Method for detecting PTH1-84 by liquid chromatography-mass spectrometry

Abstract

The invention relates to the technical field of detection, in particular to a method for detecting PTH1-84 by liquid chromatography-mass spectrometry. According to the method, an object to be detected is extracted through antibody immunocapture, PTH1-84 protein is effectively enriched, and after elution, UPLC-MS detection is carried out, so that a good detection effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a method for detecting PTH1-84 by liquid chromatography-mass spectrometry. Background Art

[0002] Parathyroid hormone (PTH) is a basic single-chain polypeptide containing 84 amino acids, which is synthesized and secreted by parathyroid chief cells. Its molecular weight is 9.4kDa and can be excreted through the kidneys. The main target organs of PTH are bones and kidneys. It mainly promotes the reabsorption of calcium ions and the excretion of phosphate by the renal tubules, thereby increasing blood calcium levels and decreasing blood phosphorus levels. Too low serum PTH levels lead to hypocalcemia. Symptoms caused by hypocalcemia include muscle cramps, hand and foot cramps, affecting heart function, and even causing myocardial damage. There are reports showing that calcium ion balance disorders caused by PTH are associated with hypertension. PTH increases renal phosphorus excretion by inhibiting the proximal tubular sodium phosphate cotransporter. Its deficiency can cause hyperphosphatemia, hyperparathyroidism, etc., can cause renal osteodystrophy, changes in the peripheral nervous system, and stubborn skin problems, which affect the patient's quality of life. Therefore, accurate measurement of PTH levels is very important for taking what treatment and evaluating the effect of treatment.

[0003] PTH secretion is regulated by many factors, such as vitamin D, calcium, phosphorus, protein kinase, gonadal steroid hormones, etc. Hypercalcemia inhibits PTH secretion, and hypocalcemia upregulates PTH expression. Active vitamin D can inhibit PTH gene transcription and reduce PTH secretion. Increased PTH is seen in primary hyperparathyroidism, ectopic hyperparathyroidism, hyperparathyroidism secondary to kidney disease, pseudohypoparathyroidism, etc. Decreased PTH is seen in hypoparathyroidism caused by thyroid surgery, renal failure, and non-parathyroid hypercalcemia caused by hyperthyroidism.

[0004] The intact PTH molecule released into the plasma can produce a variety of PTH fragments after metabolism in the liver and kidneys, forming the heterogeneity of PTH in the blood circulation. PTH exists in three forms: (1) iPTH (1-84), which is biologically active, accounting for 5%-20%, and has a very short half-life (2-4 min); (2) carboxyl-terminal PTH fragments, which are biologically inactive, such as PTH (36-84), PTH (44-84), PTH (49-84), PTH (53-84), etc., accounting for 75%-95%, and the half-life can reach 25-60 min; (3) amino-terminal PTH fragments, which are biologically active, such as PTH (1-34), with low blood levels and short half-life. Among them, iPTH can accurately reflect the function of the parathyroid gland. At the same time, PTH (1-84) and PTH (7-84) fragments play different roles in regulating bone metabolism, especially renal osteodystrophy. Therefore, whether the concentration of PTH (1-84) can be accurately detected becomes the key to correctly diagnosing renal osteodystrophy. People continue to explore new detection methods that are specific to PTH (1-84) and have no cross-reaction with (7-84). Based on this, the continuous updating of PTH detection methods is of great significance.

[0005] The methods commonly used in laboratories now include chemiluminescence with direct labeling of luminescent substances (the commonly used labeling substances are acridinium ester compounds) and electrochemiluminescence with labeling of elemental compounds (the commonly used labeling substances are terpyridine ruthenium). There are many brands of PTH detection kits on the market, such as Abbott, Roche, and Solin.

[0006] There are two common PTH test kits on the market: the second generation and the third generation. The second generation parathyroid hormone test kit uses the "sandwich method" as the detection principle, using 26-32 (N-terminal) and 37-42 (middle end) antibodies targeting the 7-84 amino acid sequence. However, since it still cross-reacts with the inactive cyclase inhibitory PTH (7-84) fragment, the test result includes the PTH (7-84) fragment, and the test result is the sum of PTH (1-84) and PTH (7-84). The second generation parathyroid hormone test kit still uses the "sandwich method" as the detection principle, using 1-6 and 44-68 antibodies targeting the 1-84 amino acid sequence. Therefore, the test result of PTH (1-84) does not include the PTH (7-84) fragment, and can specifically detect biologically active PTH (1-84), so the test result is usually lower than that of the second generation product. Literature reports showed that there were significant differences in the PTH (1-84) test results provided by different manufacturers, and the differences were statistically significant (P < 0.05).

[0007] Studies have shown that the role of PTH (7-84) in animals or cell models is opposite to that of PTH (1-84) and is produced in the parathyroid glands as blood calcium levels rise. Fehmi et al. also showed that in clinical practice, if it is used to guide the rational use of active vitamin D and avoid the occurrence of adynamic bone disease, the detection of PTH (1-84) alone is not as meaningful as the detection of the PTH (1-84) / PTH (7-84) ratio. Some scholars even suggest that the ratio of PTH (1-84) to PTH (7-84) be used as a predictive indicator for the severity of secondary hyperparathyroidism in dialysis patients.

[0008] However, there are many difficulties in the detection of PTH (1-84) and PTH (7-84), such as:

[0009] ①Difficult to standardize.

[0010] There are no recombinant or synthetic PTH standards that can be shared by various detection methods, and various detection companies continue to use homemade standards. This results in that even if the same method is used, if different manufacturers' kits are used, the antigens bound by the antibodies used in the test are different, and the PTH test results are very different. Therefore, the comparability between the test results of different methods on the market is poor.

[0011] At present, there is no unified national standard or industry standard for parathyroid hormone. The technical requirements for parathyroid hormone products of different companies are inconsistent, the normal reference values ​​are not unified, and the product quality is uneven, resulting in a lack of comparability of test results. The current solution is to monitor the patient's PTH results in the same laboratory using the same test kit, which is subject to certain restrictions on the mutual recognition of test results, resulting in increased medical costs and waste of medical resources.

[0012] ②Immunological methods are interfered with by heterophilic antibodies.

[0013] All immunoassays are susceptible to serum heterophilic antibodies (human anti-animal antibodies), and the same is true for PTH testing. 11% of the population has such antibodies, and with the wider use of monoclonal antibody drugs in inflammatory diseases, tumors, and transplantation diseases, the proportion of such antibodies in the body may be higher. Heterophilic antibodies can bind to the antibodies used, resulting in false positive results, thereby overestimating PTH levels in PTH testing.

[0014] In view of the various problems existing in PTH testing, the current solution is to monitor the patient's PTH results in the same laboratory using the same test kit, and decide on the treatment plan based on the changing trend of PTH combined with alkaline phosphatase, rather than based on the results of a single measurement.

[0015] ③Currently, there is no reference method that can accurately distinguish and quantify PTH and PTH fragments, and there is no method to detect PTH using chromatography / mass spectrometry. Summary of the invention

[0016] In view of this, the technical problem to be solved by the present invention is to provide a method for detecting PTH1-84 by liquid chromatography-mass spectrometry.

[0017] The method for detecting PTH1-84 by liquid chromatography-mass spectrometry provided by the present invention comprises:

[0018] The sample is mixed with the capture reagent, washed and eluted, the supernatant is dried and dissolved in an acetonitrile aqueous solution to obtain a sample to be tested, and the sample to be tested is detected by liquid chromatography-mass spectrometry;

[0019] The capture reagent includes: microspheres coated with PTH1-84 antibodies;

[0020] The cleaning solution contains Tween, PBS and water;

[0021] The elution solvent contains formic acid, acetonitrile and water.

[0022] The present invention adopts antibody immune capture to extract the test object, effectively enriches PTH1-84 protein, and after elution, detects through UPLC-MS to achieve good detection effect.

[0023] In the embodiment of the present invention, the core component of the capture reagent is a microsphere coated with a PTH1-84 antibody, and the material of the microsphere can be magnetic particles, polystyrene microspheres, polyacrylic acid microspheres or glass microspheres. It was found in the experiment that the effect of using polystyrene to coat the PTH1-84 antibody is the best. The polystyrene microspheres can also be polystyrene beads with a particle size of 1 μm.

[0024] In the embodiment of the present invention, in the capture reagent, the antibody coated on the microsphere is an antibody of PTH1-84, and the present invention does not limit its source or sequence, and it can be an antibody of mouse, rabbit, sheep, alpaca, donkey, or cattle. It can be a monoclonal antibody or a polyclonal antibody, and the present invention does not limit this. In some specific embodiments, a good detection effect can be achieved by coating the microsphere with a goat polyclonal anti-parathyroid hormone antibody.

[0025] In some embodiments, the immune capture reagent is polystyrene beads coated affinity purified goat polyclonal anti-parathyroid hormone antibody. The buffer in the capture reagent is tris(hydroxymethylaminomethane), wherein the mass fraction of microspheres is 0.05%.

[0026] In an embodiment of the present invention, in the cleaning solution, the mass fraction of PBS is 0.5% to 2%, and the volume fraction of Tween is 1‰ to 3‰. Wherein, Tween is Tween-20, Tween-40, Tween-60 or Tween-80. In a specific embodiment, Tween-20 is used. The preparation method of the cleaning solution comprises adding Tween-20 to a PBS buffer having a mass fraction of 0.5% to 2% until the volume fraction of Tween-20 is 1‰ to 3‰. In some embodiments, the cleaning solution is a 1% PBS solution containing 2‰ Tween-20.

[0027] The composition of the eluent affects the elution effect and ultimately interferes with the accuracy of the detection. In the present invention, the eluent solution is a mixed solution containing formic acid, acetonitrile and water. Among them, the mixed volume ratio of acetonitrile and water is 9:0.5~2; preferably, the mixed volume ratio of acetonitrile and water is 9:1. The eluent also contains formic acid with a volume percentage of 5%-15%; preferably, it contains formic acid with a volume percentage of 10%. The preparation method of the eluent includes first mixing acetonitrile and water, and then adding formic acid. Experiments show that the elution effect using an eluent with the above components and concentrations is optimal, and the target can be eluted to the greatest extent, and it is easy to blow dry. Preferably, the volume content of acetonitrile in the acetonitrile aqueous solution is 80-100%, and more preferably the volume content is 90%. More preferably, the eluent is a 90% acetonitrile aqueous solution containing 10% formic acid.

[0028] In an embodiment of the present invention, the sample to be detected is whole blood, serum or plasma, and the storage condition is -80°C to -20°C. In some embodiments, the sample to be detected is serum, and the volume ratio of the serum to the antibody capture reagent is 10:2.5~4, and more preferably the volume ratio is 10:3. In some embodiments, the volume ratio of the eluent to the capture reagent is (100~150): (200~400). Preferably, the volume ratio of the eluent to the capture reagent is 120:300.

[0029] In the embodiment of the present invention, the chromatographic column of the liquid chromatography is a Protein BEH C4 chromatographic column. Experiments have shown that the material of the chromatographic column plays a key role in the chromatographic separation of macromolecular proteins. In the previous experiments of the present invention, BEHC18 and Protein BEH C4 chromatographic columns were compared and investigated. Among them, BEH C18 is a universal ultra-high-efficiency chromatographic column widely applicable to various analytes. Because it can operate in the pH range of 1-12, these triple-bonded alkyl columns can also maintain excellent column stability under low pH and high pH conditions. However, since the pore size of the chromatographic column is 130Å, it was found in actual testing that it could not separate overly large or hydrophobic biomacromolecules, resulting in PTH1-84 residues on the chromatographic column. The Protein BEH C4 protein analysis chromatographic column has a pore size of 300Å and provides excellent separation for various polypeptides and proteins. The Protein BEH C4 chromatographic column has a strong retention of PTH1-84 in an environment with formic acid, produces thin and symmetrical peaks, and has a low system pressure. Provides excellent stability under low pH and high temperature conditions.

[0030] In the embodiment of the present invention, the mobile phase for liquid chromatography detection is an aqueous solution containing DMSO or an acetonitrile solution containing DMSO. Experiments show that the addition of DMSO is conducive to improving the detection effect, and the content of DMSO has a great influence on the effect of chromatographic separation and peak type. The previous experiments of the present invention also compared and investigated DMSO with different contents, and found that 3% DMSO helps to improve the ionization efficiency and make the ion flow coalesce into less charge state, thereby obtaining a higher quality spectrum and higher analytical sensitivity. In addition, the content of formic acid in the mobile phase also has an important influence on the detection effect. The different proportions of formic acid will affect the detection signal of the analyte. The previous experiments compared and investigated formic acid with different content ratios, and found that 0.3% formic acid content can achieve the best detection signal and is stable. In some embodiments, when the detection analysis is performed, the mobile phase A used for liquid chromatography analysis is an aqueous solution of 0.1%~0.5% formic acid and 2%~6% DMSO, and the mobile phase B is an 85%~95% acetonitrile aqueous solution containing 0.1%~0.5% formic acid and 2%~6% DMSO. The preparation method of mobile phase A is as follows: dissolving DMSO in water and then adding formic acid. The preparation method of mobile phase B is as follows: mixing acetonitrile and water, adding DMSO and then adding formic acid.

[0031] In a specific embodiment, the mobile phase A used for liquid chromatography analysis is an aqueous solution of 0.2% to 0.4% formic acid and 2% to 4% DMSO. For example, the mobile phase A is an aqueous solution containing 0.2% formic acid and 2% to 4% DMSO, or the mobile phase A is an aqueous solution containing 0.3% formic acid and 2% to 4% DMSO, or the mobile phase A is an aqueous solution containing 0.4% formic acid and 2% to 4% DMSO, or the mobile phase A is an aqueous solution containing 0.2% to 0.4% formic acid and 2% DMSO, or the mobile phase A is an aqueous solution containing 0.2% to 0.4% formic acid and 3% DMSO, or the mobile phase A is an aqueous solution containing 0.2% to 0.4% formic acid and 4% DMSO. More specifically, the mobile phase A is an aqueous solution containing 0.3% formic acid and 4% DMSO, or the mobile phase A is an aqueous solution containing 0.3% formic acid and 3% DMSO, or the mobile phase A is an aqueous solution containing 0.3% formic acid and 2% DMSO, or the mobile phase A is an aqueous solution containing 0.2% formic acid and 4% DMSO, or the mobile phase A is an aqueous solution containing 0.2% formic acid and 3% DMSO, or the mobile phase A is an aqueous solution containing 0.2% formic acid and 2% DMSO, or the mobile phase A is an aqueous solution containing 0.4% formic acid and 4% DMSO, or the mobile phase A is an aqueous solution containing 0.4% formic acid and 3% DMSO, or the mobile phase A is an aqueous solution containing 0.4% formic acid and 2% DMSO.

[0032] In a specific embodiment, the mobile phase B used for liquid chromatography analysis is an 88% to 92% acetonitrile aqueous solution containing 0.2% to 0.4% formic acid and 2% to 4% DMSO. For example: the mobile phase B is an 88%-92% acetonitrile aqueous solution containing 0.2% formic acid and 2%-4% DMSO, or the mobile phase B is an 88%-92% acetonitrile aqueous solution containing 0.3% formic acid and 2%-4% DMSO, or the mobile phase B is an 88%-92% acetonitrile aqueous solution containing 0.4% formic acid and 2%-4% DMSO, or the mobile phase B is an 88%-92% acetonitrile aqueous solution containing 0.2%-0.4% formic acid and 2% DMSO, or the mobile phase B is an 88%-92% acetonitrile aqueous solution containing 0.2%-0.4% formic acid and 3% DMSO, or the mobile phase B is an 88%-92% acetonitrile aqueous solution containing 0.2%-0.4% formic acid and 4% DMSO, or the mobile phase B is an 88%-92% acetonitrile aqueous solution containing 0.2%-0.4% formic acid and 2%-4% The mobile phase B is an 88% acetonitrile aqueous solution containing DMSO, or the mobile phase B is an 89% acetonitrile aqueous solution containing 0.2% to 0.4% formic acid and 2% to 4% DMSO, or the mobile phase B is a 90% acetonitrile aqueous solution containing 0.2% to 0.4% formic acid and 2% to 4% DMSO, or the mobile phase B is a 91% acetonitrile aqueous solution containing 0.2% to 0.4% formic acid and 2% to 4% DMSO, or the mobile phase B is a 92% acetonitrile aqueous solution containing 0.2% to 0.4% formic acid and 2% to 4% DMSO. More specifically, the mobile phase B is an 88% acetonitrile aqueous solution containing 0.3% formic acid and 4% DMSO, or the mobile phase B is an 80% acetonitrile aqueous solution containing 0.3% formic acid and 3% DMSO, or the mobile phase B is a 90% acetonitrile aqueous solution containing 0.3% formic acid and 3% DMSO, or the mobile phase B is a 92% acetonitrile aqueous solution containing 0.3% formic acid and 3% DMSO, or the mobile phase B is a 90% acetonitrile aqueous solution containing 0.4% formic acid and 3% DMSO, or the mobile phase B is a 90% acetonitrile aqueous solution containing 0.2% formic acid and 3% DMSO, or the mobile phase B is a 90% acetonitrile aqueous solution containing 0.2% formic acid and 2% DMSO, or the mobile phase B is a 90% acetonitrile aqueous solution containing 0.2% formic acid and 4% DMSO.

[0033] The percentages involved in the mobile phase of the present invention are all volume fractions.

[0034] In some specific embodiments, in the liquid chromatography detection, the chromatographic column is ACQUITY® UPLC ProteinBEH C4 (2.1×100mm ,1.7μm); the mobile phase A is an aqueous solution of 0.3% formic acid and 3% DMSO, and the mobile phase B is a 90% acetonitrile aqueous solution containing 0.3% formic acid and 3% DMSO.

[0035] In the present invention, the elution conditions play a key role in the chromatographic separation of macromolecular proteins. Since PTH is an endogenous substance and its content in the body is low, considering the influence of serum matrix background interference, it is necessary to investigate the separation effect of different gradient parameters. After optimization, in the embodiment of the present invention, the elution gradient of the liquid chromatography is:

[0036] 0~1.0min: the volume fraction of mobile phase A is 95%, and the volume fraction of mobile phase B is 5%;

[0037] 1.0~7.0min The volume fraction of mobile phase A changes from 95% to 75%,

[0038] The volume fraction of mobile phase B changes from 5% to 25%;

[0039] 7.0~11.0min The volume fraction of mobile phase A changes from 75% to 10%.

[0040] The volume fraction of mobile phase B changes from 25% to 90%;

[0041] 11.0~13.5min The volume fraction of mobile phase A is 10%, and the volume fraction of mobile phase B is 90%;

[0042] 13.5~14.5min The volume fraction of mobile phase A changes from 10% to 95%.

[0043] The volume fraction of mobile phase B changes from 90% to 5%;

[0044] 14.5~20.0min The volume fraction of mobile phase A is 95%, and the volume fraction of mobile phase B is 5%.

[0045] In addition, the conditions of liquid chromatography also include: mobile phase flow rate of 0.3 mL / min; column temperature of 60 °C, column pressure of 6380 psi, and injection volume of 40 μL.

[0046] The present invention optimized the gradient elution conditions and mass spectrometry parameters in the early experiments, and finally determined that the above-mentioned gradient elution conditions and mass spectrometry parameters can achieve accurate detection of PTH1-84. Under other conditions, it will lead to problems such as poor separation effect, peak tailing, matrix interference, and ultimately unstable detection signals, poor reproducibility, and low accuracy.

[0047] In the embodiment of the present invention, the conditions for mass spectrometry detection include:

[0048] Parent ions 629.4, 683.2 (m / z)

[0049] Product ions 660.3, 721.2 (m / z)

[0050] Collision energy 18, 19V

[0051] Cone voltage 130V

[0052] Detection mode: Positive.

[0053] In the present invention, the detection method also includes the steps of preparing a standard product, treating the standard product with the same treatment method as the sample to obtain a quality control product to be tested, and detecting, determining the value and obtaining the concentration of PTH1-84 in the standard product to be tested by liquid chromatography-mass spectrometry.

[0054] The quality control product or calibrator of the present invention includes PTH1-84 and a matrix; preferably, the matrix is ​​bovine serum after activated carbon adsorption, and the concentration of PTH1-84 is 0.005~1ng / mL. Preferably, the calibrator uses the labeled concentration as the independent variable and the measurement result as the dependent variable to obtain a linear regression equation, and then calculates the correlation coefficient (r) of the calibration curve, and uses the calibration curve to determine the value of the sample to be tested and the quality control product, thereby playing a calibration role; the value measured by the quality control product is used for quality control to evaluate the accuracy and precision of the detection process.

[0055] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0056] 1. The present invention provides for the first time a method for immunoenrichment to detect the complete peptide segment of PTH 1-84 without enzymatic hydrolysis, which greatly improves the detection specificity, detection throughput and efficiency.

[0057] 2. The present invention uses antibody immune capture to extract the test object, effectively enriches PTH1-84 protein, does not require enzymatic hydrolysis, directly measures the intact protein, and is easy to operate. The serum is further treated by activated carbon adsorption to ensure that the serum matrix sample removes interferences, thereby improving the accuracy of the test results.

[0058] 3. By adopting the chromatographic column, mobile phase, gradient elution parameters and mass spectrometry parameters selected by the present invention, the efficiency of instrument use is improved for separation and detection of PTH 1-84.

[0059] 4. The present invention further adopts quality control products and calibrators containing PTH1-84 and uses bovine serum adsorbed with activated carbon as a matrix, which has a small matrix effect and is easy to obtain, thereby achieving low-cost production and facilitating clinical application.

[0060] 6. The method of the present invention can cover samples with very different concentration levels and has a wide detection concentration range. The PTH1-84 content is at a low pg / mL level.

[0061] 7. The detection system of the present invention has lower pressure and better reproducibility, and is suitable for conventional liquid chromatography systems. The ProteinBEH C4 chromatographic column, the trifunctional bonded BEH particles with a pore size of 300Å, provide the widest available pH range for various protein-based applications. The small particle size stacking reduces dispersion and band broadening, thereby improving the resolution, sensitivity and speed in protein separation. The outstanding features of this chromatographic column are high column efficiency, low column pressure, and high-efficiency analysis under medium and low pH conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Representative chromatograms of PTH 1-84 and its isotope internal standard: (A) blank bovine serum sample spiked with IS, (B) blank bovine serum sample spiked with PTH 1-84 and IS, (C) actual patient serum sample spiked with IS;

[0063] Figure 2 Percentage of PTH1-84 (500 and 1000 pg / mL) concentration in serum samples after 6 and 24 h at room temperature;

[0064] Figure 3 Percentage of PTH1-84 (500 and 1000 pg / mL) concentration in serum samples after 6 and 24 h at 4°C;

[0065] Figure 4 The figures show the liquid chromatograms under different mobile phase detection conditions; Figure A is the chromatogram detected by adding DMSO to the mobile phase; Figure B is the chromatogram detected by not adding DMSO to the mobile phase;

[0066] Figure 5 The representative chromatograms of PTH1-84 under different mobile phase gradients are shown: Figure A is a chromatogram of PTH1-84 prepared with solvent under the gradient of Table 7; Figure B is a chromatogram of PTH1-84 prepared with blank matrix under the gradient of Table 7; Figure C is a chromatogram of PTH1-84 prepared with solvent under the gradient of Table 8; Figure D is a chromatogram of PTH1-84 prepared with blank matrix under the gradient of Table 8;

[0067] Figure 6 The following are representative chromatograms of different extraction methods: Figure A is a chromatogram of solid phase extraction; Figure B is a chromatogram of WCX magnetic bead extraction; Figure C is a chromatogram of antibody capture extraction. (The analyte concentrations shown in the figures are all 1000pg / mL PTH1-84 in blank matrix configuration);

[0068] Figure 7 The effect of different extraction reagent volumes on the recovery rate of PTH1-84 in serum is shown;

[0069] Figure 8The effect of the volume of elution solution on the recovery rate of PTH1-84 in serum is shown. DETAILED DESCRIPTION

[0070] The present invention provides a method for detecting PTH1-84 by liquid chromatography-mass spectrometry. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0071] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0072] Among them, PTH1-84 standard was purchased from Shanghai Puxin Biotechnology Co., Ltd., and polystyrene bead-coated affinity-purified goat polyclonal anti-parathyroid hormone antibody was purchased from Abbott Trading (Shanghai) Co., Ltd.

[0073] The present invention will be further described below in conjunction with embodiments:

[0074] Example 1

[0075] This embodiment provides a method for quantitatively detecting PTH1-84 by liquid chromatography-mass spectrometry, comprising the following steps:

[0076] 1) Preparation of quality control products and standards

[0077] 1.1 Accurately weigh the PTH1-84 standard and dissolve it in a 10% acetonitrile aqueous solution containing 0.1% FA and 0.1% BSA to obtain a standard stock solution with a concentration of 1 mg / mL.

[0078] 1.2 Pipette the standard stock solution in 1.1 and mix it with 0.1% FA and 0.1% BSA in 10% acetonitrile water to obtain 9 standard working solutions with different concentrations of 0.25 ng / mL, 0.50 ng / mL, 1.25 ng / mL, 2.50 ng / mL, 5.0 ng / mL, 12.5 ng / mL, 25 ng / mL, 40 ng / mL and 50 ng / mL.

[0079] 1.3 Mix 20 μL of the standard working solution described in 1.2 with 980 μL of blank bovine serum to obtain calibrators Cal-1, Cal-2, Cal-3, Cal-4, Cal-5, Cal-6, Cal-7, Cal-8, and Cal-9 at different levels.

[0080] 1.4 Pipette the standard stock solution in 1.1 and mix it with 0.1% FA and 0.1% BSA in 10% acetonitrile water to obtain three quality control standard solutions with different concentrations of 1.0 ng / mL, 15 ng / mL, and 37.5 ng / mL.

[0081] 1.5 Mix the above 20 μL quality control standard solution with 980 μL blank bovine serum to obtain the corresponding low concentration quality control product L, medium concentration quality control product M and high concentration quality control product H.

[0082] 2) Take 1 mL of each quality control and calibrator prepared in step 1) into a centrifuge tube, add 300 μL of antibody capture mixed reagent (microsphere mass fraction is 0.05%, solution is tris(hydroxymethyl)aminomethane), vortex to mix, discard the supernatant, add 500 μL of 1% PBS solution containing 2‰ Tween-20 to wash the antibody part twice, discard the supernatant, add 120 μL of 90% acetonitrile aqueous solution containing 10% formic acid for elution, collect the supernatant, blow dry with nitrogen at 37°C, add 100 μL of 10% acetonitrile aqueous solution, mix well, and set aside.

[0083] 3) placing each sample solution obtained in step 2) in a sample injector, calling up a gradient elution parameter file and a mass spectrometry parameter file for chromatographic separation, and testing each sample solution.

[0084] The specific liquid phase analysis conditions used were: using an ACQUITY® UPLC Protein BEH C4 column (2.1*100mm, 1.7μm) (Waters), the mobile phase A used was an aqueous solution containing 0.3% formic acid and 3% DMSO, the mobile phase B used was a 90% acetonitrile solution containing 0.3% formic acid and 3% DMSO, and the mobile phase gradient conditions used were shown in Table 1.

[0085] Table 1

[0086]

[0087] The mass spectrometry analysis conditions used are shown in Table 2.

[0088] Table 2

[0089]

[0090] The linear regression equation was obtained with the labeled concentration of the calibrator as the independent variable and the measurement result as the dependent variable, and then the correlation coefficient (r) of the calibration curve was calculated. The calibration curve was used to determine the concentration of the calibrator, quality control product and sample, and the concentration values ​​of PTH1-84 in the calibrator, quality control product and sample were obtained. The detection spectrum containing the minimum detection limit of PTH1-84 was as follows: Figure 1 shown.

[0091] Use weighting (w = 1 / x 2 ) The least squares method was used for regression calculation to obtain the correlation coefficient (r 2 ) are all greater than 0.99. According to the standard curve, the linear range of PTH1-84 in serum is 0.005~1 ng / mL, and the lowest detection line is 0.005 ng / mL. The typical standard curve is shown below y =0.00387x+0.00622 (r 2 =0.9962).

[0092] 4) Uniformity detection

[0093] Randomly select 10 calibrators of each level and 10 quality control samples of each level prepared by the method in step 1) for testing, and calculate the mean and CV of the test results. The uniformity should be <15%. The results are shown in Table 3.

[0094] Table 3

[0095]

[0096] 5) Accuracy analysis

[0097] Three calibrators of each level and three quality control products of each level were randomly selected for concentration testing. The deviations between the drug detection values ​​and the target values ​​at each concentration level were all within 15%. The accuracy results are shown in Table 4.

[0098] Table 4

[0099]

[0100] Example 2

[0101] This example investigates the effect of calibrators prepared with different serum matrices on the detection effect. The preparation method of the calibrators is as follows: 1) the 9 standard working solutions of different concentrations obtained in Example 1 are mixed with bovine serum adsorbed with activated carbon and commercial blank human serum in a volume ratio of 1:49 to obtain corresponding calibrators of different serum matrices and different levels, namely Cal-1, Cal-2, Cal-3, Cal-4, Cal-5 Cal-6, Cal-7, Cal-8, and Cal-9.

[0102] 2) The same method as in Example 1 was used for detection and analysis. The test results of the linear correlation coefficient and concentration deviation of the prepared calibrators with different serum matrices are shown in Table 5. The results show that the linear correlation coefficient r and concentration deviation of the calibrators prepared with different serum matrices meet the requirements.

[0103] Table 5

[0104]

[0105] In addition, this example also examines the concentration deviation and matrix effect of quality control products prepared by adding different serum matrices, and the results also show that bovine serum adsorbed with activated carbon meets the requirements of experimental accuracy and commercialization.

[0106] Example 3

[0107] This example investigates the stability of samples under different conditions. PTH 1-84 calibrators with concentrations of (500 and 1000 pg / mL) were prepared as test samples using the same preparation method as in step 1) of Example 1, and stored at room temperature and 4°C for 6 and 24 h. The test samples were treated using the method in step 2) of Example 1, and then detected using a liquid chromatography-tandem mass spectrometry system using the method in step 3) to investigate the stability of the test samples stored at room temperature and 4°C for 6 and 24 h. The results are shown in Table 1. Figure 2 , Figure 3 As shown in the figure, when stored at room temperature, the added serum samples showed a significant decrease after 6 h and 24 h, with average degradation rates of 16% and 49%, respectively. When the samples were stored at 4°C, PTH 1-84 remained stable in the serum and did not show degradation after 6 h and 24 h.

[0108] Example 4

[0109] Three different concentrations of freshly collected patient serum samples were stored in a -80°C refrigerator, and serum concentrations were measured at baseline and 1 week apart to investigate the stability of PTH in patient samples. Three different levels of patient serum samples were stored in a -20°C refrigerator to study the effect of freeze-thaw cycles on the stability of PTH1-84.

[0110] The results are shown in Table 6, which show that under the conditions of -80°C and -20°C, the detection values ​​of PTH1-84 in patient samples changed little and had good stability.

[0111] Table 6

[0112]

[0113] Example 5

[0114] Investigate the effect of mobile phase on the analyte PTH1-84:

[0115] Experiments have shown that adding DMSO to the solvent during peptide ionization can help improve the ionization efficiency of peptides and cause the ion current to coalesce into fewer charge states, resulting in higher quality spectra and greater analytical sensitivity.

[0116] First, we investigated the effect of adding DMSO or not adding DMSO to the mobile phase on the analyte PTH1-84. We found that when 3% DMSO was added to the mobile phase, the chromatographic peak height of PTH1-84 was about 3.6e4 ( Figure 4 Without adding DMSO, the peak height of PTH1-84 is about 1693 ( Figure 4 Therefore, the sensitivity of the analyte PTH1-84 was significantly improved after adding DMSO to the mobile phase.

[0117] Secondly, the effect of adding 1 vol%, 2 vol%, 3 vol%, 4 vol%, and 5 vol% DMSO to the mobile phase on PTH1-84 was investigated. The results showed that when 3% DMSO was added to the mobile phase, the chromatographic peak shape and separation of the target compound were better.

[0118] Finally, the effect of adding different amounts of formic acid (0.1 vol%, 0.2 vol%, 0.3 vol%, 0.4 vol%, 0.5 vol%) to the mobile phase on the analytes was investigated. The results showed that when the formic acid content was 0.3%, the chromatographic peak was symmetrical and sharp. Therefore, 0.3 vol% formic acid 3 vol% DMSO water-0.3 vol% formic acid 3 vol% DMSO 90% acetonitrile system was selected as the mobile phase.

[0119] Example 6

[0120] Investigating the effect of mobile phase gradient on the analyte PTH1-84

[0121] The gradient conditions of the liquid phase have a great influence on the analytes, especially those with matrix. Initially, we set the mobile phase gradient conditions as shown in Table 7. Under this gradient condition, PTH1-84 prepared with a mixed solution of 0.1% BSA, 0.1% formic acid and 10% acetonitrile showed a chromatographic peak at around 7.15 min ( Figure 5 However, PTH1-84 prepared with blank bovine serum adsorbed with activated carbon did not show a chromatographic peak within this retention time ( Figure 5 Middle B);

[0122] Table 7

[0123]

[0124] Then the gradient conditions were changed, as shown in Table 8. Under this gradient condition, the analytes prepared with solvent and the analytes prepared with blank matrix both showed chromatographic peaks within the corresponding retention time, with good peak shapes and high chromatographic peak sensitivity ( Figure 5 C and D).

[0125] Table 8

[0126]

[0127] Example 7

[0128] Investigate the effect of pretreatment conditions on analytes

[0129] First, the sample pretreatment conditions were investigated, and the extraction effects of WCX solid phase extraction, WCX magnetic bead extraction, and antibody capture extraction on the target analyte PTH1-84 were investigated. Figure 6 As shown: When using WCX solid phase extraction column and WCX magnetic beads for extraction, the sensitivity of the chromatogram of the analyte in the sample is low, while when using antibody capture to extract PTH1-84 in the sample, the sensitivity of the obtained chromatogram is high. From this, we can conclude that the recovery rate of solid phase extraction and magnetic bead extraction is low, while immune capture extraction can obtain a higher recovery rate. This may be because the relative molecular weight of PTH1-84 is low, and the chemical bond with the WCX material is not strong, resulting in the target not being completely adsorbed. Therefore, these two pretreatment methods are not suitable for the extraction of PTH1-84 in serum, so we use immune capture to extract the target.

[0130] Secondly, the effect of the volume of antibody capture reagent (microsphere mass fraction of 0.05%, solution of tris(hydroxymethyl)aminomethane) on the extraction effect was investigated. We used antibody capture reagent with volumes of 150, 200, 250, 300, 350, and 400 μL to extract PTH1-84 from 1 mL of serum. The results are shown in Figure 2. Figure 7 As shown in the figure, as the volume of the extraction reagent increases, the recovery rate gradually increases, but when the volume reaches 300 μL, the recovery rate remains basically unchanged. In order to achieve the best extraction recovery rate and save reagent costs, the antibody capture reagent volume of 300 μL was selected in this experiment.

[0131] Example 8

[0132] The volume of elution solvent is also an important parameter affecting the recovery of analytes, therefore, the composition and volume of elution solvent were investigated respectively.

[0133] First, we investigated the composition of the elution solvent. PTH1-84 is a hydrophilic compound. Based on the principle of like dissolves like, we selected formic acid, water, and acetonitrile as the composition of the elution solution. At the same time, we investigated the content of formic acid and acetonitrile in the eluent. We investigated the effects of 5%, 10%, and 15% formic acid and 80%, 90%, and 100% acetonitrile on the recovery rate of the analyte. The results showed that when the formic acid content was 10% and the acetonitrile content was 90%, the elution solution containing the analyte was easy to dry, and the recovery rate of PTH1-84 was high. Therefore, we selected 10% formic acid and 90% acetonitrile as the elution solvent.

[0134] Then the effect of the volume of the eluent on the recovery of the analyte was investigated. The analytes were eluted with 80, 100, 120, 150, and 200 μL of the elution solvent, and the eluent was analyzed. The results are shown in Figure 8 As shown in the figure, when the volume of the elution solution reached 120 μL, the recovery rate of PTH1-84 remained basically unchanged. Therefore, in order to save reagent costs and shorten the nitrogen drying time, we chose an elution solvent volume of 120 μL. Finally, we investigated the effect of the reconstitution solution on the analyte. According to the composition of the mobile phase, we chose acetonitrile and water to reconstitute the dried PTH1-84. We investigated acetonitrile aqueous solutions with a content of 5%, 10%, and 15%. According to the initial gradient conditions of the mobile phase, 10% acetonitrile aqueous solution as a reconstitution solution can obtain better chromatographic peak shape without solvent effect.

[0135] Example 9

[0136] The same method as in Example 1 was used to detect the concentration of PTH1-84 in the serum of 268 patients with normal estimated glomerular filtration rate, chronic kidney disease and dialysis. The linear regression equation was obtained by taking the labeled concentration of the calibrator as the independent variable and the measurement result as the dependent variable, and then calculating the correlation coefficient (r) of the calibration curve. The calibration curve was used to determine the test results. The results are shown in Table 9:

[0137] Table 9

[0138]

[0139] The results show that the method provided by the present invention can detect clinical samples, and its linear range can cover the concentration of clinical samples.

[0140] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting PTH1-84 by liquid chromatography-mass spectrometry, comprising: The sample is mixed with the capture reagent, washed and eluted, the supernatant is dried and dissolved in an acetonitrile aqueous solution to obtain a sample to be tested, and the sample to be tested is detected by liquid chromatography-mass spectrometry; The capture reagent includes: microspheres coated with PTH1-84 antibodies; The cleaning solution contains Tween, PBS and water; The elution solvent contains formic acid, acetonitrile and water.

2. The method according to claim 1, characterized in that The buffer in the capture reagent is tris(hydroxymethylaminomethane) containing 0.05% polystyrene beads coated with PTH1-84 antibody.

3. The method according to claim 1, characterized in that In the cleaning solution, the mass fraction of PBS is 0.5% to 2%, and the volume fraction of Tween is 1‰ to 3‰; In the eluent, the volume ratio of acetonitrile to water is 9:(0.5-2), and the volume fraction of formic acid in the eluent is 5%-15%.

4. The method according to claim 2 or 3, characterized in that: The volume ratio of the sample to the capture reagent is 10:(2.5-4), and the volume ratio of the eluent to the capture reagent is (100-150):(200-400).

5. The method according to any one of claims 1 to 4, characterized in that: The conditions of the liquid chromatography include: Protein BEH C4 column was used; Mobile phase A is an aqueous solution of 0.1%~0.5% formic acid and 2%~6% DMSO. Mobile phase B was 85%~95% acetonitrile in water containing 0.1%~0.5% formic acid and 2%~6% DMSO.

6. The method according to claim 5, characterized in that The elution gradient of the liquid chromatography is: 0~1.0min: the volume fraction of mobile phase A is 95%, and the volume fraction of mobile phase B is 5%; 1.0~7.0min The volume fraction of mobile phase A changes from 95% to 75%. The volume fraction of mobile phase B changes from 5% to 25%; 7.0~11.0min The volume fraction of mobile phase A changes from 75% to 10%. The volume fraction of mobile phase B changes from 25% to 90%; 11.0~13.5min The volume fraction of mobile phase A is 10%, and the volume fraction of mobile phase B is 90%; 13.5~14.5min The volume fraction of mobile phase A changes from 10% to 95%. The volume fraction of mobile phase B changes from 90% to 5%; 14.5~20.0min The volume fraction of mobile phase A is 95%, and the volume fraction of mobile phase B is 5%.

7. The method according to claim 5, characterized in that The conditions of the liquid chromatography also include: The chromatographic column is ACQUITY ® UPLC Protein BEH C4 (2.1×100mm,1.7μm); Mobile phase A was an aqueous solution of 0.3% formic acid and 3% DMSO. Mobile phase B was 90% acetonitrile in water containing 0.3% formic acid and 3% DMSO; The mobile phase flow rate was 0.3 mL / min; The column temperature was 60°C, the column pressure was 6380 psi, and the injection volume was 40 μL.

8. The method according to any one of claims 1 to 7, characterized in that: The conditions for mass spectrometry detection include: Parent ions 629.4, 683.2 (m / z) Product ions 660.3, 721.2 (m / z) Collision energy 18, 19V Cone voltage 130V Detection mode: Positive.

9. The method according to any one of claims 1 to 7, characterized in that: The method also includes the step of preparing a standard substance, wherein the matrix of the standard substance is bovine serum adsorbed with activated carbon, and the concentration of PTH1-84 is 0.005-1 ng / mL.

10. The method according to any one of claims 1 to 7, characterized in that: The sample is whole blood, serum or plasma, and the storage condition is -80°C to -20°C.

Images