Method for simultaneous detection of folic acid and 5-methyltetrahydrofolate in human whole blood

Abstract

The present application relates to the technical field of analytical detection, and particularly relates to a detection method for simultaneously detecting folic acid and 5-methyltetrahydrofolic acid in human whole blood. The sample to be detected is mixed with Vc, saturated zinc sulfate solution, and a mixed solution of methanol, acetonitrile and isopropyl alcohol, and then centrifugal separation supernatant is carried out for liquid chromatography detection. The present application provides a detection method with low cost, simple steps, and good separation of unmetabolized folic acid and its metabolite 5-methyltetrahydrofolic acid in whole blood samples. The method can meet the requirements of precision, accuracy and matrix effect in the verification process. Moreover, the detection method of the present application can achieve accurate detection effect without introducing internal standard. The method plays an important role in evaluating the content of folic acid and its metabolites in whole blood samples.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to a method for simultaneously detecting folic acid and 5-methyltetrahydrofolate in human whole blood. Background Technology

[0002] Folic acid is a water-soluble B vitamin that promotes the maturation of bone marrow immature cells and plays an important role in human cell growth and protein synthesis. Folic acid intake during pregnancy can effectively reduce the risk of birth defects in newborns, such as cleft lip and neural tube defects.

[0003] Folic acid testing indicators include several forms of folic acid (such as tetrahydrofolate, 5-methyltetrahydrofolate, and unmetabolized folic acid), with 5-methyltetrahydrofolate being the most biologically active and functional form of folic acid. Total folic acid in erythrocytes includes unmetabolized folic acid and other metabolites.

[0004] Currently, the main methods for folic acid detection include: microbial detection, colorimetry, radioimmunoassay, chromatography, and mass spectrometry. Microbial detection, colorimetry, and radioimmunoassay can only detect total folic acid, while mass spectrometry is more expensive in terms of instrumentation and sample preparation.

[0005] For example, in the prior art, CN112083108A discloses a precise detection method for folic acid in blood, which uses solid-phase extraction to enrich and purify folic acid in sample pretreatment, and then analyzes the purified solution using liquid chromatography-tandem mass spectrometry. This method is costly and cumbersome. CN114646713A discloses a sample pretreatment method and detection method for folic acid and its metabolites, which also uses solid-phase extraction and liquid chromatography-tandem mass spectrometry detection, but this method is also costly.

[0006] Therefore, how to provide a low-cost, simple, and accurate detection method for folic acid and 5-methyltetrahydrofolate in human whole blood has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] To address the aforementioned technical challenges, this invention provides a method for simultaneously detecting folic acid and 5-methyltetrahydrofolate in human whole blood, comprising:

[0008] The sample to be tested was mixed with Vc (L-ascorbic acid) to prepare the first mixture;

[0009] The first mixture is mixed with sample extract A and sample extract B to prepare a second mixture; sample extract A is a saturated zinc sulfate solution; sample extract B is a mixture of methanol, acetonitrile and isopropanol in a volume ratio of (1-4):(0.5-1.5):1; the volume ratio of sample extract A to sample extract B is 1:(2-4).

[0010] After centrifuging the second mixture, the supernatant was separated and analyzed by liquid chromatography.

[0011] The conditions for the liquid chromatography detection include:

[0012] The chromatographic column is a C18 column;

[0013] Mobile phase A is tetrabutylammonium hydroxide phosphate buffer; mobile phase B is acetonitrile.

[0014] The gradient elution procedure is as follows:

[0015] During the elution process, the sum of the volume percentages of mobile phase A and mobile phase B is 100%.

[0016]

[0017] Through extensive optimization experiments on various combinations of detection conditions, this invention found that selecting zinc sulfate as the erythrocyte lysis buffer, combined with a mixture of methanol, acetonitrile, and isopropanol in the aforementioned ratio as a protein precipitant, for pretreatment of whole blood samples, can achieve better erythrocyte lysis, fully release the folic acid and its derivatives to be tested, and result in a higher sample extraction rate. Moreover, by controlling the volume ratio of sample extract A and sample extract B within the aforementioned range, the supernatant after extraction can be made clear, avoiding a brown color that would affect the accuracy of subsequent detection.

[0018] In the process of liquid chromatography detection, this invention found that when tetrabutylammonium hydroxide phosphate buffer is selected as the reversed-phase mobile phase, and acetonitrile is used as another mobile phase, the problems of folic acid peak tailing and peak splitting in liquid chromatography can be effectively solved under the above elution procedure.

[0019] Preferably, the tetrabutylammonium hydroxide phosphate buffer solution is a mixed aqueous solution of potassium dihydrogen phosphate, phosphoric acid, and tetrabutylammonium hydroxide methanol solution.

[0020] Preferably, each 1L of the tetrabutylammonium hydroxide phosphate buffer contains 1-3g potassium dihydrogen phosphate, 500-800μL 75% phosphoric acid, and 2-5mL 40% tetrabutylammonium hydroxide methanol solution.

[0021] Preferably, 0.01g to 0.03g of vitamin C is added to every 200μL of the sample to be tested.

[0022] Vc is used as a protective agent in this invention.

[0023] Preferably, the volume ratio of the sample to be tested to the sample extract A is (3-5):1.

[0024] Preferably, the chromatographic column is a Svea™ HPLC Column C18 with a specification of 5 μm.

[0025] Alternatively, the chromatographic column may be a Pursuit XRs 5C18 column with dimensions of 150 × 4.6 mm.

[0026] Preferably, the column temperature of the chromatographic column is 38–42°C; and / or the flow rates of the mobile phases A and B are 0.6–1.2 mL / min.

[0027] Preferably, a UV detector in series with a fluorescence detector is used for detection after liquid chromatography.

[0028] Since folic acid detection typically uses a UV detector, which has weak fluorescence, while 5-methyltetrahydrofolate is generally detected using a fluorescence detector, UV detection is ineffective. Therefore, a preferred method is to use a UV detector in series with a fluorescence detector to detect the two compounds separately.

[0029] Furthermore, when using liquid chromatography to detect folic acid, structural analogs (such as aminopterin or folinic acid) are usually chosen as internal standards. However, this invention found that when aminopterin or folinic acid is used as an internal standard, it cannot simultaneously satisfy the calibration requirements of both UV and fluorescence detectors for folic acid and 5-methyltetrahydrofolate. Moreover, the complex whole blood matrix, with the addition of additional analytes, increases the complexity of the experiment. The detection method of this invention, after performance verification, has been shown to eliminate the need for an internal standard.

[0030] Preferably, the wavelength of the ultraviolet detector is 280–285 nm; the excitation wavelength of the fluorescence detector is 292–298 nm, and the emission wavelength is 358–364 nm.

[0031] Preferably, after centrifuging the second mixture, the separated supernatant is filtered through a 0.22 μm filter membrane and then subjected to liquid chromatography detection.

[0032] Filtration through a filter membrane can make the supernatant to be tested cleaner, thereby further improving the accuracy of the test results.

[0033] Preferably, after centrifuging the second mixture, the supernatant is centrifuged a second time, and the supernatant is then separated for liquid chromatography detection.

[0034] Preferably, the method further includes: testing the quality control sample and / or standard sample using the detection method, wherein whole rabbit blood is used as a diluent during the preparation of the quality control sample and / or standard sample.

[0035] The present invention also found that when preparing quality control samples or standards, selecting rabbit whole blood as a diluent can pass the matrix effect experiment when using the above detection method, indicating that the stability of the analyte is better.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] This invention provides a low-cost, simple method for detecting unmetabolized folic acid and its metabolite 5-methyltetrahydrofolate in whole blood samples. This method meets the requirements for precision, accuracy, and matrix effect during the validation process. Moreover, the detection method of this invention can achieve accurate detection without the introduction of an internal standard. This method plays an important role in evaluating the content of folic acid and its metabolites in whole blood samples. Attached Figure Description

[0038] Figure 1 These are photographs of supernatant samples obtained after different pretreatments.

[0039] Figure 2 This is the liquid chromatogram of folic acid detected by the method in Comparative Example 4.

[0040] Figure 3 This is a liquid chromatogram of folic acid detected by the method in Example 1.

[0041] Figure 4 This is the liquid chromatogram of 5-methyltetrahydrofolate from Example 1. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0043] Unless otherwise specified, all methods used in the examples were conventional or performed according to techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents and instruments used without specified manufacturers were all conventional products that could be purchased from legitimate channels.

[0044] Example 1

[0045] This embodiment provides a method for simultaneously detecting folic acid and 5-methyltetrahydrofolate in human whole blood, the steps of which are as follows:

[0046] 1. Instruments

[0047] Table 1

[0048] Instrument Name model brand liquid chromatograph LC 2500 Haosi Bio 96-well plate oscillator MB100-4A Hangzhou Aosheng 96-well plate centrifuge H2050R Xiangyi 10μL pipette 0.5-10μL eppendorf 200μL pipette 10-200μL eppendorf

[0049] 2. Reagents

[0050] 2.1 Preparation of compound and internal standard stock solutions

[0051] Table 2

[0052]

[0053]

[0054] 2.2 Preparation of compound working solutions and quality control working solutions

[0055] Table 3

[0056]

[0057] Take 10 μL of Sub-C1 solution and add it to 190 μL of rabbit whole blood to make the C1 working solution. C2, C3, C4, C5, C6, LQC, and HQC are prepared in the proportions shown in Table 4.

[0058] Table 4

[0059] Sub-C2 Sub-C3 Sub-C4 Sub-C5 Sub-LQC Sub-HQC Sub-C1(μL) 0.28 0.26 0.21 0.12 0.81 0.24 Sub-C6(μL) 0.01 0.03 0.08 0.17 0.06 0.63

[0060] 2.3 Preparation of other reagents

[0061] Weigh 5g of L-ascorbic acid (Vc) and dissolve it in 100mL of ultrapure water as a protective agent.

[0062] Weigh 16.67g of zinc sulfate heptahydrate and dissolve it in 100mL of ultrapure water to obtain sample extraction solution A.

[0063] Transfer 500 mL of methanol, 250 mL of acetonitrile, and 250 mL of isopropanol, mix them thoroughly, and use this as sample extraction solution B.

[0064] 2.4 Sample Pretreatment

[0065] Add sample: Transfer 200 μL of the sample to be tested into 2 mL centrifuge tubes and add 200 μL of protective agent;

[0066] Add sample extraction solution A: Transfer 50 μL of sample extraction solution A and add it to the corresponding centrifuge tubes above;

[0067] Add sample extraction solution B: Transfer 150 μL of sample extraction solution B and add it to the corresponding centrifuge tubes above. Shake well at 3000 rpm for 15 min.

[0068] Centrifugation: Centrifuge at 18000g for 10 min at 4℃; transfer 150 μL of supernatant into a new 96-well plate;

[0069] Centrifugation: Centrifuge at 3000g for 5 minutes at 4℃;

[0070] Detection: Place the 96-well plate in a liquid chromatography instrument and inject 20 μL of sample for detection.

[0071] 2.5 Chromatographic conditions

[0072] The chromatographic conditions are shown in Table 5.

[0073] Table 5

[0074]

[0075] Example 2

[0076] This embodiment evaluates the precision of the detection method in Example 1.

[0077] 1. Intra-batch precision

[0078] Validation method: A standard curve was prepared using rabbit whole blood. Quality control samples prepared from low and high concentrations of rabbit whole blood (folate concentrations of 250 ng / mL and 1200 ng / mL, respectively; 5-methyltetrahydrofolate concentrations of 150 ng / mL and 1100 ng / mL, respectively) were pretreated. Five samples were replicated for each concentration level.

[0079] Acceptance criteria: Precision is assessed using the coefficient of variation (%CV), which should be within 15.0%.

[0080] 2. Inter-batch precision

[0081] Validation method: A standard curve was prepared using rabbit whole blood. Quality control samples prepared from low and high concentrations of rabbit whole blood (folic acid concentrations of 250 ng / mL and 1200 ng / mL, respectively; 5-methyltetrahydrofolic acid concentrations of 150 ng / mL and 1100 ng / mL, respectively) were pretreated. Five samples were replicated for each concentration level. The validation period was five days, with one batch per day.

[0082] Acceptance criteria: Precision is assessed using the coefficient of variation (%CV), which should be within 15.0%.

[0083] 3. The experimental results for folic acid and 5-methyltetrahydrofolate are shown in Tables 6 and 7, respectively.

[0084] Table 6

[0085]

[0086]

[0087] Table 7

[0088]

[0089] 4. Conclusion

[0090] The coefficients of variation (%CV) of intra- and inter-batch precision at both low and high concentrations were within 15%, meeting the acceptance criteria.

[0091] Example 3

[0092] This embodiment evaluates the accuracy of the detection method in Example 1.

[0093] Validation method: A standard curve was prepared using rabbit whole blood. Low and high concentration quality control samples were prepared from human whole blood (folate concentrations were 250 ng / mL and 1200 ng / mL, respectively; 5-methyltetrahydrofolate concentrations were 150 ng / mL and 1100 ng / mL, respectively). Pretreatment was performed, with 3 samples in parallel for each concentration level. The validation period was 5 days, with one batch per day.

[0094] Acceptance criteria: Deviation from target value within 15.0%.

[0095] The experimental results for folic acid and 5-methyltetrahydrofolate are shown in Tables 8 and 9, respectively.

[0096] Table 8

[0097]

[0098] Table 9

[0099]

[0100] Conclusion: All concentration points deviated from the target value within ±15%, meeting the acceptance criteria.

[0101] Example 4

[0102] This embodiment evaluates the linearity of the detection method in Example 1.

[0103] Verification method: A certain amount of rabbit whole blood was used to prepare compound working solutions (concentrations of C1, C2, C3, C4, C5, and C6, respectively) for pretreatment for 5 consecutive days.

[0104] Acceptance criteria: Accuracy for all concentration points should be within 85%-115%, and CV should be within 20.0%. Linear regression: r ≥ 0.99.

[0105] The experimental results for folic acid and 5-methyltetrahydrofolate are shown in Tables 10 and 11, respectively.

[0106] Table 10

[0107]

[0108] Table 11

[0109]

[0110]

[0111] Conclusion: The deviations at all concentration points were within ±15.0%, meeting the acceptance criteria; linear regression: r > 0.99, meeting the acceptance criteria.

[0112] Example 5

[0113] This embodiment evaluates the matrix effect of the detection method in Example 1.

[0114] Validation method: A standard curve was prepared using rabbit whole blood. High and low concentration quality control samples were prepared from human whole blood (folic acid concentrations were 250 ng / mL and 1200 ng / mL, respectively; 5-methyltetrahydrofolic acid concentrations were 150 ng / mL and 1100 ng / mL, respectively). Rabbit whole blood quality control samples of the same concentration were also prepared. Each concentration was measured three times, and the mean concentration of each sample was calculated.

[0115] Acceptable standard: Matrix bias (%) = (mean of rabbit whole blood sample - mean of human whole blood sample) / mean of human whole blood sample (%), matrix bias < ±20%.

[0116] The experimental results for folic acid and 5-methyltetrahydrofolate are shown in Tables 12 and 13, respectively.

[0117] Table 12

[0118]

[0119]

[0120] Table 13

[0121]

[0122] Conclusion: The matrix effect is <±20%, which meets the acceptance criteria.

[0123] In summary, for folic acid detection, the coefficient of variation (%CV) of intra-assay precision ranged from 1.13% to 4.05%, and the coefficient of variation (%CV) of inter-assay precision ranged from 4.51% to 4.66%. The recoveries of high and low human whole blood samples were both between 85% and 115%. The linearity of the detection ranged from 150 ng / mL to 1600 ng / mL, with all point deviations within 15%, and the linear regression showed r > 0.99. The matrix effect for comparing human whole blood and rabbit whole blood samples ranged from -2.09% to -11.07%.

[0124] For the detection of 5-methyltetrahydrofolate, the coefficient of variation (%CV) of intra-assay precision ranged from 0.50% to 3.36%, and the coefficient of variation (%CV) of inter-assay precision ranged from 3.87% to 6.11%. The recoveries of high and low human whole blood samples ranged from 85% to 115%. The linearity of the detection ranged from 50 ng / mL to 1500 ng / mL, with all point deviations within 15%, and the linear regression result was r > 0.99. The matrix effect for comparison between human and rabbit whole blood samples ranged from -1.49% to -10.69%.

[0125] Comparative Example 1

[0126] This comparative example explores the selection of sample extract A.

[0127] The comparative experimental procedure is as follows: High and low concentration quality control points were prepared from human whole blood (folic acid concentrations: 250 ng / mL and 1200 ng / mL; 5-methyltetrahydrofolate concentrations: 150 ng / mL and 1100 ng / mL, respectively). Sample extraction solution A was prepared using ammonium chloride solution and zinc sulfate solution of the same concentration. Other pretreatment reagents, detection conditions, and procedures were the same as in Example 1. Each concentration was measured three times. The concentrations of folic acid and 5-methyltetrahydrofolate after extraction were compared.

[0128] The experimental results for folic acid and 5-methyltetrahydrofolate are shown in Tables 14 and 15, respectively.

[0129] Table 14

[0130]

[0131] Table 15

[0132]

[0133] Experimental results show that zinc sulfate has a better extraction efficiency than ammonium chloride.

[0134] Comparative Example 2

[0135] This comparative example explores the selection of sample extract solution B.

[0136] The comparative experiment steps are as follows:

[0137] In this comparative example, 100 mL of methanol, 500 mL of acetonitrile, and 500 mL of isopropanol were transferred and mixed thoroughly to prepare sample extraction solution B. High and low concentration quality control points were prepared from human whole blood (folate concentrations: 250 ng / mL and 1200 ng / mL; 5-methyltetrahydrofolate concentrations: 150 ng / mL and 1100 ng / mL). Sample extraction solutions B were prepared using both Example 1 and Comparative Example 2. Other pretreatment reagents, detection conditions, and procedures were the same as in Example 1. Each concentration was measured three times. The concentrations of folic acid and 5-methyltetrahydrofolate after extraction were compared.

[0138] The experimental results for folic acid and 5-methyltetrahydrofolate are shown in Tables 16 and 17, respectively.

[0139] Table 16

[0140]

[0141] Experimental results show that a low proportion of methanol in sample extract B will affect the extraction efficiency.

[0142] Comparative Example 3

[0143] This comparative example provides a method for simultaneously detecting folic acid and 5-methyltetrahydrofolate in human whole blood, the only difference from Example 1 being the following steps:

[0144] During sample pretreatment, add 150 μL of sample extraction solution A and 50 μL of sample extraction solution B.

[0145] The results showed that the pretreated supernatant sample in this comparative example was dark brown. Figure 1 The light gray area in the right image shows the clear and transparent supernatant sample after pretreatment in Example 1. Figure 1 (The transparent part in the left image).

[0146] Comparative Example 4

[0147] This comparative example provides a method for simultaneously detecting folic acid and 5-methyltetrahydrofolate in human whole blood, the only difference from Example 1 being the following steps:

[0148] Mobile phase A does not contain tetrabutylammonium hydroxide solution.

[0149] The results showed that the folic acid peak in the liquid chromatogram of this comparative example exhibited peak splitting. Figure 2 In contrast, the peak splitting in the liquid chromatogram of Example 1 was significantly improved. Figure 3 ), Figure 4 This is the liquid chromatogram of 5-methyltetrahydrofolate from Example 1.

[0150] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for simultaneously detecting folic acid and 5-methyltetrahydrofolate in human whole blood, characterized in that, include: The sample to be tested was mixed with Vc to prepare the first mixture; The first mixture is mixed with sample extract A and sample extract B to prepare a second mixture; sample extract A is a saturated zinc sulfate solution; sample extract B is a mixture of methanol, acetonitrile and isopropanol in a volume ratio of (1~4):(0.5~1.5):1; the volume ratio of sample extract A to sample extract B is 1:(2~4). After centrifuging the second mixture, the supernatant was separated and analyzed by liquid chromatography. The conditions for the liquid chromatography detection include: The chromatographic column was a Svea™ HPLC Column C18 with a specification of 5 μm and 110 Å. Alternatively, the chromatographic column may be a Pursuit XRs 5 C18 column with dimensions of 150 × 4.6 mm; the column temperature may be 38–42 °C; and the flow rates of mobile phases A and B may be 0.6–1.2 mL / min. Mobile phase A is tetrabutylammonium hydroxide phosphate buffer; mobile phase B is acetonitrile. The gradient elution procedure is as follows: The sum of the volume percentages of mobile phase A and mobile phase B during elution is 100%. 。 2. The detection method according to claim 1, characterized in that, Add 0.01g~0.03g of vitamin C to every 200μL of test sample.

3. The detection method according to claim 1, characterized in that, The volume ratio of the sample to be tested to the sample extract A is (3~5):

1.

4. The detection method according to claim 1, characterized in that, After liquid chromatography detection, a UV detector in series with a fluorescence detector was used for further detection.

5. The detection method according to claim 4, characterized in that, The wavelength of the ultraviolet detector is 280~285nm; the excitation wavelength of the fluorescence detector is 292~298nm, and the emission wavelength is 358~364nm.

6. The detection method according to claim 1, characterized in that, After centrifuging the second mixture, the separated supernatant was filtered through a 0.22 μm filter membrane and then analyzed by liquid chromatography.

7. The detection method according to claim 1, characterized in that, After centrifuging the second mixture, the supernatant was centrifuged a second time, and the supernatant was then separated for liquid chromatography detection.

8. The detection method according to claim 1, characterized in that, Also includes: The test involves quality control samples and / or standards, wherein whole rabbit blood is used as a diluent during the preparation of the quality control samples and / or standards.

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