CN112630340A - Detection method for determining mycophenolate mofetil concentration in human plasma by LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) - Google Patents
Detection method for determining mycophenolate mofetil concentration in human plasma by LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) Download PDFInfo
- Publication number
- CN112630340A CN112630340A CN202011509321.6A CN202011509321A CN112630340A CN 112630340 A CN112630340 A CN 112630340A CN 202011509321 A CN202011509321 A CN 202011509321A CN 112630340 A CN112630340 A CN 112630340A
- Authority
- CN
- China
- Prior art keywords
- mycophenolate mofetil
- plasma sample
- mass spectrometry
- detected
- sample
- 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
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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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
The invention belongs to the technical field of drug analysis, relates to an in-vivo drug analysis and determination method, and particularly relates to a detection method for determining mycophenolate mofetil concentration in human plasma by LC-MS/MS. The invention discloses a detection method for determining mycophenolate mofetil concentration in human plasma by LC-MS/MS, which comprises the following steps: pretreating a plasma sample to be detected; preparing a standard curve by liquid chromatography-mass spectrometry detection; and (3) detecting the concentration of mycophenolate mofetil in the plasma sample to be detected by liquid chromatography-mass spectrometry. The method is rapid, accurate, high in sensitivity and simple in pretreatment, and solves the problem of stability of mycophenolate mofetil in a plasma sample.
Description
Technical Field
The invention belongs to the technical field of drug analysis, relates to an in-vivo drug analysis and determination method, and particularly relates to a detection method for determining mycophenolate mofetil concentration in human plasma by LC-MS/MS.
Background
Mycophenolate Mofetil (MMF) is an immunosuppressive drug, is rapidly metabolized in vivo to di-morphine-acrylic acid (mycophenolic acid, MPA), is an inosinic acid dehydrogenase (IMPDH) inhibitor, can specifically inhibit the activity of inosinic acid dehydrogenized from inosine in the de novo synthesis pathway of lymphocyte purine, can selectively inhibit T, B lymphocyte proliferation, and thus has a strong function of inhibiting lymphocyte proliferation.
With the continuous and intensive research on MMF, the medicine is widely used for treating non-transplantation diseases such as systemic lupus erythematosus, immune nephropathy, rheumatoid arthritis and the like. MMF is metabolized into mycophenolic acid in vivo, the average oral bioavailability of MMF is 94%, the plasma clearance rate in healthy volunteers is 10L/min, and the plasma concentration is reduced to 0.4mg/L after administration for 10 min. MPA is metabolized into phenol glucuronide (MPAG) by the liver to lose activity, and the biological half-life is 17.9 h. MPAG is approximately 94% excreted by the renal tubules, 6% excreted from the feces, and very little (< l%) excreted as is from the urine.
Mycophenolate mofetil is extremely unstable in plasma and has low concentration, and at present, no literature report about a method capable of effectively detecting the concentration of mycophenolate mofetil in plasma exists. Therefore, a detection method for determining mycophenolate mofetil concentration in human plasma, which is simple to operate, is urgently needed.
Disclosure of Invention
The invention aims to provide a detection method for determining mycophenolate mofetil concentration in human plasma, which is simple to operate.
The invention discloses a detection method for determining mycophenolate mofetil concentration in human plasma by LC-MS/MS, which comprises the following steps:
(1) pretreating a plasma sample to be detected;
(2) preparing a standard curve by liquid chromatography-mass spectrometry detection;
(3) detecting the concentration of mycophenolate mofetil in a blood plasma sample to be detected by liquid chromatography-mass spectrometry;
the pretreatment step of the plasma sample to be detected comprises the steps of taking a certain volume of the plasma sample, adding an equal volume of deuterated internal standard solution, mixing, adding equal volume of pure water for dilution, transferring the sample into an activated Cleanert PEP-296 Wellplate, and sequentially adding H2Leaching O and 50% MeOH; and adding 100% MeOH for elution, pressing the eluent to dry by using a solid-phase extraction positive pressure device after the elution is finished, collecting all the eluent, drying the eluent under nitrogen flow, adding 50% ACN for redissolution, and mixing the mixture in a vortex manner to obtain a sample to be detected.
In a preferred embodiment, the plasma sample to be tested contains 0.2-5mg/mL of sodium fluoride.
In a preferred embodiment, the plasma sample to be tested contains an anticoagulant, such as EDTA-K2.
In a preferred embodiment, the liquid chromatography-mass spectrometry detection is as follows:
liquid chromatography determination is carried out under the conditions that: a chromatographic column: chromatography column GL Sciences inc. inertsustatin C183 μm, 2.1 × 50 mm; temperature of the chromatographic column: 40 ℃; mobile phase A: 0.1% acetic acid in water; mobile phase B: 100% ACN solution; washing liquid: 50% ACN solution and ACN/MeOH (1:1, v: v) solution; the temperature of the autosampler is 4 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10uL and analysis time of 3.5 min;
measuring mass spectrum under the conditions of: the ion source is an electrospray ion source, and the interface temperature is as follows: 300 ℃, DL temperature: 250 ℃, heating air flow: 10.00L/min, heating block temperature: 400 ℃, dryer flow: 10.00L/min; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reaction for quantitative analysis was: m/z434.20 → m/114.15.
Compared with the prior art, the method of the invention has the following advantages:
1. the method is rapid, accurate, high in sensitivity and simple in pretreatment method, the LC-MS/MS is adopted for detecting the mycophenolate mofetil in the blood plasma for the first time, and no relevant method literature reports exist at home and abroad;
2. the stability problem of mycophenolate mofetil in a plasma sample is solved by adding sodium fluoride into the plasma;
3. the method has strong applicability: the plasma sample is extracted by adopting a commercial SPE method, the required sample amount is small, and the method is suitable for measuring a large amount of samples;
4. the specificity is strong: using GL Sciences inc. inertssusin C183 μm, 2.1 × 50 mm chromatography column; aqueous phase (0.1% acetic acid in water) and 100% ACN solution as mobile phase; gradient elution with mycophenolate mofetil retention time of 2.0min
5. The sensitivity is high: the minimum quantitative limit of mycophenolate mofetil is: 10.0 pg/mL.
Drawings
FIG. 1 is a mycophenolate mofetil and mycophenolate mofetil-d 4 ion chromatogram;
FIG. 2 is a mass spectrum of human blank plasma;
FIG. 3 is a mass spectrum of human blank plasma after the addition of standard.
FIG. 4 is a graph of a linear regression equation for mycophenolate mofetil;
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Mycophenolate mofetil and deuterated internal standard mycophenolate mofetil-d 4, both provided by TRC, Canada. Acetonitrile, methanol, acetic acid, etc. were all HPLC grade from Fisher, usa, and other chemicals were analytically pure. SPE 96well plates, Cleanert PEP-2 SPE 96Wellplate, 30mg, from Agela Technologies.
CMS-8060 mass spectrometer (Shimadzu, Japan), Lab Solutions 6.84 SP1 data acquisition software (Shimadzu, Japan), Mettler XS 105DU electronic balance (Mettler, Switzerland); eppendorf Centrifuge 5424R high speed low temperature Centrifuge (Eppendorf, Germany).
Example 1 preparation of stock solutions and working solutions
Appropriate amount of mycophenolate mofetil and deuterated internal standard mycophenolate mofetil-d 4 are weighed, and a certain amount of DMSO is added to prepare stock solutions with the content of 1.00 mg/mL. Working solutions for standard curve samples and quality control samples were prepared by serially diluting separate stock solutions with 50% MeOH solutions at room temperature. All working solutions were formulated in clear polypropylene tubes and stored in a-20 ℃ freezer refrigerator.
Example 2, plasma sample pretreatment:
taking 100.0uL of plasma sample by taking sodium fluoride and EDTA-K2 as an anticoagulant and a deuterated compound as an internal standard, adding 50.0uL of deuterated internal standard solution, mixing, adding 200uL of pure water for dilution, transferring all samples to an activated Cleanert PEP-296 Wellplate, and sequentially adding 1000 muL of 100% H2O and 1000 muL of 50% MeOH for leaching; adding 1000 mu L of 100% MeOH for elution, pressing the eluent to dry by a solid phase extraction positive pressure device after the elution is finished, collecting all the eluent, drying the eluent under nitrogen flow, adding 100.0 mu L of 50% ACN for redissolution, and mixing for 10min by vortex to obtain a sample to be detected.
Example 3, detection by liquid chromatography-mass spectrometry:
liquid chromatography determination is carried out under the conditions that: chromatography column GL Sciences inc. inertsustatin C183 μm, 2.1 × 50 mm; temperature of the chromatographic column: 40 ℃; mobile phase A: 0.1% acetic acid in water; mobile phase B: 100% ACN solution; washing liquid: 50% ACN solution and ACN/MeOH (1:1, v: v) solution; the temperature of the autosampler is 4 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10uL and analysis time of 3.5 min;
measuring mass spectrum under the conditions of: the ion source is an electrospray ion source, and the interface temperature is as follows: 300 ℃, DL temperature: 250 ℃, heating air flow: 10.00L/min, heating block temperature: 400 ℃, dryer flow: 10.00L/min; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reaction for quantitative analysis was: m/z434.20 → m/114.15;
example 4, standard curve preparation:
diluting the working solution by 20 times by using plasma with sodium fluoride + EDTA-K2 as an anticoagulant, taking 100.0uL of plasma sample, adding 50.0uL of deuterated internal standard solution, mixing, adding 200uL of pure water for dilution, transferring all samples to an activated Cleanert PEP-296 Wellplate, and sequentially adding 1000 muL of 100% H2O and 1000 muL of 50% MeOH for leaching; adding 1000 mu L of 100% MeOH for elution, pressing the eluent by a solid phase extraction positive pressure device after the elution is finished, collecting all the eluent, drying the eluent under nitrogen flow, adding 100.0 mu L of 50% ACN for redissolution, and mixing for 10min by vortex to obtain a standard curve sample. Measuring standard samples with different concentrations by adopting the chromatographic and mass spectrum conditions, drawing a peak area-concentration standard curve, and obtaining a linear regression equation:
mycophenolate mofetil: y =2.24180 × x +0.00313625, R = 0.9995032;
example 5 determination of mycophenolate mofetil concentration in plasma:
preparing the plasma to be detected according to the sample preparation method in the step (1), detecting the sample to be detected according to the step (2) by adopting a liquid chromatography-mass spectrometry detection method, recording the peak area corresponding to the mycophenolate mofetil and the peak area of the internal standard, substituting the peak area ratio of the mycophenolate mofetil to the internal standard into the established standard curve, and calculating to obtain the concentration of the mycophenolate mofetil in the plasma to be detected.
Example 6 and comparative example
Precision and accuracy of the method:
an appropriate amount of mycophenolate mofetil is weighed, and a certain amount of DMSO is added to prepare stock solutions with the content of 1.00 mg/mL. Working solutions of quality control samples were prepared by sequentially diluting individual stock solutions with 50% methanol solutions, respectively, at room temperature. All working solutions were formulated in clear polypropylene tubes and stored in a-20 ℃ freezer refrigerator.
Concentration of quality control working solution (ng/mL):
| compound (I) | LLOQ QC | LQC | GMQC | MQC | HQC |
| Mycophenolate mofetil | 0.200 | 0.600 | 4.00 | 32.0 | 60.0 |
Diluting the working solution by 20 times by using plasma with sodium fluoride + EDTA-K2 as an anticoagulant, adding 100.0uL of plasma sample into a 96-deep-well plate, adding 50.0uL of deuterated internal standard solution, mixing, adding 200uL of pure water for dilution, transferring all samples into an activated Cleanert PEP-296 Wellplate, and sequentially adding 1000 muL of 100% H2O and 1000 muL of 50% MeOH for leaching; adding 1000 mu L of 100% MeOH for elution, pressing the eluent by a solid phase extraction positive pressure device after the elution is finished, collecting all the eluent, drying the eluent under nitrogen flow, adding 100.0 mu L of 50% ACN for redissolution, and mixing for 10min by vortex to obtain a quality control sample.
Determining quality control samples with different concentrations by adopting the chromatographic and mass spectrum conditions to obtain peak areas corresponding to the mycophenolate mofetil and peak areas of the internal standard, and substituting the peak area ratio of the mycophenolate mofetil to the internal standard into the established standard curve to obtain the determination concentration (n = 6) of the quality control samples:
from the data in the above table it can be seen that:
the experimental results of the method show that RE% is within 15% of acceptable range, and RSD% is within 15% of acceptable range. Therefore, the method has the advantages of good repeatability, high precision and accuracy, high sensitivity and low detection limit.
The following are stability results of mycophenolate mofetil obtained by blood collection using blood collection tubes containing EDTA-K2 without sodium fluoride. If EDTA-K2 blood collection tubes without sodium fluoride are used for blood collection, the stability is not qualified.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (5)
1. A detection method for determining mycophenolate mofetil concentration in human plasma by LC-MS/MS comprises the following steps:
pretreating a plasma sample to be detected;
preparing a standard curve by liquid chromatography-mass spectrometry detection;
detecting the concentration of mycophenolate mofetil in a blood plasma sample to be detected by liquid chromatography-mass spectrometry;
the pretreatment step of the plasma sample to be detected comprises the steps of taking a certain volume of the plasma sample, adding an equal volume of deuterated internal standard solution, mixing, adding equal volume of pure water for dilution, transferring the sample into an activated Cleanert PEP-296 Wellplate, and sequentially adding H2Leaching O and 50% MeOH; and adding 100% MeOH for elution, pressing the eluent to dry by using a solid-phase extraction positive pressure device after the elution is finished, collecting all the eluent, drying the eluent under nitrogen flow, adding 50% ACN for redissolution, and mixing the mixture in a vortex manner to obtain a sample to be detected.
2. The assay of claim 1 wherein said test plasma sample contains 0.2 to 5mg/mL sodium fluoride.
3. The assay of claim 1 wherein said test plasma sample comprises an anticoagulant.
4. The assay of claim 3 wherein said anticoagulant is EDTA-K2.
5. The detection method as claimed in claim 1, wherein the pre-treatment step of the plasma sample to be detected comprises the steps of taking 100.0uL of the plasma sample, adding 100.0uL of the deuterated internal standard solution, mixing, adding 200uL of pure water for dilution, transferring all the samples to an activated Cleanert PEP-296 Wellplate, and sequentially adding 1000 uL of 100% H2Leaching O and 1000 mu L of 50% MeOH; adding 1000 mu L of 100% MeOH for elution, pressing the eluent by a solid phase extraction positive pressure device after the elution is finished, collecting all the eluent, drying the eluent under nitrogen flow, adding 100 mu L of 50% ACN for redissolution, and mixing for 10min by vortex to obtain a sample to be detected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011509321.6A CN112630340A (en) | 2020-12-18 | 2020-12-18 | Detection method for determining mycophenolate mofetil concentration in human plasma by LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011509321.6A CN112630340A (en) | 2020-12-18 | 2020-12-18 | Detection method for determining mycophenolate mofetil concentration in human plasma by LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112630340A true CN112630340A (en) | 2021-04-09 |
Family
ID=75317723
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011509321.6A Pending CN112630340A (en) | 2020-12-18 | 2020-12-18 | Detection method for determining mycophenolate mofetil concentration in human plasma by LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112630340A (en) |
-
2020
- 2020-12-18 CN CN202011509321.6A patent/CN112630340A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| RAMBABU MADDELA ET AL.: "A novel and Rapid LC–MS/MS assay for the Determination of Mycophenolate and Mycophenolic Acid in Human Plasma", 《JOURNAL OF YOUNG PHARMACISTS》 * |
| RICARDO MARTINS DUARTE BYRRO ET AL.: "Determination of Mycophenolic acid in the vitreous humor using the HPLC–ESI-MS/MS method: Application of intraocular pharmacokinetics study in rabbit eyes with ophthalmic implantable device", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 * |
| SARAH DJABAROUTI ET AL.: "Measurement of Total and Unbound Mycophenolic Acid and Its Glucuronide by LC Coupled with MS. Application to a Pharmacokinetic Study of Mycophenolate Mofetil in Patients with Autoimmune Diseases", 《CHROMATOGRAPHIA》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kataoka et al. | Simple and rapid determination of amphetamine, methamphetamine, and their methylenedioxy derivatives in urine by automated in-tube solid-phase microextraction coupled with liquid chromatography-electrospray ionization mass spectrometry | |
| Nielen et al. | Multiresidue analysis of beta-agonists in bovine and porcine urine, feed and hair using liquid chromatography electrospray ionisation tandem mass spectrometry | |
| Zou et al. | A rapid method for determination and confirmation of the thyreostats in milk and urine by matrix solid-phase dispersion and gas chromatography–mass spectrometry | |
| Nakamoto et al. | Monolithic silica spin column extraction and simultaneous derivatization of amphetamines and 3, 4-methylenedioxyamphetamines in human urine for gas chromatographic-mass spectrometric detection | |
| Zhang et al. | Simultaneous determination of thyreostatic residues in animal tissues by matrix solid-phase dispersion and gas chromatography–mass spectrometry | |
| Kuhn et al. | Sample cleanup-free determination of mycophenolic acid and its glucuronide in serum and plasma using the novel technology of ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry | |
| CN113588804B (en) | Kit for detecting concentration of 5-hydroxytryptamine and melatonin in serum | |
| CN105136957A (en) | Detection method for simultaneously measuring OXC in human plasma and metabolite MHD and MHD-G | |
| Goutier et al. | Development and application of an LC–MS/MS method for measuring the effect of (partial) agonists on cAMP accumulation in vitro | |
| CN113049719A (en) | Method and kit for detecting free testosterone | |
| Yilmaz et al. | Determination of carvedilol in human plasma by gas chromatography-mass spectrometry method | |
| Gu et al. | Determination of 5-fluorouracil in human plasma by high-performance liquid chromatography (HPLC) | |
| Chen et al. | Technologies to improve the sensitivity of existing chromatographic methods used for bioanalytical studies | |
| CN114414696B (en) | Kit and method for determining multiple estrogens in dried blood slices | |
| Kuhn et al. | Measurement of mycophenolic acid and its glucuronide using a novel rapid liquid chromatography–electrospray ionization tandem mass spectrometry assay | |
| CN111443135A (en) | Method for determining concentrations of hydrochlorothiazide, losartan and losartan 5-carboxylate in plasma by liquid chromatography-mass spectrometry | |
| Leis et al. | Synthesis of d1-N-ethyltramadol as an internal standard for the quantitative determination of tramadol in human plasma by gas chromatography–mass spectrometry | |
| Fang et al. | Detection and identification of zeranol in chicken or rabbit liver by liquid chromatography-electrospray tandem mass spectrometry | |
| CN112630340A (en) | Detection method for determining mycophenolate mofetil concentration in human plasma by LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) | |
| Wang et al. | Rapid method for multi-residue determination of avermectins in bovine liver using high-performance liquid chromatography with fluorescence detection | |
| CN111060612B (en) | Method for determining concentration of prednisone, prednisone acetate and active metabolite prednisolone in blood plasma by liquid chromatography-mass spectrometry | |
| Nishida et al. | Miniaturized sample preparation method for determination of amphetamines in urine | |
| CN112213417A (en) | Kit and method for detecting concentration of mycophenolic acid medicine in dried blood spots | |
| Han et al. | A novel reagent pre-column derivatization method for UHPLC-MS/MS to determine four α-dicarbonyl compounds in beer | |
| CN111579690A (en) | Mass spectrum detection reagent for determining mycophenolic acid content in biological sample by using mycophenolic acid-D3 as internal standard substance and using method thereof |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210409 |
|
| RJ01 | Rejection of invention patent application after publication |