Detection method of central carbon and application thereof

Abstract

The invention provides a central carbon detection method and application thereof, and relates to the technical field of analytical chemistry. The detection method comprises the step of detecting an on-machine sample added with iminodimethyl phosphoric acid by using a liquid chromatography-tandem mass spectrometry method, and the detection method can improve peak patterns of various central carbon including 6-phosphogluconic acid, D-glucose-1-phosphoric acid, guanosine monophosphate, guanosine triphosphate, isocitric acid, malic acid and the like and improve the detection effect of the central carbon.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry, and in particular to a central carbon detection method and application thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Central carbon metabolism (CCM) is a crucial metabolic process in living organisms. Traditionally, it encompasses the adenosine phosphate pathway (EMP), the pentose phosphate pathway (PP), and the tricarboxylic acid cycle (TCA). This metabolism is the primary source of energy required by organisms and provides precursors for other metabolic processes. Central carbon metabolism is the primary source of energy required by organisms. Through processes such as glycolysis and the tricarboxylic acid cycle, it produces ATP, which powers various cellular activities and provides the carbon source required for lipid and protein synthesis, as well as for the formation of glycolipids and other physiological functions. Central carbon metabolism provides abundant precursors for other metabolic processes in the body, such as intermediates such as glucose-6-phosphate and fructose-6-phosphate in glycolysis, which participate in the synthesis of biomacromolecules such as glycogen and glycolipids. Central carbon metabolism is the hub of carbohydrate, lipid, and amino acid metabolism, enabling the mutual conversion and connection between different metabolic pathways.

[0004] The occurrence of many diseases is closely related to disorders of central carbon metabolism, such as diabetes, cardiovascular disease, and cancer. The occurrence of diseases such as favism, diabetic arteriosclerotic heart disease, gestational glucose metabolism disorders, and liver disease is closely related to abnormal glucose metabolism. Abnormalities in lactate and pyruvate are likely related to the glycolysis pathway. The important role of central carbon metabolism in all living organisms is increasingly recognized. Common methods for detecting central carbon substances include ion chromatography-mass spectrometry (HPIC-QTRAP / MS) and liquid chromatography-mass spectrometry (LC-QQQ-MS). However, because central carbon substances are difficult to detect, commonly used mass spectrometry detection methods may result in no peak or poor peak shape, which cannot meet detection requirements. Therefore, how to make central carbon substances that are difficult to detect produce measurable peaks or improve the peak shape of existing peaks to meet detection requirements is currently an unresolved problem.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The object of the present invention is to provide a method for detecting central carbon to improve the peak shape of central carbon in the detection process using liquid chromatography tandem mass spectrometry, thereby improving the detection effect of central carbon.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] In a first aspect, a method for detecting central carbon is provided, the method comprising detecting an on-machine sample added with iminodimethylphosphonic acid using liquid chromatography tandem mass spectrometry;

[0009] The central carbon includes one or more of: 6-phosphogluconate, D-glucose-1-phosphate, guanosine monophosphate, guanosine triphosphate, isocitrate, malic acid, phosphoenolpyruvate, ribose-5-phosphate, adenosine diphosphate, deoxyadenosine triphosphate, D-erythrose-4-phosphate, deoxyguanosine diphosphate, adenosine triphosphate, 5-xytosan, 7-phosphate sedoheptose, D-fructose-1,6-diphosphate, D-glucose-6-phosphate, cis-aconitic acid, glucose, glucuronic acid, guanosine diphosphate, thiamine pyrophosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, cyclic adenosine monophosphate, 2-phosphoglycerate, nicotinamide adenine dinucleotide and acetyl-CoA.

[0010] In an optional embodiment, the concentration of iminodimethylphosphonic acid in the sample is 1 to 10 mM, preferably 10 mM.

[0011] In an optional embodiment, the detection method includes pre-treating the sample to obtain the on-machine sample; the pre-treatment includes using an extraction reagent containing iminodimethylphosphonic acid to extract the central carbon in the sample to obtain the on-machine sample.

[0012] In an optional embodiment, the liquid chromatography column adopts hydrophilic interaction liquid chromatography.

[0013] In an optional embodiment, the mobile phase of the liquid chromatography includes mobile phase A and mobile phase B, wherein the mobile phase A is an aqueous solution containing ammonium acetate, and the mobile phase B is an aqueous solution containing ammonium acetate and acetonitrile.

[0014] In an optional embodiment, the elution program of the liquid chromatography is set as follows:

[0015] 0 min, mobile phase A was 5% v / v, mobile phase B was 95% v / v;

[0016] 5 min, mobile phase A 5% v / v, mobile phase B 95% v / v;

[0017] 8 min, mobile phase A 30% v / v, mobile phase B 70% v / v;

[0018] 16 min, mobile phase A: 60% v / v, mobile phase B: 40% v / v;

[0019] 21 min, mobile phase A: 60% v / v, mobile phase B: 40% v / v;

[0020] 22.1 min, mobile phase A: 5% v / v, mobile phase B: 95% v / v;

[0021] 24 min, mobile phase A is 5% v / v, mobile phase B is 95% v / v.

[0022] In an optional embodiment, the mass spectrometry parameters include: using an electrospray ionization source, an ion source temperature of 550°C, an ion source voltage negative / positive mode of -4500V / 4500V, a curtain gas of 35psi, a nebulizer gas and an auxiliary gas of 60psi, and scanning using multiple reaction monitoring.

[0023] In the second aspect, the application of the detection method described in the first aspect in the preparation of central carbon detection products is provided.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention introduces iminodimethyl phosphate into the sample on the machine, so that chromatographic peaks appear for guanosine triphosphate, isocitrate, malic acid, adenosine diphosphate, deoxyadenosine triphosphate, adenosine triphosphate, D-fructose-1,6-diphosphate and thiamine pyrophosphate, which cannot produce peaks in the original detection method; and improves the peak shapes of 6-phosphogluconate, D-glucose-1-phosphate, guanosine monophosphate, phosphoenolpyruvate, ribose-5-phosphate, D-erythrose-4-phosphate, deoxyguanosine diphosphate, xytonose 5-phosphate, sedoheptose 7-phosphate, D-glucose-6-phosphate, cis-aconitic acid, glucose, glucuronic acid, guanosine diphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, cyclic adenosine monophosphate, 2-phosphoglycerate, nicotinamide adenine dinucleotide and acetyl-CoA. The detection method can also increase the peak area of ​​the central carbon, improve the signal-to-noise ratio of the detection value, and thus improve the detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 Spectra of 6-Phosphogluconic acid-3 (6-phosphogluconic acid) with the addition of 10 mM (lower) and 0 μM (upper) iminodimethylphosphonic acid;

[0028] Figure 2The spectra of D-Glucose 1-phosphate-2 (D-glucose 1-phosphate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0029] Figure 3 Spectra of GMP-2 (uridine monophosphate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0030] Figure 4 Spectra of GTP-2 (uridine triphosphate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonate;

[0031] Figure 5 The spectra of Isocitrate-3 (isocitrate) with the addition of 10 mM (right) and 0 μM (left) imidodimethylphosphonic acid;

[0032] Figure 6 The spectra of lactic acid-1 (lactic acid) were obtained by adding 10 mM (right) and 0 μM (left) imidodimethylphosphonic acid.

[0033] Figure 7 The spectra of Malate-2 (malic acid) with the addition of 10 mM (right) and 0 μM (left) imidodimethylphosphonic acid;

[0034] Figure 8 The spectra of phosphoroenolpyruvic acid-1 (phosphoenolpyruvic acid) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid.

[0035] Figure 9 Spectra of AMP-1 (adenosine monophosphate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonate;

[0036] Figure 10 The spectra of Ribose 5-phosphate-1 (ribose-5-phosphate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0037] Figure 11 The spectra of Adenosine 5'-diphosphate-2 (adenosine diphosphate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0038] Figure 12 Spectra of dATP-1 (deoxyadenosine triphosphate) added with 10 mM (right) and 0 μM (left) iminodimethylphosphonate;

[0039] Figure 13The spectra are those of D-Erythrose 4-phosphate-1 (D-erythrose-4-phosphate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid.

[0040] Figure 14 Spectra of dGDP-1 (deoxyguanosine diphosphate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0041] Figure 15 Spectra of ATP-1 (adenosine triphosphate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonate;

[0042] Figure 16 The spectra of D-Xylulose 5-phosphate-1 (Xytonose 5-phosphate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0043] Figure 17 The spectra are for Pyruvic Acid-2 (pyruvate) with the addition of 10 mM (right) and 0 μM (left) imidodimethylphosphonate.

[0044] Figure 18 The spectra of Succinic Acid-1 (succinic acid) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0045] Figure 19 The spectra of Sedoheptulose 7-phosphate-1 (7-phosphate sedoheptulose) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0046] Figure 20 Spectra of D-Fructose 1,6-bisphosphate-1 (D-fructose-1,6-bisphosphate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0047] Figure 21 Spectra of glucose 6-phosphate-2 (D-glucose-6-phosphate) added with 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0048] Figure 22 The spectra of cis-Aconitic acid-1 (cis-aconitic acid) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0049] Figure 23Spectra of D-Glucose-1 (glucose) added with 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0050] Figure 24 Spectra of dTMP-1 (thymidine deoxynucleotide) added with 10 mM (right) and 0 μM (left) iminodimethylphosphonate;

[0051] Figure 25 The spectra of Fumaric Acid-1 (fumaric acid) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0052] Figure 26 Spectra of Glucuronic acid-2 (glucuronic acid) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0053] Figure 27 Spectra of GDP-1 (guanosine diphosphate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonate;

[0054] Figure 28 The spectra are for Thiamine diphosphate-1 (thiamine pyrophosphate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid.

[0055] Figure 29 The spectra of dihydroxyacetonephosphate-1 (dihydroxyacetone phosphate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0056] Figure 30 The spectra of 3-Phospho-D-glycerate-1 (3-phosphoglycerate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0057] Figure 31 The spectra are for Cyclic AMP-1 (cyclic adenosine monophosphate) with the addition of 10 mM (right) and 0 μM (left) imidodimethylphosphonate.

[0058] Figure 32 The spectra of 2-Phospho-D-glycerate-1 (2-phosphoglycerate) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0059] Figure 33 The spectra of NAD+-1 (nicotinamide adenine dinucleotide) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphate;

[0060] Figure 34 The spectra of Acetyl-CoA-2 (acetyl coenzyme A) with the addition of 10 mM (right) and 0 μM (left) iminodimethylphosphonic acid;

[0061] Figure 35 D-Xylulose 5-phosphate used in the test method of Experimental Group 1 in Example 3;

[0062] Figure 36 Ribose 5-phosphate (Ribose 5-phosphate) used in the test method of Experimental Group 1 in Example 3;

[0063] Figure 37 D-Xylulose 5-phosphate used in the test method of Experimental Group 2 in Example 3;

[0064] Figure 38 This is the ribose 5-phosphate used in the testing method of Experimental Group 2 in Example 3. DETAILED DESCRIPTION

[0065] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0066] In this document, unless otherwise specified, all implementation methods and preferred implementation methods mentioned herein can be combined with each other to form a new technical solution; all technical features and preferred features mentioned herein can be combined with each other to form a new technical solution; the components involved or their preferred components can be combined with each other to form a new technical solution.

[0067] Unless otherwise specified, the numerical range "a-b" is an abbreviation for any combination of real numbers between a and b, where a and b are both real numbers. For example, the numerical range "6-22" indicates that all real numbers between "6-22" are listed herein, and "6-22" is merely an abbreviation for these numerical combinations. A "range" disclosed herein in the form of lower limits and upper limits can include one or more lower limits and one or more upper limits, respectively.

[0068] Herein, unless otherwise indicated, the various reactions or process steps may be performed sequentially or in any non-sequential manner. Preferably, the reaction methods herein are performed sequentially.

[0069] As used herein, "prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0070] As used herein, the conjunction "consisting of excludes any unspecified elements, steps, or components. If used in a claim, this phrase will render the claim closed so that it excludes materials other than those recited, except for normal impurities associated therewith.

[0071] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or behavior from another entity or behavior, and does not necessarily require or imply any actual relationship, order, or importance between these entities or behaviors, such as numbering first, second, etc.

[0072] As used herein, unless otherwise stated, "optionally," "optional," "optional," or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0073] In a first aspect, a method for detecting central carbon is provided, the method comprising detecting a sample added with iminodimethylphosphonic acid using liquid chromatography-tandem mass spectrometry.

[0074] The molecular formula of iminodimethylphosphonic acid is HN[CH2PO(OH)2]2; the CAS number is 17261-34-6; and the structural formula is shown in formula (I).

[0075]

[0076] The sample on the machine refers to a sample for injection into a liquid chromatography tandem mass spectrometry system. In an optional embodiment, the sample is a sample that has been pre-treated to reduce impurities in the sample and / or enrich the central carbon in the sample.

[0077] The central carbon includes one or more of: 6-phosphogluconate, D-glucose-1-phosphate, guanosine monophosphate, guanosine triphosphate, isocitrate, malic acid, phosphoenolpyruvate, ribose-5-phosphate, adenosine diphosphate, deoxyadenosine triphosphate, D-erythrose-4-phosphate, deoxyguanosine diphosphate, adenosine triphosphate, 5-phospho-xytosyl, 7-phosphate sedoheptose, D-fructose-1,6-diphosphate, D-glucose-6-phosphate, cis-aconitic acid, glucose, glucuronic acid, guanosine diphosphate, thiamine pyrophosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, cyclic adenosine monophosphate, 2-phosphoglycerate, nicotinamide adenine dinucleotide, and acetyl-CoA. The present invention has found that adding iminodimethylphosphonic acid during liquid chromatography-tandem mass spectrometry can improve the peak shape of the above-mentioned central carbon. However, experiments have found that adding iminodimethylphosphonic acid to the mobile phase will accelerate the damage rate of the instrument chromatography equipment seal, which usually occurs within 1 to 2 weeks. However, adding imidodimethylphosphonic acid to the sample and passing it into the liquid chromatography tandem mass spectrometry detection system along with the sample will not cause damage to the instrument, and can improve the peak shape of the above-mentioned central carbon and increase the peak area and signal-to-noise ratio of most central carbons.

[0078] In an optional embodiment, the central carbon includes one or more of: guanosine triphosphate, isocitrate, malate, adenosine diphosphate, deoxyadenosine triphosphate, adenosine triphosphate, D-fructose-1,6-diphosphate and thiamine pyrophosphate.

[0079] In an optional embodiment, the concentration of iminodimethylphosphonic acid in the sample is 1 to 10 mM, for example, but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mM, preferably 10 mM.

[0080] In an optional embodiment, the detection method includes pre-treating the sample to obtain the on-machine sample; the pre-treatment includes using an extraction reagent containing iminodimethylphosphonic acid to extract the central carbon in the sample to obtain the on-machine sample containing iminodimethylphosphonic acid.

[0081] In an optional embodiment, the pretreatment method includes first extracting the analyte in the sample using a first extraction reagent, separating the supernatant, mixing the supernatant with a second extraction reagent containing iminodimethylphosphonic acid, and separating the supernatant again for chromatographic separation; the first extraction reagent is an aqueous solution containing 80-90% v / v methanol, for example, but not limited to, an aqueous solution containing 80, 81, 82, 83, 84, 85, 85.5, 86, 87, 88, 89, or 90% v / v methanol; the second extraction reagent is an aqueous solution containing 0-15% v / v methanol, for example, but not limited to, an aqueous solution containing 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% v / v methanol. When the methanol content in the second extraction reagent is 0% v / v, the second extraction reagent is pure water.

[0082] The present invention also optimizes the chromatographic and mass spectrometric conditions, and achieves better separation of various substances through optimization of liquid chromatography and mass spectrometric conditions, including liquid chromatography column, mobile phase selection, and elution gradient.

[0083] In an optional embodiment, the liquid chromatography column adopts hydrophilic interaction liquid chromatography.

[0084] In an optional embodiment, the chromatographic column is Waters Atlantis Premier BEH Z-HILIC 1.7 μM, 2.1×100 mm.

[0085] In an optional embodiment, the mobile phase of the liquid chromatography includes mobile phase A and mobile phase B, wherein the mobile phase A is an aqueous solution containing ammonium acetate, and the mobile phase B is an aqueous solution containing ammonium acetate and acetonitrile.

[0086] In an optional embodiment, the concentration of ammonium acetate in mobile phase A and mobile phase B is independently 10-20 mM, for example, but not limited to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mM, preferably 15 mM. The concentration of acetonitrile in mobile phase B is 85-95% v / v, for example, but not limited to 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95% v / v.

[0087] In an optional embodiment, according to gradient elution, the elution program is set as follows:

[0088] 0 min, mobile phase A was 5% v / v, mobile phase B was 95% v / v;

[0089] 5 min, mobile phase A 5% v / v, mobile phase B 95% v / v;

[0090] 8 min, mobile phase A 30% v / v, mobile phase B 70% v / v;

[0091] 16 min, mobile phase A: 60% v / v, mobile phase B: 40% v / v;

[0092] 21 min, mobile phase A: 60% v / v, mobile phase B: 40% v / v;

[0093] 22.1 min, mobile phase A: 5% v / v, mobile phase B: 95% v / v;

[0094] 24 min, mobile phase A is 5% v / v, mobile phase B is 95% v / v.

[0095] In an optional embodiment, the mass spectrometry parameters include: using an electrospray ionization source, an ion source temperature of 550°C, an ion source voltage negative / positive mode of -4500V / 4500V, a curtain gas of 35psi, a nebulizer gas and an auxiliary gas of 60psi, and scanning using multiple reaction monitoring.

[0096] In an optional embodiment, the central carbon detection method further comprises constructing a standard curve. The standard curve represents the relationship between the central carbon content in the linear standard and the linear standard's test value. The standard curve can be constructed using conventional construction methods or software in the art, and the present invention is not limited thereto. By substituting the test value into the standard curve, the central carbon content in the sample to be tested can be calculated.

[0097] In an optional embodiment, an internal standard method is used to construct a standard curve.

[0098] In an optional embodiment, the internal standard includes glucose-[1,3C6], [2,3,3- 2 H3]-(S)-(-)-malic acid, [2H4]-succinic acid, 15N5-deoxyadenosine-5'-monophosphate lithium salt (dAMP) and 13C6-(D)-glucose-6-phosphate disodium salt hydrate.

[0099] In an optional embodiment, the extraction reagent includes an extraction reagent containing an internal standard.

[0100] In an optional embodiment, the first extraction reagent contains an internal standard.

[0101] The present invention does not limit the source of the sample, and the sample can optionally come from a mammal, for example, but not limited to, a mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey or human. The samples of the present invention include but are not limited to blood samples, such as blood or serum, body fluid samples, tissue fluid samples, cell samples or tissue samples. The sample can also be a standard with various preset concentrations of central carbon for constructing a standard curve; the sample can also be a quality control product with various preset concentrations of central carbon for constructing a standard curve. The present invention does not limit it.

[0102] In a second aspect, the present invention also provides the use of the aforementioned detection method in the preparation of a central carbon detection product. The aforementioned detection method can be used as an evaluation standard to assess the detection performance of other central carbon detection products; or the aforementioned detection method can be pre-installed in the detection product as an operating module, and the reagents used in the aforementioned detection method can be used as supporting reagents in the detection product.

[0103] It should be noted that the detection method provided by the present invention is not for diagnostic and therapeutic purposes. Specifically, for example, the detection method of the present invention is used to detect experimental animals or animal model samples for the study of central carbon metabolism mechanisms, or for use in fields such as drug development. Moreover, knowing the content of the above-mentioned central carbon in a sample does not mean that the result can directly point to the diagnosis of the disease. Therefore, the central carbon detection method provided by the present invention is not for diagnostic and therapeutic purposes.

[0104] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0105] Example 1

[0106] 1. Reagent configuration:

[0107] (1) Preparation of internal standard solution: The internal standard is glucose-[1,3C6](Glucose-13C6), [2,3,3- 2 H3]-(S)-(-)-malic acid ((S)-(-)-MALIC-2,3,3-D3 ACID), [2H4]-succinic acid (SUCCINIC-D4 ACID), 15N5-deoxyadenosine-5'-monophosphate lithium salt (dAMP), 13C6-(D)-glucose-6-phosphate disodium salt hydrate (D-Glucose-6-phosphate disodium salt).

[0108] Use a pipette to take 200 μL of each of the five central carbon internal standard stock solutions, add 19 ml of 90% methanol aqueous solution, and mix well to obtain a mixed internal standard solution (1 μg / ml for each internal standard).

[0109] (2) Preparation of linear standards: Pipette each of the 34 central carbon stock solutions and mix thoroughly to prepare the linear stock solution 13 for the 34 standards (each central carbon concentration is 40 μg / ml). The linear stock solution dilution solution is 50% methanol in water, and after dilution, the remaining linear stock solutions 1 to 13 are obtained.

[0110] Use a pipette to transfer 80 μL of mass spectrometry water, add 20 μL of linear mother solution 1 to linear mother solution 13 respectively, then add 250 μL of internal standard solution respectively, centrifuge to obtain 100 μL of supernatant, add 100 μL (containing 20 μM iminodimethylphosphonic acid) 10% methanol water, centrifuge to obtain the supernatant, and obtain linear standard 1 to linear standard 13, where the concentrations of each central carbon are: 5, 25, 10, 125, 250, 500, 1250, 2500, 40000 ng / ml on the machine.

[0111] 2. Sample pretreatment:

[0112] 100 mg or 100 μL of a sample containing 34 central carbons was extracted with 250 μL of a mixed internal standard solution, centrifuged, and 100 μL of the supernatant was collected. 100 μL of 10% methanol water (containing 20 μM iminodimethylphosphonic acid) was added, and the supernatant was collected by centrifugation to obtain the extract, which was then loaded onto the instrument.

[0113] 3. Chromatographic and mass spectrometry conditions:

[0114] Column: Waters Atlantis Premier BEH Z-HILIC 1.7 μM, 2.1 × 100 mm.

[0115] Mobile phase: Mobile phase A: 15 mM ammonium acetate in water; Mobile phase B: 15 mM ammonium acetate in 90% v / v acetonitrile in water.

[0116] Column temperature: 40℃.

[0117] Injection volume: 2 μL.

[0118] The chromatographic gradient program is shown in the following table:

[0119] Table 1 Chromatographic gradient elution program

[0120] Time (min) Flow rate mL / min Mobile phase A% v / v Mobile phase B% v / v 0 0.4 5 95 5 0.4 5 95 8 0.4 30 70 16 0.4 60 40 21 0.4 60 40 22.1 0.4 5 95 24 0.4 5 95

[0121] Mass spectrometry detection was performed using an electrospray ionization (ESI) source at 550°C, source voltages in negative / positive modes of -4500 V / 4500 V, curtain gas at 35 psi, and nebulizer and auxiliary gas at 60 psi. Multiple reaction monitoring (MRM) was used for scanning. The mass spectrometry parameters and retention times of the 35 standards and internal standards are shown in Table 2.

[0122] Table 2 Mass spectrometry parameters and retention times

[0123]

[0124]

[0125] In Table 2, Q1 represents the precursor ion (m / z); Q3 represents the product ion (m / z); DP represents the declustering potential (V); and CE represents the collision potential (V).

[0126] The linearity of the detection method provided in this embodiment for detecting the above central carbon is shown in Table 3, and the precision results are shown in Table 4.

[0127] Table 3 Linearity results

[0128]

[0129]

[0130] Table 4 Precision results

[0131]

[0132]

[0133]

[0134] Example 2

[0135] The effects of different concentrations of imidodimethylphosphonic acid in the samples on the test results were compared. Except for the concentration of imidodimethylphosphonic acid, the other method steps and parameters were the same as those in Example 1. The difference between the control group without imidodimethylphosphonic acid and Example 1 was only that the sample pre-treatment was carried out according to the following steps: 100 mg or 100 μL of the sample was extracted with 250 μL of the mixed internal standard solution, centrifuged to obtain 100 μL of the supernatant, and then 100 μL of 10% methanol water was added, centrifuged to obtain the supernatant, and the extract was loaded on the machine. The test results are shown in Table 5. The spectra of each analyte at 0 μM (without adding imidodimethylphosphonic acid) and 10 mM imidodimethylphosphonic acid working concentrations are shown in Table 5. Figures 1 to 34 shown.

[0136] Table 5 Results of detection of central carbon at various working concentrations of iminodimethylphosphonic acid

[0137]

[0138]

[0139]

[0140] In summary, it can be seen that the addition of iminodimethyl phosphate to the extraction reagent can make chromatographic peaks appear for guanosine triphosphate, isocitrate, malic acid, adenosine diphosphate, deoxyadenosine triphosphate, adenosine triphosphate, D-fructose-1,6-diphosphate and thiamine pyrophosphate, which originally did not show peaks. For 6-phosphogluconate, D-glucose-1-phosphate, guanosine monophosphate, phosphoenolpyruvate, ribose-5-phosphate, D-erythrose-4-phosphate, deoxyguanosine diphosphate, xytonose 5-phosphate, sedoheptose 7-phosphate, D-glucose-6-phosphate, cis-aconitic acid, glucuronic acid, guanosine diphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, cyclic adenosine monophosphate, 2-phosphoglycerate, nicotinamide adenine dinucleotide and acetyl-CoA, the addition of iminodimethyl phosphate to the extraction reagent further improved the peak shape of these central carbons, increased the peak area and increased the signal-to-noise ratio. When the concentration of 1-10 mM imidodimethylphosphonate (IDMP) was used, it could effectively improve the detection of various central carbons. However, for lactate, adenosine monophosphate, pyruvate, succinate, glucose, thymidine deoxynucleotide, and fumarate, adding IDMP to the extraction reagent had no effect on the peak shape.

[0141] Example 3

[0142] Comparison of the effects of different chromatographic conditions on detection capabilities:

[0143] Experimental Group 1: The chromatographic gradient program is shown in Figure 6. The other conditions are the same as those in Example 1. The spectra of D-Xylulose 5-phosphate and Ribose 5-phosphate obtained are shown in Figure 6. Figure 35 and Figure 36 As shown, comparison Figure 16 The right figure (spectrum of 5-phosphotoluose of Example 1) and Figure 35 , and comparison Figure 10 Right (Ribose-5-phosphate of Example 1) and Figure 36 It can be seen that the chromatographic gradient program of Example 1 can obtain a larger peak area, indicating that the chromatographic gradient elution program provided by Example 1 is more optimal.

[0144] Table 6 Chromatographic gradient elution program

[0145] Time (min) Flow rate mL / min Mobile phase A% v / v Mobile phase B% v / v 1 0.4 5 95 3 0.4 30 70 11 0.4 60 40 16 0.4 60 40 18.1 0.4 5 95 20 0.4 5 95

[0146] Experimental Group 2: The chromatographic column used was a C18 column, and the chromatographic gradient program was as shown in N. The other conditions were the same as those in Example 1. The spectra of D-Xylulose 5-phosphate and Ribose 5-phosphate obtained were as shown in Figure 37 and Figure 38 As shown, comparison Figure 16 The right figure (spectrum of 5-phosphotoluose of Example 1) and Figure 37 , and comparison Figure 10 Right (Ribose-5-phosphate of Example 1) and Figure 38 It can be seen that the chromatographic gradient program of Example 1 can obtain a larger peak area, indicating that the Waters Atlantis Premier BEH Z-HILIC can achieve better detection results.

[0147] Table 7 Chromatographic gradient elution program

[0148] Time (min) Flow rate mL / min Mobile phase A% v / v Mobile phase B% v / v 4 0.3 100 0 10 0.3 0 100 12 0.3 0 100 12.1 0.3 100 0 15 0.3 100 0

[0149] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting central carbon, characterized in that: This involves the use of liquid chromatography-tandem mass spectrometry to detect samples spiked with iminodimethylphosphonic acid; The central carbon includes one or more of: 6-phosphogluconate, D-glucose-1-phosphate, guanosine monophosphate, guanosine triphosphate, isocitrate, malic acid, phosphoenolpyruvate, ribose-5-phosphate, adenosine diphosphate, deoxyadenosine triphosphate, D-erythrose-4-phosphate, deoxyguanosine diphosphate, adenosine triphosphate, 5-xytosan, 7-phosphate sedoheptose, D-fructose-1,6-diphosphate, D-glucose-6-phosphate, cis-aconitic acid, glucose, glucuronic acid, guanosine diphosphate, thiamine pyrophosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, cyclic adenosine monophosphate, 2-phosphoglycerate, nicotinamide adenine dinucleotide and acetyl-CoA.

2. The detection method according to claim 1, wherein The central carbon includes one or more of guanosine triphosphate, isocitrate, malate, adenosine diphosphate, deoxyadenosine triphosphate, adenosine triphosphate, D-fructose-1,6-diphosphate and thiamine pyrophosphate.

3. The detection method according to claim 1, wherein The concentration of iminodimethylphosphonic acid in the sample is 1-10 mM, preferably 10 mM.

4. The detection method according to claim 1, wherein The method comprises pre-treating the sample to obtain the sample for use on the machine; the pre-treatment comprises using an extraction reagent containing iminodimethylphosphonic acid to extract the central carbon in the sample to obtain the sample for use on the machine.

5. The detection method according to claim 4, characterized in that The pretreatment includes first extracting the analyte in the sample using a first extraction reagent, separating the supernatant, mixing the supernatant with a second extraction reagent containing iminodimethylphosphonic acid, and separating the supernatant again for chromatographic separation; the first extraction reagent is an aqueous solution containing 80-90% v / v methanol; the second extraction reagent is an aqueous solution containing 0-15% v / v methanol.

6. The detection method according to any one of claims 1 to 5, characterized in that: The liquid chromatography column adopts hydrophilic interaction liquid chromatography; Optionally, the chromatographic column is Waters Atlantis Premier BEH Z-HILIC 1.7 μM, 2.1×100 mm.

7. The detection method according to claim 6, characterized in that The mobile phase of the liquid chromatography comprises mobile phase A and mobile phase B, wherein the mobile phase A is an aqueous solution containing ammonium acetate, and the mobile phase B is an aqueous solution containing ammonium acetate and acetonitrile; Optionally, the concentrations of ammonium acetate in the mobile phase A and the mobile phase B are independently 10-20 mM, and the concentration of acetonitrile in the mobile phase B is 85-95% v / v.

8. The detection method according to claim 7, characterized in that The elution program of the liquid chromatography was set as follows: 0 min, mobile phase A was 5% v / v, mobile phase B was 95% v / v; 5 min, mobile phase A 5% v / v, mobile phase B 95% v / v; 8 min, mobile phase A 30% v / v, mobile phase B 70% v / v; 16 min, mobile phase A: 60% v / v, mobile phase B: 40% v / v; 21 min, mobile phase A: 60% v / v, mobile phase B: 40% v / v; 22.1 min, mobile phase A: 5% v / v, mobile phase B: 95% v / v; 24 min, mobile phase A is 5% v / v, mobile phase B is 95% v / v.

9. The detection method according to any one of claims 1 to 5, characterized in that: The mass spectrometry parameters included: using an electrospray ionization source, an ion source temperature of 550°C, an ion source voltage in negative / positive mode of -4500V / 4500V, a curtain gas of 35 psi, a nebulizer gas and an auxiliary gas of 60 psi, and scanning using multiple reaction monitoring.

10. Use of the detection method according to any one of claims 1 to 9 in the preparation of central carbon detection products.

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