Folic acid quality control product prepared by using alternative matrix

Abstract

The invention provides a folic acid quality control product prepared by using an alternative matrix, and belongs to the technical field of medical detection. The folic acid quality control product is prepared from 5-methyltetrahydrofolic acid, 5, 10-methylenetetrahydrofolic acid and a substitute matrix, the alternative matrix comprises bovine hemoglobin, L-ascorbic acid and a glycine-hydrochloric acid buffer solution (pH 3.0). The alternative matrix can meet the matrix effect requirement and can stabilize the 5-methyltetrahydrofolic acid and the 5, 10-methylenetetrahydrofolic acid for a long time, and the matrix components can be obtained from commercial ways and are low in cost, so that the folic acid quality control product provided by the invention can be widely applied to production and use of folic acid detection reagents or kits, and has wide application prospects. The folic acid detection kit has important significance on market popularization and application of the folic acid detection kit.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection, and particularly relates to a folic acid quality control product prepared using an alternative matrix. Background Art

[0002] Folate is the general name of a class of water-soluble B vitamins naturally present in foods, and there are approximately more than 100 subtypes. Its metabolic forms in the human body mainly include 5-methyltetrahydrofolate (5-MeTHF), dihydrofolate (DHF), 5,10-methylene tetrahydrofolate (5,10-CH2-THF), 5,10-methenyltetrahydrofolate (5,10-CH=THF), 5-formyltetrahydrofolate (5-FoTHF), tetrahydrofolate (THF), 10-formylfolic acid (10-FoFA), 10-formyltetrahydrofolate (10-FoTHF), and folic acid (pteroylglutamic acid, FA), etc. Among them, 5-methyltetrahydrofolate is the most stable and has the highest content in the human body, accounting for about 82% - 93%; after pteroylglutamic acid is ingested by the human body, it is successively reduced to dihydrofolate and tetrahydrofolate under the action of dihydrofolate reductase twice. The latter is a coenzyme for one-carbon unit metabolism; the N5 and N10 positions of tetrahydrofolate are occupied by one-carbon unit substituents with different oxidation levels, and then metabolites such as 5-formyltetrahydrofolate, 10-formyltetrahydrofolate, 5,10-methylene tetrahydrofolate, 5,10-methenyltetrahydrofolate, 10-formylfolic acid, and 5-methyltetrahydrofolate are formed.

[0003] As a carrier of one-carbon units, folate provides necessary precursors for the synthesis of nucleotides, proteins, and amino acids and DNA methylation modification. A large number of clinical studies have shown that folate deficiency is related to hyperhomocysteinemia, megaloblastic anemia, neural tube defects, preeclampsia, placental abruption, fetal growth restriction in utero and other birth defects. According to the "Multidisciplinary Expert Consensus on Rational Clinical Folate Supplementation in China", serum folate and red blood cell folate are specific indicators for evaluating clinical folate deficiency / insufficiency. Among them, the red blood cell folate level can reflect chronic or long-term (within 4 months) folate nutritional status and is more suitable for reflecting the folate level in tissues. It is considered by many scholars to be a better folate indicator.

[0004] The main forms of folic acid in human red blood cells include 5-methyltetrahydrofolic acid (5-MeTHF), 5,10-methylenetetrahydrofolic acid (5,10-CH2-THF), 5,10-methenyltetrahydrofolic acid (5,10-CH=THF), 5-formyltetrahydrofolic acid (5-FoTHF), tetrahydrofolic acid (THF), 10-formylfolic acid (10-FoFA), and 10-formyltetrahydrofolic acid (10-FoTHF), etc. These folic acids can be roughly divided into two categories: methylated folic acid forms and non-methylated folic acid forms. The methylated folic acid form is 5-methyltetrahydrofolic acid, and other folic acid forms are non-methylated folic acid forms. According to the existing literature, 5-methyltetrahydrofolic acid has the highest content and is the most stable. It is active folic acid that can directly enter the circulation and be absorbed and utilized by the human body; while the various forms of non-methylated folic acid can be converted into each other. Under acidic conditions, folic acid forms such as 5-formyltetrahydrofolic acid, tetrahydrofolic acid, 5,10-methylenetetrahydrofolic acid, 10-formyltetrahydrofolic acid, and 10-formylfolic acid will be partially or completely converted into 5,10-methenyltetrahydrofolic acid, and this product can stably exist under acidic conditions.

[0005] Liquid chromatography enables the quantification of single folic acid metabolites including polyglutamate folic acid. Liquid chromatography-tandem mass spectrometry is the reference method currently used internationally, which has the characteristics of high selectivity, high specificity, and high sensitivity. It can accurately quantify and detect single folic acid metabolite forms, providing more reference information for clinical practice. However, when liquid chromatography-tandem mass spectrometry is used to detect the folic acid concentration in whole blood, plasma, serum, or red blood cell samples in clinical practice, appropriate quality control products are required to evaluate the stability of the detection method and the reliability of the results. But there is no universal folic acid quality control product on the market currently. The matrices of Standard Reference Material 3949 provided by the National Institute of Standards and Technology (NIST) of the United States and WHO International Standard: 03 / 178 provided by the World Health Organization (WHO) are both serum; in addition, although the matrix of WHO International Standard Folate: 95 / 528 provided by WHO is whole blood, it only has a total folic acid value and no individual folic acid component values, which is not suitable for liquid chromatography-tandem mass spectrometry; at the same time, the above three international reference substances are expensive, have harsh storage conditions (above -70°C), and have low folic acid concentrations, and are not suitable for use as quality control for population red blood cell folic acid detection.

[0006] The patent "An alternative matrix for the detection of folic acid and 5-methyltetrahydrofolic acid and its application" (Application No.: 202411553097.9) uses rabbit whole blood, L-ascorbic acid and preservatives to prepare the alternative matrix. However, the preparation method of this matrix is complicated. It is necessary to dilute the rabbit whole blood, and the dilution ratio will affect the effect of the alternative matrix. It is difficult to control the dilution intensity, which is not conducive to industrial production. The patent "A kit and method for detecting folic acid and its metabolites" (Application No.: 202310994047.3) provides an alternative matrix composed of bovine serum albumin, PBS buffer, preservatives and stabilizers. The quality control products and calibration products prepared from this matrix need to be stored frozen and dried. After the freeze-dried powder is thawed, it needs to be diluted and dissolved with a solvent before use. The use steps are cumbersome, and its protective effect on folic acid needs to be improved. In addition, the above two alternative matrices are mainly used for the detection of folic acid and 5-methyltetrahydrofolic acid in whole blood, serum or plasma, and both use preservatives. Because the above two alternative matrices are neutral, such an environment is suitable for the survival of a large number of microorganisms, and the microorganisms will accelerate the degradation of folic acid. Therefore, preservatives are added to inhibit the growth of microorganisms and reduce the consumption of folic acid, which is beneficial to extending the effective period of folic acid quality control products. However, the preservatives may cause skin allergic reactions, and their fumes have irritating effects on the eyes and upper respiratory tract.

[0007] Therefore, there is an urgent need for a folic acid quality control product with a lower cost, less demanding storage conditions, and suitable for the detection of folic acid in red blood cell samples. Summary of the Invention

[0008] In view of the defects of the prior art, such as complex preparation / use steps, high cost, demanding storage conditions, and small application scope, etc., the present invention provides a folic acid quality control product prepared with an alternative matrix, belonging to the technical field of medical detection. The folic acid quality control product contains 5-methyltetrahydrofolic acid, 5,10-methylene tetrahydrofolic acid and an alternative matrix; the alternative matrix contains bovine hemoglobin, L-ascorbic acid and glycine-hydrochloric acid buffer (pH 3.0). The alternative matrix can not only meet the requirements of matrix effects, but also stably maintain 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid for a long time, and the matrix components can be obtained from commercial channels and have a lower cost. Therefore, the folic acid quality control product provided by the present invention can be widely used in the production and use of folic acid detection reagents or kits, which has important significance for the market promotion and application of folic acid detection kits.

[0009] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0010] On the one hand, the present invention provides an alternative matrix, and the alternative matrix contains hemoglobin.

[0011] Hemoglobin (Hb for short) is an iron-containing protein present in the red blood cells of vertebrates, mainly responsible for the transport of oxygen and partial transport of carbon dioxide. Hemoglobin is the main protein component in the cytoplasm of red blood cells, accounting for 97% of the total protein in red blood cells. Serum proteins are a class of complex protein mixtures, mainly including albumin, globulins, and fibrinogen. Serum proteins are mainly present in plasma and are one of the main components of plasma. Therefore, in the premise that it is impossible or difficult to obtain a blank true matrix, hemoglobin is often used as a substitute matrix for red blood cell samples, and serum proteins are often used as substitute matrices for plasma samples or serum samples.

[0012] In some ways, the hemoglobin is human hemoglobin and / or bovine hemoglobin, preferably bovine hemoglobin; further, the concentration of bovine hemoglobin is 0.5% (w / v) - 10% (w / v), preferably 1% (w / v).

[0013] In some ways, the substitute matrix further contains a buffer. A buffer refers to a reagent that can keep the pH environment of a solution stable.

[0014] The buffer needs to be selected according to the characteristics of different forms of folic acid. For example, 5,10-methylenetetrahydrofolic acid has better stability in a solution environment with pH = 3.0, so it is more appropriate to select a buffer system with pKa≈3. Buffers that meet this condition include potassium hydrogen phthalate-hydrochloric acid buffer, glycine-hydrochloric acid buffer, and citric acid-sodium citrate buffer.

[0015] In some ways, the buffer is selected from potassium hydrogen phthalate-hydrochloric acid buffer, glycine-hydrochloric acid buffer, and citric acid-sodium citrate buffer.

[0016] Preferably, the buffer is glycine-hydrochloric acid buffer, whose function is to maintain the pH of the solution at an acidic level, thereby preventing the conversion of 5,10-methylenetetrahydrofolic acid into other forms; the concentration of glycine in the buffer is 0.1 M - 5M, preferably 0.25 mol / L - 1.5 mol / L, more preferably 1 mol / L; the pH of the glycine-hydrochloric acid buffer is 2.5 - 3.5, preferably 3. At the same time, when the buffer is acidic, it also inhibits the growth of microorganisms and avoids the use of preservatives.

[0017] In some ways, the substitute matrix further contains an antioxidant, and the antioxidant is used to protect folic acid from oxidative degradation; the antioxidant contains one or any combination of L-ascorbic acid, β-mercaptoethanol, and citric acid, preferably L-ascorbic acid.

[0018] It is understandable that if the dosage of the antioxidant is insufficient, the stability of folic acid will be greatly reduced; if the dosage of the antioxidant is too high, the cost will increase. In some embodiments, the concentration of the antioxidant is 0.5% (w / v) - 5% (w / v), preferably 1% (w / v).

[0019] On the other hand, the present invention provides a folic acid detection reagent, which comprises the above-mentioned alternative matrix and folic acid.

[0020] In some embodiments, the folic acid comprises any one or more of 5-methyltetrahydrofolic acid, dihydrofolic acid, 5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, tetrahydrofolic acid, 10-formylfolic acid, 10-formyltetrahydrofolic acid, and folic acid.

[0021] The patent "A method for determining total folic acid in red blood cells by liquid chromatography-tandem mass spectrometry" (Patent No.: ZL202510040370.6) provides a method for calculating total folic acid in red blood cells by detecting the concentrations of 5-methyltetrahydrofolic acid and 5,10-methenyltetrahydrofolic acid based on liquid chromatography-tandem mass spectrometry technology. This method makes up for the defect that liquid chromatography-tandem mass spectrometry technology cannot detect total folic acid in red blood cells, and has certain originality and great application prospects. At present, there is no quality control product for reagents required for simultaneous detection of 5-methyltetrahydrofolic acid and 5,10-methenyltetrahydrofolic acid in liquid chromatography-tandem mass spectrometry. Based on this, the present invention provides a folic acid detection quality control product.

[0022] In some embodiments, the folic acid comprises 5-methyltetrahydrofolic acid and / or 5,10-methenyltetrahydrofolic acid.

[0023] In some embodiments, the folic acid is 5-methyltetrahydrofolic acid and 5,10-methenyltetrahydrofolic acid, and the present invention has confirmed that the alternative matrix can stably maintain the above two forms of folic acid simultaneously.

[0024] In some embodiments, the reagent is used in combination with liquid chromatography technology.

[0025] In some ways, the reagent is a quality control product, and the concentration levels of the quality control product are divided into 5 levels. Specifically, in the quality control product of concentration level 1, the concentration of 5-methyltetrahydrofolic acid is 20 - 30 ng / mL, and the concentration of 5,10-methylene tetrahydrofolic acid is 10 - 15 ng / mL; in the quality control product of concentration level 2, the concentration of 5-methyltetrahydrofolic acid is 40 - 60 ng / mL, and the concentration of 5,10-methylene tetrahydrofolic acid is 20 - 30 ng / mL; in the quality control product of concentration level 3, the concentration of 5-methyltetrahydrofolic acid is 100 - 200 ng / mL, and the concentration of 5,10-methylene tetrahydrofolic acid is 50 - 100 ng / mL; in the quality control product of concentration level 4, the concentration of 5-methyltetrahydrofolic acid is 250 - 350 ng / mL, and the concentration of 5,10-methylene tetrahydrofolic acid is 125 - 175 ng / mL; in the quality control product of concentration level 5, the concentration of 5-methyltetrahydrofolic acid is 400 - 440 ng / mL, and the concentration of 5,10-methylene tetrahydrofolic acid is 200 - 220 ng / mL.

[0026] On the other hand, the present invention provides a folic acid detection kit, which contains the above-mentioned alternative matrix and / or the above-mentioned reagent.

[0027] On the other hand, the present invention provides the application of the alternative matrix in at least one of the following aspects: detecting 5-methyltetrahydrofolic acid and / or 5,10-methylene tetrahydrofolic acid; preparing a detection reagent or kit for 5-methyltetrahydrofolic acid and / or 5,10-methylene tetrahydrofolic acid; preparing a calibration product for the detection of 5-methyltetrahydrofolic acid and / or 5,10-methylene tetrahydrofolic acid; preparing a quality control product for the detection of 5-methyltetrahydrofolic acid and / or 5,10-methylene tetrahydrofolic acid.

[0028] The beneficial effects of the present invention include:

[0029] (1) The present invention provides a new alternative matrix, the main components of which are bovine hemoglobin, ascorbic acid and glycine-hydrochloric acid (Gly-HCl) buffer solution, and these components can all be commercially purchased; among them, bovine hemoglobin avoids the ethical issues caused by using human blood as a matrix, and the Gly-HCl buffer solution can effectively maintain the pH of the solution and increase the stability of the substances to be detected (5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid).

[0030] (2) The above-mentioned alternative matrix can stably maintain 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid for a long time, so it can be used to prepare reagents for detecting the two folic acids.

[0031] (3) The present invention provides a preparation process for a quality control product, the preparation process is simple and fast, saving preparation time; and there is no need to add too many stabilizers and preservatives, reducing the preparation cost in terms of money.

[0032] (4) The present invention has formulated quality control products for detecting red blood cell folic acid at 5 concentration levels based on the above-mentioned alternative matrix. The 5 concentrations (5-methyltetrahydrofolic acid: 25 ng / mL (54.4 nmol / L) - 420 ng / mL (913.9 nmol / L); 5,10-methylene tetrahydrofolic acid: 12.5 ng / mL (27.45 nmol / L) - 210 ng / mL (461.2 nmol / L)) cover the clinical reference range of folic acid and the measurement range of the liquid chromatography tandem mass spectrometry detection method, and have certain application prospects.

[0033] (5) The quality control products can be stored and used for a long time without frequent preparation, which helps to improve the detection speed and accuracy.

[0034] (6) After the folic acid quality control products are stored at -20°C, there is no need to add additional dissolving solvents, and they can be directly used after thawing. Description of the Drawings

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0036] Figure 1 It is the chromatogram of 5-methyltetrahydrofolic acid in the quality control product sample of concentration level 1 after pretreatment;

[0037] Figure 2 It is the chromatogram of 5,10-methylene tetrahydrofolic acid in the quality control product sample of concentration level 1 after pretreatment;

[0038] Figure 3 It is the chromatogram of 5-methyltetrahydrofolic acid in the quality control product sample of concentration level 5 after pretreatment;

[0039] Figure 4 It is the chromatogram of 5,10-methylene tetrahydrofolic acid in the quality control product sample of concentration level 5 after pretreatment. Detailed Embodiments

[0040] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0041] It should be noted that in the specification and claims, certain terms are used to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. The specification and claims do not distinguish components by the difference in nouns, but by the difference in the functions of the components. As mentioned throughout the specification and claims, the terms "comprising" or "including" are open-ended terms, and should therefore be interpreted as "including but not limited to". The following description in the specification is the preferred embodiment for implementing the present invention. However, the description is for the purpose of the general principles of the specification and is not intended to limit the scope of the present invention. The protection scope of the present invention shall be determined by the scope defined in the appended claims.

[0042] The sources of the reagents used in the following examples are shown in Table 1.

[0043] Table 1 Reagents

[0044]

[0045] The detection methods for the compounds 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid in the quality control products in the following examples are as follows:

[0046] 1. Preparation of the surrogate matrix: Taking 100 mL as an example, weigh 1 g of solid bovine hemoglobin and 1 g of solid L-ascorbic acid, add 100 mL of 1 mol / L glycine-hydrochloric acid buffer (pH 3.0), vortex and mix well to prepare a surrogate matrix with a bovine hemoglobin concentration of 1% (w / v) and an L-ascorbic acid concentration of 1% (w / v).

[0047] 2. Preparation of folic acid quality control products: Dissolve 5-methyltetrahydrofolic acid solid and 5,10-methylene tetrahydrofolic acid chloride solid in 1% (w / v) L-ascorbic acid solution respectively to obtain 5-methyltetrahydrofolic acid stock solution with a concentration of 0.5-2 mg / mL (preferably 1 mg / mL) and 5,10-methylene tetrahydrofolic acid stock solution with a concentration of 0.1-0.5 mg / mL (preferably 0.25 mg / mL); mix the 5-methyltetrahydrofolic acid stock solution, 5,10-methylene tetrahydrofolic acid stock solution and 1% L-ascorbic acid solution according to the volume ratio of 1: 2: 97 to prepare a mixed stock solution of 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid. In the mixed stock solution, the concentration of 5-methyltetrahydrofolic acid is 2 times that of 5,10-methylene tetrahydrofolic acid. Specifically, the concentration of 5-methyltetrahydrofolic acid in the stock solution is 5-15 μg / mL (preferably 10 μg / mL), and the concentration of 5,10-methylene tetrahydrofolic acid is 2.5-7.5 μg / mL (preferably 5 μg / mL); mix the mixed stock solution with the alternative matrix according to the volume ratios of 1: 399, 1: 199, 3: 197, 3: 97, 21: 479 respectively to obtain 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid quality control products with 5 concentration levels. Specifically, in the quality control product of concentration level 1, the concentration of 5-methyltetrahydrofolic acid is 20-30 ng / mL (preferably 25 ng / mL), and the concentration of 5,10-methylene tetrahydrofolic acid is 10-15 ng / mL (preferably 12.5 ng / mL); in the quality control product of concentration level 2, the concentration of 5-methyltetrahydrofolic acid is 40-60 ng / mL (preferably 50 ng / mL), and the concentration of 5,10-methylene tetrahydrofolic acid is 20-30 ng / mL (preferably 25 ng / mL); in the quality control product of concentration level 3, the concentration of 5-methyltetrahydrofolic acid is 100-200 ng / mL (preferably 150 ng / mL), and the concentration of 5,10-methylene tetrahydrofolic acid is 50-100 ng / mL (preferably 75 ng / mL); in the quality control product of concentration level 4, the concentration of 5-methyltetrahydrofolic acid is 250-350 ng / mL (preferably 300 ng / mL), and the concentration of 5,10-methylene tetrahydrofolic acid is 125-175 ng / mL (preferably 150 ng / mL); in the quality control product of concentration level 5, the concentration of 5-methyltetrahydrofolic acid is 400-440 ng / mL (preferably 420 ng / mL), and the concentration of 5,10-methylene tetrahydrofolic acid is 200-220 ng / mL (preferably 210 ng / mL). The quality control products are stored at -20°C. Before use, transfer them from the low-temperature condition to the room temperature condition. After thawing, they can be detected without additional solvent dilution.

[0048] 2. Pretreatment of samples: Pipette 50 μL of the reaction solution (containing 200 ng / mL 5-methyltetrahydrofolic acid internal standard, 100 ng / mL 5,10-methylene tetrahydrofolic acid internal standard, 11.2 U / μL γ-glutamyl hydrolase, 2% L-ascorbic acid solution, and 0.5% β-mercaptoethanol solution) and 200 μL of the extraction solution (2% L-ascorbic acid solution and 0.5% β-mercaptoethanol solution) into the wells of a 96-well plate. Add 50 μL of calibration standards (5-methyltetrahydrofolic acid: 1, 5, 10, 50, 200, 500 ng / mL; 5,10-methylene tetrahydrofolic acid: 0.5, 2.5, 5, 25, 100, 250 ng / mL) and quality control samples respectively. Mix well by pipetting up and down, and incubate in the dark at 37 °C in a constant temperature water bath for 60 min. After adding 300 μL of the deproteinizing solution (15% sulfosalicylic acid solution), mix well by shaking for 30 s. Centrifuge at 2,000×g for 20 min, and then take 200 μL of the supernatant for injection analysis.

[0049] 3. Mass spectrometry analysis: Mobile phase A used is an aqueous solution containing 0.1% formic acid and 10 mM ammonium formate, and mobile phase B is an acetonitrile solution containing 0.1% formic acid and 10 mM ammonium formate. The equipment used includes a reversed-phase chromatographic column (Waters C18 3 μm 100×3.0 mm) and the Cayman Spectroscopy liquid chromatography-tandem mass spectrometry detection system CalQuant-S. The temperature of the column oven is 40 °C, and the injection volume is 20 μL. The liquid phase gradient is shown in Table 2; the mass spectrometry parameters are shown in Tables 3 - 4.

[0050] Table 2 Liquid phase gradient

[0051]

[0052] Table 3 Mass spectrometry source parameters

[0053]

[0054] Table 4 Mass spectrometry ion pair parameters

[0055]

[0056] Unless otherwise specified, the test methods used in the following examples are all conventional methods, and 3 replicates are set for each; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0057] Example 1: Preliminary exploration of alternative matrix components

[0058] Calibrators generally should have the same matrix as the test samples of the kit. However, since folic acid is an endogenous substance, it is difficult to obtain whole blood and red blood cell matrices without folic acid compounds. Therefore, it is necessary to select an alternative matrix or water as the matrix for preparing calibrators. To save costs, in this embodiment, water is selected as the matrix for preparing calibrators.

[0059] As a substance for verifying the performance of the kit, quality control samples generally should have the same matrix as the test samples of the kit. However, considering the ethical issues brought by human blood samples, the present invention selects an alternative matrix to prepare quality control samples. Based on this, in order to screen out the alternative matrix formulation with the smallest matrix effect, in this example, first, samples at 6 concentration points are prepared using fetal bovine serum (Bio-Channel), 1% human hemoglobin sample, 5% human hemoglobin sample, 1% bovine hemoglobin solution, and 5% bovine hemoglobin solution (the 6 concentrations are the same as the concentrations of 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid in the 6 calibrator solutions), and then they are mixed with the water matrix sample (the sample of the calibrator solution at the 5th concentration point) at a volume ratio of 1:1 to obtain 30 (6×5) 1:1 mixtures; finally, after pretreatment of the water matrix sample, 6 kinds of fetal bovine serum samples at 6 concentrations, 6 kinds of 1% human hemoglobin samples at 6 concentrations, 6 kinds of 5% human hemoglobin samples at 6 concentrations, 6 kinds of 1% bovine hemoglobin samples at 6 concentrations, 6 kinds of 5% bovine hemoglobin samples at 6 concentrations, and the corresponding 1:1 mixture samples, they are detected by liquid chromatography-tandem mass spectrometry technology to obtain the peak area ratios of various samples (peak area of the target compound / peak area of the corresponding internal standard), and the matrix effects of different alternative matrices are evaluated by calculating the relative deviation to ensure that there is no relative matrix effect in the detection of the calibrators prepared with water matrix for the quality control samples prepared with 5 matrices. The calculation formula of the relative deviation is as follows: , and the test results are shown in Tables 5 to 11.

[0060] Table 5 Relative matrix effect between water matrix calibrator solution and fetal bovine serum sample solution

[0061]

[0062] Table 6 Relative matrix effect between water matrix calibrator solution and 1% human hemoglobin solution sample

[0063]

[0064] Table 7 Relative matrix effect between water matrix calibrator solution and 5% human hemoglobin solution sample

[0065]

[0066] Table 8 Relative matrix effect between water matrix calibrator solution and 1% bovine hemoglobin solution sample

[0067]

[0068] Table 9 Relative matrix effects of water matrix calibration solution and 5% bovine hemoglobin solution samples

[0069]

[0070] Table 10 Peak areas of target analytes and corresponding isotope internal standards in 1% bovine hemoglobin solution

[0071]

[0072] Table 11 Peak area data of target analytes and corresponding isotope internal standards in 5% bovine hemoglobin solution

[0073]

[0074] As can be seen from Tables 5 to 9, both fetal bovine serum samples and human hemoglobin samples have relative matrix effects with the calibration product, that is, the relative deviations are all greater than ±20%. However, the bovine hemoglobin solution has no relative matrix effect with the calibration product, and the relative deviations are all less than ±20%. This indicates that the calibration product prepared with water matrix has no relative matrix effect on the quality control product prepared with bovine hemoglobin matrix. Therefore, bovine hemoglobin is preferably selected to prepare the alternative matrix for the detection of 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid. Further, from the perspective of absolute matrix effect (instrument response), the absolute matrix effect (matrix suppression) of 5% bovine hemoglobin solution is stronger than that of 1% bovine hemoglobin solution, that is, the responses of target compounds and internal standards in 5% bovine hemoglobin solution are lower (see Tables 10 and 11). This may be because some components in the bovine hemoglobin matrix compete for charges with the analytes during the ionization process, resulting in a decrease in the ionization efficiency of the analytes, thereby weakening the detection signal. And 5% bovine hemoglobin solution introduces more certain components than 1% bovine hemoglobin solution, so the detection signal is lower. At the same time, considering the raw material cost issue, 1% bovine hemoglobin solution is preferentially selected as the alternative matrix for preparing folic acid quality control products.

[0075] Example 2: Influence of alternative matrix components on the stability of folic acid

[0076] Based on the exploration results of Example 1, in this example, a 1% bovine hemoglobin solution was used as the basis, and 1% L-ascorbic acid (AA, belonging to antioxidants), 1 mol / L glycine-hydrochloric acid buffer (Gly-HCl, pH 3.0), and a combination of 1% L-ascorbic acid + 1 mol / L glycine-hydrochloric acid buffer (pH 3.0) were added respectively to prepare alternative matrices. Then, the alternative matrices were mixed with the mixed stock solution at volume ratios of 1:399 and 21:479 respectively to obtain quality control products of 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid at concentration levels 1 and 5. The quality control products were stored in a refrigerator at 2-8°C. At 0 day, 1 day, 2 days, 3 days, 5 days, 8 days, 11 days, and 15 days respectively, the quality control products were taken out of the refrigerator and tested to investigate the accelerated stability of folic acid (5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid) in quality control products of different systems. The results are shown in Tables 12 and 13. On the 15th day, the chromatogram of folic acid in the 1% bovine hemoglobin + 1% AA + 1 M Gly-HCl solution is shown in detail in Figures 1 to 4 .

[0077] Table 12 Accelerated stability of 5-methyltetrahydrofolic acid in quality control products under different systems at 2-8°C

[0078]

[0079] Note: 1d / 0d represents the ratio of the detection value of 5-methyltetrahydrofolic acid detected on the 1st day to the detection value of 5-methyltetrahydrofolic acid detected on the 0th day. The meanings of 2d / 0d, 3d / 0d, 5d / 0d, 8d / 0d, 11d / 0d, and 15d / 0d are similar. Among them, d: day.

[0080] Table 13 Accelerated stability of 5,10-methylene tetrahydrofolic acid in quality control products under different systems at 2-8°C

[0081]

[0082] Note: 1d / 0d represents the ratio of the detection value of 5,10-methylene tetrahydrofolic acid detected on the 1st day to the detection value of 5,10-methylene tetrahydrofolic acid detected on the 0th day. The meanings of 2d / 0d, 3d / 0d, 5d / 0d, 8d / 0d, 11d / 0d, and 15d / 0d are similar. Among them, d: day.

[0083] As can be seen from the results, 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid have the worst stability in the alternative matrix containing only 1% bovine hemoglobin. Followed by 1% bovine hemoglobin + 1 M Gly-HCl matrix, 1% bovine hemoglobin + 1% AA matrix, while the 1% bovine hemoglobin + 1% AA + 1 M Gly-HCl matrix can most stably maintain the above two folic acids. This is because in the presence of Gly-HCl buffer solution, the pH value of the solution is stably maintained at 3.0, which can significantly enhance the stability of L-ascorbic acid, 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid at the same time, protecting the folic acid components in the quality control product solution. It should be understood that the reason why the alternative matrix can increase the stability of 5-methyltetrahydrofolic acid is that compared with the neutral alternative matrix, the acidic alternative matrix reduces the number of microorganisms. Moreover, the concentrations of 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid do not affect the protective effect of the 1% bovine hemoglobin + 1% AA + 1 M Gly-HCl matrix on them. In summary, when using 1% bovine hemoglobin, 1 M Gly-HCl buffer solution and 1% L-ascorbic acid to prepare the alternative matrix, the stability of the quality control product is the best. The concentration ranges of 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid in the quality control product are 25 ng / mL to 420 ng / mL and 12.5 ng / mL to 210 ng / mL, respectively.

[0084] Example 3: Influence of buffer types on the stability of folic acid

[0085] Previous studies have shown that 5,10-methylene tetrahydrofolic acid is the most stable under the condition of pH = 3.0. At the same time, Example 2 also confirmed that the presence of Gly-HCl buffer solution (pH = 3.0) can improve the stability of 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid. However, it is still unclear whether the protective effects of different types of buffers with the same pH on the above two folic acids are consistent. Therefore, in this example, 1 M potassium hydrogen phthalate (KHC8H4O4)-hydrochloric acid (HCl) buffer solution (pH 3.0), glycine (Gly)-hydrochloric acid (HCl) buffer solution (pH 3.0), citric acid (C6H8O7)-sodium citrate (Na3C6H5O7) buffer solution (pH 3.0) were respectively selected and used together with bovine hemoglobin (BHGB) and L-ascorbic acid (AA) to prepare the alternative matrix (1% BHGB + 1% AA). Then, the alternative matrix was mixed with the mixed stock solution at a volume ratio of 1:399 and 21:479 respectively to obtain the quality control products of 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid at concentration level 1 and concentration level 5. The quality control products were stored in a 2-8 °C refrigerator and taken out on the 0th day, 1st day, 6th day, 10th day, and 15th day respectively. After pretreatment, the samples were analyzed by injection. The results are shown in Tables 14 and 15.

[0086] Table 14 Stability of 5-Methyltetrahydrofolic Acid in Quality Control Products with Different Buffer Systems

[0087]

[0088] Note: 1d / 0d represents the ratio of the measured value of 5-methyltetrahydrofolic acid detected on the 1st day to the measured value of 5-methyltetrahydrofolic acid detected on the 0th day. The same applies to 6d / 0d, 10d / 0d, and 15d / 0d. Here, d: day.

[0089] Table 15 Stability of 5,10-Methylenetetrahydrofolic Acid in Quality Control Products with Different Buffer Systems

[0090]

[0091] Note: 1d / 0d represents the ratio of the measured value of 5,10-methylenetetrahydrofolic acid detected on the 1st day to the measured value of 5,10-methylenetetrahydrofolic acid detected on the 0th day. The same applies to 6d / 0d, 10d / 0d, and 15d / 0d. Here, d: day.

[0092] As can be seen from Table 14 and Table 15, the ranking of buffers with good to poor stability effects is as follows: Gly-HCl buffer > KHC8H4O4-HCl buffer ≈ C6H8O7-Na3C6H5O7 buffer. Specifically, regardless of the concentration of 5-methyltetrahydrofolic acid and 5,10-methylenetetrahydrofolic acid in the quality control products, the folic acid degradation rate in the Gly-HCl group within 15 days is 2% - 4%, while the degradation rates of the other two groups are concentrated in 7% - 10%. This may be because KHC8H4O4-HCl and citric acid-sodium citrate buffers introduce a large amount of metal ions (K + , Na + ), which catalyze the oxidation reaction of folic acid to a certain extent. At the same time, it is found that the ranking of buffers with good to poor instrument response (peak area) is as follows: Gly-HCl buffer > KHC8H4O4-HCl buffer ≈ C6H8O7-Na3C6H5O7 buffer. This is because metal ions (such as K + and Na + ) can form adduct ions (such as [M+Na] + , [M+K] + ) with the target analytes (5-methyltetrahydrofolic acid, 5,10-methylenetetrahydrofolic acid, and the corresponding internal standard compounds), thereby resulting in the protonated molecular ion [M+H] +The reduction leads to a decrease in the instrument response (peak area). In summary, to improve the stability efficiency of folic acid and the instrument response, Gly-HCl buffer (pH 3.0) is preferably used to prepare the alternative matrix and quality control products of 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid.

[0093] Example 4: Effect of glycine concentration on the stability of folic acid

[0094] Example 3 demonstrated that under the condition of the same buffer pH, different buffer systems have different protective effects on 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid. Among them, glycine-hydrochloric acid buffer has the best effect, and the concentration of glycine will affect the pH value and buffering function of the buffer solution, so it is also one of the factors affecting the stability of folic acid components in the quality control products. Specifically, when the glycine concentration is low, the pH value of the buffer solution will tend to the acidic range of hydrochloric acid, that is, the pH value is low. At the same time, the buffering capacity of the low-concentration glycine-hydrochloric acid buffer solution is weak, and the pH value is easily affected by external acid-base substances and fluctuates greatly. As the glycine concentration increases, the pH value of the buffer solution will gradually increase. Correspondingly, the high-concentration glycine-hydrochloric acid buffer solution has a stronger buffering capacity and can better resist the interference of external acid-base substances, and the change of pH value is relatively small. Based on this, in this example, glycine-hydrochloric acid buffer solutions (pH 3.0) with concentrations of 2 mol / L, 1 mol / L, 0.5 mol / L, and 0.1 mol / L are used to prepare the alternative matrix, and then the alternative matrix is mixed with the mixed stock solution at volume ratios of 1:399 and 21:479 respectively to obtain quality control products of 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid at concentration levels 1 and 5. The quality control products are stored in a refrigerator at 2-8°C and taken out on the 0th day, 1st day, 6th day, 10th day, and 15th day respectively. After pretreatment, they are injected for analysis, and the results are shown in Tables 16 and 17.

[0095] Table 16 Stability of 5-methyltetrahydrofolic acid in quality control products with different glycine concentrations

[0096]

[0097] Note: 1d / 0d represents the ratio of the detected value of 5-methyltetrahydrofolic acid detected on the 1st day to the detected value of 5-methyltetrahydrofolic acid detected on the 0th day. The meanings of 6d / 0d, 10d / 0d, and 15d / 0d are similar, where d: day.

[0098] Table 17 Stability of 5,10-methylene tetrahydrofolic acid in quality control products with different glycine concentrations

[0099]

[0100] Note: 1d / 0d represents the ratio of the detected value of 5,10-methylenetetrahydrofolic acid on the 1st day to the detected value of 5,10-methylenetetrahydrofolic acid on the 0th day. The same applies to 6d / 0d, 10d / 0d, and 15d / 0d. Here, d represents day.

[0101] It can be seen from Table 16 to Table 17 that when the glycine concentration in the buffer system is 0.1 mol / L, the accelerated stability of the quality control product is the worst; as the glycine concentration increases to 1 mol / L, the accelerated stability effect is the best; subsequently, when the glycine concentration increases to 2 mol / L, the difference in folic acid stability is not significant. It can be inferred that after adding bovine hemoglobin and L-ascorbic acid to the glycine-hydrochloric acid buffer solution (pH 3.0) with a glycine concentration of 0.1 mol / L, the pH environment in the solution is affected and fluctuates greatly, that is, the pH value deviates from 3.0, resulting in the unstable existence of L-ascorbic acid and 5,10-methylenetetrahydrofolic acid, thus indirectly affecting the stability of 5-methyltetrahydrofolic acid; while when the glycine concentration increases to 1 mol / L, the buffer solution has a good buffer capacity, and the pH environment is not affected by the newly added bovine hemoglobin and L-ascorbic acid, and the pH value remains unchanged. In addition, the above results also prove that the concentrations of 5-methyltetrahydrofolic acid and 5,10-methylenetetrahydrofolic acid do not affect the protective effect of the alternative matrix on them. In summary, considering the raw material cost and the effect of glycine concentration on stabilizing folic acid, the preferred glycine concentration is 1 mol / L.

[0102] Example 5: Influence of Antioxidants on the Stability of Folic Acid

[0103] An antioxidant refers to a substance that can protect folic acid from oxidative degradation. The presence of this reagent will make the folic acid quality control product have long-term stability, usually up to several months, or even one or two years. Without the said antioxidant, the folic acid in the alternative matrix can only be maintained for dozens of hours, at most a few days, and will be oxidized and degraded. Therefore, in this example, 1% L-ascorbic acid, 1% β-mercaptoethanol, and 0.1 M citric acid were respectively used to prepare the alternative matrix and the corresponding quality control products (1% bovine hemoglobin) at concentration levels 1 and 5. The quality control products were stored in a refrigerator at 2-8°C and taken out on the 0th and 15th days. After pretreatment, they were injected for analysis, and the degradation rate of folic acid was used to evaluate the protective effect of different antioxidants on folic acid. The degradation rate = (the concentration of 5-methyltetrahydrofolic acid or 5,10-methylenetetrahydrofolic acid detected on the 0th day - the concentration of 5-methyltetrahydrofolic acid or 5,10-methylenetetrahydrofolic acid detected on the 15th day) / the concentration of 5-methyltetrahydrofolic acid or 5,10-methylenetetrahydrofolic acid detected on the 0th day × 100%. The results are shown in Table 18 to Table 19.

[0104] Table 18 Stability of 5-methyltetrahydrofolic acid in quality control products containing different oxidants

[0105]

[0106] Table 19 Stability of 5,10-methenyltetrahydrofolic acid in quality control products containing different oxidants

[0107]

[0108] It can be seen from the results that the three antioxidants, L-ascorbic acid, β-mercaptoethanol, and citric acid, can all protect 5-methyltetrahydrofolic acid and 5,10-methenyltetrahydrofolic acid from degradation to varying degrees. The protective effect of the antioxidant is independent of the concentration of folic acid and only related to the type of antioxidant. Among them, L-ascorbic acid has the best effect, followed by citric acid, and β-mercaptoethanol is the worst. This may be because β-mercaptoethanol is a reducing agent that mainly protects folic acid by destroying the activity of oxidants (such as phenolic compounds). It can reduce the disulfide bonds in proteins and denature RNase, thereby preventing RNase from degrading nucleic acids. In addition, β-mercaptoethanol can also inhibit the oxidation of phenolic compounds and reduce the damage of oxidation products to folic acid.

[0109] However, β-mercaptoethanol is volatile and highly irritating, and its protective effect can generally only last for 2-3 days. Citric acid is a weak acid, but it is not a reducing agent itself. Its mechanism of action is to adjust the pH value of the solution and maintain an acidic environment to protect folic acid. There are no active hydrogen atoms or electron pairs in the citric acid molecule, so it does not have reducibility and cannot provide electrons or hydrogen atoms like reducing agents (such as β-mercaptoethanol, L-ascorbic acid, etc.) to reduce other substances. In practical applications, citric acid is often used in combination with other antioxidants (such as L-ascorbic acid) to enhance the antioxidant effect. L-ascorbic acid is a strong reducing agent with antioxidant and free radical scavenging effects. It can protect folic acid from oxidative damage by reducing oxidants and is suitable for long-term protection of the stability of folic acid. In summary, 1% L-ascorbic acid is preferably used as an antioxidant to protect folic acid.

[0110] Example 6: Stability of folic acid in quality control products of different systems stored at -20 °C

[0111] Based on the exploration results of Example 2, in this example, based on a 1% bovine hemoglobin solution, 1% L-ascorbic acid (AA, belonging to antioxidants), 1 mol / L glycine-hydrochloric acid buffer (Gly-HCl, pH 3.0), and a combination of 1% L-ascorbic acid + 1 mol / L glycine-hydrochloric acid buffer (pH 3.0) were respectively added to prepare alternative matrices. Then, the alternative matrices were mixed with the mixed stock solution at volume ratios of 1:399 and 21:479 respectively to obtain quality control products of 5-methyltetrahydrofolic acid and 5,10-methylenetetrahydrofolic acid at concentration levels 1 and 5. The quality control products were stored in a -20°C refrigerator and taken out for testing on the 0th day, 15th day, 30th day, 3rd month, 6th month, 9th month, 12th month, and 13th month. The quality control products were taken out from the refrigerator, pretreated, and then tested to investigate the true stability of folic acid (5-methyltetrahydrofolic acid and 5,10-methylenetetrahydrofolic acid) in quality control products of different systems stored at -20°C. The results are shown in Tables 20 and 21.

[0112] Table 20 Stability of 5-methyltetrahydrofolic acid in quality control products of different systems at -20°C

[0113]

[0114] Note: 15d / 0d represents the ratio of the measured value of 5-methyltetrahydrofolic acid detected on the 15th day to the measured value of 5-methyltetrahydrofolic acid detected on the 0th day. The meanings of 30d / 0d, 3M / 0d, 6M / 0d, 9M / 0d, 12M / 0d, and 13M / 0d are similar. Among them, d: day, M: month.

[0115] Table 21 Stability of 5,10-methylenetetrahydrofolic acid in quality control products of different systems at -20°C

[0116]

[0117] Note: 15d / 0d represents the ratio of the measured value of 5,10-methylenetetrahydrofolic acid detected on the 15th day to the measured value of 5,10-methylenetetrahydrofolic acid detected on the 0th day. The meanings of 30d / 0d, 3M / 0d, 6M / 0d, 9M / 0d, 12M / 0d, and 13M / 0d are similar. Among them, d: day, M: month.

[0118] It can be seen from the results that the matrix of 1% bovine hemoglobin has the worst stability, followed by the matrix of 1% bovine hemoglobin + 1 M Gly-HCl, 1% bovine hemoglobin + 1% AA, and the most stable one is the matrix of 1% bovine hemoglobin + 1% AA + 1 M Gly-HCl. It can be seen that in the presence of Gly-HCl buffer solution, the pH of the solution is stably maintained at 3.0, and the stability of L-ascorbic acid is greatly enhanced, thus protecting the folic acid component in the control product solution. Therefore, the combination of 1% bovine hemoglobin, Gly-HCl buffer solution and L-ascorbic acid has the best stability of the control product, which is consistent with the conclusion of accelerated stability at 2-8°C in Example 2.

[0119] Example 7: An alternative matrix for detecting 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid

[0120] Based on the research results of Examples 1-6, this example proposes an alternative matrix for detecting 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid. The components of the alternative matrix are as follows: bovine hemoglobin, L-ascorbic acid and Gly-HCl buffer. Among them, the concentration of bovine hemoglobin is 0.5% (w / v) - 10% (w / v) (preferably 1% (w / v)); the concentration of L-ascorbic acid is 0.5% (w / v) - 5% (w / v) (preferably 1% (w / v)); the pH of the Gly-HCl buffer is 2.5 - 3.5 (preferably 3.0), and the concentration of glycine in the buffer system is 0.1 M - 2 M (preferably 1 M).

[0121] Example 8: A control product for 5-methyltetrahydrofolic acid and / or 5,10-methylene tetrahydrofolic acid

[0122] The alternative matrix provided by the present invention is designed for two folic acids, 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid. Among them, 5-methyltetrahydrofolic acid has relatively high stability, while 5,10-methylene tetrahydrofolic acid can be stored for a long time under acidic conditions (pH 3.0). Further, Examples 1 to 6 together prove that the matrix described in Example 7 can not only protect 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid from decomposition simultaneously; moreover, its protective effect is not affected by the folic acid concentration, that is, it can protect both high-concentration folic acid and low-concentration folic acid; the quality control product prepared with the matrix can be stored for more than 1 year at -20°C. Therefore, by summarizing the experimental results of the foregoing examples, this example provides a quality control product for 5-methyltetrahydrofolic acid and / or 5,10-methylene tetrahydrofolic acid, which contains the alternative matrix described in Example 7 and 5-methyltetrahydrofolic acid and / or 5,10-methylene tetrahydrofolic acid. Among them, the concentrations of 5-methyltetrahydrofolic acid and 5,10-methylene tetrahydrofolic acid in the quality control product are 20 ng / mL to 440 ng / mL and 10 ng / mL to 220 ng / mL respectively. In some ways, the concentration of 5-methyltetrahydrofolic acid is twice that of 5,10-methylene tetrahydrofolic acid.

[0123] It should be understood that although the present invention only provides a quality control product for folic acid prepared with the alternative matrix, under the condition that the protection mechanism of the alternative matrix is the same, the matrix can also be used to prepare corresponding calibration products or other reagents.

[0124] The above shows and describes the basic principles, main features and advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention, and any reference signs in the claims should not be regarded as limiting the claimed rights.

Claims

1. An alternative substrate, characterized in that, The alternative matrix contains hemoglobin.

2. The alternative substrate according to claim 1, wherein The hemoglobin is human hemoglobin and / or bovine hemoglobin.

3. The alternative matrix according to claim 1, wherein, The matrix further contains a buffer.

4. The alternative substrate according to claim 3, wherein The buffer is selected from potassium hydrogen phthalate-hydrochloric acid buffer, glycine-hydrochloric acid buffer, and citric acid-sodium citrate buffer.

5. The alternative substrate according to claim 3, wherein, The buffer is glycine-hydrochloric acid buffer, and the concentration of glycine in the buffer is 0.1 M to 5 M.

6. The alternative substrate according to claim 1, wherein, The matrix further contains an antioxidant.

7. A folic acid detection reagent, characterized in that, The reagent contains the alternative matrix as described in any one of claims 1 to 6 and folic acid.

8. The reagent according to claim 7, wherein The folic acid contains any one or more of 5-methyltetrahydrofolic acid, dihydrofolic acid, 5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, tetrahydrofolic acid, 10-formylfolic acid, 10-formyltetrahydrofolic acid, and folic acid.

9. A folic acid detection kit, characterized in that, The kit contains the alternative matrix as described in any one of claims 1 to 6 and / or the reagent as described in any one of claims 7 to 8.

10. Use of the alternative matrix as described in any one of claims 1 to 6 in at least one of the following aspects: detecting 5-methyltetrahydrofolic acid and / or 5,10-methenyltetrahydrofolic acid; preparing a reagent or kit for detecting 5-methyltetrahydrofolic acid and / or 5,10-methenyltetrahydrofolic acid; preparing a calibrator for detecting 5-methyltetrahydrofolic acid and / or 5,10-methenyltetrahydrofolic acid; preparing a quality control product for detecting 5-methyltetrahydrofolic acid and / or 5,10-methenyltetrahydrofolic acid.

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