Method for synchronously detecting eight catecholamines and metabolites thereof based on liquid chromatography-tandem mass spectrometry and application of method

Abstract

The invention provides a method for synchronously detecting eight catecholamines and metabolites thereof based on liquid chromatography-tandem mass spectrometry and application of the method. The detection method comprises the following steps: mixing a plasma or urine sample to be detected with an internal standard and acetonitrile; centrifuging, taking supernate, blowing liquid with nitrogen, and adjusting the pH value; mixing the supernate with a propionic anhydride solution, and carrying out derivatization reaction to obtain a test solution; performing liquid chromatography-tandem mass spectrometry detection on the test solution and the standard solution, separating dopamine, epinephrine, noradrenaline, epinephrine, noradrenaline, 3-methoxytyramine, homovanillic acid and vanillic mandelic acid by liquid chromatography, and determining the concentration of the standard substance as an X axis by using a mass spectrometry isotope internal standard quantification method. The ratio of the peak area of the standard substance to the peak area of the internal standard substance is the Y axis, establishing a standard curve, and calculating the concentrations of catecholamine and the metabolites thereof in the test solution. The method is high in detection efficiency, high in sensitivity and good in equipment compatibility, the detection time is shortened, and the cost can also be saved.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and particularly relates to a method for simultaneously detecting 8 catecholamines and their metabolites based on liquid chromatography-tandem mass spectrometry and its application. Background Art

[0002] Catecholamines (CAs) and their metabolites (such as metanephrine, vanillylmandelic acid, etc.) are important biomarkers for diagnosing diseases such as pheochromocytoma, paraganglioma, and neuroblastoma. Currently, the methods for detecting catecholamines and their metabolites mainly include: (1) Immunoassay: It can only detect terminal metabolites (such as homovanillic acid, vanillylmandelic acid), and has problems such as poor specificity, low sensitivity, and serious matrix interference (Expert Consensus on the Diagnosis and Treatment of Pheochromocytoma and Paraganglioma, Chinese Journal of Endocrinology and Metabolism, 2016). (2) Liquid chromatography-tandem mass spectrometry (LC-MS / MS): Although it partially solves the defects of immunoassay, it requires solid-phase extraction (SPE) pretreatment, which is complex and time-consuming (2 - 4 hours), and at most only 6 substances can be simultaneously detected (DA, E, NE, MN, NMN, 3-MT), and it cannot cover all 8 target substances (HVA and VMA need secondary treatment).

[0003] CN201610907368.5 discloses a method for detecting catecholamines in plasma by liquid chromatography-tandem mass spectrometry. The method includes protein precipitation with acetonitrile, derivatization reaction with dansyl chloride, and detection using a liquid chromatography-tandem triple quadrupole mass spectrometer after extraction; this method can detect 3 catecholamines of dopamine, epinephrine, and norepinephrine.

[0004] CN202110198023.8 discloses a method for detecting the content of catecholamine substances in human plasma, which includes the following steps: (1) preparing an internal standard solution; (2) derivatization and protein precipitation; (3) solid-liquid extraction; (4) on-machine detection. This method can detect 6 substances such as norepinephrine, epinephrine, dopamine, metanorepinephrine, metanephrine, and 3-methoxytyramine.

[0005] The existing liquid chromatography-tandem mass spectrometry method cannot simultaneously detect all 8 substances in one injection, and needs to be processed step by step, with low efficiency; the solid-phase extraction step is cumbersome, requires professional operation, is prone to errors and time-consuming (2 - 4 hours); the prototype compound has low mass spectrometry response, and low-concentration samples rely on mid- to high-end equipment, and the result stability is poor. Therefore, it is of important application value to provide a method with simple operation for simultaneously detecting 8 catecholamines and their metabolites. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a synchronous detection method and its application for 8 catecholamines and their metabolites based on liquid chromatography-tandem mass spectrometry. The present invention provides a detection method with simple operation and short pretreatment time (≤1 hour), realizing the synchronous detection of 8 catecholamines and their metabolites.

[0007] To achieve the purpose of this invention, the following technical solutions are adopted:

[0008] In the first aspect, the present invention provides a synchronous detection method for 8 catecholamines and their metabolites based on liquid chromatography-tandem mass spectrometry, and the detection method includes:

[0009] (1) Mix the plasma or urine sample to be tested with the internal standard and acetonitrile; centrifuge to take the supernatant and blow it with liquid nitrogen, adjust the pH; mix the supernatant with the propionic anhydride solution for derivatization reaction to obtain the test solution;

[0010] (2) Perform liquid chromatography-tandem mass spectrometry detection on the test solution and the standard solution. Among them, liquid chromatography separates dopamine, epinephrine, norepinephrine, metanephrine, normetanephrine, 3-methoxytyramine, homovanillic acid, and vanilmandelic acid, and then uses the mass spectrometry isotope internal standard quantification method. With the concentration of the standard product as the X-axis and the ratio of the peak area of the standard product to the internal standard as the Y-axis, establish a standard curve, and calculate the concentration of 8 catecholamines and their metabolites in the test solution;

[0011] The mobile phase used in liquid chromatography includes: phase A is 0.1% formic acid aqueous solution, and phase B is 0.1% formic acid methanol solution; the elution program used in liquid chromatography is:

[0012] In the 0-3 minutes, the volume fraction of mobile phase A changes uniformly from 60-61% (for example, it can be 60% or 61% etc.) to 40-41% (for example, it can be 40% or 41% etc.), and the rest is mobile phase B;

[0013] In the 3-3.1 minutes, the volume fraction of mobile phase A changes rapidly from 40-41% (for example, it can be 40% or 41% etc.) to 1-2% (for example, it can be 1% or 2% etc.), and the rest is mobile phase B;

[0014] In the 3.1-4.0 minutes, the volume fraction of mobile phase A is maintained at 1-2% (for example, it can be 1% or 2% etc.), and the rest is mobile phase B;

[0015] In the 4.0-4.1 minutes, the volume fraction of mobile phase A changes rapidly from 1-2% (for example, it can be 1% or 2% etc.) to 60-61% (for example, it can be 60% or 61% etc.), and the rest is mobile phase B;

[0016] From the 4.1st to the 5.0th minute, the volume fraction of mobile phase A is maintained at 60 - 61% (for example, it can be 60% or 61%, etc.), and the rest is mobile phase B.

[0017] The derivatization reagent used in the present invention is propionic anhydride. Using propionic anhydride for derivatization enhances the ionization efficiency of phenolic hydroxyl groups, can improve the detection sensitivity (the sensitivity is increased by 5 - 10 times), is suitable for mid - to - low - end equipment, and has a broader application prospect.

[0018] The present invention simplifies the pretreatment steps, cancels solid - phase extraction, and only requires acetonitrile extraction and nitrogen blowing. The steps are reduced to 4 steps, greatly improving the pretreatment efficiency; the pretreatment time is within 1 hour, and the detection throughput is increased by 50%.

[0019] The present invention optimizes the elution gradient. Through rapid gradient elution (only 5 minutes), efficient separation of 8 substances is achieved, avoiding matrix interference.

[0020] Preferably, in step (1), the internal standard is the isotope internal standard of 8 catecholamines and their metabolites, and the concentrations of the isotope internal standards corresponding to dopamine, epinephrine, norepinephrine, metanephrine, normetanephrine, 3 - methoxytyramine, homovanillic acid, and vanillylmandelic acid are 160 pg / mL, 800 pg / mL, 800 pg / mL, 800 pg / mL, 400 pg / mL, 400 pg / mL, 160 ng / mL, and 160 ng / mL respectively.

[0021] Preferably, in step (1), the volume ratio of the sample, internal standard, and acetonitrile is (5 - 6):1:(8 - 10). Among them, "5 - 6" can be 5, 5.5, or 6, etc.; "8 - 10" can be 8, 9, or 10.

[0022] In the present invention, if the proportion of acetonitrile is too large, it will dilute the sample and affect the final response; if the proportion of acetonitrile is too small, the matrix interference cannot be removed completely, affecting the final detection.

[0023] Preferably, in step (1), the centrifugation speed is 5000 - 6000 rpm, for example, it can be 5000 rpm, 5500 rpm, or 6000 rpm, etc., the time is 5 - 10 minutes, for example, it can be 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes, etc., and the temperature is 4 - 20 °C, for example, it can be 4 °C, 5 °C, 10 °C, 15 °C, or 20 °C, etc.

[0024] In the present invention, if the centrifugation speed is too small, the impurities cannot be removed cleanly, affecting the final detection; if the temperature is too high, it will affect the stability of the sample.

[0025] Preferably, in step (1), the time of nitrogen blowing is 15 - 20 minutes, for example, it can be 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes or 20 minutes, etc.

[0026] In the present invention, nitrogen blowing is to minimize the solvent and increase the component concentration; if the nitrogen blowing time is too short, the solvent ratio will be too large, which will affect the subsequent derivatization effect and the final response.

[0027] Preferably, in step (1), the concentration of the propionic anhydride solution is 5 - 25%, for example, it can be 5%, 10%, 15%, 20% or 25%, etc.

[0028] In the present invention, too high or too low concentration of propionic anhydride will directly affect the derivatization effect and reduce the final response.

[0029] Preferably, the volume ratio of the supernatant to the propionic anhydride solution is (5 - 6):1, where "5 - 6" can be 5, 5.5 or 6, etc.

[0030] Preferably, the time of the derivatization reaction is 5 - 10 minutes, for example, it can be 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes or 10 minutes, etc.

[0031] In the present invention, if the derivatization time is too short, the derivatization will be incomplete, affecting the derivatization effect and repeatability.

[0032] Preferably, in step (2), the standard solution includes: dopamine, epinephrine, norepinephrine, metanephrine, normetanephrine, 3 - methoxytyramine, homovanillic acid and vanilmandelic acid.

[0033] Preferably, the concentration range of dopamine in the standard solution is: 10-640 pg / mL, and the concentration gradients are 10 pg / mL, 20 pg / mL, 40 pg / mL, 80 pg / mL, 160 pg / mL, 320 pg / mL, and 640 pg / mL; the concentration range of epinephrine is: 50-3200 pg / mL, and the concentration gradients are 50 pg / mL, 100 pg / mL, 200 pg / mL, 400 pg / mL, 800 pg / mL, 1600 pg / mL, and 3200 pg / mL; the concentration range of norepinephrine is: 50-3200 pg / mL, and the concentration gradients are 50 pg / mL, 100 pg / mL, 200 pg / mL, 400 pg / mL, 800 pg / mL, 1600 pg / mL, and 3200 pg / mL; the concentration range of metanephrine is: 50-3200 pg / mL, and the concentration gradients are 50 pg / mL, 100 pg / mL, 200 pg / mL, 400 pg / mL, 800 pg / mL, 1600 pg / mL, and 3200 pg / mL; the concentration range of normetanephrine is: 25-1600 pg / mL, and the concentration gradients are 25 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 400 pg / mL, 800 pg / mL, and 1600 pg / mL; the concentration range of 3-methoxytyramine is: 25-1600 pg / mL, and the concentration gradients are 25 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 400 pg / mL, 800 pg / mL, and 1600 pg / mL; the concentration range of homovanillic acid is: 10-640 ng / mL, and the concentration gradients are 10 ng / mL, 20 ng / mL, 40 ng / mL, 80 ng / mL, 160 ng / mL, 320 ng / mL, and 640 ng / mL; the concentration range of vanillylmandelic acid is: 10-640 ng / mL, and the concentration gradients are 10 ng / mL, 20 ng / mL, 40 ng / mL, 80 ng / mL, 160 ng / mL, 320 ng / mL, and 640 ng / mL.

[0034] Preferably, in step (2), the chromatographic column used in the liquid chromatography is a C18 reversed-phase column.

[0035] Preferably, in step (2), the chromatographic conditions of the liquid chromatography further include: column temperature: 48-50 °C, for example, it can be 48 °C, 49 °C, or 50 °C, etc., injection volume: 1-1.5 μL, for example, it can be 1 μL, 1.2 μL, or 1.3 μL, etc., flow rate: 0.4-0.5 mL / min, for example, it can be 0.4 mL / min, 0.45 mL / min, or 0.5 mL / min, etc.

[0036] Preferably, in step (2), the ionization mode of the mass spectrometry detection is electrospray ionization.

[0037] Preferably, in step (2), the ion source parameters of the mass spectrometry detection include: the spray voltage is 1.0 - 3.0 kV, for example, it can be 1.0 kV, 1.5 kV, 2.0 kV, 2.5 kV or 3.0 kV, etc.; the ion source temperature is 100 - 150 °C, for example, it can be 100 °C, 110 °C, 120 °C, 130 °C, 140 °C or 150 °C, etc.; the nebulizing gas temperature is 200 - 500 °C, for example, it can be 200 °C, 250 °C, 300 °C, 350 °C, 400 °C, 450 °C or 500 °C, etc.; the nebulizing gas flow rate is 400 - 900 L / h, for example, it can be 400 L / h, 500 L / h, 600 L / h, 700 L / h, 800 L / h or 900 L / h, etc.; the cone gas flow rate is 30 - 200 L / h, for example, it can be 30 L / h, 50 L / h, 100 L / h, 150 L / h or 200 L / h, etc.

[0038] In a second aspect, the present invention provides the application of the method for synchronous detection of 8 catecholamines and their metabolites based on liquid chromatography - tandem mass spectrometry described in the first aspect in detection and analysis.

[0039] The numerical ranges described in the present invention not only include the above - listed point values, but also include any point values between the above - mentioned numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention does not exhaustively list the specific point values included in the ranges.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] (1) High detection efficiency: The pretreatment time ≤ 1 hour, and the detection throughput is increased by 50%.

[0042] (2) High sensitivity: The lowest limit of quantification (LLOQ) is as low as 10 pg / mL (blood sample).

[0043] (3) High equipment compatibility: Medium - low - end mass spectrometers (such as AB Sciex 3200) can achieve stable detection (RSD < 10%).

[0044] (4) Cost savings: The time consumed for single - sample detection is reduced by 70%. Description of the Drawings

[0045] Figure 1 It is the chromatogram of the target substances DA, E and NE.

[0046] Figure 2 It is the chromatogram of the target substances MN, NMN and 3MT.

[0047] Figure 3It is the chromatogram of the target substances HVA and VMA.

[0048] Figure 4 It is the linear result graph of dopamine (DA).

[0049] Figure 5 It is the linear result graph of adrenaline (E).

[0050] Figure 6 It is the linear result graph of noradrenaline (NE).

[0051] Figure 7 It is the linear result graph of metanephrine (MN).

[0052] Figure 8 It is the linear result graph of normetanephrine (NMN).

[0053] Figure 9 It is the linear result graph of 3-methoxytyramine (3-MT).

[0054] Figure 10 It is the linear result graph of homovanillic acid (HVA).

[0055] Figure 11 It is the linear result graph of vanillylmandelic acid (VMA).

[0056] Figure 12 It is the dansyl chloride derivative spectrum.

[0057] Figure 13 It is the dansyl chloride derivative spectrum after standing for 6 hours.

[0058] Figure 14 It is the benzoyl chloride derivative spectrum.

[0059] Figure 15 It is the benzoyl chloride derivative spectrum after standing for 6 hours.

[0060] Figure 16 It is the PAA derivative spectrum.

[0061] Figure 17 It is the PAA derivative spectrum after standing for 24 hours. Detailed implementation manners

[0062] The technical solution of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0063] For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments not indicating the manufacturers, they are all conventional products that can be obtained through regular channels of commercial purchase.

[0064] The terms in the specification are described as follows:

[0065] LC-MS / MS: Liquid Chromatography-Tandem Mass Spectrometry.

[0066] PAA: Propionic Acid Anhydride.

[0067] RSD: Relative Standard Deviation.

[0068] Dopamine: Dopamine, with the English capital abbreviation DA.

[0069] Adrenaline: adrenaline, epinephrine, with the English capital abbreviation A or E.

[0070] Norepinephrine: Norepinephrine, also known as Noradrenaline, abbreviated as NE or NA.

[0071] Metanephrine: Metanephrine, with the English capital abbreviation MN.

[0072] Normetanephrine: Normetanephrine, with the English capital abbreviation NMN.

[0073] 3-Methoxytyramine: 3-Methoxytyramine, with the English capital abbreviation 3-MT.

[0074] Homovanillic acid: homovanillic acid, with the English capital abbreviation HVA.

[0075] Vanillymandelic Acid: Vanillymandelic Acid, abbreviated as VMA.

[0076] Example 1

[0077] 1. Sample treatment for blood sample detection

[0078] (1) Take 100 μL of plasma sample, add 20 μL of internal standard (a mixed solution of isotope internal standards for 8 catecholamines and their metabolites, with the corresponding concentrations of the internal standards being 160 pg / mL, 800 pg / mL, 800 pg / mL, 800 pg / mL, 400 pg / mL, 400 pg / mL, 160 ng / mL, and 160 ng / mL) and 200 μL of acetonitrile, and vortex for 10 minutes for extraction.

[0079] (2) Centrifuge (5000 rpm, 4 - 20 °C, 5 - 10 minutes), and take the supernatant and blow it with liquid nitrogen for 15 minutes.

[0080] (3) Add pH adjustment solution (50 μL of NaOH) and derivatization solution (PAA, 20 μL of 10% concentration), and vortex for 5 minutes.

[0081] 2. Sample treatment for urine sample detection

[0082] (1) Take 100 μL of urine sample, add 20 μL of internal standard (mixed solution of isotope internal standards of 8 catecholamines and their metabolites), and 200 μL of acetonitrile, and vortex for 10 minutes for extraction.

[0083] (2) Centrifuge (5000 rpm, 4 - 20 °C, 5 - 10 minutes), take the supernatant and blow dry with liquid nitrogen for 15 minutes.

[0084] (3) Add pH adjustment solution (50 μL of NaOH) and derivatization solution (PAA, 20 μL of 10% concentration), and vortex for 5 minutes.

[0085] 3. Detection method

[0086] (1) The chromatographic conditions are as follows:

[0087] Chromatographic column: C18 reversed-phase column (2 × 100 mm, 1.7 μm).

[0088] Mobile phase: Phase A (0.1% formic acid aqueous solution), Phase B (0.1% formic acid methanol solution).

[0089] The gradient elution program is as follows:

[0090] From 0 - 3 minutes, the volume fraction of mobile phase A changes uniformly from 60% to 40%, and the rest is mobile phase B;

[0091] From 3 - 3.1 minutes, the volume fraction of mobile phase A changes rapidly from 40% to 1%, and the rest is mobile phase B;

[0092] From 3.1 - 4.0 minutes, the volume fraction of mobile phase A is maintained at 1%, and the rest is mobile phase B;

[0093] From 4.0 - 4.1 minutes, the volume fraction of mobile phase A changes rapidly from 1% to 60%, and the rest is mobile phase B;

[0094] From 4.1 - 5.0 minutes, the volume fraction of mobile phase A is maintained at 60%, and the rest is mobile phase B.

[0095] Other chromatographic conditions: Column temperature: 50 °C, injection volume: 1 μL, flow rate: 0.4 mL / min.

[0096] (2) Mass spectrometry conditions

[0097] Ionization mode: Electrospray ionization (ESI+ / -).

[0098] Ion source parameters:

[0099] Spray voltage is 1 - 3.0 kV;

[0100] Ion source temperature is 100 - 150 °C;

[0101] Nebulizing gas temperature is 200 - 500 °C;

[0102] Nebulizing gas flow rate is 400 - 900 L / h;

[0103] Skimmer gas flow rate is 30 - 200 L / h;

[0104] Target substance mass spectrometry parameters (parent ion / daughter ion):

[0105] The parent ion mass-to-charge ratio of dopamine is 279.2, the corresponding daughter ion mass-to-charge ratio is 137.1, and the collision energy is 20 V.

[0106] The parent ion mass-to-charge ratio of adrenaline is 323.2, the corresponding daughter ion mass-to-charge ratio is 145.3, and the collision energy is 20 V.

[0107] The parent ion mass-to-charge ratio of noradrenaline is 328.2, the corresponding daughter ion mass-to-charge ratio is 147.3, and the collision energy is 20 V.

[0108] The parent ion mass-to-charge ratio of metanephrine is 369.2, the corresponding daughter ion mass-to-charge ratio is 145.3, and the collision energy is 20 V.

[0109] The parent ion mass-to-charge ratio of normetanephrine is 358.3, the corresponding daughter ion mass-to-charge ratio is 234.1, and the collision energy is 20 V.

[0110] The parent ion mass-to-charge ratio of 3-methoxytyramine is 389.2, the corresponding daughter ion mass-to-charge ratio is 198.3, and the collision energy is 20 V.

[0111] The parent ion mass-to-charge ratio of homovanillic acid is 197.3, the corresponding daughter ion mass-to-charge ratio is 115.3, and the collision energy is 20 V.

[0112] The parent ion mass-to-charge ratio of vanillylmandelic acid is 232.2, the corresponding daughter ion mass-to-charge ratio is 135.2, and the collision energy is 20 V.

[0113] 4. Detection process

[0114] The injection order is shown in Table 1 below:

[0115] Table 1

[0116]

[0117]

[0118] 5. Detection Results

[0119] The linear coefficients (R2 > 0.99) of 8 substances, where the abscissa is the concentration corresponding to the compound and the ordinate is the ratio of the response of the compound to the corresponding internal standard.

[0120] Figure 1 Chromatograms of the target substances DA, E, and NE.

[0121] Figure 2 Chromatograms of the target substances MN, NMN, and 3MT.

[0122] Figure 3 Chromatograms of the target substances HVA and VMA.

[0123] Figures 1 - 3 The results show that under one - injection, the 8 substances are completely separated within 5 minutes, with symmetric peaks and no matrix interference.

[0124] Figure 4 Linear result graph of dopamine.

[0125] Figure 5 Linear result graph of adrenaline.

[0126] Figure 6 Linear result graph of noradrenaline.

[0127] Figure 7 Linear result graph of metanephrine.

[0128] Figure 8 Linear result graph of normetanephrine.

[0129] Figure 9 Linear result graph of 3 - methoxytyramine.

[0130] Figure 10 Linear result graph of homovanillic acid.

[0131] Figure 11 Linear result graph of vanillylmandelic acid.

[0132] As Figures 4 - 11 shown, the linear results of 8 catecholamine substances and their metabolites are very good, all above 0.99, thus enabling very accurate quantification. The results show that the linear regression R2 of each compound is > 0.99, verifying the accuracy of quantification.

[0133] Comparative Example 1

[0134] Detection by traditional SPE method. Solid - Phase Extraction (abbreviated as SPE)

[0135] (1) Activate the SPE plate with 200 μL of acetonitrile.

[0136] (2) Equilibrate the SPE plate with 200 μL of ultrapure water.

[0137] (3) Load 200 μL of the sample.

[0138] (4) Wash with 200 μL of ultrapure water.

[0139] (5) Wash with 200 μL of acetonitrile.

[0140] (6) Elute with 100 μL of acetonitrile (containing 1% formic acid).

[0141] (7) Dry under nitrogen using a nitrogen evaporator.

[0142] (8) Reconstitute by vortexing with 50 μL of ultrapure water, and directly detect on a mass spectrometer after reconstitution.

[0143] Comparative Example 2

[0144] In this comparative example, blood samples were detected. The steps for sample treatment refer to Example 1; the detection method refers to Example 1. The only difference in the detection method from Example 1 is that the elution program is isocratic elution, and the elution method is as follows: the volume fraction of mobile phase A is maintained at 55%, and the rest is mobile phase B, and the elution time is 15 minutes or 30 minutes.

[0145] From the result comparison between Example 1 and Comparative Example 2, it can be seen that the optimized gradient adopted in the present invention has more excellent detection results. The results are compared as shown in Table 2 below.

[0146] Table 2

[0147] Comparison Items This Application (Optimized Gradient) Existing Technology (Conventional Isocratic) Separation Time 5 minutes (while detecting 8 compounds simultaneously) 15 - 30 minutes (only separating 5 - 10 compounds) Peak Capacity 12 - 15 peaks (resolution R > 1.5) 8 - 10 peaks (resolution R < 1.2) Sensitivity Retention Peak height loss < 10% (gradient optimization) Peak height loss > 50% (isocratic elution) Method Development Cycle 1 day (orthogonal experimental design) 7 - 10 days (single - factor optimization)

[0148] Comparative Example 3

[0149] In this comparative example, blood samples were detected. The steps for sample treatment refer to Example 1. The only difference from Example 1 is that the derivatization reagent used is dansyl chloride.

[0150] The steps for sample treatment are as follows:

[0151] (1) Take 100 μL of plasma sample, add 20 μL of internal standard (a mixed solution of isotope internal standards of 8 catecholamines and their metabolites), and 200 μL of acetonitrile, and extract by vortexing for 10 minutes.

[0152] (2) Centrifuge (5000 rpm, 4 - 20 °C, 5 - 10 minutes), take the supernatant and blow dry with nitrogen for 15 minutes.

[0153] (3) Add pH adjustment solution (50 μL of NaOH) and derivatization solution (dansyl chloride, 20 μL of 10% concentration), and vortex for 15 minutes.

[0154] Comparative Example 4

[0155] In this comparative example, the blood sample was detected. The steps of sample treatment referred to Example 1, and the difference from Example 1 was only that the derivatization reagent used was benzoyl chloride.

[0156] The steps of sample treatment are as follows:

[0157] (1) Take 100 μL of plasma sample, add 20 μL of internal standard (mixed solution of isotopic internal standards of 8 catecholamines and their metabolites) and 200 μL of acetonitrile, and vortex for 10 minutes for extraction.

[0158] (2) Centrifuge (5000 rpm, 4 - 20 °C, 5 - 10 minutes), take the supernatant and blow it with liquid nitrogen for 15 minutes.

[0159] (3) Add pH adjustment solution (50 μL of NaOH) and derivatization solution (benzoyl chloride, 20 μL of 10% concentration), and vortex for 15 minutes.

[0160] The results of Comparative Examples 3 and 4 are shown in Figures 12 - 17 . Figure 12 is the dansyl chloride derivatization spectrum; Figure 13 is the dansyl chloride derivatization spectrum after standing for 6 hours; Figure 14 is the benzoyl chloride derivatization spectrum; Figure 15 is the benzoyl chloride derivatization spectrum after standing for 6 hours; Figure 16 is the PAA derivatization spectrum; Figure 17 is the PAA derivatization spectrum after standing for 24 hours.

[0161] It can be seen from the comparison of the results of Example 1 with those of Comparative Examples 3 and 4 that the use of PAA for derivatization in the present invention has more excellent detection results. The results are compared as shown in Table 3 below.

[0162] Table 3

[0163]

[0164]

[0165] In summary, the present invention provides a detection method that is simple to operate, has a short pretreatment time, and can simultaneously detect 8 catecholamines and their metabolites; the method of the present invention shortens the detection time compared with the existing technology, and the detection throughput is increased by 50%; the lowest quantification limit (LLOQ) is as low as 0.1 ng / mL (blood sample) and 0.5 ng / mL (urine sample), and the method has high equipment compatibility and can achieve stable detection for medium and low-end mass spectrometers.

[0166] The applicant declares that the above is only a specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A synchronous detection method for 8 kinds of catecholamines and their metabolites based on liquid chromatography-tandem mass spectrometry, characterized in that, The detection method includes: (1) Mix the plasma or urine sample to be tested with an internal standard and acetonitrile; centrifuge to obtain the supernatant, blow it with liquid nitrogen, and adjust the pH; mix the supernatant with a propionic anhydride solution for derivatization reaction to obtain a test solution; (2) Perform liquid chromatography-tandem mass spectrometry detection on the test solution and the standard solution. Among them, liquid chromatography separates dopamine, epinephrine, norepinephrine, metanephrine, normetanephrine, 3-methoxytyramine, homovanillic acid, and vanilmandelic acid, and then uses the mass spectrometry isotope internal standard quantification method. With the concentration of the standard product as the X-axis and the ratio of the peak area of the standard product to the internal standard as the Y-axis, establish a standard curve, and calculate the concentrations of 8 catecholamines and their metabolites in the test solution; The mobile phase used in liquid chromatography includes: phase A is an aqueous solution of 0.1% formic acid, and phase B is a methanol solution of 0.1% formic acid; the elution program used in liquid chromatography is: From 0 to 3 minutes, the volume fraction of mobile phase A changes uniformly from 60-61% to 40-41%, and the rest is mobile phase B; From 3 to 3.1 minutes, the volume fraction of mobile phase A changes rapidly from 40-41% to 1-2%, and the rest is mobile phase B; From 3.1 to 4.0 minutes, the volume fraction of mobile phase A is maintained at 1-2%, and the rest is mobile phase B; From 4.0 to 4.1 minutes, the volume fraction of mobile phase A changes rapidly from 1-2% to 60-61%, and the rest is mobile phase B; From 4.1 to 5.0 minutes, the volume fraction of mobile phase A is maintained at 60-61%, and the rest is mobile phase B.

2. The synchronous detection method for 8 kinds of catecholamines and their metabolites based on liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In step (1), the internal standard is an isotope internal standard of 8 catecholamines and their metabolites, and the concentrations corresponding to each isotope internal standard are the isotopes of dopamine, epinephrine, norepinephrine, metanephrine, normetanephrine, 3-methoxytyramine, homovanillic acid, and vanilmandelic acid, and the concentrations corresponding to the internal standard are 160 pg / mL, 800 pg / mL, 800 pg / mL, 800 pg / mL, 400 pg / mL, 400 pg / mL, 160 ng / mL, and 160 ng / mL respectively; Preferably, in step (1), the volume ratio of the sample, internal standard, and acetonitrile is (5-6):1:(8-10).

3. The synchronous detection method for 8 kinds of catecholamines and their metabolites based on liquid chromatography-tandem mass spectrometry according to claim 1 or 2, characterized in that, In step (1), the rotation speed of the centrifugation is 5000-6000 rpm, the time is 5-10 minutes, and the temperature is 4-20 °C; Preferably, in step (1), the time of nitrogen blowing is 15-20 minutes.

4. The synchronous detection method for 8 kinds of catecholamines and their metabolites based on liquid chromatography-tandem mass spectrometry according to any one of claims 1-3, characterized in that In step (1), the concentration of the propionic anhydride solution is 5-25%; Preferably, the volume ratio of the supernatant to the propionic anhydride solution is (5-6):1; Preferably, the time of the derivatization reaction is 5-10 minutes.

5. The synchronous detection method for 8 kinds of catecholamines and their metabolites based on liquid chromatography-tandem mass spectrometry according to any one of claims 1-4, characterized in that, In step (2), the standard solution includes: dopamine, epinephrine, norepinephrine, metanephrine, normetanephrine, 3-methoxytyramine, homovanillic acid, and vanilmandelic acid; Preferably, the concentration range of dopamine in the standard solution is: 10 - 640 pg / mL; the concentration range of adrenaline is: 50 - 3200 pg / mL; the concentration range of noradrenaline is: 50 - 3200 pg / mL; the concentration range of metanephrine is: 50 - 3200 pg / mL; the concentration range of normetanephrine is: 25 - 1600 pg / mL; the concentration range of 3-methoxytyramine is: 25 - 1600 pg / mL; the concentration range of homovanillic acid is: 10 - 640 ng / mL; the concentration range of vanillylmandelic acid is: 10 - 640 ng / mL.

6. The synchronous detection method for 8 kinds of catecholamines and their metabolites based on liquid chromatography-tandem mass spectrometry according to any one of claims 1-5, characterized in that, In step (2), the chromatographic column used in the liquid chromatography is a C18 reversed-phase column.

7. The synchronous detection method for 8 kinds of catecholamines and their metabolites based on liquid chromatography-tandem mass spectrometry according to any one of claims 1-6, characterized in that, In step (2), the chromatographic conditions of the liquid chromatography further include: column temperature: 48 - 50 °C, injection volume: 1 - 1.5 μL, flow rate: 0.4 - 0.5 mL / min.

8. The synchronous detection method for 8 kinds of catecholamines and their metabolites based on liquid chromatography-tandem mass spectrometry according to any one of claims 1-7, characterized in that In step (2), the ionization mode of the mass spectrometry detection is electrospray ionization.

9. The synchronous detection method for 8 kinds of catecholamines and their metabolites based on liquid chromatography-tandem mass spectrometry according to any one of claims 1-8, characterized in that, In step (2), the ion source parameters of the mass spectrometry detection include: spray voltage is 1 - 3.0 kV, ion source temperature is 100 - 150 °C, nebulizing gas temperature is 200 - 500 °C, nebulizing gas flow rate is 400 - 900 L / h, and cone gas flow rate is 30 - 200 L / h.

10. Application of the method for synchronous detection of 8 catecholamines and their metabolites based on liquid chromatography-tandem mass spectrometry according to any one of claims 1 - 9 in detection and analysis.

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