Method for detecting catecholamine in plasma

Abstract

The invention provides a method for detecting various catecholamines in plasma, which comprises the following steps: 1) taking a plasma sample, adding a catecholamine isotope internal standard solution, uniformly mixing, and performing ultrafiltration on the mixed solution to obtain filtrate; (2) carrying out liquid chromatography-tandem mass spectrometry detection on the filtrate; the contents of various catecholamines are obtained; the catecholamine is prepared from at least one of epinephrine, noradrenaline, methoxy epinephrine, dopamine, methoxy noradrenaline and 3-methoxytyramine; the isotope internal standard solution contains a stabilizer; the stabilizer solution comprises ascorbic acid, hydrochloric acid and methanol. The detection method provided by the invention is simple, can realize automation and is short in time consumption; the method can be used for rapid extraction of massive samples.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and relates to a method for detecting catecholamines in plasma. Background Art

[0002] Catecholamines (CAs) mainly include epinephrine (E), norepinephrine (NE) and dopamine (DA), and their main metabolites are 3-methoxytyramine (3-MT), methylnorepinephrine (NMN) and methylepinephrine (MN). Catecholamines and their metabolites can be used clinically to assist in the diagnosis of endocrine-related diseases such as pheochromocytoma and neuroblastoma.

[0003] Catecholamines and their metabolites are of great significance in disease diagnosis, but catecholamines and their metabolites are relatively unstable and degrade rapidly at room temperature. Currently, protein precipitation and dansyl chloride derivatization are commonly used for extraction and derivatization. However, this process is time-consuming, complicated, and cannot be automated.

[0004] The solid phase extraction method and adsorbent adsorption method commonly used in the prior art use many types of reagents, large amounts of organic reagents, and certain toxicity; the operation steps are complicated; and the cost is high.

[0005] Chinese patent publication number CN113720939A discloses a kit for automated quantitative determination of six catecholamines in plasma by magnetic bead method. The kit is mainly a kit for detecting catecholamines, but its pretreatment is complicated and time-consuming, requiring a water bath at 58°C for 20 minutes. The whole process is time-consuming and can easily cause degradation of unstable catecholamines, resulting in inconsistency between the test results and the actual results.

[0006] The Chinese patent with publication number CN109613144A discloses a method for detecting catecholamine hormones in human plasma, but in this method, the sample is subjected to protein precipitation, light protection, and solid phase extraction before detection, which is time-consuming and complicated to operate, and cannot achieve rapid detection of samples.

[0007] Chinese patent publication number CN106442837A discloses a method for detecting catecholamines in plasma using liquid chromatography tandem mass spectrometry. However, this method only detects three substances and performs LC-MS / MS detection after protein precipitation, light-proof derivatization, pH adjustment, and centrifugation. The operation is complicated and time-consuming.

[0008] Chinese patent publication number CN117554550B discloses a liquid chromatography tandem mass spectrometry detection method and a combined stabilizer for catecholamine metabolites in plasma, but the method requires multiple pre-treatment steps such as protein precipitation, derivatization and solid phase extraction, and the operation is complicated.

[0009] Chinese patent publication number CN116482248B discloses a detection kit for catecholamines and their metabolites in plasma / urine, a detection method and application thereof, but the method requires the use of multiple reagents, which are mixed in a fixed ratio and order, and the operation is complicated. Summary of the invention

[0010] The present invention aims at the above-mentioned deficiencies of the prior art and provides a method for detecting catecholamines in plasma. In the present invention, adding a specific stabilizer solution can reduce the degradation of the target compound during the pretreatment process and improve the extraction efficiency of the target compound. The method is simple, can be automated, and is time-saving; it can be used for rapid extraction and detection of a large number of samples.

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention provides a method for detecting multiple catecholamines in plasma, comprising the following steps:

[0013] 1) taking a plasma sample, adding a catecholamine isotope internal standard solution and mixing evenly, and ultrafiltrating the mixed solution to obtain a filtrate;

[0014] 2) performing liquid chromatography tandem mass spectrometry on the filtrate to obtain the contents of various catecholamines;

[0015] The catecholamines include at least one of epinephrine (E), norepinephrine (NE), metanephrine (MN), dopamine (DA), methylnorepinephrine (NMN), and 3-methoxytyramine (3-MT);

[0016] The isotope internal standard solution contains a stabilizer; the stabilizer solution includes at least one of ascorbic acid, hydrochloric acid and methanol.

[0017] The stabilizer solution contains 1-5 mg / mL ascorbic acid and a mixed solution of hydrochloric acid and pure methanol.

[0018] The mixed solution of hydrochloric acid and pure methanol is prepared by mixing 1 mol / L hydrochloric acid and HPLC grade pure methanol in a volume ratio of 5-20:80-95.

[0019] Preferably, the stabilizer solution contains 5 mg / mL ascorbic acid.

[0020] Preferably, the mixed solution of hydrochloric acid and pure methanol is prepared by mixing 1 mol / L hydrochloric acid and HPLC grade pure methanol in a volume ratio of 5:95.

[0021] Preferably, the method of ultrafiltration comprises transferring positive pressure into the ultrafiltration plate.

[0022] Preferably, the ultrafiltration plate comprises a 3KD ultrafiltration plate. The present invention uses ultrafiltration to remove macromolecules such as proteins and pigments in plasma samples, so that catecholamine substances are extracted and purified.

[0023] Preferably, the positive pressure method comprises applying positive pressure on a positive pressure device for 25-40 minutes. Preferably, the positive pressure has a pressure of 0.1-0.2 MPa.

[0024] The isotope internal standards include epinephrine-IS, norepinephrine-IS, methylepinephrine-IS, dopamine-IS, methylnorepinephrine-IS, and 3-methoxytyramine-IS.

[0025] Preferably, in the isotope internal standard solution, the concentrations of the various substances are:

[0026] Epinephrine-IS: 2000-3000 pg / mL;

[0027] Norepinephrine-IS: 3000-5000 pg / mL;

[0028] Metanephrine-IS: 2000-3000 pg / mL;

[0029] Dopamine-IS: 1000-1500 pg / mL;

[0030] Methylnorephinephrine-IS: 2000-3000 pg / mL;

[0031] 3-Methoxytyramine-IS: 1000-1500 pg / mL.

[0032] The amount of isotope internal standard added is 10-20 μL.

[0033] Liquid chromatography tandem mass spectrometry detection:

[0034] Chromatographic conditions:

[0035] Mobile phase A: aqueous solution containing 0.10 wt% formic acid

[0036] Mobile phase B: acetonitrile solution containing 0.10%wt formic acid

[0037] Chromatographic column model: E-PMN reverse phase column

[0038] The mobile phase gradient conditions were:

[0039]

[0040] Liquid chromatography tandem mass spectrometry detection:

[0041] Mass spectrometry conditions:

[0042] Ion source: ESI ion source

[0043] Detection mode: MRM acquisition mode;

[0044] The ion source parameters are: spray voltage 0.5 kV, ion source temperature 500 °C, Desolvation: 1100 L / Hr, Cone: 150 L / Hr, Nebuliser: 7.0 Bar; Q1 / Q3 ion channels are as follows:

[0045]

[0046]

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] 1. The isotope internal standard solution of the present invention uses a specific stabilizer, which can effectively reduce the degradation of the target compound during the pretreatment process and improve the extraction efficiency of the target compound. The extraction of catecholamines can be quickly achieved through ultrafiltration, and the extraction and purification can be completed within 40 minutes;

[0049] 2. Compared with the traditional SPE solid phase extraction method, it is simple to operate, saves manual operation steps, and can effectively reduce batch differences;

[0050] 3. Compared with the magnetic bead extraction method, the purification is cleaner and there is no problem of magnetic bead residue, which can greatly reduce the maintenance cost of liquid chromatography-tandem mass spectrometry;

[0051] 4. The sample is not concentrated during the sample pretreatment process, which can effectively reduce the interference of impurities and reduce the loss of the chromatographic column;

[0052] 5. Liquid chromatography-tandem mass spectrometry is used for detection, and the MRM acquisition mode is selected, which has high specificity. A pair of parent ions and daughter ions are selected for quantitative and qualitative analysis respectively, which can greatly improve the accuracy of qualitative and quantitative analysis and avoid false positives.

[0053] 6. High throughput: up to 96 samples can be processed simultaneously by selecting ultrafiltration plates of different specifications;

[0054] 7. The instrument has a short analysis time, with a sample injection time of 4 minutes. It can detect 6 catecholamine substances, which is of great significance for the detection of a large number of samples;

[0055] 8. Using isotope internal standard method for quantification can effectively eliminate matrix effect and improve the accuracy of quantitative results;

[0056] 9. High repeatability of test samples;

[0057] 10. Sample processing is simple and highly automated. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0059] Figure 1 This is the adrenaline detection spectrum of Cal 1, the lowest point of the calibrator;

[0060] Figure 2 It is the norepinephrine detection spectrum of Cal 1, the lowest point of the calibrator;

[0061] Figure 3 This is the metanephrine detection spectrum of the lowest point of the calibrator, Cal 1;

[0062] Figure 4 This is the dopamine detection spectrum of Cal 1, the lowest point of the calibrator;

[0063] Figure 5 It is the detection spectrum of methyloxynorepinephrine at the lowest point of the calibrator, Cal 1;

[0064] Figure 6 This is the 3-methoxytyramine detection spectrum of the lowest point of the calibrator, Cal 1;

[0065] Figure 7 is the linear fitting graph of adrenaline;

[0066] Figure 8 is the linear fitting graph of norepinephrine;

[0067] Fig. 9 is the linear fitting graph of metanephrine;

[0068] Fig.10 is the linear fitting graph of dopamine;

[0069] Fig.11 is the linear fitting graph of methyloxynorepinephrine;

[0070] Fig.12 This is the linear fitting plot of 3-methoxytyramine. DETAILED DESCRIPTION

[0071] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0072] The reagents and instruments used in the examples of the present invention are as follows:

[0073] Blank plasma without catecholamines: brand "Golden West";

[0074] Hydrochloric acid methanol solution containing ascorbic acid (basic stabilizer): the ascorbic acid concentration is 5 mg / mL, and the volume ratio of 1 mol / L hydrochloric acid to HPLC grade pure methanol is 5:95.

[0075] Stock solutions of standards: Weigh 1 mg of each catecholamine standard (epinephrine, norepinephrine, metanephrine, dopamine, methylnorepinephrine and 3-methoxytyramine) into a brown reagent bottle, then use a pipette to transfer 1 mL of a methanol solution of hydrochloric acid containing ascorbic acid (basic stabilizer) into each brown reagent bottle, ultrasonicate for 5 min, and vortex to mix, to obtain stock solutions of 6 catecholamine standards, the concentration of the 6 catecholamines is 1 mg / mL.

[0076] The liquid chromatograph is an ACQUITY UPLC I-Class / Xevo TQ-S IVD System from Waters Corporation.

[0077] Chromatographic conditions:

[0078] Mobile phase A: aqueous solution containing 0.10 wt% formic acid

[0079] Mobile phase B: acetonitrile solution containing 0.10%wt formic acid

[0080] Chromatographic column model: E-PMN reverse phase column

[0081] Column temperature: 40°C

[0082] Injection volume: 10 μL

[0083] Gradient elution was used, and the flow rate and gradient are shown in Table 1.

[0084] Table 1 Liquid phase elution gradient

[0085]

[0086]

[0087] Mass spectrometry conditions:

[0088] Electrospray ion source (ESI), positive ion MRM scanning analysis, ion source parameters: spray voltage 0.5 kV, ion source temperature 500 ° C, Desolvation: 1100 L / Hr, Cone: 150 L / Hr, Nebuliser: 7.0 Bar; Q1 / Q3 ion channel selection is shown in Table 2:

[0089] Table 2 Parent ion and daughter ion parameters

[0090]

[0091] Example 1 Screening of stabilizers

[0092] 1. Solution preparation

[0093] The configuration of the stabilizer in this embodiment is shown in Table 3:

[0094] Table 3

[0095]

[0096] The configuration of isotope internal standard mixed solution is shown in Table 4:

[0097] Table 4

[0098]

[0099] Preparation of spiked experimental samples:

[0100] (1) Preparation of dilution solution 1

[0101] The stock solution of each standard substance with a concentration of 1 mg / mL was diluted with methanol solution to dilute the dilution solution of each standard substance with a concentration of 100 μg / mL and 10 μg / mL;

[0102] Accurately measure the following volumes of dilution 1 of each standard, and then dilute to 1000 μL with methanol solution to obtain dilution 1:

[0103] 175 μL of 10 μg / mL epinephrine dilution;

[0104] 175 μL of 10 μg / mL metanephrine dilution;

[0105] 175 μL of 10 μg / mL methyloxynorepinephrine dilution;

[0106] 52.5 μL of 100 μg / mL norepinephrine dilution;

[0107] 70 μL of 10 μg / mL dopamine dilution;

[0108] 35 μL of 10 μg / mL 3-methoxytyramine dilution;

[0109] The concentrations of the catecholamine substances in the obtained dilution 1 are as follows: the concentrations of epinephrine, methylepinephrine and methylnorepinephrine are all 1750 ng / mL; the concentration of norepinephrine is 5250 ng / mL; the concentration of dopamine is 700 ng / mL; and the concentration of 3-methoxytyramine is 350 ng / mL.

[0110] (2) Preparation of high-concentration catecholamine standard solution

[0111] Blank plasma and diluent 1 were prepared in proportion to obtain standard solutions of catecholamines in the high-concentration group (in the experimental samples of the high-concentration group): the concentrations of epinephrine, norepinephrine, dopamine, methylepinephrine, methylnorepinephrine and 3-methoxytyramine were 3500pg / mL, 10500pg / mL, 1400pg / mL, 3500pg / mL, 3500pg / mL and 700pg / mL, respectively.

[0112] (3) Preparation of low-concentration catecholamine standard solution

[0113] Blank plasma and diluent 1 were prepared in proportion. The standard solutions of catecholamine substances in the low-concentration group (low-concentration group experimental samples) were obtained: the concentrations of epinephrine, norepinephrine, dopamine, metanephrine, metanephrine and 3-methoxytyramine were 125 pg / mL, 375 pg / mL, 50 pg / mL, 125 pg / mL, 125 pg / mL and 25 pg / mL respectively;

[0114] The standard solutions of the low-concentration group of catecholamines and the high-concentration group of catecholamines were dispensed into centrifuge tubes, 100 μL in each centrifuge tube, for later use.

[0115] 2. Experimental steps

[0116] Take the above-packaged low-concentration group experimental samples and high-concentration group experimental samples, add 10 μL of isotope internal standard mixed solution 1, isotope internal standard mixed solution 2 and isotope internal standard mixed solution 3 respectively, vortex mix evenly for 2 minutes, transfer all the liquid in the centrifuge tube to the 3KD ultrafiltration plate, pressurize on the positive pressure device for 30 minutes, receive the filtrate into the 96-well sample injection plate, and detect on the liquid chromatography-tandem mass spectrometer.

[0117] The results of mass spectrometer detection experiments in the low concentration group are shown in Tables 5 to 10 below:

[0118] Table 5

[0119]

[0120] Table 6

[0121]

[0122] Table 7

[0123]

[0124]

[0125] Table 8

[0126]

[0127] Table 9

[0128]

[0129] Table 10

[0130]

[0131] The results of mass spectrometer detection experiments of the high concentration group are shown in Tables 11 to 16 below:

[0132] Table 11

[0133]

[0134]

[0135] Table 12

[0136]

[0137] Table 13

[0138]

[0139] Table 14

[0140]

[0141] Table 15

[0142]

[0143] Table 16

[0144]

[0145] Experimental conclusion:

[0146] By comparing the above experimental results, taking into account the accuracy and CV value, stabilizer solution 2 (containing 1 mg / mL ascorbic acid, 1 mol / L hydrochloric acid and HPLC-grade pure methanol in a volume ratio of 10:90) was selected for the extraction of catecholamine targets in plasma.

[0147] Example 2 Optimization of ascorbic acid content

[0148] 1. Solution preparation

[0149] The configuration of the stabilizer in this embodiment is shown in Table 17:

[0150] Table 17

[0151] Group Components and content Stabilizer solution 2 Contains 1mg / mL ascorbic acid, 1mol / L hydrochloric acid and HPLC grade pure methanol in a volume ratio of 10:90 Stabilizer solution 4 Contains 5mg / mL ascorbic acid, 1mol / L hydrochloric acid and HPLC grade pure methanol in a volume ratio of 10:90 Stabilizer solution 5 Contains 10mg / mL ascorbic acid, 1mol / L hydrochloric acid and HPLC grade pure methanol in a volume ratio of 10:90

[0152] The configuration of the isotope internal standard mixed solution is shown in Table 18:

[0153] Table 18

[0154]

[0155] Configuration of standard product: same as Example 1.

[0156] 2. Experimental steps

[0157] Using blank plasma samples without catecholamines, standard solutions of catecholamine substances of low concentration group and high concentration group, as well as isotope internal standard mixed solution 2, isotope internal standard mixed solution 4, and isotope internal standard mixed solution 5 were added to the blank plasma respectively.

[0158] The results of mass spectrometer detection experiments in the low concentration group are shown in Tables 19 to 24 below:

[0159] Table 19

[0160]

[0161] Table 20

[0162]

[0163] Table 21

[0164]

[0165] Table 22

[0166]

[0167] Table 23

[0168]

[0169]

[0170] Table 24

[0171]

[0172] The results of mass spectrometer detection experiments of the high concentration group are shown in Tables 25 to 30 below:

[0173] Table 25

[0174]

[0175] Table 26

[0176]

[0177] Table 27

[0178]

[0179] Table 28

[0180]

[0181] Table 29

[0182]

[0183] Table 30

[0184]

[0185] Experimental conclusion:

[0186] By comparing the above experimental results and comprehensively considering the accuracy and CV value, a solution containing 5 mg / mL ascorbic acid, 1 mol / L hydrochloric acid and HPLC-grade pure methanol in a volume ratio of 10:90 was selected for the extraction of catecholamine targets in plasma.

[0187] Example 3 Confirmation of the volume ratio of hydrochloric acid to HPLC grade pure methanol

[0188] 1. Solution preparation

[0189] The configuration of the stabilizer in this embodiment is shown in Table 31:

[0190] Table 31

[0191]

[0192]

[0193] The configuration of the isotope internal standard mixed solution is shown in Table 32:

[0194] Table 32

[0195]

[0196] Configuration of standard product: same as Example 1.

[0197] 2. Experimental steps

[0198] Using blank plasma samples without catecholamines, standard solutions of low-concentration group and high-concentration group of catecholamine substances, as well as isotope internal standard mixed solution 4, isotope internal standard mixed solution 6, and isotope internal standard mixed solution 7 were added to the blank plasma respectively.

[0199] The results of mass spectrometer detection experiments in the low concentration group are shown in Tables 33 to 38 below:

[0200] Table 33

[0201]

[0202] Table 34

[0203]

[0204]

[0205] Table 35

[0206]

[0207] Table 36

[0208]

[0209] Table 37

[0210]

[0211] Table 38

[0212]

[0213] The results of mass spectrometer detection experiments of high and low concentration groups are shown in Tables 39 to 44 below:

[0214] Table 39

[0215]

[0216] Table 40

[0217]

[0218] Table 41

[0219]

[0220] Table 42

[0221]

[0222] Table 43

[0223]

[0224] Table 44

[0225]

[0226] Experimental conclusion:

[0227] By comparing the above experimental results and comprehensively considering the accuracy and CV value, a solution containing 5 mg / mL ascorbic acid, 1 mol / L hydrochloric acid and HPLC-grade pure methanol in a volume ratio of 5:95 was selected for the extraction of catecholamine targets in plasma.

[0228] Example 4 Linear range and sensitivity detection

[0229] Preparation of calibrants:

[0230] The stock solution of each standard substance with a concentration of 1 mg / mL was diluted with methanol solution to dilute the dilution solution of each standard substance with a concentration of 100 μg / mL and 10 μg / mL;

[0231] Take 50 μL of 10 μg / mL epinephrine dilution, metanephrine dilution and methylnorepinephrine dilution, 15 μL of 100 μg / mL norepinephrine dilution, 20 μL of 10 μg / mL dopamine dilution, and 10 μL of 10 μg / mL 3-methoxytyramine dilution respectively; dilute to 1000 μL with methanol solution to obtain dilution 1 (in dilution 1: the concentrations of epinephrine, metanephrine and methylnorepinephrine are 500 ng / mL, the concentration of norepinephrine is 1500 ng / mL, the concentration of dopamine is 200 ng / mL, and the concentration of 3-methoxytyramine is 100 ng / mL).

[0232] Calibrator solution CAL6: Pipette 10 μL of diluent 1, then take 990 μL of blank plasma, vortex to mix, the theoretical concentration is: epinephrine, methylepinephrine and methylnorepinephrine 5000 pg / mL, norepinephrine 15000 pg / mL, dopamine 2000 pg / mL, 3-methoxytyramine 1000 pg / mL.

[0233] Calibrator solution CAL5: Use a pipette to take 500 μL of CAL6 solution, then take 500 μL of blank plasma, vortex to mix, the theoretical concentration is: epinephrine, methylepinephrine and methylnorepinephrine 2500 pg / mL, norepinephrine 7500 pg / mL, dopamine 1000 pg / mL, 3-methoxytyramine 500 pg / mL.

[0234] Calibrator solution CAL4: Use a pipette to take 500 μL of CAL5 solution, then take 500 μL of blank plasma, vortex to mix, the theoretical concentration is: epinephrine, methylepinephrine and methylnorepinephrine 1250 pg / mL, norepinephrine 3750 pg / mL, dopamine 500 pg / mL, 3-methoxytyramine 250 pg / mL.

[0235] Calibrator solution CAL3: Use a pipette to take 200 μL of CAL4 solution, then take 800 μL of blank plasma, vortex to mix, the theoretical concentration is: epinephrine, methylepinephrine and methylnorepinephrine 250 pg / mL, norepinephrine 750 pg / mL, dopamine 100 pg / mL, 3-methoxytyramine 50 pg / mL.

[0236] Calibrator solution CAL2: Use a pipette to take 400 μL of CAL3 solution, then take 600 μL of blank plasma, vortex to mix, the theoretical concentration is: epinephrine, methylepinephrine and methylnorepinephrine 100 pg / mL, norepinephrine 300 pg / mL, dopamine 40 pg / mL, 3-methoxytyramine 20 pg / mL.

[0237] Calibrator solution CAL1: Use a pipette to take 500 μL of CAL2 solution, then take 500 μL of blank plasma, vortex to mix, the theoretical concentration is: epinephrine, methylepinephrine and methylnorepinephrine 50 pg / mL, norepinephrine 150 pg / mL, dopamine 20 pg / mL, 3-methoxytyramine 10 pg / mL.

[0238] Take 100 μL of each of the above-prepared calibrants, accurately add the isotope internal standard mixed solution 7, vortex mix evenly for 2 minutes, transfer all the liquid in the centrifuge tube to the 3KD ultrafiltration plate, pressurize on the positive pressure device for 30 minutes, receive the filtrate into the 96-well sample plate, and detect it on the liquid chromatography-tandem mass spectrometer. The linear range, standard curve equation, linear correlation coefficient and detection limit are obtained after the detection data are linearly fitted by the mass spectrometer software, as shown in Table 45. The linear fitting diagrams of the six catecholamine substances are shown in Table 45. Figure 7-12 shown.

[0239] Table 45 Linear range and regression equation of each catecholamine

[0240]

[0241] From Table 45, we can see that the curves of catecholamine substances have good correlation. (R>0.995)

[0242] Figure 1-6Shown is the spectrum of the lowest point of the six catecholamine calibrants after pretreatment, reflecting the detection limit spectrum of the method.

[0243] Example 5 Precision and Accuracy Testing

[0244] In the experiment, a blank plasma sample (brand "Golden West") without catecholamines was used. Experimental samples of catecholamines as shown in Table 46 were added to the blank plasma. Six parallel samples were prepared for each concentration. After the sample pretreatment steps in Example 4 were tested, the samples were tested on a mass spectrometer. The data are shown in Table 8.

[0245] Table 46 Recovery and precision

[0246]

[0247] As shown in Table 45, the precision and accuracy of sample detection are good (CV < 15%, recovery rate is 85%-115%).

[0248] Example 6 Detection of clinical samples

[0249] 10 plasma samples from normal subjects at the same time were selected, and after experimental treatment according to the sample pretreatment steps of Example 4, they were tested by mass spectrometer, and the data obtained are shown in Table 47.

[0250] Table 47 Clinical sample test results

[0251]

[0252]

[0253] As can be seen from Table 46, the results of the catecholamine substances in normal human plasma detected by this method are basically within the linear range.

[0254] Experimental conclusion: Through the comparison of the above experimental results, comprehensive consideration of accuracy and CV value, a solution containing 5 mg / mL ascorbic acid, 1 mol / L hydrochloric acid and HPLC-grade pure methanol in a volume ratio of 5:95 was selected for the extraction of catecholamine targets in plasma.

[0255] In summary, the present invention provides a method for extracting catecholamines in plasma based on ultrafiltration. Specifically, the present invention discloses a method for extracting epinephrine (E), norepinephrine (NE), methylepinephrine (MN), dopamine (DA), methylnorepinephrine (NMN) and 3-methoxytyramine (3-MT) in plasma based on ultrafiltration, and ultrafiltration is used to remove macromolecules such as proteins and pigments in plasma samples, so that catecholamines are extracted and purified. The present invention includes the following steps: pre-treating the plasma sample, adding an appropriate amount of isotope internal standard to the plasma sample, mixing evenly, transferring all samples to an ultrafiltration plate, positively pressurizing on a positive pressure device, receiving the filtrate into a sample injection plate, and detecting it on a liquid chromatography-tandem mass spectrometer.

[0256] It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments may be combined with each other at will.

Claims

1. A method for detecting multiple catecholamines in plasma, characterized in that: The steps include: 1) taking a plasma sample, adding a catecholamine isotope internal standard solution and mixing evenly, and ultrafiltrating the mixed solution to obtain a filtrate; 2) performing liquid chromatography tandem mass spectrometry on the filtrate to obtain the contents of various catecholamines; The catecholamines include at least one of adrenaline, norepinephrine, methylepinephrine, dopamine, methylnorepinephrine, and 3-methoxytyramine; The isotope internal standard solution contains a stabilizer; the stabilizer solution includes at least one of ascorbic acid, hydrochloric acid and methanol.

2. The method according to claim 1, characterized in that The stabilizer solution comprises ascorbic acid, hydrochloric acid and methanol; the stabilizer solution contains 1-5 mg / mL ascorbic acid and a mixed solution of hydrochloric acid and pure methanol.

3. The method according to claim 1, characterized in that The ultrafiltration method comprises transferring the mixed solution to an ultrafiltration plate under positive pressure.

4. The method according to claim 3, characterized in that The ultrafiltration plate comprises a 3KD ultrafiltration plate.

5. The method according to claim 3, characterized in that: The positive pressure method includes applying positive pressure on a positive pressure device for 25-40 minutes; the pressure of the positive pressure is 0.1-0.2 MPa.

6. The method according to claim 1, characterized in that The isotope internal standards include epinephrine-IS, norepinephrine-IS, methylepinephrine-IS, dopamine-IS, methylnorepinephrine-IS, and 3-methoxytyramine-IS.

7. The method according to claim 6, characterized in that In the isotope internal standard solution, the concentrations of the various substances are: Epinephrine-IS: 2000-3000 pg / mL; Norepinephrine-IS: 3000-5000 pg / mL; Metanephrine-IS: 2000-3000 pg / mL; Dopamine-IS: 1000-1500 pg / mL; Methylnorephinephrine-IS: 2000-3000 pg / mL; 3-Methoxytyramine-IS: 1000-1500 pg / mL.

8. The method according to claim 1, characterized in that Liquid chromatography tandem mass spectrometry detection: Chromatographic conditions: Mobile phase A: aqueous solution containing 0.10 wt% formic acid Mobile phase B: acetonitrile solution containing 0.10%wt formic acid Chromatographic column model: E-PMN reverse phase column The mobile phase gradient conditions were:

9. The method according to claim 1, characterized in that: Liquid chromatography tandem mass spectrometry detection: Mass spectrometry conditions: Ion source: ESI ion source Detection mode: MRM acquisition mode; The ion source parameters are: spray voltage 0.5 kV, ion source temperature 500 °C, Desolvation: 1100 L / Hr, Cone: 150 L / Hr, Nebuliser: 7.0 Bar; Q1 / Q3 ion channels are as follows:

10. The method according to claim 1, characterized in that The mixing method includes vortex mixing.

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