A method for simultaneous detection of multiple choline metabolites

Abstract

The application discloses a method for simultaneously detecting multiple choline metabolites, wherein the choline metabolites include TMA and one or more of GPC, CDP-Choline and PC, and specifically comprises the following steps: liquid chromatography elution separation: after sample pretreatment, gradient elution is used to elute the target objects in the sample, wherein when TMA is eluted, the content of ammonium salt in the mobile phase system is less than 1.0*10 ‑6 mol / min; when GPC, CDP-Choline or PC is eluted, the content of ammonium salt in the mobile phase system is greater than or equal to 1.0*10 ‑6 mol / min, and the target objects are eluted in sequence; and mass spectrometry is used to detect the eluted target objects, wherein the TMA detection recovery rate reaches 80%, the GPC, CDP-Choline and PC detection recovery rates are all above 97%, and the detection method has good performance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of choline metabolite detection, and more particularly relates to a method for simultaneously detecting multiple choline metabolites. BACKGROUND

[0002] Choline is a water-soluble nutrient that can be obtained from daily diet (such as fish, meat and eggs) and has been regarded as an essential dietary nutrient by the Institute of Medicine Food and Nutrition Board since 1998. Existing studies have shown that choline deficiency can cause developmental disorders, fetal brain damage, fatty liver and muscle damage, etc., and its metabolites are closely related to a variety of diseases. Therefore, choline and its metabolites have attracted widespread attention. Choline metabolism can be divided into four main pathways: acetylcholine, trimethylamine (TMA), betaine and phospholipid, which are as follows:

[0003] (1) Acetylcholine pathway: choline is used as a precursor for the synthesis of neurotransmitter acetylcholine by choline acyltransferase in the cytosol of presynaptic cholinergic neurons. Acetylcholine is then packaged into vesicles and released into the synaptic cleft, where it binds to receptors on postsynaptic neurons in the central and peripheral nervous systems. Acetylcholine is an important neurotransmitter in the central and peripheral nervous systems, and its main function is to maintain the alertness of consciousness and play an important role in learning and memory.

[0004] (2) Trimethylamine pathway: in the intestinal tract, choline is metabolized by intestinal flora into trimethylamine (TMA), which is transported to the liver and further oxidized to trimethylamine oxide (TMAO). Existing studies have shown that the concentrations of TMAO and TMA in biological samples are positively correlated with a variety of pathological conditions such as cardiovascular disease, diabetes, cancer, autoimmune disease, polycystic ovary syndrome and autism. A large amount of animal and clinical data has shown that the higher the TMAO content, the greater the risk of cardiovascular disease. However, the current gas chromatography-mass spectrometry method for detecting TMAO, although highly sensitive, is complicated to operate and time-consuming, and the sample derivatization process can easily cause TMAO to degrade, affecting the accuracy of the measurement results.

[0005] (3) Betaine pathway: choline is mainly oxidized to betaine in the liver and kidney by choline dehydrogenase and betaine aldehyde dehydrogenase through a two-step process. Betaine is a methyl donor that participates in the remethylation of homocysteine to methionine through betaine-homocysteine S-methyltransferase (BHMT), producing dimethylglycine (DMG), which is further demethylated to produce sarcosine, which is then metabolized to glycine, resulting in the transfer of carbon units to the folate pool.

[0006] (4) Phospholipid pathway: Choline is phosphorylated to phosphorylcholine (PC), which is further converted to cytidine diphosphate-choline (CDP-Choline) with cytidine triphosphate, and then to phosphatidylcholine and phosphatidylethanolamine, which are involved in the composition and function of cell membranes. Among them, PC is the main form of choline metabolites, followed by glycerophosphocholine (GPC), which is involved in the synthesis of phospholipids in vivo. Among them, CDP-Choline is a nucleoside derivative involved in the biosynthesis of phosphatidylcholine, membrane biosynthesis and myelin formation, lipid metabolism, cell division or signal molecules.

[0007] At present, choline metabolites are usually quantitatively detected by liquid chromatography tandem mass spectrometry (LC-MS / MS), but the existing methods for detecting choline metabolites generally have incomplete detection of types, especially cannot simultaneously detect TMA and GPC, CDP-Choline, PC, and the more types of choline metabolites detected simultaneously, the more beneficial for the research of related diseases in clinic. Therefore, it is of great significance to study a method capable of simultaneously detecting multiple choline metabolites, especially capable of simultaneously detecting TMA and PC, CDP-Choline, GPC. SUMMARY

[0008] In view of the above defects or improvement needs of the prior art, the present application provides a method for simultaneously detecting multiple choline metabolites, which aims to find that the ammonium salt in the mobile phase has a significant different influence on the detection results of TMA and GPC, CDP-Choline, PC when the sample is eluted, wherein the mobile phase does not contain ammonium salt or the content of ammonium salt is low (the content of ammonium salt is less than 1.0 x 10 -6 mol / min), the target TMA has good response, while the content of ammonium salt in the mobile phase is high, the target GPC, CDP-Choline, PC has good response, the present application first adopts gradient elution mode with variable flow rate of the mobile phase, by adjusting the concentration of ammonium salt in the mobile phase and the flow rate, the content of ammonium salt in the mobile phase system is less than 1.0 x 10 -6 mol / min when TMA is eluted; when GPC, CDP-Choline, or PC is eluted, the content of ammonium salt in the mobile phase system is greater than or equal to 1.0 x 10 -6 mol / min, which can realize simultaneous detection of one or more of TMA, GPC, CDP-Choline, and PC, and the sample extraction does not require derivatization treatment; thereby solving the technical problems that the existing liquid chromatography tandem mass spectrometry method for detecting choline metabolites cannot accurately detect TMA and GPC, CDP-Choline, PC simultaneously and the sample needs derivatization treatment.

[0009] To achieve the above object, according to one aspect of the present application, a method for simultaneously detecting multiple choline metabolites is provided, wherein the choline metabolites include TMA and one or more of GPC, CDP-Choline and PC, and the method is characterized by the following steps:

[0010] Liquid chromatography elution separation: after sample pretreatment, the target objects in the sample are eluted by gradient elution, wherein when TMA is eluted, the ammonium salt content in the mobile phase system is less than 1.0*10 -6 mol / min; when GPC, CDP-Choline or PC is eluted, the ammonium salt content in the mobile phase system is greater than or equal to 1.0*10 -6 mol / min, and the target objects are eluted in turn;

[0011] Mass spectrometry is used to detect the eluted target objects.

[0012] Preferably, in the method for simultaneously detecting multiple choline metabolites, when GPC, CDP-Choline or PC is eluted, the ammonium salt content in the mobile phase system is (1.0-3.0)*10 -6 mol / min.

[0013] Preferably, in the method for simultaneously detecting multiple choline metabolites, the ammonium salt concentration and / or flow rate of the mobile phase are adjusted so that when TMA or GPC, CDP-Choline or PC is eluted, the corresponding ammonium salt content in the mobile phase system is met.

[0014] Preferably, in the method for simultaneously detecting multiple choline metabolites, the ammonium salt concentration of the mobile phase is ≤10 mM, and the flow rate is 0.1-0.6 mL / min.

[0015] Preferably, in the method for simultaneously detecting multiple choline metabolites, the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is 50%-95% acetonitrile aqueous solution, and mobile phase B is 50%-95% acetonitrile aqueous solution.

[0016] Preferably, in the method for simultaneously detecting multiple choline metabolites, the ammonium salt concentration of the mobile phase is 2-3 mM, and the gradient elution program for eluting TMA, Choline, Creatinine, TMAO, Betaine, DMG, Sarcosine, GPC, CDP-Choline and PC is as follows: 0-3 min, 100% B-90% B, 0.2-0.3 mL / min; 3-5 min, 90% B-50% B, 0.4-0.5 mL / min; 5-5.5 min, 50% B-0% B, 0.5-0.55 mL / min; 5.5-8 min, 0% B, 0.55 mL / min.

[0017] Preferably, the method for simultaneously detecting multiple choline metabolites, the ammonium salt comprises ammonium formate or ammonium acetate.

[0018] Preferably, the method for simultaneously detecting multiple choline metabolites, the sample pretreatment adopts a methanol aqueous solution as a solvent for direct extraction.

[0019] Preferably, the method for simultaneously detecting multiple choline metabolites, the sample is blood, and the pretreatment is as follows:

[0020] A serum sample is taken, mixed with 90% methanol aqueous solution at a volume ratio of 1:50, an internal standard solution is added, centrifuged and filtered to obtain a sample to be eluted.

[0021] Preferably, the method for simultaneously detecting multiple choline metabolites, the sample is tissue, and the pretreatment is as follows:

[0022] A tissue sample is taken, homogenized and extracted with 90% methanol aqueous solution, the ratio of the mass of the tissue sample to the volume of the methanol aqueous solution is 5:1, an internal standard solution is added, centrifuged and filtered to obtain a sample to be eluted.

[0023] Overall, compared with the prior art, the above technical solutions conceived by the present application can achieve the following beneficial effects:

[0024] In the prior art, the flow rate of the mobile phase system is constant, while the method for simultaneously detecting choline metabolites provided by the present application adopts a gradient elution method with variable flow rate to elute the sample for the first time. When eluting trimethylamine TMA, the content of ammonium salt in the mobile phase system is less than 1.0 x 10 -6 mol / min; when eluting GPC, CDP-Choline, or PC, the content of ammonium salt in the mobile phase system is greater than or equal to 1.0 x 10 -6 mol / min. Compared with the prior art of liquid chromatography tandem mass spectrometry for detecting choline metabolites, the detection method provided by the present application can simultaneously and accurately detect TMA and GPC, CDP-Choline, and PC, wherein the recovery rate of detecting TMA reaches 80%, and the recovery rate of detecting GPC, CDP-Choline, and PC is above 97%, and the method has high detection accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a schematic diagram of a choline metabolic pathway;

[0026] Figure 2 is a chromatogram of 11 choline metabolites detected by the method of Example 1;

[0027] Figure 3 is a chromatogram of multiple choline metabolites in liver detected by the method of Example 2;

[0028] Figure 4 is the mixed standard chromatogram of 11 choline metabolites in Comparative Example 1;

[0029] Figure 5 is the mixed standard chromatogram of 11 choline metabolites in Comparative Example 2;

[0030] Figure 6 is the mixed standard chromatogram of 11 choline metabolites in Comparative Example 3. DETAILED DESCRIPTION

[0031] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0032] The choline metabolic pathway is shown in Figure 1 which includes more than ten metabolites. When a plurality of choline metabolic targets are simultaneously detected by liquid chromatography tandem mass spectrometry, one detection method is often difficult to accommodate all targets, and therefore a suitable detection method is usually selected according to the characteristics of the target. In the experimental process of detecting choline metabolites by liquid chromatography tandem mass spectrometry, it is accidentally found that when no ammonium salt is added to the mobile phase, TMA can be eluted and has a good response, and when a high concentration of ammonium salt is added to the mobile phase, PC, CDP-Choline and GPC have a good peak shape and response. However, the ammonium salt concentration in the existing detection method is a constant value, so we speculate that the existing liquid chromatography tandem mass spectrometry method is difficult to simultaneously detect TMA and GPC, CDP-Choline, PC, which may be due to the fact that the existing method cannot meet the requirement of the ammonium salt concentration in the mobile phase for the quantitative detection of TMA and GPC, CDP-Choline, PC.

[0033] In order to be able to simultaneously detect TMA and GPC, CDP-Choline, PC, the present application further studies the influence of ammonium salt in the mobile phase on the peak shape and response of TMA and GPC, CDP-Choline, PC. It is found that when the ammonium salt content in the mobile phase system is less than 1.0 x 10 -6 mol / min, TMA is eluted and has a good response, and when the ammonium salt content in the mobile phase system is greater than or equal to 1.0 x 10 -6 mol / min, GPC, CDP-Choline, PC are eluted and have a good peak shape and response. Preferably, when TMA is eluted, the ammonium salt content in the mobile phase system is controlled in the range of (0-1) x 10 -6mol / min; the ammonium salt content in the elution GPC, CDP-Choline, and PC is greater than or equal to 1.0*10 -6 mol / min, and the results show that the target object is detected after elution, and TMA and GPC, CDP-Choline, and PC have good peak shape and response.

[0034] Based on this, the application provides a method for simultaneously detecting multiple choline metabolites, which adopts a gradient elution method with variable flow rate of the mobile phase to regulate the ammonium salt content in the mobile phase system to present dynamic changes, wherein when eluting TMA, the ammonium salt content in the mobile phase system is less than 1.0*10 -6 mol / min; when eluting GPC, CDP-Choline, and PC, the ammonium salt content in the mobile phase system is greater than or equal to 1.0*10 -6 mol / min.

[0035] In some embodiments, TMA is eluted with a low flow rate first, at this time, the ammonium salt content in the mobile phase system is low, and then the flow rate is increased, so that GPC, CDP-Choline, and PC are eluted when the ammonium salt content is high, and the specific steps include the following steps:

[0036] (1) Sample pretreatment; a serum sample is taken, 90% methanol aqueous solution is added according to a volume ratio of 1:50, an internal standard solution is added, centrifugal filtration is performed, and a sample to be eluted is obtained; for example, in some embodiments, 10 μL of a serum sample is taken, an internal standard solution is added, 500 μL of 90% methanol aqueous solution is added, vortex mixing is performed, high-speed centrifugation is performed, the supernatant is taken and filtered through a 0.22 μm filter membrane, and a sample to be detected is obtained.

[0037] Or a tissue sample is taken, 90% methanol aqueous solution is added for homogenate extraction, the mass of the tissue sample is 5:1 of the volume of the methanol aqueous solution, an internal standard solution is added, centrifugal filtration is performed, and a sample to be eluted is obtained; for example, in some embodiments, 5 mg of a tissue sample is taken, an internal standard is added, 1 mL of 90% methanol aqueous solution is added for homogenate extraction, centrifugation is performed, the supernatant is taken and filtered through a 0.22 μm filter membrane, and a sample to be detected is obtained; the tissue sample is preferably a liver.

[0038] (2) Detection: 50%-95% acetonitrile aqueous solution (containing ammonium salt, and the concentration is less than or equal to 10 mM) is used as the mobile phase A, and 50%-95% acetonitrile aqueous solution (containing ammonium salt, and the concentration is less than or equal to 10 mM) is used as the mobile phase B;

[0039] The detection of choline metabolites adopts gradient elution with variable flow rate of the mobile phase, so that when eluting TMA, the ammonium salt content in the mobile phase system is (0-1)*10 -6 mol / min; when eluting GPC, CDP-Choline, and PC, the ammonium salt content in the mobile phase system is (1-3)*10 -6mol / min.

[0040] In some embodiments, when detecting TMA, Choline, Creatinine, TMAO, Betaine, DMG, Sarcosine, GPC, CDP-Choline and PC, the concentration of ammonium salt in the mobile phase is 2-3 mM, and the gradient elution program is: 0-3 min, 100% B-90% B, 0.2-0.3 mL / min; 3-5 min, 90% B-50% B, 0.4-0.5 mL / min; 5-5.5 min, 50% B-0% B, 0.5-0.55 mL / min; 5.5-8 min, 0% B, 0.55 mL / min; sequentially eluting the target and having a shorter elution time and a high recovery rate; preferably, the concentration of ammonium salt in the mobile phase is 2.5 mM.

[0041] Wherein 0-3 min, 100% B-90% B, 0.2-0.3 mL / min means that in 0-3 minutes, the proportion of mobile phase B in the mobile phase system decreases from 100% to 90%, and the flow rate increases from 0.2 ml / min to 0.3 ml / min, and TMA is eluted at this stage; 3-5 min, 90% B-50% B, 0.4-0.5 mL / min means that in 3-5 minutes, the proportion of mobile phase B in the mobile phase system decreases from 90% to 50%, and the flow rate increases from 0.4 ml / min to 0.5 ml / min; 5-5.5 min, 50% B-0% B, 0.5-0.55 mL / min means that in 5-5.5 minutes, the proportion of mobile phase B in the mobile phase system decreases from 50% to 0%, and the flow rate increases from 0.5 ml / min to 0.55 ml / min; 5.5-8 min, 0% B, 0.55 mL / min means that in 5.5-8 minutes, the proportion of mobile phase B in the mobile phase system is 0%, and the flow rate is 0.55 ml / min, and GPC, CDP-Choline and PC target are eluted at this stage.

[0042] In the present application, the ammonium salt in the mobile phase includes ammonium formate or ammonium acetate, and other suitable ammonium salts. The concentration of ammonium salt in the mobile phase and the gradient elution program need to be matched. The specific concentration of ammonium salt in the mobile phase and the flow rate and elution time can be adjusted according to the specific situation to meet the requirements of less than 1.0 x 10 -6 mol / min for eluting TMA, and greater than or equal to 1.0 x 10 - 6 mol / min for eluting GPC, CDP-Choline and PC. Preferably, the concentration of ammonium salt in the mobile phase is (1-3) x 10 -60.6 x 10

[0043] Column temperature: 45℃;

[0044] Injection volume: 1 μL;

[0045] Mass spectrometry conditions: positive ion mode, multiple reaction monitoring mode detection (MRM), dry gas temperature 300℃, dry gas flow rate 10 L / min, atomizer pressure 30 psi, sheath gas temperature 350℃, sheath gas flow rate 11 L / min, capillary voltage 4000V, nozzle voltage 500V. The mass spectrometry conditions in the present application can be adjusted according to the specific analyte or different equipment.

[0046] The following is an example:

[0047] Example 1 uses the method provided in the present application to detect a plurality of choline metabolite mixers

[0048] Chromatographic column: Waters ACQUITY UPLC BEH Amide (2.1 x 100 mm, 1.7 μm) chromatographic column is selected for separation of the target.

[0049] Mobile phase: A is 50% acetonitrile aqueous solution (0.1% formic acid, 2mM ammonium formate), B is 90% acetonitrile aqueous solution (0.1% formic acid, 2mM ammonium formate).

[0050] Gradient elution program: 0-3 min, 100% B-90% B, 0.2-0.3 mL / min; 3-5 min, 90% B-50% B, 0.4-0.5 mL / min; 5-5.5 min, 50% B-0% B, 0.5-0.55 mL / min; 5.5-8 min, 0% B, 0.55 mL / min, eluting the target in turn.

[0051] Column temperature: 45℃;

[0052] Injection volume: 1 μL;

[0053] Mass spectrometry conditions: positive ion mode, multiple reaction monitoring mode detection (MRM), dry gas temperature 300℃, dry gas flow rate 10 L / min, atomizer pressure 30 psi, sheath gas temperature 350℃, sheath gas flow rate 11 L / min, capillary voltage 4000V, nozzle voltage 500V, results as shown in Figure 2

[0054] In this example, when TMA is eluted, the ammonium salt content in the mobile phase system is less than 0.6 x 10 -6 mol / min, when GPC, CDP-Choline, PC is eluted, the ammonium salt content in the mobile phase system is 1.1 x 10 -6 ​mol / min (more than 1.0 x 10 -6 mol / min) and less than 0.75 x 10 Figure 2 It can be seen that the peak shape and response of all target substances in the mixed standard chromatogram are good.

[0055] Example 2 detects a plurality of choline metabolites in a liver sample by using the method provided in the present application

[0056] Chromatographic column: Waters ACQUITY UPLC BEH Amide (2.1 x 100 mm, 1.7 μm) chromatographic column is selected to separate the target substances.

[0057] Mobile phase: A is 50% acetonitrile aqueous solution (0.1% formic acid, 2.5 mM ammonium formate), B is 90% acetonitrile aqueous solution (0.1% formic acid, 2.5 mM ammonium formate).

[0058] Gradient elution program: 0-3 min, 100% B-90% B, 0.2-0.3 mL / min; 3-5 min, 90% B-50% B, 0.4-0.5 mL / min; 5-5.5 min, 50% B-0% B, 0.5-0.55 mL / min; 5.5-8 min, 0% B, 0.55 mL / min.

[0059] Column temperature: 45°C

[0060] Injection volume: 1 μL

[0061] Mass spectrometry conditions: positive ion mode, multiple reaction monitoring mode detection (MRM), drying gas temperature 300°C, drying gas flow rate 10 L / min, atomizer pressure 30 psi, sheath gas temperature 350°C, sheath gas flow rate 11 L / min, capillary voltage 4000 V, nozzle voltage 500 V, and the results are shown in Figure 3 .

[0062] In this example, when TMA is eluted, the ammonium salt content in the mobile phase system is less than 0.75 x 10 -6 mol / min, when GPC, CDP-choline, PC are eluted, the ammonium salt content in the mobile phase system is 1.375 x 10 -6 mol / min (more than 1.0 x 10 - 6 mol / min) and less than 0.75 x 10

[0063] mol / min) and less than 0.75 x 10 Figure 3It can be known that 10 choline metabolites are detected by using the detection method for detecting liver samples, and the peak shape and response of each target are good, and the chromatogram of TMA and GPC, CDP-Choline, PC four targets are accurately detected, which shows that the detection method provided by the application can simultaneously detect multiple choline metabolites, especially can simultaneously detect TMA and GPC, CDP-Choline, PC four targets in choline metabolites without affecting each other, and the detection accuracy is high.

[0064] Comparative Example 1: Simultaneously detecting multiple choline metabolite mixed standards by using existing LC-MS / MS

[0065] The chromatographic column: Waters ACQUITY UPLC BEH Amide (2.1x100mm, 1.7um) chromatographic column is selected for separating the target.

[0066] The mobile phase: A is 50% acetonitrile aqueous solution (0.1% formic acid, 10mM ammonium formate), and B is 90% acetonitrile aqueous solution (0.1% formic acid, 10mM ammonium formate).

[0067] Gradient elution: 0.3mL / min, the elution program is: 0-5min, 100% B-0% B; 5-8min, 0% B.

[0068] Column temperature: 45℃

[0069] Injection volume: 1uL

[0070] Mass spectrometry conditions: positive ion mode, multiple reaction monitoring mode detection (MRM), dry gas temperature 300℃, dry gas flow rate 10L / min, atomizer pressure 30psi, sheath gas temperature 350℃, sheath gas flow rate 11L / min, capillary voltage 4000V, nozzle voltage 500V, and the results are shown in Figure 4 .

[0071] From Figure 4 It can be known that the mixed standard chromatogram obtained by using the existing method for detection has the same concentration as the mixed standard detected in Example 1, but the ammonium salt concentration in the mobile phase is high (the ammonium salt content in the mobile phase system is 3x10 -6 mol / min, which is greater than 1.0x10 - 6 mol / min), and the response of TMA is poor, and the peak shape and response of other targets are good, which shows that the detection method cannot simultaneously detect TMA and GPC, CDP-Choline, PC four targets in choline metabolites.

[0072] Comparative Example 2: Simultaneously detecting multiple choline metabolite mixed standards by using existing LC-MS / MS

[0073] Chromatographic column: Waters ACQUITY UPLC BEH Amide (2.1 x 100 mm, 1.7 μm) was selected to separate the target.

[0074] Mobile phase: A was 50% acetonitrile aqueous solution (0.1% formic acid, 5 mM ammonium formate), B was 90% acetonitrile aqueous solution (0.1% formic acid, 5 mM ammonium formate).

[0075] Gradient elution 0.3 mL / min, elution program: 0-5 min, 100% B-0% B; 5-8 min, 0% B.

[0076] Column temperature: 45°C

[0077] Injection volume: 1 μL

[0078] Mass spectrometry conditions: positive ion mode, multiple reaction monitoring mode detection (MRM), drying gas temperature 300°C, drying gas flow rate 10 L / min, atomizer pressure 30 psi, sheath gas temperature 350°C, sheath gas flow rate 11 L / min, capillary voltage 4000 V, nozzle voltage 500 V, and the results are shown in Figure 5 .

[0079] As can be seen from Figure 5 , the concentration of the mixed standard detected in Comparative Example 1 is the same, but the ammonium salt concentration in the mobile phase when eluting TMA is 1.5 x 10 -6 mol / min, which is 50% lower than that of Comparative Example 1, and the TMA response is improved, but the response is still very low, and the GPC, CDP-Choline, PC peak shape and response have not changed much, although the CDP-Choline half-peak width has increased, but the peak shape and response of the other target substances are good, indicating that this detection method cannot accurately detect TMA and GPC, CDP-Choline, PC four target substances in choline metabolites at the same time.

[0080] Comparative Example 3: Simultaneous detection of multiple choline metabolite mixed standards by LC-MS / MS

[0081] Chromatographic column: Waters ACQUITY UPLC BEH Amide (2.1 x 100 mm, 1.7 μm) was selected to separate the target.

[0082] Mobile phase: A was 50% acetonitrile aqueous solution (0.1% formic acid, no ammonium salt), B was 90% acetonitrile aqueous solution (0.1% formic acid, no ammonium salt).

[0083] Gradient elution 0.3 mL / min, elution program: 0-5 min, 100% B-0% B; 5-8 min, 0% B.

[0084] Column temperature: 45℃;

[0085] Injection volume: 1 μL;

[0086] Mass spectrometry conditions: positive ion mode, multiple reaction monitoring mode detection (MRM), dry gas temperature 300℃, dry gas flow rate 10 L / min, atomizer pressure 30 psi, sheath gas temperature 350℃, sheath gas flow rate 11 L / min, capillary voltage 4000 V, nozzle voltage 500 V, wherein the mass spectrometry acquisition index is shown in Table 1, and the mass spectrum is shown in Figure 6 .

[0087] Table 1 Mass spectrometry acquisition index

[0088]

[0089] As can be seen from Figure 6 , the elution of TMA, GPC, CDP-Choline and PC is completed, the ammonium salt content in the mobile phase is 0, the peak shape and response of TMA are good, and the response and peak shape of GPC, CDP-Choline and PC are significantly poor. Although the peak shape and response of other target substances are good and are not affected, the detection method cannot accurately detect TMA and GPC, CDP-Choline and PC in choline metabolites at the same time.

[0090] It can be known from the detection results of the examples and comparative examples that the ammonium salt in the mobile phase is crucial for accurately detecting GPC, CDP-Choline and PC. In order to simultaneously detect TMA and GPC, CDP-Choline and PC, the ammonium salt in the mobile phase series needs to be controlled in a suitable range respectively.

[0091] Performance test of the method for simultaneously detecting TMA and GPC, CDP-Choline and PC in Example 3

[0092] The linear range and sample detection limit LOD of the detection method provided by the application were investigated, and the recovery rate of the method was investigated through external addition recovery test (liver sample), and the results are shown in Table 2.

[0093] Table 2 Performance index of the detection method

[0094]

[0095] As can be known from Table 1, the recovery rate of the detection method provided by the application for choline is 99.54%, the recovery rate of TMA is 80%, the recovery rate of GPC, CDP-Choline and PC is more than 97%, and the correlation coefficient R 2All are above 0.99, which indicates that the detection method provided by the application has good detection performance and high recovery rate, and in particular, TMA and GPC, CDP-choline and PC can be simultaneously detected, and the recovery rates are all above 80%, and the recovery rates of GPC, CDP-choline and PC are all above 97%, close to 100%, which indicates that the method has high detection accuracy.

[0096] Those skilled in the art can easily understand that the above description is only a preferred embodiment of the application, and is not intended to limit the application, and any modification, equivalent replacement and improvement made within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. A method for simultaneously detecting multiple choline metabolites, characterized in that, The choline metabolites include TMA, GPC, CDP-Choline, and PC, specifically including: Liquid chromatography elution separation: After sample pretreatment, gradient elution is used to elute the target analytes from the sample. When eluting TMA, the ammonium salt content in the mobile phase is less than 1.0 × 10⁻⁶. -6 mol / min; when eluting GPC, CDP-Choline and PC, the ammonium salt content in the mobile phase system is greater than or equal to 1.0 × 10⁻⁶ mol / min; -6 The target analyte was eluted sequentially at a rate of mol / min; the gradient elution process is as follows: The chromatographic column used was a Waters ACQUITY UPLC BEH Amide. Mobile phase A consisted of 50% acetonitrile aqueous solution, 0.1% formic acid, and 2-3 mM ammonium formate; mobile phase B consisted of 90% acetonitrile aqueous solution, 0.1% formic acid, and 2-3 mM ammonium formate. The gradient elution program was as follows: 0-3 min, 100%B-90%B, 0.2-0.3 mL / min; 3-5 min, 90%B-50%B, 0.4-0.5 mL / min; 5-5.5 min, 50%B-0%B, 0.5-0.55 mL / min; 5.5-8 min, 0%B, 0.55 mL / min. Mass spectrometry is used to detect the target analyte after elution.

2. The method for simultaneous detection of multiple choline metabolites as described in claim 1, characterized in that, When eluting GPC, CDP-Choline, and PC, the ammonium salt content in the mobile phase system is (1.0~3.0)×10⁻⁶. -6 mol / min.

3. The method for simultaneous detection of multiple choline metabolites as described in claim 1 or 2, characterized in that, By adjusting the concentration of ammonium salts in the mobile phase and / or the flow rate, the corresponding ammonium salt content requirements in the mobile phase system can be met when eluting TMA and GPC, CDP-Choline, and PC.

4. The method for simultaneous detection of multiple choline metabolites as described in claim 3, characterized in that, The mobile phase has an ammonium salt concentration ≤10mM and a flow rate of 0.1-0.6mL / min.

5. The method for simultaneous detection of multiple choline metabolites as described in claim 4, characterized in that, The chromatographic column used was a Waters ACQUITY UPLC BEH Amide, 2.1×100mm, 1.7μm.

6. The method for simultaneous detection of multiple choline metabolites as described in claim 5, characterized in that, The mobile phase has an ammonium salt concentration of 2-3 mM and can also elute TMA, Choline, Creatinine, TMAO, Betaine, DMG, Sarcosine, GPC, CDP-Choline, and PC. The gradient elution program is as follows: 0-3 min, 100% B-90% B, 0.2-0.3 mL / min; 3-5 min, 90% B-50% B, 0.4-0.5 mL / min; 5-5.5 min, 50% B-0% B, 0.5-0.55 mL / min; 5.5-8 min, 0% B, 0.55 mL / min.

7. The method for simultaneous detection of multiple choline metabolites as described in claim 6, characterized in that, The ammonium salt includes ammonium formate or ammonium acetate.

8. The method for simultaneous detection of multiple choline metabolites as described in claim 1, characterized in that, The sample pretreatment used methanol-water solution as a solvent for direct extraction.

9. The method for simultaneous detection of multiple choline metabolites as described in claim 8, characterized in that, The sample was blood, obtained through the following pretreatment: Take a serum sample, add 90% methanol aqueous solution at a volume ratio of 1:50, add internal standard solution, centrifuge and filter to obtain the sample to be eluted.

10. The method for simultaneous detection of multiple choline metabolites as described in claim 8, characterized in that, The sample was a tissue, obtained through the following pretreatment: Take tissue samples, add 90% methanol aqueous solution to homogenize and extract, wherein the mass ratio of tissue sample to methanol aqueous solution volume is 5:1, add internal standard solution, centrifuge and filter to obtain the sample to be eluted.

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