Liquid chromatography-mass spectrometry tandem detection method for multiple folic acid metabolism related substances in trace plasma or serum

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a method for synchronously and quantitatively detecting various folic acid metabolism related substances in trace plasma or serum, which comprises the following steps of: detecting a pretreated trace plasma or serum sample by adopting a liquid chromatography-tandem mass spectrometry method; the following nine folic acid metabolism related substances are synchronously and quantitatively detected: 5-formyl tetrahydrofolic acid, folic acid, 5-methyl tetrahydrofolic acid, S-adenosine-L-methionine, S-adenosine-L-homocysteine, vitamin B12, pyridoxamine, pyridoxine and pyridoxal. The invention further relates to application of the detection method in related detection of child neurodevelopment. According to the detection method, only trace plasma or serum is used, methotrexate is used for replacing an expensive isotope internal standard for more economical detection, and nine folate metabolism related substances can be accurately quantified at the same time. Therefore, the detection method disclosed by the invention has important clinical significance and wide market application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a liquid chromatography-mass spectrometry tandem detection method for a plurality of folic acid metabolism related substances in trace plasma or serum and application thereof. BACKGROUND

[0002] Folic acid, as an essential water-soluble vitamin B, cannot be synthesized in the body and must be taken in through food or supplemented by supplements. Its core function relies on the synergistic effect between folic acid metabolism related substances, which is realized through one-carbon unit metabolic pathways, not only providing key methyl donors for neurotransmitter synthesis, myelin formation, DNA repair, gene expression regulation and amino acid metabolism, supporting fetal neural development, but also widely participating in numerous processes such as hematopoietic system hematopoiesis, reproductive system gamete quality assurance, embryonic multi-organ growth and development, and cardiovascular system homeostasis regulation.

[0003] There are many important folic acid metabolism related substances in the body, including folic acid (FA), folinic acid (5-Formyltetrahydrofolate, 5-FTHF) and 5-methyltetrahydrofolate (5-MTHF). The transport of folic acid depends on the endocytosis mediated by folate receptor (FR), and if the function of FR is blocked by folate receptor autoantibodies, 5-FTHF can bypass the FR pathway through reduced folate carrier (RFC), not only maintaining central nervous system folic acid metabolism by crossing the blood-brain barrier, but also maintaining normal fetal growth and development by crossing the placental barrier. Folic acid mainly exists in the active form of 5-MTHF in the blood, which can enter the cerebrospinal fluid, bone marrow, reproductive glands and other key tissues through transcellular transport mechanisms, and play multiple physiological functions in the system.

[0004] 5-MTHF needs to work with Vitamin B12 (VB12) to catalyze the conversion of homocysteine to methionine: on the one hand, this process can reduce blood homocysteine levels, reduce its damage to vascular endothelial cells, and thus prevent atherosclerosis, hypertension and other cardiovascular diseases; on the other hand, the generated methionine is further synthesized into S-Adenosylmethionine (SAM), which as a core methyl donor not only supports the methylation needs of the central nervous system (such as neurotransmitter synthesis, myelin maintenance), but also provides methyl groups for DNA replication in the hematopoietic system, ensuring the normal differentiation and maturation of blood cells and avoiding megaloblastic anemia. After SAM provides a methyl donor, it is converted into S-Adenosylhomocysteine (SAH), and the ratio of the two is a core indicator of the body's methylation capacity. Vitamin B6 (VB6) has three forms of pyridoxamine (PM), pyridoxine (PN), and pyridoxal (PL), which can be converted into each other, and its active form pyridoxal 5'-phosphate not only participates in neurotransmitter synthesis, but also as a key coenzyme for serine hydroxymethyltransferase and cystathionine-β-synthase: the former catalyzes the conversion of tetrahydrofolate to 5,10-methylenetetrahydrofolate; the latter mediates the conversion of homocysteine to cystine through the cystathionine pathway, which not only helps to reduce the risk of cardiovascular disease, but also provides protection for amino acid metabolism in hematopoietic cells.

[0005] The above folate metabolic pathway involves 9 key substances (FA, 5-FTHF, 5-MTHF, SAM, SAH, VB12, PM, PN, PL), and abnormal content can cause multi-system metabolic disorders: in terms of neural development, folate deficiency in the brain, methylation damage can interfere with neural function, or high homocysteine neurotoxicity can cause children's neurodevelopmental disorders, including autism spectrum disorders, neural tube defects, schizophrenia, etc.; in the hematopoietic system, DNA synthesis caused by metabolic disorders can cause megaloblastic anemia, manifested as increased red blood cell volume and decreased hematopoietic efficiency, especially affecting pregnant women, the elderly and vegetarians; in the cardiovascular system, hyperhomocysteinemia can increase the risk of heart attack and brain infarction by damaging the vascular endothelium and promoting lipid deposition; in the field of reproduction and embryonic development, folate metabolism abnormalities can cause decreased egg quality, infertility or early pregnancy loss in women, and can also cause fetal malformations of the heart, limbs and other organs in addition to neural tube defects, and affect sperm motility and morphology in men. Current clinical and laboratory methods for detecting folate include microbial detection, chemiluminescence immunoassay, high-performance liquid chromatography, and isotope dilution liquid chromatography tandem mass spectrometry. In terms of sample selection, plasma or serum folate is considered to reflect recent folate nutritional status, and red blood cell folate reflects chronic or long-term (4 months) folate nutritional status. Compared with red blood cell folate, sample processing of plasma or serum folate is more variable, does not require complex operations such as red blood cell separation and hemolysis, can reduce sample loss and processing time, and can improve detection efficiency. In terms of detection methods, microbial detection relies on the growth characteristics of specific microorganisms for detection, which is easily affected by fluctuations in the culture environment and conditions, and has low detection precision, complicated operation, long time-consuming, and small sample throughput, making it difficult to meet the needs of large-scale sample rapid detection; chemiluminescence immunoassay has poor specificity, as the binding affinity of folate-binding proteins to different folate metabolites differs, often leading to low detection results, and its analysis range is narrow, and the matrix effect is significant when the sample is diluted, affecting accuracy; high-performance liquid chromatography has limited sensitivity when using a UV detector, and using a fluorescence detector requires post-column derivatization of folate, which is complicated; isotope dilution liquid chromatography tandem mass spectrometry has high sensitivity and specificity, and can detect multiple substances simultaneously, but the cost of isotopic internal standards is high, and most methods require a large amount of plasma or serum sample (>100 μL), limiting its application.

[0006] By detecting and regulating the levels of the nine folate metabolism-related substances, multi-dimensional health management can be achieved: for children, it can promote neural development and control the progression of neural developmental disorders; for women preparing for pregnancy and during pregnancy, it can prevent fetal malformations and anemia during pregnancy; for men preparing for pregnancy, it can improve sperm quality and increase the probability of conception; for the elderly and high-risk cardiovascular groups, it can prevent megaloblastic anemia and cardiovascular and cerebrovascular diseases; for anemia patients, it can determine the causes of folate deficiency to guide precise supplementation. By providing personalized detection and diagnosis services covering the entire population and multiple systems, precise intervention can be achieved throughout the life cycle from pre-pregnancy prevention, pregnancy care to chronic disease prevention and control in adulthood, and health maintenance in old age. SUMMARY

[0007] In view of the deficiencies of existing folate-related metabolic substance detection technologies, the present application develops a liquid chromatography mass spectrometry tandem detection method for multiple folate metabolism-related substances in trace plasma or serum. The detection method of the present application only uses trace plasma or serum (50 μL), uses methotrexate instead of expensive isotope internal standards for more economical detection, and can simultaneously accurately quantify nine folate metabolism-related substances.

[0008] Specifically, the present application is realized through the following technical solutions: The present application provides a synchronous quantitative detection method for multiple folate metabolism-related substances in trace plasma or serum, which comprises: using liquid chromatography-tandem mass spectrometry (LC-MS / MS) to detect the pretreated trace plasma or serum sample, and synchronously quantitatively detecting the following nine folate metabolism-related substances: 5-formyltetrahydrofolate (5-FTHF), folic acid (FA), 5-methyltetrahydrofolate (5-MTHF), S-adenosyl-L-methionine (SAM), S-adenosyl-L-homocysteine (SAH), vitamin B12 (VB12), pyridoxamine (PM), pyridoxine (PN), and pyridoxal (PL).

[0009] As an optional mode, in the above detection method, the pretreatment method comprises the following steps: (1) Antioxidant treatment: adding MTX as an internal standard solution to the plasma or serum sample, and then adding a dithiothreitol (DTT) solution, vortexing and incubating; (2) De-protein treatment: adding a methanol-acetonitrile mixed solution containing ascorbic acid (VC) and citric acid (CA), vortexing and then centrifuging; (3) Nitrogen blow concentration and reconstitution: freezing the supernatant, then nitrogen blowing to dryness, and then reconstituting with a reconstitution solution.

[0010] As an optional mode, in the above detection method, the reconstituting solution is a mixture of water and methanol containing 5-15 mmol / mL ammonium acetate, the trace plasma or serum is 30-60 μL of plasma or serum, the concentration of the MTX internal standard solution is 200-220 ng / mL, the concentration of the DTT solution is 5-15 mg / mL, and the concentrations of VC and CA in the methanol-acetonitrile mixed solution are both 80-120 μg / mL.

[0011] Preferably, the reconstituting solution is a mixture of water and methanol containing 10 mmol / mL ammonium acetate, and the volume ratio of water to methanol is 96:4.

[0012] Preferably, the trace plasma or serum is 50 μL of plasma or serum.

[0013] Preferably, the concentration of the MTX internal standard solution is 210 ng / mL, the concentration of the DTT solution is 10 mg / mL, and the concentrations of VC and CA in the methanol-acetonitrile mixed solution are both 100 μg / mL, and the volume ratio of methanol to acetonitrile is 1:1.

[0014] As an optional mode, in the above detection method, the liquid chromatography conditions include: a C18 reversed-phase column; a mobile phase A of 0.2% acetic acid-10 mmol / mL ammonium acetate aqueous solution and a mobile phase B of methanol; a gradient elution program of maintaining 4% B for 0-1 min, increasing to 80% B for 1-4 min, increasing to 99% B for 4-4.1 min, maintaining 99% B for 4.1-8 min, decreasing to 4% B for 8-8.1 min, and maintaining 4% B for 8.1-10 min; a flow rate of 200 μL / min, a column temperature of 40℃, and an injection volume of 10 μL.

[0015] Preferably, the C18 reversed-phase column is Agilent 858700-314 (1.8 μm, 3.0×100 mm).

[0016] As an optional mode, in the above detection method, the mass spectrometry conditions include: an ion source of positive ion electrospray (ESI+); a drying gas temperature of 200℃, a drying gas flow rate of 12 L / min, and an atomization gas pressure of 30 psi; a sheath gas temperature of 400℃, a sheath gas flow rate of 12 L / min, a capillary voltage of 2500 V (+), 3000 V (+), a nozzle voltage of 0 V (+), 1500 V (+); and Detection mode: Multi-reaction monitoring (MRM) mode.

[0017] Preferably, the MRM parameters of each substance are shown in Table 3.

[0018] As an optional mode, in the above detection method, the standard stock solution and the internal standard stock solution are both prepared with a methanol-water solution as a solvent, and the methanol-water solution contains 80-120 μg / mL VC, 80-120 μg / mL CA and 5-15 mg / mL DTT.

[0019] Preferably, the methanol-water solution contains 100 μg / mL VC, 100 μg / mL CA and 15 mg / mL DTT, and the volume ratio of methanol to water is 1:1.

[0020] As an optional mode, in the above detection method, the standard stock solution comprises: FA, SAM, SAH, VB12, PM, PN, PL stock solution (0.5 mg / mL) 5-FTHF, 5-MTHF stock solution (0.17 mg / mL) As an optional mode, in the above detection method, the internal standard stock solution is 0.5 mg / mL MTX As an optional mode, in the above detection method, the standard curve of the detection method is prepared by a matrix standard, and the dependent variable is (peak area of the sample to be tested - peak area of the blank) / peak area of the MTX internal standard, the independent variable is the theoretical concentration of the matrix standard, a quadratic polynomial is used for fitting, and the intercept is set to 0.

[0021] As an optional mode, in the above detection method, the matrix standard is prepared by fetal bovine serum.

[0022] Preferably, the preparation method comprises the following steps: adding 10 μL of 262.5 ng / mL MTX internal standard solution and 40 μL of DTT solution to 50 μL of fetal bovine serum for antioxidant treatment, and then performing deproteinization, freezing, nitrogen blowing and re-dissolution steps.

[0023] As an optional mode, in the above detection method, the detection limit and the quantification limit of the detection method are respectively 0.0015-0.4220 ng / mL and 0.0049-1.4041 ng / mL.

[0024] Compared with the prior art, the present application has the following beneficial effects: The liquid chromatography-tandem mass spectrometry method developed by the application has high sensitivity, good precision and reliable accuracy. Compared with other methods (sample consumption > 100 μL), the sample consumption of the method is reduced to 50 μL, which not only greatly reduces the use amount of precious clinical samples, but also helps multiple detection and multi-index joint detection of the same sample, significantly improves the sample utilization rate and detection efficiency; using methotrexate as an internal standard can ensure the reproducibility and stability of the detection, and compared with the expensive isotope internal standard, it has the advantages of economy and practicality. Due to the complexity of plasma or serum matrix and the serious interference of numerous endogenous substances with similar physicochemical properties on detection sensitivity and accuracy, there is currently no report on simultaneous detection of 9 kinds of folate metabolism related substances such as FA, 5-FTHF, 5-MTHF, SAM, SAH, VB12, PM, PN and PL in plasma or serum by LC-MS / MS, and the existing method can only detect part of the substances. BRIEF DESCRIPTION OF DRAWINGS

[0025] The accompanying drawings are included to provide a further understanding of the application, and constitute a part of the specification, and are used together with embodiments of the application to explain the application, and do not constitute a limitation on the application. In the drawings: Figure 1 : Standard curve of LC-MS / MS method for detecting folate metabolism related substances in human plasma or serum. DETAILED DESCRIPTION

[0026] Unless specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature or according to the product instructions are used. Unless the manufacturer of the reagent or instrument is specified, it is a conventional product that can be purchased through a regular channel.

[0027] The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.

[0028] The application will be further described below in conjunction with the examples: Examples: 1. Types of folate-related metabolic substances in plasma or serum The information of 9 kinds of folate metabolism related substances detected by the application is shown in Table 1.

[0029] Table 1 Basic information of folate-related metabolic substances 2. Solution preparation (1) Methyl alcohol-water solution (v:v=1:1, containing 100 μg / mL VC, 100 μg / mL CA, 15 mg / mL DTT) (2) Methanol-acetonitrile solution (v:v = 1:1, containing 100 pg / mL VC, 100 pg / mL CA) (3) 10 mg / mL DTT solution (4) 2000 mmol / mL ammonium acetate solution (5) 10 mmol / mL ammonium acetate solution (6) Reconstitution solution (containing 10 mmol / mL ammonium acetate solution, methanol, v:v = 96:4) (7) Standard stock solution i. FA, SAM, SAH, VB12, PM, PN, PL (0.5 mg / mL) ii. 5-FTHF, 5-MTHF (0.17 mg / mL) (8) Internal standard stock solution (0.5 mg / mL) (9) 1000 ng / mL mixed standard solution (10) 1000 ng / mL internal standard solution (11) 210 ng / mL internal standard solution (12) 262.5 ng / mL internal standard solution 3. Standard and blank control The mixed standard solution of folate-related metabolites was used as the standard, and the concentration of each standard component was 1000 ng / mL. Methanol-water solution was used as the blank control.

[0030] 4. Establishment of LC-MS / MS detection method for folate metabolism-related substances (1) Blood plasma or serum sample collection: EDTA anticoagulant blood collection tube was used to collect venous blood according to the standard procedure, and the blood plasma was collected after standing and centrifugation within 4 h after collection. The serum sample only needs to be collected after standing and centrifugation, and then stored in a frozen tube and immediately placed in a 80 °C refrigerator for subsequent detection. The whole process of transportation was kept in low temperature environment with dry ice.

[0031] (2) Preparation before detection: Take out the consumables, reagents and samples from the refrigerator 1 h in advance, and place them in a foam box covered with ice blocks. Close the box cover to avoid light during transportation. The whole experiment was carried out in a dark room under yellow light to avoid ultraviolet light causing folate decomposition. All pipetting operations were carried out on ice to maintain low temperature.

[0032] ​(3) Standard dilution: The mixed standard solution was diluted with a methanol-water solution gradient to a concentration of 1000 ng / ml, 500 ng / ml, 250 ng / ml, 125 ng / ml, 62.5 ng / ml, 31.25 ng / ml, 15.62 ng / ml, 7.81 ng / ml, 3.91 ng / ml, 1.95 ng / ml. After the following steps, the theoretical concentration of the matrix mixed standard obtained was 200 ng / ml, 100 ng / ml, 50 ng / ml, 25 ng / ml, 12.5 ng / ml, 6.25 ng / ml, 3.12 ng / ml, 1.56 ng / ml, 0.78 ng / ml, 0.39 ng / ml, respectively. The methanol-water solution was a blank control. The standard gradient dilution step is shown in Table 2.

[0033] Table 2 Standard gradient dilution table for folate-related metabolites (4) Preparation of matrix standard i. Antioxidant: Take 12 portions of 50 μL fetal bovine serum in centrifuge tubes, add 10 μL of 262.5 ng / mL internal standard (MTX) solution to each tube, then add 40 μL of 10 mg / mL DTT solution, and place in an aluminum foil incubation bag. Incubate in a multi-tube vortex mixer at 2000 rpm for 5 min, and equilibrate at 4°C for 15 min.

[0034] ii. Deproteinization: Add 500 μL of methanol-acetonitrile solution containing 100 μg / mL VC and CA (v:v=1:1), and place in an aluminum foil incubation bag. Incubate in a multi-tube vortex mixer at 2000 rpm for 10 min, and equilibrate at 4°C for 15 min. Centrifuge at 12000 rpm for 10 min at 4°C.

[0035] iii. Freezing: Freeze at 80°C for 30 min, and transfer the supernatant to a centrifuge tube.

[0036] iv. Nitrogen blow concentration: Quickly nitrogen blow the supernatant to dryness at low temperature.

[0037] v. Resuspension: Immediately add 30 μL of resuspension solution (10 mmol / mL ammonium acetate solution + methanol, v:v=96:4) after drying, and place in an aluminum foil incubation bag. Incubate in a multi-tube vortex mixer at 2000 rpm for 5 min, and centrifuge at 12000 rpm for 10 min at 4°C. Transfer the supernatant to a centrifuge tube and vortex to obtain a matrix internal standard solution (the internal standard theoretical concentration at this time is 87.5 ng / mL).

[0038] (5) Preparation of matrix standard solution: Take 11 portions of 24 μL matrix internal standard solution in centrifuge tubes, and add 6 μL of each concentration gradient mixed standard solution (1000 ng / mL-1.95 ng / mL) and a blank control (methanol-water) to each tube in turn, vortex mix, to obtain the matrix standard solution (theoretical concentration 200 ng / mL-0.39 ng / mL) and the blank control (theoretical concentration 0 ng / mL) (at this time the internal standard theoretical concentration is 70 ng / mL). Transfer the matrix standard solution to the brown sample bottle inner tube, avoid bubbles at the bottom of the tube, store at 4°C, and immediately perform LC-MS / MS detection.

[0039] (6) Sample pretreatment i. Antioxidant: Take 50 μL of sample plasma or serum in a centrifuge tube, add 10 μL of 210 ng / mL internal standard solution to each tube, then add 40 μL of 10 mg / mL DTT solution, and place in an aluminum foil heat preservation bag, and shake in a multi-tube vortex mixer at 2000 rpm for 5 min, and balance at 4°C for 15 min.

[0040] ii. De-protein: Add 500 μL of methanol-acetonitrile solution containing 100 μg / mL VC and CA (v:v=1:1), and place in an aluminum foil heat preservation bag, and shake in a multi-tube vortex mixer at 2000 rpm for 10 min, and balance at 4°C for 15 min, and centrifuge at 12000 rpm for 10 min at 4°C.

[0041] iii. Freeze: Freeze at 80°C for 30 min, and transfer the supernatant to a centrifuge tube.

[0042] iv. Nitrogen blow concentration: quickly nitrogen blow the supernatant to dryness at low temperature.

[0043] v. Redissolution: immediately add 30 μL of redissolution solution (10 mmol / mL ammonium acetate solution, methanol, v:v=96:4) after blowing dry, and place in an aluminum foil heat preservation bag, and shake in a multi-tube vortex mixer at 2000 rpm for 5 min, and centrifuge at 12000 rpm for 10 min at 4°C. Transfer the supernatant to a brown sample bottle inner tube (avoid absorbing impurities, at this time the internal standard theoretical concentration is 70 ng / mL), avoid bubbles at the bottom of the tube, store at 4°C, and immediately perform LC-MS / MS detection.

[0044] (7) Chromatographic conditions: The treated samples were analyzed for folic acid metabolism-related substances using liquid chromatography-tandem mass spectrometry (LC-MS / MS, Agilent 6495). The chromatographic column was an Agilent 858700-314 (1.8 μm, 3.0 × 100 mm); the mobile phase was 0.2% acetic acid-10 mmol / mL ammonium acetate-water (A) and methanol (B); the flow rate was 200 μL / min; the gradient elution program was: 0–1 min, 4% B; 1–4 min, 4%–80% B; 4–4.1 min, 80–99% B; 4.1–8 min, 99% B; 8–8.1 min, 99%–4% B; 8.1–10 min, 4% B; 10–10.5 min, Post Time; the column temperature was 40 °C; the injection volume was 10 μL; and the injector temperature was 4 °C.

[0045] (8) Mass spectrometry conditions: ESI ion source: positive ion mode; drying gas temperature: 200℃; drying gas flow rate: 12L / min; nebulizer gas pressure: 30psi; sheath gas temperature: 400℃; sheath gas flow rate: 12L / min; capillary voltage: 2500V (+), 3000V ( Nozzle voltage: 0V (+), 1500V ( ); Quantification was performed using the internal standard method, with the scanning type set to MRM mode. The MRM parameters for each compound are shown in Table 3.

[0046] Table 3 MRM parameters of folic acid-related metabolites (9) Constructing a standard curve: Using the theoretical concentration of the matrix standard as the independent variable (x) and (peak area of ​​standard substance - peak area of ​​blank control) / peak area of ​​internal standard substance as the dependent variable (y), fit the standard curve using a quadratic polynomial method, setting the intercept to 0. Based on the position of the peak area of ​​each analyte in the test sample / peak area of ​​the internal standard substance on the standard curve, calculate the concentration of each analyte in the reconstitution solution, and thereby estimate the concentration of each analyte in the original plasma or serum (plasma or serum concentration = reconstitution solution concentration × 30 / 50).

[0047] 5. Evaluation of LC-MS / MS detection methods for folic acid metabolism-related substances (1) Standard curve, limit of detection and limit of quantitation The standard curve was fitted by a quadratic polynomial method with the theoretical concentration of the matrix standard as the independent variable (x) and the ratio of (peak area of standard - peak area of blank control) / peak area of internal standard as the dependent variable (y). The limit of detection (LOD) and the limit of quantitation (LOQ) were represented by the concentration of each analyte corresponding to 3 times and 10 times the background signal of the instrument, respectively. The fitting equation, limit of detection and limit of quantitation of the concentration of folate-related metabolites in plasma or serum are shown in Table 4, and the standard curve is shown in Figure 1 The correlation coefficient (R²) of each standard curve was 0.9976-0.9999, and the ranges of the limit of detection and the limit of quantitation were 0.0015-0.4220 ng / mL and 0.0049-1.4041 ng / mL, respectively.

[0048] Table 4 Fitting equation, limit of detection and limit of quantitation of LC-MS / MS method for detecting folate-related metabolites in human plasma or serum (2) Precision Three plasma or serum samples at different concentrations were selected, and 3 batches were continuously detected, with 8 parallel samples for each sample in each batch to analyze the precision of the detection method. Three plasma or serum samples at different concentrations were selected, and 3 batches were continuously detected, with 6 parallel samples for each sample in each batch to analyze the precision of the detection method. The intra-batch and inter-batch differences were evaluated by relative standard deviation (RSD), and RSD = (standard deviation / mean) x 100%. After continuous repeated detection, the precision results are shown in Table 5. The intra-batch and inter-batch differences of the method for detecting folate-related metabolites in plasma or serum were ≤9.48%.

[0049] Table 5 Intra-batch and inter-batch differences of LC-MS / MS method for detecting folate-related metabolites in human plasma or serum (3) Accuracy Different concentrations of mixed standard were added to the plasma or serum matrix to prepare high, medium and low concentration spiked samples. The samples were processed according to the sample pretreatment method, and the concentration difference in plasma or serum before and after spiking was detected to calculate the spiking recovery rate, so as to evaluate the accuracy of the analysis method. The spiking recovery rate results at different concentrations are shown in Table 6. The spiking recovery rate of each folate-related metabolite in the matrix was 66.82%-106.93%.

[0050] Table 6 Spiking recovery rate of LC-MS / MS method for detecting folate-related metabolites in human plasma or serum The above merely is the preferred embodiment of the present application, it should be pointed out that, for ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for simultaneous quantitative determination of a plurality of folate metabolism-related substances in microplasma or serum, characterized in that, The detection method comprises: detecting the pretreated trace plasma or serum sample by liquid chromatography-tandem mass spectrometry, and simultaneously quantitatively detecting the following nine folate metabolism related substances: 5-formyltetrahydrofolic acid, folic acid, 5-methyltetrahydrofolic acid, S-adenosyl-L-methionine, S-adenosyl-L-homocysteine, vitamin B12, pyridoxamine, pyridoxol and pyridoxal.

2. The detection method according to claim 1, characterized in that, The pretreatment method comprises the following steps: (1) antioxidant treatment: adding MTX as an internal standard solution to the plasma or serum sample, and then adding a dithiothreitol (DTT) solution, vortexing and incubating; (2) deproteinization treatment: adding a methanol-acetonitrile mixed solution containing ascorbic acid (VC) and citric acid (CA), vortexing and then centrifuging; (3) nitrogen blow concentration and reconstitution: freezing the supernatant, and then nitrogen blowing to dryness, and then reconstituting with a reconstitution solution.

3. The detection method according to claim 2, characterized in that, The reconstitution solution is a mixed solution of water containing 5-15 mmol / mL ammonium acetate and methanol, the trace plasma or serum is 30-60 μL of plasma or serum, the concentration of the MTX internal standard solution is 200-220 ng / mL, the concentration of the DTT solution is 5-15 mg / mL, and the concentrations of VC and CA in the methanol-acetonitrile mixed solution are both 80-120 μg / mL.

4. The method of claim 1, wherein, The liquid chromatography conditions comprise: a chromatographic column: a C18 reversed-phase column; a mobile phase: A phase is 0.2% acetic acid-10 mmol / mL ammonium acetate aqueous solution, and B phase is methanol; a gradient elution program: 0-1 min, 4% B; 1-4 min, rising to 80% B; 4-4.1 min, rising to 99% B; 4.1-8 min, keeping 99% B; 8-8.1 min, falling to 4% B; 8.1-10 min, keeping 4% B.

5. The method of claim 1, wherein The mass spectrometry conditions comprise: an ion source: positive ion electrospray (ESI+); a drying gas temperature: 200°C, a drying gas flow rate: 12 L / min, and an atomization gas pressure: 30 psi; a sheath gas temperature: 400°C, a sheath gas flow rate: 12 L / min, a capillary voltage: 2500 V (+), 3000 V (+), a nozzle voltage: 0 V (+), 1500 V (+); and a detection mode: multi-reaction monitoring (MRM) mode.

6. The detection method according to claim 1, characterized in that, The standard stock solution and the internal standard stock solution are both prepared by using a methanol-water solution as a solvent, and the methanol-water solution contains 80-120 μg / mL VC, 80-120 μg / mL CA and 10-20 mg / mL DTT.

7. The method of claim 1, wherein, The standard curve of the detection method is prepared by using a matrix standard, taking (the peak area of the measured substance-the peak area of the blank) / the peak area of the MTX internal standard as the dependent variable, taking the theoretical concentration of the matrix standard as the independent variable, adopting a quadratic polynomial fitting, and setting the intercept as 0.

8. The detection method according to claim 7, characterized in that, The matrix standard is prepared by using fetal bovine serum.

9. The detection method according to claim 1, characterized in that, The detection limit and the quantification limit of the detection method range from 0.0015 to 0.4220 ng / mL and from 0.0049 to 1.4041 ng / mL, respectively.

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