CN114354822B - Method for rapidly extracting catecholamine based on magnetic adsorbent - Google Patents
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Abstract
The invention discloses a method for rapidly extracting catecholamine based on a magnetic adsorbent; the method comprises the following steps: adding the activated magnetic weak cation exchange adsorbent into a sample to be tested, carrying out vibration treatment, and removing the solution; eluting the obtained magnetic weak cation adsorbent, and removing eluting solution; adding eluent and vibrating. Sampling the solution obtained by the elution treatment, and carrying out LC-MS/MS detection; and comparing the peak area ratio of the target compound and the internal standard substance according to a standard curve to obtain the concentration of the target compound in the sample to be detected. The invention adopts the weak cation exchange adsorbent to rapidly extract catecholamine and metabolites thereof in blood plasma and urine; the extraction process can be completed within 10 min; the extraction process time is short, the trouble caused by the easy degradation of catecholamine substances is effectively avoided, and an experimental scheme is provided for the rapid extraction and detection of catecholamine substances.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for rapidly extracting catecholamine based on a magnetic adsorbent, in particular to a method for rapidly extracting catecholamine from human body fluid based on a magnetic adsorbent.
Background
Catecholamines (CAs) are an important class of neurotransmitter or hormone substances secreted by adrenal medulla and some of the sympathetic neuronal chromaffins, mainly including epinephrine (E), norepinephrine (NE) and Dopamine (DA), and their major metabolites are 3-methoxytyramine (3-MT), methoxynorepinephrine (NMN) and Methoxyepinephrine (MN). Catecholamines regulate basic physiological functions in vivo, transmit physiological signals, are important signal mediators in normal physiological processes, and also show corresponding changes in the content in pathological processes. Can be clinically used for assisting in diagnosing endocrine related diseases such as pheochromocytoma, neuroblastoma and the like.
The method for extracting catecholamine and metabolites thereof in blood plasma and urine is a solid phase extraction method (SPE), but the extraction process by the SPE is long in time consumption and complex in operation. And catecholamine and its metabolite substance stability is relatively poor, degrade fast in the room temperature environment, so in extracting plasma, in the course of catecholamine in urine, need to finish extracting fast, carry on LC-MS/MS detects, avoid the puzzlement that causes unable detection or detection result abnormality because of the substance degradation.
According to the search of the prior patent literature, CN113720939A provides a kit for automatically and quantitatively measuring 6 catecholamines in plasma by a magnetic bead method, which is mainly used for detecting catecholamines, but the pretreatment is complex and takes a long time, water bath at 58 ℃ is needed for more than 20 minutes, the combination time of the magnetic beads and a binding solution is needed for 7-10 minutes, the whole process takes a long time, unstable catecholamines are easy to degrade, the detection result is inconsistent with the actual result, and meanwhile, an organic reagent is added into a plasma sample by the method, so that the sample is denatured, a small amount of protein precipitation occurs, and the method can only be used for a sample to be detected with high concentration. According to the invention, water bath treatment is not needed, the combination of the magnetic weak cation exchange adsorbent and catecholamine substances can be realized through oscillation treatment, and then the extraction of catecholamine in plasma or urine can be completed through leaching and eluting, and the whole extraction process can be completed within 10min, and the detection requirement can be met.
CN 109613144A discloses a method for detecting catecholamine hormone in human plasma, and the sample is subjected to protein precipitation, light-shielding and solid-phase extraction, then is detected, and has long time consumption and complex operation. The rapid detection of the sample cannot be achieved. The extraction of catecholamine substances in the sample can be completed within 10 min.
CN106442837a discloses a method for detecting catecholamines in plasma by liquid chromatography tandem mass spectrometry, however, only 3 substances are detected, and LC-MS/MS detection is performed after protein precipitation, light-shielding derivatization, pH adjustment and centrifugation, which is complicated in operation and takes a long time. The invention can finish the extraction of 6 catecholamine substances within 10min without protein precipitation and pH adjustment.
Disclosure of Invention
The invention provides a method for rapidly extracting catecholamine based on a magnetic adsorbent aiming at the defects of the prior art. In particular, a method for extracting epinephrine (E), norepinephrine (NE), methoxyepinephrine (MN), dopamine (DA), methoxynorepinephrine (NMN) and 3-methoxytyramine (3-MT) from blood plasma and urine based on a magnetic adsorbent is provided. Then, sample separation is carried out through ultra-high performance liquid chromatography, and finally mass spectrum detection is carried out. The invention utilizes a magnetic weak cation exchange adsorbent, 6 series of 6 catecholamines with known concentrations and metabolites thereof standard substances and internal standard substances are extracted manually or through a full-automatic extractor, and finally detection is carried out through liquid chromatography tandem mass spectrometry, and a calibration curve is established, so that the concentrations of the 6 catecholamines and the metabolites thereof in human blood plasma and urine are calculated. The detection method can be used for non-diagnostic scientific research by rapidly extracting catecholamine and metabolites thereof from blood plasma and urine and also can be used for diagnosing and differentiating the neuroendocrine tumors from neural crest, such as pheochromocytoma, neuroblastoma, paraganglioma and other patients, and has very important significance in the development process, curative effect and prognosis evaluation of diseases, so that personalized diagnosis and treatment information is provided for the patients.
The invention aims at realizing the following technical scheme:
the invention provides a method for extracting catecholamine and metabolites thereof from a sample, which comprises the following steps:
s1, adding an activated magnetic weak cation exchange adsorbent into a sample to be tested, carrying out vibration treatment, and removing a solution;
s2, leaching the magnetic weak cation adsorbent obtained in the step S1, and removing leaching solution; adding eluent and vibrating.
As one embodiment of the present invention, the sample to be tested is an ex vivo human fluid sample, including human plasma and urine samples.
As an embodiment of the present invention, the sample to be tested is not pre-treated.
As one embodiment of the invention, the weak magnetic cation exchange adsorbent is a suspension prepared by adding 1-2 mL of water to 50mg of weak magnetic cation exchange adsorption dry powder. The magnetic weak cation exchange adsorption dry powder has a modified radical shell layer. The activation not only can improve the extraction efficiency of the magnetic beads, but also can wash away impurities brought by some magnetic beads in the production process, reduce the influence of the impurities on sample extraction, and improve the extraction precision.
As one embodiment of the invention, the activation treatment is to add methanol or acetonitrile to shake the weak cation exchange adsorbent for 10-50 s.
In one embodiment of the present invention, in step S1, the time of the shaking treatment is 10 to 50S.
As an embodiment of the present invention, in step S2, the rinsing includes rinsing with ultrapure water, and after removing the rinsing liquid, rinsing again with 50-100% aqueous methanol or 50-100% aqueous acetonitrile; each leaching is performed for 10-50 s of vibration treatment.
As an embodiment of the present invention, in step S2, the eluent is an organic reagent containing 0.1% formic acid at a concentration of 5%; the organic reagent is one or more of methanol, ethanol, acetonitrile and isopropanol.
In step S2, the time of the shaking treatment is 10 to 50 seconds.
The invention also provides a detection method based on the sample extracted by the method, which comprises the following steps:
s3, sampling the solution obtained by the elution treatment in the step S2, and carrying out LC-MS/MS detection;
s4, comparing the peak area ratio of the target compound and the internal standard substance according to a standard curve to obtain the concentration of the target compound in the sample to be detected.
The detection method may be a non-diagnostic detection method.
In step S4, the standard curve is obtained by performing the operations of steps S1 to S3 with a calibrator, wherein the concentration ratio of the standard is taken as the x-axis, and the peak area ratio of the standard and the internal standard is taken as the y-axis.
Standard solutions used include solutions of known concentrations of epinephrine, norepinephrine, dopamine, phenylephrine, norepinephrine, 3-methoxytyramine.
The detection method of the invention needs to use magnetic weak cation exchange adsorbent, activating treatment reagent, internal standard substance, eluent, standard substance solution and the like.
The weak magnetic cation exchange adsorbent is a suspension prepared by adding 1-2 mL of water into every 50mg of weak magnetic cation exchange adsorption dry powder.
The activating treatment reagent is methanol or acetonitrile.
The standard solution can be a mixed solution of epinephrine, norepinephrine, dopamine, phenylephrine, norepinephrine and 3-methoxytyramine with known series concentration.
The internal standard may be an isotopic internal standard corresponding to the calibrator.
The eluent is an organic reagent with 0.1% of formic acid concentration of 5%, and the organic reagent is one or more of methanol, ethanol, acetonitrile and isopropanol.
The leacheate comprises leacheate A and leacheate B; the leaching solution A is water; the leaching solution B is 50-100% of methanol aqueous solution or 50-100% of acetonitrile aqueous solution.
Compared with the prior art, the invention has the following beneficial effects:
1) The invention adopts the weak cation exchange adsorbent to rapidly extract catecholamine and metabolites thereof in blood plasma and urine.
2) The extraction process can be completed within 10 min; and the sample does not require pretreatment.
3) The extraction process has short time, and effectively avoids the trouble caused by the easy degradation of catecholamine substances.
4) The invention rapidly extracts catecholamine and metabolites thereof in blood plasma and urine by the weak magnetic cation exchange adsorbent, and provides an experimental scheme for rapid extraction and detection of catecholamine substances.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:
FIG. 1 is a calibration curve of epinephrine (E), norepinephrine (NE), methoxyepinephrine (MN), and Dopamine (DA) in catecholamines and their metabolites;
FIG. 2 is a calibration curve of methoxy norepinephrine (NMN) and 3-methoxy tyramine (3-MT) in catecholamines and their metabolites.
Detailed Description
The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that several modifications and improvements can be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.
Example 1
The present example relates to a method for extracting epinephrine (E), norepinephrine (NE), methoxyepinephrine (MN), dopamine (DA), methoxynorepinephrine (NMN) and 3-methoxytyramine (3-MT) from plasma, urine based on magnetic adsorbents; the method comprises the following steps:
step one: preparing a sample:
all reagents were removed from the refrigerator. The method comprises the following steps of:
0.5mL of ultrapure water was accurately removed into the calibrator with a calibrated 1000. Mu.L pipette, and vortexed for 5min.
Taking out 400 mu L of calibrator, quality control product or sample, adding 20 mu L of internal standard (the internal standard is the isotope internal standard corresponding to the calibrator) into a 1.5mL centrifuge tube, and shaking and mixing uniformly. 500. Mu.L of water or neutral sodium dihydrogen phosphate and disodium hydrogen phosphate buffer were added. Vortex for 5min.
Step two: preparing a magnetic weak cation exchange adsorbent:
taking out the glass bottle filled with the magnetic weak cation exchange adsorption dry powder (magnetic beads), and adding 1mL of water per 50mg to prepare a suspension to obtain the magnetic weak cation exchange adsorbent for later use.
Step three: the extraction process of the magnetic weak cation exchange adsorbent comprises the following steps:
the manual processing steps are as follows:
1) 10 mu L of the suspended magnetic weak cation adsorbent is taken, added into a 1.5mL centrifuge tube, 400 mu L of organic reagent is added to activate the magnetic weak cation exchange adsorbent, and the organic reagent can be methanol or acetonitrile, and acetonitrile is selected in the embodiment. Vortex shaking for 30s, sucking magnetic beads to the tube wall by using a magnetic substance, and removing the solution; and balancing by using ultrapure water, vortex oscillating for 30s, absorbing the weak cation adsorbent to the pipe wall by using a magnetic substance, and removing the solution.
2) Taking 800 mu L of the object to be detected in the centrifuge tube, vortex and shake for 30s, sucking magnetic beads to the tube wall by using a magnetic object, and removing the sample solution.
3) And (3) leaching the weak cation adsorbent with ultrapure water, vortex oscillating for 30s, sucking the magnetic beads to the pipe wall by using a magnetic substance, and removing the solution.
4) Eluting the weak cation adsorbent again with 50-100% methanol water solution or 50-100% acetonitrile water solution, vortex oscillating for 30s, sucking magnetic beads to tube wall with magnetic substance, and removing the solution.
5) And adding an eluent into the centrifugal tube, wherein the eluent comprises an organic reagent with 0.1% of formic acid and 5% of formic acid. The organic reagent may be one or more of methanol, ethanol, acetonitrile and isopropanol, and acetonitrile is selected in this embodiment. Vortex and shake for 30s, attract the magnetic bead to the tube wall with the magnetic substance.
6) The whole solution was then transferred to a sample vial or 96 Kong Jinyang plate and 10 μl was taken for LC-MS/MS detection. And (3) injection: the extraction step can be performed manually or by an automatic extractor.
7) The calibrator was tested by magnetic weak cation exchange adsorbent: and (3) establishing a calibration curve by adopting an isotope internal calibration method, taking the concentration ratio of a standard substance as an x axis and the peak area ratio of the standard substance and an internal standard substance as a y axis. The linear fitting equation of catecholamine and its metabolite in the respective concentration range is good in linearity, and the correlation coefficient is above 0.99. The obtained calibration curves are shown in fig. 1 and 2.
Example 2
The same sample is selected, the sample is extracted by using a magnetic weak cation exchange adsorbent, 3 parallel experiments are carried out, the peak area ratio of the target compound and the internal standard substance is compared, the repeatability of catecholamine and metabolites thereof in the plasma and urine extracted by the adsorbent can be better and the CV values are all less than 10 percent from the table 2.
TABLE 2 reproducibility of extraction of magnetically weak cation exchange adsorbents
| NE | E | DA | NMN | MN | 3-MT | |
| 1 | 10.334 | 6.623 | 0.124 | 2.577 | 2.278 | 14.155 |
| 2 | 9.173 | 6.668 | 0.125 | 2.652 | 2.411 | 14.759 |
| 3 | 9.253 | 7.402 | 0.125 | 2.742 | 2.307 | 14.300 |
| CV(％) | 6.76％ | 6.34％ | 0.46％ | 3.11％ | 3.00％ | 2.19％ |
Note that: the cv coefficient of variation (coefficient of variation), also known as the discrete coefficient (coefficient of dispersion) or the relative deviation (rsd), is the ratio of the standard deviation to the average, expressed as a percentage, calculated as: cv=sd/mean×100%.
Comparative example 1
The present comparative example provides a method for extracting catecholamines based on magnetic adsorbents, which is basically the same as example 1, except that: adding the magnetic bead activated organic reagent and the solution to be tested into a centrifuge tube filled with the magnetic beads. At this time, protein precipitation occurs when the plasma sample is processed, and the signal response is lower than in example 1 regardless of whether the extracted sample is plasma or urine, and the sample is detected after extraction.
The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.
Claims (6)
1. A method for extracting catecholamines and metabolites thereof from a sample, said method comprising the steps of:
s1, adding an activated magnetic weak cation exchange adsorbent into a sample to be tested, carrying out oscillation treatment for 10-50S, and removing a solution;
s2, leaching the magnetic weak cation adsorbent obtained in the step S1, and removing leaching solution; adding eluent to shake; the eluent is an organic reagent with 0.1% of formic acid and 5% of concentration; the organic reagent is one or more of methanol, ethanol, acetonitrile and isopropanol;
the weak magnetic cation exchange adsorbent is a suspension prepared by adding 1-2 mL of water into every 50mg of weak magnetic cation exchange adsorption dry powder; the activation treatment is to add methanol or acetonitrile to shake the weak cation exchange adsorbent for 10-50-s.
2. The method for extracting catecholamines and metabolites thereof from a sample of claim 1, wherein the sample to be tested is an ex vivo human fluid sample, including human plasma and urine samples; the sample to be tested is not pretreated.
3. The method for extracting catecholamines and metabolites thereof from a sample according to claim 1, wherein in step S2, the rinsing comprises rinsing with ultrapure water, and after removing the rinsing liquid, rinsing again with 50-100% aqueous methanol or 50-100% aqueous acetonitrile; each leaching is performed by shaking treatment of 10-50 and s.
4. The method according to claim 1, wherein in step S2, the shaking treatment is performed for a period of 10 to 50 to S.
5. A method of detection based on a sample extracted by the method of claim 1, comprising the steps of:
s3, sampling the solution obtained by the elution treatment in the step S2, and carrying out LC-MS/MS detection;
s4, comparing the peak area ratio of the target compound and the internal standard substance according to a standard curve to obtain the concentration of the target compound in the sample to be detected.
6. The method according to claim 5, wherein in step S4, the standard curve is established by performing the operations of steps S1 to S3 with a calibrator, wherein the concentration ratio of the calibrator to the internal standard is the x-axis, and the peak area ratio of the calibrator to the internal standard is the y-axis.
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