Method for determining content of 2, 3-diphosphoglycerate in whole blood based on liquid chromatography-tandem mass spectrometry

Abstract

The invention belongs to the field of quantitative analysis of in-vivo metabolite concentration, and particularly discloses a method for determining the content of 2, 3-diphosphoglycerate in whole blood based on liquid chromatography-tandem mass spectrometry, which comprises the following steps: (1) establishing a standard curve of 2, 3-diphosphoglycerate, and (2) determining the content of 2, 3-diphosphoglycerate in a sample to be detected. According to the method, 2, 3-DPG retention is increased by selecting an ACQUITYUPLC BEHAmide HILIC amido triple bond bonded chromatographic column, meanwhile, buffer salt ammonium bicarbonate is added into a mobile phase to improve the peak pattern, methylene diphosphoric acid and metaphosphoric acid are used for reducing adsorption and interaction of a metal pipeline, the peak pattern is further improved, and 2, 3-DPG in a whole blood sample is detected under the cooperation of a specific gradient elution program. The 3-DPG is accurately detected, and the detection sensitivity and precision are improved.

Description

Technical Field

[0001] The present invention relates to the field of quantitative analysis of metabolite concentrations in vivo, and particularly to a method for determining the content of 2,3-diphosphoglycerate in whole blood based on liquid chromatography-tandem mass spectrometry. Background Art

[0002] 2,3-Diphosphoglycerate (2,3-DPG for short) is an important intermediate in the glycolytic metabolic bypass of mammalian red blood cells including human red blood cells. It can regulate the transport and release of oxygen on hemoglobin. Generally, the concentration of 2,3-diphosphoglycerate in whole blood in vivo is about 800-1800 μg / mL. Under various physiological and pathological conditions, the concentration of 2,3-DPG will change to adapt to the aerobic metabolism of the body, which is a reference index worthy of attention in clinical diagnosis and treatment.

[0003] Currently, the concentration detection of 2,3-diphosphoglycerate usually uses an ELISA kit for detection. The double antibody sandwich enzyme-linked immunosorbent assay is adopted, and finally the absorbance is measured using an enzyme label instrument or a spectrophotometer and then calculated. The ELISA kit detection method has some inevitable disadvantages, such as low repeatability, low sensitivity, limited specificity leading to false positives, false negatives, etc.

[0004] 2,3-Diphosphoglycerate is a compound containing high-energy phosphate bonds and having a relatively large polarity. The compound is unstable and is prone to adsorption and interaction with metal substances, making the method development of liquid chromatography-tandem mass spectrometry more difficult. In view of this, it is necessary to develop a rapid and highly sensitive LC-MS / MS quantitative detection method for 2,3-diphosphoglycerate in whole blood to meet the actual needs. Summary of the Invention

[0005] To solve the above problems, the present invention provides a method for determining the content of 2,3-diphosphoglycerate in whole blood based on liquid chromatography-tandem mass spectrometry, which comprises the following steps:

[0006] (1) Establishment of a standard curve for 2,3-diphosphoglycerate

[0007] a. Preparation of standard curve samples with a series of concentrations: Take a 2,3-diphosphoglycerate standard product, dissolve it in water, and then dilute it with an acetonitrile aqueous solution into a standard curve working solution with a series of concentrations; respectively take the standard curve working solutions with a series of concentrations, add blank matrix, internal standard working solution, water and methanol in sequence, mix well, centrifuge after precipitation, take the supernatant, add acetonitrile, mix well and centrifuge again, and take the supernatant to obtain standard curve samples with a series of concentrations;

[0008] b. Respectively aspirate the standard curve samples of a series of concentrations, inject them into LC-MS / MS for analysis and detection, measure the peak areas, and obtain the standard curve of 2,3-diphosphoglycerate;

[0009] The chromatographic conditions are as follows:

[0010] Chromatographic column: ACQUITY UPLC BEH Amide HILIC amide-based triple bond-bonded chromatographic column; An aqueous solution containing methylene diphosphate and ammonium bicarbonate is used as mobile phase A, and an aqueous acetonitrile solution containing methylene diphosphate and ammonium bicarbonate is used as mobile phase B. The gradient elution program is:

[0011] 0 - 0.7 min: 90% B; 0.7 - 2.0 min: 90 - 50% B; 2.0 - 3.0 min: 50% B; 3.0 - 3.5 min: 50 - 90% B; 3.5 - 6.5 min: 90% B;

[0012] Mass spectrometry conditions: The scanning mode is ESI negative ion; The mass spectrometry detection mode is MRM multiple reaction monitoring;

[0013] (2) Determination of the content of 2,3-diphosphoglycerate in the sample to be tested:

[0014] c. Preparation of biological samples

[0015] Take the whole blood sample to be tested, successively add the internal standard working solution, water and methanol, mix well, centrifuge after precipitation, take the supernatant, add acetonitrile, mix well and centrifuge again, and take the supernatant to obtain the biological sample to be tested;

[0016] d. Determination of biological samples

[0017] Inject the biological sample to be tested into LC-MS / MS, detect it under the same conditions as in step b, and quantitatively obtain the content of 2,3-diphosphoglycerate in the whole blood sample to be tested according to the standard curve in step (1).

[0018] Furthermore, during the process of determining the content in step (2), a quality control program is also included. The specific steps are as follows:

[0019] Respectively take the quality control sample and the blank sample, inject them into LC-MS / MS for detection and analysis to monitor the interference and deviation existing in the determination process;

[0020] The preparation method of the quality control sample is as follows: Take the 2,3-diphosphoglycerate standard product, add 50% acetonitrile water to make a quality control working solution with a concentration of 4 - 20 mg / mL. Take the quality control working solution, successively add the blank matrix, the internal standard working solution, water and methanol, mix well, centrifuge after precipitation, take the supernatant, add acetonitrile, mix well and centrifuge again, and take the supernatant to obtain the quality control sample; The volume ratio of the quality control working solution, the blank matrix, the internal standard working solution, water and methanol is 1:9:20:80:550;

[0021] The preparation method of the blank sample is as follows: Take the blank matrix, add 50% acetonitrile water, water and methanol in sequence, mix well, centrifuge after precipitation, take the supernatant, add acetonitrile, mix well and centrifuge again, and take the supernatant to obtain the blank sample; the volume ratio of the blank matrix, 50% acetonitrile water, water and methanol is 9:1:100:550;

[0022] The volume ratio of the supernatant after centrifugation after precipitation to acetonitrile is 1:1.

[0023] Further, the concentration of the acetonitrile aqueous solution in step a is 50%, and the volume ratio of the standard curve working solution with a series of concentrations, the blank matrix, the internal standard working solution, water and methanol is 1:9:20:80:550; the volume ratio of the supernatant after centrifugation after precipitation to acetonitrile is 1:1.

[0024] Furthermore, the concentration of 2,3-diphosphoglyceric acid in the standard curve working solution with a series of concentrations is 2.5 - 25 mg / mL.

[0025] Further, the blank matrix is animal blank plasma or serum, preferably blank mouse plasma; the internal standard working solution is an aqueous solution containing 0.05 - 0.15 mg / mL 2,3-diphosphoglyceric acid- 13 C3 and 5% metaphosphoric acid; the internal standard working solution, water, methanol and acetonitrile are ice-bathed before use.

[0026] Further, the precipitation method is vortex oscillation for 5 - 10 min; the centrifugation is at 16000 rpm at 4°C for 3 - 8 min.

[0027] Further, the volume ratio of the whole blood sample to be tested, the internal standard working solution, water and methanol in step c is 10:20:80:550; the volume ratio of the supernatant after centrifugation after precipitation to acetonitrile is 1:1.

[0028] Further, in the chromatographic conditions in step b, the chromatographic column is Waters ACQUITY UPLC BEH Amide, 2.1×50 mm, 1.7 μm, the injection volume is 1 μL, the column temperature is 40°C, and the flow rate is 0.5 mL / min.

[0029] Further, mobile phase A is an aqueous solution containing 5 μM methylene diphosphate and 15 mM ammonium bicarbonate with a pH value of 9.00 ± 0.10; mobile phase B is an acetonitrile aqueous solution containing 5 μM methylene diphosphate and 15 mM ammonium bicarbonate with a pH value of 9.00 ± 0.10; the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 90:10.

[0030] Further, in the mass spectrometry conditions described in step b, the curtain gas is 40 psi, the ion source voltage is 5000 V, the ion source temperature is 500 °C, the nebulizing gas is 55 psi, the desolvation gas is 55 psi, and the collision gas is Medium; for the quantitative detection of 2,3-diphosphoglyceric acid, the ion pair is m / z 265.0 → m / z 167.0, the declustering voltage is -60 V, and the collision voltage is -21 V; for the qualitative detection of the ion pair, it is m / z 265.0 → m / z 177.0, the declustering voltage is -60 V, and the collision voltage is -22 V; for the quantitative detection of the internal standard ion pair, it is m / z 268.0 → m / z 170.0, the declustering voltage is -60 V, and the collision voltage is -18 V.

[0031] Further, the experimental bench operation steps of steps a and c are both carried out on wet ice; the experimental bench operation steps are dissolution and dilution.

[0032] A method for determining the content of 2,3-diphosphoglyceric acid in whole blood based on liquid chromatography-tandem mass spectrometry according to the present invention increases the retention of 2,3-DPG by selecting an ACQUITY UPLC BEH Amide HILIC amide-based triple bond-bonded chromatographic column. At the same time, ammonium bicarbonate buffer salt is added to the mobile phase to improve the peak shape, and methylene diphosphoric acid and metaphosphoric acid are used to reduce the adsorption and interaction of the metal pipeline, further improving the peak shape. With the cooperation of a specific gradient elution program, the accurate detection of 2,3-DPG in whole blood samples is realized, and the sensitivity and precision of the detection are improved. After method verification, the method of the present invention has accurate detection, high sensitivity, strong specificity, and good precision for the detection of 2,3-DPG in whole blood samples, providing a new means for effectively monitoring the change of 2,3-DPG concentration in whole blood and having practical popularization and application value.

[0033] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions, or changes can be made.

[0034] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Description of the Drawings

[0035] Figure 1 It is the total ion chromatogram (TIC) of the 2,3-diphosphoglyceric acid standard solution and the internal standard;

[0036] Figure 2 It is the extracted ion chromatogram (EIC) of the quantitative ions of the 2,3-diphosphoglyceric acid standard solution and the internal standard;

[0037] Figure 3 It is the standard curve graph of 2,3-diphosphoglycerate;

[0038] Figure 4 It is the detection chromatogram of blank plasma sample;

[0039] Figure 5 It is the detection chromatogram of whole blood sample. Detailed implementation mode

[0040] Example 1. Detection of 2,3-diphosphoglycerate content in whole blood

[0041] (I) Preparation of stock solution and internal standard

[0042] Standard curve stock solution: Weigh the standard product of 2,3-diphosphoglycerate, and dissolve it with pure water to prepare a standard curve stock solution with a concentration of 50 mg / mL;

[0043] Internal standard working solution: Weigh 2,3-diphosphoglycerate- 13 C3 isotope, dissolve it with pure water to prepare an internal standard stock solution of 1 mg / mL; Weigh metaphosphoric acid, and prepare a 10% metaphosphoric acid solution with pure water; Take 50 μL of the internal standard stock solution and 250 μL of the 10% metaphosphoric acid solution, then add 200 μL of pure water and mix well, and finally prepare an internal standard working solution containing 0.1 mg / mL 2,3-diphosphoglycerate- 13 C3 and 5% metaphosphoric acid;

[0044] (II) Establishment of the standard curve of 2,3-diphosphoglycerate

[0045] a. Preparation of standard curve samples:

[0046] Take the standard curve stock solution, and dilute it with 50% acetonitrile aqueous solution to prepare a series of concentration standard curve working solutions with concentrations of 25 mg / mL, 12.5 mg / mL, 5 mg / mL, and 2.5 mg / mL respectively;

[0047] Take 1 μL of each series of concentration standard curve working solutions respectively, mix it with 9 μL of blank matrix (blank mouse plasma), then add 20 μL of the internal standard working solution and shake for 1 min, then add 80 μL of ice water and shake for 1 min, add 550 μL of ice methanol and vortex for 5 min to precipitate proteins, centrifuge at 4 °C and 16000 rpm for 6 min on a centrifuge, take 100 μL of the supernatant after centrifugation and mix it with 100 μL of ice acetonitrile, and then centrifuge at 4 °C and 16000 rpm for 6 min again on the centrifuge, and take the supernatant to obtain a series of concentration standard curve samples;

[0048] b. Respectively aspirate the standard curve samples of a series of concentrations and inject them into the LC-MS / MS. Measure the peak areas of 2,3-diphosphoglyceric acid and the internal standard. Use the concentration of 2,3-diphosphoglyceric acid in the standard curve samples of a series of concentrations as the abscissa (X), and use the peak area ratio of 2,3-diphosphoglyceric acid to the internal standard 2,3-diphosphoglyceric acid- 13 C3 as the ordinate (Y), and plot the standard curve, then it's okay;

[0049] The chromatographic conditions are as follows:

[0050] The chromatographic column is Waters ACQUITY UPLC BEH Amide (2.1×50mm, 1.7μm); the injection volume is 1μL; the column temperature is 40°C; the flow rate is 0.5mL / min; use an aqueous solution containing 5μM methylenediphosphonic acid and 15mM ammonium bicarbonate with a pH value of 9.00±0.10 as mobile phase A, and use an acetonitrile / water (v / v = 90:10) solution containing 5μM methylenediphosphonic acid and 15mM ammonium bicarbonate with a pH value of 9.00±0.10 as mobile phase B; the gradient elution program is:

[0051] 0 - 0.7min: 90%B; 0.7 - 2.0min: 90 - 50%B; 2.0 - 3.0min: 50%B; 3.0 - 3.5min: 50 - 90%B; 3.5 - 6.5min: 90%B;

[0052] Mass spectrometry conditions: Scanning mode: ESI negative ion, MRM multiple reaction detection; curtain gas (CUR) 40psi, ion source voltage 5000V, ion source temperature 500°C, nebulizing gas (GAS1) 55psi, desolvation gas (GAS2) 55psi, collision gas (CAD) Medium;

[0053] The detection and quantification ion pair of 2,3-diphosphoglyceric acid is m / z 265.0→m / z 167.0, declustering voltage -60V, collision voltage -21V; the qualitative ion pair is m / z 265.0→m / z 177.0, declustering voltage -60V, collision voltage -22V; the internal standard 2,3-diphosphoglyceric acid- 13 The detection and quantification ion pair of C3 is m / z 268.0→m / z 170.0, declustering voltage -60V, collision voltage -18V;

[0054] (3) Determination of the content of 2,3-diphosphoglyceric acid in the sample to be tested

[0055] c. Preparation of biological samples, blank samples and quality control samples

[0056] Take 10 μL of the whole blood sample to be tested, add 20 μL of the internal standard working solution and shake for 1 min, then add 80 μL of ice water and shake for 1 min. Add 550 μL of ice methanol and vortex for 5 min to precipitate proteins. Centrifuge at 4°C and 16,000 rpm for 6 min on a centrifuge. Take 100 μL of the supernatant after centrifugation, add 100 μL of ice acetonitrile and mix well. Centrifuge again at 4°C and 16,000 rpm for 6 min, and take the supernatant to obtain the biological sample;

[0057] Take 9 μL of blank mouse plasma, add 1 μL of 50% acetonitrile aqueous solution and shake for 1 min, then sequentially add 20 μL of ice water and shake for 1 min, add 80 μL of ice water and shake for 1 min. Add 550 μL of ice methanol and vortex for 5 min to precipitate proteins. Centrifuge at 4°C and 16,000 rpm for 6 min on a centrifuge. Take 100 μL of the supernatant after centrifugation, add 100 μL of ice acetonitrile and mix well. Centrifuge again at 4°C and 16,000 rpm for 6 min, and take the supernatant to obtain the blank sample;

[0058] Take the standard curve stock solution, dilute it with 50% acetonitrile water to prepare quality control working solutions with concentrations of 20 mg / mL and 4 mg / mL respectively. Take 1 μL of each of the two concentrations of the quality control working solutions, add 9 μL of blank mouse plasma and mix well. Then add 20 μL of the internal standard working solution and shake for 1 min, then add 80 μL of ice water and shake for 1 min. Add 550 μL of ice methanol and vortex for 5 min to precipitate proteins. Centrifuge at 4°C and 16,000 rpm for 6 min on a centrifuge. Take 100 μL of the supernatant after centrifugation, add 100 μL of ice acetonitrile and mix well. Centrifuge again at 4°C and 16,000 rpm for 6 min, and take the supernatant to obtain the quality control sample;

[0059] d. Determination of biological samples

[0060] Take 1 μL of the blank sample, quality control sample and biological sample respectively and inject them into LC-MS / MS, and detect under the same conditions as in step b. Use the blank sample to monitor whether there is interference in the blank matrix, use the quality control sample to monitor whether there is deviation during sample processing and analysis detection. Quantify according to the standard curve in step (ii) to obtain the content of 2,3-diphosphoglyceric acid in the whole blood sample.

[0061] The experimental bench operation steps in step a and step c of the above detection steps are all carried out on wet ice, and the experimental bench operation steps are dissolution and dilution.

[0062] The beneficial effects of the present invention are illustrated below through test examples.

[0063] Test example 1 Verification of the methodology of the present invention

[0064] 1. Instruments and equipment

[0065] The information of the main instruments and equipment is as follows in the table:

[0066]

[0067] 2. Standard substances and main reagents

[0068] The standard substances and main reagents used: The 2,3-diphosphoglyceric acid standard substance was purchased from Sigma-Aldrich; the 2,3-diphosphoglyceric acid- 13 C3 isotope internal standard was purchased from TRC-Canada; methylenediphosphonic acid, metaphosphoric acid, and ammonia water were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; chromatographic grade acetonitrile and methanol were purchased from Merck; ammonium bicarbonate was purchased from Shanghai Anpu Experimental Technology Co., Ltd.; ultrapure water with a resistivity of 18.2 MΩ·cm was used by a pure water machine system.

[0069] 3. Preparation of standard samples

[0070] Precisely weigh a certain amount of the 2,3-diphosphoglyceric acid standard substance, dilute it with pure water to prepare a 50 mg / mL stock solution, and then gradient-dilute the stock solution with 50% acetonitrile water to standard curve working solutions with concentrations of 25 mg / mL, 12.5 mg / mL, 5 mg / mL, and 2.5 mg / mL and quality control working solutions with concentrations of 20 mg / mL and 4 mg / mL. Take 1 μL of the standard curve or quality control working solution, add 9 μL of blank mouse plasma and mix well to prepare 10 μL of standard samples. The concentration range of the standard curve samples is 250 - 2500 μg / mL, and the concentrations of the quality control samples are 400 μg / mL and 2000 μg / mL.

[0071] The dilution process is carried out in an ice bath, and the blank mouse plasma was purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0072] 4. Preparation of internal standard working solution

[0073] Precisely weigh a certain amount of 2,3-diphosphoglyceric acid- 13 C3 isotope internal standard and prepare a 1 mg / mL internal standard stock solution with pure water. Precisely weigh a certain amount of metaphosphoric acid and prepare a 10% metaphosphoric acid solution with pure water. Take 50 μL of the internal standard stock solution and 250 μL of the 10% metaphosphoric acid solution, and then add 200 μL of pure water and mix well to finally prepare an internal standard working solution containing 0.1 mg / mL 2,3-diphosphoglyceric acid- 13 C3 and 5% metaphosphoric acid.

[0074] 5. Sample pretreatment

[0075] Accurately pipette 10 μL of the whole blood sample or standard sample to be tested into a 1.5 mL centrifuge tube, add 20 μL of the internal standard working solution and shake for 1 min, then add 80 μL of ice-cold water and shake for 1 min. Add 550 μL of ice-cold methanol and vortex for 5 min to precipitate proteins. Centrifuge at 16000 rpm at 4 °C for 6 min in a centrifuge. Take 100 μL of the supernatant after centrifugation and mix it with 100 μL of ice-cold acetonitrile. Centrifuge again at 16000 rpm at 4 °C for 6 min, and then take 100 μL of the supernatant and transfer it to a vial for waiting to be injected into the instrument for analysis and detection.

[0076] All operations are carried out on an ice bath. Methanol, acetonitrile, and pure water are pre-cooled in advance before use.

[0077] 6. Sample Detection and Analysis

[0078] 6.1 Chromatographic Conditions

[0079] Mobile phase A: An aqueous solution containing 5 μM methylenediphosphonic acid and 15 mM ammonium bicarbonate; Mobile phase B: An acetonitrile / water (v / v = 90:10) solution containing 5 μM methylenediphosphonic acid and 15 mM ammonium bicarbonate.

[0080] Preparation process of the mobile phase: Weigh about 2.1345 g of ammonium bicarbonate, add 360 μL of methylenediphosphonic acid aqueous solution (25 mM stock solution), add 180 mL of pure water and shake to dissolve and mix evenly to prepare a 150 mM ammonium bicarbonate aqueous solution containing 50 μM methylenediphosphonic acid as the mother liquor; Adjust the pH of the mother liquor to 9.00 ± 0.10 with ammonia water; Take 90 mL of the mother liquor and add 810 mL of water or acetonitrile, and use an ultrasonic instrument to assist in dissolution. After preparation, the solution is clear and transparent. Use a solvent filter and a diaphragm vacuum pump equipped with a 0.45 μm aqueous filter membrane / organic filter membrane to filter the mobile phase to remove impurities, and use it after degassing with an ultrasonic instrument for 10 min.

[0081] Chromatographic column: Waters ACQUITY UPLC BEH Amide (2.1×50 mm, 1.7 μm)

[0082] Elution program: 0 - 0.7 min: 90% B; 0.7 - 2.0 min: 90 - 50% B; 2.0 - 3.0 min: 50% B; 3.0 - 3.5 min: 50 - 90% B; 3.5 - 6.5 min: 90% B;

[0083] Flow rate: 0.5 mL / min; Column temperature: 40 °C; Injection volume: 1 μL.

[0084] 6.2 Mass Spectrometry Conditions

[0085] Scanning mode: ESI negative ion, MRM multiple reaction monitoring.

[0086] Ion source parameter settings: CUR (curtain gas) 40 psi, ion source voltage 5000 V, ion source temperature 500 °C, GAS1 (spray gas) 55 psi, GAS2 (desolvation gas) 55 psi, CAD (collision gas) Medium.

[0087] Table 1 2,3-diphosphoglyceric acid and 13 Mass spectrometry parameters of C3-2,3-diphosphoglyceric acid

[0088]

[0089] Note: * indicates the quantitative daughter ion.

[0090] 7. Linearity and precision of the method

[0091] Two experimenters performed three experimental treatments and analyses each at different times according to the method content in steps 3 to 6 above, and obtained the total ion chromatograms (TIC) of the typical 2,3-diphosphoglyceric acid standard solution and the internal standard as Figure 1 , and the EIC diagrams after extracting the ion channels are as Figure 2 . The linear range is 250 - 2500 μg / mL, and the results of drawing the standard curve are as Figure 3 . The average accuracy and precision of the linear and low-high concentration quality control samples were calculated (n = 3). The measured linear regression coefficients (weight 1 / x2) were all greater than 0.995, the accuracy was between 92.69% and 109.20%, and the relative standard deviation was between 0.7% and 6.7%. The specific results are shown in Table 2 below.

[0092] Table 2 Linear range, regression equation, correlation coefficient and precision

[0093]

[0094] 8. Discussion

[0095] After at least 6 verifications of the method linearity and precision by different experimenters, there were no obvious interference peaks in the blank matrix samples ( Figure 4 ), the peak shapes of 2,3-diphosphoglyceric acid and the internal standard in the samples to be measured were good and the responses were sufficient ( Figure 5 ). The measured linear correlation coefficient R 2 (weight 1 / x 2 ) were all greater than 0.995, the accuracy was between 92.69% and 109.20%, and the relative standard deviation was between 0.7% and 6.7%. In addition, the calibration effect of the isotope internal standard can omit the verification of matrix effect and recovery rate.

[0096] In summary, the method of the present invention can achieve accurate, sensitive and rapid detection of 2,3-diphosphoglyceric acid in whole blood samples. The method has strong specificity and high sensitivity, and has good linearity and precision within the concentration range of 250-2500 μg / mL.

Claims

1. A method for determining the content of 2,3-diphosphoglyceric acid in whole blood by liquid chromatography-tandem mass spectrometry, characterized in that: It includes the following steps: (1) Establishment of the standard curve for 2,3-diphosphoglyceric acid a. Preparation of standard curve samples with a series of concentrations: Take the standard product of 2,3-diphosphoglyceric acid, dissolve it in water, and then dilute it with an acetonitrile aqueous solution to form standard curve working solutions with a series of concentrations; successively take the standard curve working solutions with a series of concentrations, add blank matrix, internal standard working solution, water, and methanol, mix well, centrifuge after precipitation, take the supernatant, add acetonitrile, mix well and centrifuge again, and take the supernatant to obtain standard curve samples with a series of concentrations; b. Respectively aspirate the standard curve samples with a series of concentrations and inject them into LC-MS / MS for analysis and detection, measure the peak area, and obtain the standard curve of 2,3-diphosphoglyceric acid; The chromatographic conditions are as follows: Chromatographic column: ACQUITY UPLC BEH Amide HILIC amide-based triple-bond-bonded chromatographic column; an aqueous solution containing methylene diphosphonic acid and ammonium bicarbonate is used as mobile phase A, and an acetonitrile aqueous solution containing methylene diphosphonic acid and ammonium bicarbonate is used as mobile phase B. The gradient elution program is: 0 - 0.7 min: 90% B; 0.7 - 2.0 min: 90 - 50% B; 2.0 - 3.0 min: 50% B; 3.0 - 3.5 min: 50 - 90% B; 3.5 - 6.5 min: 90% B; Mass spectrometry conditions: The scanning mode is ESI negative ion; the detection mode is MRM multiple reaction monitoring; (2) Determination of the content of 2,3-diphosphoglyceric acid in the sample to be tested: c. Preparation of biological samples Take the whole blood sample to be tested, successively add internal standard working solution, water, and methanol, mix well, centrifuge after precipitation, take the supernatant, add acetonitrile, mix well and centrifuge again, and take the supernatant to obtain the biological sample to be tested; d. Determination of biological samples Inject the biological sample to be tested into LC-MS / MS, detect it under the same conditions as in step b, and quantitatively obtain the content of 2,3-diphosphoglyceric acid in the whole blood sample to be tested according to the standard curve in step (1).

2. The method according to claim 1, wherein: During the process of determining the content in step (2), a quality control program is also included. The specific steps are: Respectively take quality control samples and blank samples, inject them into LC-MS / MS for detection and analysis to monitor the interference and deviation existing in the determination process; The preparation method of the quality control sample is: Take the standard product of 2,3-diphosphoglyceric acid, add 50% acetonitrile water to make a quality control working solution with a concentration of 4 - 20 mg / mL. Take the quality control working solution, successively add blank matrix, internal standard working solution, water, and methanol, mix well, centrifuge after precipitation, take the supernatant, add acetonitrile, mix well and centrifuge again, and take the supernatant to obtain the quality control sample; the volume ratio of the quality control working solution, blank matrix, internal standard working solution, water, and methanol is 1:9:20:80:550; The preparation method of the blank sample is: Take the blank matrix, successively add 50% acetonitrile water, water, and methanol, mix well, centrifuge after precipitation, take the supernatant, add acetonitrile, mix well and centrifuge again, and take the supernatant to obtain the blank sample; the volume ratio of the blank matrix, 50% acetonitrile water, water, and methanol is 9:1:100:550; The volume ratio of the supernatant after centrifugation after precipitation to acetonitrile is 1:

1.

3. The method according to claim 1, wherein: The concentration of the acetonitrile aqueous solution in step a is 50%, and the volume ratio of the calibration curve working solution with a series of concentrations, blank matrix, internal standard working solution, water, and methanol is 1:9:20:80:550; the volume ratio of the supernatant after centrifugation after precipitation to acetonitrile is 1:

1.

4. The method according to claim 3, characterized in that: The concentration of 2,3-diphosphoglyceric acid in the calibration curve working solution with a series of concentrations is 2.5 - 25 mg / mL.

5. The method according to any one of claims 1 to 3, characterized in that: The blank matrix is animal blank plasma or serum, preferably blank mouse plasma; the internal standard working solution is an aqueous solution containing 0.05 - 0.15 mg / mL 2,3-diphosphoglycerate- 13 C3 and 5% metaphosphoric acid; the internal standard working solution, water, methanol and acetonitrile are ice-bathed before use.

6. The method according to claim 1 or 2, characterized in that: The method of precipitation is vortex oscillation for 5 - 10 min; the centrifugation is at 16000 rpm at 4 °C for 3 - 8 min.

7. The method according to claim 1, wherein: In the chromatographic conditions in step b, the chromatographic column is Waters ACQUITY UPLC BEH Amide, 2.1×50 mm, 1.7 μm, the injection volume is 1 μL, the column temperature is 40 °C, and the flow rate is 0.5 mL / min; and / or: the volume ratio of the whole blood sample to be tested, internal standard working solution, water, and methanol in step c is 10:20:80:550; the volume ratio of the supernatant after centrifugation after precipitation to acetonitrile is 1:

1.

8. The method according to claim 1, wherein: The mobile phase A is an aqueous solution containing 5 μM methylenediphosphonic acid and 15 mM ammonium bicarbonate with a pH value of 9.00 ± 0.10; the mobile phase B is an acetonitrile aqueous solution containing 5 μM methylenediphosphonic acid and 15 mM ammonium bicarbonate with a pH value of 9.00 ± 0.10; the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 90:

10.

9. The method according to claim 1, wherein: In the mass spectrometry conditions in step b, the curtain gas is 40 psi, the ion source voltage is 5000 V, the ion source temperature is 500 °C, the nebulizing gas is 55 psi, the desolvation gas is 55 psi, and the collision gas is Medium; the quantitative detection ion pair of 2,3-diphosphoglyceric acid is m / z 265.0 → m / z 167.0, the declustering voltage is -60 V, and the collision voltage is -21 V; the qualitative detection ion pair is m / z 265.0 → m / z 177.0, the declustering voltage is -60 V, and the collision voltage is -22 V; the quantitative detection ion pair of the internal standard is m / z 268.0 → m / z 170.0, the declustering voltage is -60 V, and the collision voltage is -18 V.

10. The method according to claim 1, characterized in that: The experimental bench operation steps in step a and step c are both carried out on wet ice; the experimental bench operation steps are dissolution and dilution.

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