Liquid chromatography tandem mass spectrometry detection method for catecholamine and metabolite thereof

Abstract

The invention discloses a liquid chromatography-tandem mass spectrometry detection method for catecholamine and metabolites thereof, which comprises the following steps: step 1, sequentially adding a sample to be detected, an internal standard solution, a derivatization buffer solution and a derivatization solution into holes in the third / ninth column of a 96-well plate, oscillating at room temperature, replenishing water, and continuing oscillating; step 2, adding an activating solution and magnetic beads into the first / seventh column of the 96-well plate; adding equilibrium liquid into the second / eighth column; a first leacheate is added into the fourth column and the tenth column; adding a second leacheate in the fifth / eleventh column; adding the eluent into the 6 / 12th column; then placing the sample on a 32-channel sample pretreatment system for magnetic solid-phase extraction; and step 3, liquid chromatography-tandem mass spectrometry detection. According to the method, the sample is firstly derivatized and then is subjected to magnetic solid-phase extraction, so that only less sample size is needed, the sensitivity is higher, and the economical efficiency is better; according to the method for directly derivatizing the sample, the sample does not need to be derivatized after being subjected to protein precipitation and supernatant removal, and the steps are simpler, more convenient and more automatic.

Description

Technical Field

[0001] The present invention relates to the technical field of catecholamine and metabolite detection, and particularly to a liquid chromatography tandem mass spectrometry detection method for catecholamine and its metabolites. Background Art

[0002] Catecholamine is a kind of neuroactive substance containing catechol and amine groups, mainly including norepinephrine (NE), epinephrine (E) and dopamine (DA). Through a series of metabolic processes in organisms, these catecholamine substances will produce a variety of metabolites, including 3-Methoxytyramine (3-MT), Normetanephrine (NMN), Metanephrine (MN), etc.

[0003] Catecholamine and its metabolites have important clinical significance. Diseases with disorders of the catecholamine system include Parkinson's disease, depression, heart failure, hypertension and other neurocirculatory diseases. Currently, the detection of catecholamine and its metabolites is mainly used for the screening and diagnosis of tumors that produce catecholamine. For example, Normetanephrine and Metanephrine are recognized as the gold standard for the clinical diagnosis of pheochromocytomas and paragangliomas (PPGLs). Detecting 3-MT can improve the screening sensitivity of head and neck paragangliomas. When combining the detection of catecholamine metabolites and prototype substances, personalized information on disease development and treatment effects can be provided.

[0004] The concentrations of catecholamine and its metabolites in biological samples are as low as the level of dozens of pg / mL. Moreover, the catechol group on catecholamine compounds has unstable chemical properties, and it can be degraded by air, light, heat, and metal ions such as copper and iron. In addition, there are interferences from endogenous compounds in biological samples. Therefore, how to quickly and sensitively detect catecholamine and its metabolites is a major pain point in clinical practice. Compared with conventional biochemical immunoassay methods, liquid chromatography tandem mass spectrometry (LC-MS / MS) has higher detection sensitivity, stronger specificity, can basically eliminate the interference of structurally similar substances, and can detect multiple indicators simultaneously, covering the detection indicators recommended by clinical guidelines.

[0005] Patent CN116482248A, with the invention title "Detection Kit for Catecholamines and Their Metabolites in Plasma / Urine, Detection Method and Application Thereof", discloses a method for directly detecting 6 compounds in plasma and urine by magnetic solid-phase extraction and then directly loading them onto the machine for detection. This method uses a non-derivatization method, but the sample volume is 500 μL. Although the quantification limit meets the requirements for the auxiliary diagnosis of catecholamines, the large sample volume will increase sample consumption, increase the reagent dosage during pretreatment, and also increase the risk of sample cross-contamination. Summary of the Invention

[0006] In view of the problems in the existing catecholamine detection methods, such as large sample volume, harsh conditions for derivatization methods, cumbersome steps, high operation difficulty, difficulty in automation, and being not conducive to clinical promotion, the present invention provides a liquid chromatography-tandem mass spectrometry detection method for 6 catecholamines and their metabolites with a small amount of biological samples, which performs magnetic solid-phase extraction after derivatization at room temperature. This method is automated, highly specific, highly sensitive, and has a large detection throughput.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] Provide a liquid chromatography-tandem mass spectrometry detection method for catecholamines and their metabolites, wherein the catecholamines and their metabolites include dopamine (DA), epinephrine (E), norepinephrine (NE), 3-methoxytyramine (3-MT), metanephrine (MN), and normetanephrine (NMN);

[0009] The specific steps of the detection method are as follows:

[0010] Step 1, sample derivatization: Sequentially add the sample to be detected, internal standard solution, derivatization buffer solution, and derivatization solution into the wells in the 3rd / 9th columns of a 96-well plate, oscillate at room temperature, add water to make up the volume and continue to oscillate, and then set aside for use;

[0011] Step 2, magnetic solid-phase extraction: Add activation solution and magnetic beads to the 1st / 7th columns of the 96-well plate in Step 1; add equilibration solution to the 2nd / 8th columns; add the first eluent to the 4th / 10th columns; add the second eluent to the 5th / 11th columns; add elution solution to the 6th / 12th columns; place the 96-well plate after sample addition onto a 32-channel sample pretreatment system for magnetic solid-phase extraction;

[0012] Step 3, liquid chromatography-tandem mass spectrometry detection: Take out the 96-well plate after sample pretreatment in Step 2, dry the eluate in the 6th / 12th columns under nitrogen, re-dissolve it, and load it onto the machine for detection.

[0013] Furthermore, in Step 1, the sample to be detected includes calibration curve samples, quality control samples, and samples to be detected at different concentrations, and the sample volume is 100 μL.

[0014] Further, in the first step, the derivatization buffer is one of potassium hydrogen phosphate - disodium ethylenediaminetetraacetate solution, potassium hydrogen phosphate - potassium dihydrogen phosphate solution, and sodium carbonate - sodium bicarbonate solution, and its pH value is 8 - 10.

[0015] Further, in the first step, the derivatization solution is an acetonitrile solution of propionic anhydride, and the content of propionic anhydride is 5 - 85 v / v%.

[0016] Further, in the first step, the volume ratio of the sample to be tested, the internal standard solution, the derivatization buffer, and the derivatization solution is (2 - 5):1:(2 - 5):1.

[0017] Further, in the first step, the oscillation speed at room temperature is 500 - 1000 rpm, the time is 5 - 10 min, the added water volume is 50 - 100 μL, and the oscillation continues for 1 - 3 min.

[0018] Further, in the second step, the magnetic beads are hydrophilic - lipophilic balanced magnetic solid - phase extraction magnetic beads, and the added amount of magnetic beads is 1 - 3 mg; the activation solution is methanol; the equilibration solution is purified water; the first eluent is a 5 - 15 v / v% methanol aqueous solution; the second eluent is a 20 - 40 v / v% methanol aqueous solution; the elution solution is one of methanol, acetonitrile, and isopropanol.

[0019] Further, the usage amounts of the activation solution, the equilibration solution, the first eluent, and the second eluent are 200 - 500 μL; the usage amount of the elution solution is 100 - 300 μL.

[0020] Further, in the third step, the reconstitution solution is a 30 - 50 v / v% methanol aqueous solution containing 0.05 - 0.5 v / v% formic acid, and its volume is 100 - 200 μL.

[0021] Further, in the third step, the program of magnetic solid - phase extraction is as follows:

[0022]

[0023] The present invention adopts the above - mentioned technical solutions, and compared with the prior art, has the following technical effects:

[0024] 1. The method of the present invention first derivatizes the sample and then performs magnetic solid - phase extraction. After directly derivatizing 100 μL of the sample, the target compound is separated and purified by magnetic solid - phase extraction. For the existing non - derivatization or derivatization methods, the sample usage amount is 200 - 500 μL. The larger the sample usage amount, the more serious the endogenous interference and the more the amount of pretreatment reagents. The present invention only requires a smaller sample amount, has higher sensitivity and better economy; the method of directly derivatizing the sample does not need to first precipitate the protein from the sample, transfer the supernatant, and then derivatize, and the steps are simpler.

[0025] 2. The present invention adopts a magnetic solid phase extraction sample pretreatment system, which reduces the manual operation process and is more conducive to standardization. Subsequently, a 96-channel sample pretreatment system is used, and the sample throughput can be further improved.

[0026] 3. In the prior art, dansyl chloride derivatization requires high temperature; although acetaldehyde derivatization is carried out at room temperature, the sample consumption is large and the detection sensitivity is low, which cannot meet the clinical detection requirements. The derivatization method adopted by the method of the present invention not only has a small sample consumption, but also can detect 6 catecholamine compounds simultaneously during derivatization at room temperature, and the detection sensitivity also meets the trace detection requirements of catecholamine compounds.

[0027] 4. The direct derivatization of the sample in the present invention can reduce the influence of intermediate treatment steps on the stability of catecholamine substances, and the derivatized compounds are more stable. Brief Description of the Drawings

[0028] Figure 1 It is a spectrogram of the detection result of dopamine (DA).

[0029] Figure 2 It is a spectrogram of the detection result of epinephrine (E).

[0030] Figure 3 It is a spectrogram of the detection result of norepinephrine (NE).

[0031] Figure 4 It is a spectrogram of the detection result of 3-methoxytyramine (3-MT).

[0032] Figure 5 It is a spectrogram of the detection result of metanephrine (MN).

[0033] Figure 6 It is a spectrogram of the detection result of normetanephrine (NMN). Detailed Description of the Embodiments

[0034] The present invention will be further described below in conjunction with the drawings and specific embodiments, but it is not limited to the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0035] Example 1

[0036] This example provides a liquid chromatography-tandem mass spectrometry detection method for catecholamines and their metabolites, including the following steps:

[0037] (1) Preparation of standard curves and quality control samples

[0038] Dilute the primary reference standards of catecholamines and their metabolites (DA, NE, E, NMN, MN, 3-MT) with a 0.1% VC methanol solution to prepare an intermediate solution for later use. Dilute the intermediate solution and catecholamine blank human serum in different proportions to obtain calibration standards and quality control samples. The linear ranges of DA, NMN, and MN are 10 - 2000 pg / mL, the linear ranges of NE and E are 15 - 3000 pg / mL, and the linear range of 3-MT is 5 - 1000 pg / mL.

[0039] (2) Preparation of Pretreatment Reagents

[0040] Internal standard solution: Prepared by diluting dopamine-D4, norepinephrine-D6, epinephrine-D6, 3-methoxytyramine-D4, normetanephrine-D3, and metanephrine-D3 with 0.1% VC methanol;

[0041] Derivatization buffer: Potassium hydrogen phosphate - Potassium dihydrogen phosphate, pH = 8;

[0042] Derivatization solution: 50% propionic anhydride acetonitrile solution;

[0043] Activation solution: Methanol;

[0044] Equilibration solution: Purified water;

[0045] First eluent: 10% methanol aqueous solution;

[0046] Second eluent: 30% methanol aqueous solution;

[0047] Elution solution: Methanol;

[0048] Reconstitution solution: 50% methanol aqueous solution containing 0.1% formic acid.

[0049] (3) Sample Pretreatment Process

[0050] 1) Sample Derivatization

[0051] Take 100 μL each of the different concentration standard samples, quality control samples, and samples to be tested obtained in step (1) and add them to the 3rd / 9th columns of a 96-well plate. Then, successively add 50 μL of the internal standard solution, 100 μL of the derivatization buffer, 50 μL of the derivatization solution, shake at 1000 rpm for 10 min, add 100 μL of purified water, and continue shaking for 3 min for later use.

[0052] 2) Magnetic Solid Phase Extraction

[0053] Addition of solutions for magnetic solid-phase extraction in 96-well plates: Add 200 μL of activation solution and 2 mg of hydrophilic-lipophilic balanced magnetic solid-phase extraction magnetic beads to the 1st / 7th columns of the 96-well plates in step 1) above; add 200 μL of equilibration solution to the 2nd / 8th columns; add 200 μL of eluent 1 to the 4th / 10th columns; add 200 μL of eluent 2 to the 5th / 11th columns; add 200 μL of elution solution to the 6th / 12th columns. After sample addition, place the 96-well plates on a 32-channel sample pretreatment system for magnetic solid-phase extraction, and the extraction procedure is shown in Table 1.

[0054] Table 1

[0055]

[0056] 3) Dry the elution solution in the 6th / 12th columns of the 96-well plates in step 2) above under nitrogen, and redissolve it with 100 μL of reconstitution solution for subsequent injection.

[0057] (4) Use a liquid chromatography-tandem mass spectrometry detection system, LC / MS / MS System, model: AB SCIEXTriple Quad TM 4500MD for analysis and detection.

[0058] 1) Chromatographic conditions:

[0059] Chromatographic column: F5 2.6 μm LC Column 100x3.0 mm, Ea (Phenomenex) or equivalent;

[0060] Mobile phase A: Aqueous solution containing 0.1% formic acid and 2 mM ammonium acetate;

[0061] Mobile phase B: Methanol solution containing 0.1% formic acid and 2 mM ammonium acetate;

[0062] Injection volume: 20 μL;

[0063] Column temperature: 40 °C;

[0064] Liquid phase gradient (Table 2):

[0065] Table 2

[0066]

[0067] 2) Mass spectrometry conditions

[0068] Ionization Mode, ESI(+); Scan Mode, MRM; Curtain Gas, 40; Temperature, 550 °C; Collision Gas, Medium; Gas 1, 50; Gas 2, 50; IonSpray Voltage, 4500.

[0069] MRM Ion Channels (Table 3):

[0070] Table 3

[0071]

[0072]

[0073] The spectra of the detection results are as Figures 1-6 shown. It can be seen that the peak shapes of each compound are symmetric and sharp, and there are no obvious interfering peaks around the peaks.

[0074] Example 2

[0075] Verify the method of Example 1 above.

[0076] (1) Fitting of the calibration curve

[0077] Using the theoretical concentrations of the calibration standards of 6 catecholamines and their metabolites as the X-axis and the ratio of the peak areas of the calibration standards and the internal standard as the Y-axis, a calibration curve was plotted using the least squares method with a weighting factor of 1 / (x*x). Table 4 below shows the fitted linear equations and regression coefficients.

[0078] Table 4

[0079]

[0080] (2) Limit of quantitation

[0081] The reference intervals of catecholamine substances in clinical practice are shown in Table 5. The limit of quantitation that can be achieved by this method on an AB SCIEX TripleQuad TM 4500 MD instrument with a sample volume of 100 μL is shown in Table 6. By using the derivatization method, the ionization efficiency of the target compounds is significantly increased, enabling the limit of quantitation to meet the accurate quantification of trace catecholamine compounds in plasma.

[0082] Table 5

[0083]

[0084]

[0085] Table 6

[0086]

[0087] (3) Accuracy and precision

[0088] High, medium, and low concentration samples were prepared by spiking in a catecholamine blank human serum matrix, and accuracy and precision verification was carried out for three consecutive batches. The results are shown in Table 7. The within - batch and between - batch precision of catecholamines and their metabolites ≤15%, and the accuracy was between 85 - 115%.

[0089] Table 7

[0090]

[0091]

[0092] (4) Stability of the derivatized samples

[0093] Catecholamines and their metabolites are unstable under non - derivatized conditions and can only be stable for 1 day under refrigerated storage at 2 - 8°C in the dark. After derivatization, their stability can be significantly improved. Table 8 shows the stability results of the samples to be analyzed by HPLC at 2 - 8°C, indicating that the derivatized samples can be stable for 3 days.

[0094] Table 8

[0095]

[0096] The above are only the preferred embodiments of the present invention, and do not limit the implementation manners and protection scope of the present invention. Those skilled in the art should be able to realize that all equivalent substitutions and obvious changes made by using the content of the present invention's specification and drawings should be included in the protection scope of the present invention.

Claims

1. A method for detecting catecholamines and their metabolites by liquid chromatography tandem mass spectrometry, characterized in that: The catecholamines and their metabolites include dopamine, epinephrine, norepinephrine, 3-methoxytyramine, metanephrine, and methyloxynorepinephrine; The detection method specifically comprises the following steps: Step 1, sample derivatization: add the sample to be tested, internal standard solution, derivatization buffer, and derivatization solution to the wells in the 3 / 9 column of a 96-well plate in sequence, shake at room temperature, add water and continue shaking for later use; Step 2, magnetic solid phase extraction: add activation solution and magnetic beads to the 1 / 7 column of the 96-well plate in step 1; add balancing solution to the 2 / 8 column; add the first eluent to the 4 / 10 column; add the second eluent to the 5 / 11 column; add the elution solution to the 6 / 12 column; place the 96-well plate after sample addition on a 32-channel sample pretreatment system for magnetic solid phase extraction; Step 3, liquid chromatography tandem mass spectrometry detection: take out the 96-well plate after the sample pretreatment in step 2, blow dry the eluent in the 6 / 12 column under nitrogen, then re-dissolve it and put it on the machine for detection.

2. The method for detecting catecholamines and their metabolites by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In the step 1, the samples to be tested include calibration curve samples of different concentrations, quality control samples and samples to be tested, and the sample volume is 100 μL.

3. The method for detecting catecholamines and their metabolites by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: In the step 1, the derivatization buffer is one of a dipotassium hydrogen phosphate-disodium ethylenediaminetetraacetic acid solution, a dipotassium hydrogen phosphate-potassium dihydrogen phosphate solution, and a sodium carbonate-sodium bicarbonate solution, and its pH value is 8-10.

4. The method for detecting catecholamines and their metabolites by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In the step 1, the derivatization solution is an acetonitrile solution of propionic anhydride, and the content of propionic anhydride is 5-85 v / v%.

5. The method for detecting catecholamines and their metabolites by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: In the step 1, the volume ratio of the sample to be tested, the internal standard solution, the derivatization buffer solution and the derivatization solution is (2-5):1:(2-5):

1.

6. The method for detecting catecholamines and their metabolites by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In the step 1, the oscillation speed is 500-1000 rpm at room temperature for 5-10 min, 50-100 μL of water is added, and the oscillation is continued for 1-3 min.

7. The method for detecting catecholamines and their metabolites by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: In the step 2, the magnetic beads are hydrophilic-lipophilic balanced magnetic solid phase extraction magnetic beads, and the amount of magnetic beads added is 1-3 mg; the activation solution is methanol; the balance solution is purified water; the first eluent is a 5-15 v / v% methanol aqueous solution; the second eluent is a 20-40 v / v% methanol aqueous solution; and the eluent is one of methanol, acetonitrile, and isopropanol.

8. The method for detecting catecholamines and their metabolites by liquid chromatography tandem mass spectrometry according to claim 7, characterized in that: The usage amount of the activation solution, the balancing solution, the first eluent and the second eluent is 200-500 μL; the usage amount of the elution solution is 100-300 μL.

9. The method for detecting catecholamines and their metabolites by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: In the step 3, the reconstitution solution is a 30-50 v / v% methanol aqueous solution containing 0.05-0.5 v / v% formic acid, and its volume is 100-200 μL.

10. The method for detecting catecholamines and their metabolites by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In the step 3, the procedure of magnetic solid phase extraction is:

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