CN114354821A - Extraction method of catecholamine substances based on magnetic adsorbent - Google Patents
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Abstract
The invention discloses a method for extracting catecholamine substances based on a magnetic adsorbent. The invention comprises the following steps: firstly, catecholamine in blood plasma and urine is extracted by a magnetic weak cation exchange adsorbent, and derivatization is carried out in the elution process. Then, carrying out sample separation by ultra-high performance liquid chromatography, and finally carrying out mass spectrum detection. The invention utilizes a magnetic weak cation exchange adsorbent to extract 6 series of standard products and internal standards of 6 catecholamine substances with known concentrations and perform derivatization. The method can make catecholamine substances be derivatized in the extraction process, can improve the stability of the compound, and has very important significance for diagnosing and differentially diagnosing patients suffering from neuroendocrine tumors derived from neural crest, such as pheochromocytoma, neuroblastoma, paraganglioma and the like, and evaluating the development process, curative effect and prognosis of diseases, thereby providing personalized diagnosis and treatment information for the patients.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a method for extracting catecholamine substances based on a magnetic adsorbent.
Background
Catecholamines (CAs) mainly include epinephrine (E), Norepinephrine (NE) and Dopamine (DA), and their main metabolites are 3-methoxytyramine (3-MT), methoxynorepinephrine (NMN) and Methoxyepinephrine (MN). Catecholamine and metabolites thereof can be clinically used for auxiliary diagnosis of endocrine-related diseases such as pheochromocytoma and neuroblastoma.
Catecholamine and metabolites thereof have important significance in disease diagnosis, but theafenolamine and the metabolites thereof have poor stability and are degraded quickly in a room temperature environment, and protein precipitation, dansyl chloride derivatization and other methods are commonly used for extraction and derivatization at present. However, this process is time-consuming and complicated, and cannot be automated. The method is simple, can realize automation, has short time consumption, and can be used for quickly extracting a large number of samples.
In the prior patent CN113720939A, a magnetic bead method of Beijing Meis mass spectrum biotechnology Limited liability company is a kit for automatically and quantitatively determining 6 catecholamine in blood plasma, and the patent is mainly a catecholamine detection kit, but the pretreatment is complex and time-consuming, a water bath 20 at 58 ℃ is needed, the time-consuming of the whole process is long, unstable catecholamine is easily degraded, and the detection result is inconsistent with the actual result.
In the existing patent CN 109613144A, a method for detecting catecholamine hormones in human plasma of the biological medicine science and technology limited in the Shanghai Coco-Koehne field, a sample is subjected to protein precipitation, light shielding and solid phase extraction, the detection is long in time consumption and complex in operation, and the rapid detection of the sample cannot be realized.
In the prior patent CN106442837A, a method for detecting catecholamine in blood plasma by using liquid chromatography tandem mass spectrometry, which is a company limited by Hangzhou Baichen medical inspection institute, detects only 3 substances, and performs LC-MS/MS detection after protein precipitation, light-shielding derivation, pH adjustment and centrifugation, and has the disadvantages of complex operation and long time consumption.
Disclosure of Invention
The invention aims to provide a method for extracting catecholamine substances based on a magnetic adsorbent, aiming at the problems of complex operation, long time consumption and unstable extract in the prior art.
The invention discloses a method for extracting catecholamine substances based on a magnetic adsorbent. The invention particularly discloses a method for extracting and derivatizing epinephrine (E), Norepinephrine (NE), Methoxyepinephrine (MN), Dopamine (DA), methoxynorepinephrine (NMN) and 3-methoxytyramine (3-MT) in blood plasma and urine based on a magnetic weak cation exchange adsorbent.
The purpose of the invention can be realized by the following scheme:
the invention provides a method for extracting catecholamine substances based on a magnetic adsorbent, which comprises the following steps:
s1, preparing a sample reagent: adding a sample to be detected into a buffer solution and oscillating to obtain a solution to be detected for later use;
s2, activating the magnetic adsorbent: adding ultrapure water into weak cation exchange adsorption magnetic beads, and oscillating to prepare a suspension;
s3, taking the weak cation exchange adsorption magnetic bead suspension prepared in the step S2, adding an organic reagent for activation, and removing the solution after shaking; adding ultrapure water for balancing, and removing the solution after shaking to obtain an activated magnetic adsorbent for later use;
s4, extracting substances to be detected: adding the solution to be detected prepared in the step S1 into the activated magnetic adsorbent obtained in the step S3, and removing the buffer solution after shaking; adding an eluent, and removing the eluent after shaking; and adding an eluent containing the derivative solution after washing, removing the magnetic adsorbent after shaking to obtain the catecholamine substance.
As an embodiment of the present invention, the catecholamines include epinephrine, norepinephrine, methoxyepinephrine, dopamine, methoxynorepinephrine, and 3-methoxytyramine.
As an embodiment of the present invention, the sample to be tested in step S1 includes one of plasma and urine.
As one embodiment of the present invention, the buffer solution in step S1 includes one of ultrapure water, neutral sodium dihydrogen phosphate and disodium hydrogen phosphate buffer solution.
In one embodiment of the present invention, the volume ratio of the sample to be tested to the buffer solution in step S1 is 2-5: 4-6.
As an embodiment of the present invention, the time of the oscillation in step S1 is 1-5 min. The concussion was a vortex concussion.
In one embodiment of the present invention, the ratio of the weak cation exchange adsorption magnetic beads to the ultrapure water in step S2 is 20 to 50 mg: 1 mL.
As an embodiment of the present invention, the shaking time is 20-40S after adding ultrapure water in step S2, and then the mixture is left standing for 5-10 min.
As an embodiment of the present invention, the organic reagent in step S3 includes one of methanol and acetonitrile. The volume ratio of the suspension, the organic reagent and the ultrapure water is 1: 30-70: 30-70.
As an embodiment of the present invention, the shaking time after the organic reagent is added in the step S3 is 20-40S; the shaking time after adding ultrapure water is 20-40 s.
As an embodiment of the present invention, the usage ratio of the solution to be measured to the magnetic beads in step S4 is 800 μ L: 0.2-0.5 mg.
As an embodiment of the present invention, the oscillation time in step S4 is 20-40S.
As an embodiment of the present invention, the rinsing in step S4 specifically is: leaching with ultrapure water, performing vortex oscillation for 20-40s, and removing the solution; the solution was removed by rinsing again with aqueous methanol or acetonitrile and vortexing for 20-40 seconds. The methanol water solution is 50-100% by volume of methanol water solution, and the acetonitrile water solution is 50-100% by volume of acetonitrile water solution.
As an embodiment of the present invention, the eluent containing the derivative solution in step S4 is obtained by mixing an organic solution containing formic acid, a buffer solution and a derivative solution. The volume ratio of the sample to be detected to the eluent is 15-20: 8.
As an embodiment of the present invention, the formic acid-containing organic solution is an aqueous solution containing 0.1% formic acid and 5% organic reagent by volume fraction. The buffer was a commercial cyanoborohydride coupling buffer (Sigma);
as an embodiment of the invention, the derivatizing liquid comprises acetaldehyde.
The volume ratio of the organic solution containing formic acid, the buffer solution and the derivative solution is 1-2: 1: 1.
the derivatization reagent is not a conventional reagent, the conventional derivatization reagent is dansyl chloride, but a dansyl chloride solution needs to be prepared within 30min in the using process, the derivatization reagent can be stably placed without being prepared, and a signal after derivatization in the system is better without generating a large amount of impurity interference.
As an embodiment of the invention, the organic reagent comprises one or more combinations of methanol, ethanol, acetonitrile, isopropanol.
The dry powder of the magnetic bead particles used in the invention has a modified group shell layer. The activation not only can make the extraction efficiency of magnetic bead improve, can also wash away the impurity that some magnetic beads brought in process of production, reduces the influence that impurity extracted to the sample, improves the precision of extracting.
The whole process of the invention can be completed within 10 min. The extraction process is short in time consumption, and the degradation of the sample is avoided; derivatization is carried out in the extraction process, and the derivatized sample is not easy to degrade and can be stably placed for 48 hours at the temperature of 2-8 ℃. The invention has the advantages of no degradation of the sample in the treatment process, high extraction efficiency and no influence on detection.
In the present invention, the solution is removed by attracting magnetic beads to the tube wall with a magnet.
Compared with the prior art, the invention has the following beneficial effects:
1. the extraction of catecholamine substances can be quickly realized through the magnetic weak cation exchange adsorbent.
2. The extraction process can be completed within 10 min.
3. Derivatization during elution can make the catecholamines more stable.
4. Has important significance for the detection of a large number of samples.
5. By derivatizing catecholamines, the response of the target compound may be enhanced. The detection of the catecholamine substances with lower content in the sample can be realized.
6. The catecholamine substances in the blood plasma and urine are quickly extracted by the weak magnetic cation exchange adsorbent, the stability of the catecholamine substances is improved, and an experimental scheme is provided for quick extraction and detection of the catecholamine substances.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a calibration curve for epinephrine (E) in urine of catecholamines;
FIG. 2 is a calibration curve for the catecholamines Norepinephrine (NE);
FIG. 3 is a calibration curve for the catecholamines, Methoxyepinephrine (MN);
FIG. 4 is a calibration curve of the catecholamine (DA) substance;
FIG. 5 is a calibration curve for the catecholamines methoxynorepinephrine (NMN);
FIG. 6 is a calibration curve of 3-methoxytyramine (3-MT) which is a catecholamine;
FIG. 7 is an epinephrine detection profile of the lowest point Cal 1 of the calibrator;
FIG. 8 is a norepinephrine detection profile of calibrator nadir Cal 1;
FIG. 9 is a methoxyepinephrine detection profile of calibrator nadir Cal 1;
FIG. 10 is a dopamine detection profile of Cal 1 at the lowest point of the calibrator;
FIG. 11 is a methoxy norepinephrine detection profile of calibrator nadir Cal 1;
FIG. 12 is a 3-methoxytyramine detection profile for Cal 1, the lowest point of the calibrator.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments. The following examples, which are set forth to provide a detailed description of the invention and a detailed description of the operation, will help those skilled in the art to further understand the present invention. It should be noted that the scope of the present invention is not limited to the following embodiments, and that several modifications and improvements made on the premise of the idea of the present invention belong to the scope of the present invention.
Example 1
The embodiment provides a method for extracting catecholamine substances based on a magnetic adsorbent, which comprises the following specific steps
Taking 400 mu L of a sample to be detected out of a refrigerator, placing the sample in a 1.5mL centrifuge tube, accurately moving 0.5mL of ultrapure water into the sample to be detected by using a calibrated 1000 mu L pipettor, and carrying out vortex oscillation for 5min to obtain a solution to be detected for later use;
50mg of weak cation exchange adsorption magnetic beads are filled into a glass bottle, and 1mL of water is added to prepare a suspension; adding 10 mu L of suspension into a 1.5mL centrifuge tube, adding 500mL of organic reagent methanol to activate the magnetic weak cation exchange adsorbent, performing vortex oscillation for 30s, adsorbing magnetic beads to the tube wall by using a magnetic substance, and removing the solution; and then 500ml of ultrapure water is used for balancing, vortex oscillation is carried out for 30s, the magnetic weak cation adsorbent is attracted to the pipe wall by a magnetic substance, and the solution is removed to obtain the activated magnetic adsorbent for later use.
Taking 800 mu L of the prepared solution to be detected, adding the solution to be detected into a centrifugal tube of the activated magnetic adsorbent, carrying out vortex oscillation for 30s, adsorbing magnetic beads to the tube wall by using a magnetic substance, and removing the solution; leaching the magnetic weak cation adsorbent by using 500ml of ultrapure water, performing vortex oscillation for 30s, adsorbing magnetic beads to the pipe wall by using a magnetic substance, and removing the solution; and (3) leaching the magnetic weak cation adsorbent with 500ml of 50% methanol aqueous solution again, performing vortex oscillation for 30s, adsorbing magnetic beads to the tube wall by using a magnet, and removing the solution. Adding 160 mu L of prepared eluent into the centrifuge tube, wherein the eluent comprises an aqueous solution containing 0.1% of formic acid and 5% of methanol in volume fraction, a cyanoborohydride coupling buffer solution and an acetaldehyde derivative solution, and the volume ratio of the three is 1: 1: 1. vortex and shake for 30s, and attract the magnetic beads to the tube wall by a magnetic substance to obtain the extracted catecholamine substances.
The accuracy of the extraction method of catecholamines in example 1 was verified:
the verification was carried out by the same procedure as in example 1, using an isotope internal standard quantitative method. The calibrator comprises six substances of adrenaline (E), Noradrenaline (NE), Methoxyadrenaline (MN), Dopamine (DA), methoxynoradrenaline (NMN) and 3-methoxytyramine (3-MT), and the internal standard substance is an isotope internal standard substance corresponding to the calibrator.
Step one, detecting a sample to be detected
1. Preparing a sample reagent:
the same 400. mu.L of sample to be tested as in example 1 was taken out of the refrigerator, and 0.5mL of ultrapure water was accurately transferred to the sample to be tested using a calibrated 1000. mu.L pipette, followed by vortex oscillation for 5 min. Taking 400 mu L of sample solution to be detected out, placing the sample solution into a 1.5mL centrifuge tube, adding 20 mu L of internal standard, oscillating and uniformly mixing, adding 500 mu L of ultrapure water, and performing vortex oscillation for 5min to obtain solution to be detected for later use;
2. activating the magnetic adsorbent:
filling weak cation exchange adsorption magnetic beads into a glass bottle, and adding 1mL of water into the glass bottle per 50mg of weak cation exchange adsorption magnetic beads to prepare a suspension; adding 10 mu L of suspended magnetic weak cation adsorbent into a 1.5mL centrifuge tube, adding 500mL of organic reagent methanol to activate the magnetic weak cation exchange adsorbent, performing vortex oscillation for 30s, adsorbing magnetic beads to the tube wall by using a magnetic substance, and removing the solution; and then 500ml of ultrapure water is used for balancing, vortex oscillation is carried out for 30s, the magnetic weak cation adsorbent is attracted to the pipe wall by a magnetic substance, and the solution is removed to obtain the activated magnetic adsorbent for later use.
3. Extracting a substance to be detected:
adding 800 mu L of the prepared solution to be detected into a centrifugal tube for activating the magnetic adsorbent, performing vortex oscillation for 30s, adsorbing magnetic beads to the tube wall by using a magnetic substance, and removing the solution; leaching the magnetic weak cation adsorbent by using 500ml of ultrapure water, performing vortex oscillation for 30s, adsorbing magnetic beads to the pipe wall by using a magnetic substance, and removing the solution; and (3) leaching the magnetic weak cation adsorbent with 500ml of 50% methanol aqueous solution again, performing vortex oscillation for 30s, adsorbing magnetic beads to the tube wall by using a magnet, and removing the solution. And adding 160 mu L of eluent into the centrifugal tube, performing vortex oscillation for 30s, and adsorbing magnetic beads to the tube wall by using a magnetic substance to obtain a substance solution to be detected.
4. And (3) concentration detection:
then transferring all the substance solution to be detected into a sample injection 96-hole sample injection plate, and taking 10 mu L to perform LC-MS/MS detection; and (4) carrying out mass spectrometry on the substances to be detected, and substituting different detection results of the substances to be detected into the corresponding standard curves obtained in the step two to obtain the concentrations of the six substances to be detected, namely the catecholamine substances.
Step two, standard curve drawing up
All the freeze-dried calibrant and the internal standard were taken out of the refrigerator, 0.5mL of ultrapure water was accurately transferred to the freeze-dried calibrant by a calibrated 1000. mu.L pipette, and the freeze-dried calibrant was dissolved by vortex oscillation for 5 min. And taking 400 mu L of the dissolved calibrator out of a 1.5mL centrifuge tube, adding 20 mu L of internal standard, uniformly mixing by shaking, then adding 500 mu L of ultrapure water, and carrying out vortex shaking for 5 min. Extracting the prepared solution by a magnetic bead method, transferring all the solution of the substance to be detected into a sample injection 96-hole sample injection plate after extraction, taking 10 mu L of the solution for LC-MS/MS detection, measuring points Cal 1-6 with different concentrations by adopting an isotope internal standard quantitative method and taking the concentration of a standard substance as an x axis and the peak area ratio of the standard substance to an internal standard substance as a y axis, and establishing a calibration curve to obtain the calibration curve shown in figures 1-6.
Step three, standard curve verification
The calibrator is detected by the same steps as the detection of the object to be detected, the detection map of the lowest point Cal 1 of the calibration curve of the calibrator is shown in figures 7-12, and when the lowest point Cal 1 of the calibrator is detected, the signal-to-noise ratios of the 6 compounds are all larger than 10, so that the detection requirement can be met.
And calculating the deviation of the actual value and the theoretical value of the detected calibrator as shown in table 1: the deviation of the measured results is within 15%. The method for extracting and derivatizing the catecholamine substances in the blood plasma and urine based on the magnetic weak cation exchange adsorbent is feasible and reliable.
TABLE 1 calculation of the relative deviation of the actual value from the theoretical value
Note: relative deviation of 100%, (actual value-theoretical value)/theoretical value
Table 2 stability units of the samples at 2-8 ℃ after extraction: pg/mL
| E | NE | MN | DA | NMN | 3-MT | |
| 0h | 24.57 | 357.07 | 62.30 | 27.77 | 102.89 | 12.22 |
| 24h | 27.20 | 373.46 | 65.44 | 26.22 | 97.28 | 12.69 |
| 48h | 26.24 | 343.81 | 66.15 | 24.92 | 101.79 | 13.36 |
| Deviation of 24h | 10.69％ | 4.59％ | 5.05％ | -5.60％ | -5.46％ | 3.79％ |
| Deviation of 48h | 6.79％ | -3.71％ | 6.19％ | -10.25％ | -1.07％ | 9.33％ |
Note: 24h deviation 100%, (24h test value-0 h test value)/0 h test value
As shown in Table 2, the sample is derivatized in the elution process, the stability of the derivatized sample is obviously improved, the derivatized sample is placed in an environment with the temperature of 2-8 ℃, and the detection deviation of the compound is less than 15% after the derivatized sample is placed for 48 hours, which indicates that the derivatized sample can be stably placed for 48 hours, and the problems of instability and easy degradation of the underivatized sample are solved.
Example 2
The embodiment provides a method for extracting catecholamine substances based on a magnetic adsorbent, which comprises the following specific steps
Taking 400 mu L of a sample to be detected out of a refrigerator, putting the sample into a 1.5mL centrifuge tube, accurately transferring 0.5mL of neutral sodium dihydrogen phosphate and disodium hydrogen phosphate buffer solution into the sample to be detected by using a calibrated 1000 mu L pipettor, and carrying out vortex oscillation for 5min to obtain a solution to be detected for later use;
50mg of weak cation exchange adsorption magnetic beads are filled into a glass bottle, and 1mL of water is added to prepare a suspension; adding 10 mu L of suspension into a 1.5mL centrifuge tube, adding 500mL of organic reagent methanol to activate the magnetic weak cation exchange adsorbent, performing vortex oscillation for 30s, adsorbing magnetic beads to the tube wall by using a magnetic substance, and removing the solution; and then 500ml of ultrapure water is used for balancing, vortex oscillation is carried out for 30s, the magnetic weak cation adsorbent is attracted to the pipe wall by a magnetic substance, and the solution is removed to obtain the activated magnetic adsorbent for later use.
Taking 800 mu L of the prepared solution to be detected, adding the solution to be detected into a centrifugal tube of the activated magnetic adsorbent, carrying out vortex oscillation for 30s, adsorbing magnetic beads to the tube wall by using a magnetic substance, and removing the solution; leaching the magnetic weak cation adsorbent by using 500ml of ultrapure water, performing vortex oscillation for 30s, adsorbing magnetic beads to the pipe wall by using a magnetic substance, and removing the solution; the weakly magnetic cationic adsorbent was rinsed again with 500ml of 80% acetonitrile in water, vortexed for 30 seconds, and the magnetic beads were attracted to the tube wall with a magnet to remove the solution. Adding 160 mu L of eluent into the centrifuge tube, wherein the eluent comprises an aqueous solution containing 0.1% of formic acid and 5% of methanol in volume fraction, a cyanoborohydride coupling buffer solution and an acetaldehyde derivative solution, and the volume ratio of the three is 1: 1: 1. vortex and shake for 30s, and attract the magnetic beads to the tube wall by a magnetic substance to obtain the extracted catecholamine substances.
Example 3
The embodiment provides a method for extracting catecholamine substances based on a magnetic adsorbent, which comprises the following specific steps
Taking 400 mu L of a sample to be detected out of a refrigerator, placing the sample in a 1.5mL centrifuge tube, accurately moving 1mL of ultrapure water into the sample to be detected by using a calibrated 1000 mu L pipettor, and carrying out vortex oscillation for 3min to obtain a solution to be detected for later use;
filling 20mg of weak cation exchange adsorption magnetic beads into a glass bottle, and adding 1mL of water to prepare a suspension; adding 10 mu L of suspension into a 1.5mL centrifuge tube, adding 300mL of organic reagent methanol to activate the magnetic weak cation exchange adsorbent, performing vortex oscillation for 20s, adsorbing magnetic beads to the tube wall by using a magnetic substance, and removing the solution; and balancing with 700ml of ultrapure water, carrying out vortex oscillation for 40s, adsorbing the weak cation adsorbent with the magnetism by using a magnet to the pipe wall, and removing the solution to obtain the activated magnetic adsorbent for later use.
Adding 800 mu L of the prepared solution to be detected into a centrifugal tube of the activated magnetic adsorbent, performing vortex oscillation for 20s, adsorbing magnetic beads to the tube wall by using a magnetic substance, and removing the solution; leaching the magnetic weak cation adsorbent by using 500ml of ultrapure water, performing vortex oscillation for 30s, adsorbing magnetic beads to the pipe wall by using a magnetic substance, and removing the solution; and (3) rinsing the magnetic weak cation adsorbent again by using 500ml of 50% methanol aqueous solution, vortexing and shaking for 40s, adsorbing magnetic beads to the tube wall by using a magnet, and removing the solution. Adding 160 mu L of prepared eluent into the centrifuge tube, wherein the eluent comprises an aqueous solution containing 0.1% of formic acid and 5% of methanol in volume fraction, a cyanoborohydride coupling buffer solution and an acetaldehyde derivative solution, and the volume ratio of the three is 2: 1: 1. vortex and shake for 30s, and attract the magnetic beads to the tube wall by a magnetic substance to obtain the extracted catecholamine substances.
Comparative example 1
The embodiment provides a method for extracting catecholamine substances based on a magnetic adsorbent, which basically comprises the following steps of: and adding the magnetic bead activated organic reagent and the solution to be detected into a centrifugal tube filled with magnetic beads. When the plasma sample is processed, protein precipitation occurs, and whether the extracted sample is plasma or urine, the signal response of the extracted sample is lower than that of the sample in the example 1 when the sample is detected.
Comparative example 2
The embodiment provides a method for extracting catecholamine substances based on a magnetic adsorbent, which basically comprises the following steps of: the acetaldehyde eluent was replaced with dansyl chloride. And detecting the extraction result, and finding that the signal response of the dansyl chloride-derived sample is obviously lower than that of the acetaldehyde-derived sample during detection.
The invention discloses a method for extracting and derivatizing epinephrine (E), Norepinephrine (NE), Methoxyepinephrine (MN), Dopamine (DA), methoxynorepinephrine (NMN) and 3-methoxytyramine (3-MT) in blood plasma and urine based on a magnetic weak cation exchange adsorbent. The invention comprises the following steps: firstly, catecholamine in blood plasma and urine is extracted by a magnetic weak cation exchange adsorbent, and derivatization is carried out in the elution process. Then, carrying out sample separation by ultra-high performance liquid chromatography, and finally carrying out mass spectrum detection. The method comprises the steps of extracting 6 series of standard samples and internal standards of 6 catecholamine substances with known concentrations by using a magnetic weak cation exchange adsorbent, performing derivatization, detecting by liquid chromatography-tandem mass spectrometry, and establishing a calibration curve, so as to calculate the concentrations of the 6 catecholamine substances in human plasma and urine. The method can make catecholamine substances be derivatized in the extraction process, can improve the stability of the compound, and has very important significance for diagnosing and differentially diagnosing patients suffering from neuroendocrine tumors derived from neural crest, such as pheochromocytoma, neuroblastoma, paraganglioma and the like, and evaluating the development process, curative effect and prognosis of diseases, thereby providing personalized diagnosis and treatment information for the patients.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (10)
1. A method for extracting catecholamine substances based on a magnetic adsorbent is characterized by comprising the following steps:
s1, preparing a sample reagent: adding a sample to be detected into a buffer solution and oscillating to obtain a solution to be detected for later use;
s2, activating the magnetic adsorbent: adding ultrapure water into weak cation exchange adsorption magnetic beads, and oscillating to prepare a suspension;
s3, taking the weak cation exchange adsorption magnetic bead suspension prepared in the step S2, adding an organic reagent for activation, and removing the solution after shaking; adding ultrapure water for balancing, and removing the solution after shaking to obtain an activated magnetic adsorbent for later use;
s4, extracting substances to be detected: adding the solution to be detected prepared in the step S1 into the activated magnetic adsorbent obtained in the step S3, and removing the buffer solution after shaking; adding an eluent, and removing the eluent after shaking; and adding an eluent containing the derivative solution after washing, removing the magnetic adsorbent after shaking to obtain the catecholamine substance.
2. The extraction method according to claim 1, wherein the catecholamines comprise epinephrine, norepinephrine, methoxyepinephrine, dopamine, methoxynorepinephrine, and 3-methoxytyramine.
3. The extraction method according to claim 1, wherein the sample to be tested comprises one of plasma and urine.
4. The extraction method according to claim 1, wherein the buffer solution of step S1 includes one of ultrapure water, neutral sodium dihydrogen phosphate and disodium hydrogen phosphate buffer solution.
5. The extraction method according to claim 1, wherein the ratio of the weak cation exchange adsorption magnetic beads to the ultrapure water in the step S2 is 20-50 mg: 1 mL.
6. The extraction method according to claim 1, wherein the organic reagent in step S3 comprises one of methanol and acetonitrile.
7. The extraction method according to claim 1, wherein the eluent containing the derivative solution in step S4 is obtained by mixing an organic solution containing formic acid, a buffer solution and a derivative solution.
8. The extraction process according to claim 7, characterized in that said formic acid-containing organic solution is an aqueous solution containing 0.1% by volume of formic acid, 5% by volume of organic reagent.
9. The extraction process according to claim 8, wherein the organic reagent comprises one or more of methanol, ethanol, acetonitrile, isopropanol.
10. The extraction process according to claim 7, wherein the derivative liquid comprises acetaldehyde.
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| CN116482248B (en) * | 2023-03-16 | 2024-05-31 | 凯莱谱科技股份有限公司 | Detection kit for catecholamine and metabolites thereof in blood plasma/urine, detection method and application thereof |
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| 杨梦晖 等: "磁性固相萃取-高效液相色谱串联三重四级杆质谱法测定环境水样中的磺胺类抗生素残留" * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116482248A (en) * | 2023-03-16 | 2023-07-25 | 杭州凯莱谱精准医疗检测技术有限公司 | Detection kit for catecholamine and metabolites thereof in blood plasma/urine, detection method and application thereof |
| CN116482248B (en) * | 2023-03-16 | 2024-05-31 | 凯莱谱科技股份有限公司 | Detection kit for catecholamine and metabolites thereof in blood plasma/urine, detection method and application thereof |
| CN116448923A (en) * | 2023-06-20 | 2023-07-18 | 中国医学科学院北京协和医院 | Methods and kits for extracting and detecting steroid hormones and catecholamine metabolites |
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| CN114354821B (en) | 2023-05-16 |
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