CN111912920A - Method for detecting mycophenolic acid and metabolites thereof in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology - Google Patents
Method for detecting mycophenolic acid and metabolites thereof in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology Download PDFInfo
- Publication number
- CN111912920A CN111912920A CN202010703537.XA CN202010703537A CN111912920A CN 111912920 A CN111912920 A CN 111912920A CN 202010703537 A CN202010703537 A CN 202010703537A CN 111912920 A CN111912920 A CN 111912920A
- Authority
- CN
- China
- Prior art keywords
- plasma
- metabolites
- mycophenolic acid
- standard substance
- liquid chromatography
- 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.)
- Withdrawn
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
A method for detecting mycophenolic acid and metabolites thereof in plasma by an ultra performance liquid chromatography tandem mass spectrometry technology comprises the following steps: MPA and metabolites of MPA, MPAG and MPAAG; detecting the contents of mycophenolic acid and metabolites thereof in the pretreated blood plasma by adopting an ultra-high performance liquid chromatography tandem mass spectrometry, firstly separating a target object to be detected from interfering components in a blood plasma matrix by utilizing the ultra-high performance liquid chromatography, then quantifying by utilizing a mass spectrometry isotope internal standard method, establishing a calibration curve by taking the concentration ratio of a standard substance and an internal standard substance as an X axis and the peak area ratio of the standard substance and the internal standard substance as a Y axis, and calculating the content of the object to be detected; the method has the advantages of high sensitivity, strong specificity, accuracy and simple pretreatment process, completes the separation and detection of the mycophenolic acid and the metabolites thereof in the plasma within 3.0min, basically meets the requirements on matrix effect and precision, can be used for the quantitative analysis of the mycophenolic acid and the metabolites thereof in the plasma in clinic, and provides a simple and rapid detection method for the monitoring of the mycophenolic acid and the metabolites thereof in the clinic.
Description
Technical Field
The invention belongs to the technical field of blood detection, and particularly relates to a method for detecting mycophenolic acid and metabolites thereof in plasma by an ultra-high performance liquid chromatography tandem mass spectrometry technology.
Background
Mycophenolic acid (MPA) is an active metabolite of an immunosuppressant, Mycophenolate Mofetil (MMF), in vivo, and is clinically used for preventing immune rejection after organ transplantation and treating refractory rejection after allogeneic kidney transplantation. Mycophenolic acid can generate Mycophenolic acid glucoside (MPAG) through in vivo metabolism, MPGA has no pharmacological activity, MPA can be regenerated through intestinal circulation and enters the body after being absorbed again to play a role again, MPAG can influence the protein binding rate of MPA, Mycophenolic acid acyl-beta-glucoside (MPAAG) can be generated through MPA metabolism, and MPAAG is related to certain toxic and side effects of MPA, so that the simultaneous determination of MPA, MPAG and MPAAG is beneficial to better mastering the pharmacokinetic characteristics of MPA, and is beneficial to improving the curative effect and reducing adverse reactions.
At present, the blood concentration determination methods of mycophenolic acid reported at home and abroad mainly adopt high performance liquid chromatography and enzyme-linked immunosorbent assay, but the methods are often poor in specificity, low in sensitivity, large in matrix interference, long in detection time and difficult to popularize and apply.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for detecting mycophenolic acid and metabolites thereof in plasma by an ultra-high performance liquid chromatography tandem mass spectrometry technology, wherein the substances to be detected are respectively as follows: MPA and metabolites of MPA, MPAG and MPAAG; the isotope internal standard substance corresponding to the substance to be detected is MPA-d 3;
detecting contents of mycophenolic acid and metabolites thereof in pretreated blood plasma by adopting an ultra-high performance liquid chromatography tandem mass spectrometry, firstly separating target substances to be detected from interfering components in a blood plasma matrix by utilizing the ultra-high performance liquid chromatography, then quantifying by utilizing a mass spectrometry isotope internal standard method, establishing a calibration curve by taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of the substances to be detected;
(1) chromatographic conditions are as follows:
mobile phase A: water (containing 0.01-0.5% formic acid, 1-10mM ammonium formate);
mobile phase B: methanol (containing 0.01-0.5% formic acid, 1-10mM ammonium formate);
a chromatographic column: ACQUITY UPLC BEH C18(2.1×50mm,1.7μm)；
Gradient elution is carried out by adopting a mobile phase A and a mobile phase B as a mixed mobile phase, and the gradient elution is shown in a table 1;
the flow rate is 0.2-1 mL/min, the column temperature is 45-65 ℃, and the sample injection volume is 0.5-5 mu L;
TABLE 1 mobile phase gradient elution parameters
(2) Mass spectrum conditions: performing positive ion scanning in an electrospray ionization (ESI) mode by using Multiple Reaction Monitoring (MRM); the spray voltage was 1.0kV (ESI +); source temperature: 150 ℃; temperature of atomized gas: 400 ℃, atomizing gas flow rate: 800L/h, taper hole air flow rate: 150L/h; simultaneously monitoring a standard substance and internal standard parent ions, ionic ions, cluster removing voltage and collision voltage corresponding to a target object, wherein the parameters are shown in a table 2;
TABLE 2 Mass spectrometric parameters
Wherein the blood plasma is human or animal blood plasma.
Wherein the pretreated blood plasma is prepared according to the following method: putting 20 mu L of plasma into a 1.5mL centrifuge tube, adding 780 mu L of protein precipitator containing an internal standard into the centrifuge tube, and oscillating at high speed for 5 min; 14000r/min, and centrifuging for 5min at 15 ℃; transfer 70. mu.L of supernatant from the EP tube to a plastic lined tube for injection.
The standard substance is prepared according to the following steps:
weighing each standard substance to be detected, dissolving the standard substance in 50% methanol to prepare a mother solution of the standard substance, wherein the concentrations are as follows: MPA 6mg/mL, MPAG 12mg/mL, MPAAG 3mg/mL, then respectively transferring MPA50 mu L, MPAG 250 mu L and MPAAG 10 mu L, then adding 690 mu L methanol to obtain 1mL mixed standard stock solution;
preparing the mixed standard substance stock solution into six calibrator solutions with different concentration points by using a blank plasma matrix, and adding 10 mu L of the mixed standard substance stock solution into 190 mu L of the blank plasma matrix solution to be used as a first high-value concentration point (S6); diluting the first high-value concentration point (S6) with 2 times volume of blank plasma matrix solution to obtain a second high-value concentration point (S5); diluting the first high-value concentration point (S6) with 9 times volume of blank plasma matrix solution to obtain a third high-value concentration point (S4); diluting the second high-value concentration point (S5) with 9 times volume of blank plasma matrix solution to obtain a fourth high-value concentration point (S3); diluting the third high-value concentration point (S4) with 4 times volume of blank plasma matrix solution to obtain a fifth high-value concentration point (S2); diluting the fourth high concentration point (S3) with 4 times volume of blank plasma matrix solution to obtain a sixth high concentration point (S1);
the six concentration points of the standard solution are:
MPA：50ng/mL、150ng/mL、500ng/mL、1500ng/mL、5000ng/mL、15000 ng/mL；
MPAG：500ng/mL、1500ng/mL、5000ng/mL、15000ng/mL、50000ng/mL、 150000ng/mL；
MPAAG：5ng/mL、15ng/mL、50ng/mL、150ng/mL、500ng/mL、1500ng/mL。
wherein, the protein precipitant containing the internal standard is prepared by the following method: preparing MPA-d 31 mg/mL by using methanol, then transferring MPA-d 310. mu.L, adding 990. mu.L of methanol to obtain 1mL of mixed internal standard solution, and adding 20. mu.L of mixed internal standard solution to 19.98mL of protein precipitant to obtain protein precipitant containing internal standard.
Wherein the blank plasma matrix is blank plasma without mycophenolic acid and metabolites thereof.
Wherein, the protein precipitator is methanol.
Has the advantages that: the method has the advantages of high sensitivity, strong specificity, accuracy and simple pretreatment process, completes the separation and detection of the mycophenolic acid and the metabolites thereof in the plasma within 3.0min, basically meets the requirements on matrix effect and precision, can be used for the quantitative analysis of the mycophenolic acid and the metabolites thereof in the plasma in clinic, and provides a simple and rapid detection method for the monitoring of the mycophenolic acid and the metabolites thereof in the clinic.
Drawings
FIG. 1 is a chromatogram of ion current extracted from a standard mycophenolic acid and its metabolites;
FIG. 2 is a graph of ion flux of mycophenolic acid and its metabolites in plasma.
Detailed Description
For the purpose of enhancing the understanding of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.
Example 1
1. Material
Methodology samples from experiments were obtained from plasma samples collected from the heart disease hospital wuhan asia in 2019 in the outpatient clinic of 4 months.
(1) The instrument comprises the following steps: xevo TQ-S triple quadrupole mass spectrometer (Waters Corporation); UPLC I-Class ultra high performance liquid chromatography system (with autosampler, Waters Corporation); SCILOGEX D2012 high speed bench top centrifuge (usa); ultra pure water meter (ELGA LabWater, uk); multi-tube Vortex mixer (Vortex genie2, usa); an adjustable pipettor (Eppendorf 0.5-10 muL, 10-100 muL, 100-1000 muL); glassware, graduated cylinders, etc.;
(2) reagent consumables: MS grade methanol (Fisher, usa); HPLC grade methanol (Honeywell, usa); MS grade formic acid (Fisher, usa); MS grade ammonium formate (Sigma, usa); chromatography column Waters BEH C18,1.7 μm,2.1x50mm (Waters Corporation);
(3) and (3) standard substance: MPA from TCI, MPAG from Isoreag, MPAAG from Glycosci, MPA-d3 from TRC;
(4) quality control product: blank plasma matrix solution containing mycophenolic acid and metabolites thereof is divided into low, medium and high concentrations, namely QC (L), QC (M) and QC (H), and is shown in Table 3;
TABLE 3 concentration of quality control (unit: ng/mL)
2. Method of producing a composite material
(1) Chromatographic conditions are as follows: mobile phase A: water (0.1% formic acid, 4mM ammonium formate); mobile phase B: methanol (containing 0.1% formic acid, 4mM ammonium formate); a chromatographic column: ACQUITY UPLC BEH C18 (2.1X 50mm, 1.7 μm); gradient elution is carried out by adopting a mobile phase A and a mobile phase B as a mixed mobile phase, and the gradient elution is shown in a table 1; the flow rate is 0.6mL/min, the column temperature is 60 ℃, and the sample injection volume is 1 mu L;
(2) mass spectrum conditions: performing positive ion scanning in an electrospray ionization (ESI) mode by using Multiple Reaction Monitoring (MRM); the spray voltage was 1.0kV (ESI +); source temperature: 150 ℃; temperature of atomized gas: 400 ℃, atomizing gas flow rate: 800L/h, taper hole air flow rate: 150L/h; simultaneously monitoring a standard substance and internal standard parent ions, ionic ions, cluster removing voltage and collision voltage corresponding to a target object, wherein the parameters are shown in a table 2;
(3) preparation of mixed standard stock solution
Weighing each standard substance to be detected, dissolving the standard substance in 50% methanol to prepare a mother solution of the standard substance, wherein the concentrations are as follows: MPA 6mg/mL, MPAG 12mg/mL, MPAAG 3mg/mL, then respectively transferring MPA50 mu L, MPAG 250 mu L and MPAAG 10 mu L, then adding 690 mu L methanol to obtain 1mL mixed standard stock solution, the preparation method is shown in Table 4;
table 4 preparation of stock solutions for mixed standards
(4) Preparation of protein precipitant containing internal standard
Preparing MPA-d 31 mg/mL by using methanol, then transferring MPA-d 310. mu.L, adding 990. mu.L of methanol to obtain 1mL of mixed internal standard solution, and adding 20. mu.L of mixed internal standard solution to 19.98mL of protein precipitant to obtain protein precipitant containing internal standard.
(5) Preparation of quality control product
QC (L): diluting QC (M) quality control products by 10 times with blank plasma matrix;
QC (M): diluting the mixed standard stock solution by 500 times with a blank plasma matrix;
QC (H): the mixed standard stock solution was diluted 50-fold with a blank plasma matrix.
(6) Sample processing
1) Preparation of standard curve
Preparing a standard curve by adopting a gradient dilution method, and preparing the mixed standard substance stock solution into calibrator solutions with six different concentration points by using a blank plasma matrix, wherein the preparation process comprises the following steps:
adding 10 μ L of the mixed standard stock solution into 190 μ L of the blank plasma matrix solution as a first high-value concentration point (S6); diluting the first high-value concentration point (S6) with 2 times volume of blank plasma matrix solution to obtain a second high-value concentration point (S5); diluting the first high-value concentration point (S6) with 9 times volume of blank plasma matrix solution to obtain a third high-value concentration point (S4); diluting the second high-value concentration point (S5) with 9 times volume of blank plasma matrix solution to obtain a fourth high-value concentration point (S3); diluting the third high-value concentration point (S4) with 4 times volume of blank plasma matrix solution to obtain a fifth high-value concentration point (S2); diluting the fourth high concentration point (S3) with 4 times volume of blank plasma matrix solution to obtain a sixth high concentration point (S1), the preparation method is shown in Table 5;
TABLE 5 Standard Curve preparation and concentration (unit: ng/mL)
2) Plasma sample pretreatment
Putting 20 mu L of plasma sample into a 1.5mL centrifuge tube, adding 780 mu L of protein precipitant containing internal standard, then oscillating at high speed for 5min, and centrifuging at 15 ℃ for 5min under 14000 r/min; transfer 70. mu.L of supernatant from the EP tube to a plastic lined tube for injection.
3) Pretreatment of quality control product
20 μ L of each of the quality control solutions QC (L), QC (M), QC (H) were collected and placed in 1.5mL centrifuge tubes, which were followed by the plasma sample pretreatment method, and the details thereof are omitted here.
3. Method verification
1) Extracting an ion flow spectrogram: as can be seen from FIGS. 1-2, the peak shapes of the mycophenolic acid and its metabolites are relatively symmetrical and there is no interference of the peaks, which indicates that the good detection can be obtained under the conditions;
2) calibration curve: establishing a calibration curve by adopting an isotope internal standard quantitative method and utilizing TargetLynx software to calculate the concentration of the substance to be detected in the plasma by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis; the linear fitting equation of mycophenolic acid and metabolites thereof in respective concentration ranges has good linearity, the correlation coefficient is more than 0.999, and the quantitative requirements are met, which is shown in Table 6.
TABLE 6 Linear regression equation and linear correlation coefficient for mycophenolic acid and its metabolites
3) Accuracy survey: and evaluating the accuracy of the method by adopting a standard recovery rate test. A blank plasma sample is prepared, mixed standard substances with low, medium and high concentrations are respectively added, the processing and the measurement are repeated for 5 times by the same steps, the result shows that the standard addition recovery rate of the mycophenolic acid and the mycophenolate mofetil metabolites ranges from 98.05% to 112.39%, the RSD of 5 repeated tests ranges from 0.48% to 5.01%, and the statistical result is shown in the table 7.
TABLE 7 mycophenolic acid and metabolite addition recovery results
4) And (3) precision test: taking an interference-free blank plasma sample, adding mycophenolic acid and metabolite standards with different concentrations to obtain plasma samples with low, medium and high concentrations, repeatedly processing 6 batches in one day for three days continuously, quantitatively measuring the concentrations of the mycophenolic acid and the metabolite thereof by using an isotope internal standard method, wherein the batch precision is 0.53-4.65%, processing 3 batches in three days, and calculating the batch precision to be 1.67-4.72%, and the results are shown in Table 8.
TABLE 8 results of precision test between batches (unit: ng/mL)
4. Discussion of the related Art
The concentrations of mycophenolic acid and its metabolites in human plasma were determined by the ID-UPLC-MS/MS method. Meanwhile, the detection is carried out according to the peak-out time and the ion pair of the target object, the sensitivity is high, meanwhile, the matrix interference can be greatly eliminated by adopting the isotope internal standard method for quantification, and the accurate quantification can be achieved without being influenced by the conditions of pretreatment process, sample loading volume and flow and the like; the accuracy of the method is evaluated by a standard recovery test, and the result shows that the standard recovery of the mycophenolic acid and the mycophenolate mofetil metabolite is between 98.05% and 112.39%, the RSD of 5 times of repeated tests is in the range of 0.48% to 5.01%, and the accuracy is good.
The reproducibility result of the method shows that the internal precision of the mycophenolic acid and the metabolite thereof is 0.53-4.65%, the three-day internal 3-batch treatment is carried out, and the calculated batch precision is 1.67-4.72%; in the experiment, in order to obtain a target object signal which is more stable and has high sensitivity, the types and concentrations of different mobile phases and electrolytes are considered, the baseline separation of compound and matrix interference is realized as far as possible, the established pretreatment process of a plasma sample is very simple, the protein precipitation is completed in one step, and the dosage of the plasma is only 20 mu L.
In a word, the method has the advantages of high sensitivity, strong specificity, accuracy and simpler pretreatment process, can complete the separation and detection of the compound within 3min, meets the requirements on matrix effect, extraction recovery rate and precision, can be used for the quantitative analysis of the mycophenolic acid in plasma and the metabolites thereof in clinic, and provides a reliable detection method for the treatment and monitoring of the mycophenolic acid and the metabolites thereof in clinic.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (7)
1. A method for detecting mycophenolic acid and metabolites thereof in plasma by an ultra performance liquid chromatography tandem mass spectrometry technology is characterized in that the substances to be detected are respectively as follows: MPA and metabolites of MPA, MPAG and MPAAG;
detecting the contents of mycophenolic acid and metabolites thereof in the pretreated blood plasma by adopting an ultra-high performance liquid chromatography tandem mass spectrometry, firstly separating a target object to be detected from interfering components in a blood plasma matrix by utilizing the ultra-high performance liquid chromatography, then quantifying by utilizing a mass spectrometry isotope internal standard method, establishing a calibration curve by taking the concentration ratio of a standard substance and an internal standard substance as an X axis and the peak area ratio of the standard substance and the internal standard substance as a Y axis, and calculating the content of the object to be detected;
(1) chromatographic conditions are as follows:
mobile phase A: water (containing 0.01-0.5% formic acid, 1-10mM ammonium formate);
mobile phase B: methanol (containing 0.01-0.5% formic acid, 1-10mM ammonium formate);
a chromatographic column: ACQUITYUPLC BEH C18(2.1×50mm,1.7μm)；
Gradient elution is carried out by adopting a mobile phase A and a mobile phase B as a mixed mobile phase, and the gradient elution is shown in a table 1;
the flow rate is 0.2-1 mL/min, the column temperature is 45-65 ℃, and the sample injection volume is 0.5-5 mu L;
TABLE 1 mobile phase gradient elution parameters
(2) Mass spectrum conditions: performing positive ion scanning in an electrospray ionization (ESI) mode by using Multiple Reaction Monitoring (MRM); the spray voltage was 1.0kV (ESI +); source temperature: 150 ℃; temperature of atomized gas: 400 ℃, atomizing gas flow rate: 800L/h, taper hole air flow rate: 150L/h; simultaneously monitoring a standard substance and internal standard parent ions, ionic ions, cluster removing voltage and collision voltage corresponding to a target object, wherein the parameters are shown in a table 2;
TABLE 2 Mass spectrometric parameters
2. The method for detecting mycophenolic acid and its metabolites in plasma according to the ultra performance liquid chromatography tandem mass spectrometry technology of claim 1, wherein the plasma is human or animal plasma.
3. The method for detecting mycophenolic acid and its metabolites in plasma by ultra performance liquid chromatography tandem mass spectrometry as claimed in claim 1, wherein said pre-treated plasma is prepared by the following method: putting 20 mu L of plasma into a 1.5mL centrifuge tube, adding 780 mu L of protein precipitator containing an internal standard into the centrifuge tube, and oscillating at high speed for 5 min; 14000r/min, and centrifuging for 5min at 15 ℃; transfer 70. mu.L of supernatant from the EP tube to a plastic lined tube for injection.
4. The method for detecting mycophenolic acid and its metabolites in plasma by ultra performance liquid chromatography tandem mass spectrometry as claimed in claim 1, wherein said standard is prepared by the following steps: weighing each standard substance to be detected, dissolving the standard substance in 50% methanol to prepare a mother solution of the standard substance, wherein the concentrations are as follows: MPA 6mg/mL, MPAG 12mg/mL, MPAAG 3mg/mL, then respectively transferring MPA50 mu L, MPAG 250 mu L and MPAAG 10 mu L, then adding 690 mu L methanol to obtain 1mL mixed standard stock solution;
preparing the mixed standard substance stock solution into six calibrator solutions with different concentration points by using a blank plasma matrix, and adding 10 mu L of the mixed standard substance stock solution into 190 mu L of the blank plasma matrix solution to be used as a first high-value concentration point (S6); diluting the first high-value concentration point (S6) with 2 times volume of blank plasma matrix solution to obtain a second high-value concentration point (S5); diluting the first high-value concentration point (S6) with 9 times volume of blank plasma matrix solution to obtain a third high-value concentration point (S4); diluting the second high-value concentration point (S5) with 9 times volume of blank plasma matrix solution to obtain a fourth high-value concentration point (S3); diluting the third high-value concentration point (S4) with 4 times volume of blank plasma matrix solution to obtain a fifth high-value concentration point (S2); the fourth high concentration point (S3) was diluted with 4 volumes of blank plasma matrix solution to obtain a sixth high concentration point (S1).
5. The method for detecting mycophenolic acid and its metabolites in plasma by ultra performance liquid chromatography tandem mass spectrometry as claimed in claim 3, wherein said protein precipitant containing internal standard is prepared by the following method: preparing MPA-d 31 mg/mL by using methanol, then transferring MPA-d 310. mu.L, adding 990. mu.L of methanol to obtain 1mL of mixed internal standard solution, and adding 20. mu.L of mixed internal standard solution to 19.98mL of protein precipitant to obtain protein precipitant containing internal standard.
6. The method of claim 4, wherein the blank plasma matrix is blank plasma without mycophenolic acid and its metabolites.
7. The method of claim 5, wherein the protein precipitating agent is methanol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010703537.XA CN111912920A (en) | 2020-07-21 | 2020-07-21 | Method for detecting mycophenolic acid and metabolites thereof in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010703537.XA CN111912920A (en) | 2020-07-21 | 2020-07-21 | Method for detecting mycophenolic acid and metabolites thereof in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111912920A true CN111912920A (en) | 2020-11-10 |
Family
ID=73281490
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010703537.XA Withdrawn CN111912920A (en) | 2020-07-21 | 2020-07-21 | Method for detecting mycophenolic acid and metabolites thereof in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111912920A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113686984A (en) * | 2021-08-06 | 2021-11-23 | 珠海市人民医院 | Method for detecting level of alkylresorcinol metabolites in plasma by LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) |
| CN114577949A (en) * | 2022-03-15 | 2022-06-03 | 北京华氏精恒医药科技有限公司 | Method and kit for detecting 25-hydroxycholesterol based on LC-MS/MS method and application thereof |
-
2020
- 2020-07-21 CN CN202010703537.XA patent/CN111912920A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113686984A (en) * | 2021-08-06 | 2021-11-23 | 珠海市人民医院 | Method for detecting level of alkylresorcinol metabolites in plasma by LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) |
| CN114577949A (en) * | 2022-03-15 | 2022-06-03 | 北京华氏精恒医药科技有限公司 | Method and kit for detecting 25-hydroxycholesterol based on LC-MS/MS method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110542735A (en) | method for high-throughput determination of multiple fat-soluble vitamins by ultra-high performance liquid mass spectrometry | |
| CN111537648A (en) | Kit for detecting anti-tuberculosis drugs in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology | |
| CN112505179B (en) | Method for measuring isotope dilution ultra-performance liquid chromatography-mass spectrometry combination | |
| CN111766311A (en) | Method for detecting anti-tuberculosis drugs in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology | |
| CN112684075A (en) | Method for determining plasma protein binding rate of meropenem or imipenem by liquid chromatography-mass spectrometry combined ultrafiltration | |
| CN111912920A (en) | Method for detecting mycophenolic acid and metabolites thereof in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology | |
| CN111896647A (en) | Kit for detecting 21 organic acids in urine by ultra-high performance liquid chromatography tandem mass spectrometry technology | |
| CN111830153A (en) | Method for detecting concentrations of polymyxin B1and polymyxin B2 in serum | |
| CN113588804A (en) | Kit for detecting concentration of 5-hydroxytryptamine and melatonin in serum | |
| CN112730706A (en) | Method for detecting biological small molecule marker by liquid chromatography-tandem mass spectrometry | |
| CN112666273A (en) | Method for detecting concentration of methotrexate substances in erythrocytes | |
| CN115902048A (en) | Method for detecting water-soluble vitamins in serum by methyl derivatization-high performance liquid chromatography tandem mass spectrometry | |
| CN110133169A (en) | A kind of method and application using frusemide in LC-MS detection human plasma | |
| CN113341027A (en) | Method and kit for detecting testosterone in saliva by high performance liquid chromatography tandem mass spectrometry | |
| CN113607854A (en) | Method and detection kit for simultaneously detecting multiple vitamins | |
| CN111896645A (en) | Kit for detecting mycophenolic acid and metabolites thereof in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology | |
| CN111812224A (en) | Method for detecting concentration of anti-dementia drug in serum | |
| CN111812222A (en) | Method for detecting concentration of antidepressant drug in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology | |
| CN111812219A (en) | Method for detecting concentration of anticoagulant drug in blood plasma | |
| CN111413439A (en) | Method for determining metformin in blood plasma by rapid hydrophilic interaction chromatography-tandem mass spectrometry | |
| CN111665307A (en) | Kit for detecting concentrations of polymyxin B1and polymyxin B2 in serum | |
| CN111679003A (en) | Kit for detecting anti-dementia drugs in serum | |
| CN111830162A (en) | Method for detecting concentration of nucleoside antiviral drug in serum | |
| CN112213417A (en) | Kit and method for detecting concentration of mycophenolic acid medicine in dried blood spots | |
| CN112485340A (en) | Method for detecting 1, 5-sorbitan in plasma by ultra-high performance liquid chromatography tandem 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 | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20201110 |
|
| WW01 | Invention patent application withdrawn after publication |