CN114720575A - Method for detecting aldosterone in human plasma or serum - Google Patents

Method for detecting aldosterone in human plasma or serum Download PDF

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CN114720575A
CN114720575A CN202110005263.1A CN202110005263A CN114720575A CN 114720575 A CN114720575 A CN 114720575A CN 202110005263 A CN202110005263 A CN 202110005263A CN 114720575 A CN114720575 A CN 114720575A
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aldosterone
mobile phase
solution
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detection method
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钱学庆
秦炜
吴小虎
贺笑非
冯梦雪
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Shanghai Runda Rongjia Biotechnology Co ltd
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    • 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
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Abstract

The invention provides a method for detecting aldosterone in human plasma or serum, which comprises the following steps: sequentially adding aldosterone isotope internal standard solution and an extracting agent into a human plasma or serum sample, carrying out centrifugation after 30 seconds of vortex, carrying out redissolution after blowing of upper-layer extraction liquid nitrogen, and carrying out detection by using a high performance liquid chromatography-triple quadrupole mass spectrometer system. The detection method has the advantages of simple pretreatment, low detection cost, good selectivity and specificity, and further reduced quantification limit compared with the conventional method.

Description

Method for detecting aldosterone in human plasma or serum
Technical Field
The invention relates to a method for detecting aldosterone in human plasma or serum, belonging to the field of biological detection.
Technical Field
Aldosterone is a mineralocorticoid secreted by the cells of the adrenal cortico-zona. The main role of mineralocorticoids is to maintain water and electrolyte balance in the human body, and almost all metabolism of the human body is performed in water, so it is very important to maintain electrolyte balance in cells. Aldosterone is one of the strongest actions in natural mineralocorticoids, the main action is to preserve sodium and discharge potassium, and when aldosterone secretion is increased or aldosterone concentration is increased due to some diseases, obvious water retention can be caused, and diseases such as hypokalemia, hypertension and the like can also be caused.
Aldosterone synthase is encoded by the CYP11B2 gene, and besides adrenal glands, the cardiovascular system and the central nervous system also express the CYP11B2 enzyme, so aldosterone can bind to the Mineralocorticoid Receptor (MR) in local tissues to regulate the function of target organs. Aldosterone can regulate the expression of target genes and protein translation by binding to intracellular MR, and can also act on various signal-regulating pathways independently of MR, such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3), angiotensin ii (angii), Epidermal Growth Factor Receptor (EGFR), NADPH oxidase/Reactive Oxygen Species (ROS), and the like. Just because many targets of aldosterone action are involved and the involved metabolic pathways are numerous and critical, more and more studies indicate that many diseases occur and that aldosterone concentration is abnormal.
Primary aldosteronism is one of secondary hypertension, when hypertension and low potassium are used as diagnosis indexes of primary aldoses, only 0.5% of hypertensive patients are diagnosed, and when the concentration of aldosterone is used as one of the diagnosis indexes, the proportion of primary aldoses in 5-15% of hypertensive patients, especially in intractable hypertensive patients can reach more than 20%. Abnormal increases in aldosterone concentrations are also associated with insulin resistance, such as diabetes, cardiovascular disease, and the like. The existing detection method for clinically screening the primary aldehyde disease mostly adopts a radioimmunoassay, and because the content of aldosterone in a body is pg/mL magnitude, the specificity and accuracy of an radioimmunoassay are low, and misdiagnosis can be caused. Aiming at the current situation, the invention provides a method for rapidly detecting aldosterone in blood plasma or blood serum based on a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) technology, which can provide high-efficiency and accurate data for risk assessment and personalized intervention of primary aldehyde diseases and other related diseases.
Disclosure of Invention
In order to solve the technical problems, the inventor provides a method for detecting aldosterone in human plasma or serum, which is characterized by comprising the following steps of: 1) taking 100-400 mu L of human plasma or serum sample, respectively adding 5-50 mu L of 5-200ng/mL aldosterone-d 8 solution and 1.2-2.5mL extractant, uniformly mixing and carrying out vortex oscillation for 20-120 s;
2) centrifuging the solution in the step 1) (4 ℃, 3000-16000rpm) and absorbing the upper layer extract (0.8-2.2mL), and drying the solution under the condition of nitrogen flow (RT-50 ℃);
3) re-dissolving the sample in the step 2) by using a methanol aqueous solution, detecting by using a high performance liquid chromatography-triple quadrupole mass spectrometer to obtain the aldosterone analysis result, wherein the conditions of the high performance liquid chromatography method are as follows,
chromatography column Agilent poroshell 120EC-C18, 2.1 × 50mm, 2.7 μm;
the column temperature of the chromatographic column is 35-55 ℃;
the sample amount is 5-20 mu L;
the flow rate is 0.1-0.5 mL/min;
mobile phase A: 0.1-1mmol/L ammonium fluoride water; mobile phase B: 0.1-1mmol/L ammonium fluoride methanol.
The mobile phase gradient elution conditions were: the volume ratio of the mobile phase A and the mobile phase B is changed from 60:40 to 5:95 at constant speed within 0-2.5min, the ratio of 5:95 is kept to be 3.5min, and after the ratio is changed back to 60:40 within 0.1 min, the ratio of 60:40 is kept unchanged from 3.6min to 6 min.
The extractant used in the prior patent is mainly tert-butyl methyl ether or a mixed solvent of tert-butyl methyl ether and other organic solvents such as n-hexane, and the like. The inventors compared the extraction efficiency of t-butyl methyl ether and several organic solvents used alone or in combination, and found that the extraction efficiency was the highest with ethyl acetate. The ammonium fluoride is added into the mobile phase, compared with the condition that formic acid is added into the mobile phase, the response of the aldosterone is obviously increased, and the peak shape is sharp, so that the ammonium fluoride is added into the mobile phase of the chromatogram to enhance the response of the aldosterone, and the detection sensitivity is improved.
In the detection method, the mass spectrometry adopts a positive ion mode and adopts a scanning mode of multi-reaction monitoring MRM, and the parameters of the multi-reaction monitoring MRM are as follows:
Figure RE-GDA0003015134640000021
the mass spectrometry ion source parameters in the mass spectrometry are as follows:
an ionization source: ESI ionization source
Temperature of atomized gas: 300 deg.C
Flow rate of atomizing gas: 13.0L/min
Atomizing gas pressure: 50psi
Temperature of sheath gas: 350 deg.C
Flow rate of sheath gas: 12.0L/min
Capillary voltage: 3500V
Nozzle voltage: 2000V.
The solution is an aqueous solution unless otherwise specified.
The innovation points of the invention are as follows:
compared with the extraction efficiency of a plurality of organic solvents, the method has the advantages that the best efficiency result is obtained when ethyl acetate is used as the extractant, the extraction efficiency of pretreatment is improved, the detection sensitivity is improved by matching ammonium fluoride as the mobile phase additive, the quantitative limit of aldosterone is 7.5pg/mL, and the result is superior to the result of the existing literature. Compared with other reported methods for detecting aldosterone by using a liquid chromatography-triple quadrupole mass spectrometry, the method disclosed by the invention does not need solid-phase extraction, solid-phase-supported liquid-liquid extraction or derivatization operation, is low in detection cost and short in pretreatment time, and is a high-efficiency, sensitive and accurate method meeting the clinical detection requirements.
Drawings
FIG. 1 is a chromatogram and mass spectrum of aldosterone at the limit of quantitation (7.5pg/mL) concentration level.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Those skilled in the art can make appropriate modifications and alterations to the present invention, which fall within the scope of the invention.
Example 1
Preparing a standard substance stock solution and a blank matrix solution:
aldosterone stock solution (200 ng/mL): precisely weighing a proper amount of aldosterone, adding methanol to dissolve and dilute the aldosterone into a solution containing 200ng of aldosterone per ml, and storing the solution at the temperature of-20 ℃ for later use.
Stock solution of aldosterone-d 8 (200 ng/mL): an appropriate amount of aldosterone-d 8 is precisely weighed, dissolved in methanol and diluted into a solution containing 200ng of aldosterone-d 8 internal standard per milliliter, and stored at-20 ℃ for later use.
1 × PBS buffer: 10mL of 10 XPBS buffer was measured, diluted with 90mL of purified water and mixed to give 1 XPBS buffer. 4% BSA solution: 1g of bovine serum albumin was weighed, added to 25mL of 1 XPBS buffer solution, and dissolved and shaken to obtain a 4% BSA solution. Aldosterone Linear stock solution-1 (40 ng/mL): mu.L of aldosterone stock solution (200ng/mL) was removed accurately, 800. mu.L of methanol was added and mixed well.
Aldosterone Linear stock solution-2 (20 ng/mL): mu.L of aldosterone stock solution (200ng/mL) was removed accurately, 900. mu.L of methanol was added and mixed well.
Aldosterone Linear stock solution-3 (10 ng/mL): mu.L of aldosterone stock solution (200ng/mL) was removed accurately, 950. mu.L of methanol was added and mixed well.
Aldosterone Linear stock solution-4 (4000 pg/mL): accurately transferring 100. mu.L of aldosterone linear stock solution-1, adding 900. mu.L of methanol, and mixing.
Aldosterone Linear stock solution-5 (2000 pg/mL): accurately transferring 100 mu L of aldosterone linear stock solution-2, adding 900 mu L of methanol, and mixing.
Aldosterone Linear stock solution-6 (1000 pg/mL): accurately transferring 50. mu.L of aldosterone linear stock solution-2, adding 950. mu.L of methanol, and mixing.
Aldosterone Linear stock solution-7 (400 pg/mL): 40. mu.L of aldosterone linear stock solution-3 was accurately removed, 960. mu.L of methanol was added and mixed well.
Aldosterone Linear stock solution-8 (150 pg/mL): accurately transferring 75 μ L of aldosterone linear stock solution-5, adding 925 μ L of methanol, and mixing.
Aldosterone-d 8 internal standard of work solution (10 ng/mL): 250. mu.L of aldosterone-d 8 internal standard stock solution (200ng/mL) was removed accurately, 4.75mL of methanol was added and mixed well.
Blank sample solution: accurately transferring 200. mu.L of 4% BSA solution into a centrifuge tube, adding 10. mu.L of methanol solution, and mixing uniformly.
Matrix labeling solution: 190. mu.L of 4% BSA solution was accurately pipetted into a centrifuge tube, 10. mu.L of aldosterone linear stock solution-8 to-1 and 10. mu.L of aldosterone-d 8 internal standard working solution (10ng/mL) were added, and mixed well.
(II) pretreatment
Taking 200 mu L of human plasma or serum sample (190 mu L of 4% BSA solution is used for adding 10 mu L of aldosterone linear stock solution for adding a standard matrix sample), respectively adding 10 mu L of 10ng/mL aldosterone-d 8 solution and 1.6mL extracting agent, uniformly mixing and carrying out vortex oscillation for 30 s; the solution was centrifuged (4 ℃ C., 3000rpm) and 1.2mL of the upper extract was taken up and blown dry under a stream of nitrogen (40 ℃ C.); redissolving the nitrogen-blown dried sample by using a methanol-water (1:1, v: v) solution, detecting by using a high performance liquid chromatography-triple quadrupole mass spectrometer, wherein the conditions of the high performance liquid chromatography method are as follows,
chromatography column Agilent poroshell 120EC-C18, 2.1 × 50mm, 2.7 μm;
the column temperature of the chromatographic column is 40 ℃;
the sample injection amount is 20 mu L;
the flow rate is 0.3 mL/min;
the mobile phase gradient elution conditions were: the volume ratio of the mobile phase A and the mobile phase B is changed from 60:40 to 5:95 at constant speed within 0-2.5min, the ratio of 5:95 is kept to be 3.5min, and after the ratio is changed back to 60:40 within 0.1 min, the ratio of 60:40 is kept unchanged from 3.6min to 6 min.
The mass spectrometry part adopts a positive ion mode and adopts a scanning mode of multi-reaction monitoring MRM, and the parameters of the multi-reaction monitoring MRM are as follows:
Figure RE-GDA0003015134640000051
the mass spectrometry ion source parameters in the mass spectrometry are as follows:
an ionization source: ESI ionization source
Temperature of atomized gas: 300 deg.C
Flow rate of atomizing gas: 13.0L/min
Atomizing gas pressure: 50psi
Temperature of sheath gas: 350 deg.C
Flow rate of sheath gas: 12.0L/min
Capillary voltage: 3500V
Nozzle voltage: 2000V.
Example 2
The method comprises the following steps:
(1) linearity: and (3) establishing a calibration curve by adopting an isotope internal standard quantitative method, wherein the concentration ratio of the aldosterone to the internal standard is an x axis, and the peak area ratio of the aldosterone to the internal standard is a y axis, and calculating the concentration of the aldosterone in the sample. The linear fitting equation of aldosterone is that y is 6.977599x +0.003247, the linearity is good, and the correlation coefficient R is2Is 0.997.
(2) Limit of quantitation (7.5 pg/mL): under the standard adding level of 7.5pg/mL, the signal-to-noise ratio of the aldosterone chromatographic peak of each sample is above 10, and the standard adding variation coefficient is 5.0 percent, thereby meeting the requirement of quantitative limit.
Figure RE-GDA0003015134640000052
(3) Accuracy: four solutions of known concentration, low (100pg/mL), medium (500pg/mL), high (1000pg/mL), and 75% (1500pg/mL) of the upper limit of the standard were added to human plasma, and the recovery results were calculated as follows:
Figure RE-GDA0003015134640000053
Figure RE-GDA0003015134640000061
(4) and (3) selectivity: the method is characterized in that 37mmoL/L triglyceride, 342 mu mol/L bilirubin and 2g/L hemoglobin are respectively added into plasma to simulate high-fat, jaundice and hemolytic samples, MRM spectrogram shows that a blank matrix sample added with an interference substance has no response at compound retention time or the peak area is far lower than the limit of quantification, and the added interference substance has no influence on detection.
(5) Precision: taking 4 parts of human plasma as parallel samples, calculating the aldosterone concentration in the human plasma samples by using a linear standard curve, and the calculation results are as follows:
sample number Calculated concentration (pg/mL)
1 59.7236
2 64.9595
3 59.7096
4 57.7546
Mean value of 60.5368
CV 5.1％
The average value calculated by four samples is 60.5368pg/mL, the CV value is 5.1% < 15%, and the precision requirement is met.

Claims (6)

1. A method for detecting aldosterone in human plasma or serum is characterized by comprising the following steps:
1) taking 100-400 mu L of human plasma or serum sample, respectively adding 5-50 mu L of 5-200ng/mL aldosterone-d 8 solution and 1.2-2.5mL extractant, uniformly mixing and carrying out vortex oscillation for 20-120 s;
2) centrifuging the solution in the step 1) (4 ℃, 3000-16000rpm) and absorbing the upper layer extract (0.8-2.2mL), and drying the solution under the condition of nitrogen flow (RT-50 ℃);
3) re-dissolving the sample in the step 2) by using a methanol aqueous solution, detecting by using a high performance liquid chromatography-triple quadrupole mass spectrometer to obtain an aldosterone analysis detection result, wherein the conditions of the high performance liquid chromatography method are as follows,
column C18, 2.1X 50mm, 2.7 μm;
the column temperature of the chromatographic column is 35-55 ℃;
the sample amount is 5-20 mu L;
the flow rate is 0.1-0.5 mL/min;
mobile phase A: ammonium fluoride water; mobile phase B: ammonium fluoride methanol.
2. The detection method according to claim 1, wherein the extraction agent is t-butyl methyl ether, ethyl acetate or t-butyl methyl ether ethyl acetate 1-2:1-5 (v/v).
3. The detection method according to claim 1, wherein the methanol aqueous solution is 30% -70%.
4. The detection method according to claim 1, wherein the concentration of ammonium fluoride in the mobile phase is 0.1 to 1 mmol/L.
5. The detection method according to claim 1, wherein the liquid phase conditions are:
chromatography column Agilent poroshell 120EC-C18, 2.1 × 50mm, 2.7 μm;
the column temperature of the chromatographic column is 35-55 ℃;
the sample amount is 5-20 μ L;
the flow rate is 0.1-0.5 mL/min;
the mobile phase gradient elution conditions were: the volume ratio of the mobile phase A and the mobile phase B is changed from 60:40 to 5:95 at constant speed within 0-2.5min, the ratio of 5:95 is kept to be 3.5min, and after the ratio is changed back to 60:40 within 0.1 min, the ratio of 60:40 is kept unchanged from 3.6min to 6 min.
6. The detection method according to claim 1, wherein the mass spectrometry adopts a positive ion mode and adopts a scanning mode of multi-reaction monitoring MRM, and the parameters of the multi-reaction monitoring MRM are as follows:
Figure FDA0002883671670000011
。
CN202110005263.1A 2021-01-06 2021-01-06 Method for detecting aldosterone in human plasma or serum Pending CN114720575A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110865137A (en) * 2019-12-03 2020-03-06 天津国科医工科技发展有限公司 Method and kit for detecting aldosterone in blood plasma
CN111175419A (en) * 2020-03-02 2020-05-19 山东英盛生物技术有限公司 Method and kit for simultaneously detecting multiple steroid hormones in blood sample
CN111398446A (en) * 2020-03-12 2020-07-10 南京品生医学检验实验室有限公司 Method for detecting 12 kinds of steroid hormones in serum by ultra performance liquid chromatography tandem mass spectrometry technology

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110865137A (en) * 2019-12-03 2020-03-06 天津国科医工科技发展有限公司 Method and kit for detecting aldosterone in blood plasma
CN111175419A (en) * 2020-03-02 2020-05-19 山东英盛生物技术有限公司 Method and kit for simultaneously detecting multiple steroid hormones in blood sample
CN111398446A (en) * 2020-03-12 2020-07-10 南京品生医学检验实验室有限公司 Method for detecting 12 kinds of steroid hormones in serum by ultra performance liquid chromatography tandem mass spectrometry technology

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
邹继华;李全乐;沈敏;张曼;杨晓东;屠敏敏;冯东方;孙宏娜;: "血清醛固酮候选参考测量程序的建立及性能评估", 检验医学, no. 08, 30 August 2020 (2020-08-30), pages 13 - 22 *

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