A kit for detecting anemia markers, and its preparation method and application

Abstract

The present invention belongs to the field of clinical testing, and relates to a kit for detecting anemia markers, and its preparation method and application. A method for detecting anemia markers, respectively adding internal standard sample solution to the series of calibration sample liquids, the series of quality control sample liquids and serum samples, mixing, then continuing to add sample extract, mixing, taking the supernatant after centrifugation, and performing LC-MS / MS detection on the supernatant. The water-soluble vitamin detection method, folic acid detection method, methylmalonic acid detection method, etc. in the prior art are unable to achieve the simultaneous detection of homocysteine, active folic acid, vitamin B12, methylmalonic acid and vitamin C. The present invention is aimed at this technical problem and invents a detection method for simultaneously detecting the above-mentioned anemia markers.

Description

Technical Field

[0001] The present invention belongs to the field of clinical testing, and relates to a kit for detecting anemia markers, a preparation method and an application thereof. Background Art

[0002] Megaloblastic anemia is primarily caused by a deficiency of folate, vitamin B12, or vitamin C. It can also be caused by inherited or drug-induced DNA synthesis disorders. Folate deficiency leads to a significant increase in homocysteine ​​concentrations. Therefore, the "Chinese Multidisciplinary Expert Consensus on Rational Folic Acid Supplementation in Clinical Practice" and the US Centers for Disease Control and Prevention recommend a homocysteine ​​level >13 μmol / L as a nonspecific indicator of functional folate deficiency. Serum methylmalonic acid (MMA) is a specific indicator of vitamin B12 deficiency, but this may be due to renal failure. Furthermore, MMA levels can be used to monitor treatment response. MMA levels remain normal in the setting of folate deficiency, whereas they increase in the setting of vitamin B12 or folate deficiency. Therefore, simultaneous testing of folate, vitamin B12, vitamin C, homocysteine, and methylmalonic acid not only effectively diagnoses megaloblastic anemia but also identifies the specific nutrient deficiency causing the anemia.

[0003] There have been many reports on detection methods and kits for folic acid, vitamin B12, methylmalonic acid, vitamin C, etc., but they all have problems such as the inability to detect simultaneously or harsh detection conditions. For example, the currently reported methods can detect 6 or 9 water-soluble vitamins by mass spectrometry, but it is difficult to meet clinical requirements. For example, Chinese patent CN201310744507.3 provides "a detection method for the simultaneous determination of 9 water-soluble vitamins", and the detection sample is a vitamin functional beverage, not a blood sample; Chinese patent CN202010172197.2 provides a "kit for detecting 9 water-soluble vitamins in serum using ultra-high performance liquid chromatography tandem mass spectrometry technology", and the detection sample is a serum sample; Chinese patent CN202111534070.1 provides "a detection method for multiple water-soluble vitamins", which detects 5-methyltetrahydrofolate and methylmalonic acid, but lacks the detection of homocysteine, vitamin B12 and vitamin C, and cannot be used as a detection method for anemia markers; Chinese patents CN202110418069.6 and CN201610165967.4 are both vitamin B12-specific detection methods and are not compatible with the detection of other anemia markers. In addition, there are no stability reports for such detection methods or kits. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies of the existing technology and provide a kit for detecting anemia markers, as well as its preparation method and application. Based on liquid chromatography-tandem mass spectrometry, the present invention develops a method for simultaneously detecting homocysteine, 5-methyltetrahydrofolate, vitamin B12, vitamin C, and methylmalonic acid in serum. Furthermore, the present invention provides a kit with strong stability and long-term storage capability. This kit can be used as a method and detection reagent for screening and differential diagnosis of megaloblastic anemia.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] The present invention discloses a kit for detecting anemia markers, which is characterized in that:

[0007] The anemia markers are homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid;

[0008] The kit comprises the following reagents:

[0009] (1) Series of calibrators:

[0010] The series of calibrators are freeze-dried products of a series of calibrator working solutions with five different concentration levels, and the series of calibrator working solutions are calibrator working solutions with five different concentration levels prepared by diluting a standard working solution of known concentration with a second solvent;

[0011] The five concentration levels of the calibrator working solution are:

[0012] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the calibrator S1 working solution were 700 ng / mL, 1.0 ng / mL, 20 ng / mL, 0.05 ng / mL, and 400 ng / mL, respectively;

[0013] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the calibrator S2 working solution were 1400 ng / mL, 2.0 ng / mL, 40 ng / mL, 0.1 ng / mL, and 800 ng / mL, respectively;

[0014] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the calibrator S3 working solution were 7000 ng / mL, 10 ng / mL, 200 ng / mL, 0.5 ng / mL, and 4000 ng / mL, respectively;

[0015] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the calibrator S4 working solution were 28000 ng / mL, 40 ng / mL, 800 ng / mL, 2.0 ng / mL, and 16000 ng / mL, respectively;

[0016] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the calibrator S5 working solution were 56000 ng / mL, 80 ng / mL, 1600 ng / mL, 4.0 ng / mL, and 32000 ng / mL, respectively;

[0017] (2) Series of quality control products:

[0018] The series of quality control products are freeze-dried products of a series of quality control product working solutions with two different concentration levels, and the series of quality control product working solutions are quality control product working solutions with two different concentration levels prepared by diluting a standard working solution of known concentration with a second solvent;

[0019] The two concentration levels of the quality control working solution are:

[0020] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the quality control QC1 working solution were 3500 ng / mL, 5.0 ng / mL, 100 ng / mL, 0.25 ng / mL, and 2000 ng / mL, respectively;

[0021] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the quality control QC2 working solution were 35,000 ng / mL, 50 ng / mL, 1,000 ng / mL, 2.5 ng / mL, and 20,000 ng / mL, respectively;

[0022] (3) Internal standard:

[0023] The internal standard is a lyophilized product of the internal standard working solution;

[0024] The concentrations of homocysteine-d4, 5-methyltetrahydrofolate-d3, methylmalonic acid-d3, and ascorbic acid-13C6 in the internal standard working solution are 7000 ng / mL, 10 ng / mL, 200 ng / mL, and 4000 ng / mL, respectively;

[0025] (4) Protective agent: The protective agent is a lyophilized product of the protective agent working solution;

[0026] The protective agent working solution is a dithiothreitol aqueous solution, and the concentration of dithiothreitol in the solution is 5g / L;

[0027] (5) Sample extract: an aqueous solution containing metaphosphoric acid and sulfosalicylic acid, with the concentration of both metaphosphoric acid and sulfosalicylic acid in the solution being 50 g / L;

[0028] (6) Mobile phase additive: an aqueous solution containing 50% formic acid and 0.2 mol / L ammonium acetate;

[0029] The calibrator, the quality control product, the internal standard product, and the protective agent are freeze-dried using the same freeze-drying process, and the freeze-drying process sequentially includes a pre-freezing stage, sublimation drying, and desorption drying;

[0030] The operating conditions of the pre-freezing stage are shown in Table 1:

[0031] Table 1

[0032]

[0033] The operating conditions of the sublimation drying are shown in Table 2:

[0034] Table 2

[0035]

[0036] The operating conditions of the analytical drying are shown in Table 3:

[0037] Table 3

[0038]

[0039] or,

[0040] The calibrator, the quality control product, the internal standard product, and the protective agent are freeze-dried using the same freeze-drying process, and the freeze-drying process sequentially includes a pre-freezing stage, sublimation drying, and desorption drying;

[0041] The operating conditions of the pre-freezing stage are shown in Table 4:

[0042] Table 4

[0043]

[0044] The operating conditions of the sublimation drying are shown in Table 5:

[0045] Table 5

[0046]

[0047] The operating conditions of the analytical drying are shown in Table 6:

[0048] Table 6

[0049]

[0050] Wherein, when the standard working solution is mixed with the second solvent, the proportion of the standard working solution added does not exceed 5% of the total volume.

[0051] In some embodiments, the second solvent consists of bovine serum albumin, an antioxidant, a preservative, a buffer and water; the antioxidant is isoascorbic acid; the preservative is citric acid; the buffer is PBS buffer; preferably, the second solvent consists of 0.1% to 5% bovine serum albumin, 0.05% to 2% isoascorbic acid, 0.05% to 2% citric acid, 10% 10×PBS buffer and deionized water; further preferably, the second solvent consists of 0.1% bovine serum albumin, 0.1% isoascorbic acid, 0.1% citric acid, 10% 10×PBS buffer and deionized water.

[0052] In some embodiments, the standard working solution of known concentration is prepared as follows:

[0053] Weigh homocysteine ​​and mix it with 1N aqueous hydrochloric acid and water to prepare a standard stock solution with a concentration of 10,000 μg / mL; weigh 5-methyltetrahydrofolate and mix it with ammonia and water to prepare a standard stock solution with a concentration of 100 μg / mL; weigh methylmalonic acid and mix it with water to prepare a standard stock solution with a concentration of 1,000 μg / mL; weigh cyanocobalamin and mix it with water to prepare a standard stock solution with a concentration of 10 μg / mL; weigh ascorbic acid and mix it with water to prepare a standard stock solution with a concentration of 10,000 μg / mL;

[0054] The above-mentioned standard stock solutions are mixed and then prepared into a standard working solution with a first solvent; the concentration of homocysteine ​​in the standard working solution is 1120 μg / mL, the concentration of 5-methyltetrahydrofolate is 1.6 μg / mL, the concentration of methylmalonic acid is 32 μg / mL, the concentration of cyanocobalamin is 0.08 μg / mL, and the concentration of ascorbic acid is 640 μg / mL.

[0055] The mass volume ratio of homocysteine ​​to 1N hydrochloric acid aqueous solution is preferably 50 mg:20 μL.

[0056] The mass volume ratio of the 5-methyltetrahydrofolate to ammonia water is preferably 1 mg:30 μL.

[0057] In some embodiments, the internal standard working solution is prepared as follows:

[0058] Weigh homocysteine-d4 and mix it with 1N hydrochloric acid solution and water to prepare a 1000 μg / mL internal standard stock solution; weigh 5-methyltetrahydrofolate-d3 and mix it with ammonia and water to prepare a 100 μg / mL internal standard stock solution; weigh methylmalonic acid-d3 and mix it with water to prepare a 1000 μg / mL internal standard stock solution; weigh ascorbic acid-13C6 and mix it with water to prepare a 1000 μg / mL internal standard stock solution;

[0059] The above-mentioned internal standard stock solutions are mixed and then prepared into an internal standard working solution with a first solvent; the concentration of homocysteine-d4 in the internal standard working solution is 70 μg / mL, the concentration of 5-methyltetrahydrofolate-d3 is 0.1 μg / mL, the concentration of methylmalonic acid-d3 is 2.0 μg / mL, and the concentration of ascorbic acid-13C6 is 40 μg / mL;

[0060] The obtained internal standard working solution is mixed with the second solvent to prepare an internal standard product working solution.

[0061] The mass volume ratio of homocysteine-d4 to 1N hydrochloric acid aqueous solution is preferably 1 mg:20 μL.

[0062] The mass volume ratio of the 5-methyltetrahydrofolate-d3 to ammonia water is preferably 1 mg:30 μL.

[0063] In some embodiments, the first solvent consists of methanol, citric acid, dithiothreitol and deionized water; preferably, the first solvent consists of 10% methanol, 0.1% citric acid, 0.1% dithiothreitol and deionized water.

[0064] In some embodiments, preferably, the series of calibrators, the series of quality control working solutions, the internal standard working solution, and the protective agent working solution are lyophilized to obtain the series of calibrators, the series of quality control products, the internal standard, and the protective agent, respectively;

[0065] The calibrator, the quality control product, the internal standard, and the protective agent are freeze-dried using the same freeze-drying process, which sequentially includes a pre-freezing stage, sublimation drying, and desorption drying; wherein, the operating conditions of the pre-freezing stage are shown in Table 4; the operating conditions of the sublimation drying are shown in Table 5; and the operating conditions of the desorption drying are shown in Table 6.

[0066] The use of the above-mentioned kit in the simultaneous detection of anemia markers in serum using high performance liquid chromatography tandem mass spectrometry is also within the scope of protection of the present invention.

[0067] Specifically, the anemia markers are homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid;

[0068] Before performing the anemia marker test, the protective agent is redissolved with ultrapure water according to the concentration of dithiothreitol in the protective agent working solution, and then diluted 5 times with ultrapure water to obtain a protective agent-reconstituted solution;

[0069] The series of calibrators are re-dissolved with the protective agent-re-dissolving solution according to the corresponding five concentration levels of the series of calibrator working solutions, thereby obtaining a series of calibration sample solutions; the series of quality control products are re-dissolved with the protective agent-re-dissolving solution according to the corresponding two concentration levels of the series of quality control working solutions, thereby obtaining a series of quality control sample solutions; the internal standard products are re-dissolved with the protective agent-re-dissolving solution according to the concentration of each internal standard product in the internal standard working solution, thereby obtaining an internal standard sample working solution.

[0070] Specifically, the steps of detecting anemia markers in serum include:

[0071] The internal standard sample solution was added to the series of calibration sample solutions, the series of quality control sample solutions and the serum sample, mixed, and then the sample extract was added, mixed, centrifuged and the supernatant was collected for LC-MS / MS detection.

[0072] Specifically, preferably, the step of detecting anemia markers in serum includes:

[0073] 20 μL of internal standard sample solution was added to 100 μL of the series calibration sample solution, 100 μL of the series quality control sample solution and 100 μL of serum sample, mixed, and then 100 μL of sample extract was added, mixed, centrifuged and the supernatant was taken for LC-MS / MS detection.

[0074] Specifically, the chromatographic conditions for the LC-MS / MS detection are as follows:

[0075] Liquid chromatography conditions:

[0076] Chromatographic column: Feinomei Luna omega Polar C18 chromatographic column, 100mm x 2.1mm; Mobile phase A: Aqueous solution containing 0.5% formic acid and 2mmol / L ammonium acetate; Mobile phase B: Methanol solution containing 0.5% formic acid and 2mmol / L ammonium acetate; Injection volume 10μL, Gradient elution. Specific elution conditions are shown in Table 7 below:

[0077] Table 7

[0078]

[0079]

[0080] Mass spectrometry conditions:

[0081] The mass spectrometer was a Thermo scientific TSQ altis, the ion source was an electrospray source, the scanning mode was positive and negative ion switching mode, the spray voltage was 3000 V, the cone voltage was 70 V, and the nebulizer flow rate was 60 L / h;

[0082] The mobile phase A was prepared as follows: a mobile phase additive was mixed with 100 times the volume of ultrapure water to obtain mobile phase A;

[0083] The mobile phase B was prepared as follows: a mobile phase additive was mixed with 100 times the volume of methanol to obtain mobile phase B.

[0084] The technical solution research and development ideas of the present invention are:

[0085] (1) Optimization of LC-MS / MS conditions

[0086] The present invention solves the problems of difficult separation of methylmalonic acid and its isomer succinic acid, poor chromatographic retention and response, and other issues by optimizing mass spectrometry conditions, liquid phase conditions, and chromatographic columns. A method for the simultaneous detection of homocysteine, folic acid, methylmalonic acid, vitamin B12, and vitamin C is established. Among the detection indicators, folic acid is active folic acid, i.e., 5-methyltetrahydrofolate; vitamin B12 is cyanocobalamin; and vitamin C is ascorbic acid.

[0087] (2) Optimization of pretreatment conditions

[0088] The present invention solves the problem of serum sample pretreatment by optimizing the type, formula and dosage of a protein precipitant. The serum sample can be treated by a simple protein precipitation method to obtain extracts to be tested of homocysteine, 5-methyltetrahydrofolate, cyanocobalamin, methylmalonic acid and ascorbic acid.

[0089] (3) Screening of stabilizers

[0090] The present invention solves the problems of poor stability and difficult storage of folic acid and ascorbic acid by optimizing the type and concentration of stabilizers and designing experiments to verify the stability of homocysteine, 5-methyltetrahydrofolate, cyanocobalamin, methylmalonic acid and ascorbic acid calibrants.

[0091] (4) Development of freeze-drying process

[0092] The present invention optimizes the freeze-drying process of a calibrant to obtain a freeze-dried product of a calibrant for simultaneously detecting homocysteine, 5-methyltetrahydrofolate, cyanocobalamin, methylmalonic acid and ascorbic acid, and designs experiments to verify the storage stability and usage stability of the freeze-dried product.

[0093] Beneficial effects:

[0094] (1) The existing water-soluble vitamin detection methods, folic acid detection methods, methylmalonic acid detection methods, etc. are unable to achieve the simultaneous detection of homocysteine, active folic acid, vitamin B12, methylmalonic acid and vitamin C. The present invention addresses this technical problem and invents a detection method for simultaneously detecting the above anemia markers.

[0095] (2) The current method for detecting vitamin B12 is relatively complicated. The present invention simplifies the sample processing of vitamin B12 and can simultaneously detect homocysteine, active folic acid, methylmalonic acid, vitamin B12, and vitamin C. These substances are jointly involved in folic acid metabolism, and simultaneous detection is very necessary.

[0096] (3) Since active folic acid, vitamin B12, homocysteine, and vitamin C are relatively unstable, especially vitamin C, and the current reagent components cannot preserve vitamin C for a long time, the present invention has developed a detection kit with strong stability that can be stored in a liquid state for 15 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0098] Figure 1 The effect of different concentrations of formic acid as the mobile phase on the separation of methylmalonic acid and succinic acid; Figure 1 Figure a shows the liquid phase separation results when mobile phase A is an aqueous solution containing 2 mM ammonium acetate and 0.1% formic acid, and mobile phase B is a methanol solution containing 2 mM ammonium acetate and 0.1% formic acid; Figure 1 b in the figure is a liquid phase separation result diagram using mobile phase A as an aqueous solution containing 2 mM ammonium acetate and 0.5% formic acid, and mobile phase B as a methanol solution containing 2 mM ammonium acetate and 0.5% formic acid.

[0099] Figure 2 The chromatograms of methylmalonic acid and succinic acid at channels 117>73 and 117>55, respectively; Figure 2 a is the chromatogram of methylmalonic acid and succinic acid at channels 117>73; Figure 2 Figure b is the chromatogram of methylmalonic acid and succinic acid at channels 117>55.

[0100] Figure 3 To investigate the effect of sulfosalicylic acid as a precipitant in serum samples on the detection of anemia markers.

[0101] Figure 4 To investigate the effect of precipitation of serum samples with trichloroacetic acid as precipitant on the detection of anemia markers.

[0102] Figure 5 This study aimed to investigate the effect of precipitating serum samples with metaphosphoric acid as a precipitant on the detection of anemia markers.

[0103] Figure 6 This is a schematic diagram of the packaging of the anemia marker detection kit. DETAILED DESCRIPTION

[0104] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0105] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0106] Unless otherwise specified in the examples of the present invention, v / v represents volume fraction, X% means X mL / 100 mL; m / v represents mass fraction, X% means X g / 100 mL.

[0107] Unless otherwise specified, the "sulfosalicylic acid" in the embodiments of the present invention refers to 5-sulfosalicylic acid.

[0108] In the embodiment of the present invention, Hcy is homocysteine; 5-MTHF is 5-methyltetrahydrofolate; VB12 is cyanocobalamin; MMA is methylmalonic acid; and VC is ascorbic acid.

[0109] Example 1

[0110] 1.1 Preparation of stock solution and working solution

[0111] (1) Preparation of standard stock solution:

[0112] Homocysteine ​​stock solution: Weigh 50 mg of homocysteine ​​into a 5 mL volumetric flask. Add ultrapure water and 20 μL of 1N hydrochloric acid solution. Dissolve by ultrasonication. Add ultrapure water to the mark to prepare a 10,000 μg / mL homocysteine ​​stock solution.

[0113] 5-Methyltetrahydrofolate stock solution: Weigh 1 mg of 5-methyltetrahydrofolate into a 10 mL volumetric flask. Add an appropriate amount of ultrapure water and 30 μL of ammonia water. Dissolve by ultrasonication and dilute to the mark with ultrapure water to prepare a 100 μg / mL 5-methyltetrahydrofolate stock solution.

[0114] Cyanocobalamin stock solution: Weigh 1 mg of cyanocobalamin into a 100 mL volumetric flask, add ultrapure water and sonicate to dissolve, then dilute to the mark with ultrapure water to prepare a 10 μg / mL cyanocobalamin stock solution.

[0115] Methylmalonic acid stock solution: Weigh 5 mg of methylmalonic acid into a 5 mL volumetric flask, add ultrapure water and sonicate to dissolve, then dilute to the mark with ultrapure water to prepare a 1000 μg / mL cyanocobalamin stock solution.

[0116] Ascorbic acid stock solution: Weigh 50 mg of ascorbic acid into a 5 mL volumetric flask, add ultrapure water and sonicate to dissolve, then dilute to the mark with ultrapure water to prepare a 10,000 μg / mL ascorbic acid stock solution.

[0117] (2) Preparation of standard working solution:

[0118] 560 μL of homocysteine ​​stock solution, 80 μL of 5-methyltetrahydrofolate stock solution, 40 μL of cyanocobalamin stock solution, 160 μL of methylmalonic acid stock solution, and 320 μL of ascorbic acid stock solution were mixed in a 5 mL volumetric flask. The first solvent (containing 10% methanol (v / v), 0.1% citric acid (m / v), 0.1% dithiothreitol (m / v), and the balance deionized water) was added and the volume was adjusted to the mark to prepare the standard working solution. The concentration of homocysteine ​​in the standard working solution was 1120 μg / mL, 5-methyltetrahydrofolate was 1.6 μg / mL, methylmalonic acid was 32 μg / mL, cyanocobalamin was 0.08 μg / mL, and ascorbic acid was 640 μg / mL.

[0119] (3) Preparation of internal standard stock solution

[0120] Homocysteine ​​d4 stock solution: Place 1 mg of homocysteine ​​d4 in a 1 mL volumetric flask, add an appropriate amount of ultrapure water and 20 μL of 1N hydrochloric acid solution, sonicate to dissolve, and dilute to the mark with ultrapure water to prepare a 1000 μg / mL homocysteine ​​d4 stock solution.

[0121] 5-Methyltetrahydrofolate-d3 stock solution: Place 1 mg of 5-methyltetrahydrofolate-d3 in a 10 mL volumetric flask, add appropriate amount of ultrapure water and 30 μL of ammonia water, sonicate to dissolve, and dilute to the mark with ultrapure water to prepare a 100 μg / mL 5-methyltetrahydrofolate-d3 stock solution.

[0122] Methylmalonic acid-d3 stock solution: Place 1 mg of methylmalonic acid-d3 in a 1 mL volumetric flask, add appropriate amount of ultrapure water, sonicate to dissolve, and dilute to the mark with ultrapure water to prepare a 1000 μg / mL methylmalonic acid-d3 stock solution.

[0123] Ascorbic acid-13C6 stock solution: Place 1 mg of ascorbic acid-13C6 in a 1 mL volumetric flask, add appropriate amount of ultrapure water, dissolve under ultrasonication, and dilute to the mark with ultrapure water to prepare a 1000 μg / mL ascorbic acid-13C6 stock solution.

[0124] (4) Preparation of internal standard working solution

[0125] 700 μL of homocysteine-d4 stock solution, 10 μL of 5-methyltetrahydrofolate-d3 stock solution, 20 μL of methylmalonic acid-d3 stock solution, and 400 μL of ascorbic acid-13C6 stock solution were mixed and placed in a 10 mL volumetric flask. A first solvent (containing 10% methanol (v / v), 0.1% citric acid (m / v), and 0.1% dithiothreitol (m / v), with the remainder being deionized water) was added to form an internal standard working solution. In the internal standard working solution, the concentration of homocysteine-d4 was 70 μg / mL, the concentration of 5-methyltetrahydrofolate-d3 was 0.1 μg / mL, the concentration of methylmalonic acid-d3 was 2.0 μg / mL, and the concentration of ascorbic acid-13C6 was 40 μg / mL.

[0126] 1.2 Optimization of mass spectrometry conditions

[0127] The standard working solution and internal standard working solution were injected into the mass spectrometer respectively to optimize the mass spectrometry method for homocysteine, 5-methyltetrahydrofolate, cyanocobalamin, methylmalonic acid and vitamin C.

[0128] Mass spectrometry conditions were as follows: the mass spectrometer was a Thermo Scientific TSQ altis, the ion source was an electrospray ionization (ESI) source, the scan mode was positive and negative ion switching mode, the spray voltage was 3000 V, the cone voltage was 70 V, and the nebulizer flow rate was 60 L / h. The specific mass spectrometry detection results are shown in Table 1. The ion pairs for homocysteine ​​were 136>90 and 136>56; the ion pairs for 5-methyltetrahydrofolate were 460>313 and 460>180; the ion pairs for cyanocobalamin were 678.5>359 and 678.5>147; the ion pairs for methylmalonic acid were 117>73 and 117>55; and the ion pairs for ascorbic acid were 177>95 and 177>141.

[0129] The ion pair for homocysteine-d4 is 140 > 94; the ion pair for 5-methyltetrahydrofolate-d3 is 463 > 316; the ion pair for methylmalonic acid-d3 is 120 > 76; and the ion pair for ascorbic acid-13C6 is 183 > 147. 5-methyltetrahydrofolate-d3 was used as the internal standard for cyanocobalamin. Detailed ion pair parameters are shown in Table 8.

[0130] Table 8 Ion pair parameters of anemia markers

[0131]

[0132] 1.3 Optimization of liquid chromatography conditions

[0133] Dilute the standard working solution described in "1.1. Preparation of Stock and Working Solutions" 400-fold with water to prepare an analyte test solution with a homocysteine ​​concentration of 2.8 μg / mL, a 5-methyltetrahydrofolate concentration of 4 ng / mL, a methylmalonic acid concentration of 80 ng / mL, a cyanocobalamin concentration of 0.2 ng / mL, and an ascorbic acid concentration of 1.6 μg / mL.

[0134] Dilute the internal standard working solution in "1.1. Preparation of Stock and Working Solutions" 10-fold with water to prepare an internal standard test solution with a homocysteine-d4 concentration of 7 μg / mL, 5-methyltetrahydrofolate-d3 of 10 ng / mL, methylmalonic acid-d3 of 0.2 μg / mL, and ascorbic acid-13C6 of 4 μg / mL.

[0135] 100 μL of the diluted analyte test solution was mixed with 20 μL of the internal standard test solution and placed in the automatic sampler of the liquid chromatography-tandem mass spectrometer for optimization of the liquid phase conditions.

[0136] Liquid chromatography detection scheme 1:

[0137] Chromatographic conditions: The chromatographic column is a Fenome Luna omega Polar C18 column ( 100mm x 2.1mm); mobile phase A was 2mM ammonium acetate in water containing 0.5% formic acid; mobile phase B was 2mM ammonium acetate in methanol containing 0.5% formic acid; gradient elution, specific elution conditions are shown in Table 7.

[0138] Liquid chromatography detection scheme 2:

[0139] Chromatographic conditions: The chromatographic column is a Fenome Luna omega Polar C18 column ( 100mm x 2.1mm); mobile phase A was 2mM ammonium acetate in water containing 0.1% formic acid; mobile phase B was 2mM ammonium acetate in methanol containing 0.1% formic acid; gradient elution, specific elution conditions were the same as those of liquid chromatography detection protocol 1, see Table 7.

[0140] Experimental results:

[0141] like Figure 1 As shown in Figure 2, the separation between methylmalonic acid and succinic acid increases with the increase of formic acid concentration; Figure 2As shown, under the detection conditions of liquid chromatography detection scheme 2 (mobile phase A is an aqueous solution containing 2 mM ammonium acetate and 0.1% formic acid; mobile phase B is a methanol solution containing 2 mM ammonium acetate and 0.1% formic acid), it was found that methylmalonic acid has higher specificity than 117>73 under the ion pair of 117>55, and can achieve mass spectrometric separation from succinic acid without the need for chromatographic peak separation, that is, 117>55, and only methylmalonic acid peaks.

[0142] In summary, the chromatographic conditions in liquid chromatography detection scheme 1 were selected. Furthermore, 117>73 was preferred as the quantitative ion pair for methylmalonic acid. When 117>73 was interfered with, 117>55 could be selected as the quantitative ion pair.

[0143] 1.4. Optimization of pre-treatment methods

[0144] In this invention, the detection method for anemia markers is mass spectrometry. Prior to testing, the analyte must be extracted from the serum sample. Protein precipitation is a simple, convenient, and high-throughput method for processing biological samples. A protein precipitant denatures and precipitates the proteins in the serum, releasing the small molecules of the analyte. Centrifugation yields the supernatant, which is then used for liquid chromatography-tandem mass spectrometry analysis.

[0145] Protein precipitation generally uses organic solvents such as methanol and acetonitrile, but can also use inorganic salts such as zinc sulfate or acid solutions. Since the analytes in the present invention are all highly polar substances, if methanol or acetonitrile is used as a protein precipitant, the amount of protein precipitant must be at least twice the sample volume, resulting in excessive sample dilution. Furthermore, when analyzed using a C18 column, there is a significant solvent effect, resulting in the appearance of a frontal chromatographic peak.

[0146] Therefore, the present invention uses acid as a protein precipitant, and the volume of the acid solution is 0.5 to 2 times the volume of the sample to precipitate protein.

[0147] 1.4.1. Selection of precipitant

[0148] Fresh human serum samples were divided into three portions, 100 μL each, and placed in 1.5 mL centrifuge tubes. 20 μL of internal standard working solution (prepared in "1.1. Preparation of stock solution and working solution") was added to each centrifuge tube and shaken for 2 minutes to mix. Subsequently, 0.25 mL of 5% w / v sulfosalicylic acid aqueous solution, 0.25 mL of 5% w / v trichloroacetic acid aqueous solution, and 0.25 mL of 5% w / v metaphosphoric acid aqueous solution were added to each of the three samples, and shaken for 3 minutes to mix. After centrifugation, the supernatant was collected and injected into a liquid chromatography-tandem mass spectrometer for analysis. The chromatographic peaks of the analysis results are shown in Figure 2. Figure 3 、 Figure 4 and Figure 5 .

[0149] from Figure 3 It can be seen that all five target compounds were detected when sulfosalicylic acid was used as a precipitant, but the responses of methylmalonic acid and cyanocobalamin were poorer than those when metaphosphoric acid was used as a precipitant. Figure 4 It can be seen that methylmalonic acid was not detected when trichloroacetic acid was used as a precipitant; Figure 5 It can be seen that when metaphosphoric acid is used as a precipitant, all five targets are detected with good responses, but the aqueous solution of metaphosphoric acid is unstable.

[0150] Since trichloroacetic acid causes strong ion suppression on methylmalonic acid, serum treated with metaphosphoric acid and sulfosalicylic acid has better chromatograms. However, metaphosphoric acid is unstable after dissolution. In order to enhance the protein precipitation effect, sulfosalicylic acid and metaphosphoric acid are preferably selected as precipitants for sample treatment. More preferably, the mass concentrations of metaphosphoric acid and sulfosalicylic acid in the precipitant are both 5%.

[0151] 1.4.2. Optimization of precipitant dosage

[0152] Fresh human serum samples were divided into three portions, 100 μL of each portion was placed in a 1.5 mL centrifuge tube, 20 μL of internal standard working solution (prepared in "1.1, Preparation of stock solution and working solution") was added to each centrifuge tube, and the mixture was shaken for 2 minutes; then, 50 μL, 100 μL, and 200 μL of precipitant (all containing 5% (w / v) metaphosphoric acid and 5% (w / v) sulfosalicylic acid, the solvent was water) were added to the three samples, and the mixture was shaken for 3 minutes. After centrifugation, the supernatant was injected into a liquid chromatography-tandem mass spectrometer for analysis. The chromatographic detection conditions used the preferred detection conditions described above. The analysis results are shown in Table 9.

[0153] Table 9 Peak area results of analytes after treating samples with different precipitant volumes

[0154]

[0155] As shown in the table, based on the size of the analyte peak area, precipitant volumes of 50 μL and 100 μL provide higher sensitivity. Increasing the amount of precipitant dilutes the sample, resulting in decreased sensitivity. Despite a larger peak area, the protein precipitation effect is poor when the 50 μL precipitant volume is used. Therefore, a 100 μL precipitant volume is preferred for sample processing.

[0156] Example 2:

[0157] 1.1. Prepare the kit according to the following steps

[0158] The first solvent used in this experiment contained 10% methanol (v / v), 0.1% citric acid (m / v), 0.1% dithiothreitol (m / v), and the balance was deionized water.

[0159] (1) Preparation of standard stock solution:

[0160] Weigh an appropriate amount of homocysteine ​​into a volumetric flask, add ultrapure water and 20 μL of 1N hydrochloric acid, sonicate to dissolve, and dilute to the mark with ultrapure water to prepare a homocysteine ​​stock solution. Weigh an appropriate amount of 5-methyltetrahydrofolate into a volumetric flask, add an appropriate amount of ultrapure water and 30 μL of ammonia, sonicate to dissolve, and dilute to the mark with ultrapure water to prepare a 5-methyltetrahydrofolate stock solution. Dissolve cyanocobalamin, methylmalonic acid, and ascorbic acid directly in ultrapure water and dilute to the mark to prepare cyanocobalamin, methylmalonic acid, and ascorbic acid stock solutions, respectively. See Table 10 for details on the preparation of standard stock solutions.

[0161] Table 10 Preparation of standard stock solutions

[0162]

[0163] (2) Preparation of standard working solution

[0164] Take appropriate amounts of homocysteine ​​stock solution, 5-methyltetrahydrofolate stock solution, cyanocobalamin stock solution, methylmalonic acid stock solution, and ascorbic acid stock solution, mix them, and then add the first solvent to prepare a standard working solution. See Table 11 for details on the preparation of the standard working solution.

[0165] Table 11 Preparation of standard working solution

[0166]

[0167]

[0168] (3) Preparation of internal standard stock solution

[0169] Weigh homocysteine-d4, 5-methyltetrahydrofolate-d3, methylmalonic acid-d3, and ascorbic acid-13C6 separately to prepare internal standard stock solutions. The preparation method and solvent are the same as those used for the standard stock solutions. See Table 12 for details on the preparation of the internal standard stock solutions.

[0170] Table 12 Preparation of internal standard stock solution

[0171]

[0172] (4) Preparation of internal standard working solution

[0173] Take appropriate amounts of homocysteine-d4 stock solution, 5-methyltetrahydrofolate-d3 stock solution, methylmalonic acid-d3 stock solution, and ascorbic acid-13C6 stock solution, mix them, and add the first solvent to prepare the internal standard working solution. The preparation method of the internal standard working solution is detailed in Table 13.

[0174] Table 13 Preparation of internal standard working solution

[0175]

[0176] (5) Preparation of calibrators and quality control products

[0177] Bovine serum albumin, antioxidants, preservatives, etc. were mixed to prepare the second solvent. Different volumes of standard working solution were mixed with the second solvent to prepare calibrator S1 working solution, calibrator S2 working solution, calibrator S3 working solution, calibrator S4 working solution, calibrator S5 working solution, quality control QC1 working solution, and quality control QC2 working solution. The preparation methods of calibrator working solution and quality control working solution are detailed in Table 14.

[0178] The second solvent consists of bovine serum albumin, isoascorbic acid, citric acid, 10×PBS buffer and deionized water; the concentration of bovine serum albumin is 0.1% (w / v), the concentration of isoascorbic acid is 0.1% (w / v), the concentration of citric acid is 0.1% (w / v), the volume concentration of 10×PBS buffer is 10% (v / v), and the balance is deionized water.

[0179] Table 14 Preparation methods of calibrator working solution and quality control working solution

[0180]

[0181]

[0182] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the calibrator S1 working solution were 700 ng / mL, 1.0 ng / mL, 20 ng / mL, 0.05 ng / mL, and 400 ng / mL, respectively;

[0183] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the calibrator S2 working solution were 1400 ng / mL, 2.0 ng / mL, 40 ng / mL, 0.1 ng / mL, and 800 ng / mL, respectively;

[0184] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the calibrator S3 working solution were 7000 ng / mL, 10 ng / mL, 200 ng / mL, 0.5 ng / mL, and 4000 ng / mL, respectively;

[0185] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the calibrator S4 working solution were 28000 ng / mL, 40 ng / mL, 800 ng / mL, 2.0 ng / mL, and 16000 ng / mL, respectively;

[0186] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the calibrator S5 working solution were 56000 ng / mL, 80 ng / mL, 1600 ng / mL, 4.0 ng / mL, and 32000 ng / mL, respectively;

[0187] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the quality control QC1 working solution were 3500 ng / mL, 5.0 ng / mL, 100 ng / mL, 0.25 ng / mL, and 2000 ng / mL, respectively;

[0188] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin and ascorbic acid in the quality control product QC2 working solution were 35000 ng / mL, 50 ng / mL, 1000 ng / mL, 2.5 ng / mL and 20000 ng / mL, respectively.

[0189] A series of calibrator working solutions and quality control working solutions were dispensed into brown penicillin bottles at 0.5 mL each, lyophilized into powder, and stored at -20°C to obtain calibrator S1, calibrator S2, calibrator S3, calibrator S4, calibrator S5, quality control QC1, and quality control QC2, respectively.

[0190] (6) Preparation of internal standard

[0191] The internal standard working solution prepared above is mixed with a second solvent to form an internal standard working solution component; wherein the volume ratio of the internal standard working solution to the second solvent is 1:9; wherein the second solvent is composed of bovine serum albumin, isoascorbic acid, citric acid, 10×PBS buffer, and deionized water; wherein the concentration of bovine serum albumin is 0.1% (w / v), the concentration of isoascorbic acid is 0.1% (w / v), the concentration of citric acid is 0.1% (w / v), the volume concentration of 10×PBS buffer is 10% (v / v), and the balance is deionized water.

[0192] The concentrations of homocysteine-d4, 5-methyltetrahydrofolate-d3, methylmalonic acid-d3, and ascorbic acid-13C6 in the internal standard working solution were 7000 ng / mL, 10 ng / mL, 200 ng / mL, and 4000 ng / mL, respectively.

[0193] The internal standard working solution was dispensed into brown vials at 2 mL each, and lyophilized into powder, which was then stored at -20°C to obtain the internal standard.

[0194] (7) Preparation of protective agent

[0195] Dithiothreitol is dissolved in ultrapure water to serve as a protective agent working solution, wherein the protective agent working solution is a dithiothreitol aqueous solution with a dithiothreitol concentration of 5 g / L.

[0196] The protective agent working solution was dispensed into brown vials at 2 mL each, and freeze-dried into powder, which was then stored at -20°C to obtain the protective agent.

[0197] (8) Preparation of sample extract

[0198] Metaphosphoric acid and sulfosalicylic acid were dissolved in deionized water to form components of a sample extract; wherein, in the sample extract, the mass ratio of metaphosphoric acid to sulfosalicylic acid was 1:1, that is, the concentrations of metaphosphoric acid and sulfosalicylic acid were both 50 g / L.

[0199] The sample extract was divided into 20 mL portions in brown reagent bottles or HDPE plastic bottles and stored at -20°C.

[0200] (9) Preparation of mobile phase additives

[0201] Formic acid and ammonium acetate were dissolved and mixed in deionized water to prepare an aqueous solution of 50% (v / v) formic acid and 0.2 mol / L ammonium acetate as a mobile phase additive.

[0202] Dispense the mobile phase additive into reagent bottles at 5 mL each and store at -20°C.

[0203] (10) Freeze-drying process

[0204] The aforementioned series of calibrator working solutions, quality control working solutions, internal standard working solutions, and protective agent working solutions were lyophilized according to the following lyophilization process. The series of calibrator working solutions, quality control working solutions, internal standard working solutions, and protective agent working solutions were lyophilized using the same lyophilization process, which sequentially included a pre-freezing stage, sublimation drying, and desorption drying. The operating conditions for the pre-freezing stage are shown in Table 4; the operating conditions for the sublimation drying stage are shown in Table 5; and the operating conditions for the desorption drying stage are shown in Table 6.

[0205] (11) Assembly of the kit

[0206] The freeze-dried series of calibrators, series of quality control products, internal standards, protective agents, sample extracts and mobile phase additives are assembled and stored at -20°C for 15 days to obtain an anemia marker detection kit. The kit schematic is shown in Figure 6 .

[0207] 1.2. Instructions for using the kit

[0208] (1) Before use, remove the kit from -20°C and return it to room temperature before removing all reagents from the kit.

[0209] (2) Redissolution of protective agent:

[0210] Dissolve the protective agent in 2 mL of ultrapure water, place in a 50 mL centrifuge tube, add 8 mL of ultrapure water, mix thoroughly, and prepare a protective agent-reconstitution solution for use. The concentration of dithiothreitol in the protective agent-reconstitution solution is 1 g / L (i.e., 0.1% (m / v)).

[0211] (3) Reconstitution of calibrators, controls, and internal standards:

[0212] Add 0.5 mL of protective agent-reconstitution solution to each series of calibrators and quality control products; add 2 mL of protective agent-reconstitution solution to the internal standard, shake for 30 seconds to mix, and set aside.

[0213] After reconstitution:

[0214] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in calibration sample S1 were 700 ng / mL, 1.0 ng / mL, 20 ng / mL, 0.05 ng / mL, and 400 ng / mL, respectively;

[0215] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in calibration sample S2 were 1400 ng / mL, 2.0 ng / mL, 40 ng / mL, 0.1 ng / mL, and 800 ng / mL, respectively;

[0216] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in calibration sample S3 were 7000 ng / mL, 10 ng / mL, 200 ng / mL, 0.5 ng / mL, and 4000 ng / mL, respectively;

[0217] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in calibration sample S4 were 28000 ng / mL, 40 ng / mL, 800 ng / mL, 2.0 ng / mL, and 16000 ng / mL, respectively;

[0218] The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in calibration sample S5 were 56000 ng / mL, 80 ng / mL, 1600 ng / mL, 4.0 ng / mL, and 32000 ng / mL, respectively;

[0219] The homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid levels in the quality control sample QC1 were 3500 ng / mL, 5.0 ng / mL, 100 ng / mL, 0.25 ng / mL, and 2000 ng / mL, respectively;

[0220] The homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid levels in the quality control sample QC2 were 35,000 ng / mL, 50 ng / mL, 1,000 ng / mL, 2.5 ng / mL, and 20,000 ng / mL, respectively;

[0221] The concentrations of homocysteine-d4, 5-methyltetrahydrofolate-d3, methylmalonic acid-d3, and ascorbic acid-13C6 in the internal standard sample solution were 7000 ng / mL, 10 ng / mL, 200 ng / mL, and 4000 ng / mL, respectively.

[0222] (4) Preparation of mobile phase solution:

[0223] Take 2 mL of mobile phase additive (an aqueous solution containing 50% (v / v) formic acid and 0.2 mol / L ammonium acetate) and mix it with 200 mL of ultrapure water to obtain mobile phase A; mobile phase A is an aqueous solution containing 0.5% (v / v) formic acid and 2 mmol / L ammonium acetate, and set aside.

[0224] Take 2 mL of mobile phase additive (an aqueous solution containing 50% (v / v) formic acid and 0.2 mol / L ammonium acetate) and mix it with 200 mL of methanol to obtain mobile phase B; mobile phase B is a methanol solution containing 0.5% (v / v) formic acid and 2 mmol / L ammonium acetate, and set aside.

[0225] 1.3 Sample pretreatment methods

[0226] 100 μL of calibration sample S1, calibration sample S2, calibration sample S3, calibration sample S4, calibration sample S5, quality control sample QC1, quality control sample QC2, and fresh human serum were placed in 1.5 mL centrifuge tubes respectively. 20 μL of internal standard sample solution was added to each sample. After vortexing for 2 minutes, 100 μL of sample extract was added. The mixture was shaken for 3 minutes to mix. After centrifugation, the supernatant was collected as the sample to be tested.

[0227] 1.4 Testing conditions

[0228] Chromatographic conditions of liquid chromatography-tandem mass spectrometry:

[0229] Liquid chromatography conditions:

[0230] Mobile phase A was an aqueous solution containing 0.5% (v / v) formic acid and 2 mmol / L ammonium acetate; mobile phase B was a methanol solution containing 0.5% (v / v) formic acid and 2 mmol / L ammonium acetate; the chromatographic column was a Fenomega Luna Polar C18 column ( 100mm x 2.1mm); injection volume 10μL, gradient elution, specific elution conditions are shown in Table 7.

[0231] Mass spectrometry conditions:

[0232] The mass spectrometer was a Thermo scientific TSQ altis, the ion source was an electrospray ion source (ESI), the scanning mode was a positive and negative ion switching mode, the spray voltage was 3000 V, the cone voltage was 70 V, and the nebulizer flow rate was 60 L / h.

[0233] Among them, the ion pairs of homocysteine ​​are 136>90 (quantitative) and 136>56 (qualitative); the ion pairs of 5-methyltetrahydrofolate are 460>313 (quantitative) and 460>180 (qualitative); the ion pairs of cyanocobalamin are 678.5>359 (quantitative) and 678.5>147 (qualitative); the ion pairs of methylmalonic acid are 117>73 (quantitative) and 117>55 (qualitative); and the ion pairs of ascorbic acid are 177>95 (quantitative) and 177>141 (qualitative).

[0234] Among them, the ion pair of homocysteine-d4 is 140>94; the ion pair of 5-methyltetrahydrofolate-d3 is 463>316; the ion pair of methylmalonic acid-d3 is 120>76; the ion pair of ascorbic acid-13C6 is 183>147, and 5-methyltetrahydrofolate-d3 is used as the internal standard for cyanocobalamin.

[0235] 1.5 Methodological Validation

[0236] The kit calibrators were used as standard curves and the quality control QC samples were used as quality controls. The performance of the anemia marker detection method of the present invention was verified by designing experiments on the limit of quantification, precision, accuracy, and linearity. The verification results are shown in Tables 15 to 31.

[0237] 1.5.1 Linear Verification

[0238] The calibration samples S1 to S5 included with the kit were used as linearity verification samples. A 1 / X fit was used, and the correlation coefficient and slope of the linear equation were recorded. The results are shown in Table 15.

[0239] Table 15 Linearity of Analytes

[0240] Components Linear range regression curve r Homocysteine 700~56000ng / mL y=4.80766E-5x+0.00183 0.99976 5-Methyltetrahydrofolate 1–80 ng / mL y=0.00409x+0.00111 0.99974 Methylmalonic acid 20–1600 ng / mL y=0.00356x+0.01951 0.99986 Cyanocobalamin 0.05–4 ng / mL Y=0.06111x+6.287E-4 0.99710 ascorbic acid 400~32000ng / mL Y=8.433E-5x+0.01973 0.99999

[0241] The experimental results showed that the linear equation correlation coefficients of the five anemia markers were >0.99, indicating that the detection method had a good linear relationship within the concentration range of C1 to C5.

[0242] 1.5.2 Verification of Limit of Quantitation

[0243] The standard curve was used to detect samples at the lower limit of quantification concentration. Each sample was tested 5 times. The results of the lower limit of quantification are shown in Table 16.

[0244] Table 16 Quantitative lower limit results

[0245]

[0246]

[0247] The test results showed that at the lower limit of quantification (LLOQ) concentration, the mean deviation of the test results was <±15%, and the precision CV was <15%, which met the acceptance criteria. The quantitative limit concentration of this method can achieve accurate detection.

[0248] 1.5.3 Accuracy Verification

[0249] Pipette 1 mL of the standard working solution listed in Table 11 into a 5 mL volumetric flask and dilute to the mark with the primary solvent to prepare a secondary standard working solution. The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in this secondary standard working solution are 224,000 ng / mL, 320 ng / mL, 6,400 ng / mL, 16 ng / mL, and 128,000 ng / mL, respectively. Add 30 μL each of the standard working solution and the secondary standard working solution to 930 μL of human serum to prepare high and low recovery verification samples. The standard curve was used for the analysis, with three replicates for each concentration. The recovery results are shown in Tables 17 to 21.

[0250] Table 17 Homocysteine ​​accuracy results

[0251]

[0252] Table 18 Accuracy results of 5-methyltetrahydrofolate

[0253]

[0254] Table 19 Methylmalonic acid accuracy results

[0255]

[0256]

[0257] Table 20 Cyanocobalamin accuracy results

[0258]

[0259] Table 21 Ascorbic acid accuracy results

[0260]

[0261] The accuracy test results showed that the recovery rates of all indicators were within the range of 85% to 115%. The results showed that the detection method had good accuracy and met the requirements of quantitative analysis.

[0262] 1.5.4 Precision Verification

[0263] The QC samples were tested using the standard curve, with five replicates per concentration, one run per day for three consecutive days. The results of intra- and inter-run precision are shown in Tables 22–26.

[0264] Table 22 Homocysteine ​​precision results

[0265]

[0266]

[0267] Table 23 Precision results of 5-methyltetrahydrofolate

[0268]

[0269] Table 24 Methylmalonic acid precision results

[0270]

[0271] Table 25 Cyanocobalamin precision results

[0272]

[0273] Table 26 Ascorbic acid precision results

[0274]

[0275]

[0276] The precision verification results showed that the precision CV was less than 15%, indicating that the precision of this method met the requirements of quantitative detection.

[0277] In summary, through methodological verification, it is shown that the quantitative limit, linearity, accuracy and precision of the detection method of the present invention all meet the requirements of quantitative analysis.

[0278] 1.6. Optimization of protective agent

[0279] Dithiothreitol (DTT), citric acid, and tris(2-carboxyethyl)phosphine (TCEP) were each prepared into a 0.1% (w / v) aqueous solution. 0.5 mL of each of the three protective solutions and deionized water was pipetted to dissolve the lyophilized powder of quality control QC2 (concentrations: homocysteine ​​35,000 ng / mL, 5-methyltetrahydrofolate 50 ng / mL, methylmalonic acid 1,000 ng / mL, cyanocobalamin 2.5 ng / mL, ascorbic acid 20,000 ng / mL). The solution was then aliquoted and stored in a -20°C refrigerator. The samples were taken out for testing on day 1, day 3, day 5, day 10, day 15, and day 30, respectively. The test results are shown in Tables 27 to 31.

[0280] Table 27 Homocysteine ​​stability test results in freeze-dried powder after different protective agent solvents

[0281]

[0282] Table 28 Stability test results of 5-sulfosalicylic acid in freeze-dried powder after different protective agent solvents

[0283]

[0284] Table 29 Stability test results of methylmalonic acid in freeze-dried powder after different protective agent solvents

[0285]

[0286] Table 30 Cyanocobalamin stability test results in freeze-dried powder after different protective agent solvents

[0287]

[0288]

[0289] Table 31 Ascorbic acid stability test results in lyophilized powder after different protective agent solvents

[0290]

[0291] According to the above test results, dithiothreitol is preferably used as the protective agent.

[0292] The present invention provides a kit for detecting anemia markers, as well as a method for its preparation and application. Numerous methods and approaches exist for implementing this technical solution. The foregoing merely represents a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A kit for detecting anemia markers, characterized in that: The anemia markers are homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid; The kit comprises the following reagents: (1) Series of calibrators: The series of calibrators are freeze-dried products of a series of calibrator working solutions with five different concentration levels, and the series of calibrator working solutions are calibrator working solutions with five different concentration levels prepared by diluting a standard working solution of known concentration with a second solvent; The five concentration levels of the calibrator working solution are: The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the calibrator S1 working solution were 700 ng / mL, 1.0 ng / mL, 20 ng / mL, 0.05 ng / mL, and 400 ng / mL, respectively; The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the calibrator S2 working solution were 1400 ng / mL, 2.0 ng / mL, 40 ng / mL, 0.1 ng / mL, and 800 ng / mL, respectively; The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the calibrator S3 working solution were 7000 ng / mL, 10 ng / mL, 200 ng / mL, 0.5 ng / mL, and 4000 ng / mL, respectively; The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the calibrator S4 working solution were 28,000 ng / mL, 40 ng / mL, 800 ng / mL, 2.0 ng / mL, and 16,000 ng / mL, respectively; The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the calibrator S5 working solution were 56,000 ng / mL, 80 ng / mL, 1,600 ng / mL, 4.0 ng / mL, and 32,000 ng / mL, respectively; (2) Series of quality control products: The series of quality control products are freeze-dried products of a series of quality control product working solutions with two different concentration levels, and the series of quality control product working solutions are quality control product working solutions with two different concentration levels prepared by diluting a standard working solution of known concentration with a second solvent; The two concentration levels of the quality control working solution are: The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the quality control QC1 working solution were 3500 ng / mL, 5.0 ng / mL, 100 ng / mL, 0.25 ng / mL, and 2000 ng / mL, respectively; The concentrations of homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid in the quality control QC2 working solution were 35,000 ng / mL, 50 ng / mL, 1,000 ng / mL, 2.5 ng / mL, and 20,000 ng / mL, respectively; (3) Internal standard: The internal standard is a lyophilized product of the internal standard working solution; The concentrations of homocysteine-d4, 5-methyltetrahydrofolate-d3, methylmalonic acid-d3, and ascorbic acid-13C6 in the internal standard working solution are 7000 ng / mL, 10 ng / mL, 200 ng / mL, and 4000 ng / mL, respectively; (4) Protective agent: The protective agent is a lyophilized product of the protective agent working solution; The protective agent working solution is a dithiothreitol aqueous solution, and the concentration of dithiothreitol in the solution is 5 g / L; (5) Sample extract: an aqueous solution containing metaphosphoric acid and sulfosalicylic acid, with the concentration of both metaphosphoric acid and sulfosalicylic acid in the solution being 50 g / L; (6) Mobile phase additive: aqueous solution containing 50% formic acid and 0.2 mol / L ammonium acetate; The freeze-drying process of the calibrator, the quality control product, the internal standard product and the protective agent includes a pre-freezing stage, sublimation drying and analytical drying in sequence; The operating conditions of the pre-freezing stage are as follows: ； The operating conditions of the sublimation drying are as follows: ； The operating conditions of the analytical drying are as follows: ； or, The freeze-drying process of the calibrator, the quality control product, the internal standard product and the protective agent includes a pre-freezing stage, sublimation drying and analytical drying in sequence; The operating conditions of the pre-freezing stage are as follows: ； The operating conditions of the sublimation drying are as follows: ； The operating conditions of the analytical drying are as follows: 。 2. The kit according to claim 1, wherein The second solvent consists of bovine serum albumin, an antioxidant, a preservative, a buffer and water; the antioxidant is isoascorbic acid; the preservative is citric acid; and the buffer is PBS buffer.

3. The kit according to claim 1, wherein The second solvent consists of 0.1% to 5% bovine serum albumin, 0.05% to 2% isoascorbic acid, 0.05% to 2% citric acid, 10% 10×PBS buffer and deionized water.

4. The kit according to claim 1, wherein The second solvent consists of 0.1% bovine serum albumin, 0.1% isoascorbic acid, 0.1% citric acid, 10% 10×PBS buffer and deionized water.

5. The kit according to claim 1, wherein The standard working solution of known concentration was prepared as follows: Weigh homocysteine ​​and mix it with 1N aqueous hydrochloric acid and water to prepare a standard stock solution of 10,000 μg / mL; weigh 5-methyltetrahydrofolate and mix it with ammonia and water to prepare a standard stock solution of 100 μg / mL; weigh methylmalonic acid and mix it with water to prepare a standard stock solution of 1,000 μg / mL; weigh cyanocobalamin and mix it with water to prepare a standard stock solution of 10 μg / mL; weigh ascorbic acid and mix it with water to prepare a standard stock solution of 10,000 μg / mL; The above-mentioned standard stock solutions are mixed and then prepared into a standard working solution with a first solvent; the concentration of homocysteine ​​in the standard working solution is 1120 μg / mL, the concentration of 5-methyltetrahydrofolate is 1.6 μg / mL, the concentration of methylmalonic acid is 32 μg / mL, the concentration of cyanocobalamin is 0.08 μg / mL, and the concentration of ascorbic acid is 640 μg / mL.

6. The kit according to claim 1, wherein The internal standard working solution was prepared as follows: Weigh homocysteine-d4 and mix it with 1N hydrochloric acid solution and water to prepare a 1000 μg / mL internal standard stock solution; weigh 5-methyltetrahydrofolate-d3 and mix it with ammonia and water to prepare a 100 μg / mL internal standard stock solution; weigh methylmalonic acid-d3 and mix it with water to prepare a 1000 μg / mL internal standard stock solution; weigh ascorbic acid-13C6 and mix it with water to prepare a 1000 μg / mL internal standard stock solution; The above-mentioned internal standard stock solutions are mixed and then prepared into an internal standard working solution with a first solvent; the concentration of homocysteine-d4 in the internal standard working solution is 70 μg / mL, the concentration of 5-methyltetrahydrofolate-d3 is 0.1 μg / mL, the concentration of methylmalonic acid-d3 is 2.0 μg / mL, and the concentration of ascorbic acid-13C6 is 40 μg / mL; The obtained internal standard working solution is mixed with the second solvent to prepare an internal standard product working solution.

7. The kit according to claim 5 or 6, characterized in that The first solvent consists of methanol, citric acid, dithiothreitol and deionized water.

8. The kit according to claim 1, wherein Lyophilizing the series of calibrators, the series of quality control products, the internal standard product, and the protective agent working solution to obtain the series of calibrators, the series of quality control products, the internal standard product, and the protective agent, respectively; The freeze-drying process of the calibrator, the quality control product, the internal standard product and the protective agent includes a pre-freezing stage, sublimation drying and analytical drying in sequence; The operating conditions of the pre-freezing stage are as follows: ； The operating conditions of the sublimation drying are as follows: ； The operating conditions of the analytical drying are as follows: 。 9. Use of the kit according to any one of claims 1 to 8 for the simultaneous detection of anemia markers in serum using high performance liquid chromatography tandem mass spectrometry.

10. The use according to claim 9, characterized in that The anemia markers are homocysteine, 5-methyltetrahydrofolate, methylmalonic acid, cyanocobalamin, and ascorbic acid; Before performing the anemia marker test, the protective agent is redissolved with ultrapure water according to the concentration of dithiothreitol in the protective agent working solution, and then diluted 5 times with ultrapure water to obtain a protective agent-reconstituted solution; The series of calibrators are re-dissolved with the protective agent-re-dissolving solution according to the corresponding five concentration levels of the series of calibrator working solutions, thereby obtaining a series of calibration sample solutions; the series of quality control products are re-dissolved with the protective agent-re-dissolving solution according to the corresponding two concentration levels of the series of quality control working solutions, thereby obtaining a series of quality control sample solutions; the internal standard products are re-dissolved with the protective agent-re-dissolving solution according to the concentration of each internal standard product in the internal standard working solution, thereby obtaining an internal standard sample working solution.

11. The use according to claim 10, characterized in that The steps for detecting anemia markers in serum include: The internal standard sample solution was added to the series of calibration sample solutions, the series of quality control sample solutions and the serum sample, mixed, and then the sample extract was added, mixed, centrifuged and the supernatant was collected for LC-MS / MS detection.

12. The use according to claim 11, characterized in that The chromatographic conditions for the LC-MS / MS detection are as follows: Liquid chromatography conditions: Chromatographic column: Fenome Luna omega Polar C18 column, 100 Å, 100 mm × 2.1 mm; mobile phase A: aqueous solution containing 0.5% formic acid and 2 mmol / L ammonium acetate; mobile phase B: methanol solution containing 0.5% formic acid and 2 mmol / L ammonium acetate; injection volume 10 μL, gradient elution, specific elution conditions are as follows: ； Mass spectrometry conditions: The mass spectrometer was a Thermo scientific TSQ altis, the ion source was an electrospray source, the scanning mode was positive and negative ion switching mode, the spray voltage was 3000 V, the cone voltage was 70 V, and the nebulizer flow rate was 60 L / h; The mobile phase A was prepared as follows: a mobile phase additive was mixed with 100 times the volume of ultrapure water to obtain mobile phase A; The mobile phase B was prepared as follows: a mobile phase additive was mixed with 100 times the volume of methanol to obtain mobile phase B.

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