CN116298002A - Detection method for quantitatively analyzing blood concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compounds in blood plasma - Google Patents

Detection method for quantitatively analyzing blood concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compounds in blood plasma Download PDF

Info

Publication number
CN116298002A
CN116298002A CN202310032708.4A CN202310032708A CN116298002A CN 116298002 A CN116298002 A CN 116298002A CN 202310032708 A CN202310032708 A CN 202310032708A CN 116298002 A CN116298002 A CN 116298002A
Authority
CN
China
Prior art keywords
deuterated
dehydrogenated
benzoylphenyl
achyranthes
plasma
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
Application number
CN202310032708.4A
Other languages
Chinese (zh)
Inventor
赵建春
陈丹
管禄诗
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ocean University of China
Qingdao Marine Biomedical Research Institute Co Ltd
Original Assignee
Ocean University of China
Qingdao Marine Biomedical Research Institute Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Ocean University of China, Qingdao Marine Biomedical Research Institute Co Ltd filed Critical Ocean University of China
Priority to CN202310032708.4A priority Critical patent/CN116298002A/en
Publication of CN116298002A publication Critical patent/CN116298002A/en
Pending legal-status Critical Current

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
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • 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
    • G01N30/60—Construction of the column
    • G01N30/6052—Construction of the column body
    • 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
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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 discloses a detection method for quantitatively analyzing the blood concentration of deuterated dehydrogenated 3-benzoyl phenyl-achyranthes compounds in blood plasma. The detection method comprises the steps of utilizing a collected deuterated dehydrogenated 3-benzoylphenyl-type achyranthes compound treated blood sample, centrifuging to obtain plasma, mixing with acetonitrile solution (containing an internal standard), and utilizing a liquid chromatography-mass spectrometry system to analyze the sample, wherein ions quantitatively analyzed are respectively deuterated dehydrogenated 3-benzoylphenyl-type achyranthes compound: m/z 338.1→310.2, internal standard IS: m/z 260.2- & gt 116.1, the collision energy of the compound is 22V, and the collision energy of the internal standard is 15V. The invention has the advantages of high sensitivity, strong specificity, simple and efficient pretreatment of samples, short analysis period and the like, and is suitable for measuring the blood concentration of deuterated dehydrogenated 3-benzoyl phenyl achyranthes compounds in blood plasma, researching pharmacokinetics and tissue distribution.

Description

Detection method for quantitatively analyzing blood concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compounds in blood plasma
Technical Field
The invention relates to the application field of pharmacokinetics and blood concentration monitoring, in particular to a detection method for quantitatively analyzing the blood concentration of deuterated dehydrogenated 3-benzoyl phenyl achyranthes compounds in blood plasma.
Background
Plinabulin, a novel vascular blocker, initiates JNK-mediated apoptosis and inhibits angiogenesis in multiple myeloma cells (Singh, AV, band, M, raje, N et al, a novel vascular disrupting agent plinabulin triggers JNK-mediated apoptosis and inhibits angiogenesis in multiple myeloma cells. Blood 2011,117 (21), 5692-700). (3Z, 6Z) -3-benzylidene-6- [ (5-tertiary butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione is a deuterium-substituted plinabulin derivative, and shows better pharmacokinetic properties and anti-tumor effects in vitro and in vivo tests, and has the following structural formula:
Figure BDA0004047893830000011
in recent years, with the wide application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods in biological sample analysis, in order to more efficiently develop pharmacokinetic studies of animals and human bodies with relevant characteristics such as absorption, distribution, metabolism, excretion, etc. of deuterated dehydrogenated 3-benzoylphenyl-achyrans compounds in vivo, it is necessary to establish a detection method which can be quantitatively analyzed, is sensitive and efficient, and has simple pretreatment.
Disclosure of Invention
The invention aims to provide a detection method for quantitatively analyzing the blood concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compounds in blood plasma. The detection method has the advantages of simple pretreatment, less plasma consumption, strong specificity, high accuracy, short analysis time, good reproducibility and good peak shape.
In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:
the invention provides a detection method for quantitatively analyzing the blood concentration of deuterated dehydrogenated 3-benzoyl phenyl-achyranthes compounds in blood plasma, which comprises the following steps:
(1) Collection and processing of plasma samples: collecting a blood sample treated by deuterated dehydrogenated 3-benzoyl phenyl-achyranthes compounds, centrifuging to obtain a plasma sample, adding acetonitrile solution containing an internal standard to precipitate protein, centrifuging to obtain a supernatant, and obtaining the plasma sample to be detected;
(2) Plasma samples were tested using a liquid chromatography-mass spectrometry system: taking liquid chromatography as a separation system, wherein a mobile phase A is an aqueous solution containing formic acid, and a mobile phase B is an acetonitrile solution containing formic acid, and performing gradient elution; the mass spectrum is used as a detection system, ESI ion sources are adopted, the multi-stage reaction monitoring MRM mode is adopted to carry out positive ion detection, and the ions for quantitative analysis are respectively deuterated dehydrogenated 3-benzoylphenyl achyrans compounds: m/z 338.1→310.2, internal standard IS: m/z 260.1.fwdarw.116.1.
Further, upon detection in the step (2), the deuterated dehydrogenated 3-benzoylphenyl-achyrans compound has a collision energy of 22V and an internal standard collision energy of 15V.
Further, the internal standard is propranolol with the concentration of 10 ng/mL.
Further, the step (1) specifically includes: collecting 0.25mL of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compound treated blood sample, centrifuging at low temperature and high speed, and collecting 100 μl of supernatant as a blood plasma sample; to 100. Mu.L of the plasma sample, 10. Mu.L of a 60% acetonitrile aqueous solution and 200. Mu.L of a propranolol-containing acetonitrile solution were added, and after vortexing and mixing uniformly, the mixture was centrifuged at 4℃and 14000r/min for 10min, and 200. Mu.L of the supernatant was collected to obtain a plasma sample to be tested.
In the step (2), the liquid chromatography-mass spectrometry system is a liquid chromatograph and a triple quadrupole liquid chromatography-mass spectrometer.
Further, in the step (2), the column of the liquid chromatography is 2.0mm×50mm,5 μm Capcell Pak C 18 MG iii, column temperature: 30 ℃; the flow rate of the mobile phase is 400 mu L/min; sample injectionThe amount is as follows: 2. Mu.L; the formic acid concentration in the mobile phase was 0.1%.
Further, in the step (2), the change of the flow matching ratio with time is:
Figure BDA0004047893830000021
Figure BDA0004047893830000031
further, the deuterated dehydrogenated 3-benzoylphenyl-achyranthes compound has a peak time of: 1.76min; the peak time of the internal standard propranolol is as follows: 1.58min.
Further, in the step (2), the condition of the mass spectrum is a dry gas temperature: 350 ℃; drying gas flow rate: 11L/min; atomizer pressure: 30psi; capillary voltage: 4000V (+); the detection mode is as follows: MRM.
Further, the linear range of the detection method for quantitative analysis of deuterated dehydrogenated 3-benzoylphenyl-achyrans compounds is 2ng/mL-2000ng/mL, and the lowest quantitative lower limit is 2ng/mL.
Further, the deuterated dehydrogenated 3-benzoylphenyl-achyranthes compound is (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deuterated methylene ] piperazine-2, 5-dione.
In summary, the method for quantitatively analyzing the blood concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compounds in blood plasma provided by the invention comprises the following specific steps:
(1) Collecting 0.25mL of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compound treated blood sample, centrifuging by a low-temperature high-speed centrifuge, and taking 100 mu L of supernatant as a plasma sample;
adding 10 mu L of acetonitrile aqueous solution with concentration of 60% and 200 mu L of acetonitrile solution containing propranolol with concentration of 10ng/mL into 100 mu L of plasma sample, carrying out vortex mixing, centrifuging at 4 ℃ for 10min at 14000r/min, taking 2000 mu L of supernatant to obtain a plasma sample to be detected, and carrying out sample injection analysis by 2 mu L;
(2) Separating the plasma sample to be detected by using liquid chromatography as a separation system, wherein a chromatographic column adopts 2.0mm multiplied by 50mm and 5 mu m Capcell Pak C 18 MG iii, column temperature: 30 ℃; mobile phase a is an aqueous solution containing 0.1% formic acid, mobile phase B is an acetonitrile solution containing 0.1% formic acid, and the gradient elution, the change of the flow matching ratio with time is as follows:
Figure BDA0004047893830000032
wherein the mobile phase flow rate: 400 mu L min -1 The method comprises the steps of carrying out a first treatment on the surface of the Sample injection amount: 2. Mu.L;
the peak time of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compound is: 1.76min; the peak time of the internal standard propranolol is as follows: 1.58min;
(3) The mass spectrum is used as a detection system, an ion source is used as an ESI ion source, positive ion detection is carried out in a multi-stage reaction monitoring MRM mode, and ions for quantitative analysis are deuterated dehydrogenated 3-benzoyl phenyl Azithrone compounds respectively: m/z 338.1→310.2, internal standard IS: m/z 260.1 to 116.1; deuterated dehydrogenated 3-benzoyl phenyl-Acylus class compound has collision energy of 22V and internal standard collision energy of 15V; the conditions of mass spectrum are dry gas temperature: 350 ℃; drying gas flow rate: 11L/min; atomizer pressure: 30psi; capillary voltage: 4000V (+); the detection mode is as follows: MRM.
Compared with the prior art, the invention has the advantages and beneficial effects that:
1. the invention has the advantages that the dosage of the plasma is small, one sample only needs 0.25mL of blood sample, and the sample injection quantity only needs 2 mu L, which is far smaller than the dosage of the sample in the prior art;
2. the plasma sample obtained in the invention directly uses acetonitrile to precipitate protein, after vortex mixing, supernatant is centrifugally taken to directly sample, and the pretreatment method is convenient, simple and quick, is suitable for routine quantitative analysis and detection, can reduce operation steps and operation time, and is especially suitable for pretreatment of a large number of samples;
3. the linear detection range of the deuterated dehydrogenated 3-benzoylphenyl-achyranthes compounds is 2-2000ng/mL, which completely meets the measurement of dynamic analysis change of the drugs in animals and human bodies;
4. the detection method has strong selectivity, and endogenous substances in blank plasma do not interfere with the determination of the medicine and the internal standard; the sample stability is good: placing the sample at room temperature for 6 hours, repeatedly freezing and thawing for three times, storing for 10 days at-80 ℃, and placing the treated sample in an automatic sample feeding disc at 10 ℃ for 12 hours to be stable;
5. the sample in the detection method has no dilution effect and no matrix effect; the recovery rate is high, and the precision in the day and the daytime is good;
6. the whole chromatographic analysis and determination process is only required to be 4 minutes, and the detection method has short determination time, and is particularly suitable for determining the blood concentration of deuterated dehydrogenated 3-benzoyl phenyl-achyranthes compounds in animal or human blood plasma, and researching pharmacokinetics and tissue distribution.
Drawings
FIG. 1 is a mass spectrum scan of (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione detected using the quantitative analysis method of the present invention.
FIG. 2 is an ion detection (MRM) chromatogram, wherein FIG. 2A is an MRM chromatogram of a blank plasma; FIG. 2B is a MRM chromatogram of addition of 2ng/mL (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deuterated methylene ] piperazine-2, 5-dione to blank plasma; FIG. 2C is a MRM chromatogram of a plasma sample after 2mg/kg of (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione was injected intravenously at the tail of a Wistar rat for 2 min.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the specific embodiments.
In the present invention, (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione is exemplified in detail.
Example 1: detection method for quantitatively analyzing blood concentration of deuterated dehydrogenated 3-benzoyl phenyl-achyrans compounds in blood plasma
1. Instrument and reagent
(1) Instrument for measuring and controlling the intensity of light
Agilent 6460 type triple quadrupole liquid chromatograph and 1290 Infinicity II liquid chromatograph. .
(2) Reagent(s)
Acetonitrile: chromatographic grade, 99.90%, lot number: 2522021803 from the medical technology company of bard; formic acid: 50mL, lot number: 214911 from Semer Feier technology (China); propranolol, purity ∈99% (TLC), purchased from Sigma-Aldrich, lot number: 101651290 the other chemical reagents are all analytically pure.
2. Experimental procedure
(1) Pretreatment of plasma samples:
wistar rats of 6-8 weeks old and weight 180+ -20 g were subjected to tail vein injection (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione (2 mg/kg), blood was collected from the orbital venous plexus by 0.25mL, and after centrifugation of the blood by a low-temperature high-speed centrifuge, 100. Mu.L of supernatant was collected to obtain a rat plasma sample.
100. Mu.L of rat plasma sample was taken and 10. Mu.L of acetonitrile water (V Acetonitrile ：V Water and its preparation method =6: 4) 200. Mu.L of acetonitrile solution containing an internal standard (propranolol, concentration of 10 ng/mL) was added to the solution, and after vortexing and mixing, the solution was centrifuged at 14000r/min at 4℃for 10min, 200. Mu.L of the supernatant was taken and 2. Mu.L of the sample was sampled for detection.
(2) Conditions for LC-MS/MS analysis:
A. conditions of liquid chromatography
Chromatographic column: capcell Pak C 18 MG III packing, 2.0X10 mm,5 μm, purchased from Japanese senior Co., ltd., column temperature: 30 ℃; mobile phase a was water (0.1% formic acid) and mobile phase B: acetonitrile (0.1% formic acid) and the flow match over time are shown in table 1:
table 1: LC-MS/MS detection of mobile phase conditions for (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione
Figure BDA0004047893830000061
The peak time of (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deuterated methylene ] piperazine-2, 5-dione is: 1.76min; the peak time of the internal standard propranolol is as follows: 1.58min.
The flow rate of the mobile phase is 400 mu L min -1 The method comprises the steps of carrying out a first treatment on the surface of the Sample injection amount: 2. Mu.L.
B. Mass spectrometry conditions
The adopted ion source is ESI ion source, the multi-stage reaction monitoring MRM mode is used for positive ion detection, and the quantitatively analyzed ions are (3Z, 6Z) -3-benzylidene-6- [ (5-tertiary butyl-1H-imidazol-4-yl) deuteromethylidene ] piperazine-2, 5-dione: m/z 338.1→310.2, internal standard IS: m/z 260.1→116.1, (3 z,6 z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione with a collision energy of 22V and an internal standard collision energy of 15V;
the conditions of mass spectrum are dry gas temperature: 350 ℃; drying gas flow rate: 11L/min; atomizer pressure: 30psi; capillary voltage: 4000V (+); the detection mode is as follows: MRM.
The analysis conditions are typical conditions, and in practical application, various parameters can be properly adjusted according to different characteristics of different used instruments so as to obtain the best effect.
(3) LC-MS/MS analysis results
Mass spectrometry scans are shown in fig. 1, and ion detection (MRM) chromatograms of plasma samples following administration of (3 z,6 z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deuteromethylene ] piperazine-2, 5-dione are shown in fig. 2.
Example 2: detection method for quantitatively analyzing deuterated dehydrogenated 3-benzoyl phenyl-achyranthes aspera compound blood concentration in blood plasma
1. Specialization of
In the quantitative analysis detection method, the specificity of the object to be detected and the internal standard is evaluated by comparing the lowest concentration point of the standard curve with blank plasma operated by the same method. The chromatograms of the blank plasma, the blank plasma with 2ng/mL (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione and the actual plasma samples are shown in FIGS. 2A, 2B and 2C, respectively. From the figure, it can be seen that endogenous substances in the blank plasma do not interfere with the measurement of (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione and internal standard, indicating that the quantitative analysis detection method of the invention has specificity for the measurement of (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione in the plasma.
2. Standard curve
(3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene]Standard curve series solutions of piperazine-2, 5-dione were prepared from acetonitrile water (V Acetonitrile ：V Water and its preparation method =6: 4) Preparing: taking Wistar rat blank plasma, adding (3Z, 6Z) -3-benzylidene-6- [ (5-tertiary butyl-1H-imidazol-4-yl) deutero-methylene]Piperazine-2, 5-dione solution formulated to contain (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deuterated methylene]Standard plasma with piperazine-2, 5-dione concentration of 2,5, 10, 20, 50, 100, 200, 500, 1000, 2000 ng/mL. 100. Mu.L of each rat plasma sample was subjected to LC-MS/MS analysis according to the procedure of pretreatment of the plasma sample in example 1, and regression analysis was performed using a weighted least squares method to obtain a standard curve, in this example 3 batches of plasma samples were used for the experiment. The obtained linear regression equation is shown in Table 2, Y represents the ratio of the peak area of the drug to be tested to the peak area of the internal standard, and x represents the concentration of the drug to be tested. The quantitative analysis method of the invention is specific to (3Z, 6Z) -3-benzylidene-6- [ (5-tertiary butyl-1H-imidazol-4-yl) deutero-methylene]The linear range of the piperazine-2, 5-dione quantitative determination is 2-2000ng/mL, and the lowest quantitative lower limit is 2ng/mL.
Table 2: standard curve of (3 z,6 z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione in rat plasma (n=3)
Figure BDA0004047893830000071
3. Matrix effect
100. Mu.L of blank rat plasma was taken, 200. Mu.L of acetonitrile (containing internal standard) was added to precipitate the protein, and 100. Mu.L of supernatant was taken after vortex centrifugation. Then the steps of the standard curve series solution preparation method are operated to prepare the (3Z, 6Z) -3-benzylidene-6- [ (5-tertiary butyl-1H-imidazol-4-yl) deuterated methylene]The three low, medium and high concentrations of piperazine-2, 5-dione were 2, 250, 1600ng/mL, respectively, for 6 sample analyses per concentration. The matrix was then formulated as acetonitrile water (V Acetonitrile ：V Water and its preparation method =60: 40 The quality control of the low, medium and high concentrations is carried out to obtain the corresponding peak areas, and the matrix effect of the sample is inspected according to the ratio of the peak areas of the two. The analysis and measurement results are shown in Table 3, and the results show that the matrix effect has little influence on the measurement of the sample.
Table 3: matrix effects of (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione in plasma samples
Figure BDA0004047893830000081
4. Accuracy and precision
The preparation method is operated according to the steps of a standard curve series solution preparation method, so that quality control samples with low, medium and high concentrations of 5, 250 and 1600ng/mL respectively of (3Z, 6Z) -3-benzylidene-6- [ (5-tertiary butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione are prepared, and 6 sample analyses are carried out on each concentration, and 3 batches of plasma samples are used for continuous measurement for 3 days in the experiment. The precision of the quantitative analysis method is evaluated by the obtained daily and daytime RSD (%), and the accuracy is evaluated by the ratio of the actual measured value to the theoretical value. The analytical results are shown in Table 4, and the daily precision of (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deuterated methylene ] piperazine-2, 5-dione is respectively 4.39% -7.96% and 2.45% -12.90%, and the accuracy range is-1.05% -7.51%; the quantitative analysis method is smaller than +/-15%, which shows that the quantitative analysis method has good precision and accuracy.
Table 4: accuracy and precision of (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione LC-MS/MS assay method in plasma samples
Figure BDA0004047893830000082
5. Stability of
The Wsitar rat plasma sample stability test is carried out according to the steps of a standard curve series solution preparation method, and two quality control plasma samples with the concentration of 5ng/mL and 1600ng/mL of (3Z, 6Z) -3-benzylidene-6- [ (5-tertiary butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione are prepared for investigation. The stability of plasma samples was evaluated for 6 hours at room temperature, three times repeated freeze thawing, 10 days at-80 ℃ and 12 hours at 10 ℃ in an autosampler. The RSD and RE stability under various conditions were less than 15%. The plasma samples were stable both in handling and storage, indicating that the (3Z, 6Z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deuterated methylene ] piperazine-2, 5-dione plasma samples were stable under different conditions.
Table 5: stability results of (3 z,6 z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione in plasma samples (n=6)
Figure BDA0004047893830000091
6. Recovery rate of extraction
According to the preparation method steps of the standard curve series solution, preparing quality control samples with low, medium and high concentrations of 5, 250 and 1600ng/mL of (3Z, 6Z) -3-benzylidene-6- [ (5-tertiary butyl-1H-imidazol-4-yl) deutero methylene ] piperazine-2, 5-dione, and carrying out 6 sample analyses on each concentration to record chromatographic peak areas. And then preparing a pure sample solution which does not contain a matrix and is used for finally measuring the concentration of the three concentrations, analyzing the pure sample solution to obtain corresponding peak areas, and examining the extraction recovery rate of the sample according to the ratio of the peak areas and the peak areas. The analysis and measurement results are shown in Table 6, and the extraction recovery rates of three concentrations of (3Z, 6Z) -3-benzylidene-6- [ (5-tertiary butyl-1H-imidazol-4-yl) deuterated methylene ] piperazine-2, 5-dione meet the requirements.
Table 6: recovery of (3 z,6 z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione (n=6)
Figure BDA0004047893830000092
7. Dilution effect
Preparing quality control samples with the concentration of (3Z, 6Z) -3-benzylidene-6- [ (5-tertiary butyl-1H-imidazol-4-yl) deutero methylene ] piperazine-2, 5-dione of 5000ng/mL according to the steps of a standard curve series solution preparation method, diluting plasma samples 5 times and 10 times by using Wistar rat blank plasma, analyzing 6 samples for each dilution multiple, measuring the final concentration, comparing with a theoretical value, and examining the dilution effect of the samples. The analysis results are shown in Table 7, and the deviation of the actual measurement value and the theoretical value of the (3Z, 6Z) -3-benzylidene-6- [ (5-tertiary butyl-1H-imidazol-4-yl) deuterated methylene ] piperazine-2, 5-dione is within +/-15%, which shows that the influence of the dilution effect on the measurement of the sample is not great.
Table 7: dilution effect of (3 z,6 z) -3-benzylidene-6- [ (5-tert-butyl-1H-imidazol-4-yl) deutero-methylene ] piperazine-2, 5-dione (n=6)
Figure BDA0004047893830000101
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Claims (10)

1. A method for quantitatively analyzing the plasma concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compounds, comprising the steps of:
(1) Collection and processing of plasma samples: collecting a blood sample treated by deuterated dehydrogenated 3-benzoyl phenyl-achyranthes compounds, centrifuging to obtain a plasma sample, adding acetonitrile solution containing an internal standard to precipitate protein, centrifuging to obtain a supernatant, and obtaining the plasma sample to be detected;
(2) Plasma samples were tested using a liquid chromatography-mass spectrometry system: taking liquid chromatography as a separation system, wherein a mobile phase A is an aqueous solution containing formic acid, and a mobile phase B is an acetonitrile solution containing formic acid, and performing gradient elution; the mass spectrum is used as a detection system, ESI ion sources are adopted, the multi-stage reaction monitoring MRM mode is adopted to carry out positive ion detection, and the ions for quantitative analysis are respectively deuterated dehydrogenated 3-benzoylphenyl achyrans compounds: m/z 338.1→310.2, internal standard IS: m/z 260.1.fwdarw.116.1.
2. The method of claim 1, wherein the deuterated dehydrogenated 3-benzoylphenyl-achyranthes compound has a collision energy of 22V and an internal standard collision energy of 15V.
3. The method for quantitatively determining plasma deuterated dehydrogenated 3-benzoylphenyl-achyranthes-ding compound according to claim 1, wherein the internal standard is propranolol at a concentration of 10 ng/mL.
4. The method for quantitatively determining the plasma concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compound according to claim 3, wherein the step (1) comprises: collecting 0.25mL of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compound treated blood sample, centrifuging at low temperature and high speed, and collecting 100 μl of supernatant as a blood plasma sample; to 100. Mu.L of the plasma sample, 10. Mu.L of a 60% acetonitrile aqueous solution and 200. Mu.L of a propranolol-containing acetonitrile solution were added, and after vortexing and mixing uniformly, the mixture was centrifuged at 4℃and 14000r/min for 10min, and 200. Mu.L of the supernatant was collected to obtain a plasma sample to be tested.
5. The method for quantitatively determining plasma concentration of deuterated dehydrogenated 3-benzoylphenyl-achyrans compound according to claim 1, wherein in step (2), the lc system is a lc and triple quadrupole lc.
6. The method for quantitative analysis of plasma deuterated dehydrogenated 3-benzoylphenyl-achyrans compound according to claim 3, wherein in the step (2), the column of liquid chromatography is 2.0 mm. Times.50 mm,5 μm Capcell Pak C 18 MG iii, column temperature: 30 ℃; the flow rate of the mobile phase is 400 mu L/min; sample injection amount: 2. Mu.L; the formic acid concentration in the mobile phase was 0.1%.
7. The method for detecting the plasma concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compound according to claim 6, wherein the flow matching ratio is changed with time in the step (2):
Figure FDA0004047893820000021
8. the method for detecting the plasma concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compound according to claim 6, wherein the deuterated dehydrogenated 3-benzoylphenyl-achyran compound has a peak time of: 1.76min; the peak time of the internal standard propranolol is as follows: 1.58min.
9. The method for detecting the plasma concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compound according to claim 1, wherein in the step (2), the condition of mass spectrometry is dry gas temperature: 350 ℃; drying gas flow rate: 11L/min; atomizer pressure: 30psi; capillary voltage: 4000V (+); the detection mode is as follows: MRM.
10. The method for detecting the plasma concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compound according to claim 1, wherein the linear range of the deuterated dehydrogenated 3-benzoylphenyl-achyran compound is 2ng/mL to 2000ng/mL and the lowest lower limit of the quantitative analysis is 2ng/mL.
CN202310032708.4A 2023-01-10 2023-01-10 Detection method for quantitatively analyzing blood concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compounds in blood plasma Pending CN116298002A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310032708.4A CN116298002A (en) 2023-01-10 2023-01-10 Detection method for quantitatively analyzing blood concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compounds in blood plasma

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310032708.4A CN116298002A (en) 2023-01-10 2023-01-10 Detection method for quantitatively analyzing blood concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compounds in blood plasma

Publications (1)

Publication Number Publication Date
CN116298002A true CN116298002A (en) 2023-06-23

Family

ID=86787745

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310032708.4A Pending CN116298002A (en) 2023-01-10 2023-01-10 Detection method for quantitatively analyzing blood concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compounds in blood plasma

Country Status (1)

Country Link
CN (1) CN116298002A (en)

Similar Documents

Publication Publication Date Title
CN115326960B (en) Analysis method for simultaneously detecting concentration of 8 antiepileptic drugs and 1 active metabolite in human plasma
CN108072712B (en) Quantitative analysis method for blood concentration of new compound WSJ-557 in SD rat plasma
CN114019062B (en) Method for detecting related substances in rifampicin
Li et al. A comparison of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-multiplied immunoassay technique (EMIT) for the determination of the cyclosporin A concentration in whole blood from Chinese patients
CN108760920B (en) Method for determining residual quantity of cyazofamid and metabolites thereof based on HPLC-MSMS method
CN112782322A (en) Method for simultaneously determining 8 anti-tuberculosis drugs in human plasma based on LC-MS (liquid chromatography-Mass Spectrometry)
CN116183801A (en) Liquid chromatography-mass spectrometry method, kit and system for detecting insulin and C peptide
CN116298002A (en) Detection method for quantitatively analyzing blood concentration of deuterated dehydrogenated 3-benzoylphenyl-achyranthes compounds in blood plasma
CN111060612B (en) Method for determining concentration of prednisone, prednisone acetate and active metabolite prednisolone in blood plasma by liquid chromatography-mass spectrometry
CN112213417A (en) Kit and method for detecting concentration of mycophenolic acid medicine in dried blood spots
CN111307992B (en) Pre-column derivative liquid chromatography-mass spectrometry analysis method for quantitatively detecting organic acid in PM2.5
Liebich et al. Rapid quantitative microanalysis of ketones in urine by gas chromatography-mass fragmentography
CN113671064B (en) Detection method for quantitatively analyzing blood concentration of amlexanox in plasma
CN111257444A (en) Method for detecting blood concentration of candidate compound for resisting Alzheimer disease
CN109324139A (en) Ribosylzeatin liquid-liquid extraction-liquid chromatography-tandem mass spectrometry measuring method in a kind of tobacco leaf
CN114624338A (en) Method for quantitatively analyzing free amino acids in biological sample by using liquid chromatography-tandem mass spectrometry
KR100330860B1 (en) A method of analyzing a concentration of homocysteine in plasma
CN104991028B (en) The reduction method of fixedness buffer salt content in LC MS testers
CN109324140A (en) Ribosylzeatin Solid Phase Extraction-liquid chromatography-tandem mass spectrometry measuring method in a kind of tobacco leaf
CN115078621B (en) Method for measuring concentration of irinotecan derivative Dxd in blood plasma
Wu et al. Determination of betamethasone and dexamethasone in plasma by fluorogenic derivatization and liquid chromatography
KR100565939B1 (en) Method for determination of nucleosides in urine with liquid chromatography-mass spectrometry
CN106950302A (en) The quantitative analysis method of naproxen in a kind of human plasma
CN117554513A (en) HPLC-MSMS method for simultaneously measuring valproic acid, olanzapine and metabolites thereof in blood plasma
Ulmasovna ANALYSIS OF DRUGS IN PHARMACOKINETIC STUDIES

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