Method for detecting energy metabolite based on UPLC-MSMS (Ultra Performance Liquid Chromatography-Mass Spectrometry) and application

Abstract

The invention relates to a method for detecting energy metabolites based on UPLC-MSMS (ultra performance liquid chromatography-mass spectrometry) and application thereof. The method comprises the following steps: after a sample is extracted, detecting a sample extracting solution and a standard solution by adopting an ultra performance liquid chromatography-mass spectrometry combined method; a mobile phase adopted by the ultra-high performance liquid chromatography is as follows: a mobile phase A is a 5-15mM ammonium acetate aqueous solution with the pH value of 9-10; and the mobile phase B is 95-100% acetonitrile. According to the method, after the tissue sample is extracted and subjected to liquid chromatography separation, the metabolite in the energy metabolism pathway is qualitatively and quantitatively detected in a targeted mode, and due to the high selectivity of the high-resolution mass spectrum, parameter optimization does not need to be conducted on each metabolite.

Description

Technical Field

[0001] The present invention relates to the technical field of mass spectrometry detection, and in particular to a method and application of detecting energy metabolites based on UPLC-MS / MS. Background Art

[0002] Energy metabolism is the process of generating energy (ATP) and small molecule metabolites during the anaerobic glycolysis and aerobic respiration of nutrients such as glucose and fatty acids. It is one of the most basic characteristics of life and maintains the most basic life activities of living organisms. Energy metabolism is also called central carbon metabolism (CCM), including the glycolysis pathway (EMP), the tricarboxylic acid cycle (TCA) and the pentose phosphate pathway (PPP). The glycolysis pathway is a series of reactions that degrade glucose and glycogen into pyruvate accompanied by the generation of ATP. It can be carried out under anaerobic and aerobic conditions and is a common metabolic pathway for aerobic or anaerobic decomposition of glucose. The TCA cycle is a metabolic pathway that is prevalent in aerobic organisms, also known as the citric acid cycle or tricarboxylic acid cycle. It is the main way for the body to obtain energy. The tricarboxylic acid cycle is the final metabolic pathway of the three major nutrients (sugars, lipids, and amino acids), and is also the hub of the metabolic connection between sugars, lipids, and amino acids. The pentose phosphate pathway is a way of oxidative decomposition of glucose. Since this pathway starts with glucose-6-phosphate (G-6-P), it is also called the hexose phosphate bypass. Various energy metabolites are generated through glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle, which participate in cell proliferation, maturation, and apoptosis, forming a complex metabolic network in cells. Therefore, comprehensive, stable, and high-throughput detection of energy metabolite content in tissues has important guiding significance for the study of diseases related to life.

[0003] Currently, there are three main methods for detecting energy metabolites. The first is to use commercial test kits for detection, but the detection throughput is low, the cost is high, and the steps are cumbersome, making it difficult to use for large-scale sample detection; the second is to derivatize the sample and then use gas phase mass spectrometry for detection, but the derivatization steps are cumbersome and time-consuming; the third is to use liquid phase mass spectrometry in tandem with triple quadrupole detection, which has high throughput and simple steps, but the triple quadrupole uses parent and daughter ion pairs to identify metabolites, which is a low-resolution mass spectrometry and requires the collision energy and specific daughter ions of each energy substance. Therefore, a lot of time and standards are spent in the early stage of method establishment to optimize the method parameters. If the parameters are not optimal, it will cause identification errors, that is, false positive results.

[0004] Therefore, how to effectively separate and detect energy metabolites has become a problem that needs to be solved urgently. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method and application for detecting energy metabolites based on UPLC-MSMS. In the present invention, after extracting tissue samples and separating them by liquid chromatography, the metabolites in the energy metabolic pathway are qualitatively and quantitatively detected in a targeted manner. The method has the characteristics of simple pretreatment, high sensitivity, strong selectivity and good reproducibility. The method has high throughput and can detect 34 metabolites at one time.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for detecting energy metabolites based on UPLC-MSMS, the method comprising: after sample extraction, detecting the sample extract and the standard solution by ultra-high performance liquid chromatography coupled to mass spectrometry;

[0008] The mobile phase used in the ultra-high performance liquid chromatography is: mobile phase A is a 5-15mM ammonium acetate aqueous solution with a pH value of 9-10; mobile phase B is 95-100% acetonitrile. The 5-15mM can be, for example, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM or 15mM, etc. The 95-100% can be, for example, 95%, 96%, 97%, 98%, 99% or 100%, etc.

[0009] The mobile phase A used in the present invention has a pH value of 9 to 10, which can significantly improve the separation of energy metabolites within the above range, and can effectively detect a large number of energy metabolites in the sample. Ammonium acetate aqueous solution is used as mobile phase A and acetonitrile is used as mobile phase B, which can significantly improve the separation of components and greatly improve the detection throughput.

[0010] Preferably, the energy metabolites include: any one or a combination of at least two of pyruvate, lactate, glucose, cyclic adenosine monophosphate, flavin mononucleotide, flavin adenine dinucleotide, adenosine monophosphate, acetyl-CoA, nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide, α-ketoglutarate, succinate, 3-phosphoglycerate, dihydroxyacetone phosphate, ribose-5-phosphate, malic acid, ribulose-5-phosphate, adenosine diphosphate, erythrose 4-phosphate, fumaric acid, sedoheptulose-7-phosphate, trans-aconitic acid, uridine diphosphate, guanylic acid, thiamine pyrophosphate, fructose 1,6-diphosphate, glucose-6-phosphate, 5'-adenosine triphosphate, phosphoenolpyruvate, citric acid, reduced nicotinamide adenine dinucleotide phosphate, guanosine-5'-diphosphate, nicotinamide adenine dinucleotide phosphate or isocitrate.

[0011] In the present invention, 34 energy metabolites can be detected simultaneously by the above detection method, the detection flux is extremely high, and the above energy metabolites can be effectively separated.

[0012] Preferably, the sample extraction step includes: performing a first extraction after homogenizing the sample, performing a first centrifugation to obtain a supernatant and a precipitate, performing a second extraction on the precipitate and performing a second centrifugation, collecting the supernatant after the two centrifugations for incubation, performing a third centrifugation, and obtaining a sample extract after drying.

[0013] The present invention performs two sample extractions, which significantly improves the concentration of samples in high performance liquid phase detection, ensures the accuracy of detection to the greatest extent, and reduces the impact of samples on detection.

[0014] Preferably, the solvent of the homogenate includes any one of deionized water, methanol, acetonitrile or ethanol, or a combination of at least two thereof.

[0015] Preferably, the mass volume ratio of the sample to the solvent is 1:(2-100) g / mL, and the (2-100) can be, for example, 2, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100, etc.

[0016] Preferably, the first extraction reagent comprises any one of methanol, acetonitrile, ethanol, propylene glycol or water, or a combination of at least two thereof.

[0017] Preferably, the first extraction reagent comprises a combination of methanol and acetonitrile.

[0018] Preferably, the volume ratio of methanol to acetonitrile in the first extraction reagent is 1:(0.5-2). The (0.5-2) may be, for example, 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 or 2.0.

[0019] Preferably, the amount of the first extraction reagent added is 100 to 1000 μL per 50 to 150 μL of homogenate. The 50 to 150 μL may be, for example, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150, etc. The 100 to 600 μL may be, for example, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL or 1000 μL, etc.

[0020] Preferably, the first extraction time is 20 to 40 min, for example, 20 min, 25 min, 30 min, 35 min or 40 min.

[0021] Preferably, the temperature of the first extraction is 0-4°C, for example, 0°C, 1°C, 2°C, 3°C or 4°C.

[0022] Preferably, the centrifugal speed is 10000-14000 rpm, and the time is 5-15 min. The 10000-14000 rpm may be, for example, 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm or 14000 rpm, etc. The 5-15 min may be, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min, etc.

[0023] Preferably, the centrifugation temperature is 0-4°C, for example, 0°C, 1°C, 2°C, 3°C or 4°C.

[0024] Preferably, the second extraction reagent comprises any one or a combination of at least two of methanol, acetonitrile, ethanol, propylene glycol or water.

[0025] Preferably, the second extraction reagent comprises a combination of water, methanol and acetonitrile.

[0026] Preferably, the volume ratio of reagent water, methanol and acetonitrile in the second extraction is 1:(1-3):(1-3). The (1-3) may be, for example, 1, 1.5, 2, 2.5 or 3.

[0027] In the present invention, the first extraction reagent and the second extraction reagent are different. This is because the concentrations of the samples extracted twice are different. Therefore, using different reagents can better extract the energy metabolites in the residual samples, further increasing the accuracy of the detection.

[0028] Preferably, the amount of the second extraction reagent added is 0.5-1.5 mL per 50-150 μL of homogenate. The 0.5-1.5 mL may be, for example, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL or 1.5 mL, etc. The 50-150 μL may be, for example, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150, etc.

[0029] Preferably, the second extraction time is 20 to 40 min, for example, 20 min, 25 min, 30 min, 35 min or 40 min.

[0030] Preferably, the temperature of the second extraction is 0-4°C, for example, 0°C, 1°C, 2°C, 3°C or 4°C.

[0031] Preferably, the second centrifugation speed is 10000-14000 rpm, and the time is 5-15 min. The 10000-14000 rpm may be, for example, 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm or 14000 rpm, etc. The 5-15 min may be, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min, etc.

[0032] Preferably, the temperature of the second centrifugation is 0-4°C, for example, 0°C, 1°C, 2°C, 3°C or 4°C.

[0033] Preferably, the incubation temperature is -15 to -25°C, for example, it can be -25°C, -24°C, -23°C, -22°C, -21°C, -20°C, -19°C, -18°C, -17°C, -16°C or -15°C, etc.

[0034] Preferably, the incubation time is 0.5 to 2 h, for example, 0.5 h, 1 h, 1.5 h or 2 h.

[0035] Preferably, the third centrifugation speed is 10000-14000 rpm, and the time is 15-30 min. The 10000-14000 rpm may be, for example, 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm or 14000 rpm, etc. The 15-30 min may be, for example, 15 min, 20 min, 25 min or 30 min.

[0036] Preferably, the drying comprises vacuum drying.

[0037] In the present invention, vacuum drying is used to dry the sample, and the specific parameters are temperature: -50°C, time: 12 to 48 hours. The 12 to 48 hours may be, for example, 12 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours or 48 hours.

[0038] Preferably, the step of preparing the standard solution comprises: dissolving the standard in acetonitrile.

[0039] Preferably, the volume fraction of acetonitrile is 20-40%, for example, 20%, 25%, 30%, 35% or 40%.

[0040] Preferably, the concentration range of the standard solution is 1 to 20000 ng / mL, for example, it can be 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL, 5000 ng / mL, 10000 ng / mL, 15000 ng / mL or 20000 ng / mL, etc.

[0041] Preferably, before the detection, the dried sample extract is re-dissolved in acetonitrile, and the supernatant is collected after centrifugation.

[0042] Preferably, the volume fraction of acetonitrile is 20-40%, for example, 20%, 25%, 30%, 35% or 40%.

[0043] Preferably, the centrifugal speed is 10000-14000 rpm, and the time is 15-30 min. The 10000-14000 rpm may be, for example, 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm or 14000 rpm, etc. The 15-30 min may be, for example, 15 min, 20 min, 25 min or 30 min.

[0044] Preferably, the centrifugation temperature is 0-4°C, for example, 0°C, 1°C, 2°C, 3°C or 4°C.

[0045] Preferably, the stationary phase of the chromatographic column used in the ultra-high performance liquid chromatography comprises a trifunctional bonded amide stationary phase.

[0046] Preferably, the column temperature of the chromatographic column is 35-45°C, for example, it can be 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C or 45°C.

[0047] Preferably, the injection volume of the ultra-high performance liquid chromatography is 1 to 3 μL, for example, 1 μL, 1.5 μL, 2 μL, 2.5 μL or 3 μL.

[0048] Preferably, the elution procedure of the gradient elution of the ultra-high performance liquid chromatography is as follows:

[0049] From 0min to 9min, mobile phase B was changed from 90% to 40% at a constant speed;

[0050] At 9-10 minutes, mobile phase B was changed from 40% to 90% at a constant speed;

[0051] At 10.1min-12min, mobile phase B was 90%.

[0052] The above-mentioned gradient elution is adopted in the present invention because the properties of numerous energy metabolites in the sample are quite different. The above-mentioned elution procedure can promote each energy metabolite to achieve a better separation effect according to its own appropriate capacity factor.

[0053] Preferably, the flow rate of the mobile phase is 0.1-0.5 mL / min, for example, 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min or 0.5 mL / min.

[0054] Preferably, the mass spectrometer used for the mass spectrometry comprises a quadrupole-electrostatic field orbital trap high-resolution mass spectrometer.

[0055] Preferably, the scanning mode of the mass spectrometer includes full scan under negative ion conditions.

[0056] Preferably, the sheath gas of the mass spectrometer is 35 to 45 arb, for example, 35 arb, 36 arb, 37 arb, 38 arb, 39 arb, 40 arb, 41 arb, 42 arb, 43 arb, 44 arb or 45 arb.

[0057] Preferably, the auxiliary gas of the mass spectrometer is 8 to 12 arb, for example, 8 arb, 9 arb, 10 arb, 11 arb or 12 arb.

[0058] Preferably, the ion spray voltage of the mass spectrometer is -2.6 kv to -3 kv, for example, it can be -2.6 kv, -2.7 kv, -2.8 kv, -2.9 kv or -3.0 kv.

[0059] Preferably, the temperature of the ion transfer tube of the mass spectrometer is 300-350°C, for example, 300°C, 310°C, 320°C, 330°C, 340°C or 350°C.

[0060] Preferably, the primary scanning range of the mass spectrometer is 70-1000Da, and the secondary scanning range is 200-2000, wherein the 70-1000Da may be, for example, 70Da, 100Da, 200Da, 300Da, 400Da, 500Da, 600Da, 700Da, 800Da, 900Da or 1000Da, etc. The 200-2000 may be, for example, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 or 2000, etc.

[0061] Preferably, the primary resolution of the mass spectrometer is 70,000 and the secondary resolution is 17,500.

[0062] In a second aspect, the present invention provides an application of the method for detecting energy metabolites based on UPLC-MSMS according to the first aspect in the detection of energy metabolites.

[0063] Compared with the prior art, the present invention has at least the following beneficial effects:

[0064] 1. The energy metabolism analysis method based on UPLC-MSMS in the present invention extracts tissue samples and separates them by liquid chromatography, and then conducts qualitative and quantitative detection of metabolites in the energy metabolism pathway. It has the characteristics of simple pretreatment, high sensitivity, strong selectivity, low detection limit and good reproducibility. It has high throughput and can detect 34 metabolites at one time.

[0065] 2. The present invention adopts high-resolution mass spectrometry for detection, using primary mass spectrometry data for quantification and secondary mass spectrometry fragments for qualitative analysis, thereby reducing false positive detection results and simultaneously being able to obtain primary and secondary information of the compound, greatly improving both quantitative and qualitative results.

[0066] 3. The method of the present invention can be widely used in research fields such as medicine, agronomy, microbiology, and food, for screening disease biomarkers, clarifying the occurrence and treatment mechanisms of diseases, for studying plant growth and development, stress resistance, pesticide residues, and genetically modified crops, for food production and preservation, food nutrition identification, and food safety monitoring, and for studying metabolic pathways, phenotypic identification, and functional genomics. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is the extracted ion chromatogram of the pyruvate standard.

[0068] Figure 2 This is the extracted ion chromatogram of lactic acid standard.

[0069] Figure 3 This is the extracted ion chromatogram of glucose standard.

[0070] Figure 4 This is the extracted ion chromatogram of cyclic AMP standard.

[0071] Figure 5 This is the extracted ion chromatogram of the FMN standard.

[0072] Figure 6 This is the extracted ion chromatogram of the FAD standard.

[0073] Figure 7 This is the extracted ion chromatogram of AMP standard.

[0074] Figure 8 This is the extracted ion chromatogram of NADPH standard.

[0075] Fig. 9This is the extracted ion chromatogram of the acetyl-CoA standard.

[0076] Fig.10 This is the extracted ion chromatogram of NAD standard.

[0077] Fig.11 This is the extracted ion chromatogram of NADH standard.

[0078] Fig.12 This is the extracted ion chromatogram of α-ketoglutarate standard.

[0079] Fig.13 This is the extracted ion chromatogram of succinic acid standard.

[0080] Fig.14 This is the extracted ion chromatogram of 3-phosphoglyceric acid standard.

[0081] Fig.15 This is the extracted ion chromatogram of dihydroxyacetone phosphate standard.

[0082] Fig.16 This is the extracted ion chromatogram of the ribose-5-phosphate standard.

[0083] Fig.17 This is the extracted ion chromatogram of malic acid standard.

[0084] Fig.18 This is the extracted ion chromatogram of the ribulose 5-phosphate standard.

[0085] Fig.19 This is the extracted ion chromatogram of ADP standard.

[0086] Fig. 20 This is the extracted ion chromatogram of the adenosine diphosphate standard.

[0087] Fig.21 This is the extracted ion chromatogram of erythrose 4-phosphate standard.

[0088] Fig. 22 This is the extracted ion chromatogram of fumaric acid / fumaric acid standard.

[0089] Fig.23 This is the extracted ion chromatogram of Sedoheptulose-7-phosphate standard.

[0090] Fig.24 This is the extracted ion chromatogram of trans-aconitic acid standard.

[0091] Fig.25 This is the extracted ion chromatogram of UDP standard.

[0092] Fig.26 This is the extracted ion chromatogram of the GMP standard.

[0093] Fig. 27This is the extracted ion chromatogram of TPP standard.

[0094] Fig.28 This is the extracted ion chromatogram of fructose 1,6-diphosphate standard.

[0095] Fig.29 This is the extracted ion chromatogram of glucose-6-phosphate standard.

[0096] Fig.30 This is the extracted ion chromatogram of ATP standard.

[0097] Fig.31 This is the extracted ion chromatogram of NADP standard.

[0098] Fig.32 This is the extracted ion chromatogram of isocitrate standard.

[0099] Fig.33 This is the extracted ion chromatogram of phosphoenolpyruvate standard.

[0100] Fig.34 This is the extracted ion chromatogram of citric acid standard.

[0101] Fig.35 is the total ion current of the sample. DETAILED DESCRIPTION

[0102] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0103] The instrument manufacturers used in the following examples are:

[0104] Mass spectrometer: Q Exactive, Thermo Fisher Scientific, Waltham, MA, USA

[0105] Example 1

[0106] This embodiment provides a method for detecting energy metabolites based on UPLC-MS / MS

[0107] (1) Sample pretreatment

[0108] After the rice sample was fully ground with liquid nitrogen, pre-cooled deionized water was added to the sample concentration of 0.5g / mL. 800μL of a mixed solution of methanol and acetonitrile with a volume ratio of 1:1 was added to 50μL of the sample, and ultrasonic extraction was performed at 4℃ for 30min. Centrifugation was performed at 12000rpm for 10min at 4℃, and the supernatant was taken to a new centrifuge tube. 1mL of extracting solution was added to the precipitate again, which consisted of methanol, acetonitrile and water with a volume ratio of 2:2:1. Vortex mixing was performed, and extraction was performed at 0℃ for 30min. The supernatant was taken and mixed again by centrifugation, and the two supernatants were incubated at -20℃ for 1h. Centrifugation was performed at 12000rpm for 20min at 4℃, and the supernatant was taken and vacuum dried to obtain the sample extract.

[0109] (2) Preparation of standard solution

[0110] Weigh the standard powders respectively, add 25% acetonitrile aqueous solution to dissolve and prepare the corresponding standard single standard stock solution, take the same volume of each standard single standard stock solution and mix them evenly, and dilute 100 μg / mL of the standard mixed standard stock solution with 40% acetonitrile solution.

[0111] Use 30% acetonitrile aqueous solution to gradiently dilute the prepared standard solution to become standards of 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL, 5000 ng / mL, 10000 ng / mL, 15000 ng / mL, and 20000 ng / mL.

[0112] (3) Mass spectrometry

[0113] The sample extract was re-dissolved in 30% acetonitrile, and the sample concentration after re-dissolution was 0.5 g / mL. The sample was centrifuged at 12000 rpm for 20 min at 4°C, and the supernatant was taken as the sample extract. The sample extract and the standard solution were respectively subjected to mass spectrometry detection.

[0114] Liquid chromatography conditions:

[0115] The chromatographic system used Vanquish ultra-high performance liquid chromatography from Thermo. The chromatographic column was Waters BEHAmide (100×2.1mm, 1.8μm), mobile phase: phase A was 10mM ammonium acetate aqueous solution, pH 9.5, phase B was HPLC grade 99.9% pure acetonitrile; flow rate 0.3mL / min; column temperature 40°C; injection volume 2μL; elution gradient: 0-9min, phase B decreased from 90% to 40%; 9-10min, phase B changed from 40% to 90%; 10.1-12min, phase B was maintained at 90% for column balance. The sample was placed in an automatic sampler at 4°C during the entire analysis process.

[0116] Mass spectrometry conditions:

[0117] The mass spectrometry system used the Q Exactive orbital trap high-resolution mass spectrometer from Thermo. It was equipped with an electrospray ionization (ESI) source, and the scanning mode was full scan (Full MS-DDMS2) mode under negative ion conditions. The scanning conditions were as follows: sheath gas 40arb; auxiliary gas 10arb; ion spray voltage -2800V; temperature 350℃; ion transfer tube temperature 320℃. The primary scan range (scan m / z range): 70-1000Da, the secondary scan range: 200-2000, the primary resolution was 70000, and the secondary resolution was 17500.

[0118] Example 2

[0119] This embodiment provides a method for detecting energy metabolites based on UPLC-MS / MS

[0120] (1) Sample pretreatment

[0121] After the rice sample was fully ground with liquid nitrogen, pre-cooled deionized water was added to the sample concentration of 0.5g / mL. 600μL of a mixed solution of methanol and acetonitrile with a volume ratio of 1:0.5 was added to 100μL of the sample, and ultrasonic extraction was performed at 0℃ for 30min. Centrifugation was performed at 10000rpm for 5min at 2℃, and the supernatant was taken to a new centrifuge tube. 0.5mL of extracting solution was added to the precipitate again, which consisted of methanol, acetonitrile and water with a volume ratio of 1:3:1. Vortex mixing was performed, and extraction was performed at 4℃ for 20min. The supernatant was taken again by centrifugation and the two supernatants were mixed and incubated at -25℃ for 0.5h. Centrifugation was performed at 14000rpm for 15min at 2℃, and the supernatant was taken and vacuum dried to obtain the sample extract.

[0122] (2) Preparation of standard solution

[0123] Weigh the standard powders respectively, add 40% acetonitrile aqueous solution to dissolve and prepare the corresponding standard single standard stock solution, take the same volume of each standard single standard stock solution and mix them evenly, and dilute 100 μg / mL of the standard mixed standard stock solution with 35% acetonitrile solution.

[0124] Use 30% acetonitrile aqueous solution to gradient dilute the prepared standard solution to 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL, 5000 ng / mL, 10000 ng / mL, 15000 ng / mL, and 20000 ng / mL.

[0125] (3) Mass spectrometry

[0126] The sample extract was re-dissolved in 30% acetonitrile, and the sample concentration after re-dissolution was 0.5 g / mL. The sample was centrifuged at 14000 rpm for 15 min at 2°C, and the supernatant was taken as the sample extract. The sample extract and the standard solution were respectively subjected to mass spectrometry detection.

[0127] Liquid chromatography conditions:

[0128] The chromatographic system used Vanquish ultra-high performance liquid chromatography from Thermo. The chromatographic column was Waters BEHAmide (100×2.1mm, 1.8μm), mobile phase: phase A was 15mM ammonium acetate aqueous solution, pH 9, phase B was HPLC grade 99.9% pure acetonitrile; flow rate 0.5mL / min; column temperature 35°C; injection volume 3μL; elution gradient: 0-9min, phase B decreased from 90% to 40%; 9-10min, phase B changed from 40% to 90%; 10.1-12min, phase B was maintained at 90% for column balance. The sample was placed in an automatic sampler at 4°C during the entire analysis process.

[0129] Mass spectrometry conditions:

[0130] The mass spectrometry system used the Q Exactive orbital trap high-resolution mass spectrometer from Thermo. It was equipped with an electrospray ionization (ESI) source, and the scanning mode was full scan (Full MS-DDMS2) mode under negative ion conditions. The scanning conditions were as follows: sheath gas 35arb; auxiliary gas 12arb; ion spray voltage -2600V; temperature 350℃; ion transfer tube temperature 350℃. The primary scan range (scan m / z range): 70-1000Da, the secondary scan range: 200-2000, the primary resolution was 70000, and the secondary resolution was 17500.

[0131] Example 3

[0132] This embodiment provides a method for detecting energy metabolites based on UPLC-MS / MS

[0133] (1) Sample pretreatment

[0134] After the rice sample was fully ground with liquid nitrogen, pre-cooled deionized water was added to the sample concentration of 0.5g / mL. 1000μL of a mixed solution of methanol and acetonitrile with a volume ratio of 1:2 was added to 150μL of the sample, and ultrasonic extraction was performed at 2℃ for 30min. Centrifugation was performed at 14000rpm for 15min at 0℃, and the supernatant was taken to a new centrifuge tube. 1.5mL of extracting solution was added to the precipitate again, which consisted of methanol, acetonitrile and water with a volume ratio of 3:1:1. Vortex mixing was performed, and extraction was performed at 0℃ for 40min. The supernatant was taken again by centrifugation and the two supernatants were mixed and incubated at -15℃ for 2h. Centrifugation was performed at 10000rpm for 30min at 0℃, and the supernatant was taken and vacuum dried to obtain the sample extract.

[0135] (2) Preparation of standard solution

[0136] Weigh the standard powders respectively, add 30% acetonitrile aqueous solution to dissolve and prepare the corresponding standard single standard stock solution, take the same volume of each standard single standard stock solution and mix them evenly, and dilute 100 μg / mL of the standard mixed standard stock solution with 25% acetonitrile solution.

[0137] Use 40% acetonitrile aqueous solution to gradiently dilute the standard solution prepared in step 1 to make standards of 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL, 5000 ng / mL, 10000 ng / mL, 15000 ng / mL, and 20000 ng / mL.

[0138] (3) Mass spectrometry

[0139] The sample extract was re-dissolved in 30% acetonitrile, and the sample concentration after re-dissolution was 0.5 g / mL. The sample was centrifuged at 10000 rpm for 30 min at 0°C, and the supernatant was taken as the sample extract. The sample extract and the standard solution were respectively subjected to mass spectrometry detection.

[0140] Liquid chromatography conditions:

[0141] The chromatographic system used Vanquish ultra-high performance liquid chromatography from Thermo. The chromatographic column was Waters BEHAmide (100×2.1mm, 1.8μm), mobile phase: phase A was 5mM ammonium acetate aqueous solution, pH 10, phase B was HPLC grade 99.9% pure acetonitrile; flow rate 0.1mL / min; column temperature 45°C; injection volume 1μL; elution gradient: 0-9min, phase B decreased from 90% to 40%; 9-10min, phase B changed from 40% to 90%; 10.1-12min, phase B was maintained at 90% for column balance. The sample was placed in an automatic sampler at 4°C during the entire analysis process.

[0142] Mass spectrometry conditions:

[0143] The mass spectrometry system used a Thermo Q Exactive orbital trap high-resolution mass spectrometer. It was equipped with an electrospray ionization (ESI) source, and the scanning mode was full scan (Full MS-DDMS2) mode under negative ion conditions. The scanning conditions were as follows: sheath gas 45arb; auxiliary gas 8arb; ion spray voltage -3000V; temperature 350℃; ion transfer tube temperature 300℃. The primary scan range (scan m / z range): 70-1000Da, the secondary scan range: 200-2000, the primary resolution was 70000, and the secondary resolution was 17500.

[0144] Example 4

[0145] This example provides a method for detecting energy metabolites based on UPLC-MSMS, which is different from Example 1 only in that the elution procedure is:

[0146] 0min-6.5min, mobile phase B changed from 90% to 50% at a constant speed;

[0147] 6.5min-8min, mobile phase B remained unchanged at 50%;

[0148] From 8 to 12 minutes, the mobile phase B was changed from 50% to 90% at a constant speed.

[0149] The rest are consistent with Application Example 1.

[0150] Example 5

[0151] This example provides a method for detecting energy metabolites based on UPLC-MSMS, which is different from Example 1 only in that the elution procedure is:

[0152] 0min-1min, mobile phase B remained unchanged at 90%;

[0153] 1min-5min, mobile phase B was changed from 90% to 40% at a constant speed;

[0154] 5min-8min, mobile phase B remained unchanged at 40%;

[0155] From 8 to 11 minutes, mobile phase B was changed from 40% to 90% at a constant speed;

[0156] 11.1min-12min, mobile phase B remained unchanged at 90%.

[0157] The rest are consistent with Application Example 1.

[0158] Example 6

[0159] This embodiment provides a method for detecting energy metabolites based on UPLC-MSMS, which is different from Example 1 only in that the primary resolution is 60,000 and the secondary resolution is 15,000, and the rest is consistent with Application Example 1.

[0160] Example 7

[0161] This embodiment provides a method for detecting energy metabolites based on UPLC-MSMS, which is different from Example 1 only in that no second extraction is performed, and the rest is consistent with Application Example 1.

[0162] Example 8

[0163] This embodiment provides a method for detecting energy metabolites based on UPLC-MS / MS. The only difference from Example 1 is that the reagent not subjected to the second extraction is a mixed solution of methanol and acetonitrile in a volume ratio of 1:1, and the rest is consistent with Application Example 1.

[0164] Example 9

[0165] This embodiment provides a method for detecting energy metabolites based on UPLC-MSMS. The only difference from Example 1 is that the sample detected is mouse serum, and the rest is consistent with Application Example 1.

[0166] Comparative Example 1

[0167] This comparative example provides a method for detecting energy metabolites based on UPLC-MS / MS, which is different from Example 1 only in that the pH value of the ultra-high performance liquid chromatography is 8, and the rest is consistent with Application Example 1.

[0168] Comparative Example 2

[0169] This comparative example provides a method for detecting energy metabolites based on UPLC-MSMS, which is different from Example 1 only in that the pH value of the ultra-high performance liquid chromatography is 11, and the rest is consistent with Application Example 1.

[0170] Comparative Example 3

[0171] This comparative example provides a method for detecting energy metabolites based on UPLC-MSMS, which is different from Example 1 only in that mobile phase A is methanol, and the rest is consistent with Application Example 1.

[0172] Comparative Example 4

[0173] This comparative example provides a method for detecting energy metabolites based on UPLC-MSMS, which is different from Example 1 only in that mobile phase B is methanol, and the rest is consistent with Application Example 1.

[0174] Test Example 1

[0175] This test case analyzes the mass spectrometry results.

[0176] The detection results of this embodiment are further analyzed below to verify the feasibility of the method provided by the patent of the present invention: the peak area obtained from the primary mass spectrum data is quantitative, and the secondary mass spectrum fragment data is qualitative. The high-efficiency mass spectrometer used in the present invention performs baseline filtering, peak identification, peak matching, retention time correction, and peak alignment on the original spectrum obtained by analysis through the peak software, and a data matrix containing retention time, mass-to-charge ratio and peak intensity (peak area) can be obtained for the calculation of subsequent detection results.

[0177] The external standard method was used for quantification, with the concentration of 34 energy metabolism standards as the horizontal axis and the peak area value of the standard as the vertical axis to establish a standard curve. The results are shown in Table 1 below. The specific standard extracted ion current is shown in Figure 1-34 shown. Fig.35 This is the total ion current diagram of the sample in Example 1.

[0178] Table 1

[0179]

[0180]

[0181] The 34 energy metabolites showed good linear relationships within their respective concentration linear ranges. 2 ≥0.99, fully meeting the quantitative requirements. Substituting the peak areas of 34 energy metabolites in rice leaves into the standard curve, the absolute contents of 34 important energy metabolites in rice leaves can be accurately calculated with high throughput and high sensitivity. The calculation formula is: Energy metabolite content in rice sample (ng / mg) = (C×V) / M, where C is the sample concentration, in ng / mL; V is the sample extract volume, in mL; M is the sample weighing mass, in mg.

[0182] The contents of 34 energy metabolites in Examples 1-3 are shown in Table 2:

[0183] Table 2

[0184]

[0185]

[0186] In Example 1, all 34 energy metabolites were detected, with the content ranging from 34.16 to 12252.24, indicating that this method is suitable for the one-time determination of 34 energy metabolites in a sample, with a detection limit at the ng level. Compared with Example 1, the detection results of Example 2 and Example 3 showed slight differences in the contents of the 34 energy metabolites. This insignificant difference may be due to the difference in the extraction method in the pretreatment. Fructose 1,6-diphosphate was not detected in Example 2, which may be due to the low content of the compound in the sample. NADPH was not detected in Example 3 because the NADPH substance itself is unstable and easily oxidized. The above results further illustrate that this method is stable and reliable.

[0187] The contents of 34 energy metabolites in Examples 4-5 are shown in Table 3:

[0188] Table 3

[0189]

[0190]

[0191] Compared with Example 1, in Examples 4 and 5, the retention time of 34 energy metabolism target compounds changed, the peaks became more compact, and the content tended to decrease, indicating that adjusting the elution gradient of the mobile phase had a greater impact on the detection results. In Example 4, citric acid, isocitric acid and NADPH were not detected, and in Example 5, AMP, trans-aconitic acid, fructose 1,6-diphosphate and PEP were not detected. The above results all indicate that the elution gradients in Examples 4 and 5 are not more suitable than that in Example 1.

[0192] The contents of 34 energy metabolites in Examples 6-9 are shown in Table 4:

[0193] Table 4

[0194]

[0195]

[0196] Compared with the results of Example 1, there is no obvious difference in Example 6, which shows that changing the primary and secondary resolution of the mass spectrometry has little effect on the method. Compared with Example 1, the contents of the 34 target compounds in Example 7 have a downward trend as a whole, indicating that after the secondary extraction is not performed, the overall detection rate of the compound will be reduced, and the metabolites with low content in the sample itself will not be detected, such as AMP, 4-phosphoerythrose, fructose 1,6 diphosphate and NADPH, indicating that the sample pretreatment method will directly affect the detection rate of the target compound in the method. In order to ensure the sufficient extraction rate of the metabolites, secondary extraction is required. Compared with Example 1, the contents of some compounds in the 34 target compounds are reduced, and the contents of some compounds are increased, but there is no significant difference in the overall trend, among which fructose 1,6 diphosphate and NADPH are not detected, indicating that adjusting the ratio of methanol and acetonitrile in the extraction reagent will increase the extraction rate of some compounds, and at the same time, it will also reduce the extraction rate of some compounds. The overall change is not significant, so it is enough to find a suitable ratio to balance. The same method was used to determine the detection of 34 target compounds in serum samples. The results showed that compared with Example 1, the peak time of the 34 compounds did not change, and 33 of them were detected with a content range of 23.10-1095028.77 ng / mL. NADPH may not be detected due to its own instability, indicating that this method is also applicable to animal samples and liquid samples, and is stable and reliable.

[0197] The contents of 34 energy metabolites in Comparative Example 1 are shown in Table 5:

[0198] Table 5

[0199]

[0200]

[0201] Compared with Example 1, the peak times of 34 compounds in Comparative Example 1 shifted backward as a whole, and 10 metabolites were not detected, indicating that the condition of pH=8 is not suitable for the detection of some compounds.

[0202] The contents of 34 energy metabolites in Comparative Example 2 are shown in Table 6:

[0203] Table 6

[0204]

[0205]

[0206] Compared with Example 1, the peak times of 34 compounds in Comparative Example 2 drifted forward as a whole, and 10 metabolites were not detected, indicating that the condition of PH=11 is not suitable for the detection of some compounds.

[0207] The contents of 34 energy metabolites in Comparative Example 3 are shown in Table 7:

[0208] Table 7

[0209]

[0210]

[0211] Compared with Example 1, the elution times of the 34 compounds in Comparative Example 3 were more concentrated, and some metabolites were not detected, indicating that adjusting the mobile phase A from water to methanol was not applicable.

[0212] The contents of 34 energy metabolites in Comparative Example 4 are shown in Table 8:

[0213] Table 8

[0214]

[0215]

[0216] The detection results of Comparative Example 4 are similar to those of Comparative Example 3, indicating that adjusting the mobile phase B from acetonitrile to methanol is not applicable.

[0217] In summary, the energy metabolism analysis method based on UPLC-MSMS in the present invention extracts tissue samples and separates them by liquid chromatography, and then conducts qualitative and quantitative detection of metabolites in the energy metabolism pathway. It has the characteristics of simple pretreatment, high sensitivity, strong selectivity, low detection limit and good reproducibility. It has high throughput and can detect 34 metabolites at one time.

[0218] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for detecting energy metabolites based on UPLC-MSMS, characterized in that: The method comprises the following steps: after sample extraction, using ultra-high performance liquid chromatography coupled with mass spectrometry to detect the sample extract and the standard solution; The mobile phases used in the ultra-high performance liquid chromatography are: mobile phase A is a 5-15 mM ammonium acetate aqueous solution with a pH value of 9-10; and mobile phase B is 95-100% acetonitrile.

2. The method according to claim 1, characterized in that The energy metabolites include: any one or a combination of at least two of pyruvate, lactate, glucose, cyclic adenosine monophosphate, flavin mononucleotide, flavin adenine dinucleotide, adenosine monophosphate, acetyl coenzyme A, nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide, α-ketoglutarate, succinate, 3-phosphoglycerate, dihydroxyacetone phosphate, ribose-5-phosphate, malic acid, ribulose-5-phosphate, adenosine diphosphate, erythrose 4-phosphate, fumaric acid, sedoheptulose-7-phosphate, trans-aconitic acid, uridine diphosphate, guanylic acid, thiamine pyrophosphate, fructose 1,6 diphosphate, glucose-6-phosphate, 5'-adenosine triphosphate, phosphoenolpyruvate, citric acid, reduced nicotinamide adenine dinucleotide phosphate, guanosine-5'-diphosphate, nicotinamide adenine dinucleotide phosphate or isocitrate.

3. The method according to claim 1 or 2, characterized in that: The sample extraction step comprises: performing a first extraction after homogenizing the sample, performing a first centrifugation to obtain a supernatant and a precipitate, performing a second extraction on the precipitate and performing a second centrifugation, collecting the supernatant after the two centrifugations for incubation, performing a third centrifugation, and obtaining a sample extract after drying; Preferably, the solvent of the homogenate includes any one or a combination of at least two of deionized water, methanol, acetonitrile or ethanol; Preferably, the mass volume ratio of the sample to the solvent is 1:(2-100) g / mL; Preferably, the first extraction reagent used in the first extraction includes any one or a combination of at least two of methanol, acetonitrile, ethanol, propylene glycol or water; Preferably, the first extraction reagent is a combination of methanol and acetonitrile; Preferably, the volume ratio of methanol to acetonitrile in the first extraction reagent is 1:(0.5-2); Preferably, the amount of the first extraction reagent added is 100 to 1000 μL per 50 to 150 μL of homogenate; Preferably, the first extraction time is 20 to 40 minutes; Preferably, the temperature of the first extraction is 0-4°C; Preferably, the first centrifugation speed is 10000-14000 rpm, and the time is 5-15 min; Preferably, the temperature of the first centrifugation is 0-4°C.

4. The method according to claim 3, characterized in that The second extraction reagent used in the second extraction includes any one of methanol, acetonitrile, ethanol, propylene glycol or water, or a combination of at least two thereof; Preferably, the second extraction reagent is a combination of water, methanol and acetonitrile; Preferably, the volume ratio of reagent water, methanol and acetonitrile in the second extraction is 1:(1-3):(1-3); Preferably, the amount of the second extraction reagent added is 0.5 to 1.5 mL per 50 to 150 μL of homogenate; Preferably, the second extraction time is 20 to 40 minutes; Preferably, the temperature of the second extraction is 0-4°C; Preferably, the second centrifugation speed is 10000-14000 rpm, and the time is 5-15 min; Preferably, the temperature of the second centrifugation is 0-4°C; Preferably, the incubation temperature is -15 to -25°C; Preferably, the incubation time is 0.5 to 2 hours; Preferably, the third centrifugation speed is 10000-14000 rpm, and the time is 15-30 min; Preferably, the drying comprises vacuum drying.

5. The method according to any one of claims 1 to 4, characterized in that The steps of preparing the standard solution include: dissolving the standard in acetonitrile; Preferably, the volume fraction of acetonitrile is 20 to 40%; Preferably, the concentration range of the standard solution is 1 to 20000 ng / mL.

6. The method according to any one of claims 1 to 5, characterized in that Before the detection, the dried sample extract is re-dissolved with acetonitrile, and the supernatant is collected after centrifugation; Preferably, the volume fraction of acetonitrile is 20 to 40%; Preferably, the centrifugal speed is 10000-14000 rpm, and the time is 15-30 min; Preferably, the centrifugation temperature is 0-4°C.

7. The method according to any one of claims 1 to 6, characterized in that The stationary phase of the chromatographic column used in the ultra-high performance liquid chromatography comprises a trifunctional bonded amide stationary phase; Preferably, the column temperature of the chromatographic column is 35-45°C; Preferably, the injection volume of the ultra-high performance liquid chromatography is 1 to 3 μL.

8. The method according to any one of claims 1 to 7, characterized in that The elution procedure of the gradient elution of the ultra-high performance liquid chromatography is as follows: From 0min to 9min, mobile phase B was changed from 90% to 40% at a constant speed; At 9-10 minutes, mobile phase B was changed from 40% to 90% at a constant speed; 10.1min-12min, mobile phase B is 90%; Preferably, the flow rate of the mobile phase is 0.1 to 0.5 mL / min.

9. The method according to any one of claims 1 to 8, characterized in that The mass spectrometer used in the mass spectrometer includes a quadrupole-electrostatic field orbital trap high-resolution mass spectrometer; Preferably, the mass spectrometer scanning mode includes full scan under negative ion conditions; Preferably, the sheath gas of the mass spectrometer is 35 to 45 arb; Preferably, the auxiliary gas of the mass spectrometer is 8 to 12 arb; Preferably, the ion spray voltage of the mass spectrometer is -2.6kv to -3kv; Preferably, the temperature of the ion transfer tube of the mass spectrometer is 300-350°C; Preferably, the primary scanning range of the mass spectrometer is 70-1000Da, and the secondary scanning range is 200-2000; Preferably, the primary resolution of the mass spectrometer is 70,000 and the secondary resolution is 17,500.

10. Use of the method for detecting energy metabolites based on UPLC-MSMS according to any one of claims 1 to 9 in the detection of energy metabolites.

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