CN116818967A - Detection method of teprenone and detection kit of teprenone - Google Patents
Detection method of teprenone and detection kit of teprenone Download PDFInfo
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- CN116818967A CN116818967A CN202310798395.3A CN202310798395A CN116818967A CN 116818967 A CN116818967 A CN 116818967A CN 202310798395 A CN202310798395 A CN 202310798395A CN 116818967 A CN116818967 A CN 116818967A
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- DJAHKBBSJCDSOZ-AJLBTXRUSA-N (5z,9e,13e)-6,10,14,18-tetramethylnonadeca-5,9,13,17-tetraen-2-one;(5e,9e,13e)-6,10,14,18-tetramethylnonadeca-5,9,13,17-tetraen-2-one Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C(C)=C/CCC(C)=O.CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C(C)=C\CCC(C)=O DJAHKBBSJCDSOZ-AJLBTXRUSA-N 0.000 title claims abstract description 135
- 229950006156 teprenone Drugs 0.000 title claims abstract description 135
- 238000001514 detection method Methods 0.000 title claims abstract description 86
- 239000000523 sample Substances 0.000 claims abstract description 157
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 63
- 239000007788 liquid Substances 0.000 claims abstract description 45
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000012224 working solution Substances 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 37
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Classifications
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- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- 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
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- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- 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)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The application belongs to the technical field of drug detection in biological samples, and in particular relates to a method for detecting teprenone and a kit for detecting teprenone, wherein the detection method comprises pretreatment, and the pretreatment comprises the following steps: providing a plasma sample containing teprenone, mixing the plasma sample with an internal standard working solution, and then adding a precipitating agent, wherein the precipitating agent comprises methanol and acetonitrile, and performing protein precipitation to obtain an internal standard mixed solution; and (3) carrying out solid-liquid separation treatment on the internal standard mixed solution, and taking clear liquid to obtain an internal standard sample. The precipitator containing the methanol and the acetonitrile is adopted, the acetonitrile has the function of protein precipitation, a possible reaction mechanism is provided, the hydroxyl of the methanol can react with the carbonyl of the teprenone to activate the teprenone, the detection sensitivity of the teprenone is obviously improved, the treatment process is simple and quick, the loss rate of an object to be detected is low, the detection is carried out by matching with an internal standard method, the detection accuracy is greatly improved, and the sample treatment efficiency can be effectively improved.
Description
Technical Field
The application belongs to the technical field of drug detection in biological samples, and particularly relates to a method for detecting teprenone and a kit for detecting teprenone.
Background
Because most chronic gastritis patients have no symptoms, the exact prevalence rate is difficult to obtain, chronic active gastritis almost exists among helicobacter pylori symptomatic infected patients, chronic gastritis exists among most serological detection (symptomatic infection or past infection) positive patients, and besides helicobacter pylori infection, factors such as bile reflux, medicines, autoimmunity and the like can also cause the chronic gastritis. The teprenone capsule is suitable for improving gastric mucosal lesions (erosion, hemorrhage, flush and edema) in acute gastritis and acute exacerbation stage of chronic gastritis; gastric ulcer.
Teprenone belongs to gastric mucosa protectant, has remarkable anti-ulcer effect, can not influence normal physiological functions of intestines and stomach, and ensures normal secretion of gastric acid and normal gastric motility. Teprenone is expressed by promoting gastric mucosa secretion, endogenous prostaglandin production and heat shock protein HSP 70. The heat shock protein HSP70 can induce inflammatory cell apoptosis, reduce body inflammatory reaction, and repair damaged protein, thereby promoting damaged protein repair. In addition, the teprenone can enhance gastric mucosa secretion, thereby improving gastric mucosa barrier and protecting functions, stimulating gastric mucosa regeneration and promoting gastric mucosa repair. The teprenone has high fat solubility, the absorption is carried out with the help of bile acid emulsification, and the teprenone is hardly absorbed in a fasting state, so that the teprenone content in the plasma after meal administration is higher than the teprenone content in the plasma after the fasting administration, and the clinical pharmacokinetics study of the medicine is carried out under the condition of meal.
The prior detection methods mostly adopt solid-phase extraction or liquid-liquid extraction, then concentrate the extract, and the loss rate of the test substance of teprenone is high, so that the error of the detection result is large.
Disclosure of Invention
Based on the detection method and the detection kit of the teprenone, the application provides a detection method of the teprenone and a detection kit of the teprenone, and solves the technical problem that the detection result error is large due to high loss rate of the teprenone to be detected in pretreatment of the detection method in the prior art.
In order to achieve the above purpose, the application adopts the following technical scheme:
in a first aspect, a method for detecting teprenone includes a pretreatment, the pretreatment including the steps of:
providing a plasma sample containing teprenone, mixing the plasma sample with an internal standard working solution, and then adding a precipitating agent, wherein the precipitating agent comprises methanol and acetonitrile, and performing protein precipitation to obtain an internal standard mixed solution;
and (3) carrying out solid-liquid separation treatment on the internal standard mixed solution, and taking clear liquid to obtain an internal standard sample.
Optionally, the plasma sample comprises a sample to be tested and standard samples of different concentrations, and the pretreatment comprises the following steps:
mixing the sample to be measured and the standard sample with an internal standard working solution respectively, then adding a precipitator respectively, and carrying out protein precipitation to obtain an internal standard mixed solution to be measured and an internal standard mixed solution respectively;
and respectively carrying out solid-liquid separation treatment on the internal standard mixed solution and the internal standard mixed solution, and taking clear liquid to respectively obtain an internal standard sample to be detected and an internal standard sample.
Optionally, after the pretreatment, the method further comprises the following steps:
taking internal standard samples with different concentrations, performing LC-MS/MS chromatographic detection to obtain a standard map, and performing linear regression to obtain a linear regression equation and a correlation coefficient of teprenone in blood plasma;
and carrying out LC-MS/MS chromatographic detection on the internal standard sample to be detected under the same detection condition as the internal standard sample to obtain a mass spectrum analysis result of the internal standard sample to be detected, and determining the content of teprenone in the internal standard sample to be detected.
Alternatively, in LC-MS/MS chromatography detection, the detection conditions for liquid chromatography are as follows:
chromatographic column: waters ACQUITY UPLC Protein BEH C4;
autoinjector temperature: 4-10 ℃;
column temperature: 30-50 ℃;
flow rate: 0.3mL.min -1 -0.4mL.min -1 ；
Sample injection amount: 15 mu L-20 mu L;
mobile phase a phase: an aqueous solution of an organic acid having a pH of 4 to 5, the organic acid being a volatile acid;
mobile phase B phase: methanol acetonitrile solution with volume concentration of 30-60%;
the elution mode is gradient elution; and/or the number of the groups of groups,
in LC-MS/MS chromatography detection, the detection conditions for mass spectrometry are as follows:
ion source: an electrospray ion source;
ion source temperature: 400 ℃;
source injection voltage: 5000V;
inlet voltage: 10V;
collision gas: 10psi;
air curtain gas: 25psi;
ion source gas 1:75psi;
ion source gas 2:50.0psi;
ionization mode: a positive ion;
signal acquisition mode: MRM;
ionization mode: a positive ion;
ion pair:
| ion pair | Q1(Da) | Q3(Da) | Declustering voltage (V) | Collision energy (V) | Scan time(ms) |
| Object to be measured | 331.200 | 231.300 | 60.000 | 19.500 | 300.0 |
| Internal standard | 334.200 | 234.300 | 55.000 | 16.000 | 300.0 |
。
Optionally, the gradient elution procedure comprises:
providing a mobile phase A phase solution with the mass percentage of 35%, providing a mobile phase B phase solution with the mass percentage of 65%, and performing first gradient elution at 0-0.70 min;
providing a mobile phase A phase solution with the mass percentage of 35%, providing a mobile phase B phase solution with the mass percentage of 65%, and performing second gradient elution in 0.70-3.00 min;
providing a mobile phase A phase solution with the mass percentage of 5%, providing a mobile phase B phase solution with the mass percentage of 95%, and performing third gradient elution within 3.00-3.50 min;
providing a mobile phase A phase solution with the mass percentage of 5%, providing a mobile phase B phase solution with the mass percentage of 95%, and performing fourth gradient elution within 3.50-3.51 min;
providing 35% mobile phase A phase solution and 65% mobile phase B phase solution, and performing fifth gradient elution at 3.51-4.50 min.
Optionally, aStandard samples of different concentrations included a concentration of 0.75ng.ml -1 -75.0ng.mL -1 Is a standard sample of (2); and/or the number of the groups of groups,
the preparation method of the standard samples with different concentrations comprises the following steps:
providing a teprenone reference stock solution, and diluting with a diluent to obtain standard curve working solutions with different concentrations;
and mixing the standard curve working solutions with different concentrations with blank plasma according to the volume ratio of less than or equal to 1:19 to obtain standard samples with different concentrations.
Alternatively, the standard curve working fluid of different concentrations comprises a concentration of 15ng.mL -1 -1500ng.mL -1 Standard curve working solution of the teprenone standard substance; and/or the number of the groups of groups,
the diluent comprises an acetonitrile aqueous solution with the mass percentage concentration of 60-80 percent.
Optionally, the pretreatment satisfies at least one of the following conditions:
the concentration of the internal standard substance in the internal standard working solution is 0.5ng.mL -1 -0.8ng.mL -1 ；
The internal standard substance of the internal standard working solution comprises TEP- 13 C 3 -YYMMDD-IS-02；
The precipitant is methanol acetonitrile solution with volume concentration of 5% -8%;
the solid-liquid separation treatment comprises centrifugal separation; and/or the number of the groups of groups,
the detection method also comprises the pretreatment of the blank sample, and the pretreatment method of the blank sample comprises the following steps:
providing a blank sample, adding a diluent into the blank sample, diluting to a proper concentration, and then adding a precipitator for protein precipitation to obtain a blank sample mixed solution;
performing solid-liquid separation treatment on the blank sample mixed solution, and taking clear liquid to obtain a blank control sample; and/or the number of the groups of groups,
the plasma sample is replaced with a serum sample or a cerebrospinal fluid sample.
Alternatively, the volume ratio of plasma sample to precipitant is 7:15-20.
In a second aspect, a test kit of teprenone based on the test method of teprenone is provided, which is characterized in that: the detection kit comprises a teprenone standard substance, an isotope internal standard substance, a precipitant and a diluent.
The application has the beneficial effects that:
according to the method for detecting the teprenone, the precipitation agent containing the methanol and the acetonitrile is adopted to carry out protein precipitation pretreatment on a plasma sample, the acetonitrile has the function of protein precipitation, the methanol contains hydroxyl, and the teprenone contains carbonyl, and the embodiment of the application provides a possible reaction mechanism, the hydroxyl of the methanol can react with the carbonyl of the teprenone to play a role in activating the teprenone, so that the detection sensitivity of the teprenone is remarkably improved, the treatment process is simple and rapid, the loss rate of an object to be detected is low, the detection is carried out by matching with an internal standard method, the detection accuracy is greatly improved, and the sample treatment efficiency can be effectively improved; compared with the prior art, the detection method of the application adopts a protein precipitation method to pretreat the sample, does not need liquid-liquid extraction, has low loss rate of the object to be detected and high detection sensitivity, can simply, rapidly and batchly treat the sample, and improves the detection accuracy and the detection efficiency;
the test kit of the teprenone provided by the application is provided with a teprenone standard substance, an isotope internal standard substance, a precipitator and a diluent, provides necessary chemical substances for establishing a standard curve by adopting an internal standard method, can be used for preparing the required standard liquid concentration according to the detection requirement when being applied to the detection of the teprenone content in a biological sample, and establishes a standard curve in a proper range.
Drawings
The application will be further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a standard graph of a test method for teprenone according to example 1 of the present application;
FIGS. 2 (a) and 2 (b) are blank matrix sample chromatograms of a method for detecting teprenone according to an embodiment of the present application;
fig. 3 (a) and 3 (b) are quantitative lower limit chromatograms of a method for detecting teprenone according to an embodiment of the present application.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
The pretreatment of the existing teprenone detection method mostly adopts solid-phase extraction or liquid-liquid extraction, the extraction process is complex, the steps of adding an extractant, shaking uniformly, standing, taking supernatant, repeating 3 times, drying by liquid nitrogen, redissolving and the like are included, the teprenone to be detected is easy to lose, and the recovery rate of single liquid-liquid extraction is even lower than 50%.
Moreover, when the teprenone is taken by a subject in a fasting state, the teprenone content in a plasma sample is extremely low and is about 7ng/mL-10ng/mL, and after single liquid-liquid extraction, the requirement on detection precision is further improved and the detection difficulty is increased due to the limitation of recovery rate. If the teprenone is concentrated by multiple liquid-liquid extractions, a large amount of plasma samples are required, and the detection cost is greatly increased when the drug content of the plasma samples of clinical subjects is detected and analyzed.
The existing method for detecting organic matters in blood plasma, such as organic phosphorus, benzoazepine, thiophenazine and the like, adopts protein precipitation to pretreat a sample, and takes pure acetonitrile or other pure organic matters as a precipitating agent.
In the research process, protein precipitation is attempted to be carried out on a plasma sample containing the teprenone by adopting pure acetonitrile, then detection is carried out, the detection sensitivity is low, an accurate detection result is difficult to obtain, and the existing protein precipitation method is not suitable for detecting the teprenone.
Based on the above problems, the embodiment of the application provides a method for detecting teprenone, which comprises the following steps:
s1: providing a plasma sample containing teprenone, mixing the plasma sample with an internal standard working solution, then adding a precipitating agent, wherein the precipitating agent comprises methanol and acetonitrile, and carrying out protein precipitation to obtain an internal standard mixed solution.
S2: and (3) carrying out solid-liquid separation treatment on the internal standard mixed solution, and taking clear liquid to obtain an internal standard sample.
The method for detecting the teprenone provided by the application develops the pretreatment of protein precipitation on a plasma sample by adopting a precipitator containing methanol and acetonitrile, wherein the acetonitrile has the function of protein precipitation, the methanol contains hydroxyl, the teprenone contains carbonyl, and the embodiment of the application provides a possible reaction mechanism, the hydroxyl of the methanol reacts with the carbonyl of the teprenone to play a role in activating the teprenone, so that the detection sensitivity of the teprenone is obviously improved, and the detectable concentration is lower than 7ng.mL -1 The content of the teprenone is simple and quick in treatment process, the loss rate of the object to be detected is low, the detection is carried out by matching with an internal standard method, the detection accuracy is greatly improved, and the sample treatment efficiency can be effectively improved.
Compared with the prior art, the detection method provided by the application adopts the protein precipitation method to pretreat the sample, does not need liquid-liquid extraction, has low loss rate of the object to be detected, has high detection sensitivity, can simply, rapidly and batchwise treat the sample, and improves the detection accuracy and the detection efficiency.
In the step S1, in addition to precipitating the protein in the sample, the teprenone in the sample is activated, so that the response sensitivity of the teprenone is improved, and preparation is provided for subsequent chromatographic detection.
In some embodiments, the plasma sample comprises a sample to be tested and a standard sample of different concentrations. The sample to be detected is the sample containing the teprenone to be detected in the embodiment of the application. The standard sample is a standard sample prepared by adopting a teprenone standard substance, and is used for establishing a standard curve to obtain a linear regression equation and a correlation coefficient of teprenone in blood plasma, and generally, at least three standard samples with different concentrations are adopted.
At this time, the pretreatment includes the steps of:
mixing the sample to be measured and the standard sample with an internal standard working solution respectively, then adding a precipitator respectively, and carrying out protein precipitation to obtain an internal standard mixed solution to be measured and an internal standard mixed solution respectively;
and respectively carrying out solid-liquid separation treatment on the internal standard mixed solution and the internal standard mixed solution, and taking clear liquid to respectively obtain an internal standard sample to be detected and an internal standard sample.
The pretreatment steps of the sample to be detected and the standard sample are the same, the sample to be detected and the standard sample are added with the same amount of internal standard working solution and the same amount of precipitant, the same pretreatment parameters are ensured, and the conversion error is reduced.
In some embodiments, the standard sample of different concentrations comprises a concentration of 0.75ng.mL -1 -75.0ng.mL -1 Is a standard sample of (2).
In some specific embodiments, the standard samples of different concentrations comprise concentrations of 0.75ng.mL, respectively -1 、1.5ng.mL -1 、4.5ng.mL -1 、9.0ng.mL -1 、18.0ng.mL -1 、36.0ng.mL -1 、68.0ng.mL -1 、75.0ng.mL -1 When the teprenone is taken in the fasting state, the teprenone content in the blood plasma sample is about 7ng/mL-10ng/mL, and the concentration falls in the standard concentration range of the standard sample, so that the teprenone blood plasma can be used for detecting the teprenone taking in the fasting state. Of course, the user can also select a standard sample with a proper standard concentration according to the approximate concentration of the object to be detected, so as to obtain a more accurate standard curve and a calculation result.
In some embodiments, the method of preparing standard samples of different concentrations comprises the steps of:
providing a teprenone reference stock solution, and diluting with a diluent to obtain standard curve working solutions with different concentrations;
and mixing the standard curve working solutions with different concentrations with blank plasma according to the volume ratio of less than or equal to 1:19 to obtain standard samples with different concentrations.
The teprenone control stock solution is formulated using teprenone standards, and in some embodiments, the concentration (CSS, QCS) of the teprenone control stock solution may be 1mg.mL -1 And diluting with a diluent by different times to obtain standard curve working solutions with different concentrations.
The blank plasma in the examples of the present application refers to plasma without teprenone.
Blank plasma is added into the standard sample to simulate the plasma environment of the sample to be detected, so that the liquid environment of the standard sample is the same as that of the sample to be detected, and the same reaction effect is obtained in the protein precipitation reaction.
The standard curve working solution with different concentrations and blank plasma are mixed according to the volume ratio of less than or equal to 1:19, for example, the volume ratio of the standard curve working solution to the blank plasma is 1:19, 1:20, 1:21, 1:22 and the like, and the dilution ratio of the plasma matrix is not more than 5%, so that the plasma matrix is ensured not to be excessively diluted and is close to the biological matrix.
In some embodiments, the standard curve working fluid at different concentrations includes a concentration of 15ng.mL -1 -1500ng.mL -1 Standard curve working solution of the teprenone standard substance. In some specific embodiments, the standard curve working fluid of different concentrations comprises concentrations of 15ng.mL respectively -1 、30ng.mL -1 、90ng.mL -1 、180ng.mL -1 、360ng.mL -1 、720ng.mL -1 、1360ng.mL -1 、1500ng.mL -1 Standard curve working fluid of (2).
In some embodiments, the diluent comprises an aqueous acetonitrile solution having a concentration of 60% to 80% by mass.
In some embodiments, the pretreatment satisfies at least one of the following conditions:
the concentration of the internal standard substance in the internal standard working solution is 0.5ng.mL -1 -0.8ng.mL -1 ；
The internal standard substance of the internal standard working solution comprises TEP- 13 C 3 -YYMMDD-IS-02；
The precipitator is methanol acetonitrile solution with volume concentration of 5-8%, namely, in the precipitator, the volume concentration of methanol is 5-8%, and the solvent is acetonitrile. Since the usual acetonitrile gives unstable response in the quantification of teprenone, it is likely that this situation is significantly improved by the proper addition of a low proportion of methanol, depending on the molecular form of teprenone in acetonitrile.
In general, in order to obtain a more accurate standard curve, the concentration of the standard sample has at least five gradient concentrations, and the concentration of the internal standard working fluid is the same as the third to sixth concentrations of the standard sample in the order from small to large.
The internal standard substance is TEP- 13 C 3 -YYMMDD-IS-02，TEP- 13 C 3 YYMDD-IS-02 has similar physicochemical properties as teprenone, and under chromatographic conditions, TEP- 13 C 3 YYMDD-IS-02 IS capable of being substantially separated from the components of the sample.
In some embodiments, the volume ratio of plasma sample to precipitant is 7:15-20.
In some embodiments, the solid-liquid separation treatment comprises centrifugation. Alternatively, the parameters of the centrifugation may be, for example, RT,1840 g,6min.
In some embodiments, the sample mixed solution is subjected to oscillation treatment before the solid-liquid separation treatment, so that the teprenone in the mixed solution is fully dispersed into the liquid, and the teprenone is prevented from being adsorbed in protein precipitation.
In some embodiments, the plasma sample further comprises a quality control sample with different concentrations, and the pretreatment method of the quality control sample is the same as the pretreatment method of the standard sample. The quality control sample is used for checking whether the standard curve meets the acceptance standard, and the accuracy and precision.
Generally, the different concentrations of the quality control sample include at least three different concentrations of the quality control sample. In some embodiments, the concentration of the quality control sample is 2.0ng.mL -1 -60.0ng.mL -1 The concentration of the quality control sample falls between the minimum and maximum values of the standard sample concentration, and the concentration of the quality control sample is different from the concentration of the standard sample. For example, the standard samples have concentrations of 0.75ng.mL, respectively -1 、1.5ng.mL -1 、4.5ng.mL -1 、9.0ng.mL -1 、18.0ng.mL -1 、36.0ng.mL -1 、68.0ng.mL -1 、75.0ng.mL -1 The concentration of the quality control samples was 2.0ng.mL respectively -1 、5.0ng.mL -1 、25.0ng.mL -1 、60.0ng.mL -1 。
In some embodiments, the method of preparing a quality control sample comprises the steps of:
providing a teprenone reference stock solution, and diluting with a diluent to obtain quality control working solutions with different concentrations;
and mixing the quality control working solutions with different concentrations with blank plasma according to the volume ratio of less than or equal to 1:19 to obtain quality control samples with different concentrations.
In some embodiments, the concentration of the quality control working fluid is 40ng.mL -1 -1200ng.mL -1 For example, 40ng.mL may be used -1 、100ng.mL -1 、500ng.mL -1 、1200ng.mL -1 Etc.
The volume ratio of the quality control working solution to the blank plasma is less than or equal to 1:19, for example, the volume ratio of the quality control working solution to the blank plasma is 1:19, 1:20, 1:21, 1:22, etc., and the dilution ratio of the plasma matrix is not more than 5%.
In some embodiments, the detection method further comprises pretreatment of a blank sample, the pretreatment method of the blank sample comprising the steps of:
providing a blank sample, adding a diluent into the blank sample, diluting to a proper concentration, and then adding a precipitator for protein precipitation to obtain a blank sample mixed solution;
and (3) carrying out solid-liquid separation treatment on the blank sample mixed solution, and taking clear liquid to obtain a blank control sample.
The blank sample of the embodiment of the application refers to blood plasma without teprenone, only diluent is added during pretreatment, no internal standard working solution is added,
in some embodiments, after the pretreatment, the method further comprises the following steps:
s3: taking internal standard samples with different concentrations, performing LC-MS/MS chromatographic detection to obtain a standard map, and performing linear regression to obtain a linear regression equation and a correlation coefficient of teprenone in blood plasma;
s4: and carrying out LC-MS/MS chromatographic detection on the internal standard sample to be detected under the same detection condition as the internal standard sample to obtain a mass spectrum analysis result of the internal standard sample to be detected, and determining the content of teprenone in the internal standard sample to be detected.
In some embodiments, in LC-MS/MS chromatographic detection, the detection conditions for liquid chromatography are as follows:
chromatographic column: waters ACQUITY UPLC Protein BEH C4;
autoinjector temperature: 4-10 ℃;
column temperature: 30-50 ℃;
flow rate: 0.3mL min -1 -0.4mL·min -1 ；
Sample injection amount: 15 mu L-20 mu L;
mobile phase a phase: an aqueous solution of an organic acid having a pH of 4 to 5, the organic acid being a volatile acid;
mobile phase B phase: methanol acetonitrile solution with volume concentration of 30-60%;
the elution mode is gradient elution.
The teprenone is a small molecular compound, a chromatographic column for detecting small molecular organic matters is generally adopted, but the polarity of a sample is small, the adsorption of the chromatographic column for detecting the small molecular organic matters is strong, the retention time of teprenone on the chromatographic column is long, the teprenone is difficult to elute, and a high-content organic phase is required to elute, so that the content of the organic phase of an eluate is high, impurities are easy to generate, interference is easy to generate, response is inhibited, the detection time is long, and the detection efficiency is reduced.
In the research process, the chromatographic column for detecting macromolecular organic matters such as polypeptide and protein is adopted, for example, the particle size of Waters ACQUITY UPLC Protein BEH C and Waters ACQUITY UPLC Protein BEH C4 is 1.7 mu m, the inner diameter is 2.1mm, the length is 150mm, the adsorption of the chromatographic column to a sample is small, the elution speed is high, a small amount of organic phase is adopted for eluting, the interference of the organic phase is reduced, the detection time is shortened, and the detection efficiency is improved.
In some embodiments, the gradient elution procedure comprises:
providing a mobile phase A phase solution with the mass percentage of 35%, providing a mobile phase B phase solution with the mass percentage of 65%, and performing first gradient elution at 0-0.70 min;
providing a mobile phase A phase solution with the mass percentage of 35%, providing a mobile phase B phase solution with the mass percentage of 65%, and performing second gradient elution in 0.70-3.00 min;
providing a mobile phase A phase solution with the mass percentage of 5%, providing a mobile phase B phase solution with the mass percentage of 95%, and performing third gradient elution within 3.00-3.50 min;
providing a mobile phase A phase solution with the mass percentage of 5%, providing a mobile phase B phase solution with the mass percentage of 95%, and performing fourth gradient elution within 3.50-3.51 min;
providing 35% mobile phase A phase solution and 65% mobile phase B phase solution, and performing fifth gradient elution at 3.51-4.50 min.
Alternatively, the liquid chromatograph employs an shimadzu Exion LC AD.
In some embodiments, in LC-MS/MS chromatographic detection, the detection conditions for mass spectrometry are as follows:
ion source: an electrospray ion source;
ion source temperature: 400 ℃;
source injection voltage: 5000V;
inlet voltage: 10V;
collision gas: 10psi;
air curtain gas: 25psi;
ion source gas 1:75psi;
ion source gas 2:50.0psi;
ionization mode: a positive ion;
signal acquisition mode: MRM;
ion pair:
| ion pair | Q1(Da) | Q3(Da) | Declustering voltage (V) | Collision energy (V) | Scanning time (ms) |
| Object to be measured | 331.200 | 231.300 | 60.000 | 19.500 | 300.0 |
| Internal standard | 334.200 | 234.300 | 55.000 | 16.000 | 300.0 |
。
Alternatively, the mass spectrometer employs Sciex Triple Quad 6500 + 。
In some embodiments, the plasma sample may be replaced by a serum sample or a cerebrospinal fluid sample, that is, the detection method according to the embodiments of the present application is also suitable for detecting teprenone in the serum sample and the cerebrospinal fluid sample, and a detection result with high response sensitivity, high accuracy, and reliability may be obtained.
The embodiment of the application also provides a test kit of the teprenone based on the test method of the teprenone, and the test kit comprises a teprenone standard substance, an isotope internal standard substance, a precipitator and a diluent.
The test kit of the teprenone provided by the embodiment of the application is provided with the teprenone standard substance, the isotope internal standard substance, the precipitator and the diluent, provides necessary chemical substances for establishing a standard curve by adopting an internal standard method, can be used for preparing the required standard liquid concentration according to the detection requirement when being applied to the detection of the teprenone content in a biological sample, and establishes a standard curve in a proper range.
The following is illustrated by examples.
Example 1
The detection kit of the embodiment comprises a standard substance of teprenone and teprenone- 13 C 3 Standard substance, precipitant and diluent, the precipitant is 5% methanol acetonitrile solution, the diluent is 80% acetonitrile water solution.
The standard sample and the quality control sample of the embodiment are prepared from a teprenone standard in a kit.
Teprenone-containing compositions 13 C 3 The standard substance is TEP- 13 C 3 YYMDD-IS-02, and the internal standard working solution adopts teprenone in the kit 13 C 3 The concentration of the internal standard working solution is 80.000ng.mL -1 。
The sample to be tested is plasma.
The detection method of teprenone in the embodiment comprises the following steps:
s1: preparing standard sample
Taking a teprenone standard substance and a diluent to prepare 1mg.mL -1 To a stock solution of a standard teprenone.
Taking a proper amount of teprenone standard stock solution into a polypropylene EP tube, and serial dilution by taking an 80% acetonitrile aqueous solution as a solvent to obtain 8 standard curve working solutions containing teprenone, wherein the concentration of the 8 standard curve working solutions is 15ng.mL respectively -1 、30ng.mL -1 、90ng.mL -1 、180ng.mL -1 、360ng.mL -1 、720ng.mL -1 、1360ng.mL -1 、1500ng.mL -1 ；
Working solution according to a standard curve: blank plasma = 1:19 Diluting and preparing standard samples in a mode of (volume ratio) concentration of 0.75, 1.5, 4.5, 9.0, 18.0, 36.0, 68.0 and 75.0ng.mL -1 。
S2: preparing quality control samples
Taking a stock solution of a teprenone standard substance and a diluent, preparing 6 parts of teprenone quality control samples LLOQ, LQC, LMQC, HMQC, HQC, wherein the teprenone concentration in LLOQ, LQC, LMQC, HMQC, HQC teprenone quality control samples is 0.75ng.mL in sequence -1 、2.0ng.mL -1 、5.0ng.mL -1 、25.0ng.mL -1 、60.0ng.mL -1 。
S3: sample pretreatment
S31: and (3) respectively taking 140 mu L of the sample to be detected, the standard sample obtained in the step S1 and the quality control sample obtained in the step S2 into a polypropylene EP tube, respectively adding 50 mu L of an internal standard working solution, and then adding 300 mu L of a 5% methanol acetonitrile solution to correspondingly obtain an internal standard sample mixed solution, an internal standard sample mixed solution and an internal standard quality control sample mixed solution.
S32: a140. Mu.L blank plasma sample was placed in a polypropylene EP tube, 50. Mu.L of 80% acetonitrile aqueous solution was added, and 300. Mu.L of 5% methanol acetonitrile solution was further added to obtain a blank plasma sample mixture.
S33: respectively oscillating the internal standard sample mixed solution to be detected, the internal standard sample mixed solution, the internal standard quality control sample mixed solution and the blank plasma sample mixed solution for 4min, and then centrifugally separating the sample mixed solution, wherein the parameters of the centrifugal separation are as follows: and (3) taking clear liquid at RT (reverse transcription) of 1840 g for 6min, and respectively obtaining clear liquid of an internal standard sample to be detected, clear liquid of an internal standard sample, clear liquid of an internal standard quality control sample and clear liquid of a blank plasma sample.
S4: detection of samples Using LC-MS/MS
And respectively taking 300 mu L of internal standard sample clear liquid to be detected, an internal standard sample clear liquid, an internal standard quality control sample clear liquid and a blank plasma sample clear liquid, putting the sample clear liquid into a sample injector, and sampling 20 mu L.
LC-MS/MS detection parameters:
wherein the liquid chromatograph: shimadzu Exion LC AD; chromatographic column: waters ACQUITY UPLC Protein BEH C4; autoinjector temperature: 4 ℃; column temperature: 50 ℃; flow rate: 0.4mL. Min -1 The method comprises the steps of carrying out a first treatment on the surface of the Sample injection amount: 20. Mu.L; mobile phase a phase: 0.1% formic acid in water; mobile phase B phase: 50% methanol acetonitrile solution; gradient elution, gradient is shown in table 1.
TABLE 1
Detection result calculation and statistics mode
And (3) sample clear liquid is injected into a mass spectrum system for analysis, the response of the object to be detected and the internal standard is recorded, and the response ratio of the object to be detected and the internal standard is calculated. And (3) carrying out linear regression by taking the response ratio (Y) of the teprenone and the internal standard thereof as an ordinate and taking the serial standard plasma sample concentration (X) as an abscissa, so as to obtain a linear regression equation and a correlation coefficient. Substituting the response ratio of the to-be-detected object in the series of standard plasma samples to the internal standard into a linear regression equation for calculation, and comparing the concentration value obtained by testing the standard curve series concentration sample with the marked concentration value.
Establishing a standard curve
And (3) injecting clear liquid of the internal standard sample into a mass spectrum system for analysis, recording the response of the object to be detected and the internal standard, and calculating the response ratio of the object to be detected and the internal standard. The response ratio (Y) of teprenone to its internal standard is taken as the ordinate, the serial standard plasma sample concentration (X) is taken as the abscissa, and the weight factor (1/X) is used 2 ) And (3) carrying out linear regression by a least square method to obtain a linear regression equation and a correlation coefficient, and comparing a concentration value obtained by testing the standard curve series concentration sample with a marked concentration value.
The quantitative standard curve is shown in figure 1, and the result shows that the linear range of the kit for detecting the teprenone in human plasma can reach 0.75ng.mL -1 -75ng.mL -1 Meets the linear acceptance standard and has good linear quantification effect.
Accuracy and precision verification of kit
And (3) feeding the clear liquid of the internal standard quality control sample into a mass spectrum system for analysis, recording the ratio of the object to be detected to the response of the internal standard, substituting the ratio into a corresponding obtained linear regression equation, calculating the concentration of each sample, comparing the concentration of each sample with a sample marking value, and calculating the precision and accuracy in the batch according to the concentration.
Acceptance criteria: the accuracy of the LLOQ measured concentration mean value is in the range of 80% -120% of the standard value, and at least 2/3 sample accuracy deviation is within +/-20% of the standard value; the accuracy of the average value of the measured concentration of other standard urine samples with quality control concentration is in the range of 85% -115% of the standard value, and the accuracy deviation of each concentration at least 2/3 sample is within +/-15% of the standard value; the lower limit precision of the quantitative determination is not more than 20 percent, and the precision of other quality control concentrations is not more than 15 percent.
The results of the accuracy and precision verification experiments of this example are shown in table 2.
TABLE 2
The result shows that the kit repeatedly measures 5 quality control samples with different concentrations for 6 times, the mean deviation of the accuracy of each concentration of the teprenone is within +/-15% of the marked value, and the kit meets the acceptance standard, and the detection method and the kit are qualified in accuracy. In the 6 measurement results of each quality control product, the precision of the teprenone is less than or equal to 15 percent, and meets the acceptance standard, and the precision of the method and the kit is qualified.
Recovery evaluation
Taking blank plasma, preparing 6 parts of 3 quality control standard plasma samples (LQC, HMQC, HQC) respectively, wherein the concentration is 2.0ng.mL -1 、25.0ng.mL -1 、60.0ng.mL -1 According to the sample pretreatment method described in the embodiment, the quality control standard working solution with the corresponding concentration is obtained respectively. And (3) injecting 20 mu L of each quality control standard working solution to a chromatographic/mass spectrometry system for analysis, and recording the response (A) of the teprenone as the area after extraction of the to-be-detected substance.
Blank plasma was taken and treated as follows, except without analyte and internal standard: taking 140. Mu.L of blank plasma, adding 50. Mu.L of 80% acetonitrile aqueous solution and 300. Mu.L of 5% methanol acetonitrile solution, oscillating for 4min, centrifuging (RT, 1840 g,6 min),taking the supernatant to obtain an empty matrix extract. Respectively taking 7 mu L of quality control standard working solution with low concentration, high medium concentration and high concentration, and adding 5 mu L of internal standard working solution (teprenone- 13 C 3 ，800.000ng.mL -1 ) And 478 mu L of blank matrix extracting solution, and oscillating for 4min to obtain an internal standard quality control blank matrix sample with corresponding concentration. And (3) injecting 20 mu L of an internal standard quality control blank matrix sample with each concentration into a chromatographic/mass spectrometry system for analysis, and recording the response (B) of the teprenone as the unextracted area of the to-be-detected object.
The recovery rate of the teprenone in the quality control standard plasma sample is R 1 ＝A/B×100％。
The recovery rate results are shown in Table 3, the recovery rate of the teprenone with low, medium and high 3 quality control concentrations after protein precipitation extraction is 102.02% -102.25%, and the average recovery rate is 102.16%.
TABLE 3 evaluation of recovery of plasma sample samples by protein precipitation (ng.mL) -1 )
Whole blood stability evaluation
Fresh whole blood is used as a whole blood stability investigation matrix, 6 parts of quality control standard whole blood samples (LQC and HQC) are prepared, and the concentrations are 2.0ng.mL respectively -1 、60ng.mL -1 After standing at room temperature for 0h and 2h, respectively, centrifuging (1200 g, 2-8deg.C, 10 min) to obtain plasma, and processing the plasma sample according to the pretreatment method of this example to obtain whole blood quality control sample. The whole blood quality control samples of each concentration were sampled at 10 μl to chromatography/mass spectrometry system analysis. The responses of teprenone and the corresponding internal standard were recorded, and the detection results are shown in Table 4.
After 2h of standing, the sample to be tested and the internal standard response ratio of the sample to be tested are centrifugally separated and are kept for 0h, and the deviation of the response ratio is-1.77% -0.65%, which means that the sample to be tested in the embodiment can be kept in the whole blood matrix for at least 2h at room temperature. The detection method and the kit have remarkable technical innovation advantages and practical application value.
TABLE 4 stabilization of teprenone in Whole blood matricesSex (ng.mL) -1 )
Selectivity evaluation
The selection was investigated using 6 different sources of normal blank plasma, 1 source of high fat blank plasma and 1 source of hemolyzed blank plasma to prepare blank plasma samples and a lower limit of quantitation plasma sample, each source of plasma was prepared in parallel with 1 portion of each of the blank plasma samples and the lower limit of quantitation sample.
Acceptance criteria: the peak area of the interfering component at the retention time of the analyte in the blank plasma sample is less than 20% of the peak area of the analyte in the corresponding lower limit sample of the blank plasma quantification, and the peak area of the interfering component at the internal standard retention time is less than 5% of the peak area of the internal standard in the corresponding lower limit sample of the blank plasma quantification. Of the 6 individual sources of normal blank plasma formulated lower limit sample, at least 2/3 of the lower limit sample has an accuracy within + -20% of the labeled value and a precision of no more than 20%; the accuracy of the quantitative lower limit sample prepared from the high-fat blank plasma is within +/-20% of the marked value; the accuracy of the lower limit sample of the hemolysis blank plasma preparation is within + -20% of the marked value.
The results show that the interference of the blank matrix on the object to be detected and the internal standard is smaller than the corresponding acceptance standard, which indicates that the method can effectively avoid the interference in the blank matrix. Typical blank matrix sample chromatograms and lower limit quantitative chromatograms are shown in fig. 2 (a), fig. 2 (b), fig. 3 (a) and fig. 3 (b).
The foregoing description of the preferred embodiments of the application is not intended to limit the application to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the application.
Claims (10)
1. A detection method of teprenone is characterized in that: comprising a pretreatment comprising the steps of:
providing a plasma sample containing teprenone, mixing the plasma sample with an internal standard working solution, and then adding a precipitant, wherein the precipitant comprises methanol and acetonitrile, and performing protein precipitation to obtain an internal standard mixed solution;
and (3) carrying out solid-liquid separation treatment on the internal standard mixed solution, and taking clear liquid to obtain an internal standard sample.
2. The method for detecting teprenone according to claim 1, wherein: the plasma sample comprises a sample to be tested and standard samples with different concentrations, and the pretreatment comprises the following steps:
mixing the sample to be measured and the standard sample with the internal standard working solution respectively, then adding the precipitant respectively, and carrying out protein precipitation to obtain an internal standard mixed solution to be measured and an internal standard mixed solution respectively;
and respectively carrying out solid-liquid separation treatment on the internal standard mixed solution and the internal standard mixed solution, and taking clear liquid to respectively obtain an internal standard sample to be detected and an internal standard sample.
3. The method for detecting teprenone according to claim 2, wherein: after the pretreatment, the method further comprises the following steps:
taking the internal standard samples with different concentrations, performing LC-MS/MS chromatographic detection to obtain a standard map, and performing linear regression to obtain a linear regression equation and a correlation coefficient of teprenone in blood plasma;
and carrying out LC-MS/MS chromatographic detection on the internal standard sample to be detected under the same detection condition as the internal standard sample to obtain a mass spectrometry analysis result of the internal standard sample to be detected, and determining the content of teprenone in the internal standard sample to be detected.
4. A method for detecting teprenone according to claim 3, wherein: in the LC-MS/MS chromatographic detection, the detection conditions of the liquid chromatograph are as follows:
chromatographic column: waters ACQUITY UPLC Protein BEH C4;
autoinjector temperature: 4-10 ℃;
column temperature: 30-50 ℃;
flow rate: 0.3mL. Min -1 -0.4mL.min -1 ；
Sample injection amount: 15 mu L-20 mu L;
mobile phase a phase: an aqueous solution of an organic acid having a pH of 4 to 5, the organic acid being a volatile acid;
mobile phase B phase: methanol acetonitrile solution with volume concentration of 30-60%;
the elution mode is gradient elution; and/or the number of the groups of groups,
in the LC-MS/MS chromatographic detection, the detection conditions of the mass spectrum are as follows:
ion source: an electrospray ion source;
ion source temperature: 400 ℃;
source injection voltage: 5000V;
inlet voltage: 10V;
collision gas: 10psi;
air curtain gas: 25psi;
ion source gas 1:75psi;
ion source gas 2:50.0psi;
ionization mode: a positive ion;
signal acquisition mode: MRM;
ionization mode: a positive ion;
ion pair:
。
5. The method for detecting teprenone according to claim 4, wherein: the gradient elution procedure includes:
providing a mobile phase A phase solution with the mass percentage of 35%, providing a mobile phase B phase solution with the mass percentage of 65%, and performing first gradient elution at 0-0.70 min;
providing a mobile phase A phase solution with the mass percentage of 35%, providing a mobile phase B phase solution with the mass percentage of 65%, and performing second gradient elution in 0.70-3.00 min;
providing a mobile phase A phase solution with the mass percentage of 5%, providing a mobile phase B phase solution with the mass percentage of 95%, and performing third gradient elution within 3.00-3.50 min;
providing a mobile phase A phase solution with the mass percentage of 5%, providing a mobile phase B phase solution with the mass percentage of 95%, and performing fourth gradient elution within 3.50-3.51 min;
providing 35% mobile phase A phase solution and 65% mobile phase B phase solution, and performing fifth gradient elution at 3.51-4.50 min.
6. The method for detecting teprenone according to claim 2, wherein: the standard samples with different concentrations comprise the concentration of 0.75ng.mL -1 -75.0ng.mL -1 Is a standard sample of (2); and/or the number of the groups of groups,
the preparation method of the standard samples with different concentrations comprises the following steps:
providing a teprenone reference stock solution, and diluting with a diluent to obtain standard curve working solutions with different concentrations;
and mixing the standard curve working solutions with different concentrations with blank plasma according to the volume ratio of less than or equal to 1:19 to obtain standard samples with different concentrations.
7. The method for detecting teprenone according to claim 6, wherein: the standard curve working solution with different concentrations comprises 15ng.mL of concentration -1 -1500ng.mL -1 Standard curve working solution of the teprenone standard substance; and/or the number of the groups of groups,
the diluent comprises an acetonitrile aqueous solution with the mass percentage concentration of 60-80%.
8. The method for detecting teprenone according to any one of claims 1 to 7, characterized in that: the pretreatment satisfies at least one of the following conditions:
the concentration of the internal standard substance in the internal standard working solution is 0.5ng.mL -1 -0.8ng.mL -1 ；
The internal standard substance of the internal standard working solution comprises TEP- 13 C 3 -YYMMDD-IS-02；
The precipitant is methanol acetonitrile solution with volume concentration of 5% -8%;
the solid-liquid separation treatment comprises centrifugal separation; and/or the number of the groups of groups,
the detection method also comprises the pretreatment of a blank sample, and the pretreatment method of the blank sample comprises the following steps:
providing the blank sample, adding a diluent into the blank sample, diluting to a proper concentration, then adding the precipitator, and carrying out protein precipitation to obtain a blank sample mixed solution;
performing solid-liquid separation treatment on the blank sample mixed solution, and taking clear liquid to obtain a blank control sample; and/or the number of the groups of groups,
the plasma sample is replaced with a serum sample or a cerebrospinal fluid sample.
9. The method for detecting teprenone according to claim 8, wherein: the volume ratio of the plasma sample to the precipitant is 7:15-20.
10. A test kit of teprenone based on the test method of teprenone according to any one of claims 1 to 9, characterized in that: the detection kit comprises a teprenone standard substance, an isotope internal standard substance, a precipitant and a diluent.
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