A method for processing and detecting vascular calcification inhibitory factors in human blood samples.

Abstract

This invention discloses a novel method for processing and detecting CBF (vascular calcification inhibitory factor) in human blood. The sample processing steps are as follows: S1, measure the biological sample into sample tube a, add CBF internal standard working solution, 1% SDS solution, and DTT solution with a final concentration of 10 mM; shake in a water bath; add 4 times the volume of cold acetonitrile and shake thoroughly; centrifuge at 4°C; S2, transfer the supernatant from S1 to sample tube b, add 2.22 times the volume of cold acetonitrile, mix thoroughly, and let stand at -20°C; centrifuge at 4°C and discard the supernatant; S3, add 2.5 times the volume of cold acetonitrile from the blood sample and shake gently; centrifuge at 4°C, discard the supernatant, and air dry at room temperature; S4, add 0.1% FA aqueous solution, vortex, and dissolve thoroughly to obtain the sample to be tested. This method uses HPLC-MS / MS technology to perform absolute quantification of CBF in MRM mode.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for processing and detecting vascular calcification inhibitory factors in human blood samples. Background Technology

[0002] Vascular calcification is a significant contributing factor to cardiovascular disease, and its formation is largely age-related. Having vascular calcification increases the mortality rate in patients with hypertension, chronic kidney disease, or arteriosclerosis. Calcification blocking factor (CBF), derived from chromaffin secreted by the adrenal glands via the cleavage of kallikrein and calpain 1, effectively inhibits vascular calcification and participates in multiple signaling pathways that inhibit the transformation of vascular cells into osteoblasts, thus possessing medicinal research value in the prevention and treatment of vascular calcification.

[0003] Currently, the detection of vascular calcification inhibitors (CBF) has the following shortcomings and limitations: First, there is currently no literature in China describing and researching vascular calcification factors, and the detection of CBF inhibitors is also lacking in China; Second, one foreign study reported the determination of CBF, but the procedure is cumbersome, requiring multi-stage chromatographic separation to purify CBF in plasma. This process is complex, time-consuming, requires various reagents and instruments, and cannot absolutely quantify the concentration of CBF in plasma. Summary of the Invention

[0004] To address the existing technical problems, this invention has developed a novel method for processing and detecting vascular calcification inhibitory factor (CBF) samples in human blood. The method employs high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) in multiple reaction monitoring (MRM) mode to perform absolute quantification of CBF.

[0005] To achieve the above objectives, the present invention employs the following technical means:

[0006] The first aspect of the present invention is to provide a method for processing a sample for detecting vascular calcification inhibitory factors in human blood, comprising the following steps:

[0007] S1. Measure a blood sample and place it in sample tube a. Add an equal volume of 1% SDS solution and high-concentration DTT solution to the sample, with a final DTT concentration of 10 mM. Incubate in a 37°C water bath and shake for 0.8-1.2 h. Add 4 times the volume of cold acetonitrile to the blood sample and shake thoroughly to mix. Centrifuge at 4°C.

[0008] S2. Transfer the supernatant from S1 to sample tube b, add 2.22 times the volume of cold acetonitrile, mix thoroughly, and let stand in a -20℃ refrigerator for 0.8-1.2h; centrifuge at 4℃ and discard the supernatant.

[0009] S3. Add 2.5 times the volume of cold acetonitrile to the blood sample and gently shake; centrifuge at 4°C, discard the supernatant, and air dry at room temperature;

[0010] S4. Add 0.1% FA aqueous solution, vortex and shake until fully dissolved to obtain the test sample.

[0011] Preferably, CBF internal standard working solution is also added to the test sample.

[0012] Furthermore, the concentration of the CBF internal standard working solution is 1000 ng / ml, and it is prepared from CBF internal standard and blank matrix.

[0013] A second aspect of the present invention provides a method for detecting vascular calcification inhibitory factors in human plasma, wherein the method utilizes HPLC-MS / MS to detect the aforementioned test sample, and further includes the following steps:

[0014] CBF standard curve working solutions with different designed concentrations were prepared using CBF standards and blank matrix. The CBF standard curve working solutions and CBF internal standard working solutions were detected using HPLC-MS / MS. Regression analysis was performed using the ratio of the peak areas of the CBF standard curve working solutions and the CBF internal standard working solutions as the ordinate and the concentration of the CBF standard curve working solutions as the abscissa to obtain the CBF standard curve. The test sample containing the CBF internal standard working solution was injected into the HPLC-MS / MS for determination. The ratio of the peak area of ​​the test sample to the peak area of ​​the CBF internal standard working solution was substituted into the CBF standard curve to obtain the concentration of CBF in the test sample.

[0015] Furthermore, the blank matrix is ​​a 2% BSA solution.

[0016] Furthermore, the chromatographic conditions for the detection are as follows:

[0017] Chromatographic column: Phenomenex LC Column;

[0018] Mobile phase: Mobile phase A: 0.1% FA aqueous solution; Mobile phase B: 0.1% formic acid acetonitrile solution;

[0019] Flow rate: 0.3 mL / min;

[0020] Column temperature: 45℃;

[0021] Injection volume: 15 μL;

[0022] Gradient elution was employed as follows: the volume fraction of mobile phase A + the volume fraction of mobile phase B = 100%; the gradient elution time was 8 minutes.

[0023] The volume fraction of mobile phase A is 95% from 0 to 0.4 min.

[0024] Over 0.4–0.5 min, the volume fraction of mobile phase A decreased from 95% to 75%.

[0025] Within 0.5-3 minutes, the volume fraction of mobile phase A decreased from 75% to 54%.

[0026] The volume fraction of mobile phase A decreased from 54% to 2% over 3-3.1 minutes.

[0027] 3.1-6 min: The volume fraction of mobile phase A is maintained at 2%;

[0028] Over 6-6.1 min, the volume fraction of mobile phase A increased from 2% to 95%;

[0029] The volume fraction of mobile phase A was maintained at 95% for 6.1-7.9 min.

[0030] Furthermore, the mass spectrometry conditions for the detection are as follows:

[0031] Ion source: Electrospray ionization source, positive ion mode; Capillary voltage: 5500V; Ion source temperature: 500℃; Ion source atomizing gas: 55psi; Ion source heating auxiliary gas: 55psi; Curtain gas: 40psi; Collision gas: Medium; Scan mode: Scheduled MRM, MRM parameters are as follows:

[0032] Quantitative ions Q1(m / z) Q3(m / z) RT(min) DP(V) EP(V) CBF_2+_y17 748.30 1001.00 1.57 80 10 CBF_2+_H_y17 750.99 1004.92 1.57 80 10

[0033] Beneficial effects of the present invention

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] This method is the first to propose an extraction and detection method for CBF samples. It uses a dissociation agent and a precipitant to treat the sample, adds two cleaning steps, reduces the damage of the dissociation agent SDS to the instrument, and the entire extraction process takes 4-5 hours. CBF peptides in plasma / serum can then be detected on HPLC-MS / MS, which not only reduces the cost of equipment consumables but also meets the need for rapid detection of target peptides.

[0036] This method employs high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) in multiple reaction monitoring (MRM) mode to perform absolute quantification of CBF, filling a gap in domestic CBF quantitative detection. The quantitative results are accurate, highly sensitive, and specific, and are not affected by other substances. Attached Figure Description

[0037] Figure 1 The ion flow chromatogram of the CBF standard of the present invention is shown;

[0038] Figure 2 The ion flow chromatogram of the CBF heavy isotope internal standard of the present invention is shown;

[0039] Figure 3 The standard curve in Example 1 is shown;

[0040] Figure 4 An ion flow chromatogram of a CBF plasma sample from Example 1 of the present invention is shown;

[0041] Figure 5 An ion flow chromatogram of the heavy isotope internal standard of CBF plasma sample in Example 1 of the present invention is shown. Detailed Implementation

[0042] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, particularly the definitions disclosed in these documents concerning synthetic techniques, product and processing design, polymers, comonomers, initiators, or catalysts in the art. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0043] The numerical ranges in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. A numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided there is an interval of at least two units between any lower and any higher value. For example, if the stated composition, physical, or other property (such as molecular weight, melt index, etc.) is 100 to 1000, it means that all individual values, such as 100, 101, 102, etc., are explicitly listed, as well as all subranges, such as 100 to 166, 155 to 170, 198 to 200, etc. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed minimum and maximum values ​​are considered to be clearly stated in this application.

[0044] When referring to chemical compounds, unless explicitly stated otherwise, the singular includes all isomers and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively). Additionally, unless explicitly stated otherwise, nouns described with "an," "a," or "the" also include their plural forms.

[0045] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0046] To make the technical problems solved by the present invention, the technical solutions and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

[0047] Example

[0048] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials disclosed herein and cited therein are incorporated herein by reference. Many equivalent techniques of specific embodiments of the invention described herein will be recognized or can be understood by ordinary experimentation by those skilled in the art. These equivalents will be included in the claims.

[0050] The instruments, reagents, consumables, and HPLC-MS / MS detection conditions used in the following examples and comparative examples are as follows:

[0051] Instruments and equipment

[0052] AB Sciex 5500QT triple quadrupole mass spectrometer (USA, Sciex Corporation), including Shimadzu high performance liquid chromatography system (Japan, Shimadzu Scientific Corporation);

[0053] KQ-500E Ultrasonic Cleaner (China, Kunshan Ultrasonic Instrument Co., Ltd.);

[0054] Sigma 1-14K High-Speed ​​Centrifuge (Sigma, Germany)

[0055] Allsheng TMS-300 Super Constant Temperature Uniform Instrument (China, Hangzhou Aosheng Instrument Co., Ltd.);

[0056] Cascada I ultrapure water preparation system (Canada, Pallford Bioanalytical Instruments (Shanghai) Co., Ltd.);

[0057] QUINTIX125D-1CN electronic balance (China, Sartorius Scientific Instruments (Beijing) Co., Ltd.).

[0058] reagents and consumables

[0059] Standard: Vascular calcification inhibitory factor (CBF) 1 mg, sequence: N-LEGQEEEEDNRDSSMKLSF-C, purchased from Nanjing Jietai Biotechnology Co., Ltd. (China).

[0060] The acetonitrile used in the chromatography was purchased from Merck (Germany).

[0061] Chromatographic grade formic acid (FA) was purchased from Shanghai Maclean Biotechnology Co., Ltd. (China);

[0062] Bovine serum albumin (BSA) was purchased from Beyotime International Co., Ltd. (China);

[0063] SDS Ref: 436143 was purchased from Sigma-Aldrich (Germany);

[0064] DTT: 10197777001 was purchased from Roche (Germany);

[0065] 400μL 96-well plate, Boyun Company (China);

[0066] 96-hole plate gasket, Agilent Technologies (China).

[0067] Blood sample collection

[0068] If collecting plasma or serum samples, use a vacuum negative pressure blood collection tube containing EDTA anticoagulation (purple) to collect plasma, and use a vacuum negative pressure blood collection tube with a yellow cap (separating gel) to collect serum.

[0069] If random blood is collected, plasma or serum must be separated by centrifugation (3500 rpm, 8 min) within 2 hours, and then aliquoted and stored.

[0070] Plasma / serum is stored at -80℃ for a long time. When processing, it should first be taken out from -80℃ and placed at -20℃ for about 30 minutes, and then transferred to 4℃ to dissolve, which takes about 1 hour.

[0071] Reagent preparation

[0072] Prepare blank matrix (2% bovine serum albumin): 2g BSA is prepared in 100mL of ultrapure water (the volume of BSA is prepared according to the sample volume; the remaining volume can be stored in a 4℃ refrigerator for a short period of time, and needs to be prepared again for long-term use).

[0073] Cold acetonitrile: Store 100% acetonitrile in a -20°C refrigerator for later use.

[0074] To prepare a 1% SDS solution: Weigh 100 mg of SDS powder using a precision balance and place it in a 15 mL centrifuge tube. Add 10 mL of ultrapure water, mix well to dissolve the SDS, and set aside at room temperature.

[0075] Prepare a 500mM DTT solution: Accurately weigh 386mg of DTT powder into a 15mL centrifuge tube, add 5mL of ultrapure water, and mix well to dissolve the DTT. Aliquot into multiple tubes and store at -20% for later use.

[0076] Chromatography and mass spectrometry parameter settings

[0077] ①Chromatographic conditions:

[0078] Chromatographic column: Phenomenex LC Column;

[0079] Mobile phase: Mobile phase A: 0.1% FA aqueous solution; Mobile phase B: 0.1% formic acid acetonitrile solution;

[0080] Flow rate: 0.3 mL / min;

[0081] Column temperature: 45℃;

[0082] Injection volume: 15 μL;

[0083] A gradient elution method was adopted, and the elution gradient settings are shown in Table 1.

[0084] Table 1 Elution gradient

[0085]

[0086] ②Mass spectrometry conditions:

[0087] Ion source: Electrospray ionization source, positive ion mode; capillary voltage: 5500V; ion source temperature (TE M): 500℃; ion source atomizing gas (GS1): 55psi; ion source heating auxiliary gas (GS2): 55psi; curtain gas (CUR): 40psi; collision gas (CAD): Medium; scanning mode: Scheduled MRM (Enabled). MRM parameters are shown in Table 2.

[0088] Table 2. Parameters for quantitative detection of progeny ions using MRM

[0089] Quantitative ions Q1(m / z) Q3(m / z) RT(min) DP(V) EP(V) CBF_2+_y17 748.30 1001.00 1.57 80 10 CBF_2+_H_y17 750.99 1004.92 1.57 80 10

[0090] In the table above, CBF_2+_y17 is the quantitative ion of the CBF detection sample; CBF_2+_H_y17 is the quantitative ion of the CBF internal standard; Q1 is the parent ion; Q3 is the daughter ion; RT is the retention time; DP is the declustering voltage; and EP is the injection voltage.

[0091] The ion chromatogram of CBF standard is shown below. Figure 1 The ion chromatogram of the CBF standard weight isotope internal standard is shown in [reference needed]. Figure 2 .

[0092] Example 1: Determination of CBF content in plasma

[0093] I. CBF Standard Curve

[0094] (1) Preparation of standard products

[0095] ① Blank matrix: Weigh 1g of BSA powder using a precision balance, add 50mL of ultrapure water, mix thoroughly to obtain a 2% BSA solution as the blank matrix.

[0096] ② Standard stock solution: Dissolve 500 μg of CBF standard in 500 μL of blank matrix to obtain 1 μg / μL CBF stock solution, dispense and store at -80℃.

[0097] ③Standard working solution: Take 1 μL of standard stock solution and add it to 999 μL of blank matrix to obtain 1000 ng / mL CBF standard working solution.

[0098] (2) Preparation of CBF standard curve working solution:

[0099] ① Dilute the standard working solution 10 times with a blank matrix to obtain W0 solution; the concentration of W0 solution is 100 ng / mL.

[0100] ② Nine standard curve working solutions of different concentrations, named W1-W9, were prepared by diluting the WO solution with a blank matrix. The concentrations of the standard curve working solutions are shown in Table 3.

[0101] Table 3. Concentration of working solution for CBF standard curve

[0102] Standard curve working solution name Concentration (ng / mL) Transfer volume W0 (μL) 2% BSA (μL) W1 30 90 210 W2 20 60 240 W3 10 30 270 W4 5 15 275 W5 2 6 294 W6 1 6 594 Standard curve working solution name Concentration (ng / mL) Transfer W6 volume (μL) 2% BSA (μL) W7 0.5 150 150 W8 0.2 60 240 W9 0.1 30 270

[0103] (3) Preparation of CBF internal standard solution

[0104] ①CBF internal standard stock solution: Take 1 mg of CBF internal standard, add 1 mL of blank matrix to dissolve, mix well to obtain 1 mg / mL CBF internal standard stock solution, dispense and store at -80℃.

[0105] ② CBF internal standard working solution: Take 1 μL of the internal standard stock solution, add 999 μL of blank matrix for dilution, and mix well to obtain 1000 ng / ml CBF internal standard working solution.

[0106] Based on the nine known CBF concentration standard curve working solutions prepared for W1-W9, 4 μL of CBF internal standard working solution was added to each solution, followed by mass spectrometry detection. The chromatographic and mass spectrometry parameters were set as described above. A CBF standard curve was plotted with the ratio of the peak area of ​​the CBF standard curve working solution to the peak area of ​​the CBF internal standard working solution as the ordinate and the concentration of the CBF standard curve working solution as the abscissa. The linear equation obtained was: y = 0.07260x + 0.00407 (r = 0.9957). The CBF standard curve is shown below. Figure 3 As shown.

[0107] II. Blood Sample Collection

[0108] Random blood samples were collected, with a volume of 2.0 ml. Plasma was separated within 2 hours using centrifugation (3500 rpm, 8 min) for later use.

[0109] III. Sample Processing

[0110] S1. Sample preparation was performed using 1.5 mL centrifuge tubes. 200 μL of human plasma was pipetted into the tube, followed by 4 μL of internal standard working solution, 200 μL of 1% SDS solution, and 8 μL of 500 mM DTT solution (final DTT concentration 10 mM). The mixture was incubated in a 37°C water bath at 1100 rpm for 1 hour. 800 μL of -20°C chilled acetonitrile was added, and the mixture was thoroughly shaken. The mixture was then centrifuged at 13000 g for 10 minutes at 4°C (the centrifuge was pre-cooled).

[0111] S2. Take 450 μL of supernatant and add it to a new 1.5 mL centrifuge tube. Add 1 mL of cold acetonitrile, mix thoroughly, and let stand in a -20℃ refrigerator for 1 h. Centrifuge at 13000 g for 10 min at 4℃ and discard the supernatant.

[0112] S3. Add 500 μL of cold acetonitrile at -20℃ and gently shake by hand two or three times; centrifuge at 13000g for 10 min at 4℃; discard the supernatant and evaporate the residual acetonitrile at room temperature in a fume hood;

[0113] S4. Add 100 μL of 0.1% FA aqueous solution, vortex for 2 min to fully dissolve the protein in the tube.

[0114] IV. HPLC-MS / MS Quantitative Detection

[0115] The processed samples were analyzed by HPLC-MS / MS, with a sample loading volume of 15 μL each time. The chromatographic and mass spectrometry parameters were set as described above, and the detection results were as follows. Figure 4 , Figure 5 As shown in Table 4:

[0116] Ion chromatograms of CBF plasma samples are shown below. Figure 4 The ion chromatogram of the heavy isotope internal standard in CBF plasma samples is shown below. Figure 5 .

[0117] Table 4 Sample Detection Results

[0118] sample Sample peak area Internal peak area CBF concentration (ng / ml) plasma 1.05E+03 1.62E+04 0.8425

[0119] Investigation into whether dissociation agents were added during the plasma CBF extraction process in Examples 2 and 3

[0120] Example 2 uses 50 μl of 200 ng / ml standard, plasma sample, and a mixture of plasma sample and 200 ng / ml standard to conduct experiments without adding dissociation agent.

[0121] The experimental procedure is as follows:

[0122] Centrifuge tubes containing experimental samples were incubated in a 37°C water bath with shaking at 1100 rpm for 1 hour. 100 μl of 5% HAC 95% acetonitrile was added and mixed thoroughly. The mixture was allowed to stand for two minutes, then centrifuged at 13000 g for 10 minutes at 4°C. 100 μl of the mixture was added to a 96-well plate and analyzed by HPLC-MS / MS. Each sample loading was 15 μl, and the analysis was performed under the chromatographic and mass spectrometric parameter settings given above. The results are shown in Table 5.

[0123] Example 3 uses 50 μl of 200 ng / ml standard, plasma sample, and a mixed sample of plasma sample and 200 ng / ml standard, respectively, under the condition of adding 0.6% SDS as a dissociation agent.

[0124] The experimental procedure is as follows:

[0125] Centrifuge tubes containing experimental samples were incubated in a 37°C water bath with shaking at 1100 rpm for 1 hour. 100 μl of 5% HAC and 95% acetonitrile were added and mixed thoroughly. The mixture was allowed to stand for two minutes, then centrifuged at 13000 g for 10 minutes at 4°C. 100 μl of the mixture was added to a 96-well plate and analyzed by HPLC-MS / MS. Each sample loading was 15 μl, and the analysis was performed under the chromatographic and mass spectrometric parameter settings given above. The results are shown in Table 5.

[0126] Table 5. Results of the investigation regarding the addition or absence of dissociation agents.

[0127]

[0128] Recovery rate = [peak area (sample + 200 ng / ml standard) - peak area (sample)] / peak area (200 ng / ml standard).

[0129] The results showed that, compared with the addition of no dissociation agent, the spiked recovery rate was significantly improved and the peak area increased by 3.2 times after adding 0.6% SDS as a dissociation agent.

[0130] Examples 4, 5, and 6: Investigation of different types of dissociating agents in the plasma CBF extraction process

[0131] Example 4 uses 50 μl of 200 ng / ml standard, plasma sample, and a mixture of plasma sample and 200 ng / ml standard, respectively, under the condition of adding 50 μl of dissociation agent 0.6% SDS.

[0132] The experimental procedure is as follows:

[0133] Centrifuge tubes containing experimental samples were incubated in a 37°C water bath at 1100 rpm for 1 hour; 100 μl of ice-cold acetonitrile was added and mixed well; the mixture was allowed to stand for two minutes; centrifuged at 13000 g for 10 minutes at 4°C; 100 μl of the mixture was added to a 96-well plate and analyzed by HPLC-MS / MS, with a sample loading volume of 15 μl each time, under the chromatographic and mass spectrometric parameter settings given above. The detection results are shown in Table 6.

[0134] Example 5 uses 50 μl of 200 ng / ml standard, plasma sample, and a mixture of plasma sample and 200 ng / ml standard, respectively, under the condition of adding 50 μl of dissociation agent 9M Urea 50mM ABC.

[0135] The experimental procedure is as follows:

[0136] Centrifuge tubes containing experimental samples were incubated in a 37°C water bath at 1100 rpm for 1 hour; 100 μl of ice-cold acetonitrile was added and mixed well; the mixture was allowed to stand for two minutes; centrifuged at 13000 g for 10 minutes at 4°C; 100 μl of the mixture was added to a 96-well plate and analyzed by HPLC-MS / MS, with a sample loading volume of 15 μl each time, under the chromatographic and mass spectrometric parameter settings given above. The detection results are shown in Table 6.

[0137] Example 6 uses 50 μl of 200 ng / ml standard, plasma sample, and a mixture of plasma sample and 200 ng / ml standard, respectively, under the condition of adding 50 μl of dissociation agent 1% SDS.

[0138] The experimental procedure is as follows:

[0139] Centrifuge tubes containing experimental samples were incubated in a 37°C water bath at 1100 rpm for 1 hour; 100 μl of ice-cold acetonitrile was added and mixed well; the mixture was allowed to stand for two minutes; centrifuged at 13000 g for 10 minutes at 4°C; 100 μl of the mixture was added to a 96-well plate and analyzed by HPLC-MS / MS, with a sample loading volume of 15 μl each time, under the chromatographic and mass spectrometric parameter settings given above. The detection results are shown in Table 6.

[0140] Table 6. Results of the investigation into different types of dissociation agents

[0141]

[0142] Recovery rate = [peak area (sample + 200 ng / ml standard) - peak area (sample)] / peak area (200 ng / ml standard).

[0143] The results showed that the SDS dissociation system had a higher recovery rate and a larger peak area than that of 9M urea. As the SDS content increased from 0.6% to 1%, the recovery rate increased from 53.46% to 71.71%, indicating that 1% SDS had a better dissociation effect.

[0144] Investigation of the dosage of precipitant in Examples 7 and 8

[0145] Example 7 uses 50 μl of 200 ng / ml standard, plasma sample, and a mixture of plasma sample and 200 ng / ml standard, respectively, under the conditions of adding 50 μl of dissociation agent 0.6% SDS and 2 μl of 500 mM DTT (final DTT concentration 10 mM).

[0146] The experimental procedure is as follows:

[0147] Centrifuge tubes containing experimental samples were incubated in a 37°C water bath at 1100 rpm for 1 hour; 100 μl of ice-cold acetonitrile was added and mixed well; the mixture was allowed to stand for two minutes; centrifuged at 13000 g for 10 minutes at 4°C; 100 μl of the mixture was added to a 96-well plate and analyzed by HPLC-MS / MS, with each sample loading being 15 μl, under the chromatographic and mass spectrometric parameter settings given above. The results are shown in Table 7.

[0148] Example 8 uses 50 μl of 200 ng / ml standard, plasma sample, and a mixture of plasma sample and 200 ng / ml standard, respectively, with the addition of 50 μl of dissociation agent 0.6% SDS and 2 μl of 500 mM DTT (final DTT concentration 10 mM), and the amount of ice acetonitrile is varied for the experiment.

[0149] The experimental procedure is as follows:

[0150] Centrifuge tubes containing experimental samples were incubated in a 37°C water bath at 1100 rpm for 1 hour; 200 μl of ice-cold acetonitrile was added and mixed well; the mixture was allowed to stand for two minutes; centrifuged at 13000 g for 10 minutes at 4°C; 100 μl of the mixture was added to a 96-well plate for HPLC-MS / MS analysis, with a sample loading volume of 15 μl per test, under the chromatographic and mass spectrometric parameter settings given above. The results are shown in Table 7.

[0151] Table 7 Results of the study on precipitant dosage

[0152]

[0153] Recovery rate = [peak area (sample + 200 ng / ml standard) - peak area (sample)] / peak area (200 ng / ml standard).

[0154] The results showed that the amount of precipitant had a significant impact on the recovery results. When the amount of precipitant was four times that of the sample, the peak area response was enhanced by 2.9 times, and the recovery rate increased from 29.93% to 100.91%.

[0155] Summary of the investigation: The extraction efficiency of CBF under different conditions was compared using controlled single-factor and multi-factor variable analysis. This demonstrated that SDS, compared to UA, is more effective at separating the binding of various substances to CBF in plasma samples or standard matrices; a four-fold volume precipitant, compared to a two-fold volume precipitant, better removes interfering proteins from the sample or standard matrix, thus improving CBF extraction efficiency. Furthermore, the method of calculating the recovery rate by adding standards to the sample can effectively evaluate and determine the optimal experimental conditions.

[0156] During the investigation, we considered that the presence of excess SDS in the sample could contaminate the mass spectrometer and introduce some extraneous peaks. In subsequent steps, the confirmatory exploration added two steps of adding icy acetonitrile to further clean the precipitate and remove the SDS, thereby reducing the damage of SDS to the instrument.

[0157] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for processing samples for detecting vascular calcification inhibitory factors in human blood, characterized in that, The steps include the following: S1. Measure a blood sample and place it in sample tube a. Add an equal volume of 1% SDS solution and DTT solution with a final concentration of 10mM to the blood sample. Incubate in a 37°C water bath and shake for 0.8-1.2 h. Add 4 times the volume of cold acetonitrile to the blood sample and shake thoroughly to mix. Centrifuge at 4°C. S2. Transfer the supernatant from S1 to sample tube b, add 2.22 times the volume of cold acetonitrile, mix thoroughly, and let stand in a -20℃ refrigerator for 0.8-1.2 h; centrifuge at 4℃ and discard the supernatant. S3. Add 2.5 times the volume of cold acetonitrile to the blood sample and gently shake; centrifuge at 4°C, discard the supernatant, and air dry at room temperature; S4. Add 0.1% FA aqueous solution, vortex and shake until fully dissolved to obtain the test sample; The sequence of the vascular calcification inhibitor is N-LEGQEEEEDNRDSSMKLSF-C.

2. The method for processing samples for detecting vascular calcification inhibitory factors in human blood according to claim 1, characterized in that, The test sample also contains CBF internal standard working solution.

3. The method for processing samples for detecting vascular calcification inhibitory factors in human blood according to claim 2, characterized in that, The concentration of the CBF internal standard working solution is 1000 ng / ml, and it is prepared by CBF internal standard and blank matrix.

4. A method for detecting vascular calcification inhibitory factors in human plasma, characterized in that: The detection method uses HPLC-MS / MS to detect the sample described in claim 3, and includes the following steps: CBF standard curve working solutions with different designed concentrations were prepared using CBF standards and blank matrix. The CBF standard curve working solutions with added CBF internal standard were detected using HPLC-MS / MS. Regression analysis was performed using the ratio of the peak areas of the CBF standard curve working solution and the CBF internal standard working solution as the ordinate and the concentration of the CBF standard curve working solution as the abscissa to obtain the CBF standard curve. The test sample with added CBF internal standard working solution was injected into HPLC-MS / MS for determination. The ratio of the peak area of ​​the detected sample to the peak area of ​​the CBF internal standard working solution was substituted into the CBF standard curve to obtain the concentration of CBF in the test sample.

5. The method for detecting vascular calcification inhibitory factors in human plasma according to claim 4, characterized in that: The blank matrix was a 2% BSA solution.

6. The method for detecting vascular calcification inhibitory factors in human plasma according to claim 4, characterized in that: The chromatographic conditions for the detection are as follows: Chromatographic column: Phenomenex LC Column; Mobile phase: Mobile phase A: 0.1% FA aqueous solution; Mobile phase B: 0.1% formic acid acetonitrile solution; Flow rate: 0.3 mL / min; Column temperature: 45℃; Injection volume: 15 μL; A gradient elution method was used, with the following gradient elution parameters: volume fraction of mobile phase A + volume fraction of mobile phase B = 100%; the gradient elution time was 8 minutes before stopping. The volume fraction of mobile phase A is 95% from 0 to 0.4 min. The volume fraction of mobile phase A decreased from 95% to 75% in 0.4–0.5 min. Within 0.5–3 minutes, the volume fraction of mobile phase A decreased from 75% to 54%. 3-3.1 min The volume fraction of mobile phase A decreased from 54% to 2%; 3.1-6 min, the volume fraction of mobile phase A is maintained at 2%; The volume fraction of mobile phase A increased from 2% to 95% over 6-6.1 min. The volume fraction of mobile phase A was maintained at 95% for 6.1-7.9 min.

7. The method for detecting vascular calcification inhibitory factors in human plasma according to claim 4, characterized in that: The mass spectrometry conditions for the detection are as follows: Ion source: Electrospray ion source, positive ion mode; Capillary voltage: 5500 V; Ion source temperature: 500 ℃; Ion source atomizing gas: 55 psi; Ion source heating auxiliary gas: 55 psi; Air curtain gas: 40 psi; Collision gas: Medium; Scan mode: Scheduled MRM, MRM parameters are as follows:

Images