A method for detecting multiple folate metabolism related substances in trace plasma or serum by liquid chromatography-mass spectrometry

Abstract

The present application belongs to the technical field of biological detection, and particularly relates to a method for synchronously quantitatively detecting a plurality of folate metabolism related substances in trace plasma or serum, which comprises: using liquid chromatography-tandem mass spectrometry to detect the trace plasma or serum sample after pretreatment, and synchronously quantitatively detecting the following nine folate metabolism related substances: 5-formyltetrahydrofolate, folate, 5-methyltetrahydrofolate, S-adenosyl-L-methionine, S-adenosyl-L-homocysteine, vitamin B12, pyridoxamine, pyridoxol and pyridoxal. The present application also relates to the application of the detection method in the detection related to the neurological development of children. The detection method of the present application only uses trace plasma or serum, uses methotrexate instead of expensive isotope internal standard to perform more economical detection, and can simultaneously accurately quantify nine folate metabolism related substances. Therefore, the detection method of the present application has important clinical significance and broad market application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a liquid chromatography-mass spectrometry tandem detection method for a variety of folic acid metabolism-related substances in trace amounts of plasma or serum and its application. Background Technology

[0002] Folic acid, an essential water-soluble B vitamin, cannot be synthesized by the human body and must be obtained through food or supplements. Its core functions rely on the synergistic effects of substances involved in folic acid metabolism, achieved through a one-carbon unit metabolic pathway. It not only provides crucial methyl donors for neurotransmitter synthesis, myelination and DNA repair, gene expression regulation, and amino acid metabolism, supporting fetal neural development, but also participates extensively in numerous processes, including hematopoietic cell production, ensuring the quality of male and female gametes in the reproductive system, the growth and development of multiple organs in the embryo, and the homeostasis regulation of the cardiovascular system.

[0003] Several important folic acid metabolism-related substances exist in the body, including folic acid (FA), 5-formyltetrahydrofolate (5-FTHF), and 5-methyltetrahydrofolate (5-MTHF). Folic acid transport depends on endocytosis mediated by the folic acid receptor (FR). If FR function is blocked by folic acid receptor autoantibodies, 5-FTHF can bypass the FR pathway via the reduced folic acid carrier (RFC). This allows it to cross the blood-brain barrier to maintain folic acid metabolism in the central nervous system and also cross the placental barrier to maintain normal fetal growth and development. Folic acid exists primarily in the active form of 5-MTHF in the blood and can enter key tissues such as cerebrospinal fluid, bone marrow, and gonads through transcellular transport mechanisms, playing multiple physiological functions.

[0004] 5-MTHF works synergistically with vitamin B12 (VB12) to catalyze the conversion of homocysteine ​​to methionine. On the one hand, this process can lower blood homocysteine ​​levels, reducing its damage to vascular endothelial cells and thus preventing cardiovascular diseases such as atherosclerosis and hypertension. On the other hand, the generated methionine further synthesizes S-adenosylmethionine (SAM). SAM, as a core methyl donor, supports the methylation needs of the central nervous system (such as neurotransmitter synthesis and myelin maintenance) and provides methyl groups for DNA replication of blood cells in the hematopoietic system, ensuring normal differentiation and maturation of blood cells and preventing megaloblastic anemia. After providing a methyl donor, SAM is converted into S-adenosylhomocysteine ​​(SAH), and the ratio of the two is a core indicator reflecting the body's methylation capacity. Vitamin B6 (VB6) exists in three forms: pyridoxine (PM), pyridoxine (PN), and pyridoxal (PL), which can be interconverted. Its active form, pyridoxal 5'-phosphate, not only participates in neurotransmitter synthesis but also serves as a key coenzyme for serine hydroxymethyltransferase and cystathionine-β-synthase. The former catalyzes the conversion of tetrahydrofolate to 5,10-methylenetetrahydrofolate, while the latter mediates the conversion of homocysteine ​​to cystine via the cystathionine pathway. This not only helps reduce the risk of cardiovascular disease but also provides protection for the amino acid metabolism of hematopoietic cells.

[0005] The aforementioned folic acid metabolic pathway involves nine key substances (FA, 5-FTHF, 5-MTHF, SAM, SAH, VB12, PM, PN, and PL). Abnormal levels of these substances can trigger metabolic disorders in multiple systems: In neurodevelopment, folic acid deficiency in the brain and methylation damage can interfere with nerve function, or hyperhomocysteinemia can cause neurodevelopmental disorders in children, including autism spectrum disorders, neural tube defects, and schizophrenia. In the hematopoietic system, metabolic disorders leading to impaired DNA synthesis can cause megaloblastic anemia, characterized by increased red blood cell volume and decreased hematopoietic efficiency, particularly affecting pregnant women, the elderly, and vegetarians. In the cardiovascular system, hyperhomocysteinemia can increase the risk of myocardial infarction and cerebral infarction by damaging vascular endothelium and promoting lipid deposition. In the field of reproduction and embryonic development, abnormal folic acid metabolism may lead to decreased egg quality, infertility, or early miscarriage in women, and may also cause malformations of multiple organs in the fetus, in addition to neural tube defects, such as the heart and limbs, while affecting sperm motility and morphology in men. Currently, commonly used clinical and laboratory methods for folic acid detection include microbial assays, chemiluminescence immunoassay, high-performance liquid chromatography (HPLC), and isotope dilution HPLC-tandem mass spectrometry (MS / MS). Regarding sample selection, plasma or serum folic acid is considered an indicator of recent folic acid nutritional status, while erythrocyte folic acid reflects chronic or long-term (within 4 months) folic acid nutritional status. Plasma or serum folic acid requires less complex sample processing than erythrocyte folic acid, eliminating the need for complex procedures such as erythrocyte separation and hemolysis, thus reducing sample loss and processing time and improving detection efficiency. From the perspective of detection methods, microbial detection methods rely on the growth characteristics of specific microorganisms, making them susceptible to fluctuations in the culture environment and conditions. They suffer from drawbacks such as low precision, cumbersome operation, long processing time, and low sample throughput, making it difficult to meet the needs of rapid detection of large-scale samples. Chemiluminescent immunoassay has poor specificity. Due to the different binding affinity of folic acid binding proteins to different folic acid metabolites, the detection results are often low. In addition, its analytical range is narrow, and the matrix effect is significant when the sample is diluted, affecting accuracy. High-performance liquid chromatography (HPLC) has limited sensitivity when using ultraviolet detectors, and the use of fluorescence detectors requires post-column derivatization of folic acid, which is complicated. Although isotope dilution liquid chromatography-tandem mass spectrometry (ICL-MS / MS) has high sensitivity and specificity and can detect multiple substances simultaneously, the cost of isotope internal standards is high, and most methods require a large amount of plasma or serum sample (>100 μL), which limits its widespread application.

[0006] By detecting and regulating the levels of these nine folic acid metabolism-related substances, multi-dimensional health management can be achieved: for children, it can promote neurodevelopment and control the progression of neurodevelopmental 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 groups for cardiovascular disease, it can prevent megaloblastic anemia and cardiovascular and cerebrovascular diseases; for patients with anemia, it can identify the cause of folic acid deficiency to guide precise supplementation. By providing personalized testing and treatment services covering the entire population and multiple systems, precise intervention throughout the entire life cycle can be achieved, from preconception prevention and prenatal care to chronic disease prevention and control in adulthood and health maintenance in old age. Summary of the Invention

[0007] To address the shortcomings of existing folic acid-related metabolite detection technologies, this invention develops a liquid chromatography-mass spectrometry tandem detection method for multiple folic acid metabolism-related substances in trace amounts of plasma or serum. This method uses only a trace amount of plasma or serum (50 μL), replaces expensive isotope internal standards with methotrexate for more economical detection, and can simultaneously and accurately quantify nine folic acid metabolism-related substances.

[0008] Specifically, the present invention is achieved through the following technical solutions:

[0009] This invention provides a method for the simultaneous quantitative detection of multiple folic acid metabolism-related substances in trace amounts of plasma or serum. The method includes: using liquid chromatography-tandem mass spectrometry (LC-MS / MS) to detect pretreated trace amounts of plasma or serum samples, and simultaneously quantitatively detecting the following nine folic acid 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), pyridoxine (PM), pyridoxine (PN), and pyridoxal (PL).

[0010] Alternatively, in the above detection method, the preprocessing method includes the following steps:

[0011] (1) Antioxidant treatment: Add MTX as an internal standard solution to plasma or serum samples, then add dithiothreitol (DTT) solution and vortex incubate;

[0012] (2) Protein removal treatment: Add a methanol-acetonitrile mixed solution containing ascorbic acid (VC) and citric acid (CA), vortex and then centrifuge;

[0013] (3) Nitrogen blowing concentration and resolution: Take the supernatant, freeze it, blow it dry with nitrogen, and then resolution with resolution solution.

[0014] Alternatively, in the above detection method, the reconstitution 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.

[0015] Preferably, the complex solution is a mixture of water and methanol containing 10 mmol / mL ammonium acetate, with a water to methanol volume ratio of 96:4.

[0016] Preferably, the trace amount of plasma or serum is 50 μL of plasma or serum.

[0017] Preferably, the concentration of the MTX internal standard solution is 210 ng / mL, the concentration of the DTT solution is 10 mg / mL, 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.

[0018] As an optional approach, the liquid chromatography conditions in the above detection methods include:

[0019] Column: C18 reversed-phase column;

[0020] Mobile phase: Phase A is 0.2% acetic acid-10 mmol / mL ammonium acetate aqueous solution, and Phase B is methanol;

[0021] Gradient elution program: 0-1 min maintain 4% B, 1-4 min increase to 80% B, 4-4.1 min increase to 99% B, 4.1-8 min maintain 99% B, 8-8.1 min decrease to 4% B, 8.1-10 min maintain 4% B;

[0022] Flow rate: 200 μL / min, column temperature: 40℃, injection volume: 10 μL.

[0023] Preferably, the chromatographic column is an Agilent 858700-314 (1.8 μm, 3.0 × 100 mm).

[0024] As an optional approach, the mass spectrometry conditions in the above detection methods include:

[0025] Ion source: Positive ion electrospray (ESI+);

[0026] Drying gas temperature: 200℃, drying gas flow rate: 12L / min, atomizing gas pressure: 30psi;

[0027] Sheath gas temperature: 400℃, sheath gas flow rate: 12L / min, capillary voltage: 2500V (+), 3000V (+), nozzle voltage: 0V (+), 1500V (+).

[0028] Detection mode: Multi-reaction monitoring (MRM) mode.

[0029] Preferably, the MRM parameters for each substance are shown in Table 3.

[0030] As an optional approach, in the above detection method, both the standard stock solution and the internal standard stock solution are prepared using methanol-water solution as the solvent, wherein the methanol-water solution contains 80~120 μg / mL VC, 80~120 μg / mL CA and 5~15 mg / mL DTT.

[0031] 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.

[0032] Alternatively, in the above detection method, the standard stock solution includes:

[0033] FA, SAM, SAH, VB12, PM, PN, PL stock solutions (0.5 mg / mL)

[0034] 5-FTHF and 5-MTHF stock solutions (0.17 mg / mL)

[0035] Alternatively, in the above detection method, the internal standard stock solution is 0.5 mg / mL MTX.

[0036] As an optional approach, in the above detection method, the standard curve of the detection method is prepared by matrix standards, with (analyte peak area - blank peak area) / MTX internal standard peak area as the dependent variable and the theoretical concentration of matrix standards as the independent variable, and is fitted by a quadratic polynomial with the intercept set to 0.

[0037] Alternatively, in the above detection method, the matrix standard is prepared from fetal bovine serum.

[0038] Preferably, the preparation method includes 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, followed by deproteinization, freezing, nitrogen blowing and reconstitution steps.

[0039] Alternatively, in the above detection method, the detection limit and quantitation limit ranges are 0.0015–0.4220 ng / mL and 0.0049–1.4041 ng / mL, respectively.

[0040] Compared with the prior art, the present invention has the following advantages:

[0041] The liquid chromatography-tandem mass spectrometry (LC-MS / MS) method developed in this invention, using methotrexate as an internal standard, exhibits high sensitivity, good precision, and reliable accuracy. Compared to other methods (sample volume > 100 μL), this method reduces sample consumption to 50 μL, significantly reducing the amount of precious clinical samples used and facilitating multiple tests of the same sample and combined detection of multiple indicators, thus significantly improving sample utilization and detection efficiency. Using methotrexate as an internal standard ensures reproducibility and stability of the detection, offering both economic and practical advantages compared to expensive isotope internal standards. Due to the complexity of plasma or serum matrices, numerous endogenous substances with similar physicochemical properties severely interfere with detection sensitivity and accuracy. Currently, there are no reports of simultaneous detection of nine folic acid metabolism-related substances (FA, 5-FTHF, 5-MTHF, SAM, SAH, VB12, PM, PN, and PL) in plasma or serum using LC-MS / MS; existing methods can only detect some of these substances. Attached Figure Description

[0042] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0043] Figure 1 Standard curves for detecting folic acid metabolism-related substances in human plasma or serum using LC-MS / MS methods. Detailed Implementation

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0045] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0046] The present invention will be further illustrated below with reference to the embodiments:

[0047] Example:

[0048] 1. Types of folic acid-related metabolites in plasma or serum

[0049] Information on the nine folic acid metabolism-related substances detected in this invention is shown in Table 1.

[0050] Table 1 Basic Information on Folic Acid-Related Metabolites

[0051]

[0052] 2. Solution preparation

[0053] (1) Methanol-water solution (v:v=1:1, containing 100 μg / mL VC, 100 μg / mL CA, and 15 mg / mL DTT)

[0054] (2) Methanol-acetonitrile solution (v:v=1:1, containing 100 μg / mL VC and 100 μg / mL CA)

[0055] (3) 10 mg / mL DTT solution

[0056] (4) 2000 mmol / mL ammonium acetate solution

[0057] (5) 10 mmol / mL ammonium acetate solution

[0058] (6) Reconstituted solution (containing 10 mmol / mL ammonium acetate solution and methanol, v:v = 96:4)

[0059] (7) Standard stock solution

[0060] i. FA, SAM, SAH, VB12, PM, PN, PL (0.5mg / mL)

[0061] ii. 5-FTHF, 5-MTHF (0.17mg / mL)

[0062] (8) Internal standard stock solution (0.5 mg / mL)

[0063] (9) 1000 ng / mL mixed standard solution

[0064] (10) 1000 ng / mL internal standard solution

[0065] (11) 210 ng / mL internal standard solution

[0066] (12) 262.5 ng / mL internal standard solution

[0067] 3. Standards and blank controls

[0068] A mixed standard solution of folic acid-related metabolites was used as the standard, with each component having a concentration of 1000 ng / mL. A methanol-water solution was used as a blank control.

[0069] 4. Establishment of LC-MS / MS detection method for folic acid metabolism-related substances

[0070] (1) Collection of plasma or serum samples: Collect venous blood using EDTA anticoagulant blood collection tubes according to standard procedures. Centrifuge within 4 hours after collection to collect the supernatant plasma. For serum samples, centrifuge only to collect the supernatant serum. Aliquot into cryovials and immediately place them in the cryovials. An 80°C freezer was used for subsequent testing, and dry ice was used throughout the transportation process to maintain a low-temperature environment.

[0071] (2) Preparation before testing: Take out consumables, reagents, and samples from the refrigerator 1 hour in advance and place them in a foam box filled with ice. Close the box lid to protect them from light during transport. The entire experiment should be conducted in a dark room under a yellow light lamp to avoid ultraviolet light causing the decomposition of folic acid and other substances. All pipetting operations should be performed on ice to maintain a low temperature.

[0072] (3) Standard dilution: The mixed standard solution was serially diluted with methanol-water solution to concentrations 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, and 1.95 ng / ml. After the following steps, the theoretical concentrations of the matrix mixed standard were 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, and 0.39 ng / ml, respectively. The methanol-water solution served as a blank control. The serial dilution steps for the standard are shown in Table 2.

[0073] Table 2. Serial Dilution Table of Standards for Folic Acid-Related Metabolites

[0074]

[0075] (4) Preparation of matrix standards

[0076] i. Antioxidant: Take 12 50 μL fetal bovine serum samples into 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, put them into aluminum foil insulated bags, place them in a multi-tube vortex mixer and incubate at 2000 rpm for 5 min, then equilibrate at 4℃ for 15 min.

[0077] ii. Protein removal: Add 500 μL of methanol-acetonitrile solution containing 100 μg / mL VC and CA (v:v=1:1), place in an aluminum foil insulated bag, place in a multi-tube vortex mixer and incubate at 2000 rpm for 10 min, equilibrate at 4℃ for 15 min, and centrifuge at 12000 rpm at 4℃ for 10 min.

[0078] iii. Freezing: Freeze at 80℃ for 30 minutes, then transfer the supernatant to a centrifuge tube.

[0079] iv. Nitrogen blowing concentration: The supernatant is rapidly blown dry with nitrogen at low temperature.

[0080] v. Reconstitution: After drying, immediately add 30 μL of the reconstitution solution (10 mmol / mL ammonium acetate solution + methanol, v:v = 96:4), place in an aluminum foil insulated bag, and vortex at 2000 rpm for 5 min in a multi-tube vortex mixer. Centrifuge at 12000 rpm for 10 min at 4℃. Transfer the supernatant to a centrifuge tube and vortex to obtain the matrix internal standard solution (the theoretical concentration of the internal standard at this time is 87.5 ng / mL).

[0081] (5) Preparation of matrix standard solutions: Take 11 aliquots of 24 μL matrix internal standard solution into centrifuge tubes. Add 6 μL of mixed standard solutions of various concentration gradients (1000 ng / mL~1.95 ng / mL) and blank control (methanol-water) to each tube sequentially. Vortex to mix, obtaining matrix standard solutions (theoretical concentration 200 ng / mL~0.39 ng / mL) and blank control (theoretical concentration 0 ng / mL) (at this time, the theoretical concentration of internal standard is 70 ng / mL). Transfer the matrix standard solutions to brown sample vials lined with tubes, avoiding air bubbles at the bottom of the tubes, and store at 4℃. Perform LC-MS / MS detection immediately.

[0082] (6) Sample pretreatment

[0083] i. Antioxidant: Take 50 μL of sample plasma or serum into 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, put it into an aluminum foil insulated bag, place it in a multi-tube vortex mixer and incubate at 2000 rpm for 5 min, then equilibrate at 4℃ for 15 min.

[0084] ii. Protein removal: Add 500 μL of methanol-acetonitrile solution containing 100 μg / mL VC and CA (v:v=1:1), place in an aluminum foil insulated bag, place in a multi-tube vortex mixer and incubate at 2000 rpm for 10 min, equilibrate at 4℃ for 15 min, and centrifuge at 12000 rpm at 4℃ for 10 min.

[0085] iii. Freezing: Freeze at 80℃ for 30 minutes, then transfer the supernatant to a centrifuge tube.

[0086] iv. Nitrogen blowing concentration: The supernatant is rapidly blown dry with nitrogen at low temperature.

[0087] v. Reconstitution: After drying, immediately add 30 μL of reconstitution solution (10 mmol / mL ammonium acetate solution, methanol, v:v = 96:4), place in an aluminum foil insulated bag, and vortex at 2000 rpm for 5 min in a multi-tube vortex mixer. Centrifuge at 12000 rpm for 10 min at 4℃. Transfer the supernatant to a brown sample vial lined with a tube (avoid aspirating impurities; the theoretical internal standard concentration is 70 ng / mL at this point), ensuring no air bubbles appear at the bottom of the tube. Store at 4℃ and immediately perform LC-MS / MS analysis.

[0088] (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.

[0089] (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.

[0090] Table 3 MRM parameters of folic acid-related metabolites

[0091]

[0092] (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).

[0093] 5. Evaluation of LC-MS / MS detection methods for folic acid metabolism-related substances

[0094] (1) Standard curve, limit of detection and limit of quantitation

[0095] 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), a standard curve was fitted using a quadratic polynomial method. The limits of detection (LOD) and quantitation (LOQ) were expressed as the concentrations of each analyte corresponding to 3 times and 10 times the instrument background signal, respectively. The fitting equations, LOD, and LOQ for the concentrations of folic acid-related metabolites in plasma or serum are shown in Table 4, and the standard curves are shown in [Table 4]. Figure 1 The correlation coefficients (R²) of the standard curves ranged from 0.9976 to 0.9999, and the limits of detection and quantitation ranged from 0.0015 to 0.4220 ng / mL and 0.0049 to 1.4041 ng / mL, respectively.

[0096] Table 4. Fitting equations, limits of detection, and limits of quantitation for LC-MS / MS methods for detecting folate metabolism-related substances in human plasma or serum.

[0097]

[0098] (2) Precision

[0099] Three plasma or serum samples at different concentrations were selected and tested consecutively for three batches, with eight parallel samples per batch. The precision of the detection method was analyzed. Alternatively, three plasma or serum samples at different concentrations were tested consecutively for three batches, with six parallel samples per batch. The precision of the detection method was analyzed. Intra-batch and inter-batch differences were assessed using relative standard deviation (RSD), RSD = (standard deviation / mean) × 100%. The precision results after repeated testing are shown in Table 5. The intra-batch and inter-batch differences in the detection of folic acid metabolism-related substances in plasma or serum using this method were ≤9.48%.

[0100] Table 5. Intra-assay and inter-assay variability in the detection of folate metabolism-related substances in human plasma or serum using LC-MS / MS methods.

[0101]

[0102] (3) Accuracy

[0103] Spiked samples at high, medium, and low concentrations were prepared by adding mixed standards of different concentrations to plasma or serum matrices. Samples were processed according to the sample pretreatment method, and the spiked recoveries were calculated by detecting the concentration differences in plasma or serum before and after spiking to evaluate the accuracy of the analytical method. The spiked recoveries at different concentrations are shown in Table 6. The spiked recoveries of various folic acid metabolism-related substances in the matrix ranged from 66.82% to 106.93%.

[0104] Table 6. Spiked recoveries of folate metabolism-related substances in human plasma or serum by LC-MS / MS method.

[0105]

[0106] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for simultaneous quantitative detection of multiple folic acid metabolism-related substances in trace amounts of plasma or serum, characterized in that, The detection method includes: using liquid chromatography-tandem mass spectrometry to detect pretreated trace amounts of plasma or serum samples, simultaneously and quantitatively detecting the following nine folic acid metabolism-related substances: 5-formyltetrahydrofolate, folic acid, 5-methyltetrahydrofolate, S-adenosyl-L-methionine, S-adenosyl-L-homocysteine, vitamin B12, pyridoxine, pyridoxine, and pyridoxal. The preprocessing method includes the following steps: (1) Antioxidant treatment: Methotrexate (MTX) was added to plasma or serum samples as an internal standard solution, followed by dithiothreitol (DTT) solution, and the mixture was vortexed and incubated. (2) Protein removal treatment: Add a methanol-acetonitrile mixed solution containing ascorbic acid (VC) and citrate (CA), vortex and then centrifuge; (3) Nitrogen blowing concentration and redissolution: Take the supernatant, freeze it, blow it dry with nitrogen, and then redissolve it with a redissolution solution. Liquid chromatography conditions include: Column: C18 reversed-phase column; Mobile phase: Phase A is 0.2% acetic acid-10 mmol / mL ammonium acetate aqueous solution, and Phase B is methanol; Gradient elution program: 0-1 min maintain 4% B, 1-4 min increase to 80% B, 4-4.1 min increase to 99% B, 4.1-8 min maintain 99% B, 8-8.1 min decrease to 4% B, 8.1-10 min maintain 4% B.

2. The detection method according to claim 1, characterized in that, The reconstitution 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.

3. The detection method according to claim 1, characterized in that, Mass spectrometry conditions include: Ion source: Positive ion electrospray (ESI+); Drying gas temperature: 200℃, drying gas flow rate: 12 L / min, atomizing gas pressure: 30psi; Sheath gas temperature: 400℃, sheath gas flow rate: 12 L / min, capillary voltage: 2500V+, 3000V+, nozzle voltage: 0V+, 1500V+; Detection mode: Multi-reaction monitoring (MRM) mode.

4. The detection method according to claim 1, characterized in that, Both the standard stock solution and the internal standard stock solution were prepared using methanol-water solution as the solvent, wherein the methanol-water solution contained 80-120 μg / mL VC, 80-120 μg / mL CA and 10-20 mg / mL DTT.

5. The detection method according to claim 1, characterized in that, The standard curve of the detection method is prepared by matrix standards. The curve is fitted with a quadratic polynomial with (analyte peak area - blank peak area) / MTX internal standard peak area as the dependent variable and the theoretical concentration of matrix standards as the independent variable, and the intercept is set to 0.

6. The detection method according to claim 5, characterized in that, The matrix standard was prepared from fetal bovine serum.

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