A method for determining total folate in red blood cells by liquid chromatography-tandem mass spectrometry

Abstract

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: firstly, using liquid chromatography-tandem mass spectrometry to detect the concentrations of two folate metabolites, 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate, in red blood cells; and then substituting the measured concentrations into the formula (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 , correction coefficient a=3), the total folate concentration of red blood cells is calculated, and the formula is corrected by the detection result of the folate protein binding method. In summary, the present invention fills the gap that liquid chromatography-tandem mass spectrometry cannot detect total folate of red blood cells, and the detection method has simple steps and accurate results.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a method for determining total folate in red blood cells by liquid chromatography-tandem mass spectrometry. 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. 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%~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.

[0003] 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, the red blood cell folate level can reflect chronic or long-term (within 4 months) folate nutritional status, is more suitable for reflecting the folate level in tissues, and is considered by many scholars to be a better folate indicator.

[0004] At present, the main methods for detecting folic acid are microbiological method, folate protein binding method, high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. Among them, the microbiological method and folate protein binding method can only detect the total folate concentration, and cannot distinguish a single form of folate. Among them, the folate protein binding method has the following two calculation methods for measuring the total folate concentration of red blood cells: one is to simultaneously detect the total folate concentration and hematocrit of whole blood, and calculate the total folate concentration of red blood cells according to the formula "total folate concentration of red blood cells = total folate concentration of whole blood / hematocrit"; the other is to simultaneously detect the total folate concentration of whole blood, the total folate concentration of plasma and the hematocrit, and then obtain the total folate concentration of red blood cells according to the formula "total folate concentration of red blood cells = [total folate concentration of whole blood - total folate concentration of plasma × (1-hematocrit)] / hematocrit".

[0005] 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 and provide more reference information for clinical use. Existing literature and patents ("A sample pretreatment method for folate and its metabolites, a detection method and a kit", application number CN202210474366.7) usually simply add up 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 types of folate forms in the human body, some folate forms are very unstable, and there are problems of mutual conversion between various folate forms, so that liquid chromatography-tandem mass spectrometry cannot detect all folate forms, but can only detect specific folate forms, and cannot obtain the total folate concentration in the strict sense. Summary of the invention

[0006] In view of the above problems, the present invention provides a method for determining total folate in red blood cells by liquid chromatography-tandem mass spectrometry, wherein the total folate refers to the sum of all folate concentrations, rather than merely the sum of the concentrations of several known forms of folate. The steps of the method are as follows: first, the concentrations of two folate metabolites, 5-methyltetrahydrofolate and 5,10-methytetrahydrofolate, in red blood cells are detected by liquid chromatography-tandem mass spectrometry; then, the measured concentrations are substituted into the formula (C total folate in red blood cells = C 5-methyltetrahydrofolate in red blood cells + a × C 5,10-methytetrahydrofolate in red blood cells, with a correction factor a = 3) to calculate the total folate concentration in red blood cells, wherein the formula is corrected by the detection results of the folate protein binding method.

[0007] The method is designed based on the existing forms of folic acid in the body and the conversion relationship between folic acid. The folic acid forms in human red blood cells mainly 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), etc. 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 that 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, 5-formyltetrahydrofolate, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 10-formyltetrahydrofolate and 10-formylfolate and other folic acid forms will be fully or partially converted into 5,10-methylenetetrahydrofolate, which can exist stably under acidic conditions.

[0008] Therefore, the present invention uses liquid chromatography-tandem mass spectrometry to detect the concentrations of erythrocyte 5-methyltetrahydrofolate and 5,10-methytetrahydrofolate, with the erythrocyte 5-methyltetrahydrofolate concentration representing the methylated folate concentration of erythrocytes, and the erythrocyte 5,10-methytetrahydrofolate concentration representing the concentration of part of the erythrocyte non-methylated folate. The concentration of the missing part of the erythrocyte non-methylated folate is obtained by comparing and correcting the total erythrocyte folate concentration obtained by the folate protein binding method or the microbial method, and then the total erythrocyte folate concentration by liquid chromatography-tandem mass spectrometry is obtained by calculation, and the calculation formula is: total erythrocyte folate concentration = original erythrocyte 5-methyltetrahydrofolate concentration + a×original erythrocyte 5,10-methytetrahydrofolate concentration, where a is the correction coefficient of the total amount of erythrocyte 5,10-methytetrahydrofolate and erythrocyte non-methylated folate, preferably, a = 3.

[0009] 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 types of folic acid into a formula, and then calculating the concentration of total folate in red blood cells.

[0010] From the above detection principle, it can be seen that there is still a part of the concentration of non-methylated folate in red blood cells that 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.

[0011] 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...

[0012] The present invention uses liquid chromatography-tandem mass spectrometry and folate protein binding method (chemiluminescence method) to determine the concentration of folic acid in 210 red blood cell samples, respectively, draws linear regression equations under different correction coefficients (such as a = 1~4), analyzes the linear correlation coefficient of the regression equation, and determines that under the condition of a = 3, the best linear correlation coefficient r can be obtained; on the other hand, based on the experimental results, it can also be inferred that the total concentration of non-methylated folic acid that cannot be converted into 5,10-methylenetetrahydrofolate is about twice the total concentration of non-methylated folic acid that can be converted into 5,10-methylenetetrahydrofolate.

[0013] 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 .

[0014] 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.

[0015] Furthermore, the reaction solution contains 1.6 U / μL ~ 24 U / μL of γ-glutamyl hydrolase; although the hydrolases within the above activity range can hydrolyze polyglutamate well, for the accuracy of the test results, 11.2 U / μL is preferred; specifically, when the total volume of the reaction solution is 50 μL, 7 μL of 80 U / μL of γ-glutamyl hydrolase needs to be added.

[0016] Furthermore, the volume of the extract is 50 μL to 300 μL; although the extracts within the above volume range can rupture red blood cells and protect folic acid well, 200 μL is preferred for the accuracy of the test results.

[0017] Furthermore, the deproteinizing solution contains one or more of sulfosalicylic acid, trichloroacetic acid and perchloric acid; for the accuracy of the test results, 300 μL of 15% sulfosalicylic acid is preferred, because sulfosalicylic acid can not only provide acidic conditions, which is conducive to the conversion of various non-methylated folic acids into 5,10-methylenetetrahydrofolate, but also has the highest efficiency in removing impurities.

[0018] Furthermore, after the sample is treated with the extract, the sample needs to be incubated for 15 to 120 min; the incubation time is more than 60 min, so that the polyglutamate form of folic acid can be completely converted into the monoglutamate form of folic acid. Therefore, in order to ensure the accuracy of the test results, preferably, 60 min to 90 min; more preferably, 60 min.

[0019] 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.

[0020] In another aspect, the present invention provides a method for determining the total folate concentration of red blood cells by liquid chromatography-tandem mass spectrometry, wherein the total folate concentration of red blood cells is obtained by not merely adding up a plurality of known and common folate concentrations.

[0021] The present invention provides a method for detecting total folate in red blood cells, the detection accuracy of which is as high as 100%, and the method comprises the following steps:

[0022] (1) Sample pretreatment: including sequentially treating the red blood cell saline suspension sample with reaction solution, extraction solution, and deproteinization solution;

[0023] (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;

[0024] 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 process, 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.

[0025] (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 mass spectrometry. Specifically, the kit is the folic acid determination kit (chemiluminescence method) Access Folate produced by Beckman Coulter, Inc.

[0026] 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.

[0027] (4) Calculate the total red blood cell folate concentration by liquid chromatography-tandem mass spectrometry: Compare the red blood cell 5-methyltetrahydrofolate and red blood cell 5,10-methytetrahydrofolate concentrations obtained by liquid chromatography-tandem mass spectrometry with the total red blood cell folate obtained by the folate protein binding method or the microbial method to obtain the correction factor a; substitute the factor into the calculation formula (total red blood cell folate concentration = red blood cell 5-methyltetrahydrofolate concentration + a × red blood cell 5,10-methytetrahydrofolate concentration) to calculate the total red blood cell folate concentration by liquid chromatography-tandem mass spectrometry.

[0028] In some specific embodiments, the correction coefficient a = 3.

[0029] 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, 11.2U / 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 above.

[0030] The beneficial effects of the present invention include:

[0031] 1. The concentrations of two common forms of folic acid are measured by liquid chromatography-tandem mass spectrometry. The calculation formula provided by the present invention can be used to obtain the total folate concentration of red blood cells 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 up, which could not represent the true total folate concentration of red blood cells.

[0032] 2. The method is simple to operate and the test results are accurate, with an accuracy rate of up to 100%;

[0033] 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 of red blood cells, such as the activity of the hydrolase in the reaction solution, the incubation time, the volume of the extraction solution, the deproteinized solution and the antioxidant components, are confirmed, which provides a theoretical basis for improving the repeatability and accuracy of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative work.

[0035] Figure 1 : Chromatograms of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate and the corresponding internal standard at concentration point S6 of the standard curve;

[0036] Figure 2 : Chromatograms of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate and the corresponding internal standards in a representative sample of red blood cell saline suspension;

[0037] Figure 3 : Linear regression equation for the detection of total folate concentration in red blood cells by liquid chromatography-tandem mass spectrometry and chemiluminescence when the correction coefficient a=1;

[0038] Figure 4 : Linear regression equation for total folate concentration in red blood cells detected by liquid chromatography-tandem mass spectrometry and chemiluminescence when the correction coefficient a=2;

[0039] Figure 5 : Linear regression equation for total folate concentration in red blood cells detected by liquid chromatography-tandem mass spectrometry and chemiluminescence when the correction coefficient a=3;

[0040] Figure 6 : Linear regression equation for the detection of total folate concentration in red blood cells by liquid chromatography-tandem mass spectrometry and chemiluminescence when the correction coefficient a=4;

[0041] Figure 7 : Bland-Altman scatter plot of total folate concentration in erythrocytes detected by liquid chromatography-tandem mass spectrometry and chemiluminescence. DETAILED DESCRIPTION

[0042] The present invention is further elaborated in detail below in conjunction with the accompanying drawings and specific embodiments of the specification. 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 the field without making creative work are within the scope of protection of the present invention.

[0043] The chromatographic column used in the present invention is a reverse phase chromatographic column (Waters C18 3μm 100×3.0 mm); the liquid chromatography-mass spectrometry instrument is a CalQuant-S liquid chromatography tandem mass spectrometry detection system.

[0044] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0045] Example 1: Determination of the concentration of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate in erythrocytes by liquid chromatography-tandem mass spectrometry

[0046] This embodiment provides a method for detecting the concentration of 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate in red blood cells by liquid chromatography-tandem mass spectrometry, and the specific steps are as follows:

[0047] (1) Preparation of red blood cell saline suspension sample: Centrifuge the whole blood sample (1200 × g for 10 min); after removing the upper plasma, resuspend the red blood cells at the bottom with saline, slowly invert upside down for 3 to 5 times to mix, centrifuge again at 1200 × g for 10 min, remove the upper liquid, and repeat washing with saline twice; resuspend the red blood cells at the bottom with twice the volume of saline, slowly invert upside down for 3 to 5 times to mix, and obtain the red blood cell saline suspension sample (RBC sample).

[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), 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), low value quality control (5-methyltetrahydrofolate: 7.5 ng / mL; 5,10-methylenetetrahydrofolate: 3.75 ng / mL), high-value quality control (5-methyltetrahydrofolate: 250 ng / mL; 5,10-methylenetetrahydrofolate: 125 ng / mL), and red blood cell saline suspension sample (RBC sample) were mixed by pipetting up and down with a pipette tip, 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 and mixed for 30 s, and after centrifugation at 2,000×g for 20 min, 200 μL of the supernatant was taken for injection analysis.

[0049] (3) Instruments and related parameters: The mobile phase A used in this example is an aqueous solution containing 0.1% formic acid and 10 mM ammonium formate, and the mobile phase B is an acetonitrile solution containing 0.1% formic acid and 10 mM ammonium formate. The equipment used includes a reverse phase chromatographic column (Waters C18 3 μm 100×3.0 mm) and a CalQuant-S liquid chromatography tandem mass spectrometry detection system. The temperature of the column oven is 40°C and the injection volume is 20 μL. The liquid phase gradient used is shown in Table 1. The mass spectrometry parameters are shown in Tables 2 and 3.

[0050] Table 1 Liquid phase gradient

[0051]

[0052] Table 2 Mass spectrometry source parameters

[0053]

[0054] Table 3 Mass spectrometry ion pair parameters

[0055]

[0056] (4) Sample detection and concentration calculation: The above samples were detected using liquid chromatography tandem mass spectrometry and the specific conditions in the technical solution. 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 ( Figure 1~Figure 2 ); Taking the theoretical concentration of the standard curve as the independent variable x, and the compound peak area corresponding to the concentration / stable isotope peak internal standard area as the dependent variable y, the least squares method was used to fit the linear regression equation of each compound (5-methyltetrahydrofolate: y = 0.0113x+0.00048, r = 0.9998; 5,10-methylenetetrahydrofolate: y = 0.0256 x+0.00535, r = 0.9992); Substitute the compound peak area / stable isotope peak internal standard area corresponding to the sample into the linear regression equation to obtain the 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate concentrations of the diluted red blood cell saline suspension sample; then multiply by the dilution factor 3 to obtain the original red blood cell 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate concentrations.

[0057] Example 2: Determination of total folate concentration in red blood cells by chemiluminescence

[0058] This embodiment provides a chemiluminescence method for detecting the total folate concentration of red blood cells. This embodiment uses the folic acid determination kit (chemiluminescence method) Access Folate produced by Beckman Coulter, Inc., the folic acid calibrator Access Folate Calibrators, and the matching quality control product Lyphochek Whole Blood Control produced by Bio-Rad Laboratories, Inc., and detects the corresponding whole blood sample in the above embodiment 1 and the upper plasma sample obtained by centrifugation on the Access2 immunoassay analyzer, and the method is as follows:

[0059] (1) After whole blood sampling (more than 3 mL), gently invert to 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 in a low-speed centrifuge. After centrifugation, turn on the fully automatic hematocrit 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.

[0060] (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 (hemolyzing agent), gently mix the hemolyzed blood several times, and let it stand upright at room temperature (18-25°C) for at least 90 min; after standing, determine the folic acid content in the hemolyzed blood within 1.5 h.

[0061] (3) The remaining whole blood was centrifuged at 1200 × g for 10 min, and 500 μL of the upper plasma was taken out for the detection of plasma folate content.

[0062] (4) Detection method

[0063] A. Turn on the Beckman Access2 Immunoassay Analyzer and the computer.

[0064] B. In the main menu, press F3 for consumables, 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.

[0065] 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, put in the luminescent agent, and press F1 DONE to confirm.

[0066] 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 forcefully, and press F1 to confirm.

[0067] E. In the main menu, press F5 to calibrate, then press F5 to set calibration, press F1 to add calibration solution, when the dialog box appears, scan the barcode of the calibration solution or enter characters manually, and press OK to confirm.

[0068] F. Press F1 in the main menu, enter the sample rack number, press Enter, press F3 to apply for testing, enter the starting sample number, select the item to be done (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 open. Just enter the sample number (calibrator, quality control product, whole blood sample, plasma sample). 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 product, whole blood sample, plasma sample has been added to the measuring cup on the sample rack), press F1, place it in the sample rack, press OK to confirm, and press RUN to run.

[0069] G. Press F2 in the main menu, press F1 to select the result screening method, OK to display the test results, select the result, and press F5 to transfer the result to the PC for data processing.

[0070] (5) Calculate the result according to the following formula:

[0071] Plasma folate value (nmol / L) = Plasma folate value (ng / mL) × 2.266 (unit conversion)

[0072] Red blood cell folate value (nmol / L) = (hemolyzed folate value × 21-plasma folate value × (1-hematocrit)) / hematocrit

[0073] It should be understood that in order to ensure the accuracy of the test results, this embodiment uses a kit for detecting total folate in red blood cells by chemiluminescence method certified by the Food and Drug Administration. Other methods (such as microbiological method, radioanalysis method) or kits that can accurately detect total folate in red blood cells can also be used for the correction described in the present invention.

[0074] Example 3: Determination of correction coefficient a

[0075] 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 correction, and 210 human whole blood samples (third-party testing company) are tested using liquid chromatography-tandem mass spectrometry (Example 1) and chemiluminescence method (Example 2). Through comparative analysis, the correction factor a in the calculation formula for detecting total folate in red blood cells by liquid chromatography-tandem mass spectrometry is obtained.

[0076] The red blood cell 5-methyltetrahydrofolate concentration ① and red blood cell 5,10-methylenetetrahydrofolate concentration ② obtained by liquid chromatography-tandem mass spectrometry, by substituting different coefficients into the formula, the correlation and consistency of total folate under different coefficients and total folate measured by chemiluminescence can be calculated. The following examples are given with coefficients (a) of 1, 2, 3 and 4, and the data fitted according to ①+②, ①+2×②, ①+3×② and ①+4×② are as follows Figure 3~Figure 6 And as shown in Table 4.

[0077] 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 a slope of 100.09%; and there were no outlier samples (relative deviation exceeded ±15%), thus determining the correction coefficient a = 3.

[0078] Table 4 Linear fitting correlation coefficient, slope and outlier sample statistics (n = 210)

[0079]

[0080] 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 was drawn ( Figure 7 ).Depend on Figure 7The 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 results of the two methods, and the detection results of the two methods were consistent.

[0081] Example 4: Determination of total folate concentration in red blood cells by liquid chromatography-tandem mass spectrometry

[0082] Based on the research results of the above embodiments, this embodiment will describe a method for detecting the total folate concentration of red blood cells using liquid chromatography-tandem mass spectrometry, and the specific steps are as follows:

[0083] (1) Determine the concentration of erythrocyte 5-methyltetrahydrofolate and erythrocyte 5,10-methylenetetrahydrofolate according to the method described in Example 1;

[0084] (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.

[0085] The method is simple to operate and has accurate test results with an accuracy rate of up to 100%. It can accurately measure the concentrations of two common folic acids at one time and obtain the total folic acid concentration in red blood cells by further calculation. Moreover, the total folic acid concentration is not just the superposition of multiple known folic acid concentrations. It further expands the application scope of liquid chromatography-tandem mass spectrometry, that is, it fills the gap that liquid chromatography-tandem mass spectrometry could not measure the total folic acid concentration in the past.

[0086] Example 5: Effect of hydrolase on test results

[0087] 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 (Example 1) provided by the present invention to detect total folate in red blood cells, 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 quantitative detection result of folic acid, the present embodiment uses a reaction solution (total volume of 50 μL) containing hydrolases with different total activities to pretreat two red blood cell saline suspension samples (50 μL) with known total folic acid concentrations (1140 nmol / L and 731 nmol / L), respectively. The reaction solution contains 1, 3, 5, 7, 10, and 15 μL of 80 U / μL hydrolase, respectively, that is, the final activity of the hydrolase in the reaction solution is 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 folic acid concentration in the sample is then detected. The specific experimental operation is the same as that described in Examples 1 and 4. The test results are shown in Table 5, where accuracy (%) = calculated total folic acid concentration / theoretical folic acid concentration × 100%.

[0088] Table 5 Effect of hydrolase volume on test results

[0089]

[0090] The results show 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 detected folic acid concentrations 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 folate concentration of red blood cells is closest to the theoretical value. As the volume of the hydrolase increases (i.e., the final activity increases), the concentrations of 5-methyltetrahydrofolate and 5,10-methytetrahydrofolate obtained by hydrolysis become higher, until the volume of the hydrolase in the 50 μL reaction solution is 7 μL (i.e., the final activity of the hydrolase in the reaction solution is 11.2 U / μL), the total folate concentration detected is the highest, closest to the theoretical value, and has the highest accuracy; after continuing to increase the volume of the hydrolase (increasing the final activity of the hydrolase), the actual detected concentration of 5,10-methytetrahydrofolate is low, which may be because the hydrolase is stored in an alkaline solution. Adding too much hydrolase will cause the pH value of the reaction system to increase. Although this will not affect the detection of 5-methyltetrahydrofolate, it will inhibit the conversion of other non-methylated folates into 5,10-methytetrahydrofolate, thus causing the concentration of 5,10-methytetrahydrofolate to decrease, and then the detection value becomes smaller. 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.

[0091] Example 6: Effect of incubation time on test results

[0092] Folic acid exists in the form of polyglutamic acid in red blood cells, but liquid chromatography-tandem mass spectrometry can only detect folic acid in the form of monoglutamic acid. Therefore, in order to determine the accuracy of the results, it is necessary to convert the polyglutamic acid in the red blood cells into the monoglutamic acid as much as possible. The conversion process is completed in the incubation step after the red blood cells are broken and before the protein is removed. Therefore, this embodiment optimizes the incubation time (15 min, 30 min, 60 min, 90 min and 120 min), and the detection object is 2 red blood cell saline suspension samples (50 μL) with known folic acid concentrations (740 nmol / L and 1370 nmol / L). The remaining detection steps are the same as those described in Examples 1 and 4. The test results are shown in Table 6, where accuracy (%) = calculated total folic acid concentration / theoretical folic acid concentration × 100%.

[0093] Table 6 Effect of incubation time on test results

[0094]

[0095] It can be seen from the results that the accuracy of the total folate detection value changes with the extension of the incubation time. When the incubation time is 15 min, the detection value accuracy is 90.6% and 91.7% respectively; when the incubation time is 60 min, the accuracy is close to 100%, and it can be considered that the polyglutamic acid form of folate has been completely converted into monoglutamic acid form of folate; when the incubation time is extended to 120 min, the detection accuracy decreases, and the detected 5,10-methylenetetrahydrofolate concentration decreases significantly, which may be because the longer the incubation time, the more 5,10-methylenetetrahydrofolate is degraded, so for the accuracy of the detection of the method provided by the present invention, the preferred water incubation time is 60~90 min, more preferably, 60 min.

[0096] Example 7: Effect of Extract Volume on Detection Results

[0097] 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, but the polyglutamate metabolites of folic acid are mainly present in red blood cells, so it is necessary to fully lyse the red blood cells, release the polyglutamate metabolites of folic acid, and then hydrolyze. 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 have the effect of protecting the stability of folic acid metabolites in addition to promoting the lysis of red blood cells. 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 added extract will affect the rupture of red blood cells, the degree of folate release, and the stability of various folate metabolites. Therefore, the volume of the extract (50 μL, 100 μL, 150 μL, 200 μL, 250 μL, and 300 μL) was optimized in this example. The test objects were 2 samples (50 μL) of red blood cell saline suspensions with known folate concentrations (480 nmol / L and 1300 nmol / L). The remaining test 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%.

[0098] Table 7 Effect of extraction volume on test results

[0099]

[0100] 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.

[0101] Specifically, as the volume of the extract increases, the degree of red blood cell rupture increases, and the protection of folic acid also increases. When the volume of the extract is 200 μL, the calculated total folic acid concentration is the highest and closest to the theoretical value, that is, the accuracy is the highest. As the volume of the extract continues to increase (> 200 μL), the calculated total folic acid concentration is lower. Specifically, this is because the extract is an acidic solution, and the reaction solution system is too acidic, which will cause tetrahydrofolate to be destroyed and form small molecules. Therefore, the 5,10-methylenetetrahydrofolate converted from tetrahydrofolate is reduced, resulting in a decrease in the concentration of 5,10-methylenetetrahydrofolate. Therefore, for the accuracy of the detection of the method provided by the present invention, the volume of the extract is preferably 200 μL.

[0102] Example 8: Effect of deproteinized solution on test results

[0103] Existing studies have shown that a variety of non-methylated folic acid will be 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, and the concentration of 5,10-methylenetetrahydrofolate represents the concentration of part of the 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, sulfosalicylic acid, trichloroacetic acid and perchloric acid of different concentrations (5%, 10%, 15% and 20%) were selected as deproteinizing solutions, and two red blood cell saline samples (50 μL) with known folic acid concentrations (1270 nmol / L and 700 nmol / L) were pretreated to explore the effect of the deproteinizing solution on the accurate results. The specific operation was the same as that described in Examples 1 and 4. The test results are shown in Table 8, where accuracy (%) = calculated total folic acid concentration / theoretical folic acid concentration × 100%.

[0104] Table 8 Effect of deproteinization solution on test results

[0105]

[0106] From the results, it can be seen that no matter what the composition of the deproteinizing solution is, the optimal concentration of the deproteinizing solution is 15%; on the other hand, from the perspective of the components of the deproteinizing solution, the protein removal effect of perchloric acid and trichloroacetic acid is not as good as sulfosalicylic acid, that is, they cannot completely dissociate folic acid from protein, and then the accuracy of the test results does not meet the requirements; 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 protein removal effect is better, until the concentration of sulfosalicylic acid is 15%, the folic acid detection concentration reaches the maximum value, which is closest to the theoretical value, that is, the highest accuracy. When the concentration of sulfosalicylic acid is 20%, the acidity of the solution system increases, which will cause tetrahydrofolate to be destroyed and form small molecules. Therefore, 5,10-methylenetetrahydrofolate converted from tetrahydrofolate is reduced, making the detected 5,10-methylenetetrahydrofolate concentration low; therefore, preferably, 300 μL 15% sulfosalicylic acid is the most suitable deproteinizing solution. The detection value under this condition is closest to the theoretical value of total folate in red blood cells and has the highest detection accuracy.

[0107] Example 9: Effect of antioxidant components on test results

[0108] In combination with the above optimization results, this example optimizes the antioxidants (2% L-ascorbic acid, 0.5% β-mercaptoethanol) in the reaction solution and the extract in the 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 detection objects. 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%.

[0109] Table 9 Effects of L-ascorbic acid and β-mercaptoethanol on test results

[0110]

[0111] From the above results, it can be seen that when L-ascorbic acid and β-mercaptoethanol are added to the reaction solution and the extract, the detected total folate concentration of red blood cells is closest to the theoretical value, and the accuracy of detecting the total folate concentration of red blood cells using the method provided by the present invention is the highest, indicating that the combination of antioxidants L-ascorbic acid and β-mercaptoethanol has the best effect in protecting 5-methyltetrahydrofolate and non-methylated folate, so 2% L-ascorbic acid and 0.5% β-mercaptoethanol are preferably used as antioxidant components in the reaction solution and the extract.

[0112] 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 solution, the extraction solution and the deproteinized solution. Therefore, in order to ensure the accuracy of the detection results, it is necessary to use the above-mentioned optimized reagents.

[0113] The basic principles and main features of the present invention and the advantages of the present invention are shown and described above. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and 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 attached claims rather than the above description. Therefore, it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention, and any figure marks in the claims should not be regarded as limiting the claims involved.

Claims

1. 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 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 then the concentration of total folate in red blood cells is calculated; Before using liquid chromatography-tandem mass spectrometry to determine the red blood cell folate concentration, the red blood cell physiological saline suspension sample needs to be pretreated; the reagents required for the pretreatment include a reaction solution, an extraction solution, and a deproteinized solution; The reaction solution contains 1.6 U / μL to 24 U / μL of γ-glutamyl hydrolase activity; The volume of the extract is 50 μL to 300 μL; The deproteinizing solution contains one or more of sulfosalicylic acid, trichloroacetic acid and perchloric acid.

2. The method according to claim 1, characterized in that After treating the sample with the extraction solution, the sample needs to be incubated for 15 to 120 minutes.

3. The method according to claim 2, characterized in that The reaction solution and the extract both contain L-ascorbic acid and beta-mercaptoethanol.

4. 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 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 then the total folate concentration of red blood cells was calculated.

5. The method according to claim 4, characterized in that Before using liquid chromatography-tandem mass spectrometry to determine the red blood cell folate concentration, the red blood cell physiological saline suspension sample needs to be pretreated; the reagents required for the pretreatment include a reaction solution, an extraction solution, and a deproteinized solution.

6. The method according to claim 5, characterized in that The reaction solution contains 1.6 U / μL to 24 U / μL of γ-glutamyl hydrolase activity.

7. The method according to claim 5, characterized in that The volume of the extract is 50 μL to 300 μL.

8. The method according to claim 5, characterized in that The deproteinizing solution contains one or more of sulfosalicylic acid, trichloroacetic acid and perchloric acid.

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