A method for simultaneous detection of multiple amatoxins

Abstract

This application relates to a method for the simultaneous detection of multiple amatoxins, belonging to the technical field of analytical detection. The method includes the following steps: S1: preparing a sample solution to be tested; S2: detecting amatoxins in the sample solution using liquid chromatography-mass spectrometry (LC-MS); wherein the chromatographic separation conditions of the LC-MS method are: column: Kinetex 2.6μm XB-C18 100A, LC column 50x3mm; mobile phase: phase A is formic acid-water, phase B is methanol, gradient elution. This application has the ability to simultaneously detect four target analytes (α-amatoxin, β-amatoxin, γ-amatoxin, and dihydroxyphalamydin) in a short time, exhibiting very high sensitivity, meeting the requirements of toxic substance residue analysis, and providing strong technical support for emergency detection in public health emergencies.

Description

Technical Field

[0001] This application relates to the field of analytical detection technology, and in particular to a method for simultaneously detecting multiple amatoxins. Background Technology

[0002] Amatoxins are cyclic peptide compounds composed of 7-8 amino acids. Based on their structure, they can be classified into three categories: amatoxins (AMA 0.01%), phalloidin, and amatoxins. There is currently no evidence of harmful effects of amatoxins on humans, phalloidin exhibits immediate toxicity, and AMA exhibits delayed toxicity. Nine types of AMA have been isolated and identified from nature, among which α-AMA is the most abundant and toxic, and is considered a major cause of liver damage and death in patients. Furthermore, in the life sciences, α-AMA is used as a specific inhibitor of eukaryotic RNAPII, and is widely used in research on the transcription process from DNA to mRNA, which is of significant value.

[0003] Currently, amatoxins in blood are mainly detected by liquid chromatography-mass spectrometry / mass spectrometry. However, due to the large and close molecular weight of amatoxins (approximately 900-1000 Da), and the lack of easily protonated structures, the positive ion scanning mode response is low, resulting in low sensitivity and significant matrix influence.

[0004] To solve the above technical problems, it is necessary to develop a detection reagent and detection method for detecting amatoxins in blood with high sensitivity, accuracy, precision and recovery rate. Summary of the Invention

[0005] This application addresses the challenges of existing technologies by developing a method for the simultaneous detection of multiple amatoxins based on liquid chromatography-tandem mass spectrometry (LC-MS / MS). The method employs a negative ion mode combined with a specific mobile phase and gradient elution to achieve baseline separation of four amatoxins. This method can complete the simultaneous detection of four amatoxins within 10 minutes, exhibiting very high sensitivity and meeting the requirements for toxic substance residue analysis. It provides strong technical support for emergency detection in public health emergencies.

[0006] This application provides a method for simultaneously detecting multiple amatoxins, comprising the following steps:

[0007] S1: Prepare the sample solution to be tested;

[0008] S2: High performance liquid chromatography-mass spectrometry was used to quantitatively detect amatoxins in the sample solution to be tested;

[0009] The chromatographic separation conditions of the liquid chromatography-mass spectrometry method are as follows: chromatographic column: Kinetex 2.6μmXB-C18 100A, LC Column 50x3mm; mobile phase: phase A is formic acid and water, phase B is methanol, gradient elution.

[0010] Optionally, the amatoxin is one or more of α-amatoxin, β-amatoxin, γ-amatoxin, and dihydroxyphalazotoxin.

[0011] Optionally, step S1 specifically involves: taking a plasma sample, adding concentrated hydrochloric acid, and shaking to mix for 5-15 minutes; adding an acetonitrile:methanol (ratio = 9:1) solution, shaking to mix, centrifuging to obtain the supernatant to obtain the sample solution to be tested.

[0012] Optionally, the mass spectrometry conditions for the high performance liquid chromatography-mass spectrometry method are as follows: ESI ion source; scanning mode: negative ion scanning mode; ionization voltage (IS): -4500V; temperature: 500℃; spray gas (GS1): 55psi; auxiliary heating gas (GS2): 45psi.

[0013] Optionally, the parent ion-daughter ion pairs for mass spectrometry analysis in the quantitative detection are: α-amanita peptide, m / z 917.2→899.0; β-amanita peptide, m / z 918.2→900.3; γ-amanita peptide, m / z 901.2→883.3; and dihydroxyphalangeal peptide, m / z 787.7→743.5.

[0014] Optionally, phase A is 0.01% formic acid water, and phase B is methanol.

[0015] Optionally, the gradient elution procedure is as follows:

[0016] Time (min) Flow (mL / min) A(%) B(%) 0.00 0.30 99.0 1.0 5.00 0.30 10.0 90.0 7.00 0.30 10.0 90.0 8.00 0.30 90.0 10.0 .

[0017] Optionally, the quantitative detection is an external standard method.

[0018] Optionally, the external standard method for quantitative detection includes the following steps:

[0019] a) Prepare a series of standard solutions of amatoxins at different concentrations;

[0020] b) The series of standard solutions were measured using the liquid chromatography-mass spectrometry method, and the chromatographic peak area of ​​each standard solution was recorded.

[0021] c) Plot a standard curve with the concentration of the standard solution on the x-axis and the corresponding peak area on the y-axis;

[0022] d) Substitute the chromatographic peak area of ​​the target analyte in the sample solution to be tested into the standard curve to calculate the content of amatoxin in the sample solution to be tested.

[0023] Optionally, the standard solution in step a) is prepared using an acidified blank matrix; the blank matrix is ​​plasma that does not contain the target amatoxin.

[0024] In summary, this application includes at least one of the following beneficial technical effects:

[0025] 1. During sample processing, the added hydrochloric acid can adjust the pH to provide a slightly acidic environment, which helps the target analyte release and improves the sample extraction efficiency. At the same time, acidic conditions help improve the mass spectrometry response and separation.

[0026] 2. This application employs a negative ion mode to address the issue of low response in positive ion scanning modes caused by the large molecular weight (approximately 900-1000 Da) and lack of easily protonated structures of amatoxins. However, negative ions themselves have low sensitivity. These toxins are extremely toxic, with toxic doses in humans ranging from milligrams to micrograms. This application features refined optimization and development of the chromatographic mobile phase and elution gradient, using formic acid water and methanol as the mobile phase and setting a reasonable elution gradient. Detection is completed within 10 minutes of analysis, with a linear range covering 8-400 ng / mL, exhibiting very high sensitivity. Attached Figure Description

[0027] Figure 1 The total ion current chromatogram was obtained using Example 1.

[0028] Figure 2 This is a graph showing the results of β-amanita peptide detection using Comparative Example 1.

[0029] Figure 3 This is a graph showing the results of the detection of dihydroxyphalangeal peptide using Comparative Example 1.

[0030] Figure 4 These are chromatograms of α-amanita peptides from this application, wherein A is the chromatogram of α-amanita peptides detected in Example 1, and B is the chromatogram of α-amanita peptides detected in Comparative Example 2.

[0031] Figure 5 These are chromatograms of β-amatoxins from this application, where A is the chromatogram of β-amatoxins detected in Example 1, and B is the chromatogram of β-amatoxins detected in Comparative Example 2.

[0032] Figure 6 These are chromatograms of γ-amatoxins from this application, wherein A is the chromatogram of γ-amatoxins detected in Example 1, and B is the chromatogram of γ-amatoxins detected in Comparative Example 2.

[0033] Figure 7 These are chromatograms of the dihydroxyphalangeal peptide of this application, wherein A is the chromatogram of the dihydroxyphalangeal peptide detected in Example 1, and B is the chromatogram of the dihydroxyphalangeal peptide detected in Comparative Example 2. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific implementation examples, but the invention is not limited thereto.

[0035] Example 1 Detection Method

[0036] 1. Plasma sample pretreatment

[0037] Take 1 mL of mixed plasma, add 2.5 μL of concentrated hydrochloric acid, and shake to mix for 10 minutes.

[0038] 2. Preparation of standard solutions

[0039] Using a 1 / 100,000 balance, weigh appropriate amounts of α-amatoxin, β-amatoxin, γ-amatoxin, and dihydroxyphalazoxin standards into 2 mL brown vials, dilute and dissolve them with methanol to prepare a stock solution with a concentration of 10 μg / mL, and store at -80℃ for later use. When using, dilute with methanol in a gradient manner, adding it to the blank sample matrix at a volume ratio of 1:10 to obtain standard solutions of different concentrations, with concentrations of 400, 160, 80, 40, 16, and 8 ng / mL. The blank sample matrix is ​​mixed plasma without the tested mushroom toxins, which is used after the pretreatment step in step 1.

[0040] 3. Testing

[0041] Take 50 μL of the above standard solution and 50 μL of the pretreated plasma sample and add them to a 1.5 mL centrifuge tube. Add 250 μL of acetonitrile:methanol (ratio = 9:1) solution, shake and mix for 10 minutes, centrifuge at 13000 rpm for 5 minutes, take 150 μL of the supernatant and add it to a 96-well plate. Detect the sample using liquid chromatography-mass spectrometry (LC and triple quadrupole tandem mass spectrometry).

[0042] 4. Construction of Standard Curve

[0043] Standard working curves were plotted with the peak areas of quantitative ion pairs of standard solutions of α-amanita peptide, β-amanita peptide, γ-amanita peptide, and dihydroxyphalamydin as the ordinate and the concentration as the abscissa.

[0044] 5. Sample quantification

[0045] The concentration of each target substance in the sample is calculated by substituting the peak area of ​​the target substance in the plasma sample into the standard curve equation obtained by the standard solution.

[0046] The data acquisition instrument system used in this invention mainly includes high-performance liquid chromatography (HPLC) (Jasper). TM The assay was performed using tandem mass spectrometry (MS / MS) (AB SCIEX Triple Quad™ 4500MD). Mass spectrometry detection parameters are shown in Table 1. Multiple reaction monitoring parameters and quantitative internal standards are shown in Table 2 (Scheduled Multiple Reaction Monitoring, sMRM). Liquid chromatography detection conditions are shown in Table 3. Liquid chromatography elution gradients are shown in Table 4. Detection results are as follows: Figure 1 As shown.

[0047] Table 1

[0048] Ion Mode - Curtain Gas 10 IS -4500 Temperature 500 Ion Source Gas1 55 Ion Source Gas2 45 Collision Gas 8 Scan type MRM

[0049] Table 2

[0050] Analytes Mother ion daughter ions Acquisition window (s) α-Amanita peptide 917.2 899.0 30 β-Amanita peptide 918.2 900.3 30 γ-Amanita peptide 901.2 883.3 30 Dihydroxypharmacin 787.7 743.5 30

[0051] Table 3

[0052] chromatographic column Kinetex 2.6μm XB-C18 100A, LC Column 50x3mm Mobile phase A 0.01% formic acid solution Mobile phase B methanol Needle washing solution Acetonitrile:water = 1:1 Column temperature 45℃ Flow rate 0.300 mL / min Injection volume 3uL

[0053] Table 4

[0054] Time (min) Flow (mL / min) A(%) B(%) 0.00 0.30 99.0 1.0 5.00 0.30 10.0 90.0 7.00 0.30 10.0 90.0 8.00 0.30 90.0 10.0

[0055] Example 2 Methodological Validation

[0056] (1) Linear range and linear equation

[0057] The standard curves configured above were tested according to the test conditions of Example 1, and the linear ranges and linear equations of each amatoxin were obtained as shown in Table 5.

[0058] Table 5

[0059] Analytes Linear range (ng / mL) Linear equations Correlation coefficient α-Amanita peptide 8-400 Y = 0.0000965X + 0.000312 0.9943 β-Amanita peptide 8-400 Y = 0.000107X + 0.000005 0.9959 γ-Amanita peptide 8-400 Y = 0.00044X + 0.000154 0.9934 Dihydroxypharmacin 8-400 Y = 0.000112X + 0.000361 0.9941

[0060] (2) Recovery rate

[0061] To determine the concentrations of various indicators in plasma, a human sample with a known concentration (0.00 ng / mL) was selected as the original sample. Different volumes of standard solution (less than 10% of the total volume) were added to this sample to prepare three spiked plasma samples with high, medium, and low concentrations. Each spiked sample was independently measured five times. The recovery rate was calculated by measuring the concentrations in the plasma before and after spiking to evaluate the accuracy of this method. The recovery rates of various amatoxins in plasma are shown in Tables 6-9.

[0062] Table 6. Recovery rates of α-amanita peptides in plasma

[0063]

[0064] Table 7 Recovery rates of β-Amanita peptides in plasma

[0065]

[0066] Table 8 Recovery rates of γ-amatoxins in plasma

[0067]

[0068] Table 9. Recovery rates of spiked dihydroxyphalantin in plasma

[0069]

[0070] (3) Precision

[0071] Intra-batch precision: Within the same analytical batch (on the same day), samples at high, medium, and low concentration levels were tested five times independently.

[0072] Inter-batch precision: Samples at high, medium, and low concentration levels were tested independently 5 times per day, with 3 batches tested, resulting in a total of 45 test data points. Precision is shown in Tables 10-13.

[0073] Table 10 Precision of α-Amanita peptides in plasma

[0074]

[0075] Table 11 Precision of β-Amanita peptides in plasma

[0076]

[0077] Table 12 Precision of γ-Amanita peptides in plasma

[0078]

[0079] Table 13 Precision of dihydroxyphalantide in plasma

[0080]

[0081] In summary, the above experiments verify that the linearity, recovery rate, and precision of the detection method in Example 1 all meet the requirements, and accurate results can be obtained by using the method of the present invention to detect amatoxins in plasma.

[0082] Comparative Example 1

[0083] Take 40 ng / mL of intermediate standard solutions of α-amatoxins, β-amatoxins, γ-amatoxins, and dihydroxyphalangeal peptide and add them to the blank matrix and mix well. The blank matrix is ​​mixed plasma that has not been acidified. The rest is the same as in Example 1.

[0084] The detection results of β-amanita peptide in Comparative Example 1 are as follows: Figure 2 As shown, the detection results of dihydroxyphalazoline in Comparative Example 1 are as follows: Figure 3 As shown, from Figure 1 and Figure 2 The results comparison shows that the detection method in Example 1 has better separation and higher response for β-amatoxins. Figure 3 and Figure 1 The results show that the detection method of Example 1 detected a higher response of dihydroxyphalazotoxin than that of Comparative Example 1.

[0085] Comparative Example 2

[0086] The mobile phase A in Example 1 was replaced with 5 mmol / L ammonium acetate, and the rest was the same as in Example 1.

[0087] The comparison graph of the test results with those of Example 1 is shown below. Figures 4-7 As shown in the results, the ammonium acetate mobile phase significantly inhibited the mass spectrometry signal of amatoxins, and the resolution of the mobile phase used was not as good as that of the formic acid water in Example 1 (0.01%).

[0088] The molecular weights of α-amanita and β-amanita differ by only 1 Da. The M+1 isotope peak of α-amanita interferes with the detection of β-amanita, thus requiring chromatographic separation for effective separation. Commonly used additives in the mobile phase under negative ion mode include ammonia and ammonium acetate, as reported in the literature (…). [J]. Chromatography, 2022, 40(05):443-451. Ammonia, as an additive, significantly enhances the mass spectrometry signal of amatoxins, but the mass spectrometry response is highly unstable. During our research, we found that the addition of formic acid or ammonium acetate inhibited the response, but the inhibitory effect weakened as the formic acid concentration decreased. Simultaneously, the separation between α-amatoxins and β-amatoxins gradually improved. Therefore, a lower concentration of formic acid (0.01%) was chosen, which both improved the separation between α-amatoxins and β-amatoxins while meeting the required sensitivity.

[0089] Of course, the above description is only a specific embodiment of this application and is not intended to limit the scope of the invention. All equivalent changes or modifications made in accordance with the features and principles described in the claims of this invention should be included in the scope of the claims of this invention.

Claims

1. A method for simultaneously detecting multiple amatoxins, characterized in that, Includes the following steps: S1: Take a plasma sample, add concentrated hydrochloric acid, shake and mix for 5-15 minutes; add acetonitrile methanol solution with a volume ratio of 9:1, shake and mix, centrifuge and take the supernatant to obtain the sample solution to be tested; S2: The amatoxin in the sample solution to be tested was detected by liquid chromatography-mass spectrometry. The chromatographic separation conditions of the liquid chromatography-mass spectrometry method are as follows: chromatographic column: Kinetex 2.6μm XB-C18 100A, LC Column 50x3mm; mobile phase: phase A is 0.01% formic acid water, phase B is methanol, gradient elution; The mass spectrometry conditions for the liquid chromatography-mass spectrometry method are as follows: ESI ion source; scanning mode: negative ion scanning mode; ionization voltage: -4500V; temperature: 500℃; spray gas: 55psi; auxiliary heating gas: 45psi. The gradient elution procedure is as follows: ； The various amatoxins are composed of α-amatoxin, β-amatoxin, γ-amatoxin, and dihydroxyphalazotoxin.

2. The method for simultaneously detecting multiple amatoxins according to claim 1, characterized in that, The parent ion-daughter ion pairs in the liquid chromatography-mass spectrometry method are as follows: α-Amanita peptide, m / z 917.2→899.0; β-Amanita peptide, m / z 918.2→900.3; γ-Amanita peptide, m / z 901.2→883.3; and dihydroxyphalangeal peptide, m / z 787.7→743.

5.

3. The method for simultaneously detecting multiple amatoxins according to claim 1, characterized in that, The detection method is a quantitative detection using the external standard method.

4. The method for simultaneously detecting multiple amatoxins according to claim 3, characterized in that, The external standard method for quantitative detection includes the following steps: a) Prepare a standard solution of amatoxins; b) The amatoxin standard solution was measured using the liquid chromatography-mass spectrometry method, and the chromatographic peak area of ​​each standard solution was recorded. c) Plot a standard curve with the concentration of the standard solution on the x-axis and the corresponding peak area on the y-axis; d) Substitute the chromatographic peak area of ​​the target analyte in the sample solution to be tested into the standard curve to calculate the content of amatoxin in the sample solution to be tested.

5. The method for simultaneously detecting multiple amatoxins according to claim 4, characterized in that, The standard solution in step a) is prepared using an acidified blank matrix; the blank matrix is ​​plasma that does not contain the target amatoxin.

Images