CN115980248A - Reagent set for detecting methotrexate, folic acid, 5-formyltetrahydrofolic acid and 5-methyltetrahydrofolic acid - Google Patents
Reagent set for detecting methotrexate, folic acid, 5-formyltetrahydrofolic acid and 5-methyltetrahydrofolic acid Download PDFInfo
- Publication number
- CN115980248A CN115980248A CN202310112697.0A CN202310112697A CN115980248A CN 115980248 A CN115980248 A CN 115980248A CN 202310112697 A CN202310112697 A CN 202310112697A CN 115980248 A CN115980248 A CN 115980248A
- Authority
- CN
- China
- Prior art keywords
- acid
- solution
- methotrexate
- folic acid
- methyltetrahydrofolic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a reagent kit for detecting methotrexate, folic acid, 5-formyltetrahydrofolic acid and 5-methyltetrahydrofolic acid, which can simultaneously and quantitatively detect the four components and consists of a solid phase extraction plate, a standard solution, an internal standard solution, an activation solution, a solution A, a solution B and a quality control sample. The invention also provides a method for simultaneously detecting methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid by using the reagent kit or the kit. The method effectively enriches the substances in the blood through a specific solid phase extraction plate; meanwhile, the mixed solution containing ascorbic acid, citric acid, 2-mercaptoethanol and dithiothreitol is used as a protective agent of the folic acid metabolites, so that the stability of the folic acid metabolites is improved, and the reliability of quantitative detection of the folic acid metabolites is improved; thereby realizing the high-efficiency, stable and accurate detection of the methotrexate, the folic acid, the 5-leucovorin and the 5-methyltetrahydrofolic acid.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and relates to a reagent kit for detecting methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid, in particular to a reagent kit for detecting methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid, a kit and application thereof.
Background
Folic acid is also called pteroylglutamic acid and is an important exogenous B vitamin. Folic acid absorbed by the human body is converted in the liver to tetrahydrofolic acid and other reduced derivatives. Among them, 5-methyltetrahydrofolate is the most active metabolite, accounting for about 98% of the folate content in plasma, and its main biological function is as a one-carbon unit carrier in biochemical reactions in vivo, participating in the synthesis of DNA and RNA and the metabolism of amino acids.
Methotrexate is an anti-tumor drug with a structure similar to that of folic acid, inhibits tumor cell proliferation by interfering folic acid metabolism, and has important effects on improving disease remission rate and prolonging life. At present, a high-dose methotrexate treatment strategy is widely applied to treatment of diseases such as acute lymphocytic leukemia, osteosarcoma and non-Hodgkin lymphoma. However, methotrexate acts on tumor cells and also affects normal cellular metabolism, resulting in toxic and side effects such as bone marrow suppression, impairment of liver and kidney functions, gastrointestinal reactions, mucosal damage, and nervous system damage. Clinically, the toxic and side effects are generally relieved by using calcium 5-formyltetrahydrofolate (existing in a human body in the form of 5-formyltetrahydrofolate). The calcium 5-formyltetrahydrofolate is calcium salt of 5-formyltetrahydrofolate, and can be directly converted into 5-methyltetrahydrofolate to exert physiological effect by crossing the process of converting folic acid into tetrahydrofolic acid when being supplemented into the body as antidote of large dose methotrexate, thus being not interfered by large dose methotrexate.
In the process of using the leucovorin calcium to save the toxic and side effects of the methotrexate, the frequency of the rescue is different from patient to patient. For example, in the case of acute lymphoblastic leukemia, the blood concentration of methotrexate is reduced to a safe range after 1 to 5 rescues in most children, and the blood concentration of methotrexate is reduced to an acceptable range after 30 rescues in few children. Therefore, when a high-dose methotrexate therapy/calcium 5-formyltetrahydrofolate rescue strategy is adopted, the measurement of the levels of methotrexate, folic acid, calcium 5-formyltetrahydrofolate and 5-methyltetrahydrofolate before and after the drug administration is of great significance for the treatment of acute lymphoblastic leukemia, osteosarcoma and non-Hodgkin's lymphoma.
At present, methods for detecting folic acid and metabolites thereof of a human body comprise a radioimmunoassay, a chemiluminescence analysis method, a microbiological method, a gas chromatography-tandem mass spectrometry method and the like, and the methods generally have the limitations of low sensitivity, poor stability and the like. The ultra performance liquid chromatography-tandem mass spectrometry is a detection method combining ultra performance liquid chromatography and mass spectrometry, can simultaneously present molecular weight and structural information of each component, and improves the sensitivity and accuracy of clinical detection.
Patent CN 112083108B discloses a method for quantitatively detecting folic acid and main metabolites thereof (5-methyltetrahydrofolic acid and 5-formyltetrahydrofolic acid) in a human body by utilizing a high performance liquid chromatography-tandem mass spectrometry combined technology. Patent CN 112834677A discloses a liquid chromatography-tandem mass spectrometry combined technology simultaneously detecting homocysteine and metabolism related substances (methionine pyridoxine, folic acid, and 5-methyltetrahydrofolic acid). In addition, patent CN 105181829B discloses a method for synchronously detecting the content of total folic acid and folic acid derivatives (tetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, 5, 10-methylenetetrahydrofolic acid, 10-formylfolic acid, 10-formyltetrahydrofolic acid) in plant leaves by using ultra-high performance liquid chromatography-tandem mass spectrometry. Patent CN110146626A discloses a method for measuring the contents of folic acid and 5-methyltetrahydrofolic acid in blood spots by a high performance liquid chromatography-tandem mass spectrometry combined technology. Patent CN 11309033A discloses a method for measuring the contents of folic acid, tetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, 5, 10-methylenetetrahydrofolic acid, S-adenosylmethionine, S-adenosylhomocysteine and homocysteine in a human body by using a high performance liquid chromatography-tandem mass spectrometry combined technology. The method can measure folic acid and metabolites thereof, but fails to carry out combined detection on the therapeutic methotrexate with similar structure to folic acid. Thus, the prior art fails to address the simultaneous monitoring of the in vivo levels of therapeutic drugs, rescue drugs and related substances during the treatment of diseases such as acute lymphocytic leukemia, osteosarcoma and non-hodgkin's lymphoma.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a reagent kit and a kit for detecting methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid aiming at the defects of the prior art and the major needs of clinical treatment, and the reagent kit or the kit can be used for simultaneously and quantitatively detecting methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid.
The invention also provides the application of the reagent kit in the manufacture of the kit, and the kit can be used for accurately mastering the in-vivo levels of therapeutic drugs, rescue drugs and related substances.
To this end, the invention provides, in a first aspect, a reagent set for detecting methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid, which is capable of simultaneously and quantitatively detecting methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid, and which consists of a solid phase extraction plate, a standard solution, an internal standard solution, an activation solution, a solution A, a solution B and a quality control sample.
In the present invention, the solid phase extraction plate is a mixed anion type solid phase extraction plate, which is capable of specifically binding an acidic compound.
According to the invention, the solution A is prepared from a solvent A and an antioxidant; wherein the solvent A is 99.9% pure water and 0.1% ammonia water, and the antioxidant is ascorbic acid, citric acid, 2-mercaptoethanol and dithiothreitol; in the solution A, the concentrations of the ascorbic acid, the citric acid, the 2-mercaptoethanol and the dithiothreitol are respectively and independently 20-100 mu g/mL.
In some embodiments of the invention, the standard solution is a standard solution of methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid formulated with solution A, wherein the concentration of the standard of methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid ranges from 0.1ng/mL to 5000ng/mL.
In other embodiments of the present invention, the internal standard solution is an isotope standard solution of methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid prepared from the solution A, wherein the concentration range of the isotope standard of methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid is 0.1 ng/mL-500 ng/mL.
According to the invention, the solution B is prepared from a solvent B and an antioxidant; wherein the solvent B is 98% methanol +2% formic acid, and the antioxidant is ascorbic acid, citric acid, 2-mercaptoethanol and dithiothreitol; in the solution B, the concentrations of the ascorbic acid, the citric acid, the 2-mercaptoethanol and the dithiothreitol are respectively and independently 20-100 mu g/mL.
In the invention, the mixed solution of the oxidant containing ascorbic acid, citric acid, 2-mercaptoethanol and dithiothreitol is used as the protective agent of the folic acid metabolites, so that the stability of the folic acid metabolites is increased, and the reliability of quantitative detection of the folic acid metabolites is improved.
In some embodiments of the invention, the activation solution is methanol; and/or the quality control sample is a serum sample containing methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid, and 3 samples with low concentration, medium concentration and high concentration are obtained.
It will be appreciated by those skilled in the art that the low, medium and high concentration ranges of methotrexate, folic acid, 5-formyltetrahydrofolic acid and 5-methyltetrahydrofolic acid in serum samples used as quality control samples are not all identical.
For example, in some cases, the serum sample used as the quality control sample contains folic acid at a low concentration of 5ng/mL, a medium concentration of 20ng/mL, and a high concentration of 100ng/mL.
In other examples, the serum samples used as quality control samples contain 5 methyltetrahydrofolate, 5 formyltetrahydrofolate and methotrexate at low and medium concentrations of 50, 200 and 1000ng/mL, respectively.
In a second aspect, the invention provides a kit for detecting methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolate, which contains the kit of reagents according to the first aspect of the invention.
In a third aspect, the present invention provides an application of a reagent kit according to the first aspect of the present invention in the preparation of a kit for detecting methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid in a human body, which comprises:
step L, uniformly mixing a sample to be detected or a standard solution with an internal standard solution and the solution A to obtain a first mixed solution;
step M, sequentially activating and balancing the solid-phase extraction plate by using an activating solution and a solution A; adding the first mixed solution in the step L for sample loading; washing the solid phase extraction plate with solution a; eluting with the solution B to obtain a target component solution; blowing nitrogen to dry; adding the solution A for redissolution to obtain a solution to be detected;
and N, measuring the solution to be measured obtained in the step M by adopting an ultra performance liquid chromatography-tandem mass spectrometry combined technology, and calculating the contents of methotrexate, folic acid, 5-formyltetrahydrofolic acid and 5-methyltetrahydrofolic acid in the sample solution by adopting an internal standard and external standard curve method.
In some embodiments of the invention, the ultra performance liquid chromatography conditions in step M are as follows: a chromatographic column: HSS T3.1 (inner diameter) × 100mm (length), particle size 1.8 μm; column temperature: 40 ℃; the mobile phase A is water and 0.1 percent formic acid; the mobile phase B is acetonitrile; gradient elution: 0 to 0.5min, A92 to 92 percent; 0.5-2.5min, A92% -75%; 2.5-2.6min, A75-5%; 2.6-3.0min, A5% -5%; 3.0-4.0min, A is 5% -92%;
and/or, the mass spectrometry conditions in step M are as follows: ionization mode: ESI +, multiple reaction monitoring mode. Capillary voltage: 1KV, taper hole voltage: 50V, taper hole airflow rate: 50L/Hr, desolventizing temperature: 500 ℃, desolventizing flow rate: 1000L/Hr (hours).
The detection parameters of the target substance were as follows:
TABLE 1 table of parameters for detection of target substance
The above application of the invention can be understood as a method for detecting methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid by using the reagent kit or the kit provided by the invention.
The term "precision within the day" as used herein refers to the closeness of the results when the same test object is subjected to secondary parallel measurements at different times within the same day.
The term "day precision" as used herein refers to the degree of agreement between repeated measurements of the same test subject on different days.
The invention provides a reagent kit and a kit for simultaneously detecting methotrexate, folic acid, 5-formyltetrahydrofolic acid and 5-methyltetrahydrofolic acid. The invention also provides a method for simultaneously detecting methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid by using the reagent kit or the kit. Compared with the prior art scheme of the background technology part, the method effectively enriches the substances in the blood through the specific solid phase extraction plate; meanwhile, the mixed solution containing ascorbic acid, citric acid, 2-mercaptoethanol and dithiothreitol is used as a protective agent of the folic acid metabolites, so that the stability of the folic acid metabolites is improved, and the accuracy and reliability of quantitative detection of the folic acid metabolites are improved; thereby realizing the high-efficiency, stable and accurate detection of the methotrexate, the folic acid, the 5-leucovorin and the 5-methyltetrahydrofolic acid. The method has the advantages of wide linear range, high precision, good reproducibility, simple and convenient operation, easy popularization and the like, and has important significance for promoting the updating and upgrading of the drug monitoring technology in the treatment process of acute lymphocytic leukemia, osteosarcoma and non-Hodgkin lymphoma and the treatment safety of patients.
Drawings
The invention is described in further detail below with reference to the attached drawing figures:
FIG. 1 is a quantitative limit chromatogram of four standards for analytes, wherein a) is methotrexate, b) is folic acid, c) is 5-formyltetrahydrofolic acid, and d) is 5-methyltetrahydrofolic acid.
FIG. 2 shows standard curves for four assays, where a) is methotrexate, b) folic acid, c) 5-formyltetrahydrofolic acid, and d) 5-methyltetrahydrofolic acid.
FIG. 3 shows the QC serum sample isolation results for four assays.
FIG. 4 shows the results of the separation of the standard solutions of the four analytes.
Detailed Description
In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to the appended drawings. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Examples
The present invention will be specifically described below with reference to specific examples. The experimental methods described below are, unless otherwise specified, all routine laboratory procedures. The experimental materials described below, unless otherwise specified, are commercially available.
Example 1:
a kit for accurately measuring methotrexate, folic acid, 5-formyltetrahydrofolic acid and 5-methyltetrahydrofolic acid in blood comprises the following components:
TABLE 2 kit composition
The quality control sample comprises 3-concentration methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid serum samples; wherein the low concentration of the folic acid is 5ng/mL, the medium concentration is 20ng/mL, and the high concentration is 100ng/mL; the low, medium and high concentrations of 5 methyltetrahydrofolate, 5 formyltetrahydrofolate and methotrexate are, independently, 50, 200 and 1000ng/mL, respectively.
The concentrations of methotrexate, 5-leucovorin and 5-methyltetrahydrofolate in the standard solutions were independently as follows: 5. 10, 20, 50, 100, 500, 1000, and 2000ng/mL; the concentrations of folic acid were as follows: 1. 2, 5,10, 20, 50, 100 and 200ng/mL.
Example 2:
a method for accurately determining methotrexate, folic acid, 5-formyltetrahydrofolic acid, and 5-methyltetrahydrofolic acid in blood comprising:
A. uniformly mixing 100 mu L of quality control sample or standard solution with 20 mu L of internal standard solution and 300 mu L of solution A to obtain a first mixed solution;
B. sequentially activating and balancing the solid phase extraction plate by using 200 mu L of activating solution and 200 mu L of solution A; adding the first mixed solution in the step A for sample loading; wash the solid phase extraction plate with 200 μ L of solution a; eluting with 40 μ L of solution B to obtain target component solution; blowing nitrogen to dry; adding 80 mu L of solution A for redissolving to obtain a solution to be detected.
C. And D) measuring the solution to be measured obtained in the step B) by adopting an ultra performance liquid chromatography-tandem mass spectrometry combined technology, and calculating the contents of methotrexate, folic acid, 5-formyltetrahydrofolic acid and 5-methyltetrahydrofolic acid in the sample solution by adopting an internal standard external standard curve method.
The chromatographic conditions were as follows: a chromatographic column: HSS T3.1 (inner diameter) × 100mm (length), particle size 1.8 μm; column temperature: at 40 ℃; the mobile phase A is water and 0.1 percent formic acid; the mobile phase B is acetonitrile; gradient elution: 0 to 0.5min, A92 to 92 percent; 0.5-2.5min, A92% -75%; 2.5-2.6min, A75-5%; 2.6-3.0min, A5% -5%; 3.0-4.0min, A is 5% -92%.
The mass spectrometry conditions were as follows: ionization mode: ESI +, multiple reaction monitoring mode. Capillary voltage: 1KV, taper hole voltage: 50V, taper hole airflow rate: 50L/Hr, desolvation temperature: 500 ℃, desolventizing flow rate: 1000L/Hr. The detection parameters of the target substance were as follows:
TABLE 3 table of parameters for detection of target substance
Remarking: quantitation of 5-formyltetrahydrofolate was performed using isotopic internal standards for folate.
The performance of the method in aspects of quantitative limit, linear range, precision, accuracy, specificity, interference test and the like is evaluated by referring to a CLSI C62-A file of standard procedures for development and verification of mass spectrometry published by the American society for clinical laboratory standards and a file of development and verification of a liquid chromatography tandem mass spectrometry clinical detection method published by the society for testing physicians of China, and the like, and the clinical applicability of the method is examined.
Limit of quantification
And (3) carrying out sample injection detection on the standard substance solution to be detected of the methotrexate, the folic acid, the 5-leucovorin and the 5-methyltetrahydrofolic acid, and according to a standard curve of the substance to be detected, defining the concentration of the substance to be detected in the corresponding standard substance as the limit of quantitation of the detection method when the average signal-to-noise ratio is larger than 10. The quantitative limits of the four analytes are shown in Table 4, and the chromatograms of the four analytes are shown in FIG. 1, which all meet the clinical detection requirements.
TABLE 4 quantitation limits for four analytes
Linear range
And respectively injecting standard solutions with different concentrations to be detected. The result shows that the concentrations and the response values of the four substances to be tested are linear, and the linear correlation coefficient R is 2 >0.998, meets the verification standard. The results of the linear range of the three analytes are shown in Table 5, and the standard curve is shown in FIG. 2.
TABLE 5 Linear Range of four lines of test object
For 3 consecutive days, 3 concentrations (low, medium, high) of the quality control sample were measured, and each concentration was measured 5 times in parallel to calculate the intra-day precision and the inter-day precision. The results are shown in Table 6, where the daily and daytime precision of the test substances were within acceptable ranges (+ -15%).
TABLE 6 methods for determining the day precision and day precision of the test substances
Accuracy of
Respectively measuring 3 concentrations (low, medium and high concentrations) of quality control samples, wherein the low concentration of folic acid is 5ng/mL, the medium concentration is 20ng/mL, and the high concentration is 100ng/mL; the concentrations of 5-methyltetrahydrofolate, CF and methotrexate were 50, 200 and 1000ng/mL, respectively. And 5 parallel samples are respectively arranged at each concentration, the samples are subjected to quantitative analysis after sample injection detection, and the standard addition recovery rate is calculated. The results are shown in Table 7, the recovery rates of the samples within the acceptable range (plus or minus 15%) are verified to meet the requirements.
TABLE 7 method for determining accuracy of test substance
Specificity and interference test
The low-concentration quality control sample and the standard solution to be detected are respectively subjected to sample injection and determination, the QC serum sample separation results of the four objects to be detected are shown in figure 3, the standard solution separation results of the four objects to be detected are shown in figure 4, and the comparison between the figure 3 and the figure 4 shows that methotrexate, folic acid, 5-formyltetrahydrofolic acid and 5-methyltetrahydrofolic acid can be completely separated through ultra-high performance liquid chromatography, so that the detection method can effectively avoid matrix interference in the serum sample and mutual interference among the objects to be detected, and has strong specificity.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are used for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
Claims (10)
1. A reagent kit for detecting methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid comprises a solid phase extraction plate, a standard solution, an internal standard solution, an activation solution, a solution A, a solution B and a quality control sample, and can simultaneously and quantitatively detect the methotrexate, the folic acid, the 5-leucovorin and the 5-methyltetrahydrofolic acid.
2. The kit of claim 1, wherein the solid phase extraction plate is a mixed anionic solid phase extraction plate capable of specifically binding an acidic compound.
3. The reagent kit of claim 1, wherein the solution a is prepared from a solvent a and an antioxidant; wherein the solvent A is 99.9% pure water and 0.1% ammonia water, and the antioxidant is ascorbic acid, citric acid, 2-mercaptoethanol and dithiothreitol; in the solution A, the concentrations of the ascorbic acid, the citric acid, the 2-mercaptoethanol and the dithiothreitol are respectively and independently 20-100 mu g/mL.
4. The kit of claim 3, wherein the standard solution is a standard solution of methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid formulated from solution A, wherein the concentration of the methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid standard ranges from 0.1ng/mL to 5000ng/mL.
5. The reagent set of claim 3, wherein the internal standard solution is an isotope standard solution of methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid prepared from solution A, wherein the concentration range of the isotope standard solution of methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid is 0.1 ng/mL-5000 ng/mL.
6. The kit according to claim 1, wherein the solution B is prepared from a solvent B and an antioxidant; wherein the solvent B is 98% methanol +2% formic acid, and the antioxidant is ascorbic acid, citric acid, 2-mercaptoethanol and dithiothreitol; in the solution B, the concentrations of the ascorbic acid, the citric acid, the 2-mercaptoethanol and the dithiothreitol are respectively and independently 20-100 mu g/mL.
7. The kit of claim 1, wherein the activation solution is methanol; and/or the quality control sample is a serum sample containing methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolic acid, and 3 samples with low concentration, medium concentration and high concentration are obtained.
8. A kit for the detection of methotrexate, folic acid, 5-leucovorin and 5-methyltetrahydrofolate comprising a kit of reagents according to any one of claims 1 to 7.
9. Use of a kit of reagents according to any one of claims 1 to 7 for the preparation of a kit for the detection of methotrexate, folic acid, 5-formyltetrahydrofolic acid and 5-methyltetrahydrofolic acid in humans comprising:
step L, uniformly mixing a sample to be detected or a standard solution with an internal standard solution and the solution A to obtain a first mixed solution;
step M, sequentially activating and balancing the solid-phase extraction plate by using an activating solution and a solution A; adding the first mixed solution in the step L for sample loading; washing the solid phase extraction plate with solution a; eluting with the solution B to obtain a target component solution; blowing nitrogen to dry; adding the solution A for redissolution to obtain a solution to be detected;
and step N, measuring the solution to be measured obtained in the step M by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry combined technology, and calculating the contents of methotrexate, folic acid, 5-formyltetrahydrofolic acid and 5-methyltetrahydrofolic acid in the sample solution by adopting an internal standard and external standard curve method.
10. The use according to claim 9, wherein the ultra high performance liquid chromatography conditions in step M are as follows: a chromatographic column: HSS T3.1 (inner diameter) × 100mm (length), particle size 1.8 μm; column temperature: 40 ℃; the mobile phase A is water and 0.1 percent formic acid; the mobile phase B is acetonitrile; gradient elution: 0 to 0.5min, A92 to 92 percent; 0.5-2.5min, A92% -75%; 2.5-2.6min, A75-5%; 2.6-3.0min, A5% -5%; 3.0-4.0min, A5-92%;
and/or, the mass spectrometry conditions in step M are as follows: ionization mode: ESI +, multiple reaction monitoring mode; capillary voltage: 1KV, taper hole voltage: 50V, taper hole airflow rate: 50L/Hr, desolvation temperature: 500 ℃, desolventizing flow rate: 1000L/Hr.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310112697.0A CN115980248A (en) | 2023-02-14 | 2023-02-14 | Reagent set for detecting methotrexate, folic acid, 5-formyltetrahydrofolic acid and 5-methyltetrahydrofolic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310112697.0A CN115980248A (en) | 2023-02-14 | 2023-02-14 | Reagent set for detecting methotrexate, folic acid, 5-formyltetrahydrofolic acid and 5-methyltetrahydrofolic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115980248A true CN115980248A (en) | 2023-04-18 |
Family
ID=85970359
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310112697.0A Pending CN115980248A (en) | 2023-02-14 | 2023-02-14 | Reagent set for detecting methotrexate, folic acid, 5-formyltetrahydrofolic acid and 5-methyltetrahydrofolic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115980248A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116223693A (en) * | 2023-05-06 | 2023-06-06 | 南京品生医学检验实验室有限公司 | Method for measuring folic acid and metabolite thereof in erythrocytes by high performance liquid chromatography tandem mass spectrometry |
| CN116718710A (en) * | 2023-08-09 | 2023-09-08 | 江苏硕世生物科技股份有限公司 | Kit and method for detecting folic acid and metabolite thereof |
| CN117665183A (en) * | 2023-12-07 | 2024-03-08 | 大连博源医学科技有限公司 | Preparation method of folic acid substance freeze-dried standard substance and stabilizing composition |
-
2023
- 2023-02-14 CN CN202310112697.0A patent/CN115980248A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116223693A (en) * | 2023-05-06 | 2023-06-06 | 南京品生医学检验实验室有限公司 | Method for measuring folic acid and metabolite thereof in erythrocytes by high performance liquid chromatography tandem mass spectrometry |
| CN116223693B (en) * | 2023-05-06 | 2023-08-18 | 南京品生医学检验实验室有限公司 | Method for measuring folic acid and metabolite thereof in erythrocytes by high performance liquid chromatography tandem mass spectrometry |
| CN116718710A (en) * | 2023-08-09 | 2023-09-08 | 江苏硕世生物科技股份有限公司 | Kit and method for detecting folic acid and metabolite thereof |
| CN117665183A (en) * | 2023-12-07 | 2024-03-08 | 大连博源医学科技有限公司 | Preparation method of folic acid substance freeze-dried standard substance and stabilizing composition |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115980248A (en) | Reagent set for detecting methotrexate, folic acid, 5-formyltetrahydrofolic acid and 5-methyltetrahydrofolic acid | |
| Rafii et al. | High-throughput and simultaneous measurement of homocysteine and cysteine in human plasma and urine by liquid chromatography–electrospray tandem mass spectrometry | |
| Schofield et al. | Development and validation of a turbulent flow chromatography and tandem mass spectrometry method for the quantitation of methotrexate and its metabolites 7-hydroxy methotrexate and DAMPA in serum | |
| Alam et al. | Measurement of homocysteine: a historical perspective | |
| CN106770819B (en) | A kind of method of folic acid concentration in LC-MS quantitative detection rat plasma | |
| CN113341012B (en) | Method and kit for simultaneously detecting multiple metabolites on homocysteine metabolic pathway and application of kit | |
| CN112505179B (en) | Method for measuring isotope dilution ultra-performance liquid chromatography-mass spectrometry combination | |
| Nelson et al. | Affinity extraction combined with stable isotope dilution LC/MS for the determination of 5-methyltetrahydrofolate in human plasma | |
| CN111551654B (en) | Method for detecting cefixime polymer impurities | |
| CN108072712B (en) | Quantitative analysis method for blood concentration of new compound WSJ-557 in SD rat plasma | |
| Guo et al. | A rapid, sensitive, and widely applicable method for quantitative analysis of underivatized amino acids in different biological matrices by UHPLC‐MS/MS | |
| CN111812220A (en) | Method for detecting concentration of antitumor drug in blood plasma | |
| CN113125600B (en) | Method for simultaneously determining concentration of 3 vitamin B12 in serum | |
| RU2702998C1 (en) | Method for monitoring the content of anti-tuberculosis preparations of the main line and their toxic metabolites in blood plasma | |
| van Haandel et al. | Comprehensive quantitative measurement of folate polyglutamates in human erythrocytes by ion pairing ultra‐performance liquid chromatography/tandem mass spectrometry | |
| CN116087373B (en) | Detection method and pretreatment method for folic acid and 5-methyltetrahydrofolate in erythrocytes | |
| Rao et al. | Development of a validated LC-MS/MS method for determination of doxofylline on rat dried blood spots and urine: application to pharmacokinetics | |
| CN110806458A (en) | Method for simultaneously detecting leucine, isoleucine and valine contents in blood | |
| Sun et al. | A rapid and sensitive HPLC-MS/MS method for determination of endogenous creatine biosynthesis precursors in plasma of children with viral myocarditis | |
| CN114994213A (en) | Kit and method for determining blood concentration of anti-tumor drug tyrosine kinase inhibition in human plasma | |
| CN113030312B (en) | Method for simultaneously determining concentration of anticoagulant drug and active metabolite in blood plasma | |
| CN112362765B (en) | Solid-phase extraction and detection method and kit for doxepin and metabolite N-nor doxepin thereof | |
| CN112611814B (en) | Method for determining 1, 5-anhydroglucitol in dried blood slices | |
| CN114264765B (en) | Analytical method for determining related substances in glimepiride intermediate by utilizing HPLC | |
| Xu et al. | A rapid and sensitive method for the quantification of ganciclovir in plasma using liquid chromatography/selected reaction monitoring/mass spectrometry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |