calculate the concentrations of glutamic acid, β-citrylglutamic acid, and citric acid in the sample to be detected.

[0135] Table 4. Conditions for tandem detection by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry

[0136]

[0137]

[0138] Table 5. Gradient elution program

[0139] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0～2 95→95 5→5 2～2.5 95→90 5→10 2.5～3.5 90→5 10→95 3.5～4 5→95 95→5

[0140] Table 6. Multiple reaction monitoring parameters

[0141]

[0142] 4. Methodology investigation

[0143] 4.1 Precision

[0144] Prepare plasma samples at low, medium, and high concentration levels. Within one batch, continuously detect the plasma samples at low, medium, and high concentration levels 3 times each as the within-batch precision detection data; select plasma samples at low, medium, and high concentration levels for between-batch precision verification. Detect the above two-level samples 3 times a day for 3 consecutive days as the between-batch precision detection data (precision < 15% is considered normal). The results are shown in Table 7.

[0145] Table 7. Precision detection results

[0146]

[0147] 4.2 Recovery rate

[0148] Add standard solutions with high, medium, and low concentrations to the sample matrix (the volume of the added standard solution does not exceed 5% of the total volume), and the final concentration after spiking needs to cover the entire medical decision level. Parallel process 3 samples for each of the matrix and each concentration after spiking, calculate the average value of the matrix, and the recovery rate (R) of the corresponding matrix for each sample after spiking. R = (measured value after spiking - average measured value of the unspiked matrix) / theoretical spiking value × 100% (an R value between 85% and 115% is considered normal), and the results are shown in Table 8.

[0149] Table 8. Detection Results of Spiking Recovery

[0150]

[0151] According to the above results of the methodological investigation, various technical indicators of the detection method of the embodiments of the present application, such as the limit of quantification, precision, and spiking recovery rate, all meet the requirements. It can simultaneously detect the contents of glutamic acid, β-citrylglutamic acid, and citric acid in plasma, with good reproducibility, high spiking recovery rate, and accurate detection results.

[0152] All the documents mentioned in the present application are incorporated herein by reference as if each document was individually incorporated by reference. Unless it conflicts with the application purpose and / or technical solution of the present application, the cited documents involved in the present application are incorporated by reference in their entirety and for all purposes. When the present application involves citing documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also incorporated by reference. When the present application involves citing documents, the examples and preferred methods of the relevant technical features cited can also be incorporated as references into the present application, but only to the extent that the present application can be implemented. It should be understood that when the cited content conflicts with the description in the present application, the present application shall prevail or be modified adaptively according to the description in the present application.

[0153] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0154] The embodiments described above merely represent several implementation manners of the present application, but should not be construed as limiting the scope of the patent application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can be made, and these all fall within the protection scope of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the technical solutions provided by the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims, and the specification can be used to explain the content of the claims.

Claims

1. A detection method for simultaneously detecting glutamic acid, citric acid, and β-citrylglutamic acid, characterized in that, It includes the following steps: After adding an internal standard solution to the plasma to be tested and mixing, perform pretreatment to obtain a solution of the sample to be tested; Use an ultra-high performance liquid chromatograph-triple quadrupole tandem mass spectrometer to perform tandem detection on the solution of the sample to be tested to obtain the contents of glutamic acid, citric acid, and β-citrylglutamic acid in the solution of the sample to be tested; Among them, the internal standard solution contains isotope-labeled substances of glutamic acid, citric acid, and β-citrylglutamic acid.

2. The detection method according to claim 1, wherein The conditions for detection by the ultra-high performance liquid chromatograph include: using a C18-bonded silica reversed-phase chromatographic column; using gradient elution; the mobile phase includes mobile phase A and mobile phase B. Mobile phase A is an aqueous solution containing ammonium acetate and ammonia water, and mobile phase B is an aqueous solution of acetonitrile containing ammonium acetate and ammonia water. The volume concentration of acetonitrile in the aqueous solution of acetonitrile is 94% - 96%; the molar concentration of ammonium acetate in mobile phase A and mobile phase B is independently 8 mmol / L - 12 mmol / L, and the mass concentration of ammonia water is independently 0.03% - 0.05%.

3. The detection method according to claim 2, wherein The total volume concentration of mobile phase A and mobile phase B is 100%; the program for gradient elution includes: From 0 to 2 min, the volume concentration of mobile phase A is maintained at 95%; From 2 to 2.5 min, the volume concentration of mobile phase A is reduced from 95% to 90%; From 2.5 to 3.5 min, the volume concentration of mobile phase A is reduced from 90% to 5%; From 3.5 to 4 min, the volume concentration of mobile phase A is increased from 5% to 95%.

4. The detection method according to any one of claims 2 to 3, characterized in that The conditions for detection by the ultra-high performance liquid chromatograph further include at least one of the following (1) - (3): (1) The flow rate of the mobile phase is 0.3 mL / min - 0.4 mL / min; (2) The injection volume is 0.5 μL - 1.5 μL; (3) The length of the C18-bonded silica reversed-phase chromatographic column is 40 mm - 60 mm, the diameter is 2 mm - 2.2 mm, and the particle size of the packing is 1.6 μm - 1.8 μm.

5. The detection method according to claim 1, wherein The conditions for detection by the triple quadrupole tandem mass spectrometer include: using multiple reaction monitoring scanning; using an electrospray ionization source; the spray voltage is 3000 V - 3500 V; the gas flow rate is 8 L / min - 12 L / min.

6. The detection method according to claim 5, characterized in that The conditions for detection by the triple quadrupole tandem mass spectrometer further include at least one of the following (1) - (2): (1) The nebulizing gas is 15 psi - 25 psi; (2) The temperature is 380 °C - 420 °C.

7. The detection method according to any one of claims 1, 5, and 6, characterized in that Use the multiple reaction monitoring scanning mode to perform quantitative and qualitative detection on the glutamic acid, the citric acid, and the β-citrylglutamic acid; the quantitative ion pair of glutamic acid is 148.1 → 84, and the qualitative ion pair is 148.1 → 130.1; the quantitative ion pair of citric acid is 191 → 87, and the qualitative ion pair is 191 → 111; the quantitative ion pair of β-citrylglutamic acid is 320.1 → 284.1, and the qualitative ion pair is 322.1 → 276.

1.

8. The detection method according to claim 1, characterized in that, The internal standard solution includes 13 C and 15 N-labeled glutamic acid, deuterium-labeled citric acid and 13 C-labeled β-citrylglutamic acid.

9. The detection method according to claim 1, characterized in that, The step of adding an internal standard solution to the plasma to be tested and mixing, and then performing pretreatment to obtain a solution of the sample to be tested, includes: After adding the internal standard solution to the plasma to be tested and mixing, add the first solvent and mix, then vortex and ultrasonicate for 10 min to 20 min, and then add the second solvent and mix to obtain the first mixture; After centrifuging the first mixture at 12,000 rpm to 13,000 rpm for 8 min to 12 min, take the lower layer liquid and mix it with the third solvent to obtain the second mixture; After incubating the second mixture at -25°C to -15°C for 0.5 h to 1.5 h, centrifuge it at 12,000 rpm to 13,000 rpm for 8 min to 12 min, dry the upper clear liquid, and re-dissolve it with a solvent to obtain the solution of the sample to be tested; The first solvent is a mixed solution of methyl tert-butyl ether and methanol, the second solvent is a mixed solution of water and methanol, and the third solvent is methanol.

10. The detection method according to claim 9, wherein Meet at least one of the following conditions: (1) The volume ratio of methyl tert-butyl ether to methanol in the first solvent is (2 to 4):1; (2) The volume ratio of water to methanol in the second solvent is (2 to 4):1; (3) The volume ratio of the plasma to be tested to the first solvent is 1:(1 to 2); (4) The volume ratio of the first mixture to the second solvent is 1:(0.1 to 0.3); (5) The volume ratio of the second mixture to the third solvent is 1:(0.73 to 0.83).

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