Method for separating and detecting G3P and DHAP in blood sample

Abstract

The invention discloses a method for separating and detecting G3P and DHAP in a blood sample, which replaces the traditional derivatization method with simple operation steps of protein precipitation and concentration, simplifies the pretreatment process, saves the use cost of reagents and consumables, and greatly shortens the sample pretreatment time. G3P and DHAP in a blood sample are analyzed through liquid chromatography-tandem mass spectrometry, the concentrations of G3P and DHAP can be well separated and accurately quantified, the test result is more accurate, and higher specificity is achieved. The method for separating and detecting G3P and DHAP in the blood sample is strong in specificity, high in accuracy, good in stability, low in cost, high in sensitivity, high in efficiency and simple and rapid to operate, and can meet dual requirements of clinical blood sample detection on timeliness and accuracy.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and particularly relates to a method for separating and detecting G3P and DHAP in a blood sample. Background Art

[0002] Glycolytic metabolism is one of the main ways for biological cells to obtain energy (ATP, adenosine triphosphate). Monitoring glycolytic products (such as glucose, lactic acid, glyceraldehyde 3-phosphate [G3P], dihydroxyacetone phosphate [DHAP], etc.) is of great significance in bioenergy metabolism assessment, disease diagnosis and monitoring, metabolic pathway research, bioengineering and industrial applications, basic biological process research, as well as drug development and evaluation.

[0003] Glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) are a very critical pair of isomeric intermediate products in glycolysis and energy metabolism. Glucose is converted into G3P and DHAP during glycolysis and is further metabolized into ATP in cells to provide and store energy for organism activities. The reversible cyclic conversion between G3P and DHAP dynamically regulated by cells is of great significance for internal metabolic regulation.

[0004] Currently, the main monitoring methods for G3P / DHAP in the laboratory are high-performance liquid chromatography (HPLC), enzyme-linked method, chemiluminescence method, immunoassay method, and mass spectrometry. Among them, the most commonly used method is high-performance liquid chromatography. However, before the sample is analyzed by HPLC using this method, it usually needs to go through complex extraction and derivatization steps. At the same time, there are also disadvantages such as long analysis time, insufficient specificity, and high reagent consumption. In the detection and analysis of G3P and DHAP by the enzyme-linked method, chemiluminescence method, and immunoassay method, due to factors such as unstable enzyme activity, complex pretreatment process, and poor anti-interference ability, it often fails to meet the requirements of laboratory sample analysis. In recent years, mass spectrometry has gradually become one of the important means for the detection and analysis of G3P and DHAP due to its advantages such as high separation, accurate quantification, short analysis time, and high sensitivity.

[0005] Chinese patent document CN118112133A discloses a method and a detection kit for detecting energy metabolism-related compounds, which provides various detection methods and kits for energy metabolism-related compounds. However, its detection method has a long analysis time and cannot achieve good separation and accurate quantification of G3P and DHAP, and cannot meet the detection and analysis of glycolytic metabolites in actual samples.

[0006] Chinese Patent Document CN202310263449 discloses a kit and method of use for metabolizing neutralization and metabolic flux covering multiple metabolites, which provides a method for detecting and analyzing multiple metabolites in biological samples. However, the pretreatment of this detection and analysis method uses derivatization technology, and the pretreatment reagents and steps are complex, costly, and time-consuming, so it is relatively limited in clinical applications.

[0007] Chinese Patent Document CN115616112A discloses a method for measuring mitochondrial energy metabolome, which provides a method for detecting and analyzing related metabolites including tricarboxylic acid cycle, glycolysis, amino acid metabolism, fatty acid metabolism, and nucleotide metabolism in mitochondrial metabolome. The sensitivity of target metabolites in chromatography is improved by derivatization method. However, the pretreatment method of this detection and analysis method is complex, consumes a lot of reagents, and cannot meet the separation and detection of this pair of isomers of G3P and DHAP.

[0008] Chinese Patent Document CN108303475A discloses a method for detecting energy substances, which provides a liquid chromatography method for simultaneously measuring multiple glycolytic metabolites. However, this detection method does not include the separation means and quantitative analysis method of glycolytic metabolites G3P and DHAP, and cannot meet the requirements of separating and accurately quantifying G3P and DHAP in clinical samples. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a method for separating and detecting G3P and DHAP in blood samples, which has strong specificity, high accuracy, good stability, low cost, high sensitivity, high efficiency, simple and rapid operation, and can meet the dual requirements of timeliness and accuracy for clinical blood sample detection.

[0010] To solve the above technical problems, the method for separating and detecting G3P and DHAP in blood samples provided by the present invention includes the following steps:

[0011] S1. Take a blood sample in a first centrifuge tube and add an internal standard working solution;

[0012] S2. Add a pre-cooled precipitant to the first centrifuge tube, mix well by shaking, and centrifuge at high speed and low temperature;

[0013] S3. Take the supernatant of the sample prepared in step S2 in a second centrifuge tube, thoroughly dry it, add a reconstitution solution, mix well by shaking, and centrifuge at high speed and low temperature;

[0014] S4. Take all the supernatant of the sample prepared in step S3, and analyze G3P and DHAP in the blood sample by LC-MS / MS according to the G3P calibration curve and the DHAP calibration curve.

[0015] Preferably, the blood sample is serum or plasma.

[0016] Preferably, in step S1, the first centrifuge tube is 1.5 mL;

[0017] In step S2, add a pre-cooled precipitant to the first centrifuge tube, mix well by shaking for 10 min, and centrifuge at high speed and low temperature for 10 min;

[0018] In step S3, the second centrifuge tube is 1.5 mL;

[0019] In step S3, take the supernatant of the sample prepared in step S2 into the second centrifuge tube. After thorough drying, add a reconstitution solution, mix well by shaking for 10 min, and centrifuge at high speed and low temperature for 2 min.

[0020] Preferably, in step S1, the internal standard working solution is one of lactic acid - 13C3, succinic acid - d6, or citric acid - d4, with an added volume of 5 - 100 μL and a concentration of 0.1 - 5 μg / mL;

[0021] In step S2, the precipitant is one of methanol, acetonitrile, or a mixed solution of methanol / acetonitrile. The added volume ratio to the sample is 5 - 20, the shaking speed is 500 - 2000 rpm, the centrifugation speed is 4000 - 14000 rpm, and the temperature is 4 - 25 °C;

[0022] In step S3, take 400 - 1500 μL of the supernatant of the sample prepared in step S2 into the second centrifuge tube; use vacuum drying or nitrogen blowing to dry, with the drying temperature not exceeding 35 °C; then add the reconstitution solution, mix well by shaking, and centrifuge at high speed and low temperature;

[0023] The reconstitution solution is one of an aqueous methanol solution or an aqueous acetonitrile solution. The ratio of methanol or acetonitrile to ultrapure water is (0.2 - 5):1, the shaking speed is 500 - 2000 rpm, the centrifuge speed is 4000 - 14000 rpm, and the temperature is 4 - 25 °C.

[0024] Preferably, in step S4, the liquid chromatography conditions are as follows:

[0025] The chromatographic column model is one of an Amide chromatographic column or a HILIC chromatographic column;

[0026] Mobile phase A: An aqueous solution containing ammonium acetate (5 - 25 mM) and ammonium hydroxide (0.2% - 1.2%);

[0027] Mobile phase B: An acetonitrile solution containing ammonium acetate (5 - 25 mM) and ultrapure water (5% - 20%);

[0028] The column temperature is set at 30 °C - 45 °C.

[0029] Preferably, the mobile phase uses gradient elution, and the elution program is as follows: mobile phase A + mobile phase B = 100%;

[0030] The relevant gradients are as follows: from 0 to 0.8 min, mobile phase B is 70 - 90%; from 0.8 to 3.8 min, mobile phase B linearly changes from 70 - 90% to 35 - 50%; from 3.8 to 4.2 min, mobile phase B linearly changes from 35 - 50% to 25 - 35%; from 4.2 to 5 min, mobile phase B remains at 25 - 35%; from 5 to 5.01 min, mobile phase B linearly changes from 25 - 35% to 70 - 90%; from 5.01 to 6 min, mobile phase B is maintained at 70 - 90%;

[0031] The flow rate is: 0.2 - 0.6 mL / min;

[0032] The injection volume is 2 - 30 μL.

[0033] Preferably, in step S4, the mass spectrometry conditions are as follows:

[0034] Ion source: electrospray ionization source;

[0035] Ion mode: negative ion mode;

[0036] Detection mode: multiple reaction monitoring;

[0037] The ion source temperature is 350 °C - 550 °C;

[0038] The spray voltage for negative ions is 4500 V - 5500 V;

[0039] Curtain gas is 25 - 40 psi;

[0040] The pressure of nebulizing gas Gas1 and auxiliary heating gas pressure Gas2 is 30 - 60 psi.

[0041] Preferably, in step S1, the blood sample is a human serum or plasma sample;

[0042] In step S3, the drying method of the supernatant in the second centrifuge tube is vacuum drying at 0 °C;

[0043] The selection process of the drying method of the supernatant in the second centrifuge tube in step S3 is as follows:

[0044] a). Take 100 μL of serum samples from different sources into 1.5 mL centrifuge tubes, prepare 3 replicates in parallel, accurately add 1000 μL of pre-cooled precipitant respectively, mix by shaking at 2000 rpm for 10 min, place the centrifuge tubes in a centrifuge, and centrifuge at 4 °C and 14000 rpm for 10 min;

[0045] b). Transfer the supernatant in each centrifuge tube in step a) to another 1.5 mL centrifuge tube, and concentrate the supernatant to complete dryness using the conditions of nitrogen blowing at 25°C, vacuum drying at 25°C, and freeze-vacuum drying at 0°C respectively;

[0046] c). Pipette 60 μL of the reconstitution solution into each of the other centrifuge tubes in step b), shake at 2000 rpm for 10 min, centrifuge at 14000 rpm for 2 min at 4°C;

[0047] d). Collect the supernatant in each of the other centrifuge tubes in step c) into a 96-well injection plate, analyze and compare on the machine, and screen out the drying method with the highest G3P peak area as the actually adopted drying method;

[0048] In step S4, a Shimadzu LC-30A ultra-high performance liquid chromatography system is used, and the liquid chromatography conditions are as follows:

[0049] Column model: Amide column;

[0050] Column temperature: 35°C;

[0051] Injection volume: 5 μL;

[0052] Mobile phase A: An aqueous solution containing 25 mM ammonium acetate and 0.6% ammonium hydroxide;

[0053] Mobile phase B: An acetonitrile solution containing 25 mM ammonium acetate and 10% ultrapure water;

[0054] The above mobile phases are used for gradient elution;

[0055] Elution program: Mobile phase A + mobile phase B = 100%;

[0056] Elution conditions are as follows:

[0057] Flow rate: 0.4 mL / min;

[0058] 0 - 0.8 min, mobile phase B is 87%, 0.8 - 3.8 min, mobile phase B linearly changes from 87% to 45%; 3.8 - 4.2 min, mobile phase B linearly changes from 45% to 35%; 4.2 - 5 min, mobile phase B remains at 35%, 5 - 5.01 min linearly changes from 35% to 87%, 5.01 - 6 min, mobile phase B remains at 87%.

[0059] Preferably, 9. According to the method for separating and detecting G3P and DHAP in a blood sample according to claim 1, characterized in that

[0060] The calibration product is a surrogate matrix added with a set amount of G3P and DHAP standard products;

[0061] The alternative matrix is a 5% BSA solution;

[0062] The G3P calibration curve and the DHAP calibration curve are obtained through the following steps:

[0063] P1. Take the calibration sample in an A calibration centrifuge tube and add the internal standard working solution;

[0064] P2. Add the pre-cooled precipitant to the A calibration centrifuge tube, mix well by shaking, and centrifuge at high speed and low temperature;

[0065] P3. Take the supernatant of the sample prepared in step P2 in a B calibration centrifuge tube, thoroughly dry it, add the reconstitution solution, mix well by shaking, and centrifuge at high speed and low temperature;

[0066] P4. Take all the supernatant of the sample prepared in step P3, analyze G3P and DHAP in the calibration sample by LC-MS / MS, so as to obtain the G3P calibration curve and the DHAP calibration curve.

[0067] 10. The method for separating and detecting G3P and DHAP in a blood sample according to claim 9, characterized in that the alternative matrix screening process is as follows:

[0068] 1). Prepare 90% acetonitrile / water solution, 50% acetonitrile / 1×PBS solution, 0.1% BSA solution and 5% BSA solution respectively as the candidate matrices for the calibration curve;

[0069] 2). Use a pipette to accurately pipette 10 μL of the G3P standard secondary stock solution into a 1.5 mL centrifuge tube, pipette 4 portions in parallel, and then add 90 μL of each candidate matrix solution prepared in step 1) respectively;

[0070] 3). Add 1000 μL of the pre-cooled precipitant to each centrifuge tube in step 2), mix at 2000 rpm for 10 min by shaking, place the centrifuge tube in a centrifuge, and centrifuge at 4°C and 14000 rpm for 10 min;

[0071] 4). Transfer the supernatant in each centrifuge tube in step 3) to another 1.5 mL centrifuge tube respectively, and blow it to complete dryness with nitrogen;

[0072] 5). Pipette 60 μL of the reconstitution solution into each of the other 1.5 mL centrifuge tubes in step 4), mix at 2000 rpm for 10 min, and centrifuge at 4°C and 14000 rpm for 2 min;

[0073] 6). Collect the supernatant in each of the other 1.5 mL centrifuge tubes in step 5) into a 96-well injection plate, analyze and compare on the machine, and screen out the candidate matrix solution with the best G3P peak shape and response as the alternative matrix.

[0074] The method for separating and detecting G3P and DHAP in blood samples of the present invention replaces the traditional derivatization method with simple protein precipitation and concentration operation steps, simplifies the pretreatment process, saves the use cost of reagents and consumables, and greatly shortens the sample pretreatment time (the pretreatment process only takes 90 minutes); by analyzing the glycolytic metabolites G3P and DHAP in blood samples by liquid chromatography-tandem mass spectrometry, it can well separate and accurately quantify the concentrations of the glycolytic metabolites G3P and DHAP, and the test results are more accurate and have higher specificity. The method for separating and detecting G3P and DHAP in blood samples is highly specific, accurate, stable, low-cost, highly sensitive, efficient, simple and fast to operate, and can meet the dual requirements of timeliness and accuracy for clinical blood sample detection. Description of the Drawings

[0075] In order to more clearly illustrate the technical solutions of the present invention, the following briefly introduces the drawings required for the present invention. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0076] Figure 1 It is the chromatogram of G3P in different candidate matrices in an embodiment of the method for separating and detecting G3P and DHAP in blood samples of the present invention;

[0077] Figure 2 It is the chromatographic peak area of G3P under different concentration conditions in an embodiment of the method for separating and detecting G3P and DHAP in blood samples of the present invention;

[0078] Figure 3 It is the chromatogram of G3P and DHAP under different elution conditions in an embodiment of the method for separating and detecting G3P and DHAP in blood samples of the present invention;

[0079] Figure 4 It is the total chromatogram of G3P, DHAP and internal standard in an embodiment of the method for separating and detecting G3P and DHAP in blood samples of the present invention;

[0080] Figure 5 It is the chromatogram of G3P in an embodiment of the method for separating and detecting G3P and DHAP in blood samples of the present invention;

[0081] Figure 6 It is the chromatogram of DHAP in an embodiment of the method for separating and detecting G3P and DHAP in blood samples of the present invention;

[0082] Figure 7It is the internal standard chromatogram in an embodiment of the method for separating and detecting G3P and DHAP in a blood sample of the present invention;

[0083] Figure 8 It is the standard curve of G3P in an embodiment of the method for separating and detecting G3P and DHAP in a blood sample of the present invention;

[0084] Figure 9 It is the standard curve of DHAP in an embodiment of the method for separating and detecting G3P and DHAP in a blood sample of the present invention;

[0085] Figure 10 It is the flow chart of an embodiment of the method for separating and detecting G3P and DHAP in a blood sample of the present invention. Detailed implementation manners

[0086] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the accompanying drawings. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0087] Embodiment 1

[0088] A method for separating and detecting G3P and DHAP in a blood sample is as Figure 10 shown, and includes the following steps:

[0089] S1. Take a blood sample in a first centrifuge tube and add an internal standard working solution;

[0090] S2. Add a pre-cooled precipitant to the first centrifuge tube, mix well by shaking, and centrifuge at high speed and low temperature;

[0091] S3. Take the supernatant of the sample prepared in step S2 in a second centrifuge tube, thoroughly dry it, add a reconstitution solution, mix well by shaking, and centrifuge at high speed and low temperature;

[0092] S4. Take all the supernatant of the sample prepared in step S3, and analyze G3P and DHAP in the blood sample by LC-MS / MS (Liquid chromatography-mass spectrometry) according to the G3P calibration curve and the DHAP calibration curve.

[0093] Preferably, the blood sample is serum or plasma, and serum is preferred.

[0094] The method for separating and detecting G3P and DHAP in a blood sample of Example 1 replaces the traditional derivatization method with simple protein precipitation and concentration operation steps, simplifies the pretreatment process, saves the usage cost of reagents and consumables, and greatly shortens the sample pretreatment time (the pretreatment process only takes 90 minutes); by analyzing the glycolytic metabolite pair glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) in a blood sample by liquid chromatography-tandem mass spectrometry, it can well separate and accurately quantify the concentrations of the glycolytic metabolites G3P and DHAP, and the test results are more accurate and have higher specificity. The method for detecting G3P and DHAP in this blood sample has strong specificity, high accuracy, good stability, low cost, high sensitivity, high efficiency, simple and rapid operation, and can meet the dual requirements of timeliness and accuracy for clinical blood sample detection.

[0095] Example 2

[0096] Based on the method for separating and detecting G3P and DHAP in a blood sample of Example 1, in step S1, the first centrifuge tube is 1.5 mL;

[0097] In step S2, add the precooled precipitant to the first centrifuge tube, mix well by shaking for 10 min, and centrifuge at high speed and low temperature for 10 min;

[0098] In step S3, the second centrifuge tube is 1.5 mL;

[0099] In step S3, take the supernatant of the sample prepared in step S2 into the second centrifuge tube, thoroughly dry it, add the reconstitution solution, mix well by shaking for 10 min, and centrifuge at high speed and low temperature for 2 min.

[0100] Example 3

[0101] Based on the method for separating and detecting G3P and DHAP in a blood sample of Example 2, in step S1, the internal standard working solution uses one of lactic acid-13C3 or succinic acid-d6 or citric acid-d4, the added volume is 5 - 100 μL, and the concentration is 0.1 - 5 μg / mL.

[0102] Preferably, in step S2, the precipitant uses one of methanol or acetonitrile or a mixed solution of methanol / acetonitrile, the added volume ratio to the sample is 5 - 20, the shaking speed is 500 - 2000 rpm, the centrifugation speed is 4000 - 14000 rpm, and the temperature is 4 - 25 °C.

[0103] Preferably, in step S3, take 400 - 1500 μL of the supernatant of the sample prepared in step S2 into the second centrifuge tube.

[0104] Preferably, in step S3, the supernatant of the sample prepared in step S2 is taken into a second centrifuge tube, and is dried by vacuum drying or blowing with nitrogen, and the drying temperature is not higher than 35°C; then a reconstitution solution is added, shaken and mixed evenly, and centrifuged at high speed and low temperature.

[0105] Preferably, in step S3, the reconstitution solution is one of a methanol aqueous solution or an acetonitrile aqueous solution, and the ratio of methanol or acetonitrile to ultrapure water is (0.2-5):1, the shaking speed is 500-2000 rpm, the centrifuge speed is 4000-14000 rpm, and the temperature is 4-25°C.

[0106] Example 4

[0107] Based on the method for separating and detecting G3P and DHAP in a blood sample of Example 1, in step S4, the liquid chromatography conditions are as follows:

[0108] The chromatographic column model is one of an Amide chromatographic column or a HILIC chromatographic column;

[0109] Mobile phase A: An aqueous solution containing ammonium acetate (5-25 mM) and ammonium hydroxide (0.2%-1.2%);

[0110] Mobile phase B: An acetonitrile solution containing ammonium acetate (5-25 mM) and ultrapure water (5%-20%);

[0111] The column temperature is set at 30°C to 45°C.

[0112] Preferably, the mobile phase is gradient eluted, and the elution program is: mobile phase A + mobile phase B = 100%;

[0113] The relevant gradients are as follows: 0-0.8 min, mobile phase B is 70-90%; 0.8-3.8 min, mobile phase B linearly changes from 70-90% to 35-50%; 3.8-4.2 min, mobile phase B linearly changes from 35-50% to 25-35%; 4.2-5 min, mobile phase B remains at 25-35%; 5-5.01 min, mobile phase B linearly changes from 25-35% to 70-90%; 5.01-6 min, mobile phase B remains at 70-90%;

[0114] The flow rate is: 0.2-0.6 mL / min;

[0115] The injection volume is 2-30 μL.

[0116] Preferably, in step S4, the mass spectrometry conditions are as follows:

[0117] Ion source: Electrospray ionization source;

[0118] Ion mode: Negative ion mode;

[0119] Detection mode: Multiple reaction monitoring;

[0120] The ion source temperature is 350 °C to 550 °C. Preferably, the ion source temperature is 500 °C;

[0121] The spray voltage for negative ions is 4500 V to 5500 V. Preferably, the spray voltage for negative ions is 5500 V;

[0122] The curtain gas is 25 - 40 psi. Preferably, the curtain gas is 35 psi;

[0123] The pressure of the nebulizing gas Gas1 and the auxiliary heating gas pressure Gas2 is 30 - 60 psi, preferably 50 psi.

[0124] Example Five

[0125] Based on the method for separating and detecting G3P and DHAP in a blood sample in Example One, the calibrator is a surrogate matrix added with a set amount of G3P and DHAP standards;

[0126] The surrogate matrix is a 5% BSA solution.

[0127] Preferably, the G3P calibration curve and the DHAP calibration curve are obtained through the following steps:

[0128] P1. Take the calibrator sample in an A calibration centrifuge tube and add the internal standard working solution;

[0129] P2. Add the pre-cooled precipitant to the A calibration centrifuge tube, shake well, and centrifuge at high speed and low temperature;

[0130] P3. Take the supernatant of the sample prepared in step P2 in a B calibration centrifuge tube, thoroughly dry it, add the reconstitution solution, shake well, and centrifuge at high speed and low temperature;

[0131] P4. Take all the supernatant of the sample prepared in step P3, analyze G3P and DHAP in the calibrator sample by LC-MS / MS, so as to obtain the G3P calibration curve and the DHAP calibration curve.

[0132] Preferably, the surrogate matrix screening process is as follows:

[0133] 1). Prepare 90% acetonitrile / water solution, 50% acetonitrile / 1×PBS solution, 0.1% BSA solution and 5% BSA solution respectively as the candidate matrices for the calibration curve;

[0134] 2). Use a pipette to accurately transfer 10 μL of the G3P standard secondary stock solution into a 1.5 mL centrifuge tube, transfer 4 aliquots in parallel, and then add 90 μL of each candidate matrix solution prepared in step 1);

[0135] 3). Add 1000 μL of pre-cooled precipitant to each centrifuge tube in step 2), mix by shaking at 2000 rpm for 10 min, place the centrifuge tubes in a centrifuge, and centrifuge at 14000 rpm for 10 min at 4°C;

[0136] 4). Transfer the supernatant in each centrifuge tube from step 3) to another 1.5 mL centrifuge tube respectively, and blow it to complete dryness with nitrogen;

[0137] 5). Pipette 60 μL of the reconstitution solution into each of the other 1.5 mL centrifuge tubes in step 4), shake at 2000 rpm for 10 min, and centrifuge at 14000 rpm for 2 min at 4°C;

[0138] 6). Collect the supernatant in each of the other 1.5 mL centrifuge tubes in step 5) into a 96-well sample injection plate, analyze and compare it on the machine, and screen out the candidate matrix solution with the best G3P peak shape and response as the alternative matrix

[0139] Result analysis: Since the G3P peak shape and response vary greatly in different matrices, it is necessary to first screen out the matrix with better peak shape and response for the preparation of the standard curve. The chromatograms of G3P in four different candidate matrices are as Figure 1 shown. Among them, the 5% BSA solution is used as the alternative matrix, and both the G3P peak shape and response are the best. Therefore, the 5% BSA solution is preferably used as the initially screened alternative matrix.

[0140] Example Six

[0141] Based on the method for separating and detecting G3P and DHAP in a blood sample in Example 1, in step S1, the blood sample is a human serum or plasma sample;

[0142] In step S3, the drying method of the supernatant in the second centrifuge tube is vacuum drying at 0°C,

[0143] Preferably, the selection process of the drying method of the supernatant in the second centrifuge tube in step S3 is as follows:

[0144] a). Take 100 μL of serum samples from different sources into 1.5 mL centrifuge tubes, prepare 3 parallel samples, accurately add 1000 μL of pre-cooled precipitant to each, mix by shaking at 2000 rpm for 10 min, place the centrifuge tubes in a centrifuge, and centrifuge at 14000 rpm for 10 min at 4°C;

[0145] b). Transfer the supernatant in each centrifuge tube in step a) to another 1.5 mL centrifuge tube, and concentrate the supernatant to complete dryness under the conditions of nitrogen blowing at 25°C, vacuum drying at 25°C, and freeze-vacuum drying at 0°C respectively;

[0146] c). Pipette 60 μL of the reconstituted solution into each of the additional centrifuge tubes in step b), shake at 2000 rpm for 10 min, centrifuge at 14000 rpm for 2 min at 4 °C;

[0147] d). Collect the supernatant in each of the additional centrifuge tubes in step c) into a 96-well sample plate, analyze and compare on the machine, and select the drying method with the highest G3P peak area as the actual drying method used.

[0148] Result analysis: The comparison results of the sample drying and concentration conditions are as Figure 2 shown. Among the samples treated under the 0 °C vacuum drying condition, the G3P peak area is higher than that of the samples treated under other conditions. It is preferred to complete the drying and concentration of the samples under the 0 °C vacuum condition.

[0149] Example Seven

[0150] Based on the method for separating and detecting G3P and DHAP in a blood sample in Example One, in step S4, a Shimadzu LC-30A ultra-high performance liquid chromatography system is used, and the liquid chromatography conditions are as follows:

[0151] Column model: Amide column;

[0152] Column temperature: 35 °C;

[0153] Injection volume: 5 μL;

[0154] Mobile phase A: An aqueous solution containing 25 mM ammonium acetate and 0.6% ammonium hydroxide;

[0155] Mobile phase B: An acetonitrile solution containing 25 mM ammonium acetate and 10% ultrapure water;

[0156] The above mobile phases are used for gradient elution.

[0157] Preferably, the elution program is: Mobile phase A + Mobile phase B = 100%;

[0158] Select the elution conditions with the best separation effect and peak shape of the isomers in the mass spectrum as the actual elution conditions.

[0159] Elution condition 1 (flow rate: 0.3 mL / min):

[0160] 0 - 0.8 min, 80% of mobile phase B; 0.8 - 3.8 min, mobile phase B linearly changes from 80% to 45%; 3.8 - 4.2 min, mobile phase B linearly changes from 45% to 35%; 4.2 - 5 min, mobile phase B remains at 35%; 5 - 5.01 min linearly changes from 35% to 80%; 5.01 - 6 min, mobile phase B is maintained at 80%;

[0161] Elution condition 2 (flow rate: 0.4 mL / min):

[0162] From 0 - 0.8 min, mobile phase B is 87%; from 0.8 - 3.8 min, mobile phase B linearly changes from 87% to 55%; from 3.8 - 4.2 min, mobile phase B linearly changes from 55% to 45%; from 4.2 - 5 min, mobile phase B remains at 45%; from 5 - 5.01 min, it linearly changes from 45% to 87%; from 5.01 - 6 min, mobile phase B is maintained at 87%.

[0163] Elution condition 3 (flow rate: 0.4 mL / min):

[0164] From 0 - 0.8 min, mobile phase B is 87%; from 0.8 - 3.8 min, mobile phase B linearly changes from 87% to 45%; from 3.8 - 4.2 min, mobile phase B linearly changes from 45% to 35%; from 4.2 - 5 min, mobile phase B remains at 35%; from 5 - 5.01 min, it linearly changes from 35% to 87%; from 5.01 - 6 min, mobile phase B is maintained at 87%.

[0165] Result analysis: The separation effects of G3P and DHAP under different elution gradients are as Figure 3 shown. In the mass spectrometry diagram of elution condition 3, the separation effect and peak shape of the isomers are the best, and elution condition 3 is preferably selected as the elution condition.

[0166] Example 8

[0167] Based on the method for separating and detecting G3P and DHAP in a blood sample in Example 1, in step S1, 100 μL of the sample is taken in a 1.5 mL centrifuge tube, and 20 μL of the internal standard working solution is accurately added.

[0168] In step S2, 1000 μL of pre-cooled methanol is accurately added to the centrifuge tube in step S1 using a pipette, shaken at 2000 rpm for 10 min, the centrifuge tube is placed in a refrigerated centrifuge, and centrifuged at 4°C and 14000 rpm for 10 min.

[0169] In step S3, 900 μL of the supernatant of the sample prepared in step S2 is transferred to a new 1.5 mL centrifuge tube, placed in a vacuum dryer, and completely dried under the condition of 0°C.

[0170] 60 μL of 50% acetonitrile / water solution is pipetted into the new centrifuge tube, shaken at 2000 rpm for 10 min, and centrifuged at 4°C and 14000 rpm for 2 min.

[0171] In step S4, all the supernatant of the sample prepared in step S3 is collected into a 96-well injection plate, and LC-MS / MS (Liquid chromatography-mass spectrometry) is used to analyze the glycolytic metabolites in the blood sample.

[0172] Example Nine

[0173] Based on the method for separating and detecting G3P and DHAP in the blood sample of Example 1, in step S4, a Shimadzu LC-30A ultra-high performance liquid chromatography system is used, and the liquid chromatography conditions are as follows:

[0174] Column model: Amide column;

[0175] Column temperature: 35 °C;

[0176] Mobile phase A: An aqueous solution containing 25 mM ammonium acetate and 0.6% ammonium hydroxide;

[0177] Mobile phase B: An acetonitrile solution containing 25 mM ammonium acetate and 10% ultrapure water;

[0178] The above mobile phases are subjected to gradient elution.

[0179] Preferably, the elution program is: mobile phase A + mobile phase B = 100%;

[0180] 0 - 0.8 min, mobile phase B 87%, 0.8 - 3.8 min, mobile phase B linearly changes from 87% to 45%; 3.8 - 4.2 min, mobile phase B linearly changes from 45% to 35%; 4.2 - 5 min, mobile phase B remains at 35%, 5 - 5.01 min linearly changes from 35% to 87%, 5.01 - 6 min, mobile phase B is maintained at 87%;

[0181] Flow rate: 0.4 mL / min;

[0182] Injection volume: 5 μL.

[0183] Preferably, in step S4, an AB SCIEX API 6500+ mass spectrometer is used, and the mass spectrometry conditions are as follows:

[0184] Ion source: Electrospray ionization source, ion mode: negative ion mode; detection mode: multiple reaction monitoring (parameters are shown in Table 1);

[0185] Ion source temperature is 500 °C;

[0186] Negative ion spray voltage is 5500 V;

[0187] Curtain gas is 35 psi;

[0188] The atomizing gas Gas1 and the auxiliary heating gas have a pressure of 50 psi.

[0189] The total chromatograms of G3P, DHAP and the internal standard are as Figure 4 shown; the chromatogram of G3P is as Figure 5 shown; the chromatogram of DHAP is as Figure 6 shown; the chromatogram of the internal standard is as Figure 7 shown. It can be seen that the chromatograms of G3P, DHAP and the internal standard obtained by the detection method of the present invention have good peak shapes, the detection baseline is stable, and there is no obvious interference.

[0190] Table 1 Multiple reaction monitoring mass spectrometry parameters of G3P, DHAP and the internal standard

[0191]

[0192] By the labeled concentrations (X) of each calibrator, and the ratios (Y) of the peak areas of G3P and DHAP to the internal standard in the series of calibrators, calibration curves were plotted respectively and the calibration curve equations were fitted to obtain the linear regression equations and correlation coefficients. As Figure 8 - 9 and Table 2 show, G3P and DHAP of the present invention have good linear relationships, and the linear correlation coefficient r 2 values are all ≥ 0.995.

[0193] Table 2 Linear regression equations and correlation coefficients of the calibration curves of G3P and DHAP

[0194] Serial number Single metabolite name Linear range Linear regression equation <![CDATA[Correlation coefficient r 2 <!-- 9 -->]]> 1 G3P 200 - 50000 ng / mL y = 0.0008x - 0.2447 0.9998 2 DHAP 200 - 50000 ng / mL y = 0.0003x - 0.0844 0.9998

[0195] Example Ten

[0196] Based on the method for separating and detecting G3P and DHAP in a blood sample in Example One, mixed human serum (obtained by mixing a certain number of human serum samples, used to reflect the common biochemical properties of human serum and exclude the influence of individual differences, and the specific number can be determined according to experimental requirements) was selected as a conventional sample. A solvent without the analyte in an amount equal to that of the recovery sample was added to the conventional sample (mixed human serum) to prepare a basic sample; different amounts of the analyte standard were added to the conventional sample (mixed human serum) (the volume of the added standard solution does not exceed 5% of the total sample volume) to prepare 3 recovery samples with different added concentrations (low, medium and high concentrations). The recovery samples and the basic sample were measured by the detection method of the present invention. The sample treatment method was the same as that in Example Eight, and the chromatographic conditions and mass spectrometry parameters were the same as those in Example Nine. Each concentration level sample was analyzed 3 times repeatedly and completed in the same analysis batch, and the mean value was taken for calculation. The detection results of the detection method of the present invention are shown in Tables 3 to 4. The recovery rates of G3P and DHAP are both in the range of 85% - 115%, meeting the technical requirements, indicating that the detection results of the detection method of the present invention are accurate and reliable.

[0197] Table 3 Spike recovery rate of G3P

[0198]

[0199] Table 4 Spike recovery rate of DHAP

[0200]

[0201] Example XI

[0202] Based on the method for separating and detecting G3P and DHAP in a blood sample of Example I, a standard solution of the target compound at a certain concentration was added to a surrogate matrix to prepare quality control samples at certain concentrations: low, medium, and high concentrations. Five replicates were measured for each concentration level per day, and the measurement was continuously carried out for 3 days, namely 3 batches. The sample treatment method was the same as that of Example III, and the chromatographic conditions and mass spectrometry parameters were the same as those of Example IV. The coefficient of variation (CV%) of each concentration level was calculated, and the within-batch and between-batch precision was represented by this CV% (within-batch precision formula: CV% = (within-batch standard deviation / within-batch mean) × 100%. Between-batch precision CV% = (standard deviation of 3 batches of data / mean of 3 batches of data) × 100%). The detection results and calculation results are shown in Tables 5 to 6. The within-batch and between-batch precision of G3P and DHAP are both within 15.0%, indicating that the detection method of the present invention has good precision.

[0203] Table 5 Within-batch and between-batch precision of G3P

[0204]

[0205] Table 6 Within-batch and between-batch precision of DHAP

[0206]

[0207]

[0208] Example XII

[0209] Based on the method for separating and detecting G3P and DHAP in a blood sample of Example I, a high-concentration sample was prepared by adding a standard product of the analyte to a mixed human serum sample (the same as the mixed human serum sample of Example X).

[0210] It is evaluated by continuously injecting 5 blank samples after injecting a high-concentration sample; continuously measuring for 3 days, the sample treatment method is the same as in Example 8, the chromatographic conditions and mass spectrometry parameters are the same as in Example 9, and the detection results are shown in Tables 7 to 9 below. The residual peak areas of G3P and DHAP in the blank sample compounds are both less than 20% of the peak area of the compound at the lower limit of quantification. It meets the technical requirements (peak area of the blank solution < 20% of the peak area of C1), and the residual peak area of the internal standard in the blank sample is less than 5% of the peak area corresponding to the working concentration of the internal standard.

[0211] Table 7 G3P Carryover Contamination

[0212]

[0213] Table 8 DHAP Carryover Contamination

[0214]

[0215]

[0216] Table 9 Internal Standard Carryover Contamination

[0217]

[0218] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for separating and detecting G3P and DHAP in a blood sample, characterized in that, It includes the following steps: S1. Take a blood sample in a first centrifuge tube and add an internal standard working solution; S2. Add a pre-cooled precipitant to the first centrifuge tube, mix well by oscillation, and centrifuge at high speed and low temperature; S3. Take the supernatant of the sample prepared in step S2 in a second centrifuge tube. After thorough drying, add a reconstitution solution, mix well by oscillation, and centrifuge at high speed and low temperature; S4. Take all the supernatant of the sample prepared in step S3 and analyze G3P and DHAP in the blood sample by LC-MS / MS according to the G3P calibration curve and the DHAP calibration curve.

2. The method for separating and detecting G3P and DHAP in a blood sample according to claim 1, wherein the blood sample is serum or plasma.

3. The method for separating and detecting G3P and DHAP in a blood sample according to claim 1, wherein in step S1, the first centrifuge tube is 1.5 mL; in step S2, add a pre-cooled precipitant to the first centrifuge tube, mix well by oscillation for 10 min, and centrifuge at high speed and low temperature for 10 min; in step S3, the second centrifuge tube is 1.5 mL; in step S3, take the supernatant of the sample prepared in step S2 in a second centrifuge tube. After thorough drying, add a reconstitution solution, mix well by oscillation for 10 min, and centrifuge at high speed and low temperature for 2 min.

4. The method for separating and detecting G3P and DHAP in a blood sample according to claim 3, wherein in step S1, the internal standard working solution uses one of lactic acid-13C3 or succinic acid-d6 or citric acid-d4, the added volume is 5-100 μL, and the concentration is 0.1-5 μg / mL; in step S2, the precipitant uses one of methanol or acetonitrile or a mixed solution of methanol / acetonitrile, the added volume ratio to the sample is 5-20, the oscillation speed is 500-2000 rpm, the centrifugation speed is 4000-14000 rpm, and the temperature is 4-25 °C; in step S3, take 400-1500 μL of the supernatant of the sample prepared in step S2 in a second centrifuge tube; use vacuum drying or nitrogen blowing to dry, and the drying temperature is not higher than 35 °C; then add a reconstitution solution, mix well by oscillation, and centrifuge at high speed and low temperature; the reconstitution solution is one of an aqueous methanol solution or an aqueous acetonitrile solution, the ratio of methanol or acetonitrile to ultrapure water is (0.2-5):1, the oscillation speed is 500-2000 rpm, the centrifuge speed is 4000-14000 rpm, and the temperature is 4-25 °C.

5. The method for separating and detecting G3P and DHAP in a blood sample according to claim 1, wherein in step S4, the liquid chromatography conditions are as follows: the chromatographic column model is one of an Amide chromatographic column or a HILIC chromatographic column; mobile phase A: an aqueous solution containing ammonium acetate (5-25 mM) and ammonium hydroxide (0.2%-1.2%); mobile phase B: an acetonitrile solution containing ammonium acetate (5-25 mM) and ultrapure water (5%-20%); the column temperature is set at 30 °C-45 °C.

6. The method for separating and detecting G3P and DHAP in a blood sample according to claim 5, wherein The mobile phase adopts gradient elution, and the elution program is: Mobile phase A + Mobile phase B = 100%; The relevant gradient is as follows: from 0 to 0.8 min, Mobile phase B is 70 - 90%; from 0.8 to 3.8 min, Mobile phase B linearly changes from 70 - 90% to 35 - 50%; from 3.8 to 4.2 min, Mobile phase B linearly changes from 35 - 50% to 25 - 35%; from 4.2 to 5 min, Mobile phase B remains at 25 - 35%; from 5 to 5.01 min, Mobile phase B linearly changes from 25 - 35% to 70 - 90%; from 5.01 to 6 min, Mobile phase B remains at 70 - 90%; The flow rate is: 0.2 - 0.6 mL / min; The injection volume is 2 - 30 μL.

7. The method for separating and detecting G3P and DHAP in a blood sample according to claim 1, characterized in that In step S4, the mass spectrometry conditions are: Ion source: Electrospray ionization source; Ion mode: Negative ion mode; Detection mode: Multiple reaction monitoring; The ion source temperature is 350 °C - 550 °C; The spray voltage for negative ions is 4500 V - 5500 V; The curtain gas is 25 - 40 psi; The pressure of the nebulizing gas Gas1 and the auxiliary heating gas pressure Gas2 is 30 - 60 psi.

8. The method for separating and detecting G3P and DHAP in a blood sample according to claim 1, characterized in that In step S1, the blood sample is a human serum or plasma sample; In step S3, the drying method of the supernatant in the second centrifuge tube is vacuum drying at 0 °C; The selection process of the drying method of the supernatant in the second centrifuge tube in step S3 is as follows: a). Take 100 μL of serum samples from different sources into 1.5 mL centrifuge tubes, prepare 3 parallel samples, accurately add 1000 μL of pre-cooled precipitant to each, mix by shaking at 2000 rpm for 10 min, place the centrifuge tubes in a centrifuge, and centrifuge at 4 °C and 14000 rpm for 10 min; b). Transfer the supernatant in each centrifuge tube in step a) to another 1.5 mL centrifuge tube, and concentrate the supernatant to complete dryness using the conditions of nitrogen blowing at 25 °C, vacuum drying at 25 °C, and freeze vacuum drying at 0 °C respectively; c). Pipette 60 μL of the reconstitution solution into each of the other centrifuge tubes in step b), shake at 2000 rpm for 10 min, and centrifuge at 4 °C and 14000 rpm for 2 min; d). Collect the supernatant in each of the other centrifuge tubes in step c) into a 96-well injection plate, perform on-machine analysis and comparison, and select the drying method with the highest peak area of G3P as the actually adopted drying method; In step S4, a Shimadzu LC-30A ultra-high performance liquid chromatography system is adopted, and the liquid chromatography conditions are: Chromatographic column model: Amide chromatographic column; Column temperature: 35 °C; Injection volume: 5 μL; Mobile phase A: An aqueous solution containing 25 mM ammonium acetate and 0.6% ammonium hydroxide; Mobile phase B: An acetonitrile solution containing 25 mM ammonium acetate and 10% ultrapure water; The above mobile phase adopts gradient elution; The elution program is: Mobile phase A + Mobile phase B = 100%; The elution conditions are: Flow rate: 0.4 mL / min; From 0 - 0.8 min, mobile phase B is 87%; from 0.8 - 3.8 min, mobile phase B linearly changes from 87% to 45%; from 3.8 - 4.2 min, mobile phase B linearly changes from 45% to 35%; from 4.2 - 5 min, mobile phase B remains at 35%; from 5 - 5.01 min, it linearly changes from 35% to 87%; from 5.01 - 6 min, mobile phase B is maintained at 87%.

9. The method for separation and detection of G3P and DHAP in a blood sample according to claim 1, characterized in that the calibration standard is a surrogate matrix added with a set amount of G3P and DHAP standard substances; the surrogate matrix is a 5% BSA solution; the G3P calibration curve and the DHAP calibration curve are obtained through the following steps: P1. Take a calibration standard sample in an A calibration centrifuge tube and add an internal standard working solution; P2. Add a pre-cooled precipitant to the A calibration centrifuge tube, mix well by shaking, and centrifuge at high speed and low temperature; P3. Take the supernatant of the sample prepared in step P2 in a B calibration centrifuge tube, thoroughly dry it, add a reconstitution solution, mix well by shaking, and centrifuge at high speed and low temperature; P4. Take all the supernatant of the sample prepared in step P3, analyze G3P and DHAP in the calibration standard sample by LC-MS / MS, so as to obtain the G3P calibration curve and the DHAP calibration curve.

10. The method for separation and detection of G3P and DHAP in a blood sample according to claim 9, characterized in that the surrogate matrix screening process is as follows: 1). Respectively prepare 90% acetonitrile / water solution, 50% acetonitrile / 1×PBS solution, 0.1% BSA solution and 5% BSA solution as candidate matrices for the calibration curve; 2). Use a pipette to accurately transfer 10 μL of the secondary stock solution of the G3P standard into a 1.5 mL centrifuge tube, transfer 4 aliquots in parallel, and then add 90 μL of each candidate matrix solution prepared in step 1) respectively; 3). Add 1000 μL of pre-cooled precipitant to each centrifuge tube in step 2), mix at 2000 rpm for 10 min by shaking, place the centrifuge tube in a centrifuge, and centrifuge at 14000 rpm for 10 min at 4°C; 4). Transfer the supernatant in each centrifuge tube in step 3) to another 1.5 mL centrifuge tube respectively, and blow it to complete dryness with nitrogen; 5). Pipette 60 μL of the reconstitution solution into each of the other 1.5 mL centrifuge tubes in step 4), mix at 2000 rpm for 10 min by shaking, and centrifuge at 14000 rpm for 2 min at 4°C; 6). Collect the supernatant in each of the other 1.5 mL centrifuge tubes in step 5) into a 96-well sample injection plate, analyze and compare on the machine, and screen out the candidate matrix solution with the best peak shape and response of G3P as the surrogate matrix.

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