CN117554550B - Liquid chromatography tandem mass spectrometry detection method for catecholamine metabolites in plasma and combined stabilizer - Google Patents
Liquid chromatography tandem mass spectrometry detection method for catecholamine metabolites in plasma and combined stabilizer Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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- Life Sciences & Earth Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a liquid chromatography tandem mass spectrometry detection method of catecholamine metabolites in blood plasma, a combined stabilizer and application thereof. The detection method comprises the steps of firstly adding a combined stabilizer into a blood collection tube, wherein the combined stabilizer comprises the following components in percentage by mass: 0.5-2% of ascorbic acid, 0.15-1% of 5-hydroxy ferulic acid, 0.5-2% of formic acid and pure water, wherein the pure water is complemented to 100%; therefore, the stability of a sample before analysis is ensured, and the derivatization efficiency is improved by derivatizing the sample and adding the derivatization stabilizer L-ascorbic acid in the derivatization process, so that the detection sensitivity, the specificity, the detection flux and the detection accuracy are improved, and the method has higher application value.
Description
Technical Field
The invention relates to the technical field of catecholamine metabolite detection, in particular to a liquid chromatography tandem mass spectrometry detection method of catecholamine metabolites in blood plasma, a combined stabilizer and application thereof.
Background
Pheochromocytoma and paraganglioma (pheochromocytoma andparaganglioma, PPGL) are rare endocrine diseases and PPGL disease data are not yet available in China. The prevalence of PPGL in common hypertension clinics is reported to be 0.2% -0.6% abroad, 1.7% in children's hypertensive patients, and about 5% in adrenal gland accidental tumors. The disease can occur in all ages, the disease peak is 30-50 years old, and the incidence rate of men and women is basically the same.
Methoxyepinephrine (MN), methoxynorepinephrine (NMN) and 3-methoxytyramine (3-MT) are intermediate metabolites of catecholamines, and are important bases for qualitative diagnosis of Pheochromocytoma and Paraganglioma (PPGL). PPGL is a substance which is derived from adrenal medulla or adrenal outer sympathetic nerve chain respectively, synthesizes and secretes Catecholamine (CA) and the like, is easy to cause a series of clinical symptoms such as blood pressure rise and the like of patients, and causes serious complications such as heart, brain, kidney and the like. MN and NMN are respectively epinephrine (E), intermediate metabolites of Norepinephrine (NE) (collectively MNs) are only metabolized in adrenal medullary pheochromocyte or PPGL tumor, and persist at high concentration level, and half-life of MNs is longer than CA, and is also more stable, and its specificity and sensitivity are high, and can reflect PPGL tumor functional state. MN and NMN in plasma and urine are measured as the first detection index for screening pheochromocytoma in the diagnosis and treatment expert consensus of pheochromocytoma and paraganglioma published by the society of endocrinology of the chinese medical society in 2016 and 2020. 3-MT is an intermediary metabolite of Dopamine (DA), and the 2020 edition PPGL guide indicates that detection of 3-MT can increase sensitivity in screening for head and neck Paragangliomas (PGL); a significant increase in plasma 3-MT concentration in PPGL patients is highly indicative of metastatic tumors. The 3 compounds are all of pg grade with low in vivo content.
At present, in the method for detecting MN, NMN and 3-MT in blood plasma, the most commonly used method is a high performance liquid chromatography and electrochemical combined detection method, but the method has the defects of low sensitivity, large sample demand, long pretreatment time, strong component interference and the like, and cannot meet the clinical detection demand. The detection accuracy is low due to cross reaction and nonspecific combination based on the immune detection method, and the results of different immune tests cannot be compared with each other, so that the immune detection method has no universality. Liquid chromatography tandem mass spectrometry (LC-MS/MS) is an analysis technique for mass spectrometry detection by liquid chromatography separation, and has the characteristics of high sensitivity, high specificity and high selectivity. Several documents report that LC-MS/MS is used to detect catecholamine metabolites, and currently, non-derivative ion exchange SPE concentration is generally used to extract the analyte, but when the sample is treated by the method, the moderately and severely hemolyzed sample has different effects on the analyte. In addition, the polarity of the to-be-detected substance is large, the to-be-detected substance is difficult to be retained, and the non-derivative method is adopted to detect the to-be-detected substance, so that the retention is enhanced by using an HILIC chromatographic column, but some clinical samples contain interference peaks, and the interference peaks are difficult to separate from the to-be-detected substance, so that the detection result is abnormal.
For accurate quantification of catecholamine metabolites, in addition to improving the accuracy and precision of the detection method, another challenge to be addressed is the stability of catecholamine metabolites prior to analysis. Because catecholamine substances dopamine, epinephrine and norepinephrine are easily metabolized into 3-MT, MN and NMN by Catechol Oxygen Methyl Transferase (COMT) respectively, the content of an object to be detected in a sample is increased. Furthermore, the chemical structure of catecholamine metabolites shows that the structure has one phenolic hydroxyl group and one side chain with amino group, which is easily oxidized into quinone under neutral or alkaline conditions, resulting in reduced content of the analyte. Thus, the catecholamine metabolite concentration in patient samples may rise or fall to varying degrees prior to analysis.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a liquid chromatography tandem mass spectrometry detection method of catecholamine metabolites in plasma, a combined stabilizer and application thereof. According to the detection method, the stability of a sample before analysis is ensured by adding the combined stabilizer into the blood collection tube, and the derivatization efficiency is improved by derivatizing the sample and adding the derivatization stabilizer in the derivatization process, so that the detection sensitivity, the specificity, the detection flux and the detection accuracy are improved.
Specifically, the invention is realized through the following technical schemes:
In a first aspect, the present invention provides a liquid chromatography tandem mass spectrometry detection method of catecholamine metabolites in plasma, comprising the steps of:
(1) Collecting 3-4 mL of whole blood sample of a patient, and placing the whole blood sample in a blood collection tube containing an anticoagulant;
(2) Adding 9-11 mu L of combined stabilizer into the blood collection tube, and then gently reversing the blood collection tube for a plurality of times to mix uniformly;
(3) Placing the uniformly mixed blood collection tube in the step (2) at 3000rpm and 25 ℃, centrifugally separating for 10min, and placing the supernatant in a 1.5mLEP tube to obtain a plasma sample to be tested;
(4) Placing the plasma sample to be tested obtained in the step (3) into a centrifuge tube, adding 10-20 mu L of internal standard working solution, mixing, adding 300-900 mu L of precipitant for protein precipitation, and performing centrifugal separation to obtain supernatant;
(5) Sequentially adding 5-10 mu L of a derivatization stabilizer aqueous solution, 50-200 mu L of an acetonitrile solution of a derivatization agent and 100-300 mu L of a buffer solution into the supernatant obtained in the step (4), mixing, and performing derivatization reaction at the temperature of 30-90 ℃ for 5-30 min to obtain a derivatization sample;
(6) Adding the derivatization sample obtained in the step (5) into a solid phase extraction small column which is activated by methanol and balanced by deionized water for column passing, eluting, collecting eluent, and centrifugally separating to obtain supernatant;
(7) Carrying out LC-MS/MS detection on the supernatant obtained in the step (6);
(8) Substituting the ratio of the peak area of the catecholamine metabolite in the plasma sample to be detected obtained in the step (7) to the peak area of the internal standard into a standard curve equation, and calculating to obtain the concentration of the catecholamine metabolite in the plasma sample to be detected;
wherein the combined stabilizer consists of the following components in percentage by mass: 0.5-2% of ascorbic acid, 0.15-1% of 5-hydroxy ferulic acid, 0.5-2% of formic acid and pure water, wherein the pure water is complemented to 100%;
the derivative stabilizer in the step (5) is L-ascorbic acid, and the concentration of the L-ascorbic acid aqueous solution is 0.05% -0.2%;
The catecholamine metabolites are methoxyepinephrine, methoxynorepinephrine and 3-methoxytyramine.
In the step (4):
The internal standard working solution is prepared by mixing methoxyepinephrine-d 3, methoxynorepinephrine-d 3 and 3-methoxytyramine-d 4 and diluting the mixture with a 50% acetonitrile aqueous solution containing 0.1% -0.5% glutathione.
The precipitant is acetonitrile, methanol or a mixed solution of methanol and acetonitrile.
In the step (5):
The derivatization agent is dansyl chloride, and the concentration of the acetonitrile solution of the dansyl chloride is 1-10 mg/mL.
The buffer solution is one selected from Na2CO3-NaHCO3、Na2HPO4-NaH2PO4、K2HPO4-KH2PO4, and the pH value is 8-13.
In the step (6):
The packing materials of the solid phase extraction column are C8 and C18.
The leaching is carried out by leaching the leaching solution 1 and the leaching solution 2 respectively; the eluent 1 is selected from one of 0.1% formic acid aqueous solution, acetonitrile-water (1:9, v/v) and methanol-water (1:9, v/v), and the dosage of the eluent 1 is 100-300 mu L; the leaching solution 2 is selected from one of 50% acetonitrile water solution, acetonitrile and methanol, and the dosage of the leaching solution 2 is 20-100 mu L.
The eluent for eluting is selected from one of methanol containing 0.1-0.3% of formic acid by volume fraction, acetonitrile water (8:2, v/v) solution containing 0.1-0.3% of formic acid by volume fraction and acetonitrile containing 0.1-0.3% of formic acid by volume fraction, and the using amount of the eluent is 100-300 mu L.
In the step (7):
The conditions of the liquid chromatography are as follows:
Chromatographic column: ACQUITY UPLC BEH Phenyl,1.7 μm, 2.1X10 mm; column temperature 40 ℃; the temperature of the sample injection chamber is 10 ℃; the sample injection amount is 5 mu L; carrying out gradient elution by using a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.5-5 mmol/L ammonium formate aqueous solution, the mobile phase B is acetonitrile solution, and the gradient elution procedure is as follows:
The mass spectrum parameters are as follows:
Ionization mode: esi+; air curtain gas (CUR): 30psi; collision gas (CAD): 9psi; spray voltage (IS) 5500V; temperature (TEM) 450 ℃; gas1:60psi; gas2:60psi; multiple reaction detection (MRM).
In the step (8), the establishment of the standard curve equation includes the following steps:
1) The catecholamine metabolites of methoxyepinephrine, methoxynorepinephrine and 3-methoxytyramine are diluted to a plurality of different concentrations by using 50% acetonitrile water solution containing 0.1% -0.5% of glutathione, the combined stabilizer is added into each concentration standard solution, and the volume ratio of the standard solution to the combined stabilizer is 3-4 mL: 9-11 mu L; then adding an internal standard working solution, mixing, adding a precipitator for protein precipitation, and carrying out centrifugal separation to obtain a supernatant;
2) Sequentially carrying out the same treatment operation on the supernatant obtained in the step 1) by adopting the step (5) and the step (6), so as to obtain a plurality of standard samples, and carrying out LC-MS/MS detection on the standard samples;
3) And (3) carrying out linear regression on the standard line sample obtained in the step (2) by taking the peak area of the object to be detected and the internal standard peak area ratio (y) as an ordinate and the concentration (x) of the object to be detected as an abscissa, wherein the weight is 1/x 2, so as to obtain a standard curve equation.
In a second aspect, the present invention provides a combination stabilizer, which consists of the following components in mass fraction: 0.5 to 2 percent of ascorbic acid, 0.15 to 1 percent of 5-hydroxy ferulic acid, 0.5 to 2 percent of formic acid and pure water, wherein the pure water is complemented to 100 percent.
In a third aspect, the present invention provides the use of a combination stabilizer as described above for the liquid chromatography tandem mass spectrometry detection of catecholamine metabolites in plasma, said combination stabilizer being used for stabilizing catecholamine metabolites in plasma.
Compared with the prior art, the invention has the following beneficial effects:
(1) The combined stabilizer provided by the invention can avoid the oxidative degradation of catecholamine metabolites in blood plasma, avoid the conversion of other endogenous substances, ensure the stability of a sample before analysis, solve the problem that the catecholamine metabolites can be placed in a blood collection tube only for a short time after being collected under the existing condition, and improve the accuracy of a detection result.
(2) According to the liquid chromatography tandem mass spectrometry detection method of catecholamine metabolites in plasma, the properties of the to-be-detected substances are changed by derivatizing the samples. Compared with a non-derivative method, the polarity of the to-be-detected object is reduced, so that the retention of the to-be-detected object on a chromatographic column is enhanced, the to-be-detected object can be separated from complex interference peaks in a sample, the analysis time is short, and the sample flux is high. Secondly, the structure after derivatization contains primary amine groups, so that the ionization efficiency of the object to be detected can be effectively increased, and the detection sensitivity is improved.
(3) According to the liquid chromatography tandem mass spectrometry detection method of catecholamine metabolites in plasma, provided by the invention, the derivatization efficiency can be remarkably improved, the detection sensitivity is improved, and the response of three substances to be detected can be improved by about 4-8 times by adding the derivatization stabilizer L-ascorbic acid in the derivatization process of the sample. In addition, the detection method can accurately quantify Methoxyepinephrine (MN), methoxynorepinephrine (NMN) and 3-methoxytyramine (3-MT) in blood plasma at the same time by only 5 mu L of sample injection amount, so that the solvent effect generated during large-volume sample injection and pollution to chromatographic columns and instruments are reduced.
(4) According to the liquid chromatography tandem mass spectrometry detection method of catecholamine metabolites in plasma, complex interfering substances in plasma samples are removed to a large extent through the steps of protein precipitation, derivatization, solid phase extraction and the like, so that the influence of a hemolysis sample on a detection result is effectively avoided, and the detection accuracy of clinical abnormal hemolysis samples is improved. Therefore, the detection method can provide more timely and accurate reference basis for diagnosis of Pheochromocytoma and Paraganglioma (PPGL).
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is an ion flow chromatogram of methoxyepinephrine and its internal standard in a plasma sample.
FIG. 2 is an ion flow chromatogram of methoxynorepinephrine and its internal standard in a plasma sample.
FIG. 3 is an ion flow chromatogram of 3-methoxytyramine and its internal standard in a plasma sample.
FIG. 4 shows the separation of the analyte from the interference peak under the derivatization condition of the present invention and the separation of the analyte from the interference peak under the non-derivatization condition. Wherein the MN retention time in figure a is 2.37min and the interference peak retention time is 2.30min; in FIG. B, the MN retention time was 2.58min and the interference peak retention time was 2.23min.
FIG. 5 is a graph of comparative chromatograms of ionic strength of three catecholamine metabolites without and after addition of a derivatizing stabilizer during derivatization in accordance with the present invention. Wherein figure a is an ionic strength chromatogram with the addition of a derivatized stabilizer; panel B is an ionic strength chromatogram without the addition of a derivatized stabilizer.
Detailed Description
The following detailed description of the embodiments of the present invention is provided for better illustration of the present invention, but is not to be construed as limiting the invention.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
The test materials used in the examples described below, unless otherwise specified, are all commercially available products.
Example 1
A liquid chromatography tandem mass spectrometry detection method of catecholamine metabolites in plasma, comprising the following steps:
(1) Standards of catecholamine metabolites Methoxyepinephrine (MN), methoxynorepinephrine (NMN) and 3-methoxytyramine (3-MT) were diluted with 0.2% glutathione in 50% acetonitrile in water to the concentrations shown in table 1:
table 1: standard solutions of different concentrations
(2) Preparation of pretreatment reagent
And (3) a precipitant: methanol-acetonitrile (1:1)
Derivatizing agent solution: acetonitrile solution of 2mg/mL dansyl chloride
Buffer solution: na 2CO3-NaHCO3, pH 11
Derivatizing the stabilizer solution: 0.2% ascorbic acid aqueous solution
Internal standard working solution: mixing methoxyepinephrine-d 3, methoxynorepinephrine-d 3 and 3-methoxytyramine-d 4, and diluting with 50% acetonitrile aqueous solution containing 0.2% glutathione
Activating solution: methanol
Balancing solution: deionized water
Eluent 1:0.1% formic acid aqueous solution
Eluent 2:50% acetonitrile aqueous solution
Eluent: aqueous 80% acetonitrile solution containing 0.2% formic acid
(3) Collecting 3mL of whole blood sample of a patient, placing the whole blood sample into a blood collection tube containing an anticoagulant, adding 9 mu L of a combined stabilizer consisting of 1% of ascorbic acid, 0.5% of 5-hydroxyferulic acid, 0.5% of formic acid and pure water, and gently reversing the blood collection tube for a plurality of times to mix uniformly; then placing the uniformly mixed blood collection tube at 3000rpm and 25 ℃, centrifugally separating for 10min, and taking supernatant to obtain a plasma sample to be tested;
(4) Respectively adding a combined stabilizer consisting of 1% of ascorbic acid, 0.5% of 5-hydroxyferulic acid, 0.5% of formic acid and pure water into the standard substance solutions with different concentrations, and uniformly mixing, wherein the volume ratio of the standard substance solution to the combined stabilizer is 1mL:3 μL;
(5) Sample pretreatment process
1) Protein precipitation
Respectively taking 300 mu L of standard substance solutions with different concentrations obtained in the step (4) and 300 mu L of plasma sample to be detected obtained in the step (3), respectively adding into different 1.5mLEP pipes, respectively adding 15 mu L of internal standard working solution, adding 600 mu L of precipitant after vortex mixing, and centrifuging at 4 ℃ and 15000rpm for 5min.
2) Derivatization
Taking 800 mu L of supernatant obtained after centrifugal separation in the step 1), sequentially adding 10 mu L of derivative stabilizer aqueous solution, 200 mu L of derivative agent solution and 200 mu L of buffer solution, mixing uniformly by vortex, and carrying out derivatization reaction for 5min at 70 ℃.
3) Solid phase extraction
(A) 96 Kong Guxiang extraction plate activation, equilibration
300 Mu L of methanol is added into each hole of a 96 Kong Guxiang extraction plate to naturally drain, and 300 mu L of deionized water is added into each hole of the 96 extraction plate to naturally drain.
(B) Loading sample
And adding the sample solution after derivatization into the corresponding holes of the 96 Kong Guxiang extraction plates after activation and balancing, and naturally draining.
(C) Rinsing
300 Mu L of leacheate 1 is added into the corresponding holes of the 96 Kong Guxiang extraction plates to naturally drain, and 100 mu L of leacheate 2 is added into each hole of the 96 extraction plates to naturally drain.
(D) Elution
The 96 Kong Guxiang extraction plate was combined with a new 96 well collection plate, 200 μl of eluent was added to the wells corresponding to the 96 Kong Guxiang extraction plate to allow it to drain naturally, and then a positive pressure device was used to provide a certain pressure to allow it to drain rapidly.
Placing the collected eluent in a centrifuge, centrifuging at 4 ℃ and 15000rpm for 5min, taking the supernatant, placing the supernatant in a 96-well sample plate, sealing a membrane, and detecting on the machine.
4) On-machine detection
Preparing a mobile phase A: and adding 0.315g of ammonium formate into 1000mL of deionized water, and uniformly mixing by ultrasonic waves for later use.
Preparing a mobile phase B:1000mL acetonitrile solution, sonicated for use.
(A) Conditions of liquid chromatography
An ultra-high performance liquid chromatography tandem mass spectrometry detection system is adopted: AB SCIEX Triple Quad TM 4500MD column: ACQUITYUPLC BEH Phenyl 1.7.7 um (2.1. Times.50 mm)
Column temperature: 40 DEG C
Sample introduction chamber temperature: 10 ℃;
sample injection amount: 5 mu L
The gradient elution procedure is shown in table 2:
Table 2: gradient elution procedure
(B) The mass spectrum parameters are
Ionization mode: a positive ion electrospray ion source (esi+);
Scanning mode: multiple reactive ion monitoring mode (MRM);
Curtain Gas(CUR)：30psi；
Collision Gas(CAD)：9psi；
IonSpray Voltage(IS)：5500V；
Temperature(TEM)：450℃；
Gas1：60psi；
Gas2：60psi；
The specific mass spectral parameters of the compounds are shown in table 3:
Table 3: mass spectral parameters of compounds
* To quantify ions
5) Calculation of detection results
In the standard series sample, linear regression is carried out by taking the peak area of the object to be detected and the ratio (y) of the area of the internal standard peak as the ordinate and the concentration (x) of the object to be detected as the abscissa, and the weight is 1/x2, so that a standard curve equation is obtained. Substituting the ratio of the peak area of catecholamine metabolite in the plasma sample to be detected to the internal standard peak area into a standard curve equation, and calculating to obtain the concentration of catecholamine metabolite in the plasma sample to be detected, wherein the detection result is shown in table 4, and the ion flow chromatograms of the catecholamine metabolite are shown in fig. 1-3.
Table 4: LC-MS/MS method for detecting concentration result of catecholamine metabolite in blood plasma
Comparative example 1
A method for the non-derivatization detection of catecholamine metabolites in plasma, the steps comprising:
Non-derivatization pretreatment method:
300 mu L of the plasma sample to be detected is taken and added into a 1.5mL EP tube, 15 mu L of internal standard working solution is added, after vortex mixing, 30 mu L of 1% ammonia water is added for dilution, vortex mixing is carried out, and solid phase extraction is carried out. The solid phase extraction procedure is as in example 1.
Non-derivatized chromatographic conditions:
Chromatographic column: waters, ACQUITYUPLC@BEH HILIC, 2.1X100 mm,1.7 μm
Mobile phase: (A) 10mmol/L ammonium formate, water-acetonitrile (95:5, v/v), pH 3; (B) acetonitrile solution column temperature: 35 ℃;
Sample introduction chamber temperature: 10 ℃;
sample injection amount: 10 mu L
The gradient elution procedure is shown in table 5:
Table 5: gradient elution procedure
Non-derivatized mass spectrometry conditions:
ionization mode: a positive ion electrospray ion source (esi+);
Scanning mode: multiple reactive ion monitoring mode (MRM);
Curtain Gas(CUR)：30psi；
Collision Gas(CAD)：9psi；
IonSpray Voltage(IS)：5500V；
Temperature(TEM)：450℃；
Gas1：60psi；
Gas2：60psi；
The specific mass spectral parameters of the compounds are shown in table 6:
Table 6: mass spectral parameters of compounds
* To quantify ions
The pair of derivatization process detection chromatograms of example 1 of the present invention and the pair of non-derivatization process detection chromatograms of comparative example 1 are shown in fig. 4. As can be seen from fig. 4, the properties of the test object are changed by derivatizing the sample. Compared with a non-derivative method, the polarity of the to-be-detected object is reduced, so that the retention of the to-be-detected object on a chromatographic column is enhanced, the to-be-detected object can be separated from complex interference peaks in a sample, the analysis time is short, the sample flux is high, and the detection sensitivity is improved.
Example 2
The derivatization stabilizer is added in the derivatization process of the invention to improve the derivatization efficiency, and the specific improvement effect is shown in table 7 and fig. 5.
Table 7: influence of derivatization stabilizer on ionic strength of to-be-detected object and internal standard in derivatization process
As can be seen from table 7 and fig. 5, by adding the derivatization stabilizer L-ascorbic acid in the derivatization process of the sample, the derivatization efficiency can be significantly improved, the detection sensitivity can be improved, and the response of three test substances can be improved by about 4 to 8 times.
Example 3
The accuracy and precision of the liquid chromatography tandem mass spectrometry detection method of catecholamine metabolites in plasma were examined, and the results are shown in table 8.
Table 8: accuracy and precision investigation of detecting concentration of catecholamine metabolite in plasma by LC-MS/MS method
As can be seen from Table 8, the liquid chromatography tandem mass spectrometry detection method of catecholamine metabolites in plasma provided by the invention has high detection accuracy and precision.
Example 4
Abnormal sample interference data of the liquid chromatography tandem mass spectrometry detection method of catecholamine metabolites in plasma are examined, the concentration of the interferents is shown in table 9, and the interference results are shown in table 10.
Table 9: concentration of severe interferents in plasma samples
Table 10: severe interference result of catecholamine metabolite liquid chromatography tandem mass spectrometry detection method
From table 10, it can be seen that the concentration deviation of severe combined jaundice, severe non-combined jaundice, severe lipidemia and severe hemolysis plasma samples in the experimental group and the plasma samples in the control group is calculated to be less than +/-15%, which indicates that the interference plasma samples have no interference to the measurement result and meet the requirement of the expected use.
Example 5
Combined stabilizer for improving stability of catecholamine metabolite in blood plasma and stabilizing effect thereof
A combined stabilizer comprises 1% of ascorbic acid, 0.5% of 5-hydroxy ferulic acid, 0.5% of formic acid and pure water. The stabilizing effect is shown in tables 11 and 12.
Table 11: stability data and test results of catecholamine metabolite 6h before centrifugation
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Table 12: stability data and test results of catecholamine metabolites 16h after centrifugation
As can be seen from tables 11 and 12, after adding the combination stabilizer to the catecholamine metabolite, the catecholamine metabolite was allowed to stand stably at room temperature for at least 6 hours before centrifugation of the blood collection tube, and after centrifugation, was allowed to stand stably in the blood collection tube for 16 hours. The combined stabilizer disclosed by the invention can obviously improve the stability of catecholamine metabolites in blood plasma, ensure the stability of a sample before analysis and improve the accuracy of a detection result.
It is apparent that the above examples are only illustrative of the present invention and are not limiting of the embodiments of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (9)
1. A liquid chromatography tandem mass spectrometry detection method of catecholamine metabolites in plasma, characterized by comprising the following steps:
(1) Collecting 3-4 mL of whole blood sample of a patient, and placing the whole blood sample in a blood collection tube containing an anticoagulant;
(2) Adding 9-11 mu L of combined stabilizer into the blood collection tube, and then gently reversing the blood collection tube for a plurality of times to mix uniformly;
(3) Placing the uniformly mixed blood collection tube in the step (2) at 3000rpm and 25 ℃, centrifugally separating for 10min, and placing the supernatant in a 1.5mLEP tube to obtain a plasma sample to be tested;
(4) Placing the plasma sample to be tested obtained in the step (3) into a centrifuge tube, adding 10-20 mu L of internal standard working solution, mixing, adding 300-900 mu L of precipitant for protein precipitation, and performing centrifugal separation to obtain supernatant;
(5) Sequentially adding 5-10 mu L of a derivatization stabilizer aqueous solution, 50-200 mu L of an acetonitrile solution of a derivatization agent and 100-300 mu L of a buffer solution into the supernatant obtained in the step (4), mixing, and performing derivatization reaction at the temperature of 30-90 ℃ for 5-30 min to obtain a derivatization sample;
(6) Adding the derivatization sample obtained in the step (5) into a solid phase extraction small column which is activated by methanol and balanced by deionized water for column passing, eluting, collecting eluent, and centrifugally separating to obtain supernatant;
(7) Carrying out LC-MS/MS detection on the supernatant obtained in the step (6);
(8) Substituting the ratio of the peak area of the catecholamine metabolite in the plasma sample to be detected obtained in the step (7) to the peak area of the internal standard into a standard curve equation, and calculating to obtain the concentration of the catecholamine metabolite in the plasma sample to be detected;
wherein the combined stabilizer consists of the following components in percentage by mass: 0.5-2% of ascorbic acid, 0.15-1% of 5-hydroxy ferulic acid, 0.5-2% of formic acid and pure water, wherein the pure water is complemented to 100%;
the derivative stabilizer in the step (5) is L-ascorbic acid, and the concentration of the L-ascorbic acid aqueous solution is 0.05% -0.2%;
The catecholamine metabolites are methoxyepinephrine, methoxynorepinephrine and 3-methoxytyramine.
2. The method for liquid chromatography tandem mass spectrometry detection of catecholamine metabolites in blood plasma according to claim 1, wherein in the step (4): the internal standard working solution is prepared by mixing methoxyepinephrine-d 3, methoxynorepinephrine-d 3 and 3-methoxytyramine-d 4, and diluting with 50% acetonitrile aqueous solution containing 0.1% -0.5% glutathione; the precipitant is acetonitrile, methanol or a mixed solution of methanol and acetonitrile.
3. The method for detecting catecholamine metabolites in plasma by liquid chromatography tandem mass spectrometry according to claim 1, wherein in the step (5), the derivatization agent is dansyl chloride, and the concentration of the acetonitrile solution of the dansyl chloride is 1-10 mg/mL; the buffer solution is one selected from Na2CO3-NaHCO3、Na2HPO4-NaH2PO4、K2HPO4-KH2PO4, and the pH value is 8-13.
4. The method for detecting catecholamine metabolites in plasma by liquid chromatography tandem mass spectrometry according to claim 1, wherein in the step (6), the packing of the solid phase extraction column is C8, C18;
the leaching is carried out by leaching the leaching solution 1 and the leaching solution 2 respectively; the eluent 1 is selected from one of 0.1% formic acid aqueous solution, acetonitrile-water (1:9, v/v) and methanol-water (1:9, v/v), and the dosage of the eluent 1 is 100-300 mu L; the leaching solution 2 is selected from one of 50% acetonitrile water solution, acetonitrile and methanol, and the dosage of the leaching solution 2 is 20-100 mu L;
The eluent for eluting is selected from one of methanol containing 0.1-0.3% of formic acid by volume fraction, acetonitrile water (8:2, v/v) solution containing 0.1-0.3% of formic acid by volume fraction and acetonitrile containing 0.1-0.3% of formic acid by volume fraction, and the using amount of the eluent is 100-300 mu L.
5. The method for tandem mass spectrometry for detecting catecholamine metabolites in plasma according to claim 1, wherein in the step (7), the conditions of the liquid chromatography are as follows:
Chromatographic column: ACQUITY UPLC BEH Phenyl,1.7 μm, 2.1X10 mm; column temperature 40 ℃; the temperature of the sample injection chamber is 10 ℃; carrying out gradient elution by using a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.5-5 mmol/L ammonium formate aqueous solution, the mobile phase B is acetonitrile solution, and the gradient elution procedure is as follows:
the sample injection amount was 5. Mu.L.
6. The method for tandem liquid chromatography mass spectrometry detection of catecholamine metabolites in blood plasma according to claim 1, wherein in said step (7), said mass spectrometry parameters are: ionization mode: esi+; air curtain gas (CUR): 30psi; collision gas (CAD): 9psi; spray voltage (IS) 5500V; temperature (TEM) 450 ℃; gas1:60psi; gas2:60psi; multiple reaction detection (MRM).
7. The method for tandem mass spectrometry for detecting catecholamine metabolites in blood plasma according to claim 1, wherein in the step (8), the establishment of the standard curve equation comprises the steps of:
1) The catecholamine metabolites of methoxyepinephrine, methoxynorepinephrine and 3-methoxytyramine are diluted to a plurality of different concentrations by using 50% acetonitrile water solution containing 0.1% -0.5% of glutathione, the combined stabilizer is added into each concentration standard solution, and the volume ratio of the standard solution to the combined stabilizer is 3-4 mL: 9-11 mu L; then adding an internal standard working solution, mixing, adding a precipitator for protein precipitation, and carrying out centrifugal separation to obtain a supernatant;
2) Sequentially carrying out the same treatment operation on the supernatant obtained in the step 1) by adopting the step (5) and the step (6), so as to obtain a plurality of standard samples, and carrying out LC-MS/MS detection on the standard samples;
3) And (3) carrying out linear regression on the standard line sample obtained in the step (2) by taking the peak area of the object to be detected and the internal standard peak area ratio (y) as an ordinate and the concentration (x) of the object to be detected as an abscissa, wherein the weight is 1/x 2, so as to obtain a standard curve equation.
8. The combined stabilizer is characterized by comprising the following components in percentage by mass: 0.5 to 2 percent of ascorbic acid, 0.15 to 1 percent of 5-hydroxy ferulic acid, 0.5 to 2 percent of formic acid and pure water, wherein the pure water is complemented to 100 percent.
9. Use of a combination stabilizer according to claim 8 for liquid chromatography tandem mass spectrometry detection of catecholamine metabolites in plasma, wherein the combination stabilizer is used for stabilizing catecholamine metabolites in plasma.
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