Kit for determining total folic acid in red blood cells by liquid chromatography-tandem mass spectrometry

Abstract

The invention provides a kit for determining total folic acid in red blood cells by liquid chromatography-tandem mass spectrometry, the kit comprises a reaction solution, an extract and a deproteinized solution, and can be used in combination with the liquid chromatography-tandem mass spectrometry to simultaneously detect the concentrations of two folic acid metabolites, namely 5-methyltetrahydrofolic acid and 5, 10-methylenetetrahydrofolic acid in the red blood cells; next, the measured concentration is substituted into a formula (C red blood cell total folic acid = C red blood cell 5-methyltetrahydrofolic acid + a * C red blood cell 5, 10-methylenetetrahydrofolic acid, a correction factor is 3), the red blood cell total folic acid concentration is obtained through calculation, and the formula is obtained through correction of a detection result of a folic acid protein binding method. In a word, the kit is designed on the basis of the transformation relation between different folic acid forms, the vacancy that the total folic acid in the red blood cells cannot be detected by a liquid chromatography-tandem mass spectrometry method is filled, and the kit is simple in use step and accurate in result.

Description

[0001] This application is a divisional application of the parent application, the parent application number is 202510040370.6, and the application date is January 10, 2025 Technical Field

[0002] The present invention relates to the technical field of biological detection, and in particular to a kit for determining total folate in red blood cells by liquid chromatography-tandem mass spectrometry. Background Art

[0003] 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. The metabolic forms of folic acid 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% to 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 being 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 the metabolites formed include 5-formyltetrahydrofolate, 10-formyltetrahydrofolate, 5,10-methylenetetrahydrofolate, 5,10-methylenetetrahydrofolate, 10-formylfolate and 5-methyltetrahydrofolate, etc.

[0004] As a carrier of one-carbon units, folic acid provides the necessary precursors for nucleotide synthesis, protein and DNA methylation, and amino acid synthesis. A large number of clinical studies have shown that folic acid 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 Clinical Folic Acid Supplementation", serum folate and red blood cell folate are specific indicators for evaluating clinical folate deficiency / insufficiency. Among them, red blood cell folate levels can reflect chronic or long-term (within 4 months) folate nutritional status, are more suitable for reflecting folate levels in tissues, and are considered by many scholars to be better folate indicators.

[0005] Currently, the main methods for folate detection include microbiological methods, folate protein binding methods, high-performance liquid chromatography, and liquid chromatography-tandem mass spectrometry. Microbiological methods and folate protein binding methods can only detect total folate concentration and cannot distinguish between individual folate forms. The folate protein binding method can be used to measure total folate concentration in red blood cells. There are two methods for calculating total folate concentration in red blood cells: one is to simultaneously detect the total folate concentration in whole blood and the hematocrit, and calculate the total folate concentration in red blood cells according to the formula "total folate concentration in red blood cells = total folate concentration in whole blood / hematocrit"; the other is to simultaneously detect the total folate concentration in whole blood, plasma, and hematocrit, and then calculate the total folate concentration in red blood cells according to the formula "total folate concentration in red blood cells = [total folate concentration in whole blood - total folate concentration in plasma × (1-hematocrit)] / hematocrit."

[0006] Liquid chromatography makes it possible to quantify single folate metabolites, including polyglutamic acid folate, but its sensitivity and detection speed are lower than those of liquid chromatography-tandem mass spectrometry. Liquid chromatography-tandem mass spectrometry is a reference method currently used internationally. It has the characteristics of high selectivity, high specificity and high sensitivity. It can accurately and quantitatively detect single folate metabolite forms, providing more reference information for clinical practice. Existing literature and patents ("A sample pretreatment method, detection method and kit for folate and its metabolites", application number CN202210474366.7) usually simply add together the detected folates as the total folate concentration, which is not the total folate concentration in the strict sense. At the same time, due to the many forms of folate in the human body, some folate forms are very unstable, and there is a problem of mutual conversion between various folate forms, which makes it impossible for liquid chromatography-tandem mass spectrometry to detect all folate forms. It can only detect a few specific folate forms and cannot obtain the total folate concentration in the strict sense. Summary of the Invention

[0007] To address the above problems, the present invention provides a method for determining total erythrocyte folate by liquid chromatography-tandem mass spectrometry. The total folate refers to the sum of all folate concentrations, rather than simply the sum of the concentrations of several known forms of folate. The method comprises the following steps: first, detecting the concentrations of two folate metabolites, 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate, in erythrocytes by liquid chromatography-tandem mass spectrometry; then, substituting the measured concentrations into the formula (C total erythrocyte folate = C 5-methyltetrahydrofolate + a × C 5,10-methylenetetrahydrofolate, with a correction factor a = 3) to calculate the total erythrocyte folate concentration. The formula is corrected based on the detection results of the folate protein binding method.

[0008] The method is designed based on the forms of folic acid in the body and the conversion relationship between folic acid. The main forms of folic acid in human red blood cells include 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 folate and unmethylated folate. The methylated folate is 5-methyltetrahydrofolate, and the other folate forms are unmethylated folate. 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 fully or partially converted into 5,10-methylenetetrahydrofolate, which can exist stably under acidic conditions.

[0009] Therefore, the present invention uses liquid chromatography-tandem mass spectrometry to detect the concentrations of 5-methyltetrahydrofolate and 5,10-methynyltetrahydrofolate in red blood cells. The 5-methyltetrahydrofolate concentration in red blood cells represents the concentration of methylated folate in red blood cells, and the 5,10-methynyltetrahydrofolate concentration in red blood cells represents the concentration of partial non-methylated folate in red blood cells. The concentration of the missing non-methylated folate in red blood cells is obtained by comparing and correcting the total folate concentration in red blood cells obtained by folate protein binding method or microbial method. The total folate concentration in red blood cells obtained by liquid chromatography-tandem mass spectrometry is then calculated. The calculation formula is: total folate concentration in red blood cells = original 5-methyltetrahydrofolate concentration in red blood cells + a × original 5,10-methynyltetrahydrofolate concentration in red blood cells, where a is the correction coefficient for the total amount of 5,10-methynyltetrahydrofolate in red blood cells and non-methylated folate in red blood cells. Preferably, a=3.

[0010] On the one hand, the present invention provides a method for determining total folate in red blood cells by liquid chromatography-tandem mass spectrometry, the method comprising the following steps: determining the concentrations of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate in red blood cells by liquid chromatography-tandem mass spectrometry; substituting the measured concentrations of the two folic acids into a formula, and then calculating the concentration of total folate in red blood cells.

[0011] From the above detection principle, it can be seen that the concentration of non-methylated folate in red blood cells cannot be detected by liquid chromatography-tandem mass spectrometry. Therefore, the concentration of 5,10-methylenetetrahydrofolate detected by liquid chromatography-tandem mass spectrometry can be corrected to obtain the concentration of non-methylated folate in red blood cells.

[0012] Based on this, it can be assumed that the formula is C 红细胞总叶酸 =C 红细胞5-甲基四氢叶酸 +a×C 红细胞5,10-次甲基四氢叶酸 , where a is the correction factor, such as 1, 2, 3...

[0013] The present invention uses liquid chromatography-tandem mass spectrometry and a folate protein binding method (chemiluminescence method) to measure the folic acid concentration in 210 red blood cell samples, respectively. Linear regression equations are drawn under different correction coefficients (e.g., a=1 to 4), and the linear correlation coefficients of the regression equations are analyzed. It is determined that the best linear correlation coefficient r is obtained when a=3. On the other hand, based on the experimental results, it can also be inferred that the total concentration of unmethylated folic acid that cannot be converted into 5,10-methylenetetrahydrofolate is approximately twice the total concentration of unmethylated folic acid that can be converted into 5,10-methylenetetrahydrofolate.

[0014] Furthermore, the formula is C 红细胞总叶酸 =C 红细胞5-甲基四氢叶酸 +3×C 红细胞5,10-次甲基四氢叶酸 .

[0015] Furthermore, before using liquid chromatography-tandem mass spectrometry to determine the folic acid concentration, the red blood cell saline suspension sample needs to be pretreated; the reagents required for the pretreatment include a reaction solution, an extraction solution, and a deproteinized solution.

[0016] Furthermore, the reaction solution contains 1.6U / μL to 24U / μL of γ-glutamyl hydrolase; although hydrolases within the above activity range can hydrolyze polyglutamate well, for the accuracy of the test results, 11.2U / μL is preferred; specifically, when the total volume of the reaction solution is 50μL, 7μL of 80U / μL of γ-glutamyl hydrolase needs to be added.

[0017] Furthermore, the volume of the extract is 50 μL to 300 μL. Although the extracts within the above volume range can effectively rupture red blood cells and protect folic acid, 200 μL is preferred for the accuracy of the test results.

[0018] Furthermore, the deproteinizing solution contains one or more of sulfosalicylic acid, trichloroacetic acid, and perchloric acid. To ensure the accuracy of the test results, 300 μL of 15% sulfosalicylic acid is preferred because sulfosalicylic acid not only provides acidic conditions that are beneficial for the conversion of various non-methylated folic acids into 5,10-methylenetetrahydrofolate, but also has the highest efficiency in removing impurities.

[0019] Furthermore, after the sample is treated with the extract, the sample needs to be incubated for 15 to 120 minutes; incubation for more than 60 minutes allows the polyglutamic acid form of folic acid to be completely converted into the monoglutamic acid form of folic acid. Therefore, in order to ensure the accuracy of the test results, preferably, 60 minutes to 90 minutes; more preferably, 60 minutes.

[0020] Furthermore, both the reaction solution and the extract contain L-ascorbic acid and β-mercaptoethanol; the chemical reagents L-ascorbic acid and β-mercaptoethanol are antioxidants that can effectively prevent 5-methyltetrahydrofolate and non-methylated folate from being oxidized to pteroylglutamate.

[0021] In another aspect, the present invention provides a method for determining the total folate concentration in red blood cells by liquid chromatography-tandem mass spectrometry, wherein the total folate concentration in red blood cells is obtained by not merely adding together multiple known and common folate concentrations.

[0022] The present invention provides a method for detecting total folate in red blood cells, which has a detection accuracy rate of up to 100%. The method comprises the following steps:

[0023] (1) Sample pretreatment: This includes sequentially treating the red blood cell saline suspension sample with reaction solution, extraction solution, and deproteinization solution;

[0024] (2) Liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode was used to measure the concentrations of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate in red blood cell samples suspended in saline;

[0025] In some specific embodiments, the original red blood cell sample is diluted to 3 times with physiological saline before mass spectrometry detection. This is because after the dilution treatment, the red blood cells are suspended in the physiological saline and are easier to transfer, further ensuring the accuracy and repeatability of the test results.

[0026] (3) Using chemiluminescence or microbiological methods to detect the total folate concentration in the corresponding whole blood samples and plasma samples; in some specific embodiments, chemiluminescence is used to detect the total folate concentration in the sample. This is because such kits with registration certificates can be purchased on the market. The registration certificate means that the kit has been certified by the Food and Drug Administration and the test results are reliable. Therefore, chemiluminescence is used to correct the test results of the mass spectrometry. Specifically, the kit is the folic acid determination kit (chemiluminescence method) Access Folate produced by Beckman Coulter, Inc.

[0027] Similarly, as long as the test results of the selected kit are accurate and reliable, they can be used to correct the test results of liquid chromatography-tandem mass spectrometry.

[0028] (4) Calculate the total erythrocyte folate concentration by liquid chromatography-tandem mass spectrometry: Compare the erythrocyte 5-methyltetrahydrofolate and erythrocyte 5,10-methytetrahydrofolate concentrations obtained by liquid chromatography-tandem mass spectrometry with the total erythrocyte folate obtained by the folate protein binding method or the microbial method to obtain the correction factor a; substitute the formula (total erythrocyte folate concentration = erythrocyte 5-methyltetrahydrofolate concentration + a × erythrocyte 5,10-methytetrahydrofolate concentration) to calculate the total erythrocyte folate concentration by liquid chromatography-tandem mass spectrometry.

[0029] In some specific embodiments, the correction coefficient a=3.

[0030] On the other hand, the present invention provides a kit for determining the total folate concentration of red blood cells, the kit comprising a 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), an extraction solution (2% L-ascorbic acid solution and 0.5% β-mercaptoethanol solution) and a deproteinized solution (15% sulfosalicylic acid solution); the method for using the kit is the same as described above.

[0031] The beneficial effects of the present invention include:

[0032] 1. Liquid chromatography-tandem mass spectrometry was used to measure the concentrations of two common forms of folic acid. The calculation formula provided by this invention can be used to obtain the total red blood cell folate concentration by liquid chromatography-tandem mass spectrometry. Previously, liquid chromatography-tandem mass spectrometry could only measure the concentrations of several known forms of folic acid and then simply add them together, which did not represent the true total red blood cell folate concentration.

[0033] 2. This method is simple to operate and has accurate test results, with an accuracy rate of up to 100%;

[0034] 3. The factors that affect the results of the liquid chromatography-tandem mass spectrometry method provided by the present invention for detecting the total folate concentration in red blood cells, such as the activity of the hydrolase in the reaction solution, incubation time, extraction volume, deproteinized solution and antioxidant components, were confirmed, providing a theoretical basis for improving the repeatability and accuracy of the detection results. 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 : Chromatograms of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate and the corresponding internal standards at concentration point S6 of the standard curve;

[0037] Figure 2 : Chromatograms of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate and the corresponding internal standards in a representative sample of red blood cell suspension in normal saline;

[0038] Figure 3 : Linear regression equation for total folate concentration in red blood cells determined by liquid chromatography-tandem mass spectrometry and chemiluminescence when the correction coefficient a=1;

[0039] Figure 4 : Linear regression equation for total folate concentration in red blood cells determined by liquid chromatography-tandem mass spectrometry and chemiluminescence assay when the correction coefficient a=2;

[0040] Figure 5 : Linear regression equation for total folate concentration in red blood cells determined by liquid chromatography-tandem mass spectrometry and chemiluminescence when the correction coefficient a=3;

[0041] Figure 6 : Linear regression equation for total folate concentration in red blood cells determined by liquid chromatography-tandem mass spectrometry and chemiluminescence assay when the correction coefficient a=4;

[0042] Figure 7 : Bland-Altman scatter plot of total folate concentration in erythrocytes detected by liquid chromatography-tandem mass spectrometry and chemiluminescence. DETAILED DESCRIPTION

[0043] The present invention is further elaborated below in conjunction with the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0044] The chromatographic column used in the present invention is a reverse phase chromatographic column (Waters C18 3 μm 100×3.0 mm); the liquid chromatography-mass spectrometry instrument is a CalQuant-S liquid chromatography tandem mass spectrometry detection system.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0046] Example 1: Determination of erythrocyte 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate concentrations by liquid chromatography-tandem mass spectrometry

[0047] This example provides a method for detecting the concentrations of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate in red blood cells by liquid chromatography-tandem mass spectrometry, the specific steps of which are as follows:

[0048] (1) Preparation of red blood cell saline suspension sample: The whole blood sample was centrifuged (1200×g for 10 min); after removing the upper plasma layer, the red blood cells at the bottom were resuspended in saline, and the mixture was slowly inverted upside down 3 to 5 times, and then centrifuged again at 1200×g for 10 min, the upper liquid was removed, and the sample was washed twice with saline; the red blood cells at the bottom were resuspended in twice the volume of saline, and the mixture was slowly inverted upside down 3 to 5 times to obtain the red blood cell saline suspension sample (RBC sample).

[0049] (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), and add 50 μL of standard curve concentration points S1-S6 (5-methyltetrahydrofolate: 1, 5, 10, 50, 200, 500 ng / mL; 5,10-methylenetetrahydrofolate: 0.5, 2.5, 5, 25, 100, 250 ng / mL) and low-value quality control (5-methyltetrahydrofolate: 7.5 ng / mL; 5,10-methylenetetrahydrofolate: 3.75 ng / mL) respectively. , high-value quality control products (5-methyltetrahydrofolate: 250 ng / mL; 5,10-methylenetetrahydrofolate: 125 ng / mL) and red blood cell saline suspension samples (RBC samples) were pipetted up and down with a pipette tip to mix, and incubated in a 37°C constant temperature water bath in the dark for 60 min; after adding 300 μL of deproteinized solution (15% sulfosalicylic acid solution), the mixture was shaken for 30 s, centrifuged at 2,000 × g for 20 min, and 200 μL of the supernatant was taken for sample injection and analysis.

[0050] (3) Instruments and related parameters: The mobile phase A used in this example was an aqueous solution containing 0.1% formic acid and 10 mM ammonium formate, and the mobile phase B was an acetonitrile solution containing 0.1% formic acid and 10 mM ammonium formate. The equipment used included a reversed-phase chromatography 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 1. The mass spectrometry parameters are shown in Tables 2 and 3.

[0051] Table 1 Liquid phase gradient

[0052] Time (min) Mobile phase B ratio (%) Flow rate (mL / min) 0.00 5 0.6 2.00 95 0.6 2.50 95 0.6 2.52 5 0.6 4.00 5 0.6

[0053] Table 2 Mass spectrometry source parameters

[0054] parameter Value Curtain Air (CUR) 35psi Collision gas (CAD) 9psi Ion spray voltage (IS) 5500V Temperature (TEM) 500℃ Ion source gas 1 (GS1) 50psi Ion source gas 2 (GS2) 50psi Ion source type Electrospray ionization (ESI+) Scanning method Multiple reaction monitoring (MRM)

[0055] Table 3 Mass spectrometry ion pair parameters

[0056] Compound parent ion daughter ions Declustering voltage Fragmentation voltage 5-MeTHF-1 460.2 313.1 60 27 5-MeTHF-2 460.1 194.1 80 46 IS-5-MeTHF 465.2 313.0 55 31 5,10-CH=THF-1 456.2 412.2 190 43 5,10-CH=THF-2 456.2 327.2 190 47 IS-5,10-CH=THF 460.2 416.2 190 43

[0057] (4) Sample detection and concentration calculation: The above samples were detected using liquid chromatography tandem mass spectrometry and the specific conditions in the technical plan. The peak areas of the test compounds 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate and their stable isotope internal standards were determined by selecting the ion pairs detected by reaction monitoring and the corresponding retention times ( Figures 1 and 2 ); the least-squares method was used to fit the linear regression equation for each compound, with the theoretical concentration of the standard curve as the independent variable x and the corresponding compound peak area / stable isotope peak internal standard area as the dependent variable y (5-methyltetrahydrofolate: y = 0.0113x + 0.00048, r = 0.9998; 5,10-methylenetetrahydrofolate: y = 0.0256x + 0.00535, r = 0.9992). The 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate concentrations of the diluted red blood cell saline suspension were calculated by substituting the compound peak area / stable isotope peak internal standard area corresponding to the sample into the linear regression equation. This was then multiplied by the dilution factor of 3 to obtain the original red blood cell 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate concentrations.

[0058] Example 2: Chemiluminescence determination of erythrocyte total folate concentration

[0059] This example provides a chemiluminescence method for detecting total folate concentration in red blood cells. This example uses the Access Folate folate assay kit (chemiluminescence method) manufactured by Beckman Coulter, Inc., Access Folate Calibrators, and the Lyphochek Whole Blood Control (Bio-Rad Laboratories, Inc.). The whole blood sample and the centrifuged plasma sample corresponding to Example 1 above were tested on an Access2 immunoassay analyzer as follows:

[0060] (1) After whole blood sampling (more than 3 mL), gently invert and mix, take out 1.3 mL of whole blood, add it to an empty packed cell tube, and centrifuge it at 1200 × g for 10 min. After centrifugation, turn on the fully automatic erythrocyte sedimentation dynamic analyzer (Chongqing Nanfang CNC Equipment Co., Ltd.) and the corresponding software, insert the packed cell tube into the instrument, wait for the packed cell result, and record the hematocrit (HCT) of each sample.

[0061] (2) Gently mix the remaining whole blood sample to ensure that the sample is fully mixed, take out 50 μL of whole blood, and add 1 mL of 0.15% L-ascorbic acid aqueous solution (hemolytic agent), gently mix the hemolyzed blood several times, and let it stand upright at room temperature (18-25°C) for at least 90 minutes; after standing, determine the folic acid content in the hemolyzed blood within 1.5 hours.

[0062] (3) The remaining whole blood was centrifuged at 1200 × g for 10 min, and 500 μL of the upper plasma was removed for the detection of plasma folic acid content.

[0063] (4) Detection method

[0064] A. Turn on the Beckman Access2 immunoassay analyzer and computer.

[0065] B. In the main menu, press F3 for consumables, then press F1 for reagent loading. Wait until a dialog box appears on the screen: Scan the barcode on the reagent kit or enter the barcode manually, insert the reagent kit, and press F1 DONE to confirm.

[0066] C. In the main menu, press F3 for consumables, press F5 to replace the luminescent agent, and wait until a dialog box appears on the screen: scan the barcode on the reagent kit or enter the barcode manually, add the luminescent agent, and press F1 DONE to confirm.

[0067] D. In the main menu, press F3 for consumables, press F4 to load the reaction cup, put the reaction cup in (do not touch the wall of the reaction cup), press the upper cover, open the upper cover, take out the empty reaction cup rack, close the upper cover firmly, and press F1 to confirm.

[0068] E. Press F5 to calibrate in the main menu, then press F5 to set calibration, press F1 to add calibration solution, and when the dialog box appears, scan the barcode of the calibration solution or enter characters manually, and press OK to confirm.

[0069] F. Press F1 on the main menu, enter the sample rack number, press Enter, press F3 to apply for testing, enter the starting sample number, select the project to be performed (plasma FOLW or whole blood RBCW), press F8 to display the options, move the cursor to the first item Trun Batch Request On, press Enter, and the batch mode is now turned on. Only the sample number (calibrator, quality control, whole blood sample, plasma sample) needs to be entered. After editing, check that all sample items are edited, return to the previous screen, select the sample rack to be placed (the corresponding calibrator, quality control, whole blood sample, plasma sample has been added to the measuring cup on the sample rack), press F1, place the sample in the sample rack, press OK to confirm, and press RUN to run.

[0070] G. Press F2 in the main menu, press F1 to select the result filtering method, OK to display the test results, select the result, and press F5 to transfer the result to the PC for data processing.

[0071] (5) Calculate the result according to the following formula:

[0072] Plasma folate value (nmol / L) = Plasma folate value (ng / mL) × 2.266 (unit conversion)

[0073] Red blood cell folate value (nmol / L) = (hemolytic folate value × 21 - plasma folate value × (1 - hematocrit)) / hematocrit

[0074] It should be understood that in order to ensure the accuracy of the test results, this embodiment uses a chemiluminescence method kit for detecting total red blood cell folate certified by the State Food and Drug Administration. Other methods (such as microbiological methods, radioanalysis methods) or kits that can accurately detect total red blood cell folate can also be used for the correction described in the present invention.

[0075] Example 3: Determination of correction coefficient a

[0076] Liquid chromatography-tandem mass spectrometry only detects the concentrations of two types of folic acid, 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate, and a portion of non-methylated folic acid is not detected. The total concentration of this portion of non-methylated folic acid requires correction analysis of the total folic acid concentration detection results of liquid chromatography-tandem mass spectrometry and chemiluminescence. According to the detection principles and results of liquid chromatography-tandem mass spectrometry and chemiluminescence, multiple correction is more appropriate than additive constant or n-th power correction. Therefore, in this embodiment, C 红细胞总叶酸 =C 红细胞5-甲基四氢叶酸 +a×C 红细胞5,10-次甲基四氢叶酸 (a is the correction factor) is the basic formula for the correction. 210 human whole blood samples (from a third-party testing company) were tested using liquid chromatography-tandem mass spectrometry (Example 1) and chemiluminescence (Example 2). Through comparative analysis, the correction factor a in the calculation formula for total red blood cell folate detection by liquid chromatography-tandem mass spectrometry was obtained.

[0077] Using the erythrocyte 5-methyltetrahydrofolate concentration ① and erythrocyte 5,10-methylenetetrahydrofolate concentration ② obtained by liquid chromatography-tandem mass spectrometry, different coefficients can be substituted into the formula to calculate the correlation and consistency between the total folate under different coefficients and the total folate measured by chemiluminescence. The following examples are given with coefficients (a) of 1, 2, 3, and 4. The data are fitted according to ①+②, ①+2×②, ①+3×②, and ①+4×②, respectively. Figures 3 to 6 and as shown in Table 4.

[0078] After screening, when a=3, the correlation coefficient was the highest, reaching 0.9966, indicating that the linear regression equation under this condition had the highest degree of fit; at the same time, the slope of the linear regression equation was closest to 1, representing the best accuracy, with the slope reaching 100.09%; and there were no outlier samples (relative deviation exceeded ±15%), thus determining the correction coefficient a=3.

[0079] Table 4 Linear fitting correlation coefficient, slope and outlier sample statistics (n=210)

[0080] formula ①+② ①+2×② ①+3×② ①+4×② Correlation coefficient r <![CDATA[0.9611(R 2 =0.9238)]]> <![CDATA[0.9885(R 2 =0.9771)]]> <![CDATA[0.9966(R 2 =0.9932)]]> <![CDATA[0.9892(R 2 =0.9786)]]> Slope (accuracy) 90.1％ 95.1％ 100.09％ 105.09％ Number of outlier samples 38 15 0 21

[0081] In the case of correction coefficient a=3, this embodiment also performed Bland-Altman analysis, with the ratio of the difference and the average value of the total folate concentration of red blood cells detected by liquid chromatography-tandem mass spectrometry and chemiluminescence as the Y axis, and the average value of the total folate concentration of red blood cells detected by liquid chromatography-tandem mass spectrometry and chemiluminescence as the X axis, and a Bland-Altman scatter plot ( Figure 7 ).Depend on Figure 7 The results showed that the 95% consistency limits of the liquid chromatography-tandem mass spectrometry and chemiluminescence detection data were (-10.7226%, 11.4559%), and only nine data points fell outside the limit, accounting for 4.29% (9 / 210), that is, no less than 95% of the points were within d±1.96Sd, indicating that there was no statistical difference in the determination results of the two methods, and the detection results of the two methods were highly consistent.

[0082] Example 4: Determination of total folate concentration in red blood cells by liquid chromatography-tandem mass spectrometry

[0083] Based on the research results of the above examples, this example will describe a method for detecting the total folate concentration of red blood cells using liquid chromatography-tandem mass spectrometry. The specific steps are as follows:

[0084] (1) Determine the concentrations of erythrocyte 5-methyltetrahydrofolate and erythrocyte 5,10-methylenetetrahydrofolate according to the method described in Example 1;

[0085] (2) Substitute the measured folic acid concentration into formula C 红细胞总叶酸=C 红细胞5-甲基四氢叶酸 +3×C 红细胞5,10-次甲基四氢叶酸 , and the total folate concentration of red blood cells was calculated.

[0086] This method is simple to operate and provides accurate results with a 100% accuracy rate. It can accurately measure the concentrations of two common folic acids in a single assay and, through further calculation, derive the total folate concentration in red blood cells. Furthermore, the total folate concentration is not simply the sum of multiple known folate concentrations. This method further expands the application scope of liquid chromatography-tandem mass spectrometry, addressing the previous limitation of liquid chromatography-tandem mass spectrometry in determining total folate concentration.

[0087] Example 5: Effect of hydrolases on test results

[0088] γ-Glutamyl hydrolase is one of the components in the reaction solution described in Example 1. Its function is to hydrolyze polyglutamic acid folate into monoglutamic acid folate. Liquid chromatography-tandem mass spectrometry can only detect the latter. Therefore, when using the liquid chromatography-tandem mass spectrometry method provided by the present invention (Example 1) to detect total erythrocyte folate, the activity of the hydrolase used will have a certain impact on the hydrolysis efficiency and the detection results. To ensure the accuracy of the folic acid quantitative test results, in this example, two samples (50 μL) of red blood cell saline suspensions with known total folate concentrations (1140 nmol / L and 731 nmol / L) were pretreated using reaction solutions (total volume 50 μL) containing hydrolases with different total activities. The reaction solutions contained 1, 3, 5, 7, 10, and 15 μL of 80 U / μL hydrolase, respectively, resulting in final hydrolase activities of 1.6 U / μL, 4.8 U / μL, 8 U / μL, 11.2 U / μL, 16 U / μL, and 24 U / μL, respectively. The total folate concentrations in the samples were then tested using the same experimental procedures as described in Examples 1 and 4. The test results are shown in Table 5, where accuracy (%) = calculated total folate concentration / theoretical folate concentration × 100%.

[0089] Table 5 Effect of hydrolase volume on test results

[0090]

[0091] From the results, it can be seen that when the volume of the hydrolase is 7 μL (i.e. the final activity of the hydrolase in the reaction solution is 11.2 U / μL), the two folic acid concentrations detected are substituted into the formula C 红细胞总叶酸 =C 红细胞5-甲基四氢叶酸 +3×C 红细胞5,10-次甲基四氢叶酸The calculated total erythrocyte folate concentration is closest to the theoretical value. As the volume of hydrolase increases (i.e., its final activity increases), the concentrations of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate obtained by hydrolysis increase. When the volume of hydrolase in a 50-μL reaction solution is 7 μL (i.e., its final activity is 11.2 U / μL), the total folate concentration measured is the highest, closest to the theoretical value and most accurate. Further increases in the volume of hydrolase (increasing its final activity) lead to lower concentrations of 5,10-methylenetetrahydrofolate. This is likely because the hydrolase is stored in an alkaline solution. Adding too much hydrolase increases the pH of the reaction system. While this does not affect the detection of 5-methyltetrahydrofolate, it inhibits the conversion of other non-methylated folates to 5,10-methylenetetrahydrofolate, resulting in a decrease in the 5,10-methylenetetrahydrofolate concentration and a subsequent decrease in the measured value. Therefore, in order to ensure the accuracy of the detection method provided by the present invention, when the volume of the reaction solution is 50 μL, the volume of the hydrolase is preferably 7 μL, that is, the final activity of the hydrolase in the reaction solution is 11.2 U / μL.

[0092] Example 6: Effect of incubation time on test results

[0093] Folic acid exists in the form of polyglutamic acid within red blood cells, but liquid chromatography-tandem mass spectrometry can only detect the monoglutamic acid form. Therefore, to ensure accurate measurement results, it is necessary to convert the polyglutamic acid form in red blood cells to the monoglutamic acid form as completely as possible. This conversion process is completed during the incubation step after red blood cell disruption and before protein removal. Therefore, this example optimized the incubation times (15 min, 30 min, 60 min, 90 min, and 120 min). The test subjects were two samples (50 μL) of red blood cell saline suspensions with known folate concentrations (740 nmol / L and 1370 nmol / L). The remaining testing steps were the same as those described in Examples 1 and 4. The test results are shown in Table 6, where accuracy (%) = calculated total folate concentration / theoretical folate concentration × 100%.

[0094] Table 6 Effect of incubation time on test results

[0095]

[0096] The results show that the accuracy of total folate detection changes with the extension of incubation time. When the incubation time is 15 minutes, the detection accuracy is 90.6% and 91.7%, respectively; when the incubation time is 60 minutes, the accuracy is close to 100%, which can be considered that the polyglutamic acid form of folate has been completely converted to the monoglutamic acid form of folate. When the incubation time is extended to 120 minutes, the detection accuracy decreases, and the detected 5,10-methylenetetrahydrofolate concentration decreases significantly. This may be because the longer the incubation time, the more 5,10-methylenetetrahydrofolate is degraded. Therefore, in order to ensure the accuracy of the detection method provided by the present invention, the incubation time is preferably 60 to 90 minutes, and more preferably 60 minutes.

[0097] Example 7: Effect of Extract Volume on Detection Results

[0098] The polyglutamate metabolites of folic acid in the red blood cell sample need to be hydrolyzed into monoglutamate metabolites under the action of hydrolases before subsequent detection can be carried out. However, the polyglutamate metabolites of folic acid are mainly present in red blood cells, so it is necessary to fully lyse the red blood cells to release the polyglutamate metabolites of folic acid and then hydrolyze them. Specifically, for red blood cell samples, in addition to adding a certain volume of internal standard and hydrolase, a certain volume of lysis solution needs to be added to cause the red blood cells to rupture. Since folic acid is unstable, antioxidants need to be added for protection. Specifically, L-ascorbic acid and β-mercaptoethanol not only have the effect of promoting the lysis of red blood cells, but also have the effect of protecting the stability of folic acid metabolites. According to literature research, this embodiment selects a 2% L-ascorbic acid solution and a 0.5% β-mercaptoethanol solution as the extraction liquid. The volume of the extract added affects red blood cell rupture, the degree of folate release, and the stability of various folate metabolites. Therefore, in this example, the extract volumes (50 μL, 100 μL, 150 μL, 200 μL, 250 μL, and 300 μL) were optimized. Two samples (50 μL) of red blood cell saline suspensions with known folate concentrations (480 nmol / L and 1300 nmol / L) were tested. The remaining testing steps were the same as those described in Examples 1 and 4. The test results are shown in Table 7, where accuracy (%) = calculated total folate concentration / theoretical folate concentration × 100%.

[0099] Table 7 Effect of extraction volume on test results

[0100]

[0101] The results show that when the volume of the extract is 200 μL, the calculated total folate concentration in red blood cells is closest to the theoretical value.

[0102] Specifically, as the volume of the extract increases, the degree of rupture of red blood cells increases, and the protection of folic acid is also enhanced. The volume of the extract is 200 μL, and the calculated total folic acid concentration is the highest, closest to the theoretical value, that is, the highest accuracy. As the volume of the extract continues to increase (>200 μL), the calculated total folic acid concentration is on the small side. Specifically, this is because the extract is an acidic solution, and the acidity of the reaction solution system is too high, which will cause tetrahydrofolate to be destroyed and form small molecules. Therefore, the 5,10-methylenetetrahydrofolate converted from tetrahydrofolate is reduced, which reduces the concentration of 5,10-methylenetetrahydrofolate. Therefore, for the accuracy of the detection method provided by the present invention, the volume of the extract is preferably 200 μL.

[0103] Example 8: Effect of deproteinized solution on test results

[0104] Studies have shown that various non-methylated folic acids are converted into 5,10-methylenetetrahydrofolate under acidic conditions. Therefore, the present invention utilizes this feature. Before detecting the sample concentration, it is necessary to convert non-methylated folic acid into 5,10-methylenetetrahydrofolate to the greatest extent possible. The concentration of 5,10-methylenetetrahydrofolate represents the concentration of some non-methylated folic acid. The process of converting non-methylated folic acid into 5,10-methylenetetrahydrofolate is assisted by a deproteinized solution. Therefore, in this example, different concentrations (5%, 10%, 15%, and 20%) of sulfosalicylic acid, trichloroacetic acid, and perchloric acid were used as deproteinizing solutions. Two samples (50 μL) of normal saline with known folate concentrations (1270 nmol / L and 700 nmol / L) of red blood cells were pretreated to investigate the effect of the deproteinizing solution on the accuracy of the test results. The specific procedures were the same as those described in Examples 1 and 4. The test results are shown in Table 8, where accuracy (%) = calculated total folate concentration / theoretical folate concentration × 100%.

[0105] Table 8 Effect of deproteinized solution on test results

[0106]

[0107] The results show that regardless of the composition of the deproteinizing solution, the optimal concentration of the deproteinizing solution is 15%. On the other hand, from the perspective of the components of the deproteinizing solution, perchloric acid and trichloroacetic acid are not as effective as sulfosalicylic acid in removing protein, that is, they cannot completely dissociate folic acid from protein, and then the accuracy of the test results does not meet the requirements. However, using sulfosalicylic acid as a deproteinizing solution can completely release folic acid bound to protein, and as the concentration of sulfosalicylic acid gradually increases, the deproteinizing effect becomes better, until the concentration of sulfosalicylic acid reaches 15%. The folic acid detection concentration reaches the maximum value, which is closest to the theoretical value, that is, the highest accuracy. When the sulfosalicylic acid concentration is 20%, the acidity of the solution system increases, which will cause the tetrahydrofolate to be destroyed and form small molecules. Therefore, the 5,10-methylenetetrahydrofolate converted from tetrahydrofolate is reduced, making the detected 5,10-methylenetetrahydrofolate concentration low; therefore, preferably, 300 μL of 15% sulfosalicylic acid is most suitable as the deproteinized solution. The detection value under this condition is closest to the theoretical value of total folate in red blood cells, and the detection accuracy is the highest.

[0108] Example 9: Effect of antioxidant components on test results

[0109] In light of the above optimization results, this example optimizes the antioxidants (2% L-ascorbic acid and 0.5% β-mercaptoethanol) in the reaction solution and extract during sample pretreatment. The antioxidants L-ascorbic acid and β-mercaptoethanol protect 5-methyltetrahydrofolate and non-methylated folate. In this example, two red blood cell saline samples (50 μL) with known folate concentrations (650 nmol / L and 1300 nmol / L) were selected as test subjects. The specific experimental steps were the same as those described in Examples 1 and 4. The specific results are shown in Table 9, where accuracy (%) = calculated total folate concentration / theoretical folate concentration × 100%.

[0110] Table 9 Effects of L-ascorbic acid and β-mercaptoethanol on test results

[0111]

[0112] The above results show that when L-ascorbic acid and β-mercaptoethanol are added to the reaction solution and the extract, the detected total erythrocyte folate concentration is closest to the theoretical value. The accuracy of detecting the total erythrocyte folate concentration using the method provided by the present invention is the highest, indicating that the combination of the antioxidants L-ascorbic acid and β-mercaptoethanol is most effective in protecting 5-methyltetrahydrofolate and non-methylated folate. Therefore, 2% L-ascorbic acid and 0.5% β-mercaptoethanol are preferred as antioxidant components in the reaction solution and the extract.

[0113] In summary, the optimization results of all the above embodiments show that the pH environment in the reaction system during pretreatment is crucial to the detection accuracy of the method provided by the present invention, and the stability of the pH environment is jointly maintained by the reaction liquid, extraction liquid and deproteinization liquid. Therefore, in order to ensure the accuracy of the test results, it is necessary to use the above-mentioned optimized reagents.

[0114] 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. A kit for determining the total folate concentration in red blood cells, characterized in that: The kit comprises a reaction solution, an extraction solution and a deproteinization solution; the deproteinization solution comprises one or more of sulfosalicylic acid, trichloroacetic acid and perchloric acid.

2. The kit according to claim 1, wherein The protein solution contains 15% sulfosalicylic acid solution.

3. The kit according to claim 1, wherein The reaction solution contains 1.6 U / μL to 24 U / μL of γ-glutamyl hydrolase activity.

4. The kit according to claim 1, wherein The reaction solution contains any one or both of 5-methyltetrahydrofolate internal standard and 5,10-methylenetetrahydrofolate internal standard.

5. The kit according to claim 1, wherein The reaction solution and the extract both contain L-ascorbic acid and beta-mercaptoethanol.

6. The kit according to claim 1, wherein The reaction solution contains 5-methyltetrahydrofolate internal standard, 5,10-methylenetetrahydrofolate internal standard, gamma-glutamyl hydrolase, L-ascorbic acid solution and beta-mercaptoethanol solution.

7. The kit according to claim 1, wherein The reaction solution contains 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; the extract contains 2% L-ascorbic acid solution and 0.5% β-mercaptoethanol solution; and the protein solution contains 15% sulfosalicylic acid solution.

8. A kit for detecting the concentration of 5-methyltetrahydrofolate and / or 5,10-methylenetetrahydrofolate, characterized in that: The kit is the same as that described in any one of claims 1 to 7.

9. A method for determining total folate in red blood cells by liquid chromatography-tandem mass spectrometry, characterized in that: The method comprises the following steps: using liquid chromatography-tandem mass spectrometry to determine the concentrations of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate in red blood cells; substituting the measured concentrations of the two folic acids into a formula, and then calculating the total folate concentration in red blood cells.

10. The method according to claim 8, wherein The formula is C 红细胞总叶酸 =C 红细胞5-甲基四氢叶酸 +a×C 红细胞5,10-次甲基四氢叶酸 , a is the correction coefficient.

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