Method for detecting parathyroid hormone

Abstract

The invention relates to the technical field of parathyroid hormone detection, in particular to a parathyroid hormone detection method which comprises the following steps: fixing a cation exchange stationary phase on the inner wall of a capillary column with a porous interface end to obtain a pretreated capillary column, and placing the porous interface end of the pretreated capillary column above a sample inlet of a mass spectrometer; the method comprises the following steps: carrying out balance activation treatment on a cation exchange stationary phase in a pretreated capillary column, loading a sample through an inlet end of the pretreated capillary column, carrying out elution treatment on the capillary column adsorbing serum by using a detergent and an eluent in sequence to obtain a target object, and finally detecting the target object by using a mass spectrometer. According to the detection method, the problem of parathyroid hormone metabolic fragment interference caused by antibody recognition limitation in the prior art is solved, and the problems of high detection cost, complicated operation and large sample consumption are solved, so that high-sensitivity, high-specificity and low-cost detection of parathyroid hormone is realized.

Description

Technical Field

[0001] The invention relates to the technical field of parathyroid hormone detection, and in particular to a method for detecting parathyroid hormone. Background Art

[0002] Parathyroid hormone, or PTH, is an alkaline single-chain polypeptide hormone secreted by parathyroid chief cells. The whole parathyroid hormone consists of 84 amino acids, namely PTH1-84. PTH7-84 is a metabolic fragment of parathyroid hormone, which consists of amino acids 7 to 84 of the whole parathyroid hormone. In the human body, PTH mainly acts on bones and kidneys, and participates in regulating the metabolism of calcium and phosphorus in the body. Studies have shown that abnormal PTH levels often indicate the occurrence of hyperparathyroidism and chronic kidney disease. However, due to the extremely low PTH content in blood samples, generally as low as pg / mL, the complex matrix components and the interference of a large number of PTH metabolic fragments, PTH quantification is currently difficult, and clinical testing still lacks standardized PTH detection methods. Therefore, the development of standardized PTH quantification methods is of great significance in the diagnosis of clinical diseases.

[0003] At present, among the developed PTH detection methods, chemiluminescent immunoassay, or CLIA, has the characteristics of fast detection speed and high specificity, and is currently the most commonly used method for detecting PTH. However, the CLIA method has a significant defect, that is, its antibody recognition of PTH is only based on a partial sequence of the polypeptide, which makes it unable to effectively exclude the interference of PTH metabolic fragments in the sample matrix. In contrast, mass spectrometry has shown unique advantages. Based on the action of electromagnetic field, mass spectrometry can ionize the components in the PTH sample to generate ions with different charge-to-mass ratios. These ions are efficiently separated and detected by accelerating electric fields and mass analyzers, thereby achieving the resolution of target objects and interfering fragments. However, although mass spectrometry has many theoretical advantages, it faces many challenges in practical applications. These methods usually require antibody capture of the target before detection, and offline enzyme digestion treatment, and quantification by detecting specific enzyme-cut fragments. In this process, the high price of antibodies and biological enzymes directly leads to a significant increase in detection costs. In addition, the offline pretreatment steps are cumbersome and complicated, time-consuming, and the sample consumption is large, generally in milliliters. More importantly, this method cannot eliminate the interference of PTH metabolic fragments on the test results. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method for detecting parathyroid hormone. The present invention adopts a solid phase microextraction-mass spectrometry method, enriches and extracts parathyroid hormone in a capillary column, and then determines the parathyroid hormone in a mass spectrometer. The method of the present invention not only overcomes the interference problem of PTH metabolic fragments caused by the limitation of antibody recognition in traditional detection methods, but also solves the problems of high detection cost, cumbersome pretreatment operation and large sample consumption caused by the existing mass spectrometry, thereby achieving high sensitivity, high specificity and low cost detection of parathyroid hormone.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: The first object of the present invention is to provide a method for detecting parathyroid hormone, comprising the following steps: S1. Etching the outlet end of the capillary column to form a porous interface end with an electrospray function, thereby obtaining a capillary column with a porous interface end.

[0006] S2. Carboxyl-modified polystyrene microspheres are used as cation exchange stationary phase and fixed on the inner wall of a capillary column with a porous interface end to obtain a pretreated capillary column.

[0007] S3. Place the porous interface end of the pretreated capillary column above the injection port of the mass spectrometer.

[0008] S4. Using an activator to perform equilibrium activation treatment on the cation exchange stationary phase in the pretreated capillary column to activate the cation exchange stationary phase and obtain a capillary column with activated stationary phase.

[0009] S5. After the equilibrium activation treatment is completed, the inlet end of the capillary column activated by the stationary phase is loaded with a sample, the sample is serum, and a capillary column adsorbing serum is obtained; the capillary column adsorbing serum is first washed with a detergent to remove non-specifically adsorbed impurities; and then the capillary column adsorbing serum is eluted with an eluent to obtain a target substance, which contains parathyroid hormone. The target substance enters into a mass spectrometer through the porous interface end of the capillary column adsorbing serum, and the mass spectrometer is used to detect the target substance.

[0010] Preferably, the flow rate for serum loading is 0.25 μL / min to 0.45 μL / min; wherein, too large a flow rate or too small a flow rate will lead to a decrease in the extraction efficiency of the serum.

[0011] Preferably, the flow rate for serum loading is 0.3 μL / min to 0.4 μL / min.

[0012] Preferably, the capillary column has a length of 90 cm and an inner diameter of 30 μm, the entire tube is filled with polystyrene microspheres, and the serum loading volume is 1 μL to 5 μL.

[0013] Preferably, the adsorption capacity of parathyroid hormone by carboxyl-modified polystyrene microspheres is 0.03 ng / cm2-0.04 ng / cm2.

[0014] Preferably, the serum loading volume is 3 μL~4 μL.

[0015] Preferably, the washing agent is acetamine solution and acetonitrile, and the washing is first performed with acetonitrile and then with acetamine solution.

[0016] Preferably, the eluent is a mixed solution of acetonitrile and acetic acid, and the volume ratio of acetonitrile to acetic acid is 7 to 9:1; within this volume ratio range, the extraction efficiency can be improved.

[0017] Preferably, the pressure of the eluent is 15 psi to 40 psi.

[0018] Preferably, the pressure of the eluent is 25 psi to 30 psi.

[0019] Preferably, the ratio of serum loading volume to eluent usage is 3 μL:5 nL~25 nL.

[0020] Preferably, among parathyroid hormones, the minimum quantification limit of PTH1-84 is 13.0 pg / mL, and the minimum quantification limit of PTH7-84 is 20.0 pg / mL.

[0021] Preferably, among parathyroid hormones, the detection limit of PTH1-84 is 6 pg / mL, and the detection limit of PTH7-84 is 9 pg / mL.

[0022] Preferably, the pH value of the ammonium acetate solution is 6.8-7.2; within this range, the extraction efficiency can be improved.

[0023] Preferably, the activator is selected from ammonium acetate solution or ammonium formate solution.

[0024] Preferably, the particle size of the carboxyl-modified polystyrene microspheres is 40 nm to 50 nm; if the polystyrene microspheres are too large, the specific surface area is small, resulting in reduced solid phase extraction efficiency; if the polystyrene microspheres are too small, they are difficult to wash and centrifuge.

[0025] Preferably, the conditions for the equilibrium activation treatment are: flushing the pre-treated capillary column with an activator at 80 psi to 90 psi for 10 min to 20 min. The purpose of the equilibrium activation treatment is to improve the ion exchange capacity, optimize the selectivity, remove impurities and residues, stabilize the stationary phase structure, and adjust the pH and ionic strength.

[0026] Preferably, the cation exchange stationary phase is fixed by: First, a capillary column with a porous interface end is subjected to amino modification treatment using 3-aminopropyldimethoxymethylsilane to obtain an amino-modified capillary column; for amino modification of the inner wall of a capillary column with a porous interface end, the most effective and most commonly used reagent is 3-aminopropyldimethoxymethylsilane.

[0027] The carboxyl-modified polystyrene microspheres are mixed with an ethanol solution containing carbodiimide hydrochloride and N-hydroxysuccinimide, and then injected into an amino-modified capillary column and allowed to stand. During the standing period, under the action of carbodiimide hydrochloride and N-hydroxysuccinimide, the carboxyl groups on the surface of the polystyrene microspheres undergo condensation reaction with the amino groups on the inner wall of the capillary column, and the carboxyl-modified polystyrene microspheres are fixed as a cation exchange stationary phase on the amino-modified layer on the inner wall of the capillary column with a porous interface end.

[0028] Preferably, the molar ratio of carbodiimide hydrochloride to N-hydroxysuccinimide is 1:1.

[0029] Preferably, before the capillary column with the porous interface end is subjected to amino modification treatment, it is also subjected to washing treatment, and the washing treatment operation is: firstly washing with NaOH, and then washing with water and ethanol, wherein the concentration of NaOH is 0.1 mol / L~0.2 mol / L.

[0030] Preferably, the etching process is as follows: in an inert atmosphere, the outlet end of the capillary column is first polished smooth, the coating at the outlet is stripped, and then the capillary column is inserted into a centrifuge tube containing a hydrofluoric acid solution and octanol for etching. By etching the outer wall of the capillary column with a hydrofluoric acid solution, a porous structure can be formed. During the etching process, the material of the outer wall of the capillary is gradually corroded to form tiny holes. The size of these holes is usually at the nanometer level, which can allow liquid to pass through and form tiny droplets.

[0031] Preferably, the volume ratio of hydrofluoric acid solution to octanol is 6-7:0.1, and the mixed solution of hydrofluoric acid solution and octanol is used to corrode the outlet end of the capillary column to form a porous layer that is conductive but not liquid-conductive; if the proportion of hydrofluoric acid is too large, the corrosion speed is too fast and it is difficult to control the time; if the proportion is too small, the corrosion time is too long.

[0032] Preferably, the mass fraction of hydrofluoric acid in the hydrofluoric acid solution is 48% to 51%.

[0033] Preferably, after the capillary column outlet is etched, it is also washed; the washing process is as follows: the capillary column outlet with a porous interface is first soaked in a NaOH solution, and then soaked in ultrapure water; wherein the concentration of NaOH is 2.5 mol / L. Ultrapure water contains almost no impurity ions, organic matter, and microorganisms, which can prevent these impurities from remaining in the capillary column with a porous interface or reacting with the target, thereby affecting the accuracy and repeatability of the detection results.

[0034] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a method for detecting parathyroid hormone, which adopts solid phase microextraction-mass spectrometry, shows a high degree of automation, and can automatically enrich and extract parathyroid hormone. Compared with the traditional offline processing method, the detection method of the present invention does not require a complex serum pretreatment step, thereby significantly simplifying the detection process. In particular, the present invention introduces carboxyl-modified polystyrene microspheres as a cation exchange stationary phase, which not only effectively avoids the use of expensive antibodies, but also greatly reduces the detection cost. In addition, the present invention further reduces the demand for sample loading by applying a capillary column, and only 1 μL~5 μL serum is required to complete efficient detection. In particular, the detection method of the present invention can quickly and accurately quantitatively analyze the whole parathyroid hormone, i.e., PTH1-84, in human serum, and can also effectively eliminate the interference of parathyroid hormone metabolic fragments when analyzing PTH1-84 with high sensitivity, thereby achieving a significant improvement in the accuracy of the test results.

[0035] 2. The present invention provides a method for detecting parathyroid hormone, wherein mass spectrometry has a strong qualitative ability. Based on the effect of electromagnetic field, it can ionize the components of parathyroid hormone-containing serum to generate ions with different charge-to-mass ratios, which are then separated and detected by an accelerating electric field and a mass analyzer, thereby achieving the ability to distinguish between the target and the interfering fragments. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The figures are the result graphs of water contact angle characterization of the capillary column of the present invention, wherein (a) is the result graph of water contact angle of the capillary column, (b) is the result graph of water contact angle of the capillary column modified with amino group, and (c) is the result graph of water contact angle of the capillary column with cation exchange stationary phase.

[0037] Figure 2 The figures are scanning electron microscope characterization results of the capillary column of the present invention, wherein (a) is a scanning electron microscope image of polystyrene microspheres, (b) is a scanning electron microscope image of a capillary column, and (c) is a scanning electron microscope characterization result image of a capillary column with a cation exchange stationary phase.

[0038] Figure 3 The feasibility results of the method for detecting parathyroid hormone of the present invention are shown in Figure 1, wherein (a) is a graph showing the detection results of a blank serum matrix with protein removed and spiked with PTH1-84 and PTH7-84, and a graph showing the detection results of a blank serum matrix without spiked with PTH1-84 and PTH7-84; (b) is a graph showing the detection results after running a test process once; the illustration in (b) is a graph showing the detection results of an eluent.

[0039] Figure 4 These are graphs of the detection results of Examples 1 to 19 of the present invention, wherein (a) is a graph of sample flow rate, (b) is a graph of sample volume, (c) is a graph of elution pressure, and (d) is a graph of the volume of a mixed solution of acetonitrile and acetic acid.

[0040] Figure 5 The results of the method for detecting parathyroid hormone of the present invention for online detection of PTH are shown in FIG. 1 , wherein (a) is a standard curve diagram of the detection method of the present invention for detecting PTH1-84, and (b) is a standard curve diagram of the detection method of the present invention for detecting PTH7-84. DETAILED DESCRIPTION

[0041] The technical solution of the present invention will be clearly and completely described below in combination with the data in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] It should be noted that the professional terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, the various raw materials, reagents, instruments and equipment used in the following embodiments of the present invention can be purchased from the market or prepared by existing methods.

[0043] In the prior art, although CLIA is widely used in PTH detection, its inherent limitations, such as the inability to effectively eliminate the interference of PTH metabolic fragments, limit the accuracy of its detection. Although mass spectrometry can theoretically distinguish between the target and the interfering fragments, the reported PTH mass spectrometry analysis methods often involve complex pretreatment steps, high costs, and large sample consumption, which are not conducive to its widespread application in clinical testing. In addition, these mass spectrometry methods usually require offline processing of serum samples, including antibody capture and enzyme cleavage, which not only increases the difficulty of operation, but also introduces additional errors.

[0044] In view of the problems existing in the above-mentioned prior art, the present invention provides a method for detecting parathyroid hormone, which adopts a solid phase microextraction-mass spectrometry method, comprising the following steps: etching the outlet end of a capillary column to form a porous interface end with an electrospray function, thereby obtaining a capillary column with a porous interface end; using carboxyl-modified polystyrene microspheres as cation exchange stationary phases, and fixing them on the inner wall of the capillary column with a porous interface end, thereby obtaining a pretreated capillary column; placing the porous interface end of the pretreated capillary column above the injection port of a mass spectrometer; and using an activator to activate the cation exchange stationary phases in the pretreated capillary column. The capillary column is subjected to equilibrium activation treatment to activate the cation exchange stationary phase to obtain a capillary column with activated stationary phase; after the equilibrium activation treatment, a sample is loaded through the inlet end of the capillary column with activated stationary phase, and the sample is serum to obtain a capillary column with adsorbed serum; the capillary column with adsorbed serum is first washed with a detergent to remove non-specifically adsorbed impurities; and the capillary column with adsorbed serum is then eluted with an eluent to obtain a target object, which contains parathyroid hormone. The target object enters a mass spectrometer through the porous interface end of the capillary column with adsorbed serum, and the target object is detected by the mass spectrometer.

[0045] In view of the problems of antibody capture, off-line enzyme digestion, high cost, complicated operation, time-consuming, large sample consumption and inability to eliminate interference from metabolic fragments in the reported PTH mass spectrometry, the present invention adopts carboxyl-modified polystyrene microspheres as cation exchange stationary phase, which not only greatly simplifies the pretreatment process, but also realizes efficient enrichment and purification of parathyroid hormone, completely abandons the steps of antibody capture and off-line enzyme digestion, thereby significantly reducing the detection cost, shortening the detection cycle, and greatly reducing the consumption of serum samples. More importantly, through the optimization of elution conditions, the present invention can accurately perform high-sensitivity quantitative analysis of PTH1-84, while effectively eliminating the interference of parathyroid hormone metabolic fragments, so that the accuracy and reliability of the detection results are significantly improved.

[0046] In the CLIA method, the antibody recognition of PTH is based only on a partial sequence of the polypeptide, and the problem of being unable to eliminate the interference of PTH metabolic fragments in the sample matrix is ​​solved. The present invention realizes the accurate detection of the whole parathyroid hormone and specific fragments through the unique design of solid phase microextraction-mass spectrometry. In the mass spectrometry analysis process, since the ions generated after the ionization of each component have different charge-to-mass ratios, the accelerating electric field and mass analyzer are used for efficient separation and detection, so that the present invention can accurately distinguish the target and the interfering fragments, thereby overcoming the limitation that the CLIA method cannot effectively eliminate the interference of PTH metabolic fragments.

[0047] S1. Online coupling interface of capillary column solid phase microextraction-mass spectrometry: A fused silica capillary column with a length of 90 cm, an inner diameter of 30 μm, and an outer diameter of 150 μm was selected, referred to as a capillary column. The outlet end of the capillary column was polished smooth and flush with sandpaper, and the polyimide coating on the surface of the capillary column was peeled off by flame burning. The length of the polyimide coating was 4 cm. The polyimide coating was close to the outlet end of the capillary column, and its outer wall was wiped clean with ethanol. Then, the outlet end of the capillary column was inserted into a mixed solution containing 6 mL of a 48% hydrofluoric acid solution and 100 μL of octanol. The mixed solution was located in a 10 mL measuring cylinder, and N was continuously introduced into the capillary column. 2 , the capillary column was corroded for 20 minutes; after the corrosion treatment, the outlet end of the capillary column was first immersed in a 2.5 mol / L NaOH solution for 1 hour, and then immersed in ultrapure water for 1 hour to obtain an online coupling interface of capillary column solid phase microextraction-mass spectrometry, that is, a capillary column with a porous interface end.

[0048] S2, Capillary column with cation exchange stationary phase: The capillary column with a porous interface end was first rinsed with 0.1 mol / L NaOH for 20 min, then rinsed with water and ethanol for 10 min; then, rinsed with a mixed solution of 3-aminopropyldimethoxymethylsilane and ethanol for 3 h, rinsed with ethanol for 10 min and dried in a nitrogen flow for 30 min; wherein the volume ratio of 3-aminopropyldimethoxymethylsilane to ethanol was 1:100; and an amino-modified capillary column was obtained.

[0049] 1.5 mL of an ethanol solution containing 0.015 mol / L of carbodiimide hydrochloride and 0.03 mol / L of N-hydroxysuccinimide was added to carboxyl-modified polystyrene microspheres with an average particle size of 50 nm. After sufficient dilution and ultrasonic dispersion, the solution was injected into an amino-modified capillary column and allowed to stand for 1 hour. The column was then rinsed with ethanol for 30 minutes to obtain a pretreated capillary column, i.e., a capillary column with a cation exchange stationary phase.

[0050] S3, balanced activation treatment of cation exchange stationary phase: Under the pressure of 80 psi, the capillary column with the cation exchange stationary phase was flushed with 50 mmol / L amine acetate solution with a pH value of 7.0 for 15 minutes to obtain a capillary column with activated stationary phase.

[0051] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0052] Among them, the serum samples in Examples 1 to 19 are: a mixed standard of PTH1-84 and PTH7-84, the mass concentration of the mixed standard is 500 ng / mL; the mixed standard is obtained by mixing equal volumes of PTH1-84 with a mass concentration of 1 μg / mL and PTH7-84 with a mass concentration of 1 μg / mL; clinical serum, from the First Hospital of Jilin University; blank serum, i.e., a blank serum sample with protein removed from the serum; 1 μg / mL PTH 15 N-1-84 standard stock solution, i.e. internal standard.

[0053] Example 1 A method for detecting parathyroid hormone, comprising the following steps: 3 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column for adsorbing serum. After the loading, the capillary column for adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then rinsed with 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column for adsorbing serum at 30 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0054] Example 2 A method for detecting parathyroid hormone, comprising the following steps: 3 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.25 μL / min to obtain a capillary column for adsorbing serum. After the loading, the capillary column for adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then rinsed with 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column for adsorbing serum at 30 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0055] Example 3 A method for detecting parathyroid hormone, comprising the following steps: 3 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.40 μL / min to obtain a capillary column adsorbing serum. After the loading was completed, the capillary column adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then the capillary column was rinsed with 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column adsorbing serum at 30 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0056] Example 4 A method for detecting parathyroid hormone, comprising the following steps: 3 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.45 μL / min to obtain a capillary column for adsorbing serum. After the loading, the capillary column for adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then rinsed with 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column for adsorbing serum at 30 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0057] Example 5 A method for detecting parathyroid hormone, comprising the following steps: 1 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column for adsorbing serum. After the loading, the capillary column for adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then rinsed with 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column for adsorbing serum at 30 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0058] Example 6 A method for detecting parathyroid hormone, comprising the following steps: 2 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column adsorbing serum. After the loading, the capillary column adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then the capillary column was rinsed with 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column adsorbing serum at 30 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0059] Example 7 A method for detecting parathyroid hormone, comprising the following steps: 4 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column for adsorbing serum. After the loading was completed, the capillary column for adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then the capillary column was rinsed with a 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column for adsorbing serum at 30 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0060] Example 8 A method for detecting parathyroid hormone, comprising the following steps: 5 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column for adsorbing serum. After the loading was completed, the capillary column for adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then the capillary column was rinsed with a 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column for adsorbing serum at 30 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0061] Example 9 A method for detecting parathyroid hormone, comprising the following steps: 3 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column adsorbing serum. After the loading, the capillary column adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then the capillary column was rinsed with a 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column adsorbing serum at 15 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0062] Example 10 A method for detecting parathyroid hormone, comprising the following steps: 3 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column for adsorbing serum. After the loading was completed, the capillary column for adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then the capillary column was rinsed with a 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column for adsorbing serum at 20 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0063] Embodiment 11 A method for detecting parathyroid hormone, comprising the following steps: 3 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column for adsorbing serum. After the loading, the capillary column for adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then rinsed with 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column for adsorbing serum at 25 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0064] Example 12 A method for detecting parathyroid hormone, comprising the following steps: 3 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column for adsorbing serum. After the loading was completed, the capillary column for adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then the capillary column was rinsed with a 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column for adsorbing serum at 35 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0065] Example 13 A method for detecting parathyroid hormone, comprising the following steps: 3 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column for adsorbing serum. After the loading, the capillary column for adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then rinsed with 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column for adsorbing serum at 40 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0066] Embodiment 14 A method for detecting parathyroid hormone, comprising the following steps: 3 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column for adsorbing serum. After the loading was completed, the capillary column for adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then the capillary column was rinsed with a 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 5 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column for adsorbing serum at 30 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0067] Embodiment 15 A method for detecting parathyroid hormone, comprising the following steps: 3 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column for adsorbing serum. After the loading, the capillary column for adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then rinsed with 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column for adsorbing serum at 30 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0068] Example 16 A method for detecting parathyroid hormone, comprising the following steps: 3 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column for adsorbing serum. After the loading, the capillary column for adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then rinsed with 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 20 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column for adsorbing serum at 30 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0069] Embodiment 17 A method for detecting parathyroid hormone, comprising the following steps: 3 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column for adsorbing serum. After the loading was completed, the capillary column for adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then the capillary column was rinsed with a 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 25 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column for adsorbing serum at 30 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 8:1.

[0070] Embodiment 18 A method for detecting parathyroid hormone, comprising the following steps: 3 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column adsorbing serum. After the loading, the capillary column adsorbing serum was first rinsed with acetonitrile at a pressure of 80 psi for 5 min, and then the capillary column was rinsed with 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column adsorbing serum at 30 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 9:1.

[0071] Embodiment 19 A method for detecting parathyroid hormone, comprising the following steps: 3 μL of serum sample was injected into the capillary column activated by the stationary phase at a flow rate of 0.35 μL / min to obtain a capillary column for adsorbing serum. After the loading was completed, the capillary column for adsorbing serum was first rinsed with acetonitrile at a pressure of 90 psi for 15 min, and then the capillary column was rinsed with a 50 mmol / L acetamide solution with a pH value of 7.0 for 5 min, and then 16 nL of a mixed solution of acetonitrile and acetic acid was used to elute the capillary column for adsorbing serum at 30 psi, and mass spectrometry analysis was performed. The whole analysis time was 42 min. Among them, the volume ratio of acetonitrile to acetic acid was 7:1.

[0072] Depend on Figure 1 Figures (a), (b), and (c) show that the contact angle of the capillary column increased from 30° to 93° before and after amino modification, indicating that the hydrophilicity of the inner wall of the capillary column was significantly reduced.

[0073] Depend on Figure 2 Figures (a), (b), and (c) show that the inner wall of the amino-modified capillary column is covered with a large number of uniform polystyrene microsphere nanoparticles, whose size and morphology are basically consistent with those of polystyrene microspheres, proving that the chemical modification is successful, that is, the cation exchange stationary phase is successfully fixed.

[0074] Based on the detection method of the present invention, mass spectrometry analysis was performed on blank serum containing only internal standard, i.e., without PTH1-84 and PTH7-84, blank serum containing PTH1-84 and internal standard, and blank serum containing PTH7-84 and internal standard, i.e., with PTH1-84 and PTH1-84. The results are as follows: Figure 3 shown.

[0075] observe Figure 3 Figure (a) shows that when PTH1-84 and PTH7-84 were added to the blank serum matrix containing the internal standard, significant mass spectrometry signals were generated in the ion channel, proving that the method for detecting parathyroid hormone proposed in the present invention is feasible.

[0076] After a blank serum sample containing 2000.0 pg / mL of standard parathyroid hormone is injected into the pretreated capillary column, it is subjected to elution treatment, and then the eluent is tested using the method for detecting parathyroid hormone of the present invention. Figure 3 Figure (b) shows that no mass spectrometry signal was detected. This result strongly proves that the method for detecting parathyroid hormone of the present invention does not have the problem of carryover contamination and can ensure the accuracy in clinical serum continuous detection.

[0077] Depend on Figure 4 Figure (a) shows that the sample loading flow rate affects the reaction time of PTH1-84 and PTH7-84 with the cation exchange stationary phase. The slower the flow rate, the more complete the reaction, which is beneficial to improving the extraction efficiency. However, too slow a flow rate will prolong the loading time, so 0.35 μL / min is selected as the optimal sample loading flow rate. When the volume of the mixed solution of acetonitrile and acetic acid remains unchanged, increasing the volume of PTH1-84 and PTH7-84 can increase the enrichment multiple, thereby improving the final signal response.

[0078] Depend on Figure 4 Figure (b) shows that the signal response is strongest when the sample volume is 3 μL, and due to the limitation of the pretreatment capillary column capacity, further increasing the sample volume will not have a significant effect on the final signal. According to the paradigm equation, the greater the flow rate, the smaller the longitudinal diffusion of the sample in the mobile phase, so increasing the thrust is beneficial to improving the mass spectrometry signal.

[0079] Depend on Figure 4 Figure (c) shows that in the method for detecting parathyroid hormone of the present invention, the injection process is the elution process. Excessive thrust during the elution process will cause the flow rate of the mixed solution of acetonitrile and acetic acid to be too fast, resulting in incomplete elution and weakening of the signal. Finally, 30psi is selected as the optimal auxiliary pressure in the detection process.

[0080] Depend on Figure 4 Figure (d) shows that if the mixed solution of acetonitrile and acetic acid is too little, the elution will be incomplete, while if it is too much, the enrichment efficiency will be reduced, resulting in a decrease in signal response. Therefore, 16μL is selected as the optimal volume of the mixed solution of acetonitrile and acetic acid.

[0081] Practical Application: Based on the method for detecting parathyroid hormone of the present invention, parathyroid hormone in healthy human serum samples is detected. By adding an internal standard to blank serum, samples with different concentrations are prepared. The specific method is:

[0082] First, take 1 μg / mL of PTH 15 N-1-84 standard stock solution was diluted with blank serum to a PTH concentration of 100 pg / mL 15N-1-84 sample; then take PTH1-84 with a mass concentration of 1 μg / mL and PTH7-84 standard stock solution with a mass concentration of 1 μg / mL, and dilute them with blank serum samples to 8 concentration levels of 26pg / mL, 40pg / mL, 100pg / mL, 800pg / mL, 1600pg / mL, 2000pg / mL, 3200pg / mL, and 4000pg / mL, respectively, to obtain PTH1-84 sample and PTH7-84 sample; then, dilute 10μL, 100pg / mL of PTH 15 The N-1-84 sample was mixed with various concentrations of PTH1-84 samples and PTH7-84 samples to prepare PTH standard spiked samples with mass concentrations of 13 pg / mL, 20 pg / mL, 50 pg / mL, 400 pg / mL, 800 pg / mL, 1000 pg / mL, 1600 pg / mL, and 2000 pg / mL, respectively, for evaluating the standard curve in the serum matrix.

[0083] Subsequently, the concentrations of the PTH1-84 samples and the PTH7-84 samples were used as the X-axis, and the peak area ratio of the target, i.e., PTH, to the internal standard was used as the Y-axis to draw the standard curve. The target concentrations corresponding to 3 times and 10 times the signal-to-noise ratio were used as the detection limit and quantification limit of the method for detecting parathyroid hormone of the present invention, respectively. The detection limit was recorded as LOD, and the quantification limit was recorded as LOQ.

[0084] Depend on Figure 5 It was concluded that the linear ranges of the method for detecting parathyroid hormone of the present invention for PTH1-84 and PTH7-84 were 13.0 pg / mL to 1600 pg / mL and 20.0 pg / mL to 2000 pg / mL, respectively, and the LODs were 6 pg / mL and 9 pg / mL, respectively.

[0085] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiment, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiment and all changes and modifications that fall within the scope of the present invention.

Claims

1. A method for detecting parathyroid hormone, characterized in that: The solid phase microextraction-mass spectrometry method is used, including the following steps: The outlet end of the capillary column is etched to form a porous interface end with an electrospray function, thereby obtaining a capillary column with a porous interface end; Carboxyl-modified polystyrene microspheres are used as cation exchange stationary phase and fixed on the inner wall of a capillary column with a porous interface end to obtain a pretreated capillary column; Place the porous interface end of the pretreated capillary column above the injection port of the mass spectrometer; Using an activator to perform equilibrium activation treatment on the cation exchange stationary phase in the pretreated capillary column to activate the cation exchange stationary phase and obtain a capillary column with activated stationary phase; After the equilibrium activation treatment is completed, a sample is loaded through the inlet end of the capillary column activated by the stationary phase, and the sample is serum, thereby obtaining a capillary column adsorbing serum; First, a detergent is used to wash the capillary column that has adsorbed serum to remove non-specifically adsorbed impurities; then an eluent is used to elute the capillary column that has adsorbed serum to obtain a target substance, which contains parathyroid hormone; the target substance enters a mass spectrometer through the porous interface end of the capillary column that has adsorbed serum, and the mass spectrometer is used to detect the target substance.

2. A method for detecting parathyroid hormone according to claim 1, characterized in that: The flow rate for serum loading is 0.25μL / min~0.45μL / min.

3. A method for detecting parathyroid hormone according to claim 2, characterized in that: The flow rate for serum loading is 0.3μL / min~0.4μL / min.

4. A method for detecting parathyroid hormone according to claim 1, characterized in that: The capillary column is 90 cm long and has an inner diameter of 30 μm. The entire tube is filled with carboxyl-modified polystyrene microspheres. The serum loading volume is 1 μL~5 μL. The adsorption capacity of parathyroid hormone by carboxyl-modified polystyrene microspheres is 0.03 ng / cm~0.04 ng / cm.

5. A method for detecting parathyroid hormone according to claim 1, characterized in that: The detergents are acetamine solution and acetonitrile, and the acetonitrile is used for washing first, and then the acetamine solution is used for washing.

6. A method for detecting parathyroid hormone according to claim 1, characterized in that: The eluent is a mixed solution of acetonitrile and acetic acid, and the volume ratio of acetonitrile to acetic acid is 7~9:

1.

7. A method for detecting parathyroid hormone according to claim 1, characterized in that: The pressure of the eluent is 15psi~40psi.

8. A method for detecting parathyroid hormone according to claim 1, characterized in that: The ratio of serum loading volume to eluent usage is 3μL:5nL~25nL.

9. A method for detecting parathyroid hormone according to claim 1, characterized in that: Among parathyroid hormones, the minimum quantification limits of PTH1-84 and PTH7-84 were 13.0 pg / mL and 20.0 pg / mL, respectively.

10. A method for detecting parathyroid hormone according to claim 1, characterized in that: Among parathyroid hormones, the detection limits of PTH1-84 and PTH7-84 were 6 pg / mL and 9 pg / mL, respectively.

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