A folic acid quality control formulated using an alternative matrix

Abstract

The present invention provides a folic acid quality control product prepared using an alternative matrix, belonging to the field of medical testing technology. The folic acid quality control product comprises 5-methyltetrahydrofolate, 5,10-methylenetetrahydrofolate, and an alternative matrix; the alternative matrix comprises bovine hemoglobin, L-ascorbic acid, and glycine-hydrochloric acid buffer (pH 3.0). The alternative matrix can not only meet the matrix effect requirements, but also stabilize 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate for a long time. In addition, the matrix components can be obtained from commercial channels at a low 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, and is of great significance for the market promotion and application of folic acid detection kits.

Description

Technical Field

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

[0002] Folic acid (Folate) is a general term for a class of water-soluble B vitamins naturally present in food. There are about 100 subtypes of folic acid. Its metabolic forms in the human body mainly include 5-methyltetrahydrofolate (5-MeTHF), dihydrofolate (DHF), 5,10-methylenetetrahydrofolate (5,10-CH2-THF), 5,10-methylenetetrahydrofolate (5,10-CH=THF), 5-formyltetrahydrofolate (5-FoTHF), tetrahydrofolate (THF), 10-formylfolate (10-FoFA), 10-formyltetrahydrofolate (10-FoTHF) and folic acid (pteroylglutamate, FA). Among them, 5-methyltetrahydrofolate is the most stable and has the highest content in the human body, accounting for about 82%~93%; after pteroylglutamate is ingested by the human body, it is reduced to dihydrofolate and tetrahydrofolate successively under the two reduction actions of dihydrofolate reductase. The latter is a coenzyme for one-carbon unit metabolism; the N5 and N10 positions of tetrahydrofolate will be occupied by one-carbon unit substituents with different oxidation levels, and then form metabolites such as 5-formyltetrahydrofolate, 10-formyltetrahydrofolate, 5,10-methylenetetrahydrofolate, 5,10-methylenetetrahydrofolate, 10-formylfolate and 5-methyltetrahydrofolate.

[0003] As a carrier of one-carbon units, folic acid provides an essential precursor for the synthesis of nucleotides, proteins, and amino acids, as well as for DNA methylation modification. Numerous clinical studies have shown that folate deficiency is associated with birth defects such as hyperhomocysteinemia, megaloblastic anemia, neural tube defects, preeclampsia, placental abruption, and intrauterine growth restriction. According to the "Chinese Multidisciplinary Expert Consensus on Rational Folic Acid Supplementation in Clinical Practice," serum folate and red blood cell folate are specific indicators for evaluating clinical folate deficiency / insufficiency. Red blood cell folate levels can reflect chronic or long-term (within four months) folate nutritional status and are more suitable for reflecting tissue folate levels, and are considered by many scholars to be a better folate indicator.

[0004] The main forms of folic acid in human red blood cells are 5-methyltetrahydrofolate (5-MeTHF), 5,10-methylenetetrahydrofolate (5,10-CH2-THF), 5,10-methylenetetrahydrofolate (5,10-CH=THF), 5-formyltetrahydrofolate (5-FoTHF), tetrahydrofolate (THF), 10-formylfolate (10-FoFA) and 10-formyltetrahydrofolate (10-FoTHF). These folic acids can be roughly divided into two categories: methylated folic acid form and non-methylated folic acid form. The methylated folic acid form is 5-methyltetrahydrofolate, and the other folic acid forms are non-methylated folic acid forms. According to existing literature, 5-methyltetrahydrofolate has the highest content and is the most stable. It is the active folic acid and can directly enter the circulation and be absorbed and utilized by the human body. However, various forms of non-methylated folic acid will convert into each other. Under acidic conditions, folic acid forms such as 5-formyltetrahydrofolate, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 10-formyltetrahydrofolate and 10-formylfolate will be partially or completely converted into 5,10-methylenetetrahydrofolate, which can exist stably under acidic conditions.

[0005] Liquid chromatography makes it possible to quantify individual folate metabolites, including polyglutamic acid folate. Liquid chromatography-tandem mass spectrometry is the reference method currently used internationally. It has the characteristics of high selectivity, high specificity, and high sensitivity. It can accurately and quantitatively detect individual folate metabolite forms, providing more reference information for clinical use. However, when liquid chromatography-tandem mass spectrometry is used in clinical testing of folate concentrations in whole blood, plasma, serum, or red blood cell samples, appropriate quality control products are required to evaluate the stability of the detection method and the reliability of the results. However, there are currently no universal folate quality control products on the market. The matrices for the Standard Reference Material 3949 provided by the National Institute of Standards and Technology (NIST) and the WHO International Standard: 03 / 178 provided by the World Health Organization (WHO) are both serum. Furthermore, although the matrix for the WHO International Standard Folate: 95 / 528 provided by the WHO is whole blood, it only contains a total folate value and no individual folate fractions, making it unsuitable for liquid chromatography-tandem mass spectrometry. Furthermore, the aforementioned three international reference materials are expensive, have demanding storage conditions (above -70°C), and have low folate concentrations, making them unsuitable for quality control of red blood cell folate testing in the general population.

[0006] The patent "A substitute matrix for folic acid and 5-methyltetrahydrofolate detection and its application" (application number: 202411553097.9) uses rabbit whole blood, L-ascorbic acid and preservatives to prepare the substitute matrix, but the matrix preparation method is complicated and requires dilution of rabbit whole blood. The dilution ratio will affect the effect of the substitute matrix, and it is difficult to control the dilution strength, which is not conducive to industrial production; the patent "A kit and method for detecting folic acid and its metabolites" (application number: 202310994047.3) provides a substitute matrix composed of bovine serum albumin, PBS buffer, preservatives, and stabilizers. The quality control products and calibrators prepared from this matrix need to be freeze-dried for storage. 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-methyltetrahydrofolate 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 microorganisms will accelerate the degradation of folic acid. Therefore, adding preservatives can inhibit microbial growth and reduce folic acid consumption, which is beneficial to extend the shelf life of folic acid quality control products. However, preservatives may cause skin allergic reactions, and their smoke is irritating to the eyes and upper respiratory tract.

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

[0008] To address the shortcomings of existing technologies, such as complex preparation and use procedures, high costs, demanding storage conditions, and a limited scope of application, the present invention provides a folic acid quality control product formulated using an alternative matrix, belonging to the field of medical testing technology. The folic acid quality control product comprises 5-methyltetrahydrofolate, 5,10-methylenetetrahydrofolate, and an alternative matrix; the alternative matrix comprises bovine hemoglobin, L-ascorbic acid, and glycine-hydrochloric acid buffer (pH 3.0). The alternative matrix not only meets matrix effect requirements but also provides long-term stability for 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate. Furthermore, the matrix components are commercially available at a low 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, and is of great 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] In one aspect, the present invention provides a surrogate matrix comprising hemoglobin.

[0011] Hemoglobin (Hb) is an iron-containing protein found in vertebrate red blood cells. It is primarily responsible for oxygen transport and partial carbon dioxide transport. Hemoglobin is the primary protein component of the erythrocyte cytoplasm, accounting for 97% of total red blood cell protein. Serum proteins are a complex mixture of proteins, primarily including albumin, globulins, and fibrinogen. Serum proteins are primarily found in plasma and are one of its main components. Therefore, when a blank, authentic matrix is ​​unavailable or difficult to obtain, hemoglobin is often used as a surrogate matrix for red blood cell samples, while serum proteins are often used as a surrogate matrix for plasma or serum samples.

[0012] In some embodiments, 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 embodiments, the surrogate matrix further comprises a buffer. A buffer is a reagent that can maintain a stable pH environment of a solution.

[0014] The buffer solution should be selected based on the characteristics of different forms of folic acid. For example, 5,10-methylenetetrahydrofolic acid is more stable in a solution environment with a pH of 3.0, so a buffer system with a pKa of 3 is more appropriate. Buffer solutions that meet this condition include potassium hydrogen phthalate-hydrochloric acid buffer, glycine-hydrochloric acid buffer, and citric acid-sodium citrate buffer.

[0015] In some embodiments, 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 a glycine-hydrochloric acid buffer, which functions to maintain the pH of the solution at an acidic level, thereby preventing the conversion of 5,10-methylenetetrahydrofolate to other forms. The concentration of glycine in the buffer is 0.1 M to 5 M, preferably 0.25 mol / L to 1.5 mol / L, and more preferably 1 mol / L. The pH of the glycine-hydrochloric acid buffer is 2.5-3.5, preferably 3. Furthermore, when the buffer is acidic, the growth of microorganisms is inhibited, avoiding the use of preservatives.

[0017] In some embodiments, the replacement matrix further comprises an antioxidant, which is intended to protect folic acid from oxidative degradation; the oxidant comprises one or more of L-ascorbic acid, β-mercaptoethanol, and citric acid, preferably L-ascorbic acid.

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

[0019] In another aspect, the present invention provides a folic acid detection reagent, comprising the above-mentioned replacement matrix and folic acid.

[0020] In some embodiments, the folic acid comprises any one or more of 5-methyltetrahydrofolate, dihydrofolate, 5,10-methylenetetrahydrofolate, 5,10-methylenetetrahydrofolate, 5-formyltetrahydrofolate, tetrahydrofolate, 10-formylfolate, 10-formyltetrahydrofolate and folic acid.

[0021] The patent "A Method for Determining Total Red Blood Cell Folate by Liquid Chromatography-Tandem Mass Spectrometry" (Patent No.: ZL202510040370.6) provides a method for calculating total red blood cell folate by detecting the concentrations of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate based on liquid chromatography-tandem mass spectrometry. This method overcomes the limitation of liquid chromatography-tandem mass spectrometry in not being able to detect total red blood cell folate, and is groundbreaking and has great application prospects. However, currently, there is no quality control product suitable for the reagents required for the simultaneous detection of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate in liquid chromatography-tandem mass spectrometry. Based on this, the present invention provides a quality control product for folate detection.

[0022] In some aspects, the folate comprises 5-methyltetrahydrofolate and / or 5,10-methylenetetrahydrofolate.

[0023] In some embodiments, the folic acid is 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate, and the present invention demonstrates that the alternative matrix can simultaneously stabilize the two forms of folic acid.

[0024] In some embodiments, the reagents are used in conjunction with liquid chromatography techniques.

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

[0026] In another aspect, the present invention provides a folic acid detection kit, comprising the above-mentioned alternative matrix and / or the above-mentioned reagent.

[0027] On the other hand, the present invention provides the use of the alternative matrix in at least one of the following aspects: detecting 5-methyltetrahydrofolate and / or 5,10-methylenetetrahydrofolate; preparing a reagent or kit for detecting 5-methyltetrahydrofolate and / or 5,10-methylenetetrahydrofolate; preparing a calibrator for detecting 5-methyltetrahydrofolate and / or 5,10-methylenetetrahydrofolate; and preparing a quality control product for detecting 5-methyltetrahydrofolate and / or 5,10-methylenetetrahydrofolate.

[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, all of which are commercially available. Among them, bovine hemoglobin avoids the ethical issues caused by using human blood as a matrix, and Gly-HCl buffer can effectively maintain the pH of the solution and increase the stability of the substances to be detected (5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate).

[0030] (2) The above-mentioned alternative matrix can stabilize 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate for a long time, and thus 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, which has a simple and fast preparation process, saving preparation time; and does not require the addition of excessive stabilizers and preservatives, thereby reducing the monetary cost of preparation.

[0032] (4) Based on the above-mentioned alternative matrix, the present invention prepares five concentration levels of quality control products suitable for detecting red blood cell folate. The five concentrations (5-methyltetrahydrofolate: 25 ng / mL (54.4 nmol / L) - 420 ng / mL (913.9 nmol / L); 5,10-methylenetetrahydrofolate: 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 liquid chromatography tandem mass spectrometry detection method, and have certain application prospects.

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

[0034] (6) After the folic acid quality control product is stored at -20°C, it can be used directly after thawing without adding additional dissolving solvent. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 This is the chromatogram of 5-methyltetrahydrofolate in the quality control sample at concentration level 1 after pretreatment;

[0037] Figure 2 This is the chromatogram of 5,10-methylenetetrahydrofolate in the quality control sample at concentration level 1 after pretreatment;

[0038] Figure 3 This is the chromatogram of 5-methyltetrahydrofolate in the quality control sample at concentration level 5 after pretreatment;

[0039] Figure 4 This is the chromatogram of 5,10-methylenetetrahydrofolate in the quality control sample at concentration level 5 after pretreatment. DETAILED DESCRIPTION

[0040] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0041] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. For example, "including" or "comprising" mentioned throughout the specification and claims are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present invention, but the description is based on the general principles of the specification and is not intended to limit the scope of the invention. The scope of protection of the present invention shall be as defined in the attached 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 method of the compounds 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate in the quality control products in the following examples is as follows:

[0046] 1. Preparation of 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-HCl buffer (pH 3.0), and vortex to mix to prepare a surrogate matrix with a bovine hemoglobin concentration of 1% (w / v) and a L-ascorbic acid concentration of 1% (w / v).

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

[0048] 2. Sample pretreatment: Pipette 50 μL of reaction solution (containing 200 ng / mL 5-methyltetrahydrofolate internal standard, 100 ng / mL 5,10-methylenetetrahydrofolate internal standard, 11.2 U / μL γ-glutamyl hydrolase, 2% L-ascorbic acid solution and 0.5% β-mercaptoethanol solution) and 200 μL of extract (2% L-ascorbic acid solution and 0.5% β-mercaptoethanol solution) into the wells of a 96-well plate, add 50 μL of calibrators (5-methyltetrahydrofolate: 1, 5, 10, 50, 200, 500 ng / mL; 5,10-methylenetetrahydrofolate: 0.5, 2.5, 5, 25, 100, 250 ng / mL) and quality control respectively, mix by pipetting up and down with a pipette tip, incubate in a 37°C water bath in the dark for 60 min; add 300 μL of 5-methyltetrahydrofolate and 0.5% β-mercaptoethanol. After adding μL of deproteinized solution (15% sulfosalicylic acid solution), the mixture was shaken for 30 s and centrifuged at 2,000 × g for 20 min. 200 μL of the supernatant was injected for analysis.

[0049] 3. Mass Spectrometry Analysis: Mobile phase A was an aqueous solution containing 0.1% formic acid and 10 mM ammonium formate, and mobile phase B was an acetonitrile solution containing 0.1% formic acid and 10 mM ammonium formate. The equipment used included a reversed-phase chromatographic column (Waters C18 3 μm 100 × 3.0 mm) and a CalQuant-S liquid chromatography-tandem mass spectrometry detection system. The column oven temperature was 40°C, and the injection volume was 20 μL. The liquid phase gradient used is shown in Table 2. Mass spectrometry parameters are shown in Tables 3 and 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 experimental methods used in the following examples are conventional methods, and three replicates were performed. The materials and reagents used are commercially available unless otherwise specified.

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

[0058] Calibrators should generally be prepared using the same matrix as the test kit samples. However, since folic acid is an endogenous substance, it is difficult to obtain whole blood and red blood cell matrices that do not contain folic acid compounds. Therefore, an alternative matrix or water is used as the matrix for preparing the calibrator. To save costs, water is selected as the matrix for preparing the calibrator in this embodiment.

[0059] As a substance for verifying the performance of the test kit, the quality control product should generally be the same as the sample matrix of the test kit. However, considering the ethical issues brought about by human blood samples, the present invention chooses an alternative matrix to prepare the quality control product. Based on this, in order to screen and obtain the alternative matrix formula with the smallest matrix effect, this embodiment first uses fetal bovine serum (Bio-Channel), 1% human hemoglobin sample, 5% human hemoglobin sample, 1% bovine hemoglobin solution, and 5% bovine hemoglobin solution to prepare samples at 6 concentration points (the 6 concentrations are respectively the same as the concentrations of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate in the 6 calibration solution), and then mixes them with water matrix samples (calibrator solution sample at the 5th concentration point) at a volume ratio of 1:1 to obtain 30 (6×5) 1:1 mixtures; finally, the water matrix samples, 6 kinds of After pretreatment, samples of fetal bovine serum (FBS) at 6 different concentrations, samples of 1% human hemoglobin at 6 different concentrations, samples of 5% human hemoglobin at 6 different concentrations, samples of 1% bovine hemoglobin at 6 different concentrations, and samples of the corresponding 1:1 mixtures were detected by liquid chromatography-tandem mass spectrometry to obtain the peak area ratios of various samples (peak area of ​​target compound / peak area of ​​corresponding internal standard). The matrix effects of different surrogate matrices were evaluated by calculating the relative deviations to ensure that the calibrators prepared with water matrix had no relative matrix effect on the quality control products prepared with the five matrices. The calculation formula for the relative deviations is as follows: The test results are shown in Table 5 to Table 11.

[0060] Table 5 Relative matrix effects of water matrix calibrant solutions and fetal bovine serum sample solutions

[0061]

[0062] Table 6 Relative matrix effects of water matrix calibrant solution and 1% human hemoglobin solution sample

[0063]

[0064] Table 7 Relative matrix effects of water matrix calibrant solution and 5% human hemoglobin solution sample

[0065]

[0066] Table 8 Relative matrix effects of water matrix calibrant solution and 1% bovine hemoglobin solution sample

[0067]

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

[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 shown in Tables 5 to 9, both fetal bovine serum and human hemoglobin samples had relative matrix effects with the calibrators, with relative deviations greater than ±20%. However, there was no relative matrix effect with the bovine hemoglobin solution with the calibrators, with relative deviations less than ±20%. This indicates that the calibrators prepared in water matrix have no relative matrix effect on the quality control materials prepared in bovine hemoglobin matrix. Therefore, bovine hemoglobin is the preferred surrogate matrix for the preparation of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate. Furthermore, from the perspective of absolute matrix effects (instrument response), the 5% bovine hemoglobin solution exhibited a stronger absolute matrix effect (matrix suppression) than the 1% bovine hemoglobin solution. Specifically, the 5% bovine hemoglobin solution exhibited lower responses for the target compounds and internal standard (see Tables 10 and 11). This is likely due to the fact that certain components in the bovine hemoglobin matrix competed with the analytes for charge during ionization, resulting in reduced ionization efficiency and a weakened detection signal. Furthermore, the 5% bovine hemoglobin solution introduced more of these components than the 1% bovine hemoglobin solution, leading to a lower detection signal. Considering raw material cost, the 1% bovine hemoglobin solution was the preferred alternative matrix for the preparation of folic acid quality control.

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

[0076] Based on the results of Example 1, this example is based on a 1% bovine hemoglobin solution, and 1% L-ascorbic acid (AA, an antioxidant), 1 mol / L glycine-hydrochloric acid buffer (Gly-HCl, pH 3.0), 1% L-ascorbic acid + 1 mol / L glycine-hydrochloric acid buffer (pH 3.0) combination to prepare a surrogate matrix, which was then mixed with the mixed stock solution at a volume ratio of 1:399 and 21:479, respectively, to obtain quality control products of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate at concentration levels 1 and 5, respectively. The quality control products were stored in a refrigerator at 2-8°C. The quality control products were taken out of the refrigerator and tested at 0 days, 1 day, 2 days, 3 days, 5 days, 8 days, 11 days, and 15 days, respectively, to investigate the accelerated stability of folic acid (5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate) in the quality control products of different systems. The results are shown in Tables 12 and 13. On day 15, the chromatographic detection diagram of folic acid in 1% bovine hemoglobin + 1% AA + 1 M Gly-HCl solution is shown in detail. Figures 1 to 4 .

[0077] Table 12 Accelerated stability of 5-methyltetrahydrofolate 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-methyltetrahydrofolate detected on day 1 to the detection value of 5-methyltetrahydrofolate detected on day 0. The meanings of 2d / 0d, 3d / 0d, 5d / 0d, 8d / 0d, 11d / 0d, and 15d / 0d are similar, where d means day.

[0080] Table 13 Accelerated stability of 5,10-methylenetetrahydrofolic 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-methylenetetrahydrofolate detected on day 1 to the detection value of 5,10-methylenetetrahydrofolate detected on day 0. The meanings of 2d / 0d, 3d / 0d, 5d / 0d, 8d / 0d, 11d / 0d, and 15d / 0d are similar, where d means day.

[0083] The results show that 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate have the worst stability in a surrogate matrix containing only 1% bovine hemoglobin, followed by 1% bovine hemoglobin + 1 M Gly-HCl matrix and 1% bovine hemoglobin + 1% AA matrix. The 1% bovine hemoglobin + 1% AA + 1 M Gly-HCl matrix is ​​the most stable for the two folic acids. This is because, in the presence of Gly-HCl buffer, the pH of the solution is stably maintained at 3.0, which can significantly enhance the stability of L-ascorbic acid, 5-methyltetrahydrofolate, and 5,10-methylenetetrahydrofolate, thereby protecting the folic acid components in the quality control solution. It should be understood that the reason why the surrogate matrix can increase the stability of 5-methyltetrahydrofolate is that the acidic surrogate matrix reduces the number of microorganisms compared to the neutral surrogate matrix. Furthermore, the concentrations of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate did not affect the protective effect of the 1% bovine hemoglobin + 1% AA + 1 M Gly-HCl matrix. In summary, the stability of the quality control was best when the surrogate matrix was prepared using 1% bovine hemoglobin, 1 M Gly-HCl buffer, and 1% L-ascorbic acid. The concentrations of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate in this quality control ranged from 25 ng / mL to 420 ng / mL and 12.5 ng / mL to 210 ng / mL, respectively.

[0084] Example 3: Effect of buffer type on folic acid stability

[0085] Studies have shown that 5,10-methylenetetrahydrofolate is most stable at pH = 3.0. Example 2 also demonstrates that the presence of a Gly-HCl buffer solution (pH = 3.0) can improve the stability of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate. However, it is unclear whether different types of buffers with the same pH have the same protective effect on the two folic acids. Therefore, in this example, 1 M potassium hydrogen phthalate (KHC8H4O4)-hydrochloric acid (HCl) buffer (pH 3.0), glycine (Gly)-hydrochloric acid (HCl) buffer (pH 3.0), and citric acid (C6H8O7)-sodium citrate (Na3C6H5O7) buffer (pH 3.0) were used to prepare a surrogate matrix (1% BHGB + 1% AA) with bovine hemoglobin (BHGB) and L-ascorbic acid (AA). The surrogate matrix was then mixed with the mixed stock solution at a volume ratio of 1:399 and 2:1, respectively. The quality control samples of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate at concentration level 1 and concentration level 5 were obtained respectively. The quality control samples were stored in a refrigerator at 2-8°C and taken out on day 0, 1 day, 6 days, 10 days and 15 days respectively. The samples were injected and analyzed after pretreatment. The results are shown in Tables 14 and 15.

[0086] Table 14 Stability of 5-methyltetrahydrofolate in quality control products in different buffer systems

[0087]

[0088] Note: 1d / 0d represents the ratio of the detection value of 5-methyltetrahydrofolate detected on day 1 to the detection value of 5-methyltetrahydrofolate detected on day 0. The meanings of 6d / 0d, 10d / 0d, and 15d / 0d are similar, where d means day.

[0089] Table 15 Stability of 5,10-methylenetetrahydrofolate in quality control products in different buffer systems

[0090]

[0091] Note: 1d / 0d represents the ratio of the detection value of 5,10-methylenetetrahydrofolate detected on day 1 to the detection value of 5,10-methylenetetrahydrofolate detected on day 0. The meanings of 6d / 0d, 10d / 0d, and 15d / 0d are similar, where d means day.

[0092] As shown in Tables 14 and 15, the ranking of buffers with the best to worst stabilization effects is as follows: Gly-HCl buffer > KHC8H4O4-HCl buffer ≈ C6H8O7-Na3C6H5O7 buffer. Specifically, regardless of the concentrations of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate in the quality control product, the folic acid degradation rate in the Gly-HCl group within 15 days was between 2% and 4%, while the degradation rates in the other two groups were concentrated between 7% and 10%. This may be because the KHC8H4O4-HCl and citric acid-sodium citrate buffers introduced a large amount of metal ions (K + 、Na + ), which catalyzed the oxidation reaction of folic acid to a certain extent. At the same time, it was found that the buffers ranked from best to worst in instrument response (peak area) were 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] + ), resulting in the protonated molecular ion [M+H] +In summary, to improve the stability efficiency of folic acid and the instrument response, Gly-HCl buffer (pH 3.0) is preferred for the preparation of surrogate matrices and quality control products for 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate.

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

[0094] Example 3 demonstrates that when the buffer pH is the same, different buffer systems have different protective effects on 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate, among which glycine-hydrochloric acid buffer has the best effect. The concentration of glycine affects the pH value and buffering function of the buffer solution, and is therefore one of the factors affecting the stability of the folic acid component in the quality control product. 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 gradually increases. Correspondingly, the high-concentration glycine-hydrochloric acid buffer solution has a strong buffering capacity and can better resist the interference of external acid-base substances, and the pH value changes relatively little. Based on this, in this example, 2 mol / L, 1 mol / L, 0.5 mol / L, and 0.1 mol / L glycine-hydrochloric acid buffer solutions (pH 3.0) were used to prepare surrogate matrices. The surrogate matrices were then mixed with the mixed stock solution at volume ratios of 1:399 and 21:479, respectively, to obtain quality control products of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate at concentration levels 1 and 5, respectively. The quality control products were stored in a refrigerator at 2-8°C, taken out on day 0, 1 day, 6 days, 10 days, and 15 days, and sampled and analyzed after pretreatment. The results are shown in Tables 16 and 17.

[0095] Table 16 Stability of 5-methyltetrahydrofolate in quality control samples with different glycine concentrations

[0096]

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

[0098] Table 17 Stability of 5,10-methylenetetrahydrofolate in quality control samples with different glycine concentrations

[0099]

[0100] Note: 1d / 0d represents the ratio of the detection value of 5,10-methylenetetrahydrofolate detected on day 1 to the detection value of 5,10-methylenetetrahydrofolate detected on day 0. The meanings of 6d / 0d, 10d / 0d, and 15d / 0d are similar, where d means day.

[0101] Tables 16 and 17 show that the accelerated stability of the control product was the worst when the glycine concentration in the buffer system was 0.1 mol / L. As the glycine concentration increased to 1 mol / L, the accelerated stability effect was the best. Subsequently, when the glycine concentration increased to 2 mol / L, there was little difference in the stability of folic acid. This suggests that the addition of bovine hemoglobin and L-ascorbic acid to a 0.1 mol / L glycine-HCl buffer solution (pH 3.0) affected the pH environment and caused significant fluctuations. Specifically, the pH value deviated from 3.0, causing L-ascorbic acid and 5,10-methylenetetrahydrofolate to become unstable, thereby indirectly affecting the stability of 5-methyltetrahydrofolate. However, when the glycine concentration increased to 1 mol / L, the buffer solution maintained a good buffering capacity, and the pH environment was unaffected by the newly added bovine hemoglobin and L-ascorbic acid, remaining constant. Furthermore, the above results also demonstrate that the concentrations of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate do not affect the protective effect of the alternative matrix. In summary, considering the cost of raw materials and the effect of glycine concentration on stabilizing folic acid, the glycine concentration is preferably 1 mol / L.

[0102] Example 5: Effect of antioxidants on the stability of folic acid

[0103] Antioxidants are substances that protect folic acid from oxidative degradation. The presence of these agents provides long-term stability for folic acid quality control products, typically lasting several months, or even a year or two. Without these antioxidants, folic acid in the surrogate matrix would only last for a few dozen hours, or at most a few days, before oxidative degradation. Therefore, in this example, 1% L-ascorbic acid, 1% β-mercaptoethanol, and 0.1 M citric acid were used to prepare surrogate matrices and corresponding quality control materials (1% bovine hemoglobin) at concentration levels 1 and 5, respectively. The quality control materials were stored in a refrigerator at 2-8°C and taken out on days 0 and 15. After pretreatment, the samples were injected and analyzed. The degradation rate of folic acid was used to evaluate the protective effect of different antioxidants on folic acid. The degradation rate = (concentration of 5-methyltetrahydrofolate or 5,10-methylenetetrahydrofolate detected on day 0 - concentration of 5-methyltetrahydrofolate or 5,10-methylenetetrahydrofolate detected on day 15) / concentration of 5-methyltetrahydrofolate or 5,10-methylenetetrahydrofolate detected on day 0 × 100%. The results are shown in Tables 18 and 19.

[0104] Table 18 Stability of 5-methyltetrahydrofolate in quality control samples containing different oxidants

[0105]

[0106] Table 19 Stability of 5,10-methylenetetrahydrofolate in quality control samples containing different oxidants

[0107]

[0108] The results show that all three antioxidants—L-ascorbic acid, β-mercaptoethanol, and citric acid—all protected 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate from degradation to varying degrees. The protective effect of the antioxidants was independent of folate concentration and only related to the type of antioxidant. L-ascorbic acid was the most effective, followed by citric acid, and β-mercaptoethanol was the least effective. This may be because β-mercaptoethanol is a reducing agent that primarily protects folate by inactivating oxidants (such as phenolic compounds). It can reduce disulfide bonds in proteins and denature RNases, thereby preventing RNase degradation of nucleic acids. β-mercaptoethanol also inhibits the oxidation of phenolic compounds, reducing the damage to folate by oxidation products.

[0109] However, β-mercaptoethanol is volatile and highly irritating, and its protective effects 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 regulate the pH of the solution, maintaining an acidic environment to protect folic acid. Citric acid molecules lack active hydrogen atoms or electron pairs and lack reducing properties. Unlike reducing agents such as β-mercaptoethanol and L-ascorbic acid, it cannot donate electrons or hydrogen atoms to reduce other substances. In practical applications, citric acid is often used in combination with other antioxidants, such as L-ascorbic acid, to enhance its antioxidant effect. L-ascorbic acid is a strong reducing agent with antioxidant and free radical scavenging properties. It can protect folic acid from oxidative damage by reducing oxidants, making it suitable for long-term maintenance of folic acid stability. Therefore, 1% L-ascorbic acid is the preferred antioxidant for folic acid protection.

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

[0111] Based on the results of Example 2, this Example further prepared surrogate matrices using a 1% bovine hemoglobin solution as the base, adding 1% L-ascorbic acid (AA, an antioxidant), 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), respectively. The surrogate matrices were then mixed at a volume ratio of 1:399 and 21: 479 was mixed with the mixed stock solution to obtain quality control products of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate at concentration level 1 and concentration level 5, respectively. The quality control products were stored in a -20°C refrigerator and taken out from the refrigerator for testing at 0 days, 15 days, 30 days, 3 months, 6 months, 9 months, 12 months and 13 months, respectively. The quality control products were taken out from the refrigerator and tested after pretreatment to investigate the true stability of folic acid (5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate) 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-methyltetrahydrofolate at -20℃ in quality control products of different systems

[0113]

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

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

[0116]

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

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

[0119] Example 7: A surrogate matrix for the detection of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate

[0120] Based on the research findings of Examples 1-6, this example proposes a surrogate matrix for the detection of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate. The surrogate matrix comprises the following components: bovine hemoglobin, L-ascorbic acid, and Gly-HCl buffer. The concentration of bovine hemoglobin is 0.5% (w / v) to 10% (w / v) (preferably 1%); the concentration of L-ascorbic acid is 0.5% (w / v) to 5% (w / v) (preferably 1%); the pH of the Gly-HCl buffer is 2.5 to 3.5 (preferably 3.0); and the concentration of glycine in the buffer system is 0.1 M to 2 M (preferably 1 M).

[0121] Example 8: A quality control product for 5-methyltetrahydrofolate and / or 5,10-methylenetetrahydrofolate

[0122] The surrogate matrix provided by this invention is designed for two types of folic acid: 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate. 5-methyltetrahydrofolate is highly stable, while 5,10-methylenetetrahydrofolate can be stored for long periods in an acidic environment (pH 3.0). Furthermore, Examples 1-6 demonstrate that the matrix described in Example 7 not only protects both 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate from decomposition, but also demonstrates that its protective efficacy is unaffected by folic acid concentration, effectively protecting both high and low concentrations. A quality control sample formulated with this matrix can be stored at -20°C for over a year. Therefore, this example summarizes the experimental results of the previous examples and provides a quality control sample for 5-methyltetrahydrofolate and / or 5,10-methylenetetrahydrofolate. The quality control sample comprises the surrogate matrix described in Example 7 and 5-methyltetrahydrofolate and / or 5,10-methylenetetrahydrofolate. The concentrations of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate in the quality control sample are 20 ng / mL to 440 ng / mL and 10 ng / mL to 220 ng / mL, respectively. In some embodiments, the concentration of 5-methyltetrahydrofolate is twice the concentration of 5,10-methylenetetrahydrofolate.

[0123] It should be understood that although the present invention only provides a method for preparing a quality control product of folic acid using the surrogate matrix, the matrix can also be used to prepare corresponding calibrators or other reagents if the protection mechanism of the surrogate matrix is ​​the same.

[0124] The basic principles, main features and advantages of the present invention are shown and described above. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the appended claims rather than the above description. Therefore, it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention, and any figure signs in the claims should not be regarded as limiting the claims involved.

Claims

1. Use of a surrogate matrix in at least one of the following aspects: detecting 5-methyltetrahydrofolate and / or 5,10-methylenetetrahydrofolate; preparing a reagent or kit for detecting 5-methyltetrahydrofolate and / or 5,10-methylenetetrahydrofolate; preparing a calibrator for detecting 5-methyltetrahydrofolate and / or 5,10-methylenetetrahydrofolate; preparing a quality control for detecting 5-methyltetrahydrofolate and / or 5,10-methylenetetrahydrofolate; the surrogate matrix comprises bovine hemoglobin, glycine-hydrochloric acid buffer, and L-ascorbic acid.

2. A folic acid detection reagent, characterized in that: The reagent comprises a replacement matrix and folic acid; the replacement matrix comprises bovine hemoglobin, glycine-hydrochloric acid buffer and L-ascorbic acid; the folic acid comprises any one or more of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate.

3. A folic acid detection kit, characterized in that: The kit contains a substitute matrix and / or the reagent according to claim 2; the substitute matrix comprises bovine hemoglobin, glycine-hydrochloric acid buffer and L-ascorbic acid; the folic acid is any one or more of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate.

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