Method for rapidly detecting catecholamine and metabolite content of catecholamine in blood

Abstract

The invention relates to a method for rapidly detecting the content of catecholamine and metabolites thereof in blood, and belongs to the technical field of biological sample analysis. According to the method, a target object is extracted through a solid-liquid extraction method (SPE), and then liquid chromatography and tandem mass spectrometry (LC-MS / MS) are used for detection. Through specific liquid phase conditions and elution gradient, rapid separation and detection of six substances including noradrenaline, epinephrine, dopamine, noradrenaline, epinephrine and 3-methoxytyramine are realized. According to the method, the formula of the mobile phase is simplified, the elution gradient is optimized, and the detection efficiency and accuracy are improved.

Description

Technical Field

[0001] The present application relates to the technical field of biological sample analysis, and in particular to a method for rapidly testing the content of catecholamines and their metabolites in blood. Background Art

[0002] Catecholamines include dopamine (DA), norepinephrine (NE) and epinephrine (E), generally referred to as catecholamine prototype substances, which are neurotransmitters or hormones present in the central and peripheral nervous systems. These neurotransmitters are involved in a variety of regulatory systems and metabolic processes and can be used as important biomarkers for the screening, diagnosis, treatment and prognosis of a variety of neuroendocrine and cardiovascular diseases. Catecholamine metabolites mainly include metanephrine (MN), methylnormetanephrine (NMN), and 3-methoxytyramine (3MT). When measured in combination with catecholamine prototype substances, they can provide personalized information on disease development and treatment effects. The expert consensus on pheochromocytoma and paraganglioma (PPGL) emphasizes that the combined detection of the first-line indicator MNs with other indicators can help improve the sensitivity and specificity of disease detection.

[0003] Liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS) has rapidly become a new technique for the quantification of catecholamines, following different sample purification methods. In the presence of drugs that normally interfere with HPLC analysis, the selective quantification of catecholamines can be achieved by liquid-liquid extraction or solid-liquid extraction (SPE) based on diphenylborate (PBA), followed by analysis using LC-MS / MS. However, the existing methodologies have time-consuming extraction processes and long chromatographic run times, and there are still challenges in the accurate determination of catecholamine concentrations in biological samples, limiting their widespread application in clinical laboratories.

[0004] For example, a Chinese patent with publication number CN114814044A discloses a method for detecting the content of catecholamines and their metabolites in blood, which discloses that the mobile phase is phase A: 0.1% formic acid + 5mmol / L ammonium formate water, phase B: pure methanol; the elution gradient is: 0.00-2.00min: 98% A; 2.01-3.05min: 80% A; 3.51-5.50min: 98% A; the analysis time is 5.5min. Among them, the NE peak time is 1.25min, the E peak time is 1.68min, the DA peak time is 2.01min, the NMN peak time is 1.95min, the MN peak time is 3.05min, and the 3-MT peak time is 4.05min. The detection time is long and the detection results have mixed peaks. A Chinese patent with publication number CN115327008A discloses a method for detecting catecholamines and their metabolites in plasma and urine, and the detection time is 5 minutes. A Chinese patent with publication number CN112782328A discloses a method, a kit and its application for detecting catecholamines and their metabolites in urine, and one injection also takes 5 minutes, and there is also the problem of long detection time. In addition, this method can only detect five items at a time. A Chinese patent with publication number CN116642975A discloses a six-item hormone-like mass spectrometry detection method and application, and the detection time is also 5 to 6 minutes.

[0005] In addition, the existing methodological extraction process still has the difficulty in accurately measuring the concentration of catecholamines in biological samples, which limits its widespread application in clinical laboratories. Summary of the invention

[0006] The present invention aims to develop and verify a rapid, specific and sensitive LC-MS / MS quantitative method for the determination of catecholamines and their metabolites. The present application provides an A+ grade solar simulation filter for photovoltaics and a coating method thereof.

[0007] In the first aspect, the present application provides a method for rapidly testing the content of catecholamines and their metabolites in blood using the following technical solution:

[0008] A method for rapidly testing the content of catecholamines and their metabolites in blood, characterized by comprising the following steps:

[0009] (1) Sample preparation: mixing a standard working solution of catecholamines and their metabolites and an internal standard working solution to obtain a standard product, wherein the standard working solution comprises six concentration gradients; the catecholamines and their metabolites comprise norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine and 3-methoxytyramine;

[0010] (2) The target compounds were extracted from the standard samples by SPE method, and the standard samples were detected by LC-MS / MS to establish standard curves for the six compounds;

[0011] (3) extracting the target substance from the substance to be tested by SPE; testing the sample to be tested by LC-MS / MS, and determining the content of each compound in the substance to be tested by using the standard curve established in step (2).

[0012] Through the above technical solution: the detection of six substances, namely norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine and 3-methoxytyramine, can be achieved simultaneously without repeated sampling, which can shorten the detection time and improve the detection efficiency.

[0013] As a preferred embodiment, the liquid chromatography parameters include: the chromatographic column mobile phase A is an aqueous solution containing 0.05-0.1% formic acid, B is an acetonitrile solution containing 0-0.1% formic acid, the flow rate is 0.35-0.4 mL / min, and the elution gradient is as follows:

[0014] 0.5min, 98% A, 2% B;

[0015] 1.2min, 65% A, 35% B;

[0016] 1.7min, 65% A, 35% B;

[0017] 2.0min, 5% A, 95% B;

[0018] 3.0min, 5% A, 95% B;

[0019] 3.1min, 98% A, 2% B;

[0020] 4.0min, 98% A, 2% B.

[0021] Through the above technical scheme: a specific gradient elution mode, wherein 0-0.5min 98%A is for compounds with strong hydrophilicity such as NE and E to have a better elution premise, 0.5-1.2min rapid transition from 98%A to 65%A advances the elution time of other less hydrophilic substances to achieve the purpose of rapid elution, 1.2-1.7min 65%A isocratic allows the gradient change rate to remain gentle, which is conducive to the stable elution of the compound, 2.0-3.0min 5%A isocratic can stably elute the less hydrophilic substances, 3.01-4.00min 98%A isocratic is used to balance the chromatographic column to maintain the stability of the next injection. Compared with the gradient elution of CN114814044 A patent, Example 1 uses the effect of rapid change of the mobile phase to quickly and effectively elute various compounds, achieving the purpose of good separation and short detection time. .

[0022] As a preferred embodiment, the chromatographic column used in step (2) is Phenomenex Kinetex PFP or Shim-pack Velox PFPP.

[0023] As a preferred solution, the detection time is less than or equal to 4 minutes.

[0024] As a preferred embodiment, the SPE sample extraction in step (2) and step (3) comprises the following steps:

[0025] (a) Activation, adding eluent for activation, and draining;

[0026] (b) Balance, add ultrapure water to balance, and drain;

[0027] (c) Loading, transferring samples to the plate and draining;

[0028] (d) elution 1, adding eluent 1, and draining;

[0029] (e) Eluent 2, add eluent 2, and drain;

[0030] (f) Replace the waste liquid tank of the negative pressure device and insert an elution plate;

[0031] (g) Elution.

[0032] As a preferred solution, the eluent 1 is water containing 0.1-0.5% formic acid; the eluent 2 is a mixed solution of isopropanol and acetonitrile, with a volume ratio of 1:0.5-1.

[0033] As a preferred solution, after SPE extraction in step (2) and step (3), the product is concentrated, re-dissolved in ultrapure water and then subjected to LC-MS / MS detection.

[0034] As a preferred solution, the concentration temperature is 30±5°C and the time is 1.5 to 2 hours.

[0035] In summary, the present application includes at least one of the following beneficial technical effects:

[0036] The method involved in the present application can simultaneously detect six substances including norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine and 3-methoxytyramine without the need for repeated sampling, which can shorten the detection time and improve the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the LC-MS / MS chromatogram of the method involved in Example 1 of the present application.

[0038] Figure 2 This is the linear spectrum of the method for testing norepinephrine (NE) according to Example 1 of the present application.

[0039] Figure 3 This is the linear spectrum of the method for testing epinephrine (E) in Example 1 of the present application.

[0040] Figure 4 This is a linear graph of dopamine (DA) tested by a method according to Example 1 of the present application.

[0041] Figure 5 This is a linear graph of the method for testing norepinephrine (NMN) according to Example 1 of the present application.

[0042] Figure 6 This is the linear spectrum of the method for testing methylephrine (MN) according to Example 1 of the present application.

[0043] Figure 7 This is the linear spectrum of the method for testing 3-methoxytyramine (3-MT) according to Example 1 of the present application. DETAILED DESCRIPTION

[0044] The following is combined with Figures 1 to 7 The present invention is further illustrated by the following embodiments.

[0045] Example 1

[0046] A method for rapidly testing the content of catecholamines and their metabolites in blood comprises the following steps:

[0047] (1) Sample preparation: prepare standard working solutions and internal standard working solutions of catecholamines and their metabolites, wherein the standard working solutions include six concentration gradients for detecting norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine and 3-methoxytyramine, respectively, for subsequent establishment of standard curves; mix the standard working solutions and the internal standard working solutions to obtain standard products;

[0048] (2) The target compound was extracted from the standard by SPE method, concentrated at 30°C for 1.5 h, and reconstituted with ultrapure water. The standard was then detected by LC-MS / MS to establish standard curves for the six compounds. The extraction steps were as follows:

[0049] (a) Activation: prepare 80% acetonitrile water containing 0.5% formic acid as eluent, add the eluent to the sample prepared in step (1) for activation, and drain;

[0050] (b) Balance, add ultrapure water to balance, and drain;

[0051] (c) Loading, transferring samples to the plate and draining;

[0052] (d) Elution 1: prepare 0.1% formic acid water as eluent 1, add eluent 1 in step (c), and drain;

[0053] (e) washing 2, preparing a mixed solution of isopropanol and acetonitrile in a ratio of 1:1 as the washing liquid 2, adding the product of step (2) to the washing liquid 2, and draining;

[0054] (f) Replace the waste liquid tank of the negative pressure device and insert an elution plate;

[0055] (g) elution;

[0056] (4) Place the sample from step 3 into a vacuum concentrator and concentrate at 30°C for 1.5 h;

[0057] (5) Reconstitute with ultrapure water;

[0058] (6) LC-MS / MS on-machine testing.

[0059] The detection parameters of LC-MS / MS are as follows: Use Phenomenex Kinetex PFP or Shim-pack VeloxPFPP chromatographic columns and set liquid chromatography parameters. Mobile phase A is an aqueous solution containing 0.05% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid. The flow rate is set to 0.38mL / min. Elution is performed using a specific elution gradient:

[0060] Table 1 Standard elution gradient

[0061] Time / min A / ％ B / ％ 0.5 98 2 1.2 65 35 1.7 65 35 2.0 5 95 3.0 5 95 3.1 98 2 4.0 98 2

[0062] The response value of each compound in the standard was obtained by LC-MS / MS detection, and standard curves of the six compounds were established respectively.

[0063] (3) Extracting the target substance from the substance to be tested by SPE; detecting the sample to be tested by LC-MS / MS, and determining the content of each compound in the substance to be tested by using the standard curve established in step (2); the extraction steps are as follows:

[0064] (a) Activation: prepare an eluent of 80% acetonitrile in water containing 0.5% formic acid, add the eluent to the sample prepared in step (1) for activation, and drain;

[0065] (b) Balance, add ultrapure water to balance, and drain;

[0066] (c) Loading, transferring samples to the plate and draining;

[0067] (d) Elution 1: prepare 0.1% formic acid water as eluent 1, add eluent 1 in step (c), and drain;

[0068] (e) washing 2, preparing a mixed solution of isopropanol and acetonitrile in a ratio of 1:1 as the washing liquid 2, adding the product of step (2) to the washing liquid 2, and draining;

[0069] (f) Replace the waste liquid tank of the negative pressure device and insert an elution plate;

[0070] (g) elution;

[0071] (4) Place the sample from step 3 into a vacuum concentrator and concentrate at 30°C for 1.5 h;

[0072] (5) Reconstitute with ultrapure water;

[0073] (6) LC-MS / MS on-machine testing.

[0074] The detection parameters of LC-MS / MS are as follows: Use Phenomenex Kinetex PFP or Shim-pack VeloxPFPP chromatographic columns and set liquid chromatography parameters. Mobile phase A is an aqueous solution containing 0.05% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid. The flow rate is set to 0.38mL / min. Elution is performed using a specific elution gradient:

[0075] Table 2 Target elution gradient table

[0076] Time / min A / ％ B / ％ 0.5 98 2 1.2 65 35 1.7 65 35 2.0 5 95 3.0 5 95 3.1 98 2 4.0 98 2

[0077] The content of each compound in the test object was determined by the standard curve.

[0078] The mass spectra of catecholamines and their metabolites are shown in Table 3 , and the mass spectrometry conditions of the mass spectrometer in ESI (+) detection mode are shown in Table 4 .

[0079] Table 3 Mass spectrometry parameters of catecholamines and their metabolites

[0080]

[0081] Table 4 Mass spectrometry conditions for mass spectrometry detector in ESI (+) detection mode

[0082]

[0083] The test results can be found in Figure 1 The peak times of catecholamines and their metabolites, namely norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine and 3-methoxytyramine, are shown in Table 5.

[0084] Table 5 Peak timetable of catecholamines and their metabolites

[0085] index Peak time (min) NE 1.30 NE-d6 1.30 E 1.69 E-d6 1.69 DA 1.90 DA-d4 1.90 NMN 1.84 NMN-d3 1.84 MN 2.64 MN-d3 2.64 3-MT 2.77 3-MT-d4 2.77

[0086] The peak times of catecholamines and their metabolites, namely norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine, and 3-methoxytyramine, are shown in Figure 2 to Figure 7 .

[0087] Compared with CN114814044 A, Example 1 adopts a gradient elution method, wherein 0-0.5min 98% A is for compounds with strong hydrophilicity such as NE and E to have a better elution premise, 0.5-1.2min quickly transitions from 98% A to 65% A to advance the elution time of other less hydrophilic substances to achieve the purpose of rapid elution, 1.2-1.7min 65% A isocratic allows the gradient change rate to remain gentle, which is conducive to the stable elution of the compound, 2.0-3.0min 5% A isocratic can stably elute the less hydrophilic substances, 3.01-4.00min 98% A isocratic is used to balance the chromatographic column to maintain the stability of the next injection. Compared with the gradient elution of CN114814044A patent, Example 1 uses the effect of rapid change of mobile phase to quickly and effectively elute various compounds, achieving the purpose of good separation and short detection time.

[0088] Example 2

[0089] A method for rapidly testing the content of catecholamines and their metabolites in blood comprises the following steps:

[0090] (1) Sample preparation: prepare standard working solutions and internal standard working solutions of catecholamines and their metabolites, wherein the standard working solutions include six concentration gradients for detecting norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine and 3-methoxytyramine, respectively, for subsequent establishment of standard curves; mix the standard working solutions and the internal standard working solutions to obtain standard products; the standard working solutions include six concentration gradients;

[0091] (2) The target compounds were extracted from the standard by SPE method, and the standard compounds were detected by LC-MS / MS to establish standard curves for the six compounds. The extraction steps were as follows:

[0092] (a) Activation: prepare 85% acetonitrile water containing 1% formic acid as an eluent, add the eluent to the sample prepared in step (1) for activation, and drain;

[0093] (b) Balance, add ultrapure water to balance, and drain;

[0094] (c) Loading, transferring samples to the plate and draining;

[0095] (d) Elution 1: prepare 0.3% formic acid water as eluent 1, add eluent 1 in step (c), and drain;

[0096] (e) washing 2, preparing a mixed solution of isopropanol and acetonitrile in a ratio of 0.8: as washing liquid 2, adding the product of step (2) to the washing liquid 2, and draining;

[0097] (f) Replace the waste liquid tank of the negative pressure device and insert an elution plate;

[0098] (g) elution;

[0099] (4) LC-MS / MS on-machine detection.

[0100] The detection parameters of LC-MS / MS are as follows: Use Phenomenex Kinetex PFP or Shim-pack VeloxPFPP chromatographic columns and set liquid chromatography parameters. Mobile phase A is an aqueous solution containing 0.05% formic acid, and mobile phase B is an acetonitrile solution containing 0.01% formic acid. The flow rate is set to 0.38mL / min. Elution is performed using a specific elution gradient:

[0101] Table 6 Standard elution gradient

[0102] Time / min A / ％ B / ％ 0.5 98 2 1.2 65 35 1.7 65 35 2.0 5 95 3.0 5 95 3.1 98 2 4.0 98 2

[0103] The response value of each compound in the standard was obtained by LC-MS / MS detection, and standard curves of the six compounds were established respectively.

[0104] (3) Extracting the target substance from the substance to be tested by SPE; detecting the sample to be tested by LC-MS / MS, and determining the content of each compound in the substance to be tested by using the standard curve established in step (2); the extraction steps are as follows:

[0105] (a) Activation: prepare an eluent of 85% acetonitrile in water containing 1% formic acid, add the eluent to the sample prepared in step (1) for activation, and drain;

[0106] (b) Balance, add ultrapure water to balance, and drain;

[0107] (c) Loading, transferring samples to the plate and draining;

[0108] (d) Elution 1: prepare 0.3% formic acid water as eluent 1, add eluent 1 in step (c), and drain;

[0109] (e) washing 2, preparing a mixture in a ratio of 0.8:1 as eluent 2, adding the product of step (2) to the eluent 2, and draining;

[0110] (f) Replace the waste liquid tank of the negative pressure device and insert an elution plate;

[0111] (g) elution;

[0112] (4) LC-MS / MS on-machine testing;

[0113] The detection parameters of LC-MS / MS are as follows: Use Phenomenex Kinetex PFP or Shim-pack VeloxPFPP chromatographic columns and set liquid chromatography parameters. Mobile phase A is an aqueous solution containing 0.05% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid. The flow rate is set to 0.38mL / min. Elution is performed using a specific elution gradient:

[0114] Table 7 Target elution gradient table

[0115] Time / min A / ％ B / ％ 0.5 98 2 1.2 65 35 1.7 65 35 2.0 5 95 3.0 5 95 3.1 98 2 4.0 98 2

[0116] The content of each compound in the test object was determined by the standard curve.

[0117] Experiments have shown that, in this embodiment, through specific liquid phase conditions, the separation of the target six catecholamines and their metabolites can be achieved in 4 minutes with one injection.

[0118] Example 3

[0119] The difference from Example 1 is that the mobile phase A is adjusted. See Table 8 for the signal-to-noise ratio comparison of the mobile phase A containing 0.05% formic acid and the aqueous solution containing 1% formic acid.

[0120] Table 8 Comparison of signal-to-noise ratio of different mobile phases A

[0121]

[0122] Referring to Table 8, it can be seen that when the mobile phase A is an aqueous solution containing 0.05% formic acid, the signal-to-noise ratios of the first five indicators are lower than when the mobile phase A is an aqueous solution containing 1% formic acid, while the signal-to-noise ratio of 3-MT is the opposite.

[0123] Experiments have shown that this embodiment can detect six catecholamines and their metabolites in one injection in four minutes.

[0124] Formic acid plays a major role in adjusting pH and improving peak shape in the mobile phase. By adjusting the concentration of formic acid, the chromatographic separation effect and mass spectrometry detection sensitivity can be further optimized.

[0125] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, any equivalent changes made according to the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for rapidly testing the content of catecholamines and their metabolites in blood, characterized in that: The following steps are involved: (1) Sample preparation: mixing a standard working solution of catecholamines and their metabolites and an internal standard working solution to obtain a standard product, wherein the standard working solution comprises six concentration gradients; the catecholamines and their metabolites comprise norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine and 3-methoxytyramine; (2) The target compounds were extracted from the standard samples by SPE method, and the standard samples were detected by LC-MS / MS to establish standard curves for the six compounds; (3) Extracting the target substance from the substance to be tested by SPE; testing the sample to be tested by LC-MS / MS, and determining the content of each compound in the substance to be tested by using the standard curve established in step (2).

2. The method for rapidly testing the content of catecholamines and their metabolites in blood according to claim 1, characterized in that: Liquid chromatography parameters include: column mobile phase A is an aqueous solution containing 0.05-0.1% formic acid, B is an acetonitrile solution containing 0-0.1% formic acid, the flow rate is 0.35-0.4 mL / min, and the elution gradient is as follows: 0-0.5min, 98% A, 2% B; 0.5-1.2min, 65% A, 35% B; 1.2-1.7 min, 65% A, 35% B; 1.7-2.0min, 5% A, 95% B; 2.0-3.0min, 5% A, 95% B; 3.0-3.1min, 98% A, 2% B; 3.1~4.0min, 98% A, 2% B.

3. The method for rapidly testing the content of catecholamines and their metabolites in blood according to claim 1 or 2, characterized in that: The chromatographic column used in step (2) is Phenomenex Kinetex PFP or Shim-pack Velox PFPP.

4. The method for rapidly testing the content of catecholamines and their metabolites in blood according to claim 3, characterized in that: The detection time is less than or equal to 4 minutes.

5. The method for rapidly testing the content of catecholamines and their metabolites in blood according to claim 1, characterized in that: The SPE sample extraction in step (2) and step (3) comprises the following steps: (a) Activation, adding eluent for activation, and draining; (b) Balance, add ultrapure water to balance, and drain; (c) Loading, transferring samples to the plate and draining; (d) elution 1, adding eluent 1, and draining; (e) Eluent 2, add eluent 2, and drain; (f) Replace the waste liquid tank of the negative pressure device and insert an elution plate; (g) Elution.

6. The method for rapidly testing the content of catecholamines and their metabolites in blood according to claim 5, characterized in that: The eluent 1 is water containing 0.1-0.5% formic acid; the eluent 2 is a mixed solution of isopropanol and acetonitrile, with a volume ratio of 1:0.5-1.

7. The method for rapidly testing the content of catecholamines and their metabolites in blood according to claim 1, characterized in that: After SPE extraction in step (2) and step (3), the product is concentrated, re-dissolved in ultrapure water, and then subjected to LC-MS / MS detection.

8. The method for rapidly testing the content of catecholamines and their metabolites in blood according to claim 7, characterized in that: The concentration temperature is 30±5℃ and the time is 1.5~2h.

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