Substitute matrix for detecting folic acid and 5-methyltetrahydrofolic acid and application of substitute matrix

Abstract

The invention relates to the technical field of analysis and detection, in particular to a substitute matrix for detecting folic acid and 5-methyltetrahydrofolic acid and application of the substitute matrix. The alternative substrate comprises a preservative and a rabbit whole blood diluent; the rabbit whole blood diluent comprises rabbit whole blood and normal saline; the rabbit whole blood accounts for 25%-75% of the volume of the rabbit whole blood diluent. The invention provides a substitute matrix for detecting folic acid and 5-methyltetrahydrofolic acid, which not only can meet the matrix effect requirement, but also can be stably stored for a long time, can be applied to the production and use of folic acid detection reagents or kits, and has important significance on the market-oriented popularization and application of whole blood folic acid detection kits.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a substitute matrix for detecting folic acid and 5-methyltetrahydrofolate and an application thereof. Background Art

[0002] In the human body, folic acid is related to the synthesis of nucleic acids. When folic acid is deficient, DNA synthesis is inhibited, DNA synthesis in bone marrow megaloblasts is reduced, and the rate of cell division is reduced, resulting in larger cell size and loose chromatin in the nucleus, called megaloblasts. Most of the megaloblasts in the bone marrow are destroyed before maturity, causing anemia symptoms and megaloblastic anemia. Folic acid deficiency or deficiency generally goes through multiple stages, including insufficient dietary folic acid intake or increased requirements, decreased serum folic acid levels, decreased red blood cell folate levels, increased serum homocysteine ​​levels, changes in giant cells in the bone marrow and other rapidly proliferating tissues, and megaloblastic anemia.

[0003] Serum folate is considered to be an indicator of recent folate nutritional status. Testing serum folate levels alone cannot distinguish between one-time folate insufficiency and chronic folate deficiency. However, red blood cell folate levels can reflect chronic or long-term (within 4 months) folate nutritional status and are more suitable for evaluating the effectiveness of folate intervention.

[0004] In the prior art, CN 117589895 A discloses a method for detecting folic acid and 5-methyltetrahydrofolate in human whole blood, which obtains a substitute matrix that can meet the requirements of the matrix effect experiment by screening a substitute matrix, and realizes accurate detection of folic acid and 5-methyltetrahydrofolate in human whole blood. However, the storage stability of the substitute matrix still needs to be further improved.

[0005] Therefore, how to provide an alternative matrix for the detection of folic acid and 5-methyltetrahydrofolate that can meet the matrix effect requirements and can be stored stably for a long time has become a technical problem that urgently needs to be solved in this field. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an alternative matrix, including a preservative and a rabbit whole blood diluent; the rabbit whole blood diluent includes rabbit whole blood and physiological saline; the volume percentage of the rabbit whole blood in the rabbit whole blood diluent is 25% to 75% (for example, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%).

[0007] Preferably, the volume percentage of the rabbit whole blood in the rabbit whole blood dilution is 25% to 70%, preferably 25% to 65%, more preferably 40% to 60%, and even more preferably 45% to 55%.

[0008] Preferably, the volume percentage of the physiological saline in the rabbit whole blood dilution is 25% to 75% (for example, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%), preferably 30% to 75%, more preferably 40% to 60%, and more preferably 45% to 55%.

[0009] Preferably, the replacement matrix also includes L-ascorbic acid.

[0010] Preferably, the weight ratio of the L-ascorbic acid to the rabbit whole blood diluent is (0.5-1.5):100.

[0011] Preferably, the preservative is a proclin series preservative.

[0012] Preferably, the preservative is at least one of proclin300 and proclin950.

[0013] Preferably, the volume ratio of the preservative to the rabbit whole blood diluent is (0.05-0.3):100.

[0014] More preferably, the volume ratio of the preservative to the rabbit whole blood diluent is (0.05-0.2):100.

[0015] Most preferably, the preservatives include proclin300 and proclin950, and the volume ratio of proclin300, proclin950 to rabbit whole blood diluent is (0.05-0.15):100.

[0016] Preferably, the replacement matrix includes proclin300, proclin950, L-ascorbic acid and rabbit whole blood diluent; the rabbit whole blood diluent includes rabbit whole blood and physiological saline; wherein the volume ratio of proclin300 and proclin950 to the rabbit whole blood diluent is (0.05~0.15):100; the weight ratio of L-ascorbic acid to the rabbit whole blood diluent is (0.8~1.2):100; the volume percentage of the rabbit whole blood in the rabbit whole blood diluent is 25%~75% (or the volume percentages listed above), and the volume percentage of the physiological saline in the rabbit whole blood diluent is 25%~75% (or the volume percentages listed above).

[0017] Most preferably, the replacement matrix includes proclin300, proclin950, L-ascorbic acid and rabbit whole blood diluent; the rabbit whole blood diluent includes rabbit whole blood and physiological saline; wherein the volume ratio of proclin300 and proclin950 to the rabbit whole blood diluent is (0.05~0.15):100; the weight ratio of L-ascorbic acid to the rabbit whole blood diluent is (0.8~1.2):100; the volume percentage of the rabbit whole blood in the rabbit whole blood diluent is 45%~55%, and the volume percentage of the physiological saline in the rabbit whole blood diluent is 45%~55%.

[0018] Furthermore, the present invention provides a folic acid and 5-methyltetrahydrofolate detection reagent or kit, which contains the replacement matrix.

[0019] Preferably, the reagents include but are not limited to calibrators and quality control products.

[0020] Furthermore, the present invention provides the use of the alternative matrix in at least one of the following aspects: (1) detecting folic acid and 5-methyltetrahydrofolate; (2) preparing folic acid and 5-methyltetrahydrofolate detection reagents or kits; (3) preparing folic acid and 5-methyltetrahydrofolate detection calibrators; (4) preparing folic acid and 5-methyltetrahydrofolate detection quality control products.

[0021] When detecting folic acid and 5-methyltetrahydrofolate, samples include, but are not limited to, dried blood spots, serum or whole blood.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an alternative matrix for detecting folic acid and 5-methyltetrahydrofolate, which can meet the matrix effect requirements and can be stored stably for a long time. The alternative matrix can be used in the production and use of folic acid detection reagents or kits, and has important significance for the market promotion and application of whole blood folic acid detection kits. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific technology or conditions are not indicated in the embodiments, they are all conventional methods or carried out according to the technology or conditions described in the literature in the field, or according to the product instructions. The reagents and instruments used, etc., which do not indicate the manufacturer, are all conventional products that can be purchased through regular channels. The percentage of proclin300 and proclin950 in the following embodiments represents the volume ratio of preservatives to rabbit whole blood / rabbit whole blood diluent (rabbit whole blood diluent is a mixture of rabbit whole blood and saline, or a mixture of rabbit whole blood and phosphate buffer). The percentage of rabbit whole blood or saline or phosphate buffer represents the volume percentage of each component in the rabbit whole blood diluent. The percentage of bovine serum albumin or L-ascorbic acid represents the weight ratio of each component to the rabbit whole blood diluent.

[0024] The detection methods of the compounds folic acid and 5-methyltetrahydrofolate in the following examples are as follows: 1. The instruments are shown in Table 1.

[0025] Table 1

[0026] 2. Detection method steps

[0027] 2.1 Experimental procedures

[0028] 2.1.1 The preparation of compound and internal standard stock solutions is shown in Table 2.

[0029] Table 2

[0030] 2.1.2 The preparation of compound working solution and quality control working solution are shown in Table 3 and Table 4.

[0031] Table 3

[0032] Table 4

[0033] Take 20 μL of Sub-C1 solution and add it to 180 μL of the treated surrogate matrix to make the C1 working solution. C2, C3, C4, C5, C6, LQC, MQC, and HQC are prepared in this ratio.

[0034] 2.1.3 Preparation of other reagents

[0035] Weigh 5 g of L-ascorbic acid and dissolve it in 100 mL of ultrapure water as a protective agent; weigh 16.67 g of zinc sulfate heptahydrate and dissolve it in 100 mL of ultrapure water as sample extract A; transfer 500 mL of methanol, 250 mL of acetonitrile, and 250 mL of isopropanol, mix them evenly, and use them as sample extract B.

[0036] 2.2 Sample processing method

[0037] (1) Add sample: Pipette 200 μL of sample into 2 mL centrifuge tubes and add 200 μL of protective agent; (2) Add sample extract A: Pipette 50 μL of sample extract A and add it to the corresponding centrifuge tubes above; (3) Add sample extract B: Pipette 150 μL of sample extract B and add it to the corresponding centrifuge tubes above, shake thoroughly at 3000 rpm for 15 min; (4) Centrifugation: Centrifuge at 4°C and 18,000 g for 10 min. Take 150 μL of the supernatant and place it in a new 96-well plate; (5) Centrifugation: Centrifuge at 3000 g for 5 min at 4 °C.

[0038] (6) Detection: Place the 96-well plate in the instrument and inject 15 μL of sample for detection.

[0039] 2.3 Chromatographic conditions are shown in Table 5.

[0040] Table 5

[0041] Example 1 Preparation of a surrogate matrix

[0042] This embodiment provides a variety of alternative matrices, the specific formula is as follows: Alternative matrix 1: 0.1% proclin300, 0.1% proclin950, rabbit whole blood

[0043] Alternative matrix 2: 0.1% proclin300, 0.1% proclin950, 50% rabbit whole blood, 50% saline

[0044] Alternative matrix 3: 0.1% proclin300, 0.1% proclin950, 50% rabbit whole blood, 50% 0.01M phosphate buffer

[0045] Alternative matrix 4: 0.1% proclin300, 0.1% proclin950, 5%wt bovine serum albumin, 50% rabbit whole blood, 50% 0.01M phosphate buffer

[0046] Alternative matrix 5: 0.1% proclin300, 0.1% proclin950, 1%wt L-ascorbic acid, 50% rabbit whole blood, 50% saline

[0047] Alternative matrix 6: 0.1% proclin300, 0.1% proclin950, 20% rabbit whole blood, 80% saline

[0048] Alternative matrix 7: 0.1% proclin300, 0.1% proclin950, 40% rabbit whole blood, 60% saline

[0049] Alternative matrix 8: 0.1% proclin300, 0.1% proclin950, 60% rabbit whole blood, 40% saline

[0050] Alternative matrix 9: 0.1% proclin300, 0.1% proclin950, 80% rabbit whole blood, 20% saline

[0051] Alternative matrix 10: 0.1% proclin300, 0.1% proclin950, 0.5%wt L-ascorbic acid, 20% rabbit whole blood, 80% saline

[0052] Alternative matrix 11: 0.1% proclin300, 0.1% proclin950, 0.5%wt L-ascorbic acid, 40% rabbit whole blood, 60% saline

[0053] Alternative matrix 12: 0.1% proclin300, 0.1% proclin950, 0.5%wt L-ascorbic acid, 60% rabbit whole blood, 40% saline

[0054] Alternative matrix 13: 0.1% proclin300, 0.1% proclin950, 0.5%wt L-ascorbic acid, 80% rabbit whole blood, 20% saline

[0055] The preparation method of the above-mentioned replacement matrix is ​​to mix the components uniformly according to the proportion.

[0056] Example 2 Stability test of alternative matrix

[0057] This example tests the storage stability of the alternative matrix in Example 1, and the steps are as follows: (1) Verification method: Different surrogate matrices were used to prepare high and low concentration points, where the concentrations of 5-methyltetrahydrofolate were 125 ng / mL and 425 ng / mL, and the concentrations of folic acid were 245 ng / mL and 425 ng / mL, respectively. The stability of the compounds was investigated when stored at 37°C for 1 day, 3 days, 5 days, and at 2-8°C for 1 day, 3 days, 7 days, and 14 days. The degradation rate was calculated as follows: the detection values ​​of folic acid and 5-methyltetrahydrofolate were detected at different time points according to the above detection method, and the degradation rate (%) = (detection value - initial detection value) / initial detection value × 100%; the initial detection value is the detection value at storage day 0.

[0058] (2) Acceptance criteria: The degradation rates at high and low concentration points are within ±15%.

[0059] The degradation rates of 5-methyltetrahydrofolate by alternative matrices 1 to 5 at 37°C and 2 to 8°C are shown in Table 6.

[0060] Table 6

[0061] The degradation rates of folic acid in alternative matrices 1 to 5 at 37°C and 2 to 8°C are shown in Table 7.

[0062] Table 7

[0063] It can be seen that the degradation rates of surrogate matrix 2 and surrogate matrix 5 at 37°C and 2-8°C, folic acid and 5-methyltetrahydrofolate all meet the acceptance criteria, indicating that the surrogate matrix using a mixture of rabbit whole blood and normal saline as a diluent has better stability.

[0064] The degradation rates of 5-methyltetrahydrofolate by alternative matrices 6 to 13 at 37°C and 2 to 8°C are shown in Table 8.

[0065] Table 8

[0066] The folic acid degradation rates of alternative matrices 6-13 at 37°C and 2-8°C are shown in Table 9.

[0067] Table 9

[0068] It can be seen that the degradation rates of alternative matrices 6, 7, 8, 10, 11 and 12 at 37°C and 2-8°C, folic acid and 5-methyltetrahydrofolate all meet the acceptance criteria; while alternative matrices 9 and 13 fail to meet the acceptance criteria after storage at 2-8°C for 14 days, indicating that when the diluent contains too high a proportion of rabbit whole blood, the stability of the alternative matrix will deteriorate.

[0069] Example 3 Matrix effect experiment

[0070] In this example, the matrix effects of alternative matrices 2, 5, 6, 7, 8, 10, 11, and 12 with good storage stability were tested.

[0071] (1) Verification method: Take the above-mentioned surrogate matrix and pure solvent (solvent is water) samples, prepare high, medium and low concentration points, and prepare solvent samples, matrix samples, and mixed samples of solvent samples and matrix samples in a ratio of 1:1. Detect the peak area of ​​all samples, and repeat the measurement of each sample 3 times. The peak area of ​​the solvent sample is BP, the peak area of ​​the matrix sample is A, and the peak area ratio of the mixed sample of solvent sample and matrix sample in a ratio of 1:1 is CP.

[0072] (2) Acceptable standard: Matrix deviation (%) = (CP-(A+BP) / 2) / ((A+BP) / 2) (%), relative matrix deviation ≤ 20%.

[0073] The results of the 5-methyltetrahydrofolate matrix effect test are shown in Table 10.

[0074] Table 10

[0075] The results of the folic acid matrix effect test are shown in Table 11.

[0076] Table 11

[0077] It can be seen that alternative matrix 2, alternative matrix 5, alternative matrix 7, alternative matrix 8, alternative matrix 11, and alternative matrix 12 can meet the acceptance criteria for matrix effect, which means that when the diluent contains too low a proportion of rabbit whole blood, the matrix effect will not meet the requirements.

[0078] Example 4 Long-term stability

[0079] (1) Verification method: Prepare low, medium and high concentration samples with alternative matrix 5. At a certain time interval, randomly select samples stored at normal storage temperature (-20±5℃) for testing at each time point. Take 2 samples each time and test each sample 3 times. Perform statistical processing on these stability study data and use formula (1) to formula (4) to perform trend significance test.

[0080]

[0081] When |b1|<t0.05, n-2·, it means the trend is not significant, otherwise the trend is significant.

[0082] (2) Acceptability criteria: Statistical processing of real-time stability study data, including data from samples stored at normal storage temperature (-20 ± 5 °C) for 13 months, is performed on these data for trend significance test, and the trend should not be significant.

[0083] The results are shown in Table 12.

[0084] Table 12

[0085] The results showed that the samples prepared with alternative matrix 5 could be stably stored for more than 12 months under normal storage temperature conditions (-20±5℃).

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A replacement matrix, characterized in that It comprises a preservative and a rabbit whole blood diluent; the rabbit whole blood diluent comprises rabbit whole blood and physiological saline; the volume percentage of the rabbit whole blood in the rabbit whole blood diluent is 25% to 75%.

2. The replacement matrix according to claim 1, characterized in that The volume percentage of the rabbit whole blood in the rabbit whole blood dilution solution is 25% to 70%.

3. The replacement matrix according to claim 1, characterized in that The volume percentage of the physiological saline to the rabbit whole blood dilution solution is 25% to 75%.

4. The replacement matrix according to claim 1, characterized in that The replacement matrix also includes L-ascorbic acid.

5. The replacement matrix according to claim 4, characterized in that The weight ratio of the L-ascorbic acid to the rabbit whole blood diluent is (0.5-1.5):

100.

6. The surrogate matrix according to any one of claims 1 to 5, characterized in that The preservative is a proclin series preservative.

7. The replacement matrix according to claim 6, characterized in that The preservative is at least one of proclin300 and proclin950; and / or the volume ratio of the preservative to the rabbit whole blood diluent is (0.05-0.3):

100.

8. The replacement matrix according to claim 7, characterized in that The invention comprises proclin300, proclin950, L-ascorbic acid and rabbit whole blood diluent; the rabbit whole blood diluent comprises rabbit whole blood and physiological saline; wherein the volume ratio of proclin300 and proclin950 to the rabbit whole blood diluent is (0.05-0.15):100 respectively; the weight ratio of L-ascorbic acid to the rabbit whole blood diluent is (0.8-1.2):100; the volume percentage of the rabbit whole blood in the rabbit whole blood diluent is 45%-55%, and the volume percentage of the physiological saline in the rabbit whole blood diluent is 45%-55%.

9. A folic acid and 5-methyltetrahydrofolate detection reagent or kit, characterized in that: It contains the substitute matrix according to any one of claims 1 to 8.

10. Use of the surrogate matrix according to any one of claims 1 to 8 in at least one of the following aspects: (1) detecting folic acid and 5-methyltetrahydrofolate; (2) preparing folic acid and 5-methyltetrahydrofolate detection reagents or kits; (3) preparing folic acid and 5-methyltetrahydrofolate detection calibrators; (4) preparing folic acid and 5-methyltetrahydrofolate detection quality control products.

Images