A method for detecting parathyroid hormone

Abstract

The present invention relates to the technical field of parathyroid hormone detection, and specifically to a method for detecting parathyroid hormone, comprising the following steps: fixing a cation exchange stationary phase on the inner wall of a capillary column with a porous interface end, obtaining a pretreated capillary column, and placing the porous interface end above the inlet of a mass spectrometer; after equilibrium activation treatment of the cation exchange stationary phase in the pretreated capillary column, loading the sample through the inlet end of the pretreated capillary column, and sequentially using a detergent and an eluent to elute the capillary column adsorbed with serum to obtain a target, and finally using a mass spectrometer to detect the target. The detection method of the present invention not only overcomes the problem of parathyroid hormone metabolic fragment interference caused by the limitations of antibody recognition in the prior art, but also solves the problems of high detection cost, cumbersome operation and large sample consumption, thereby achieving high sensitivity, high specificity and low-cost detection of parathyroid hormone.

Description

Technical Field

[0001] The present 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 (PTH) is a basic, single-chain polypeptide hormone secreted by the parathyroid chief cells. The full PTH fragment consists of 84 amino acids, PTH1-84. PTH7-84 is a metabolic fragment of PTH, consisting of amino acids 7 to 84 of the full PTH fragment. In humans, PTH primarily acts on bones and kidneys, regulating calcium and phosphate metabolism. Studies have shown that abnormal PTH levels often indicate the development of hyperparathyroidism and chronic kidney disease. However, PTH quantification is currently difficult due to extremely low PTH levels in blood samples, typically as low as pg / mL, complex matrix composition, and the presence of numerous interferences from PTH metabolic fragments. Standardized PTH detection methods are still lacking for clinical testing. Therefore, the development of standardized PTH quantification methods is of great significance in clinical disease diagnosis.

[0003] Among the currently developed PTH detection methods, chemiluminescent immunoassay (CLIA) is the most commonly used method for PTH detection due to its rapid detection and high specificity. However, a significant drawback of CLIA is that its antibodies recognize PTH based only on a partial peptide sequence, which makes it unable to effectively eliminate interference from PTH metabolites in the sample matrix. In contrast, mass spectrometry offers unique advantages. Based on electromagnetic fields, mass spectrometry can ionize components in a PTH sample to form ions with varying charge-to-mass ratios. These ions are efficiently separated and detected by an accelerating electric field and a mass analyzer, enabling resolution of the target from interfering fragments. However, despite its numerous theoretical advantages, mass spectrometry faces numerous challenges in practical application. These methods typically require antibody capture of the target prior to detection, followed by offline enzymatic digestion, with quantification performed by detecting the specific fragments cleaved. The high cost of antibodies and enzymes in this process significantly increases the cost of testing. Furthermore, offline pretreatment steps are tedious and time-consuming, and require large sample volumes, typically in the milliliter range. More importantly, this method cannot eliminate the interference of PTH metabolic fragments on the test results. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention provides a method for detecting parathyroid hormone. Using solid-phase microextraction (SPM)-mass spectrometry, the present invention enriches and extracts PTH within a capillary column, followed by mass spectrometry analysis. This method not only overcomes the interference of PTH metabolic fragments caused by antibody recognition limitations in traditional detection methods, but also addresses the high detection costs, cumbersome pretreatment procedures, and high sample consumption associated with existing mass spectrometry methods, thereby achieving highly sensitive, specific, and low-cost detection of PTH.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A first object of the present invention is to provide a method for detecting parathyroid hormone, comprising the following steps:

[0007] 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.

[0008] S2. Carboxyl-modified polystyrene microspheres are used as a 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.

[0009] S3. Place the porous interface end of the pretreatment capillary column above the injection port of the mass spectrometer.

[0010] S4. Using an activating agent, 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.

[0011] S5. After the equilibrium activation treatment is completed, the sample is loaded on the inlet end of the capillary column activated by the stationary phase, and the sample is serum, to obtain a capillary column that adsorbs serum; the capillary column that adsorbs serum is first washed with a detergent to remove non-specifically adsorbed impurities; and the capillary column that adsorbs serum is then eluted with an eluent 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 adsorbs serum, and is detected by the mass spectrometer.

[0012] 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 result in a decrease in serum extraction efficiency.

[0013] Preferably, the flow rate for serum loading is 0.3 μL / min to 0.4 μL / min.

[0014] Preferably, the capillary column has a length of 90 cm and an inner diameter of 30 μm, is filled with polystyrene microspheres, and the serum loading volume is 1 μL to 5 μL.

[0015] Preferably, the adsorption capacity of parathyroid hormone by the carboxyl-modified polystyrene microspheres is 0.03 ng / cm3 to 0.04 ng / cm3.

[0016] Preferably, the serum loading volume is 3 μL to 4 μL.

[0017] Preferably, the detergent is acetamine solution and acetonitrile, and the washing is first performed with acetonitrile and then with acetamine solution.

[0018] 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.

[0019] Preferably, the pressure of the eluent is 15 psi to 40 psi.

[0020] Preferably, the pressure of the eluent is 25 psi to 30 psi.

[0021] Preferably, the ratio of serum sample volume to eluent volume is 3 μL:5 nL~25 nL.

[0022] 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.

[0023] 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.

[0024] Preferably, the pH value of the ammonium acetate solution is 6.8-7.2; within this range, the extraction efficiency can be improved.

[0025] Preferably, the activator is selected from ammonium acetate solution or ammonium formate solution.

[0026] 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.

[0027] Preferably, the equilibrium activation treatment conditions are: flushing the pre-treated capillary column with the activator at 80 psi to 90 psi for 10 to 20 minutes. The purpose of the equilibrium activation treatment is to improve ion exchange capacity, optimize selectivity, remove impurities and residues, stabilize the stationary phase structure, and adjust pH and ionic strength.

[0028] Preferably, the cation exchange stationary phase is fixed by:

[0029] First, a capillary column with a porous interface end is amino-modified 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.

[0030] 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.

[0031] Preferably, the molar ratio of carbodiimide hydrochloride to N-hydroxysuccinimide is 1:1.

[0032] Preferably, the capillary column with the porous interface end is washed before being amino-modified. The washing operation is: first washing with NaOH, then washing with water and ethanol, wherein the concentration of NaOH is 0.1 mol / L to 0.2 mol / L.

[0033] Preferably, the etching process involves polishing the outlet of the capillary column in an inert atmosphere, stripping the coating therefrom, and then inserting the capillary column 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 capillary outer wall is gradually corroded, forming tiny pores. These pores are typically nanometer-sized, allowing liquid to pass through and form fine droplets.

[0034] 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.

[0035] Preferably, the mass fraction of hydrofluoric acid in the hydrofluoric acid solution is 48% to 51%.

[0036] Preferably, after etching, the capillary column outlet is also washed. The washing step involves soaking the capillary column outlet with the porous interface in a NaOH solution and then in ultrapure water. The concentration of NaOH is 2.5 mol / L. Ultrapure water contains virtually no impurity ions, organic matter, or microorganisms, preventing these impurities from remaining in the capillary column with the porous interface or reacting with the target, thereby affecting the accuracy and repeatability of the test results.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. The present invention provides a method for detecting parathyroid hormone (PTH) using solid-phase microextraction (SPM)-mass spectrometry, exhibiting a high degree of automation and capable of automatically enriching and extracting PTH. Compared to traditional offline processing methods, the detection method of the present invention does not require complex serum pretreatment steps, thereby significantly simplifying the detection process. In particular, the present invention introduces carboxyl-modified polystyrene microspheres as a cation exchange stationary phase, effectively avoiding the use of expensive antibodies and significantly reducing detection costs. Furthermore, the present invention further reduces the sample load requirement by utilizing a capillary column, requiring only 1 μL to 5 μL of serum for efficient detection. In particular, the detection method of the present invention not only rapidly and accurately quantitatively analyzes intact PTH (PTH1-84) in human serum, but also effectively eliminates interference from PTH metabolic fragments during high-sensitivity analysis of PTH1-84, thereby significantly improving the accuracy of test results.

[0039] 2. The present invention provides a method for detecting parathyroid hormone, wherein mass spectrometry has a strong qualitative capability. Based on the action of the electromagnetic field, it can ionize the components of the 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 the target object from interfering fragments. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The figures show the water contact angle characterization results of the capillary column of the present invention, wherein (a) shows the water contact angle result of the capillary column, (b) shows the water contact angle result of the amino-modified capillary column, and (c) shows the water contact angle result of the capillary column with a cation exchange stationary phase.

[0041] Figure 2 These are scanning electron microscopy characterization results of the capillary column of the present invention, wherein (a) is a scanning electron microscopy image of polystyrene microspheres, (b) is a scanning electron microscopy image of the capillary column, and (c) is a scanning electron microscopy characterization result of the capillary column with a cation exchange stationary phase.

[0042] Figure 3 Figures 1 and 2 show the feasibility results of the method for detecting parathyroid hormone according to the present invention. (a) shows the detection results of a protein-removed blank serum matrix spiked with PTH1-84 and PTH7-84, and without spiked with PTH1-84 and PTH7-84. (b) shows the detection results after running the test process once. The illustration in (b) shows the detection results of the eluent.

[0043] Figure 4 These are graphs showing the test results of Examples 1 to 19 of the present invention, wherein (a) is a graph showing the sample flow rate, (b) is a graph showing the sample volume, (c) is a graph showing the elution pressure, and (d) is a graph showing the volume of a mixed solution of acetonitrile and acetic acid.

[0044] Figure 5 The figures are the results of online detection of PTH by the method for detecting parathyroid hormone of the present invention, wherein (a) is the standard curve diagram of the detection method of the present invention for detecting PTH1-84, and (b) is the standard curve diagram of the detection method of the present invention for detecting PTH7-84. DETAILED DESCRIPTION

[0045] The following will clearly and completely describe the technical solution of the present invention in conjunction with the data in the embodiments of the present invention. Obviously, the embodiments described 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 making creative efforts are within the scope of protection of the present invention.

[0046] 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 specified, 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.

[0047] While CLIA is widely used in PTH detection, its inherent limitations, such as the inability to effectively eliminate interference from PTH metabolic fragments, limit its accuracy. While mass spectrometry can theoretically distinguish between the target substance and interfering fragments, reported PTH mass spectrometry analysis methods often involve complex pretreatment steps, high costs, and high sample consumption, all of which hinder their widespread application in clinical testing. Furthermore, these mass spectrometry methods typically require offline processing of serum samples, including antibody capture and enzymatic digestion, which not only increases operational complexity but also introduces additional errors.

[0048] In response to 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 a cation exchange stationary phase, 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 phase 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 substance, which contains parathyroid hormone. The target substance enters a mass spectrometer through the porous interface end of the capillary column with adsorbed serum, and the target substance is detected by the mass spectrometer.

[0049] In response to the problems of previously reported PTH mass spectrometry analysis methods, which include antibody capture, offline enzymatic digestion, high cost, complex operation, time consumption, large sample consumption, and the inability to eliminate interference from metabolic fragments, the present invention uses carboxyl-modified polystyrene microspheres as a cation exchange stationary phase. This not only greatly simplifies the pretreatment process, but also achieves efficient enrichment and purification of parathyroid hormone, completely eliminating the steps of antibody capture and offline enzymatic digestion, thereby significantly reducing detection costs, shortening the detection cycle, and significantly reducing the consumption of serum samples. More importantly, by optimizing the elution conditions, the present invention can accurately perform high-sensitivity quantitative analysis of PTH1-84 while effectively eliminating interference from parathyroid hormone metabolic fragments, significantly improving the accuracy and reliability of the test results.

[0050] In the CLIA method, antibodies recognize PTH based only on a partial polypeptide sequence, failing to eliminate interference from PTH metabolic fragments in the sample matrix. The present invention, through the unique design of a solid-phase microextraction-mass spectrometry method, achieves precise detection of intact parathyroid hormone and specific fragments. During mass spectrometry analysis, since the ions generated by the ionization of each component have different charge-to-mass ratios, efficient separation and detection are achieved through an accelerating electric field and a mass analyzer. This enables the present invention to accurately distinguish between the target and interfering fragments, thus overcoming the limitation of the CLIA method that cannot effectively eliminate interference from PTH metabolic fragments.

[0051] S1. Online coupling interface of capillary column solid phase microextraction and mass spectrometry:

[0052] 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 48% hydrofluoric acid solution and 100 μL of octanol in a 10 mL graduated cylinder. At the same time, N2 was continuously introduced into the capillary column to corrode the capillary column 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.

[0053] S2. Capillary column with cation exchange stationary phase:

[0054] The capillary column with a porous interface end was first flushed with 0.1 mol / L NaOH for 20 minutes, then flushed with water and ethanol for 10 minutes; then, flushed with a mixed solution of 3-aminopropyldimethoxymethylsilane and ethanol for 3 hours, flushed with ethanol for 10 minutes and dried in a nitrogen flow for 30 minutes; wherein the volume ratio of 3-aminopropyldimethoxymethylsilane to ethanol was 1:100; thereby obtaining an amino-modified capillary column.

[0055] To carboxyl-modified polystyrene microspheres with an average particle size of 50 nm, 1.5 mL of an ethanol solution containing 0.015 mol / L carbodiimide hydrochloride and 0.03 mol / L N-hydroxysuccinimide was added. 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.

[0056] S3, equilibrium activation treatment of cation exchange stationary phase:

[0057] Under a pressure of 80 psi, a capillary column having a cation exchange stationary phase was flushed with a 50 mmol / L amine acetate solution having a pH value of 7.0 for 15 minutes to obtain a capillary column with an activated stationary phase.

[0058] 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 with reference to specific embodiments.

[0059] 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, obtained 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, also known as internal standard.

[0060] Example 1

[0061] A method for detecting parathyroid hormone, comprising the following steps:

[0062] 3 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 30 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0063] Example 2

[0064] A method for detecting parathyroid hormone, comprising the following steps:

[0065] 3 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.25 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 30 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0066] Example 3

[0067] A method for detecting parathyroid hormone, comprising the following steps:

[0068] 3 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.40 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 30 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0069] Example 4

[0070] A method for detecting parathyroid hormone, comprising the following steps:

[0071] 3 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.45 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 30 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0072] Example 5

[0073] A method for detecting parathyroid hormone, comprising the following steps:

[0074] 1 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 30 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0075] Example 6

[0076] A method for detecting parathyroid hormone, comprising the following steps:

[0077] 2 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 30 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0078] Example 7

[0079] A method for detecting parathyroid hormone, comprising the following steps:

[0080] 4 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 30 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0081] Example 8

[0082] A method for detecting parathyroid hormone, comprising the following steps:

[0083] 5 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 30 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0084] Example 9

[0085] A method for detecting parathyroid hormone, comprising the following steps:

[0086] 3 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 15 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0087] Example 10

[0088] A method for detecting parathyroid hormone, comprising the following steps:

[0089] 3 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 20 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0090] Example 11

[0091] A method for detecting parathyroid hormone, comprising the following steps:

[0092] 3 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 25 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0093] Example 12

[0094] A method for detecting parathyroid hormone, comprising the following steps:

[0095] 3 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 35 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0096] Example 13

[0097] A method for detecting parathyroid hormone, comprising the following steps:

[0098] 3 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 40 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0099] Example 14

[0100] A method for detecting parathyroid hormone, comprising the following steps:

[0101] 3 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 5 nL of a mixed solution of acetonitrile and acetic acid at 30 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0102] Example 15

[0103] A method for detecting parathyroid hormone, comprising the following steps:

[0104] 3 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 30 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0105] Example 16

[0106] A method for detecting parathyroid hormone, comprising the following steps:

[0107] 3 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 20 nL of a mixed solution of acetonitrile and acetic acid at 30 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0108] Example 17

[0109] A method for detecting parathyroid hormone, comprising the following steps:

[0110] 3 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 25 nL of a mixed solution of acetonitrile and acetic acid at 30 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 8:1.

[0111] Example 18

[0112] A method for detecting parathyroid hormone, comprising the following steps:

[0113] 3 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 80 psi for 5 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 30 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 9:1.

[0114] Example 19

[0115] A method for detecting parathyroid hormone, comprising the following steps:

[0116] 3 μL of serum sample was injected into the stationary phase-activated capillary column at a flow rate of 0.35 μL / min to obtain a capillary column that adsorbed serum. After sample loading, the capillary column that adsorbed serum was first flushed with acetonitrile at 90 psi for 15 min, followed by flushing with 50 mmol / L acetamide solution with a pH of 7.0 for 5 min. The capillary column that adsorbed serum was then eluted with 16 nL of a mixed solution of acetonitrile and acetic acid at 30 psi, and mass spectrometry analysis was performed. The entire analysis time was 42 min. The volume ratio of acetonitrile to acetic acid was 7:1.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] observe Figure 3Figure (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.

[0121] After injecting a blank serum sample containing 2000.0 pg / mL standard parathyroid hormone into the pretreated capillary column, the pretreated capillary column was eluted, and then the eluent was 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.

[0122] 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. Slower flow rates result in more complete reactions, which improves extraction efficiency. However, excessively slow flow rates prolong sample loading time, so 0.35 μL / min was selected as the optimal sample loading flow rate. While maintaining the same volume of the acetonitrile and acetic acid mixture, increasing the volume of PTH1-84 and PTH7-84 improves the enrichment factor, thereby increasing the final signal response.

[0123] Depend on Figure 4 Figure (b) shows that the signal response is strongest when the sample volume is 3 μL. However, due to the limited capacity of the pretreatment capillary column, further increasing the sample volume has no significant effect on the final signal. According to the paradigm equation, a higher flow rate reduces the longitudinal diffusion of the sample in the mobile phase, so increasing the thrust is beneficial for improving the mass spectrometry signal.

[0124] 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, 30 psi was selected as the optimal auxiliary pressure in the detection process.

[0125] Depend on Figure 4 Figure (d) shows that too little acetonitrile and acetic acid mixed solution will result in incomplete elution, while too much will reduce the enrichment efficiency and lead to a decrease in signal response. Therefore, 16 μL is selected as the optimal volume of the acetonitrile and acetic acid mixed solution.

[0126] Practical Application:

[0127] Based on the method of detecting parathyroid hormone of the present invention, parathyroid hormone in serum samples of healthy subjects was detected. By adding an internal standard to blank serum, samples with different concentrations were prepared. The specific method is as follows:

[0128] 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 15 N-1-84 sample; then, PTH1-84 with a mass concentration of 1 μg / mL and PTH7-84 standard stock solutions with a mass concentration of 1 μg / mL were taken respectively, and the blank serum samples were also diluted to 8 concentration levels of 26 pg / mL, 40 pg / mL, 100 pg / mL, 800 pg / mL, 1600 pg / mL, 2000 pg / mL, 3200 pg / mL, and 4000 pg / mL, respectively, to obtain PTH1-84 samples and PTH7-84 samples; then, 10 μL and 100 pg / mL of PTH 15 The N-1-84 sample was mixed with various concentrations of PTH1-84 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 evaluation of the standard curve in the serum matrix.

[0129] Subsequently, standard curves were plotted using the concentrations of the PTH1-84 and PTH7-84 samples as the X-axis and the peak area ratio of the target substance (PTH) to the internal standard as the Y-axis. The target concentrations corresponding to 3x and 10x the signal-to-noise ratios, respectively, were used as the limits of detection (LOD) and quantification (LOQ), respectively, for the method of detecting parathyroid hormone of the present invention.

[0130] Depend on Figure 5 It was found 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.

[0131] It should be noted that when the present invention involves numerical ranges, it should be understood that both 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 embodiments, in order to avoid redundancy, the present invention describes preferred embodiments. Although preferred embodiments of the present invention have been described, those skilled in the art will be able to make additional changes and modifications to these embodiments once they understand the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments 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 includes 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 mass spectrometer inlet; An activating agent is used 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; The capillary column adsorbing the serum is first washed with a detergent to remove non-specifically adsorbed impurities; the capillary column adsorbing the serum is then eluted with an eluent to obtain the target; the target enters the mass spectrometer through the porous interface end of the capillary column adsorbing the serum, and the mass spectrometer is used to detect the target; The stationary operation of the cation exchange stationary phase is: 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; 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 a condensation reaction with the amino groups on the inner wall of the capillary column, thereby immobilizing the carboxyl-modified polystyrene microspheres as a cation exchange stationary phase on the amino-modified layer on the inner wall of the capillary column having a porous interface end; The target is the complete parathyroid hormone PTH1-84 and its metabolic fragment PTH7-84; The particle size of carboxyl-modified polystyrene microspheres is 40nm~50nm.

2. The method for detecting parathyroid hormone according to claim 1, wherein: The flow rate for serum loading was 0.25 μL / min~0.45 μL / min.

3. The method for detecting parathyroid hormone according to claim 2, wherein: The flow rate for serum loading was 0.3 μL / min~0.4 μL / min.

4. The method for detecting parathyroid hormone according to claim 1, wherein: 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 carboxyl-modified polystyrene microspheres for parathyroid hormone is 0.03 ng / cm~0.04 ng / cm.

5. The method for detecting parathyroid hormone according to claim 1, wherein: 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. The method for detecting parathyroid hormone according to claim 1, wherein: 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. The method for detecting parathyroid hormone according to claim 1, wherein: The pressure of the eluent is 15psi~40psi.

8. The method for detecting parathyroid hormone according to claim 1, wherein: The ratio of serum sample volume to eluent volume is 3μL:5nL~25nL.

9. The method for detecting parathyroid hormone according to claim 1, wherein: For parathyroid hormone, 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.

10. The method for detecting parathyroid hormone according to claim 1, wherein: For parathyroid hormone, the detection limit of PTH1-84 is 6 pg / mL, and the detection limit of PTH7-84 is 9 pg / mL.

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