A method and kit for detecting catecholamines and their metabolites in urine

Abstract

The application discloses a method and a kit for detecting catecholamine and its metabolites in urine, and comprises the following steps: adding an internal standard working solution to a urine sample to be detected, mixing, then adding butyric anhydride solution, mixing, and then oscillating and mixing at 20-35 DEG C for 5-30 min to perform a derivatization reaction; after the derivatization reaction is completed, centrifugation is performed, the supernatant is taken, and liquid chromatography tandem mass spectrometry is used for detection. The toxicity and danger of butyric anhydride are low, the by-product after derivatization is only butyric acid, and the by-product does not interfere with catecholamine substance detection, and a neutral or weakly alkaline environment with a pH of 7.0-8.0 can meet the derivatization conditions. Since the pretreatment time is short, and the structure is changed after derivatization, the stability of catecholamine prototype substances is improved, meanwhile, the structure after derivatization is more easily ionized in a mass spectrometry ion source, the substance response intensity is also increased, and the detection result is more reliable.

Description

Technical Field

[0001] This invention belongs to the field of catecholamine detection technology, specifically relating to a method and kit for detecting catecholamines and their metabolites in urine. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Pheochromocytoma and paraganglioma (PPGL) are neuroendocrine tumors originating from the adrenal glands and possessing hormone-secreting functions. They primarily synthesize, secrete, and release large amounts of catecholamines, which can cause a series of diseases in patients, including hypertension, cardiovascular, cerebrovascular, and renal complications, and metabolic changes. In severe cases, they can lead to death. Catecholamines, as neurotransmitters and hormones, have strong physiological activity and play an important role in the brain and nerve signal transduction.

[0004] While testing for catecholamines in plasma or urine is recommended, plasma catecholamine levels are more susceptible to patient emotions and blood collection position than urine levels, affecting the stability of test results. Urine catecholamines (dopamine (DA), norepinephrine (NE), adrenaline (E)) and their metabolites (3-methoxytyramine (3-MT), norepinephrine (NMN), metanephrine (MN), homovanillic acid (HVA), vanillylmandelic acid (VMA)) have significant diagnostic value for catecholamine-secreting tumors such as PPGL. Currently, clinical or laboratory diagnostic methods mainly include radioimmunoassay, liquid chromatography-electrochemical detection, and liquid chromatography-tandem mass spectrometry, with liquid chromatography-tandem mass spectrometry being the most commonly used.

[0005] The selection of pretreatment is very important in the method development process. The reported methods include solid phase extraction (SPE), magnetic bead method, derivatization method, and direct dilution method, each of which has certain shortcomings. For example, the SPE method uses the principle of WCX weak cation exchange, and needs to go through the processes of activation, sample loading, elution, elution, nitrogen blowing, and redissolution to enrich and purify the analyte of the sample. Although the purification effect is good, the pretreatment process is too complicated, the reproducibility between laboratories is poor, the data results are unstable, and the phenolic amine structure of catecholamine itself is easy to oxidize and degrade. In addition, the two end metabolites, homovanillic acid (HVA) and vanillylmandelic acid (VMA), are not suitable for the SPE method due to their own properties, and the SPE plate also brings high cost.

[0006] The sensitivity of the magnetic bead method is not high, and the lower limit of quantification or the lowest point of the calibration curve is difficult to detect, especially for catecholamines (DA, NE, E) with low content, which affects the effective development of the project in the clinic. In addition, high-quality magnetic beads are expensive, and if the quality of the magnetic beads is not good, it will affect the capture of the target substance, and a full-automatic extraction instrument is also required, which is a large overall investment.

[0007] Derivatization method is mainly based on the characteristics of catecholamine substances, such as chemical instability and low content in the human body. The catecholamine substances are derivatized into more stable substances by specific chemical reagents through chemical reaction, and higher response can be obtained in the instrument, thereby improving the detection sensitivity. The commonly used derivatization reagents include dansyl chloride, acetaldehyde, benzoyl chloride, TMBB-Su, etc. However, most of the derivatization reagents have certain risks, which limit their application in the field of clinical testing, and the derivatization effect is uneven. In addition, the production of fluorescent substances may cause certain damage to the instrument.

[0008] Direct dilution method is generally used for detecting total catecholamines in urine. In addition to catecholamine prototypes (DA, NE, E), the content of other five metabolite forms of total catecholamines is high, and direct dilution method can be used. The operation is simple and the cost is low, but the detection of prototypes is difficult. Catecholamines in urine are generally divided into free and bound forms. The bound form is the sulfate salt combined form of free catecholamines under the action of gastrointestinal sulfotransferase. This combined form generally needs to be hydrolyzed by high temperature acid to open, so the free form of catecholamines in urine is generally detected in the clinic.

[0009] In summary, the above several common catecholamine pretreatment methods are basically developed based on the LC-MS / MS platform, mainly using the high sensitivity and high specificity of the platform, but due to the objective problems of catecholamine substances, such as instability, easy degradation, low content, and the presence of interfering substances, it is found that the detection results are greatly different among laboratories in actual use, and the EQA activity results of urine catecholamine show that the CV among laboratories is very large, which shows that the consistency of urine catecholamine detection results is poor. SUMMARY

[0010] In view of the deficiencies in the prior art, the purpose of the present application is to provide a method and kit for detecting catecholamines and their metabolites in urine.

[0011] In order to achieve the above-mentioned purpose, the present application is realized by the following technical solutions:

[0012] In a first aspect, the present application provides a method for detecting catecholamines and their metabolites in urine, comprising the following steps:

[0013] An internal standard working solution is added to the urine sample to be detected, and after mixing, butyric anhydride solution is added and mixed, and then derivatization reaction is carried out at 20-35℃ for 5-30min.

[0014] After the derivatization reaction is completed, centrifugation is performed, and the supernatant is taken for detection by liquid chromatography tandem mass spectrometry.

[0015] In some embodiments, the volume ratio of the urine sample to be detected, the internal standard working solution and the butyric anhydride solution is 50:100-200:150-250.

[0016] Preferably, the volume ratio of the urine sample to be detected, the internal standard working solution and the butyric anhydride solution is 50:150:200.

[0017] Preferably, the concentration of butyric anhydride in the butyric anhydride solution is 10-30%, preferably 15-25%.

[0018] In some embodiments, the centrifugation speed is 10000-15000rpm, and the centrifugation time is 2-10min.

[0019] Preferably, the centrifugation speed is 11000-13000rpm, and the centrifugation time is 4-7min.

[0020] In some embodiments, the liquid chromatography conditions are as follows: flow rate: 0.1-0.5mL / min; column temperature: 30-40℃; mobile phase A: 0.01-0.05% formic acid 1-5mM ammonium formate 5-15% methanol aqueous solution; mobile phase B: acetonitrile; chromatographic column: InertSustain C18 2.1mm*100mm*3μm.

[0021] Formic acid can improve the ionization efficiency of analyte in positive ion scanning mode of ESI source, but the proportion should not be too high, because the analyte VMA is in negative ion scanning mode; ammonium formate can improve the chromatographic peak shape, making the peak shape more smooth, symmetric and sharp; the mobile phase A is an aqueous phase, as a component of the kit, when the storage or transportation temperature is too high, it is easy to be contaminated, adding a certain proportion of organic solvent methanol can prevent the growth of bacteria, on the other hand, methanol is a protic solvent, which belongs to a weak eluent, and can enhance the retention of compounds to obtain chromatographic peaks with better specificity.

[0022] Preferably, the liquid chromatography conditions are as follows: flow rate: 0.4 mL / min; column temperature: 40℃; mobile phase A: 0.01% formic acid 1mM ammonium formate 10% methanol aqueous solution; mobile phase B: acetonitrile.

[0023] In a second aspect, the present application provides a kit for detecting catecholamine and its metabolites in urine, comprising a derivatization buffer, a derivatization reagent, a mobile phase A, a mobile phase B, a catecholamine calibrator, a catecholamine calibrator diluent, a catecholamine quality control I, a catecholamine quality control II, a catecholamine internal standard, a 96-well sample plate and a silica gel cover plate.

[0024] The derivatization reagent is a solution of butyric anhydride in acetonitrile with a concentration of 10-30%.

[0025] In some embodiments, the mobile phase A is a 0.01-0.05% formic acid 1-5mM ammonium formate 5-15% methanol aqueous solution.

[0026] Experiments have found that butyric anhydride in the derivatization reagent has good effect, butyric anhydride is an organic solvent with a molecular formula of C8H 14 O3, which is formed by the combination of two molecules of butyric acid in anhydride form. In an alkaline environment, this structure is easy to esterify with phenolic hydroxyl or amino group, thereby changing the chemical structure of the original substance and changing the physical and chemical properties.

[0027] Taking dopamine as an example, the derivatization process is as follows: in an alkaline environment, the two phenolic hydroxyl groups and one amino group of dopamine esterify with butyric acid groups, respectively, to produce dopamine derivatives as the main product and butyric acid as the byproduct. The optimal ion pair of dopamine derivative under mass spectrometry detection is 364→137.

[0028]

[0029] Danger: Compared with commonly used dansyl chloride, acetaldehyde, benzoyl chloride and TMBB-Su, butyric anhydride has lower toxicity and danger, and is more suitable for use in clinical testing sites.

[0030] Whether to introduce interference: such as the derivative reagent dansyl chloride, which is a yellow crystalline powder, is a fluorescent derivative reagent, mainly in the strong alkaline environment with biological amines for derivatization reaction, commonly used in amino acid modification analysis, the derivative product is generally blue or blue-green fluorescent substance. But for the detection instrument like mass spectrometry, the instrument itself is relatively expensive, and the maintenance cost is also high, so it is necessary to reduce the introduction of complex structure or fluorescent substance, and to reduce the influence on the instrument to the minimum. In addition, due to the existence of strong alkaline derivatization environment, generally pH is about 11.0, before sample analysis, the pH environment needs to be adjusted, otherwise it is not friendly to the subsequent chromatographic separation. The by-product of butyric anhydride derivatization is only butyric acid, which will not interfere with the detection of catecholamine substances, and the derivatization environment is generally pH 7.0-8.0, neutral or weakly alkaline environment can meet the derivatization conditions.

[0031] Simplify the operation steps: without complex SPE pretreatment process and time-consuming nitrogen blowing process, after derivatization, centrifugation and supernatant can be detected on the machine, which greatly saves the pretreatment time, and the operation is simple, the steps are less, and the standardization and standardization of the pretreatment method are realized.

[0032] Reduce the cost: without SPE pretreatment plate and supporting instruments and magnetic bead fillers required by magnetic bead method, the cost is effectively reduced.

[0033] Improve the sensitivity: it can ensure the effective detection of catecholamine prototypes (DA, NE, E) in urine, and the effective detection of end metabolites VMA with good peak type.

[0034] Improve the stability of the substance: because the pretreatment time is short, and the structure is changed after derivatization, the stability of catecholamine prototypes (DA, NE, E) is improved, and the structure after derivatization is more easily ionized in the mass spectrometry ion source, the response intensity of the substance is also increased, and the detection result is more reliable. BRIEF DESCRIPTION OF DRAWINGS

[0035] The drawings accompanying the specification of this application form a part of this application and serve to further understand the application. The schematic embodiments of the application and their descriptions are used to explain the application, and do not constitute an improper limitation on the application.

[0036] Figure 1 is the chromatogram of 8 kinds of catecholamine derivatives in the embodiment of the application;

[0037] Figure 2 is the chromatogram of VMA measured by SPE method in the embodiment of the application;

[0038] Figure 3 is the chromatogram of VMA measured by derivatization method in the embodiment of the application. DETAILED DESCRIPTION

[0039] It should be noted that the following detailed description is illustrative only and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0040] The application is further described below in conjunction with examples.

[0041] Examples

[0042] Sample: 24h urine sample;

[0043] Reagents: standard DA, NE, E, 3-MT, NMN, MN, HVA, VMA; internal standard DA-D4, NE-D6, E-D3, 3-MT-D3, NMN-D3, MN-D3, HVA-D3, VMA-D3; methanol, acetonitrile, formic acid, hydrochloric acid, ammonium formate, butyric anhydride, PBS solution, etc.

[0044] Instrument: high performance liquid chromatography tandem mass spectrometry detection system YS EXACT 9900MD.

[0045] Reagent preparation:

[0046] 1) Preparation of calibrators: weigh DA, E, NE, 3-MT, MN, NMN, HVA, VMA standard dry powder respectively, add to clean light-resistant plastic reagent bottles (glass bottles have adsorption), take a certain amount of methanol containing 5% 1M hydrochloric acid (v / v) to dissolve DA, E, NE standards, take a certain amount of methanol to dissolve 3-MT, MN, NMN, HVA, VMA standards, shake well and mix until completely dissolved, prepare high-concentration standard stock solution. And mark, -70℃ or below for storage for future use. The concentration of each analyte standard stock solution is shown in Table 1:

[0047] Table 1

[0048]

[0049] Add the standard stock solution to the blank urine, and stir well to obtain the concentration shown in Table 2. The calibration curve is prepared using the calibrators.

[0050] Table 2

[0051] Analyte Calibrator target concentration DA (pg / L) 1500 NE (pg / L) 750 E (pg / L) 300 3-MT (pg / L) 750 NMN (pg / L) 450 MN (pg / L) 450 HVA (mg / L) 60 VMA (mg / mL) 60

[0052] 2) Preparation of quality control: add the above standard stock solution to the blank urine, and stir well to obtain the concentration shown in Table 3. The quality control is prepared.

[0053] Table 3

[0054] Analyte Low control target High control target DA (pg / L) 30 750 NE (pg / L) 15 375 E (pg / L) 6 150 3-MT (pg / L) 15 375 NMN (pg / L) 9 225 MN (pg / L) 9 225 HVA (mg / L) 1.2 30 VMA (mg / mL) 1.2 30

[0055] 3) Internal standard preparation: DA-D4, NE-D6, E-D3, 3-MT-D3, NMN-D3, MN-D3, HVA-D3, VMA-D3 internal standard standard dry powder were weighed respectively and added into clean light-protected plastic reagent bottles (glass bottles have adsorption), a certain amount of methanol was added to dissolve the internal standard, and the internal standard stock solution was prepared, and the internal standard stock solution was prepared. The concentration of each internal standard stock solution is shown in Table 4:

[0056] Table 4

[0057]

[0058] Internal standard mixture preparation: The internal standard stock solution was taken out from the-18°C refrigerator, and diluted with methanol to the concentration of the internal standard mixture in Table 5.

[0059] Table 5

[0060] Internal standard species Formulated concentration (pg / mL) DA-D4 30 NE-D6 6 E-D3 4 3-MT-D3 3 NMN-D3 8 MN-D3 4 HVA-D3 75 VMA-D3 150

[0061] 4) Derivative reagent: 20% butyric anhydride in acetonitrile solution;

[0062] Derivative environment: PBS buffer containing 1.0 mM EDTA, pH 7.0-8.0.

[0063] Pre-treatment and derivation process: preparation of calibration curve: take the above prepared calibration sample, dilute with blank urine in proportion to obtain 7 concentrations (S1-S7), and the concentration points are shown in Table 6:

[0064] Table 6

[0065]

[0066] Derivation process: ① The internal standard mixture was diluted 100 times with the above prepared PBS buffer containing 1.0 mM EDTA to obtain the internal standard working solution; ② 50 μL of urine sample was taken and added to a 1.5 mL centrifuge tube, 150 μL of internal standard working solution was added, and the mixture was mixed well; ③ 200 μL of derivative reagent was added and mixed well; ④ room temperature oscillation mixing for 15 min; ④ 12,000 rpm centrifugation for 5 min, and the supernatant was taken for sample analysis. The preparation process of the calibration sample and the quality control sample is the same as above.

[0067] Instrument conditions:

[0068] ① Liquid phase conditions: mobile phase A: 0.01% formic acid 1 mM ammonium formate 10% methanol aqueous solution; mobile phase B: acetonitrile; chromatographic column: InertSustain C18 2.1 mm*100 mm*3 um; injection volume: 5 μL; flow rate: 0.4 mL / min; column temperature: 40°C; liquid phase gradient as shown in Table 7:

[0069] Table 7

[0070]

[0071] ② Mass spectrometry conditions: positive and negative ion mixed scanning mode was used, YS EXACT 9900MD: ion source temperature was 300℃, spray voltage was 3500V (+), 2500V (-). Data was collected by MRM mode, conditions were as shown in Table 8:

[0072] Table 8

[0073]

[0074]

[0075] 1) Limit of quantification

[0076] The limit of quantification of each analyte was shown in Table 9:

[0077] Table 9

[0078]

[0079] Each catecholamine standard was diluted with blank matrix to obtain five samples at the limit of quantification concentration, each sample was repeatedly measured five times, and the measurement results were recorded. According to the following formula, the coefficient of variation (CV) of the five measurement results of each limit of quantification concentration sample and the relative deviation of the average concentration of the five times from the theoretical concentration were obtained. The coefficient of variation (CV) of the measurement results was ≤20%, and the relative deviation of the measured value from the specified value was within ±15%, then the concentration was the limit of quantification concentration of the analyte.

[0080] CV = SD / M x 100%;

[0081] Deviation = |M-LOQ| / LOQ x 100%;

[0082] Where: CV: coefficient of variation; SD: standard deviation of measurement results; M: average value of measurement results; LOQ: limit of quantification test results as shown in Table 10:

[0083] Table 10

[0084]

[0085]

[0086] 2) Linear range

[0087] Dilute each catecholamine standard with blank matrix to obtain high-value samples close to the upper limit of the linear range. Prepare different concentration level samples from the high-value samples in turn, and test each level sample 3 times to obtain the mean value (yi) of each concentration gradient determination result. Perform linear regression analysis with the dilution concentration (xi) as the independent variable and the mean value (yi) of the determination result as the dependent variable, and obtain the estimated value by substituting the dilution concentration (xi) into the regression equation. Calculate the relative deviation of y i i from the estimated value, and require that the correlation coefficient (r) should not be less than 0.9900, the concentration deviation of the minimum point should be within ±20%, and the concentration deviation of the remaining points should be within ±15%.

[0088] The test results are shown in Table 11:

[0089] Table 11

[0090]

[0091] 3) Recovery rate

[0092] Select a clinical sample, and add low and high concentrations of each catecholamine standard solution to the sample respectively to prepare a recovery sample. The volume of the standard solution added should not exceed 10% of the volume of the clinical sample. Divide each concentration sample into 5 parts, and determine each part 3 times. Calculate the mean value of 3 determinations for each sample, and calculate the recovery rate according to the formula, which should meet the requirement of 85%-115%.

[0093]

[0094] wherein V is the volume of the standard solution added, V0 is the volume of the clinical sample, C is the mean value of the detection concentration of the clinical sample after adding the standard solution of the analyte, C0 is the mean value of the detection concentration of the analyte in the clinical sample, and C s is the concentration of the analyte in the standard solution. The test results are shown in Table 12:

[0095] Table 12

[0096]

[0097] 4) Intra-day precision

[0098] Use low and high concentration level samples, and determine each 10 times to calculate the coefficient of variation (CV) of 10 measurement results, which should meet the requirement of CV≤15%, as shown in Table 13.

[0099] Table 13

[0100]

[0101] 5) Inter-day precision ​

[0102] Using low and high concentration levels of samples, test for 3 days, repeat 10 times a day, calculate the coefficient of variation (CV) of 30 measurements, which should meet the requirement of CV≤15%, see Table 14.

[0103] Table 14

[0104]

[0105] Kit and sample testing

[0106] 1. Product name

[0107] Eight catecholamine assay kit (high performance liquid chromatography-tandem mass spectrometry)

[0108] 2. Package size

[0109] 96 servings per box, 480 servings per box.

[0110] 3. Intended use

[0111] The kit is used for in vitro quantitative detection of dopamine, norepinephrine, adrenaline, 3-methoxytyramine, normetanephrine, metanephrine, homovanillic acid, and vanillylmandelic acid in human urine samples.

[0112] Catecholamines as neurotransmitters and hormone substances have strong physiological activity and play an important role in brain and nerve signal transmission. The levels of catecholamines (dopamine (DA), norepinephrine (NE), adrenaline (E)) and their metabolites (3-methoxytyramine (3-MT), normetanephrine (NMN), metanephrine (MN), homovanillic acid (HVA), and vanillylmandelic acid (VMA)) in urine are closely related to various physiological and pathological phenomena in the human body. The detection of catecholamine content in the human body is clinically used for the auxiliary diagnosis of catecholamine metabolism-related diseases such as hypertension and adrenal medulla hyperplasia, and the auxiliary evaluation of treatment effect.

[0113] The main clinical manifestations of patients with increased secretion of catecholamines and their metabolites are hypertension and vascular complications of the heart, brain, and kidneys, as well as metabolic changes. Patients have the following multisystem clinical manifestations:

[0114] I. Blood pressure changes: patients can be paroxysmal, persistent or paroxysmal exacerbation on the basis of persistent hypertension, some patients with orthostatic hypotension.

[0115] II. Associated symptoms: patients with hypertension accompanied by headache, palpitations, sweating and other symptoms, myocardial contractility, heart rate or palpitations, irritability and other symptoms.

[0116] Hypertension can be accompanied by heart, brain, kidney and other organ function or organic damage, and needs early diagnosis and early treatment. Therefore, the detection of catecholamine and its metabolite concentration in urine has very important clinical significance.

[0117] 4. Detection principle

[0118] First, the sample (including calibration, quality control, urine samples) is derived, centrifuged, and then the supernatant is detected by high performance liquid chromatography-tandem mass spectrometry. Data is collected in multiple reaction monitoring mode (MRM / SRM), and the chromatogram and peak area of each analyte and corresponding internal standard in the sample are recorded. The ratio of the peak area of each analyte in the sample to the corresponding internal standard is calculated, and the target concentration of the calibration is taken as the abscissa, and the peak area ratio of the calibration and the internal standard is taken as the ordinate. The standard curve is drawn and the linear equation is fitted, and the concentration of the sample (quality control, clinical sample) to be measured is calculated by substituting the peak area ratio of the sample (quality control, clinical sample) to be measured and the corresponding internal standard into the standard curve equation.

[0119] 5. Kit composition, see Table 15.

[0120] Table 15

[0121]

[0122]

[0123] Note: The calibration, calibration diluent, quality control and internal standard are provided in lyophilized powder form for easy transportation and storage.

[0124] 6. Storage conditions and shelf life

[0125] The calibration, quality control, internal standard and calibration diluent are stored at -18°C or below in the dark and sealed, and the other components are stored at 2°C-30°C in the dark and sealed. The kit is valid for 12 months from the date of production.

[0126] 7. Operation method

[0127] Preparation of internal standard stock and working solutions

[0128] (1) Take 200 μL of methanol and add it to the internal standard dry powder vial, vortex for 1 min to prepare the internal standard concentrate.

[0129] (2) Dilute the internal standard stock solution with derivatization buffer at a ratio of 1:150 to prepare the internal standard working solution. The internal standard working solution needs to be prepared immediately before use.

[0130] Preparation of calibrator working solutions

[0131] (1) Preparation of high concentration calibrator solution (S1): Take 0.4 mL of pure water and add it to the calibrator dry powder vial, dissolve and mix well.

[0132] (2) Preparation of calibrator diluent: Take 1.5 mL of pure water and add it to the calibrator diluent dry powder vial, dissolve and mix well.

[0133] (3) Preparation of calibrator working solution: Take the high concentration calibrator solution and dilute it with the calibrator diluent in proportion to form 7 concentrations (S1-S7). The dilution method and the corresponding theoretical concentrations of each calibrator working solution are as follows in Table 16:

[0134] Table 16

[0135]

[0136] Preparation of control solutions

[0137] Take 0.4 mL of pure water and add it to the quality control I and quality control II dry powder vials, dissolve and mix well.

[0138] Sample preparation

[0139] (1) Take 50 μL of urine sample and add it to a 1.5 mL centrifuge tube, add 150 μL of internal standard working solution, mix well;

[0140] (2) Add 200 μL of derivatization reagent to the above mixture and mix well;

[0141] (3) Mix well at room temperature for 15 min;

[0142] (4) Centrifuge at 12000 rpm for 5 min, take 100 μL of supernatant to a 96-well injection plate, cover with a silica gel cover plate, for injection analysis;

[0143] (5) The preparation process of calibrators and quality controls is the same as above.

[0144] Instrument method

[0145] (1) Liquid chromatography conditions

[0146] Mobile phase A: 0.01% formic acid 1 mM ammonium formate 10% methanol in water; mobile phase B: acetonitrile; column: InertSustain C18 2.1 mm*100 mm*3 μm; injection volume: 5 μL; flow rate: 0.4 mL / min; column temperature: 40 °C.

[0147] Liquid phase gradient as shown in Table 17:

[0148] Table 17

[0149]

[0150] (2) Mass spectrometry conditions

[0151] The positive and negative ion mixed scanning mode was used with an ESI source, YS EXACT 9900 MD: ion source temperature was 300 °C, and the spray voltage was 3500 V (+), 2500 V (-). The data were collected in MRM mode, and the conditions were as shown in Table 18:

[0152] Table 18

[0153]

[0154] 8. Data processing

[0155] The instrument analysis software was opened, and the corresponding data were selected for result analysis. First, the standard curve was drawn with the calibration standard concentration as the abscissa and the peak area ratio of the calibration standard to the internal standard as the ordinate, to obtain the linear equation y = bx + a. The peak area ratio of the sample to the internal standard was substituted into the equation to calculate the concentration of the analyte in the sample.

[0156] 9. Calibration procedure

[0157] (1) It is recommended to use the kit to calibrate multiple points with the target concentration of the calibration standard as the abscissa and the peak area ratio of the calibration standard to the corresponding internal standard as the ordinate to draw a standard curve. The calibration needs to be performed for each experiment.

[0158] (2) The correlation coefficient r of the standard curve should be greater than or equal to 0.99 to carry out the detection.

[0159] (3) The calibration standard should be prepared in the same way as the sample, and the reference sample should be prepared.

[0160] 10. Quality control procedure

[0161] (1) The quality control sample should be involved in the whole process of extraction and detection. The quality control should be in control to ensure the reliability of the detection results. The quality control sample should be detected for each experiment.

[0162] (2) Each laboratory should establish its own quality control average value and allowable error range based on the indicated quality control target value.

[0163] (3) It is suggested to use matched quality control products for indoor quality control. The control value of determination should be within the determined limit range. If the control is out of control, the laboratory should find out the reason and take appropriate corrective measures.

[0164] The liquid chromatogram of sample determination is shown in Figure 1. Figure 1

[0165] Comparative Example 1

[0166] The magnetic bead method was used to detect catecholamine in urine. The urine sample to be detected was the same as that in Example 1.

[0167] 1) Magnetic adsorbent: prepare 0.2 g / mL WCX magnetic adsorbent suspension;

[0168] 2) Take 50 μL of sample, add 150 μL of internal standard working solution, mix well, and then transfer all into the hole position of the 96-hole plate of the magnetic automatic solid-phase extraction device;

[0169] 3) As shown in the following table, take the solution in each hole position, and set the magnetic solid-phase extraction automatic extraction method. Add 10 μL of completely mixed magnetic adsorbent suspension in the corresponding hole. The device running process is shown in Table 19:

[0170] Table 19

[0171]

[0172] 4) After processing, take 40 μL of supernatant for sample analysis.

[0173] Comparative Example 2

[0174] The SPE method was used to detect catecholamine in urine. The urine sample to be detected was the same as that in Example 1.

[0175] 1) Sample loading: take 50 μL of sample, add 150 μL of internal standard working solution, and mix well. Transfer all the above liquid to the 96-hole SPE plate, and press the sample into the hole through the positive pressure device.

[0176] 2) Elution: add 100 μL of elution liquid to the hole of the 96-hole SPE plate, and press the liquid into the 96-hole plate through the positive pressure device.

[0177] 3) Elution: add 75 μL of elution liquid to the hole of the 96-hole SPE plate, and collect the liquid through the 96-hole receiving plate. Repeat the elution liquid operation step twice.

[0178] 4) Nitrogen blowing: place the 96-hole receiving plate on the nitrogen blowing instrument, and blow the liquid in the hole with nitrogen.

[0179] ​5) Reconstitution: Add 50 μL reconstitution solution to each well of the 96-well receiving plate, and mix well by blowing.

[0180] 6) On-machine: Cover the silica gel cover plate, and prepare for detection.

[0181] 7) The preparation of calibrators and quality controls is consistent with the sample.

[0182] Comparative Example 3

[0183] Danusil chloride derivation:

[0184] 1) Take 50 μL of the sample, add 150 μL of the internal standard working solution, and mix well by blowing.

[0185] 2) Freeze centrifugation at 13000 rpm for 5 min.

[0186] 3) Take the supernatant, and dry it under nitrogen.

[0187] 4) Add 50 μL of acetonitrile solution containing 5 mg / mL of danusil chloride, and 100 μL of 0.1 mol / L Na2CO3 buffer with a pH value of 11.0, mix well, and then place in a 50°C water bath for 30 min.

[0188] 5) After the derivation reaction is completed, adjust the pH to about 7.0 with formic acid, and freeze centrifuge at 13000 rpm for 5 min.

[0189] 6) Take the supernatant for sample injection analysis.

[0190] The response values of the analytes in the urine samples are shown in Table 19.

[0191] Table 19

[0192]

[0193] Improved stability of the substance: Since the pretreatment time is short, and the structure is also changed after derivation, the stability of the catecholamine prototype (DA, NE, E) substance is improved. At the same time, the structure after derivation is more easily ionized in the mass spectrometry ion source, and the response intensity of the substance is also increased, so the detection result is more reliable.

[0194] Comparative Example 3 is also a derivation, but there is a nitrogen blowing process in the pretreatment process, and the derivation time is long, which significantly increases the pretreatment time. In addition, for NE, E, and 3-MT with relatively low content, although the signal response is improved compared with the magnetic bead method and the SPE method, the improvement effect is obviously inferior to that of Example 1.

[0195] Improved peak shape: The end metabolite VMA by the SPE method is affected by two states, and the peak shape is wide and tailing, and the overall peak shape is poor. See Figure 2And the structure of the VMA derivative changes, and the physicochemical properties change accordingly, the VMA is effectively detected and the peak shape is good, see Figure 3 , the peak shape of the derivative method is better.

[0196] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for detecting catecholamines and their metabolites in urine, characterized in that: Includes the following steps: Add internal standard working solution to the urine sample to be tested, mix well, add butyric anhydride solution, mix well, and then carry out derivatization reaction by shaking and mixing at 20-35℃ for 5-30 minutes. The concentration of butyric anhydride in the butyric anhydride solution is 20%. After the derivatization reaction was completed, the mixture was centrifuged, and the supernatant was collected and analyzed by liquid chromatography-tandem mass spectrometry. The derivatization environment is a PBS buffer containing 1.0 mM EDTA, pH 7.0-8.0; The liquid chromatography conditions were as follows: flow rate: 0.1-0.5 mL / min; column temperature: 30-40℃; mobile phase A: 0.01-0.05% formic acid, 1-5mM ammonium formate, and 5-15% methanol aqueous solution; mobile phase B: acetonitrile; chromatographic column: InertSustain C18 2.1mm*100mm*3μm; The volume ratio of the urine sample to be tested, the internal standard working solution, and the butyric anhydride solution is 50:150:

200.

2. The method for detecting catecholamines and their metabolites in urine according to claim 1, characterized in that: The centrifugation speed is 10000-15000 rpm, and the centrifugation time is 2-10 min.

3. The method for detecting catecholamines and their metabolites in urine according to claim 2, characterized in that: The centrifugation speed is 11000-13000 rpm, and the centrifugation time is 4-7 min.

4. The method for detecting catecholamines and their metabolites in urine according to claim 1, characterized in that: The liquid chromatography conditions were as follows: flow rate: 0.4 mL / min; column temperature: 40℃; mobile phase A: 0.01% formic acid, 1mM ammonium formate, and 10% methanol aqueous solution; mobile phase B: acetonitrile.

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