Kit for determination of folate and its metabolites in erythrocytes by high performance liquid chromatography tandem mass spectrometry

Abstract

The present invention relates to a kit for determining folic acid and its metabolites in red blood cells by high performance liquid chromatography tandem mass spectrometry. The kit solves the problem of quantifying red blood cells by detecting the content of folic acid and its metabolites in red blood cells. The kit has a simple pretreatment process for the entire sample to be tested, high sensitivity, small sample volume, and strong specificity. Five types of folic acid and their metabolites in red blood cells can be simultaneously detected within 7.0 minutes. The kit can be used for clinical diagnosis of the five types of folic acid and their metabolites in red blood cells and health assessment.

Description

Technical Field

[0001] The present invention belongs to the technical field of blood testing, and specifically relates to a kit for determining folic acid and its metabolites in red blood cells by high performance liquid chromatography tandem mass spectrometry, and the use of the kit in determining folic acid and its metabolites in red blood cells by high performance liquid chromatography tandem mass spectrometry, wherein the folic acid and its metabolites are: folic acid, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, flavin adenine dinucleotide and vitamin B3. Background Art

[0002] Folic acid (VB9) is a heat-labile, low-density, water-soluble B vitamin that participates in the metabolism of nucleic acids, amino acids, proteins, and phospholipids. It is closely related to cell differentiation, proliferation, and function. The human body cannot synthesize or convert folic acid and must obtain it from food. Appropriate folic acid supplementation for women of childbearing age can reduce the incidence of neural tube defects, trisomy 21, cleft lip and palate, heart disease, and leukemia in fetuses, and reduce the likelihood of miscarriage. Red blood cell folate is the most commonly used indicator of folate status. Folate exists primarily in erythrocytes as polyglutamic acid (1-7 glutamic acids). Under the action of γ-glutamyl hydrolase in plasma, the active monoglutamic acid form is released into the serum. Therefore, erythrocyte folate testing can be used to assess human folate reserves. The key to erythrocyte folate testing is the complete conversion of polyglutamic acid into monoglutamic acid compounds.

[0003] Flavin adenine dinucleotide (FAD, the active form of vitamin B2 in the body) and vitamin B3 (VB3) both play a corresponding auxiliary role in the folate metabolic pathway. Measuring them simultaneously can help clinicians analyze folate metabolism more comprehensively. However, there are currently no methods reported in the literature that can simultaneously detect folate, FAD, and VB3 in a single test. Therefore, if it is necessary to simultaneously understand the amount of folate, FAD, and VB3 in a patient's red blood cells, it is usually necessary to perform multiple tests on the same sample, which is cumbersome and costly in practice.

[0004] At present, the main detection methods for red blood cell folate are mainly immunoassays, electrochemical methods, fluorescence methods and other technologies, but these methods have poor specificity, and the detection has not yet been standardized. The same sample may get inconsistent results using the detection systems of different manufacturers. The huge difference in the test results may be caused by the differences in the detection capabilities of the calibrators and folic acid glutamate derivatives (glutamate antigen substances) used by different manufacturers, and it is also difficult for the immunoassay to measure multiple compounds at the same time. High performance liquid chromatography tandem mass spectrometry is a technology that separates the target compound and its interfering components from each other through the differences in the polarity, mass-to-charge ratio and characteristic fragments contained in the compound's structure, and ultimately achieves precise quantification. Compared with immunoassays, electrochemical methods, fluorescence methods and other technologies, high performance liquid chromatography tandem mass spectrometry has higher sensitivity, precision and accuracy.

[0005] Although this method detects vitamin compounds, it is essentially different from conventional serum vitamin detection items. When using conventional serum / liquid phase mass spectrometry detection methods to measure vitamin compounds in red blood cells, the concentration of each compound in the red blood cells cannot be fully measured. This is because the substances in the red blood cells are tightly bound to the cells and proteins in the red blood cells. Using conventional serum pretreatment methods, the vitamins contained in the red blood cells cannot be effectively extracted, resulting in the measurement results being lower than the true value. Therefore, when measuring folic acid and its metabolites in red blood cells, certain measures need to be taken to fully lyse the red blood cells so that the substances to be tested are fully released.

[0006] Some literature on erythrocyte folate uses indirect measurement as an alternative to direct erythrocyte measurement. These methods measure the concentration of each compound in whole blood and plasma, as well as the hematocrit (the ratio of erythrocyte volume to whole blood volume, HCT%), to calculate the analyte concentration in erythrocytes. The whole blood method is similar to the erythrocyte method, involving issues such as erythrocyte lysis, hydrolysis of folate polyglutamic acid, and sample extraction. Therefore, it is no simpler than direct erythrocyte measurement. Furthermore, whole blood and plasma measurements are essentially two different samples, requiring separate assay development and validation, making them less convenient than direct erythrocyte measurement.

[0007] In the analysis of red blood cells, the key pretreatment steps are the dissociation of folate glutamyl derivatives and the extraction of the analyte from the sample. The dissociation process of folate glutamyl derivatives is typically time-consuming, often lasting several hours or even overnight. Due to the complex matrix, solid-phase extraction (SPE) cartridges are often used to purify and concentrate the sample using appropriate wash and eluent solutions. However, the SPE cartridges used for sample solution purification in the SPE method are currently quite expensive, resulting in a high assay cost for a single sample, often accounting for more than half of the sample assay cost.

[0008] In summary, there is an urgent need for a method for measuring folic acid, FAD, and vitamin B3 in red blood cells that is highly specific, accurate, simple and convenient to operate, short in measurement time, and relatively inexpensive, so as to help improve the quality of clinical folic acid testing and provide a more comprehensive analysis of folic acid metabolism. Summary of the Invention

[0009] The purpose of the present invention is to provide a kit for determining folic acid and its metabolites in red blood cells by high performance liquid chromatography tandem mass spectrometry on the basis of the existing technology.

[0010] Another object of the present invention is to provide a use of the above kit in the determination of folic acid and its metabolites in red blood cells using high performance liquid chromatography tandem mass spectrometry.

[0011] The technical solutions of the present invention are as follows:

[0012] A kit for determining folic acid and its metabolites in red blood cells by high performance liquid chromatography tandem mass spectrometry, wherein:

[0013] Folic acid and its metabolites are: folic acid (VB9), 5-methyltetrahydrofolate (5-MTHF), 5-formyltetrahydrofolate (5-FoTHF), flavin adenine dinucleotide (FAD) and vitamin B3 (VB3);

[0014] The isotopic internal standards corresponding to the above-mentioned folic acid and its metabolites are: folic acid-d4 (VB9--d4), 5-methyltetrahydrofolate-d4 (5-MTHF-d4), 5-formyltetrahydrofolate-d4 (5-FoTHF-d4), flavin adenine dinucleotide-d5 (FAD-d5) and vitamin B3-d4 (VB3-d4);

[0015] The kit comprises the following reagents:

[0016] (1) Electrolyte concentrate A: 40-60% formic acid-water solution;

[0017] (2) Electrolyte concentrate B: 40-60% formic acid-methanol;

[0018] (3) Mixed standard SO solution:

[0019] A methanol-water solution containing 2 μmol / L folic acid, 20 μmol / L 5-methyltetrahydrofolate, 4 μmol / L 5-formyltetrahydrofolate, 40 μmol / L flavin adenine dinucleotide, and 2000 μmol / L vitamin B3;

[0020] (4) Internal standard SI solution:

[0021] A methanol-water solution containing 0.2 μmol / L folic acid-d4, 2 μmol / L 5-methyltetrahydrofolate-d4, 0.4 μmol / L 5-formyltetrahydrofolate-d4, 4 μmol / L flavin adenine dinucleotide-d5, and 40 μmol / L vitamin B3-d4;

[0022] (5) Protective agent: 1% to 3% VC solution;

[0023] (6) pH adjuster: 1M to 5M NaOH solution;

[0024] (7) Lysis solution: an aqueous solution containing 0.3-1.0 units / ml of glutamyl hydrolase.

[0025] In a preferred embodiment, electrolyte concentrate A is a 50% formic acid-water solution, and electrolyte concentrate B is a 50% formic acid-methanol solution. When the kit is used, the high-volume concentration formic acid-water solution is diluted to a lower-volume concentration mobile phase A, for example, to a 0.05-0.2% formic acid-water solution as mobile phase A; preferably, to a 0.1-0.15% formic acid-water solution as mobile phase A, and more preferably, to a 0.1% formic acid-water solution as mobile phase A. Similarly, when the kit is used, the high-volume concentration formic acid-methanol solution is diluted to a lower-volume concentration mobile phase B, for example, to a 0.05-0.2% formic acid-methanol solution as mobile phase B; preferably, to a 0.1-0.15% formic acid-methanol solution as mobile phase B, and more preferably, to a 0.1% formic acid-methanol solution as mobile phase B.

[0026] In the present invention, the protective agent is a 1% to 3% VC solution, which may be but is not limited to a 1% VC solution, a 1.5% VC solution, a 2% VC solution, a 2.5% VC solution or a 3% VC solution. Preferably, the protective agent is a 2% VC solution.

[0027] Furthermore, the pH adjuster is a 1M to 5M NaOH solution, which may be but is not limited to a 1M NaOH solution, a 2M NaOH solution, a 3M NaOH solution, a 4M NaOH solution or a 5M NaOH solution. Preferably, the pH adjuster is a 2M to 3M NaOH solution, and more preferably, the pH adjuster is a 2M NaOH solution.

[0028] Furthermore, the lysis solution is an aqueous solution containing 0.3 to 1.0 units / ml of glutamyl hydrolase, wherein the concentration of glutamyl hydrolase can be, but is not limited to, 0.4 units / ml, 0.5 units / ml, 0.6 units / ml, 0.7 units / ml, 0.8 units / ml, 0.9 units / ml, or 1.0 units / ml. Preferably, the lysis solution is an aqueous solution containing 0.4 to 0.7 units / ml of glutamyl hydrolase. More preferably, the lysis solution is an aqueous solution containing 0.5 units / ml of glutamyl hydrolase.

[0029] In a preferred embodiment, the mixed standard SO solution mentioned in the present invention is prepared according to the following method: weighing each analyte standard, including folic acid, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, flavin adenine dinucleotide and vitamin B3, respectively, adding methanol aqueous solution to completely dissolve them, and preparing a standard stock solution with a concentration of 1 mmol / L folic acid, 10 mmol / L 5-methyltetrahydrofolate, 1 mmol / L 5-formyltetrahydrofolate, 1 mmol / L flavin adenine dinucleotide and 20 mmol / L vitamin B3;

[0030] The above-mentioned standard stock solutions were further prepared with methanol-water solution to prepare a mixed standard S0 solution containing 2 μmol / L folic acid, 20 μmol / L 5-methyltetrahydrofolate, 4 μmol / L 5-formyltetrahydrofolate, 40 μmol / L flavin adenine dinucleotide and 2000 μmol / L vitamin B3.

[0031] In a preferred embodiment, the internal standard SI solution mentioned in the present invention is prepared according to the following method: weighing each isotopic internal standard, including folic acid-d4, 5-methyltetrahydrofolate-d4, 5-formyltetrahydrofolate-d4, flavin adenine dinucleotide-d5 and vitamin B3-d4, respectively, adding methanol aqueous solution to completely dissolve them, and preparing isotope stock solutions with concentrations of 0.1 mmol / L folic acid-d4, 1 mmol / L 5-methyltetrahydrofolate-d4, 0.1 mmol / L 5-formyltetrahydrofolate-d4, 1 mmol / L flavin adenine dinucleotide-d5 and 1 mmol / L vitamin B3-d4;

[0032] The above-mentioned isotope internal standard stock solutions were further prepared with methanol-water solution to prepare an isotope mixed internal standard SI solution containing 0.2 μmol / L folic acid-d4, 2 μmol / L 5-methyltetrahydrofolate-d4, 0.4 μmol / L 5-formyltetrahydrofolate-d4, 4 μmol / L flavin adenine dinucleotide-d5 and 40 μmol / L vitamin B3-d4.

[0033] When preparing the mixed standard S0 solution and the internal standard SI solution, the methanol aqueous solution is 50-90% methanol aqueous solution, preferably 80% methanol aqueous solution.

[0034] In a preferred embodiment, a kit for determining folic acid and its metabolites in red blood cells by high performance liquid chromatography tandem mass spectrometry comprises the following reagents:

[0035] (1) Electrolyte concentrate A: 50% formic acid-water solution;

[0036] (2) Electrolyte concentrate B: 50% formic acid-methanol;

[0037] (3) Mixed standard SO solution:

[0038] Weigh each analyte standard, including folic acid, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, flavin adenine dinucleotide, and vitamin B3, and dissolve them completely in 80% methanol solution to prepare standard stock solutions with concentrations of 1 mmol / L folic acid, 10 mmol / L 5-methyltetrahydrofolate, 1 mmol / L 5-formyltetrahydrofolate, 1 mmol / L flavin adenine dinucleotide, and 20 mmol / L vitamin B3;

[0039] The above-mentioned standard stock solutions were further prepared with 80% methanol aqueous solution to prepare a mixed standard S0 solution containing 2 μmol / L folic acid, 20 μmol / L 5-methyltetrahydrofolate, 4 μmol / L 5-formyltetrahydrofolate, 40 μmol / L flavin adenine dinucleotide and 2000 μmol / L vitamin B3;

[0040] (4) Internal standard SI solution:

[0041] Weigh each isotopic internal standard, including folic acid-d4, 5-methyltetrahydrofolate-d4, 5-formyltetrahydrofolate-d4, flavin adenine dinucleotide-d5, and vitamin B3-d4, and completely dissolve them in 80% methanol solution to prepare isotope stock solutions with concentrations of 0.1 mmol / L folic acid-d4, 1 mmol / L 5-methyltetrahydrofolate-d4, 0.1 mmol / L 5-formyltetrahydrofolate-d4, 1 mmol / L flavin adenine dinucleotide-d5, and 1 mmol / L vitamin B3-d4;

[0042] The above-mentioned isotope internal standard stock solutions were further prepared with 80% methanol aqueous solution to prepare an isotope mixed internal standard SI solution containing 0.2 μmol / L folic acid-d4, 2 μmol / L 5-methyltetrahydrofolate-d4, 0.4 μmol / L 5-formyltetrahydrofolate-d4, 4 μmol / L flavin adenine dinucleotide-d5 and 40 μmol / L vitamin B3-d4;

[0043] (6) pH adjuster: 2M-3M NaOH solution;

[0044] (7) Lysis solution: an aqueous solution containing 0.4-0.7 units / ml of glutamyl hydrolase.

[0045] In a more preferred embodiment, the kit for determining folate and its metabolites in red blood cells by high performance liquid chromatography tandem mass spectrometry comprises the following reagents:

[0046] (1) Electrolyte concentrate A: 50% formic acid-water solution;

[0047] (2) Electrolyte concentrate B: 50% formic acid-methanol;

[0048] (3) Mixed standard SO solution:

[0049] Weigh each analyte standard, including folic acid, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, flavin adenine dinucleotide, and vitamin B3, and dissolve them completely in 80% methanol solution to prepare standard stock solutions with concentrations of 1 mmol / L folic acid, 10 mmol / L 5-methyltetrahydrofolate, 1 mmol / L 5-formyltetrahydrofolate, 1 mmol / L flavin adenine dinucleotide, and 20 mmol / L vitamin B3;

[0050] The above-mentioned standard stock solutions were further prepared with 80% methanol aqueous solution to prepare a mixed standard S0 solution containing 2 μmol / L folic acid, 20 μmol / L 5-methyltetrahydrofolate, 4 μmol / L 5-formyltetrahydrofolate, 40 μmol / L flavin adenine dinucleotide and 2000 μmol / L vitamin B3;

[0051] (4) Internal standard SI solution:

[0052] Weigh each isotopic internal standard, including folic acid-d4, 5-methyltetrahydrofolate-d4, 5-formyltetrahydrofolate-d4, flavin adenine dinucleotide-d5, and vitamin B3-d4, and completely dissolve them in 80% methanol solution to prepare isotope stock solutions with concentrations of 0.1 mmol / L folic acid-d4, 1 mmol / L 5-methyltetrahydrofolate-d4, 0.1 mmol / L 5-formyltetrahydrofolate-d4, 1 mmol / L flavin adenine dinucleotide-d5, and 1 mmol / L vitamin B3-d4;

[0053] The above-mentioned isotope internal standard stock solutions were further prepared with 80% methanol aqueous solution to prepare an isotope mixed internal standard SI solution containing 0.2 μmol / L folic acid-d4, 2 μmol / L 5-methyltetrahydrofolate-d4, 0.4 μmol / L 5-formyltetrahydrofolate-d4, 4 μmol / L flavin adenine dinucleotide-d5 and 40 μmol / L vitamin B3-d4;

[0054] (6) pH adjuster: 2M NaOH solution;

[0055] (7) Lysis buffer: an aqueous solution containing 0.5 unit / ml glutamyl hydrolase.

[0056] In a more preferred embodiment, the mixed standard SO solution mentioned in the present invention is prepared as follows:

[0057] Prepare the standard stock solutions of each analyte in the table below with 80% methanol aqueous solution, and then prepare the mixed standard S0 solutions (see Table 1 for details) with 80% methanol aqueous solution. Mix well and set aside.

[0058] Table 1 Preparation of mixed standard solution S0

[0059]

[0060] When the kit of the present invention is used, the mixed standard SO solution is prepared with a blank matrix solution to prepare eight calibration solutions at different concentrations. The eight concentrations of the calibration solution are:

[0061] The concentrations of folic acid were: 0.5 nmol / L, 1 nmol / L, 2 nmol / L, 4 nmol / L, 10 nmol / L, 20 nmol / L, 50 nmol / L, and 100 nmol / L;

[0062] The concentrations of 5-methyltetrahydrofolate were: 5 nmol / L, 10 nmol / L, 20 nmol / L, 40 nmol / L, 100 nmol / L, 200 nmol / L, 500 nmol / L, and 1000 nmol / L;

[0063] The concentrations of 5-formyltetrahydrofolate were: 1 nmol / L, 2 nmol / L, 4 nmol / L, 8 nmol / L, 20 nmol / L, 40 nmol / L, 100 nmol / L, and 200 nmol / L;

[0064] The concentrations of flavin adenine dinucleotide were: 10 nmol / L, 20 nmol / L, 40 nmol / L, 80 nmol / L, 200 nmol / L, 400 nmol / L, 1000 nmol / L, and 2000 nmol / L;

[0065] The concentrations of vitamin B3 are: 0.5μmol / L, 1μmol / L, 2μmol / L, 5μmol / L, 10μmol / L, 20μmol / L, 50μmol / L and 100μmol / L.

[0066] Wherein, the blank matrix solution is a methanol aqueous solution of 50-90% methanol aqueous solution, preferably 80% methanol aqueous solution.

[0067] In a preferred embodiment, the preparation process of the calibration solution is as follows:

[0068] Take 20 μL of the mixed standard solution S0 to a 1.5 mL centrifuge tube, add 380 μL of blank matrix (80% methanol in water) to obtain the first high concentration point S8, and then dilute it step by step to S1 (see Table 2 for details). The concentrations of each calibration point are listed in Table 5.

[0069] Table 2 Preparation and concentration of standard curve

[0070] Marking the song Solution (μL) Blank matrix (μL) VB9 5-MTHF 5-FoTHF FAD VB3 S8 20μL S0 380 100 1000 200 2000 100000 S7 100 μL S7 100 50 500 100 1000 50000 S6 40 μL S7 160 20 200 40 400 20000 S5 40 μL S6 160 10 100 20 200 10000 S4 40 μL S5 160 5 40 8 80 5000 S3 40 μL S4 160 2 20 4 40 2000 S2 50 μL S3 150 1 10 2 20 1000 S1 50 μL S2 150 0.5 5 1 10 500

[0071] (Note: Concentration units are all nmol / L)

[0072] For each concentration point sample, 50 μL was taken into a 1.5 mL centrifuge tube, 20 μL of the mixed internal standard working solution, 100 μL of 2% VC solution and 10 μL of 2M NaOH solution were added, vortexed for 5 seconds, 10 μL of 0.5 unit / ml glutamyl hydrolase aqueous solution was added, vortexed for 10 seconds, incubated at 37 ° C in the dark for 1 hour, 400 μL of a methanol-acetonitrile mixed solution with a liquid volume ratio of 2:1 was added, oscillated for 5 minutes, centrifuged at a speed of 14800 r / min and 4 ° C for 5 minutes, and 80 μL of the supernatant was taken for injection.

[0073] In a more preferred embodiment, the internal standard SI solution mentioned in the present invention is prepared as follows:

[0074] Each isotope internal standard was prepared with 80% methanol aqueous solution to prepare the isotope stock solution in the table below, and then the isotope stock solution of each concentration was prepared with 80% methanol aqueous solution to prepare the isotope mixed internal standard SI solution (see Table 3 for details).

[0075] Table 3 Preparation of mixed internal standard SI solution

[0076]

[0077] When using the kit of the present invention, the internal standard SI solution is diluted with methanol to form a mixed internal standard working solution. The specific preparation method is as follows: take 100 μL of the mixed internal standard SI solution, add 900 μL of methanol, and mix evenly to obtain the mixed internal standard working solution. The mixed internal standard working solution contains 0.02 μmol / L folic acid-d4, 0.2 μmol / L 5-methyltetrahydrofolate-d4, 0.04 μmol / L 5-formyltetrahydrofolate-d4, 0.4 μmol / L flavin adenine dinucleotide-d5, and 4 μmol / L vitamin B3-d4.

[0078] The concentration of the methanol aqueous solution mentioned in the present invention generally refers to the volume concentration.

[0079] In the present invention, when using a kit for determining folic acid and its metabolites in red blood cells using high performance liquid chromatography tandem mass spectrometry, the sample to be tested is a blood sample containing an EDTA anticoagulant, which is centrifuged to remove plasma to obtain red blood cells.

[0080] When used, the kit of the present invention also includes a quality control product, which contains a mixed solution of red blood cells containing five types of folic acid and its metabolites, divided into three concentrations: low, medium, and high, namely QC-L, QC-M, and QC-H. The specific preparation method is as follows:

[0081] QC-L consists of the mixed standard S0 solution diluted 500-fold with a pre-measured red blood cell sample. The volume ratio of S0 solution to red blood cells is 1:499. The following compounds are added to QC-L: 4 nmol / L folic acid, 40 nmol / L 5-methyltetrahydrofolate, 8 nmol / L 5-formyltetrahydrofolate, 80 nmol / L flavin adenine dinucleotide, and 4000 nmol / L vitamin B3.

[0082] QC-M consists of the mixed standard S0 solution diluted 100-fold with a pre-measured red blood cell sample. The volume ratio of S0 solution to red blood cells is 1:99. The following compounds are added to QC-M: 20 nmol / L folic acid, 200 nmol / L 5-methyltetrahydrofolate, 40 nmol / L 5-formyltetrahydrofolate, 400 nmol / L flavin adenine dinucleotide, and 20,000 nmol / L vitamin B3.

[0083] QC-H consists of the mixed standard S0 solution diluted 25-fold with a pre-measured red blood cell sample. The volume ratio of S0 solution to red blood cells is 1:24. The following compounds are added to QC-H: 80 nmol / L folic acid, 800 nmol / L 5-methyltetrahydrofolate, 160 nmol / L 5-formyltetrahydrofolate, 1600 nmol / L flavin adenine dinucleotide, and 80,000 nmol / L vitamin B3.

[0084] The amount of each compound added to the quality control product at each concentration is shown in Table 4:

[0085] Table 4 Addition amount of various compounds in quality control products (unit: nmol / L)

[0086] Compound QC-L QC-M QC-H folic acid 4 20 80 5-Methyltetrahydrofolate 40 200 800 5-Formyltetrahydrofolate 8 40 160 Flavin adenine dinucleotide 80 400 1600 Vitamin B3 4000 2000 8000

[0087] Since the background concentration of red blood cells used in the preparation of quality control varies between each batch, the red blood cell quality control products in each batch of the kit need to be measured using this patented method to determine the specific concentration of each compound before use.

[0088] The use of the above kit in the determination of folic acid and its metabolites in red blood cells using high performance liquid chromatography tandem mass spectrometry is also within the scope of protection of the present invention.

[0089] The above kit is used to determine folic acid and its metabolites in red blood cells using high performance liquid chromatography tandem mass spectrometry. The specific process is as follows:

[0090] After pretreatment of the sample, the target analyte is separated from the interfering components in the sample matrix using high performance liquid chromatography. The mass-to-charge ratio response of the target analyte and its corresponding isotope internal standard is then detected by mass spectrometry. The isotope internal standard method is used for quantification, and the contents of five types of red blood cell folate and their metabolites are calculated. The specific chromatographic conditions are as follows:

[0091] (1) HPLC conditions:

[0092] Mobile phase A: 0.05-0.2% formic acid-water solution;

[0093] Mobile phase B: 0.05-0.2% formic acid-methanol;

[0094] Chromatographic column: Kinetex XB-C18;

[0095] Gradient elution was performed using mobile phase A and mobile phase B as a mixed mobile phase, and the gradient elution process was as follows: within 0-0.5 minutes, the volume ratio of mobile phase A to mobile phase B was 70:30; within 0.5-2.0 minutes, the volume ratio of mobile phase A to mobile phase B was gradually changed from 70:30 to 2:98 at a constant speed; within 3.0-5.0 minutes, the volume ratio of mobile phase A to mobile phase B was 2:98; within 5.0-5.01 minutes, the volume ratio of mobile phase A to mobile phase B was gradually changed from 2:98 to 70:30 at a constant speed; within 5.01-7.0 minutes, the volume ratio of mobile phase A to mobile phase B was 70:30;

[0096] (2) Mass spectrometry conditions:

[0097] In electrospray ionization mode, multiple reaction monitoring was used for positive ion mode scanning; the capillary voltage was 3.0 kV, the nozzle voltage was 200 V, the drying gas temperature was 300°C, the sheath gas temperature was 350°C, the drying gas flow rate was 10 L / min, the sheath gas flow rate was 11 L / min, and the nebulizing gas pressure was 45 psi; folic acid and its metabolites in five types of red blood cells were monitored simultaneously with the corresponding isotopic internal standards.

[0098] In chromatography, the selection of a chromatographic column is very important, and the requirements for a chromatographic column are: high column efficiency, good selectivity, and fast analysis speed. The present invention uses 0.05-0.2% formic acid-water solution and 0.05-0.2% formic acid-methanol as the mobile phase, and the chromatographic column is a Kinetex XB-C18. Under the cooperation of other conditions, endogenous substances do not interfere with the determination of the sample. The method has high sensitivity, strong specificity, and low cost. Separation and detection can be completed within 7.0 minutes, and both precision and accuracy meet the requirements. In a preferred embodiment, the length of the chromatographic column is 100 mm, the diameter is 3.0 mm, and the filler particle size is 2.6 μm. For example, the chromatographic column is a Kinetex XB-C18 (2.6 μm, 3.0 mm × 100 mm).

[0099] In order to improve the selectivity of chromatographic separation, it is possible to consider adjusting the polarity of the mobile phase. The present invention adds formic acid to mobile phase A and mobile phase B, which can effectively improve the ionization efficiency of certain target compounds. Under the cooperation of other conditions, compared with the prior art method of using LC-MS / MS method to detect folic acid and its metabolites in red blood cells, the pretreatment process is simple, the sensitivity is higher, the sample amount is small, the specificity is strong, the cost is low, and 5 types of folic acid and its metabolites can be detected simultaneously within 7.0 minutes. Without affecting the effect of the present invention, in a preferred embodiment, mobile phase A is 0.1-0.15% formic acid-water solution, and mobile phase B is 0.1-0.15% formic acid-methanol. In a more preferred embodiment, mobile phase A is 0.1% formic acid-water solution, and mobile phase B is 0.1% formic acid-methanol.

[0100] When using the internal standard method, the selection of an internal standard is crucial. An ideal internal standard should be able to be added to a sample in an accurate, known amount and possess essentially the same or as close to identical physicochemical properties, chromatographic behavior, and response characteristics as the sample being analyzed. Under chromatographic analysis conditions, the internal standard must be fully separable from the components in the sample. The present invention utilizes folic acid-d4 (VB9-d4), 5-methyltetrahydrofolate-d4 (5-MTHF-d4), 5-formyltetrahydrofolate-d4 (5-FoTHF-d4), flavin adenine dinucleotide-d5 (FAD-d5), and vitamin B3-d4 (VB3-d4) as internal standards. These deuterated internal standards have the same retention time, chemical properties, and matrix effects as the analyte, resulting in excellent reproducibility and accuracy in the determination of folic acid and its metabolites in red blood cells.

[0101] In a preferred embodiment, the flow rate is 0.2 to 0.4 mL / min, preferably 0.3 mL / min.

[0102] Furthermore, the column temperature is 30-45°C, preferably 40°C.

[0103] Furthermore, the injection volume is 1 to 10 μL, preferably 2 μL, for example, 1 μL, 2 μL, 5 μL, or 10 μL.

[0104] In a preferred embodiment, the above kit is used to determine folic acid and its metabolites in red blood cells using high performance liquid chromatography tandem mass spectrometry, and the specific process is as follows:

[0105] (1) HPLC conditions:

[0106] Mobile phase A: 0.1% formic acid-water solution;

[0107] Mobile phase B: 0.1% formic acid-methanol;

[0108] Chromatographic column: Kinetex XB-C18 (2.6 μm, 3.0 mm × 100 mm);

[0109] Mobile phase A and mobile phase B were used as a mixed mobile phase for gradient elution, and the gradient elution process was as follows: within 0-0.5 minutes, the volume ratio of mobile phase A and mobile phase B was 70:30; within 0.5-2.0 minutes, the volume ratio of mobile phase A and mobile phase B was gradually changed from 70:30 to 2:98 at a uniform rate; within 3.0-5.0 minutes, the volume ratio of mobile phase A and mobile phase B was 2:98; within 5.0-5.01 minutes, the volume ratio of mobile phase A and mobile phase B was gradually changed from 2:98 to 70:30 at a uniform rate; within 5.01-7.0 minutes, the volume ratio of mobile phase A and mobile phase B was 70:30; the specific gradient elution mode is shown in Table 5, the flow rate is 0.3 mL / min, the column temperature is 40°C, and the injection volume is 2 μL.

[0110] Table 5 Mobile phase gradient elution parameters

[0111]

[0112] (2) Mass spectrometry conditions:

[0113] In electrospray ionization mode, multiple reaction monitoring was used for positive ion mode scanning; the capillary voltage was 3.0 kV, the nozzle voltage was 200 V, the drying gas temperature was 300°C, the sheath gas temperature was 350°C, the drying gas flow rate was 10 L / min, the sheath gas flow rate was 11 L / min, and the nebulizer gas pressure was 45 psi; folic acid and its metabolites in five types of red blood cells and the corresponding isotopic internal standards were monitored simultaneously. The mass spectrometry acquisition parameters for each target analyte are shown in Table 6.

[0114] Table 6 Mass spectrometry parameters of erythrocyte folate and its metabolites

[0115]

[0116] In the present invention, the sample to be tested is a blood sample containing EDTA anticoagulant, and the sample to be tested is red blood cells obtained by centrifuging and removing plasma.

[0117] For the present invention, the pretreatment of the sample to be tested is prepared according to the following method: take the red blood cell sample to be tested, add the mixed internal standard working solution, protective agent and pH adjuster, vortex and add lysis solution, incubate in the dark, then add protein precipitant, shake and centrifuge, and take the supernatant for injection.

[0118] The protective agent is a 1% to 3% VC solution, which may be but is not limited to a 1% VC solution, a 1.5% VC solution, a 2% VC solution, a 2.5% VC solution or a 3% VC solution. Preferably, the protective agent is a 2% VC solution.

[0119] Furthermore, the pH adjuster is a 1M to 5M NaOH solution, which may be but is not limited to a 1M NaOH solution, a 2M NaOH solution, a 3M NaOH solution, a 4M NaOH solution or a 5M NaOH solution. Preferably, the pH adjuster is a 2M to 3M NaOH solution, and more preferably, the pH adjuster is a 2M NaOH solution.

[0120] Furthermore, the lysis solution is an aqueous solution containing 0.3 to 1.0 units / ml of glutamyl hydrolase, wherein the concentration of glutamyl hydrolase can be, but is not limited to, 0.4 units / ml, 0.5 units / ml, 0.6 units / ml, 0.7 units / ml, 0.8 units / ml, 0.9 units / ml, or 1.0 units / ml. Preferably, the lysis solution is an aqueous solution containing 0.4 to 0.7 units / ml of glutamyl hydrolase. More preferably, the lysis solution is an aqueous solution containing 0.5 units / ml of glutamyl hydrolase.

[0121] In a preferred embodiment, the pretreatment of the sample to be tested is prepared according to the following method: 50 μL of red blood cells of the sample to be tested is taken into a 1.5 mL EP tube, 20 μL of the mixed internal standard working solution is added, 100 μL of 2% VC solution and 10 μL of 2M~3M NaOH solution are added, vortexed for 5 seconds, 10 μL of 0.4~0.7 unit / ml glutamyl hydrolase aqueous solution is added, vortexed for 10 seconds, incubated at 37°C in the dark for 0.5~2 hours, 400 μL of a methanol-acetonitrile mixed solution with a liquid volume ratio of 1~2:1 is added, shaken for 5 minutes, centrifuged at a speed of 14000~15000 r / min and 4°C for 4~10 minutes, and the supernatant is taken for injection.

[0122] In a more preferred embodiment, the pretreatment of the sample to be tested is prepared as follows: 50 μL of red blood cells of the sample to be tested is placed in a 1.5 mL EP tube, 20 μL of the mixed internal standard working solution is added, 100 μL of 2% VC solution and 10 μL of 2M NaOH solution are added, vortexed for 5 seconds, 10 μL of 0.5 unit / ml glutamyl hydrolase aqueous solution is added, vortexed for 10 seconds, incubated at 37° C. in the dark for 1 hour, 400 μL of a methanol-acetonitrile mixed solution with a liquid volume ratio of 2:1 is added, shaken for 5 minutes, centrifuged at a speed of 14800 r / min and 4° C. for 5 minutes, and 80 μL of the supernatant is taken for injection.

[0123] The present invention establishes a simple, efficient and reliable method for detecting folic acid and its metabolites in red blood cells, which can better meet the needs of clinical application compared with existing technologies.

[0124] Adopt the technical scheme of the present invention, the advantages are as follows:

[0125] The kit of the present invention is used to detect the content of folic acid and its metabolites in red blood cells, which solves the problem of quantification of red blood cells. The entire pretreatment process of the sample to be tested is simple, with high sensitivity, small sample dosage, and strong specificity. Five types of folic acid and their metabolites in red blood cells can be simultaneously detected within 7.0 minutes, and can be used for clinical diagnosis of five types of folic acid and their metabolites in red blood cells and health assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0126] Figure 1 Selected ion chromatograms of five folic acid and metabolite standards;

[0127] Figure 2 Selected ion chromatograms of five types of folic acid and their metabolites in red blood cell samples;

[0128] Figure 3 This is the selected ion chromatogram of the five folic acids and their metabolites in the red blood cell sample in Comparative Example 3. DETAILED DESCRIPTION

[0129] In order to more clearly understand the technical solution of the present invention, the present invention is further described through the following examples, but these examples do not constitute any limitation to the present invention.

[0130] The components of the assay kit are shown in Table 7.

[0131] Table 7 Components of the folic acid and its metabolite analysis kit (100 servings)

[0132]

[0133] Example 1

[0134] 1. Experimental Materials and Instruments

[0135] 1. Materials

[0136] Methodology The samples for the research experiments were freshly collected blood samples containing EDTA anticoagulant and the red blood cells were obtained by centrifugation to remove the plasma.

[0137] (1) Instruments: Qlife Lab 9000plus triple quadrupole mass spectrometer (PinSheng Medical); Qlife Lab 9000 high-performance liquid chromatography system (with G7167A autosampler, PinSheng Medical); system working software: MS quantitative analysis 10.0; SCILOGEX D2012 high-speed desktop centrifuge (USA); ultrapure water meter (ELGA LabWater, UK); multi-tube vortex mixer (Vortex genie2, USA); adjustable pipettes (Eppendorf 0.5-10 μL, 10-100 μL, 100-1000 μL); glassware, graduated cylinder, etc.

[0138] (2) Reagents and consumables: MS-grade methanol (Fisher, USA); HPLC-grade methanol (Honeywell, USA); ascorbic acid (Sigma, USA); sodium hydroxide (Sigma, USA); chromatographic column Kinetex XB-C18 (2.6 μm, 3.0 × 100 mm, Phenomenex).

[0139] (3) Standards: 5-MTHF, VB9, 5-FoTHF, VB3, and FAD standards were purchased from Sigma, and the corresponding isotope internal standards were purchased from TRC.

[0140] 2. Liquid quality conditions

[0141] (1) Chromatographic conditions: Mobile phase A: 0.1% formic acid-water; Mobile phase B: 0.1% formic acid-methanol; Chromatographic column model: Kinetex XB-C18 (2.6 μm, 3.0 mm × 100 mm); Gradient elution was used (see Table 5 for details). Flow rate: 0.3 ml / min; Column temperature: 40°C; Injection volume: 2 μL.

[0142] (2) In electrospray ionization (ESI) mode, multiple reaction monitoring (MRM) was used for positive ion mode scanning; the capillary voltage was 3.0 kV, the nozzle voltage was 200 V; the drying gas temperature was 300 °C, the sheath gas temperature was 350 °C, the drying gas flow rate was 10 L / min, the sheath gas flow rate was 11 L / min, and the nebulizer gas pressure was 45 psi; folic acid and its metabolites in five types of red blood cells and the corresponding isotopic internal standards were monitored simultaneously. The mass spectrometry acquisition parameters for each target analyte are shown in Table 6.

[0143] 3. Experimental Process

[0144] (1) Preparation of standard products:

[0145] Prepare the standard stock solution of each analyte with 80% methanol aqueous solution, and then prepare the mixed standard S0 solution (see Table 1 for details) with 80% methanol aqueous solution. Mix them well and set aside.

[0146] Take 20 μL of the mixed standard solution S0 to a 1.5 mL centrifuge tube, add 380 μL of blank matrix (80% methanol in water) to obtain the first high concentration point S8, and then dilute it step by step to S1 (see Table 2 for details). The concentrations of each calibration point are listed in Table 5.

[0147] (2) Preparation of mixed internal standard working solution:

[0148] Each isotopic internal standard was prepared into an isotope stock solution using 80% methanol in water. The isotope stock solution at each concentration was then prepared into an isotope mixed internal standard SI solution using 80% methanol in water (see Table 3 for details). Finally, 100 μL of the mixed internal standard SI solution was added to 900 μL of methanol and mixed thoroughly to obtain the mixed internal standard working solution. The mixed internal standard working solution contained 0.02 μmol / L folic acid-d4, 0.2 μmol / L 5-methyltetrahydrofolate-d4, 0.04 μmol / L 5-formyltetrahydrofolate-d4, 0.4 μmol / L flavin adenine dinucleotide-d5, and 4 μmol / L vitamin B3-d4.

[0149] (3) Preparation of quality control products:

[0150] The mixed red blood cell solution containing five types of folic acid and its metabolites is divided into three concentrations: low, medium and high. See Table 4 for details, namely QC-L, QC-M and QC-H.

[0151] QC-L is the mixed standard S0 solution diluted 500 times with a red blood cell sample of pre-determined concentration, and the volume ratio of S0 solution to red blood cells is 1:499.

[0152] QC-M is the mixed standard S0 solution diluted to 100 times with a red blood cell sample of a pre-determined concentration, and the volume ratio of S0 solution to red blood cells is 1:99.

[0153] QC-H is the above-mentioned mixed standard S0 solution diluted to 25 times with a red blood cell sample of a pre-determined concentration, and the volume ratio of S0 solution to red blood cells is 1:24.

[0154] (4) Sample processing

[0155] 1) Standard sample pretreatment:

[0156] For each concentration point sample, 50 μL was taken into a 1.5 mL centrifuge tube, 20 μL of the mixed internal standard working solution, 100 μL of 2% VC solution and 10 μL of 2M NaOH solution were added, vortexed for 5 seconds, 10 μL of 0.5 unit / ml glutamyl hydrolase aqueous solution was added, vortexed for 10 seconds, incubated at 37 ° C in the dark for 1 hour, 400 μL of a methanol-acetonitrile mixed solution with a liquid volume ratio of 2:1 was added, oscillated for 5 minutes, centrifuged at a speed of 14800 r / min and 4 ° C for 5 minutes, and 80 μL of the supernatant was taken for injection.

[0157] 2) Sample pretreatment:

[0158] Take 50 μL of red blood cells of the sample to be tested and place them in a 1.5 mL EP tube. Add 20 μL of mixed internal standard working solution, 100 μL of 2% VC solution and 10 μL of 2M NaOH solution, vortex for 5 seconds, add 10 μL of 0.5 unit / ml glutamyl hydrolase aqueous solution, vortex for 10 seconds, incubate at 37°C in the dark for 1 hour, add 400 μL of methanol-acetonitrile mixed solution with a liquid volume ratio of 2:1, shake for 5 minutes, centrifuge at a speed of 14800 r / min and 4°C for 5 minutes, and take 80 μL of the supernatant for injection.

[0159] 3) Pre-treatment of quality control products:

[0160] Take 50 μL of each of the quality control solutions QC-L, QC-M and QC-H in 1.5 mL centrifuge tubes, and then pre-treat them in the same way as the red blood cell samples, which will not be repeated here.

[0161] 4. Experimental Results

[0162] One actual human blood sample was taken, and the concentrations of five erythrocyte folates and their metabolites in erythrocytes were determined according to the chromatographic conditions described in Example 1. The results are shown in Table 8 below:

[0163] Table 8 Concentrations of five erythrocyte folates and their metabolites in erythrocytes

[0164] Red blood cells VB9 (nM) 5-MTHF (nM) 5-FoTHF (nM) FAD (nM) VB3 (μM) Sample 1 1.58 295.4 2.53 345.9 73.9

[0165] 5. Method Validation

[0166] 1. Exclusivity

[0167] Figure 1 and Figure 2 The following are selected ion chromatograms of standard solutions of five folate and its metabolites in red blood cells and red blood cell samples. As can be seen, the chromatographic retention times of these five compounds are consistent, both in the standard solutions and in the actual samples, with no interference near the peaks of each compound, indicating excellent specificity.

[0168] 2. Standard Curve

[0169] Using the isotope internal standard quantification method, MS Quantitative Analysis 10.0 software was used to establish a calibration curve, with the concentration ratio of the standard to the internal standard as the X-axis and the peak area ratio of the standard to the internal standard as the Y-axis. The following linear fitting equations for the five erythrocyte folates and their metabolites within their respective concentration ranges showed good linearity, with correlation coefficients exceeding 0.99. See Table 9 for details.

[0170] Table 9 Retention time and linear range of five red blood cell folates and their metabolites

[0171]

[0172] 3. Investigation of spike recovery

[0173] A mixed standard SO solution of known concentration was added to red blood cells to prepare three reference concentrations: low, medium, and high. Six parallel runs were performed, and recoveries were calculated. See Tables 10-1, 10-2, 10-3, 10-4, and 10-5. The results show that the recoveries of the five erythrocyte folates and their metabolites in the red blood cell samples ranged from 85% to 115%, meeting all requirements.

[0174] Table 10-1 Recovery results of spiked VB9 in quality control samples

[0175]

[0176] Table 10-2 Recovery results of 5-MTHF spiked in quality control samples

[0177]

[0178] Table 10-3 Recovery results of 5-FoTHF spiked in quality control samples

[0179]

[0180] Table 10-4 Recovery results of spiked FAD in quality control samples

[0181]

[0182] Table 10-5 Recovery results of spiked VB3 in quality control samples

[0183]

[0184] 4. Precision test

[0185] Red blood cell quality control samples of various concentrations were processed six times daily for three days. The concentrations of folate and its metabolites in the five red blood cell samples were quantitatively determined using the isotope internal standard method. The intra-batch precision was calculated daily for three consecutive days. Three batches were processed within three days, and the inter-batch precision was calculated (see Table 11). The results showed that the intra-batch and inter-batch precision of the five red blood cell folate and its metabolites in the red blood cell samples were all below 15%, meeting the requirements.

[0186] Table 11 Intra-batch and inter-batch precision test results of quality control samples

[0187]

[0188]

[0189] 6. Discussion

[0190] The kit provided by the present invention uses liquid chromatography-tandem mass spectrometry to detect the concentrations of folic acid and its metabolites in human erythrocytes. The isotope internal standard method for quantification can greatly eliminate matrix interference and is not affected by conditions such as pretreatment process, sample volume and mobile phase, thereby achieving accurate quantification.

[0191] This method investigated the spiked recoveries of folic acid and its metabolites in five types of red blood cells, all of which were between 85% and 115%, meeting the requirements. The reproducibility results of the method showed that the intra-batch and inter-batch precisions of folic acid and its metabolites in the five types of red blood cells were both within 15%, indicating good reproducibility of the method.

[0192] This method has higher sensitivity and specificity than other LC-MS / MS methods. It can simultaneously detect 5 types of folic acid and its metabolites in red blood cells within 7.0 minutes, which can meet the daily clinical needs for folic acid detection in red blood cells and health assessment.

[0193] Example 2

[0194] In this example, the LC-MS conditions were the same as those in Example 1, and the sample pretreatment process was similar to that in Example 1, except that during the red blood cell sample pretreatment, the protein precipitant was adjusted from a methanol-acetonitrile mixed solution with a volume ratio of 2:1 to a methanol-acetonitrile mixed solution with a volume ratio of 1:1.

[0195] Standard solutions of various concentrations and quality control products QC-L, QC-M, and QC-H were used to determine the concentrations of five erythrocyte folates and their metabolites in erythrocytes according to the chromatographic conditions described in Example 2. The results for the compounds in each quality control product were essentially consistent with those in Example 1, with the main difference being the peak area of ​​each compound. Specific differences are shown in Table 12.

[0196] Table 12 Comparison of peak areas of samples in Example 1 and Example 2

[0197]

[0198]

[0199] As can be seen from Table 12, in Example 2, the peak areas of the various compounds in the standard sample solution are not much different from those in Example 1, while in the quality control sample, except for VB3, the peak areas of the various compounds are reduced to varying degrees relative to Example 1, with a reduction of about 10%.

[0200] Example 3

[0201] In this embodiment, the LC-MS conditions are the same as those in Example 1, and the sample pretreatment process is similar to that in Example 1, except that during the red blood cell sample pretreatment, the pH adjuster is adjusted from 2M NaOH solution to 3M NaOH solution.

[0202] Three aliquots of standard solution at each concentration and three red blood cell samples were collected and the concentrations of five erythrocyte folates and their metabolites were determined in the red blood cells according to the chromatographic conditions described in Example 3. The results for 5-methyltetrahydrofolate in each red blood cell sample were slightly lower than those in Example 1, but the overall differences were not significant. Specific differences are shown in Table 13.

[0203] Table 13 Comparison of the measurement results of each sample in Example 1 and Example 3

[0204]

[0205] Example 4

[0206] In this example, the LC-MS conditions were the same as those in Example 1, and the sample pretreatment process was similar to that in Example 1, except that during the red blood cell sample pretreatment, the lysis buffer was adjusted from a 0.5 unit / ml aqueous solution of glutamyl hydrolase to a 0.7 unit / ml aqueous solution of glutamyl hydrolase.

[0207] Three aliquots of standard solution at each concentration and three red blood cell samples were taken and the concentrations of five erythrocyte folates and their metabolites in the erythrocytes were determined according to the chromatographic conditions described in Example 4. The results for 5-methyltetrahydrofolate in each erythrocyte sample were slightly lower than those in Example 1, but the overall differences were not significant. Specific differences are shown in Table 14.

[0208] Table 14 Comparison of the measurement results of each sample in Example 1 and Example 4

[0209]

[0210] Comparative Example 1

[0211] In this comparative example, the LC-MS conditions were the same as those in Example 1, and the sample pretreatment process was the same as in Example 1, except that no pH adjuster (2M NaOH solution) was added during the red blood cell sample pretreatment.

[0212] Three portions of standard solution and red blood cell samples at each concentration were collected and the concentrations of five erythrocyte folates and their metabolites were determined in red blood cells using the chromatographic conditions described in Comparative Example 1. The results for folate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate in each red blood cell sample were significantly lower than those in Example 1. Specific differences are shown in Table 15.

[0213] Table 15 Comparison of the measurement results of each sample in Example 1 and Comparative Example 1

[0214]

[0215] Comparative Example 2

[0216] In this comparative example, the LC-MS conditions were the same as those in Example 1, and the sample pretreatment process was the same as in Example 1, except that during the red blood cell sample pretreatment, the lysis buffer was adjusted from a 0.5 unit / ml aqueous solution of glutamyl hydrolase to a 0.2 unit / ml aqueous solution of glutamyl hydrolase.

[0217] Three aliquots of standard solution at each concentration and three red blood cell samples were collected and the concentrations of five erythrocyte folates and their metabolites were determined in the red blood cells according to the chromatographic conditions described in Example 3. The results for folate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate in each red blood cell sample were lower than those in Example 1, with no significant overall differences. Specific differences are shown in Table 16.

[0218] Table 16 Comparison of the measurement results of each sample in Example 1 and Comparative Example 1

[0219]

[0220] Comparative Example 3

[0221] In this comparative example, the LC-MS conditions were the same as those in Example 1, and the sample pretreatment process was similar to that in Example 1, except that mobile phase A was 0.01% formic acid-water solution, and mobile phase B was 0.01% formic acid-methanol.

[0222] Take one portion of the standard solution of each concentration and one portion of the red blood cell sample, and determine the concentrations of five erythrocyte folates and their metabolites in the red blood cells according to the chromatographic conditions described in Comparative Example 3. The measured values ​​of each compound in the red blood cell sample are basically consistent with the results of Example 1, but the peak shapes of the ion current chromatograms of VB9, VB3, and FAD are worse than those in Example 1, as shown in the following figure. Figure 3 , wherein the ion chromatograms of the five compounds in Example 1 of this application are shown in Figure 2 .

[0223] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that it is still possible to modify the technical solutions described in the aforementioned embodiments, or to make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of the kit in the determination of folic acid and its metabolites in red blood cells using high performance liquid chromatography tandem mass spectrometry, The folic acid and its metabolites are: folic acid, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, flavin adenine dinucleotide and vitamin B3; The isotopic internal standards corresponding to the above-mentioned folic acid and its metabolites are: folic acid-d4, 5-methyltetrahydrofolate-d4, 5-formyltetrahydrofolate-d4, flavin adenine dinucleotide-d5 and vitamin B3-d4; The kit comprises the following reagents: (1) Electrolyte concentrate A: 40~60% formic acid-water solution; (2) Electrolyte concentrate B: 40-60% formic acid-methanol; (3) Mixed standard S0 solution: A methanol-water solution containing 2 μmol / L folic acid, 20 μmol / L 5-methyltetrahydrofolate, 4 μmol / L 5-formyltetrahydrofolate, 40 μmol / L flavin adenine dinucleotide, and 2000 μmol / L vitamin B3; (4) Internal standard SI solution: A methanol-water solution containing 0.2 μmol / L folic acid-d4, 2 μmol / L 5-methyltetrahydrofolate-d4, 0.4 μmol / L 5-formyltetrahydrofolate-d4, 4 μmol / L flavin adenine dinucleotide-d5, and 40 μmol / L vitamin B3-d4; (5) Protective agent: 1%~3% VC solution; (6) pH adjuster: 1M~5M NaOH solution; (7) Lysis buffer: an aqueous solution containing 0.3–1.0 unit / ml glutamyl hydrolase; When using the kit, add methanol to dilute the internal standard SI solution to form a mixed internal standard working solution. The specific preparation method is as follows: take 100 μL of the mixed internal standard SI solution, add 900 μL of methanol, and mix well to obtain the mixed internal standard working solution; After pretreatment of the sample, the target analyte is separated from the interfering components in the sample matrix using high performance liquid chromatography. The mass-to-charge ratio response of the target analyte and its corresponding isotope internal standard is then detected by mass spectrometry. The isotope internal standard method is used for quantification, and the contents of five types of red blood cell folate and their metabolites are calculated. The specific chromatographic conditions are as follows: (a) HPLC conditions: Mobile phase A: 0.05~0.2% formic acid-water solution; Mobile phase B: 0.05–0.2% formic acid-methanol; Chromatographic column: Kinetex XB-C18, the length of the chromatographic column is 100 mm, the diameter is 3.0 mm, and the filler particle size is 2.6 μm; Gradient elution was performed using mobile phase A and mobile phase B as a mixed mobile phase, and the gradient elution process was as follows: within 0-0.5 minutes, the volume ratio of mobile phase A and mobile phase B was 70:30; within 0.5-2.0 minutes, the volume ratio of mobile phase A and mobile phase B was gradually changed from 70:30 to 40:60 at a uniform speed; within 2.0-3.0 minutes, the volume ratio of mobile phase A and mobile phase B was gradually changed from 40:60 to 2:98 at a uniform speed; within 3.0-5.0 minutes, the volume ratio of mobile phase A and mobile phase B was 2:98; within 5.0-5.01 minutes, the volume ratio of mobile phase A and mobile phase B was gradually changed from 2:98 to 70:30 at a uniform speed; within 5.01-7.0 minutes, the volume ratio of mobile phase A and mobile phase B was 70:30; (b) Mass spectrometry conditions: Electrospray ionization was performed in positive ion mode with multiple reaction monitoring. The capillary voltage was 3.0 kV, the nozzle voltage was 200 V, the drying gas temperature was 300°C, the sheath gas temperature was 350°C, the drying gas flow rate was 10 L / min, the sheath gas flow rate was 11 L / min, and the nebulizer gas pressure was 45 psi. Folate and its metabolites were simultaneously monitored in five erythrocytes along with the corresponding isotopic internal standards. The sample to be tested is a blood sample containing EDTA anticoagulant, which is centrifuged to remove plasma to obtain red blood cells; wherein, The pretreatment of the sample to be tested was prepared as follows: a red blood cell sample to be tested was taken, a mixed internal standard working solution, a protective agent, and a pH adjuster were added, vortexed, and a lysis solution was added. After incubation in the dark, a protein precipitant was added, and after oscillation and centrifugation, the supernatant was collected for injection; the protein precipitant was a methanol-acetonitrile mixed solution with a volume ratio of 1 to 4:

1.

2. The use according to claim 1, characterized in that The protective agent is a 2% VC solution; the pH regulator is a 2M~3M NaOH solution; the lysate is an aqueous solution containing 0.4~0.7 unit / ml glutamyl hydrolase; and the protein precipitant is a 1~2:1 methanol-acetonitrile mixed solution.

3. The use according to claim 2, characterized in that The pH regulator is a 2M NaOH solution; and the lysate is an aqueous solution containing 0.5 unit / ml glutamyl hydrolase.

4. The use according to claim 1, characterized in that The pretreatment of the sample to be tested was prepared as follows: 50 μL of the red blood cells of the sample to be tested was placed in a 1.5 mL EP tube, 20 μL of the mixed internal standard working solution was added, 100 μL of 2% VC solution and 10 μL of 2M-3M NaOH solution were added, and the tube was vortexed for 5 seconds. 10 μL of 0.4-0.7 unit / mL glutamyl hydrolase aqueous solution was added, and the tube was vortexed for 10 seconds. Incubated at 37°C in the dark for 0.5-2 hours, 400 μL of a methanol-acetonitrile mixture with a volume ratio of 1-2:1 was added, and the tube was shaken for 5 minutes. The tube was centrifuged at 14000-15000 r / min and 4°C for 4-10 minutes, and the supernatant was collected for injection. The mobile phase A is 0.1-0.15% formic acid-water solution; the mobile phase B is 0.1-0.15% formic acid-methanol; the flow rate is 0.2-0.4 mL / min; the column temperature is 30-45°C; and the injection volume is 1-10 µL.

5. The use according to claim 4, characterized in that The pretreatment of the sample to be tested was prepared as follows: 50 μL of the red blood cells of the sample to be tested was taken into a 1.5 mL EP tube, 20 μL of the mixed internal standard working solution was added, 100 μL of 2% VC solution and 10 μL of 2M NaOH solution were added, and the tube was vortexed for 5 s. 10 μL of 0.5 unit / mL glutamyl hydrolase aqueous solution was added, and the tube was vortexed for 10 s. Incubated at 37°C in the dark for 1 h, 400 μL of a methanol-acetonitrile mixture with a liquid volume ratio of 2:1 was added, and the tube was shaken for 5 min. The tube was centrifuged at 14800 r / min and 4°C for 5 min, and 80 μL of the supernatant was collected for injection. The mobile phase A was 0.1% formic acid-water solution; the mobile phase B was 0.1% formic acid-methanol; the flow rate was 0.3 mL / min; the column temperature was 40°C; and the injection volume was 2 µL.

6. The use according to claim 1, characterized in that The mixed standard SO solution was prepared as follows: Weigh each standard substance to be analyzed, including folic acid, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, flavin adenine dinucleotide, and vitamin B3, and add each standard substance to a methanol-water solution to completely dissolve them. Prepare standard stock solutions with concentrations of 1 mmol / L folic acid, 10 mmol / L 5-methyltetrahydrofolate, 1 mmol / L 5-formyltetrahydrofolate, 1 mmol / L flavin adenine dinucleotide, and 20 mmol / L vitamin B3. The above-mentioned standard stock solutions were further prepared with methanol-water solution to prepare a mixed standard S0 solution containing 2 μmol / L folic acid, 20 μmol / L 5-methyltetrahydrofolate, 4 μmol / L 5-formyltetrahydrofolate, 40 μmol / L flavin adenine dinucleotide and 2000 μmol / L vitamin B3; The internal standard SI solution was prepared as follows: Weigh each isotopic internal standard, including folic acid-d4, 5-methyltetrahydrofolate-d4, 5-formyltetrahydrofolate-d4, flavin adenine dinucleotide-d5, and vitamin B3-d4, and completely dissolve them in methanol-water solution to prepare isotope stock solutions with concentrations of 0.1 mmol / L folic acid-d4, 1 mmol / L 5-methyltetrahydrofolate-d4, 0.1 mmol / L 5-formyltetrahydrofolate-d4, 1 mmol / L flavin adenine dinucleotide-d5, and 1 mmol / L vitamin B3-d4; The above-mentioned isotope internal standard stock solutions were further prepared with methanol-water solution to prepare an isotope mixed internal standard SI solution containing 0.2 μmol / L folic acid-d4, 2 μmol / L 5-methyltetrahydrofolate-d4, 0.4 μmol / L 5-formyltetrahydrofolate-d4, 4 μmol / L flavin adenine dinucleotide-d5 and 40 μmol / L vitamin B3-d4.

7. The use according to claim 6, characterized in that When preparing the mixed standard SO solution and the internal standard SI solution, the methanol aqueous solution is 50-90% methanol aqueous solution.

8. The use according to claim 7, characterized in that When preparing the mixed standard SO solution and the internal standard SI solution, the methanol aqueous solution is 80% methanol aqueous solution.

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