Method and kit for detecting folate and 5-methyltetrahydrofolate in human whole blood

Abstract

The present application relates to the technical field of analytical detection, and in particular to a method and kit for detecting folate and 5-methyltetrahydrofolate in human whole blood. The kit comprises a protective agent Vc solution, folate calibrators and / or quality control products, 5-methyltetrahydrofolate calibrators and / or quality control products, rabbit whole blood diluent, sample extraction solution A containing saturated zinc sulfate solution, sample extraction solution B containing a mixture of methanol, acetonitrile and isopropyl alcohol, tetrabutylammonium hydroxide phosphate buffer and acetonitrile. The kit not only meets the performance indicators, but also has good stability. The detection method and the matching kit have the advantages of low detection cost, simple detection steps and important role in evaluating the content of folate and its metabolites in human whole blood samples.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to a method and kit for detecting folic acid and 5-methyltetrahydrofolate in human whole blood. Background Technology

[0002] Folic acid is not a single molecule but is composed of a series of derivatives. After folic acid is ingested by the human body, it is reduced twice by dihydrofolate reductase to dihydrofolate (DHF) and tetrahydrofolate (THF), the latter being a coenzyme for the metabolism of one-carbon units. The N5 and N10 positions of THF are occupied by one-carbon unit substituents with different oxidation levels, forming the main metabolites including 5-formyltetrahydrofolate (5-FoTHF), 10-formyltetrahydrofolate, 5,10-methylenetetrahydrofolate, 5,10-methylenetetrahydrofolate, and 5-methyltetrahydrofolate (5-MTHF). Among these, 5-MTHF has the highest content in the human body, accounting for approximately 82% to 93%.

[0003] Total folate in erythrocytes includes unmetabolized folate and other metabolites. Folate testing indicators also include several forms of folate (such as tetrahydrofolate, 5-methyltetrahydrofolate, unmetabolized folate, etc.), with 5-methyltetrahydrofolate being the most biologically active and functional form of folate.

[0004] Currently, folic acid test kits utilize methods such as fluorescence immunochromatography, chemiluminescence immunoassay, and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Fluorescence immunochromatography and chemiluminescence immunoassay are both immunoassays and can only detect total folic acid in the blood, not unmetabolized folic acid or its metabolites. While LC-MS / MS can simultaneously detect unmetabolized folic acid and its metabolites, the equipment is expensive and maintenance costs are high.

[0005] Therefore, providing a low-cost and simple method for detecting folic acid and 5-methyltetrahydrofolate in human whole blood, along with a corresponding reagent kit, is a technical challenge that needs to be addressed in this field. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention is proposed.

[0007] First, this invention provides a method for detecting folic acid and 5-methyltetrahydrofolate in human whole blood, comprising:

[0008] The test sample was mixed with vitamin C and incubated at 35–40°C to obtain the first mixture; the calibrator and / or quality control were diluted with rabbit whole blood and then mixed with vitamin C to obtain the calibrator mixture and / or quality control mixture.

[0009] The first mixture, the calibrator mixture, and / or the quality control mixture are mixed with sample extract A and sample extract B, respectively. After centrifugation, the supernatant is separated and analyzed by liquid chromatography. The sample extract A is a saturated zinc sulfate solution, and the sample extract B is a mixture of methanol, acetonitrile, and isopropanol.

[0010] The conditions for the liquid chromatography detection include:

[0011] The chromatographic column is a C18 column;

[0012] Mobile phase A is tetrabutylammonium hydroxide phosphate buffer; mobile phase B is acetonitrile.

[0013] The gradient elution procedure is as follows:

[0014] During the elution process, the sum of the volume percentages of mobile phase A and mobile phase B is 100%.

[0015]

[0016] In the detection method of this invention, the inventors discovered that when using commonly used alternative matrices such as bovine serum, bovine serum albumin solutions of different concentrations, and bovine whole blood as calibrators and / or quality control samples, the matrix effect test failed. When rabbit whole blood was used as an alternative matrix, the compound was directly added to the rabbit whole blood for storage as a calibrator or quality control sample before application, but the compound exhibited poor stability. Furthermore, the inventors attempted to mix the compound, rabbit whole blood, and a stabilizer for use in the matrix effect test, but this also failed. Finally, the inventors discovered that using rabbit whole blood as a diluent to dilute the calibrator and / or quality control sample before detection enabled the above detection method to pass the matrix effect test.

[0017] Furthermore, through extensive experimentation with various combinations of conditions, the inventors discovered that using zinc sulfate as the erythrocyte lysis buffer, combined with a mixture of methanol, acetonitrile, and isopropanol as the protein precipitant, for whole blood sample pretreatment resulted in better erythrocyte lysis, fully releasing the target folic acid and 5-methyltetrahydrofolate, leading to a higher sample extraction rate. Simultaneously, when using tetrabutylammonium hydroxide phosphate buffer as the reversed-phase mobile phase in liquid chromatography detection, combined with acetonitrile as another mobile phase, the aforementioned elution procedure effectively resolved issues such as folic acid peak tailing and peak splitting in the liquid chromatography.

[0018] Preferably, 0.01 g to 0.03 g of vitamin C is added to every 200 μL of test sample, diluted calibrator and / or quality control sample.

[0019] Preferably, the incubation is carried out in the dark.

[0020] Preferably, the sample extract B is a mixture of methanol, acetonitrile, and isopropanol in a volume ratio of (1-4):(0.5-1.5):1.

[0021] And / or, the volume ratio of the sample extract A to the sample extract B is 1:(2-4).

[0022] Preferably, the volume ratio of the sample to be tested to the sample extract A is (3-5):1.

[0023] Preferably, the centrifugation speed is above 15000g and the time is above 5min.

[0024] More preferably, the centrifugation speed is 18000g or higher, and the time is 10 minutes or higher.

[0025] Preferably, the supernatant separated by centrifugation is filtered under positive pressure through a 0.22 μm filter plate and then detected by liquid chromatography.

[0026] Preferably, the chromatographic column is a Pursuit XRs 5C18 column with dimensions of 150×4.6mm.

[0027] Preferably, the column temperature of the chromatographic column is 38–42°C; and / or, the flow rates of the mobile phases A and B are 1–1.5 mL / min.

[0028] Preferably, the tetrabutylammonium hydroxide phosphate buffer solution is a mixed aqueous solution of potassium dihydrogen phosphate, phosphoric acid, and tetrabutylammonium hydroxide methanol solution.

[0029] Preferably, each 1L of the tetrabutylammonium hydroxide phosphate buffer contains 1-3g potassium dihydrogen phosphate, 500-800μL 75% phosphoric acid, and 2-5mL 40% tetrabutylammonium hydroxide methanol solution.

[0030] Preferably, a UV detector in series with a fluorescence detector is used for detection after liquid chromatography.

[0031] Preferably, the wavelength of the ultraviolet detector is 280–285 nm; the excitation wavelength of the fluorescence detector is 292–298 nm, and the emission wavelength is 358–364 nm.

[0032] Furthermore, the present invention provides a kit for implementing the detection method in any of the above schemes, comprising:

[0033] Protective agent vitamin C solution, folic acid calibrator and / or quality control, 5-methyltetrahydrofolate calibrator and / or quality control, rabbit whole blood diluent, sample extract A containing saturated zinc sulfate solution, sample extract B containing a mixture of methanol, acetonitrile and isopropanol, potassium dihydrogen phosphate aqueous solution, phosphoric acid solution, and tetrabutylammonium hydroxide methanol solution.

[0034] Preferably, the rabbit whole blood diluent is composed of rabbit whole blood and a preservative.

[0035] Preferably, the preservative is at least one of proclin 300 and proclin 950.

[0036] More preferably, the rabbit whole blood diluent contains 0.1-0.2 mL of preservative proclin 300 and 0.1-0.2 mL of preservative proclin 950 per 100 mL of rabbit whole blood.

[0037] Preferably, the concentration of the protective agent Vc solution is 1-6%; the sample extract B is a mixture of methanol, acetonitrile and isopropanol in a volume ratio of (1-4):(0.5-1.5):1.

[0038] Preferably, the concentration of potassium dihydrogen phosphate aqueous solution in the kit is 0.15-0.25 g / mL, the concentration of phosphoric acid solution is 70%-80%, and the concentration of tetrabutylammonium hydroxide methanol solution is 35%-45%.

[0039] Preferably, the concentration range of folic acid in the folic acid calibrator is 2000–11000 ng / mL; and the concentration range of 5-methyltetrahydrofolate in the 5-methyltetrahydrofolate calibrator is 500–11000 ng / mL.

[0040] Preferably, the concentration range of the folic acid quality control sample is 3500–8600 ng / mL; the concentration range of the 5-methyltetrahydrofolic acid quality control sample is 2250–8200 ng / mL.

[0041] Preferably, within the above concentration range, the volume ratio of the calibrator and / or quality control sample to the rabbit whole blood diluent is 1:(8-10). At this volume ratio, the matrix effect experiment results are better.

[0042] More preferably, the folic acid calibrator includes C1 to C6, and the concentrations of C1 to C6 are 2000 ng / mL, 2600 ng / mL, 3800 ng / mL, 4700 ng / mL, 7400 ng / mL, and 11000 ng / mL, respectively.

[0043] More preferably, the 5-methyltetrahydrofolate calibrator includes C1 to C6, and the concentrations of C1 to C6 are 500 ng / mL, 1200 ng / mL, 2600 ng / mL, 3650 ng / mL, 6800 ng / mL, and 11000 ng / mL, respectively.

[0044] In some embodiments, the kit also includes a syringe wash additive, which is a 3% to 8% aqueous solution of sodium hydroxide.

[0045] Furthermore, the present invention provides the application of the kits in any of the above-described schemes in the detection of folic acid and 5-methyltetrahydrofolate in human whole blood; the application is for non-disease diagnosis and treatment purposes.

[0046] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0047] This invention addresses the limitations of existing folic acid and its metabolite detection kits on the market, which only offer two methods: immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS / MS). It develops a detection method and matching kit using liquid chromatography to detect unmetabolized folic acid and its metabolite 5-methyltetrahydrofolate. This kit not only meets the performance requirements of the method but also exhibits good stability. The detection method and kit of this invention have low detection costs, simple detection steps, and play an important role in evaluating the content of folic acid and its metabolites in human whole blood samples. Attached Figure Description

[0048] Figure 1 The image shows the chromatogram of folic acid in a human whole blood sample detected by the detection method in Example 1.

[0049] Figure 2 The chromatogram of 5-methyltetrahydrofolate in human whole blood samples is shown in Example 1. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0051] Unless otherwise specified, all methods used in the examples were conventional or performed according to techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents and instruments used without specified manufacturers were all conventional products that could be purchased from legitimate channels.

[0052] Example 1

[0053] This embodiment provides a method for detecting folic acid and 5-methyltetrahydrofolate in human whole blood, the steps of which are as follows:

[0054] 1. Instruments

[0055] Table 1

[0056] Instrument Name model brand liquid chromatograph LC 2500 Haosi Bio 96-well plate oscillator MB100-4A Hangzhou Aosheng 96-well plate centrifuge H2050R Xiangyi 10μL pipette 0.5-10μL eppendorf 200μL pipette 10-200μL eppendorf

[0057] 2. Reagents

[0058] 2.1 Preparation of compound and internal standard stock solutions

[0059] Table 2

[0060]

[0061] 2.2 Preparation of compound working solutions and quality control working solutions

[0062] Table 3

[0063]

[0064] Take 20 μL of C1 solution and add it to 180 μL of rabbit whole blood to make the C1 working solution. C2, C3, C4, C5, C6, LQC, MQC, and HQC are prepared in the proportions shown in Table 4.

[0065] Table 4

[0066] C2 C3 C4 C5 LQC MQC HQC C1(mL) 1.4 1.2 1.05 0.6 1.25 0.8 0.4 C6(mL) 0.1 0.3 0.45 0.9 0.25 0.7 0.1

[0067] 2.3 Preparation of other reagents

[0068] Take 0.1 mL of proclin 300 and 0.1 mL of proclin 950 and add them to 100 mL of rabbit whole blood as a diluent.

[0069] Weigh 5g of L-ascorbic acid (Vc) and dissolve it in 100mL of ultrapure water as a protective agent.

[0070] Weigh 16.67g of zinc sulfate heptahydrate and dissolve it in 100mL of ultrapure water to obtain sample extraction solution A.

[0071] Transfer 500 mL of methanol, 250 mL of acetonitrile, and 250 mL of isopropanol, mix them thoroughly, and use this as sample extraction solution B.

[0072] 2.4 Sample Pretreatment

[0073] Add sample: Transfer 200 μL of the sample to be tested into 2 mL centrifuge tubes, add 200 μL of protective agent, and incubate at 37 °C in the dark for 2 hours;

[0074] Add calibrators and quality control samples: Transfer 20 μL of calibrator and quality control sample solution, add 180 μL of diluent, and add them to 2 mL centrifuge tubes respectively. Add 200 μL of protective agent (calibrators and quality control samples do not need to be incubated).

[0075] Add sample extraction solution A: Transfer 50 μL of sample extraction solution A and add it to the corresponding centrifuge tubes above;

[0076] Add sample extraction solution B: Transfer 150 μL of sample extraction solution B and add it to the corresponding centrifuge tubes above. Shake well at 3000 rpm for 15 min.

[0077] Centrifugation: Centrifuge at 4℃ and 18000g for 10 min;

[0078] Filtration: Take 300 μL of supernatant and place it in a 0.22 μm filter plate, place a new 96-well plate below, and filter under positive pressure;

[0079] Detection: Place the 96-well plate in a liquid chromatography instrument and inject 20 μL of sample for detection.

[0080] 2.5 Chromatographic conditions

[0081] The chromatographic conditions are shown in Table 5.

[0082] Table 5

[0083]

[0084] The chromatogram of a human whole blood sample detected using the above method is shown below. Figure 1 and Figure 2 As shown.

[0085] In addition, this embodiment also provides a supporting kit for implementing the above detection method, the kit comprising:

[0086] Protectant: 5% Vitamin C solution; 25mL per bottle;

[0087] Rabbit whole blood diluent: Mix 100 mL of rabbit whole blood, 0.1 mL of proclin 300, and 0.1 mL of proclin 950, then dispense into 6.5 mL vials.

[0088] Sample extraction solution A: saturated zinc sulfate solution; 6.5 mL per bottle;

[0089] Sample extract solution B: a mixture of methanol, acetonitrile, and isopropanol in a volume ratio of 2:1:1; 19 mL per bottle;

[0090] Mobile phase additive 1: Add 20g of potassium dihydrogen phosphate to 100mL of water, mix well and dispense into 24mL bottles;

[0091] Mobile phase additive 2: 75% phosphoric acid solution; 1.5 mL per bottle;

[0092] Mobile phase additive 3: 40% tetrabutylammonium hydroxide methanol solution; 8mL per bottle;

[0093] Additive for injection washing solution: Add 5g of sodium hydroxide to 100mL of water, mix well and dispense; each bottle contains 1.2mL.

[0094] Folic acid calibrators C1 to C6 have concentrations of 2000 ng / mL, 2600 ng / mL, 3800 ng / mL, 4700 ng / mL, 7400 ng / mL, and 11000 ng / mL, respectively; each bottle contains 60 μL.

[0095] 5-Methyltetrahydrofolate C1-C6, with concentrations of 500 ng / mL, 1200 ng / mL, 2600 ng / mL, 3650 ng / mL, 6800 ng / mL, and 11000 ng / mL respectively; 60 μL per vial;

[0096] Folic acid quality control products LQC, MQC, and HQC have concentrations of 3500 ng / mL, 6200 ng / mL, and 8600 ng / mL, respectively; each bottle contains 60 μL.

[0097] 5-Methyltetrahydrofolate quality control products LQC, MQC, and HQC have concentrations of 2250 ng / mL, 5400 ng / mL, and 8200 ng / mL, respectively; the specification is 60 μL per bottle.

[0098] Example 2

[0099] This embodiment evaluates the linearity of the detection method in Example 1.

[0100] 1. Experimental Design

[0101] Based on the series of working solutions provided in the kit, prepare a standard curve. The analyte concentrations in the calibration standard and quality control samples will be plotted with the analyte-to-internal-standard peak area ratio as the ordinate, weighted (W = 1 / X). 2 The least squares method is used to perform linear regression between the concentration (X) of the analyte and the peak area ratio (Y). The resulting regression equation (Y = aX + b) is the standard curve. The standard curves of three analytical batches are evaluated, the correlation coefficient R of the linear regression is calculated, and the relative deviation between the concentration of the calibrator and the labeled concentration is calculated.

[0102] 2. Judgment criteria

[0103] (1) The r value is greater than 0.99;

[0104] (2) The accuracy of all concentration points should be within 100±15%.

[0105] 3. The experimental results for folic acid and 5-methyltetrahydrofolate are shown in Tables 6 and 7, respectively.

[0106] Table 6

[0107]

[0108] Table 7

[0109]

[0110] 4. Conclusion

[0111] The deviations at all concentration points were within ±15.0%, meeting the acceptance criteria; linear regression: r > 0.99, meeting the acceptance criteria.

[0112] Example 3

[0113] This embodiment evaluates the precision of the detection method in Example 1.

[0114] 1. Experimental Design

[0115] Precision was evaluated by measuring human blood samples at three concentration levels: low, medium, and high. The method was used to perform repeated measurements on samples at the three levels over three days, with five parallel tubes set up each day, and the concentration should cover the quantitative range.

[0116] 2. Judgment criteria

[0117] Acceptable range for intra-batch and inter-batch precision evaluation experiments: CV% ≤ 15%, with a minimum value of ≤ 20%.

[0118] 3. The experimental results for folic acid and 5-methyltetrahydrofolate are shown in Table 8 and Table 9, respectively.

[0119] Table 8

[0120]

[0121]

[0122] Table 9

[0123]

[0124] 4. Conclusion: Both intra-batch precision and inter-batch precision are within acceptable ranges.

[0125] Example 4

[0126] This example evaluates the open-bottle stability of the reagent kit from Example 1.

[0127] 1. Experimental Design

[0128] The standard curve stored at -80℃ was used to test three quality control samples (low, medium, and high) after opening the bottle at 0, 7, 14, and 28 days, with three parallel tubes set up each day.

[0129] 2. Judgment criteria

[0130] The accuracy of all quality control points should be within 100 ± 15%.

[0131] 3. The experimental results for folic acid and 5-methyltetrahydrofolate are shown in Tables 10 and 11, respectively.

[0132] Table 10

[0133]

[0134] Table 11

[0135]

[0136]

[0137] 4. Conclusion: The accuracy of the quality control samples in the kit was within acceptable range when stored at 2–8℃ for 28 days after opening.

[0138] Example 5

[0139] This example evaluates the freeze-thaw stability of the kit from Example 1.

[0140] 1. Experimental Design

[0141] Low, medium, and high quality control samples placed at -20℃ were frozen for at least 12 hours and then thawed at room temperature. This was considered one freeze-thaw experiment. The effects of 1, 2, 3, 4, and 5 freeze-thaw cycles on the quality control concentration were investigated.

[0142] 2. Judgment criteria

[0143] The concentration of samples after 2, 3, 4, and 5 freeze-thaw cycles deviated from that of samples after 1 freeze-thaw cycle by within ±15%.

[0144] 3. The experimental results for folic acid and 5-methyltetrahydrofolate are shown in Table 12 and Table 13, respectively.

[0145] Table 12

[0146]

[0147]

[0148] Table 13

[0149]

[0150] 4. Conclusion: After 5 freeze-thaw cycles, the deviations of the quality control samples after 1 freeze-thaw cycle were all within 15%.

[0151] Example 6

[0152] This example evaluates the freeze-thaw stability of the kit from Example 1.

[0153] 1. Experimental Design

[0154] The linearity and accuracy of the kits stored at -20°C were tested at months 0, 1, 3, 6, and 7.

[0155] 2. Judgment criteria

[0156] (1) The r value is greater than 0.99;

[0157] (2) The accuracy of each concentration point of the calibrator should be within 100±15%;

[0158] (3) The accuracy of all concentration points should be within 100±15%.

[0159] 3. The experimental results for folic acid and 5-methyltetrahydrofolate are shown in Tables 14 and 15, respectively.

[0160] Table 14

[0161]

[0162]

[0163] Table 15

[0164]

[0165]

[0166] 4. Conclusion: The linearity and accuracy of the kit were within acceptable ranges after being stored at -20℃ for 7 months.

[0167] Example 7

[0168] This embodiment evaluates the matrix effect of the detection method in Example 1.

[0169] Verification method: Three quality control samples with low, medium and high concentrations were prepared using human whole blood, and rabbit whole blood samples with the same concentration were also prepared. Each concentration was measured three times, and the mean concentration of each sample was calculated.

[0170] Acceptable standard: Matrix bias (%) = (mean of rabbit whole blood sample - mean of human whole blood sample) / mean of human whole blood sample (%), matrix bias < ±20%.

[0171] The experimental results for folic acid and 5-methyltetrahydrofolate are shown in Tables 16 and 17, respectively.

[0172] Table 16

[0173]

[0174] Table 17

[0175]

[0176] Comparative Example 1

[0177] In this comparative example, the diluent in Example 1 was replaced with whole bovine blood to verify the detection method.

[0178] Verification method: Three quality control samples with low, medium and high concentrations were prepared using human whole blood, and bovine whole blood samples with the same concentration were also prepared. Other detection conditions were the same as in Example 1. Each concentration was measured three times, and the mean concentration of each sample was calculated.

[0179] Acceptable standard: Matrix bias (%) = (mean of bovine whole blood sample - mean of human whole blood sample) / mean of human whole blood sample (%), matrix bias < ±20%.

[0180] The experimental results for folic acid and 5-methyltetrahydrofolate are shown in Tables 18 and 19, respectively.

[0181] Table 18

[0182]

[0183]

[0184] Table 19

[0185]

[0186] Comparative Example 2

[0187] This comparative example uses samples containing stabilizer V. C The detection method was validated by replacing the diluent in Example 1 with rabbit whole blood.

[0188] Verification method: Take 50mL of rabbit whole blood and 50mL of 5% vitamin D. C Aqueous solutions were thoroughly mixed to form a matrix for preparing low, medium, and high quality control samples. Human whole blood samples of the same concentration were also prepared. Other testing conditions were the same as in Example 1. Each concentration was measured three times, and the mean concentration of each sample was calculated.

[0189] Acceptable criteria: Matrix bias (%) = (mean of mixed rabbit whole blood sample - mean of human whole blood sample) / mean of human whole blood sample (%), matrix bias < ±20%.

[0190] The experimental results for folic acid and 5-methyltetrahydrofolate are shown in Tables 20 and 21, respectively.

[0191] Table 20

[0192]

[0193]

[0194] Table 21

[0195]

[0196] The results showed that the matrix effect experiment failed when using bovine whole blood and rabbit whole blood with added stabilizers as diluents to prepare calibrators and quality control samples. Furthermore, the inventors also tried using different concentrations of bovine serum albumin as alternative matrices, but all attempts failed the matrix effect experiment. Only representative comparative examples are provided here, and not all examples are listed here.

[0197] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting folic acid and 5-methyltetrahydrofolate in human whole blood, characterized in that, include: The sample to be tested was mixed with vitamin C and incubated at 35-40°C to obtain the first mixture; the calibrator and / or quality control were diluted with rabbit whole blood and then mixed with vitamin C to obtain the calibrator mixture and / or quality control mixture. The first mixture, the calibrator mixture, and / or the quality control mixture are mixed with sample extract A and sample extract B, respectively. After centrifugation, the supernatant is separated and analyzed by liquid chromatography. The sample extract A is a saturated zinc sulfate solution. The sample extract B is a mixture of methanol, acetonitrile, and isopropanol in a volume ratio of (1~4):(0.5~1.5):1; the volume ratio of sample extract A to sample extract B is 1:(2~4). The conditions for the liquid chromatography detection include: The chromatographic column was a Pursuit XRs 5 C18 column with dimensions of 150 × 4.6 mm; Mobile phase A is tetrabutylammonium hydroxide phosphate buffer; mobile phase B is acetonitrile. The gradient elution procedure is as follows: The sum of the volume percentages of mobile phase A and mobile phase B during elution is 100%. 。 2. The detection method according to claim 1, characterized in that, The supernatant separated by centrifugation was filtered under positive pressure through a 0.22 μm filter plate and then analyzed by liquid chromatography.

3. A kit for implementing the detection method according to claim 1 or 2, characterized in that, include: The sample contains: a vitamin C protective solution, folic acid calibrators and / or quality control samples, 5-methyltetrahydrofolate calibrators and / or quality control samples, rabbit whole blood diluent, sample extract A containing saturated zinc sulfate solution, sample extract B containing a mixture of methanol, acetonitrile, and isopropanol, potassium dihydrogen phosphate aqueous solution, phosphoric acid solution, and tetrabutylammonium hydroxide methanol solution; the concentration of the vitamin C protective solution is 1%–6%; the sample extract B is a mixture of methanol, acetonitrile, and isopropanol in a volume ratio of (1–4):(0.5–1.5):1; the concentration of the potassium dihydrogen phosphate aqueous solution is 0.15–0.25 g / mL, the concentration of the phosphoric acid solution is 70%–80%, and the concentration of the tetrabutylammonium hydroxide methanol solution is 35%–45%.

4. The reagent kit according to claim 3, characterized in that, The rabbit whole blood diluent consists of rabbit whole blood and preservatives.

5. The reagent kit according to claim 4, characterized in that, The rabbit whole blood diluent contains 0.1-0.2 mL of preservative proclin 300 and 0.1-0.2 mL of preservative proclin 950 per 100 mL of rabbit whole blood.

6. The reagent kit according to claim 3, characterized in that, The folic acid concentration range in the folic acid calibrator is 2000~11000 ng / mL; the concentration range of 5-methyltetrahydrofolate in the 5-methyltetrahydrofolate calibrator is 500~11000 ng / mL. And / or, the concentration range of the folic acid quality control sample is 3500~8600 ng / mL; the concentration range of the 5-methyltetrahydrofolic acid quality control sample is 2250~8200 ng / mL.

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