CN114994215A - Application of chlorzoxazone, method for detecting concentration of enzyme inhibitor antibiotic drug, kit and application - Google Patents
Application of chlorzoxazone, method for detecting concentration of enzyme inhibitor antibiotic drug, kit and application Download PDFInfo
- Publication number
- CN114994215A CN114994215A CN202210928389.0A CN202210928389A CN114994215A CN 114994215 A CN114994215 A CN 114994215A CN 202210928389 A CN202210928389 A CN 202210928389A CN 114994215 A CN114994215 A CN 114994215A
- Authority
- CN
- China
- Prior art keywords
- concentration
- internal standard
- enzyme inhibitor
- detecting
- chlorzoxazone
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The application relates to the technical field of blood concentration monitoring, in particular to application of chlorzoxazone, a method for detecting concentration of enzyme inhibitor antibiotic drugs, a kit and application. The detection method and the kit provided by the application have the advantages that the property of the compound is known, chlorzoxazone is selected as an internal standard and used for calibrating deviation generated in sample extraction, HPLC sample injection, chromatographic separation and detection, so that accurate quantification is realized, the problem that the internal standard of the existing enzyme inhibitor compound is lack or the internal standard of an isotope is expensive is solved, and the method and the kit can be used for monitoring the blood concentration of the compound.
Description
Technical Field
The application relates to the technical field of blood concentration monitoring, in particular to application of chlorzoxazone, a method for detecting concentration of enzyme inhibitor antibiotic drugs, a kit and application.
Background
Currently, the common analytical methods for Therapeutic Drug Monitoring (TDM) mainly include immunological methods, High Performance Liquid Chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS/MS). Among them, the conventional analytical technique of TDM mainly includes an immunological method. The immunological method has the advantages of high sensitivity, high analysis speed, high automation degree, simple and convenient operation and the like, is suitable for measuring a large number of samples, but is limited by the types of drug antibodies, most TDM drugs do not have proper antibody supply, and a plurality of drugs or metabolites can not be measured simultaneously. The HPLC has the advantages of strong specificity, wide linear range, relatively good stability, low cost, high popularization rate and the like, but the sensitivity of the HPLC is low, the sample pretreatment analysis period is long, and the simultaneous detection of various substances cannot be considered sometimes. LC-MS/MS has the advantages of high sensitivity, strong specificity, high analysis speed, high flux and the like, and becomes a gold standard for biological sample analysis and quantification.
In the related technology, the LC-MS/MS mainly adopts an isotope internal standard method and an isotope external standard method for quantitative detection in TDM. However, when the blood concentration in human serum is detected by using an isotope internal standard method, an isotope internal standard compound is needed, on one hand, because the synthesis cost of the stable isotope internal standard compound is too high or the synthesis difficulty is too large, commercialization is difficult to realize, and no commercialized isotope internal standard compound exists in most blood concentration monitoring; on the other hand, when the blood concentration in human serum is detected, most of the blood concentration is higher, and for the existence of the commercialized isotope internal standard compound, the usage amount of the isotope internal standard compound in the blood concentration monitoring process is larger, so that expensive cost is caused for the blood concentration monitoring. When the blood concentration in human serum is quantitatively detected by an external standard method, sample purification is required in the early detection stage, protein in the serum is removed by using a protein precipitation method (PPT) in the sample purification, and then multi-step dilution is carried out to carry out sample injection detection; however, the multistep dilution introduces a large error, so that the quantitative result of the blood concentration in human serum is inaccurate, and great difficulty is caused to the quantitative detection of an external standard method.
Disclosure of Invention
In order to provide a detection method which is low in cost and can be used for simultaneously detecting the concentration of a plurality of enzyme inhibitor antibiotic medicines, application of chlorzoxazone, a method for detecting the concentration of the enzyme inhibitor antibiotic medicines, a kit and application.
In a first aspect, the present application provides the use of chlorzoxazone as an internal standard compound in a method for detecting the concentration of an enzyme inhibitor antibiotic drug.
Preferably, the enzyme inhibitor antibiotic drug is selected from tazobactam and sulbactam.
Preferably, the chlorzoxazone can be used for simultaneously detecting two or more enzyme inhibitor antibiotic drugs.
In the detection of the drug concentration, an internal standard method is generally used. Isotopically internal standard compounds (internal standards) are used as a chemical species and are generally closely related to the compound to be detected. In the internal standard method, a sample is a sample to be detected, and a compound to be detected exists in the sample; samples were prepared after a series of treatments. And a proper internal standard is added in the sample preparation process, so that the quantitative accuracy of the compound to be detected can be improved. Before the analysis starts, an internal standard is added to a standard curve solution prepared by a sample and a specific matrix according to a known fixed concentration, and then sample processing is carried out, so that a sample to be detected is obtained and is detected on a mass spectrometer. The suitability of the internal standard can compensate for deviations generated in sample extraction, HPLC sample introduction, and chromatographic separation and detection. The internal standard is considered to correct for the effects of the matrix effect if the matrix of the standard curve is different from the matrix of the sample. The normalized response of the compound to be detected is obtained by the ratio of the response of the compound to be detected (expressed as peak area) to the response of the internal standard. And fitting the analyte with known concentration and the ratio of the analyte to the internal standard response in the standard curve to the standard curve, and calculating the concentration of the compound to be detected in the sample according to the response value of the unknown sample.
In a second aspect, the present application provides a method for detecting the concentration of an enzyme inhibitor antibiotic drug, which adopts the following technical scheme:
a method for detecting the concentration of an enzyme inhibitor antibiotic drug, which comprises the steps of preparing an internal standard working solution; the internal standard compound in the internal standard working solution is chlorzoxazone.
Preferably, the enzyme inhibitor antibiotic drug is at least one selected from tazobactam and sulbactam.
Preferably, the method for detecting the concentration of the drug is performed by LC-MS/MS.
Preferably, the chromatographic conditions of the LC-MS/MS are as follows:
a chromatographic column: waters acquisition UPLC BEH C8, 2.1x50mm, particle size 1.7 μm;
mobile phase A: water containing 0.1% formic acid;
mobile phase B: methanol with 0.1% formic acid: the volume ratio of acetonitrile is 1: 1;
flow rate: 0.35-0.45 mL/min;
sample introduction volume: 4-6 μ L;
column temperature: 35-45 ℃;
the mass spectrum conditions of the LC-MS/MS are as follows:
the ion source is an ESI-ion source:
the detection mode is negative ion detection;
the scanning mode is multi-reaction monitoring MRM.
Preferably, the gradient elution procedure is as follows:
preferably, the concentration of the internal standard compound in the internal standard working solution is 150 ng/mL.
In one embodiment, the internal standard working solution is specifically formulated as follows: weighing 10.000mg of the standard internal standard compound in a glass sample bottle, adding 50% methanol aqueous solution, and obtaining internal standard stock solution with the concentration of 1.000mg/mL after complete dissolution. Taking a proper amount of internal standard stock solution, diluting the internal standard stock solution with methanol, and preparing into an internal standard working solution with the concentration of 150 ng/mL.
In a third aspect, the present application provides a kit for detecting the concentration of an enzyme inhibitor antibiotic drug, which adopts the following technical scheme:
a kit for detecting the concentration of an enzyme inhibitor antibiotic drug, which comprises an internal standard compound of chlorzoxazone.
Preferably, the kit also comprises reagents necessary for detecting the concentration of the drug.
Preferably, the enzyme inhibitor antibiotic drug is at least one selected from tazobactam and sulbactam.
In a fourth aspect, the application relates to a kit for detecting the concentration of an enzyme inhibitor antibiotic drug.
Preferably, the kit is applied to detecting the concentration of the enzyme inhibitor antibiotic drugs in human serum.
In summary, the present application has the following beneficial effects:
the application finds the application of chlorzoxazone as an internal standard substance in detecting the concentration of an enzyme inhibitor antibiotic drug. The application finds that when chlorzoxazone is used as an internal standard substance to detect the concentration of the enzyme inhibitor antibiotic drugs, the deviation generated in sample extraction, HPLC sample injection, chromatographic separation and detection can be calibrated, so that accurate quantification is realized, the blank that the existing enzyme inhibitor compounds lack the internal standard substance is filled, in addition, chlorzoxazone is cheap compared with the expensive existing isotope internal standard substance, and the cost of the internal standard substance is reduced.
Drawings
FIG. 1 is a chromatogram of chlorzoxazone.
Fig. 2 is a chromatogram of flurbiprofen.
FIG. 3 is a chromatogram showing the concentration of tazobactam of 10.54. mu.g/ml (0 deviation) in example 1.
FIG. 4 is a chromatogram of sulbactam concentration of 89.82 μ g/ml (0 deviation) in example 2.
FIG. 5 is a chromatogram for simultaneous detection of multiple drug classes (tazobactam concentration of 10.54. mu.g/ml (0 bias) and sulbactam concentration of 89.82. mu.g/ml (0 bias)) in example 3.
Detailed Description
In methods for detecting drug concentration (e.g., LC-MS/MS detection), exogenously ionization-stable compounds can be selected as internal standard compounds based on their polarity and solubility to compensate for variations in sample extraction, HPLC sampling, and chromatographic separation and detection.
According to the method, a substitute internal standard compound, namely Chlorzoxazone (Chlorzoxazone), which is stable in ionization and can be used for TDM is screened out according to the polarity and the solubility of the compound, so that the problems that the internal standard compound is lacked, the price of the isotope internal standard compound is high and the like in the TDM detection process are solved.
The application provides the application of chlorzoxazone as an internal standard compound in a method for detecting the concentration of an enzyme inhibitor antibiotic drug (particularly an LC-MS/MS detection method). The enzyme inhibitor is selected from tazobactam and sulbactam. The chlorzoxazone can be used for simultaneously detecting multiple (such as two, three or more) enzyme inhibitor antibiotic drugs, can be simultaneously used for detecting multiple compounds, and can meet the clinical combined medication requirement. In addition, chlorzoxazone can also be used in other assay items, depending on the nature of the compound to be detected.
In the related technology, tazobactam-D4 is used as an isotope internal standard substance of tazobactam, and sulbactam-D3 is used as an isotope internal standard substance of sulbactam. However, tazobactam-D4 and sulbactam-D3 are both expensive.
The method and the kit for detecting the concentration of the drug based on LC-MS/MS can be suitable for detecting compounds such as enzyme inhibitors at present, and can be widely applied to other therapeutic drugs with similar solubility and polarity. The detection method can be used for sample types such as serum, plasma, whole blood and the like.
Reagents such as methanol (chromatographically pure), acetonitrile (chromatographically pure), formic acid (MS grade) and the like, which are referred to in the present application, are commercially available.
The application also provides a method for detecting the concentration of the enzyme inhibitor antibiotic drugs based on LC-MS/MS. The method specifically comprises the following steps:
first, solution preparation
(1) Preparation of internal standard working solution
Precisely weighing 10.000mg of the standard internal standard compound in a glass sample bottle, adding 50% methanol aqueous solution, and obtaining internal standard stock solution with the concentration of 1.000mg/mL after complete dissolution. Taking a proper amount of internal standard stock solution, diluting the internal standard stock solution with methanol, and preparing internal standard working solution with the concentration of 150 ng/mL.
(2) Treatment of samples to be tested
The sample to be tested is serum obtained by treating human whole blood after oral administration or intravenous injection of a drug to be tested with a yellow separation gel conventional serum blood collection tube, separating, storing in a refrigerator at-80 deg.C, and naturally thawing at room temperature before use.
Taking 50 mu L of a sample to be detected, putting the sample into a 96-hole sample plate, adding 150 mu L of internal standard working solution (150 ng/mL), vortexing for 10min, centrifuging for 10min at 4000g and 4 ℃, taking 20 mu L of supernatant, adding 180 mu L of ultrapure water, mixing uniformly, and then injecting a sample for LC-MS/MS analysis.
(3) Standard Curve sample and quality control sample processing
1. Preparation of standard substance stock solution
Weighing 10mg of a drug standard to be detected in a glass sample bottle, adding 50% methanol solution, and preparing into a standard stock solution with the concentration of 1 mg/mL. The stock solution of the standard was vortexed thoroughly until completely dissolved and stored at-20 ℃ as a stock solution of the linear standard.
The above operations were repeated to prepare a stock solution of standard substance having a concentration of 1 mg/mL. And fully swirling the standard substance stock solution until the standard substance stock solution is completely dissolved, and storing the standard substance stock solution at-20 ℃ to serve as a quality control sample stock solution.
2. Preparation of standard curve working solution and quality control working solution
Standard curve working solution: diluting the linear standard sample stock solution prepared in the step 1 into 8 concentration points by using methanol to prepare a series of standard curve working solutions with the concentrations of 5.80 mu g/ml, 11.60 mu g/ml, 23.20 mu g/ml, 46.40 mu g/ml, 92.80 mu g/ml, 185.60 mu g/ml, 371.20 mu g/ml and 742.60 mu g/ml;
quality control working solution: and (2) diluting the quality control sample stock solution prepared in the step (1) by using methanol to prepare a series of quality control working solutions with the concentrations of 210.8 mu g/ml and 575.8 mu g/ml.
3. Preparation of standard curve sample and quality control serum sample
Standard curve sample: adding 10 μ L of each standard curve working solution into blank human serum matrix to obtain a series of standard curve samples with concentrations of 0.29 μ g/ml, 0.58 μ g/ml, 1.16 μ g/ml, 2.32 μ g/ml, 4.64 μ g/ml, 9.28 μ g/ml, 18.56 μ g/ml and 37.13 μ g/ml;
quality control of serum samples: adding 10 mu L of each quality control working solution into a blank biological matrix to obtain quality control samples, wherein the quality control samples comprise high-value samples (28.79 mu g/ml) and low-value samples (10.54 mu g/ml);
wherein the blank biological matrix is obtained by treating whole blood of healthy people with yellow separating gel conventional serum blood collecting tube, separating to obtain serum, storing in a refrigerator at-80 deg.C, and naturally thawing at room temperature before use.
4. Standard Curve sample and quality control sample processing
And (3) putting 50 mu L of each standard curve sample and quality control sample into a 96-hole sample plate, adding 150 mu L of internal standard working solution (150 ng/mL), vortexing for 10min, centrifuging for 10min at 4000g and 4 ℃, taking 20 mu L of supernatant, adding 180 mu L of ultrapure water, mixing uniformly, and then injecting a sample for LC-MS/MS analysis.
Second, detecting the conditions
Using a SCIEX Triple Quad TM 4500 detection by liquid chromatography tandem mass spectrometry (SCIEX, USA).
(1) The chromatographic conditions were as follows:
a chromatographic column: waters acquisition UPLC BEH C8, 2.1x50mm, particle size 1.7 μm;
mobile phase A: water containing 0.1% formic acid;
mobile phase B: methanol with 0.1% formic acid: the volume ratio of acetonitrile is 1: 1;
flow rate: 0.35-0.45 mL/min; sample introduction volume: 4-6 μ L; column temperature: 35-45 ℃;
the gradient elution procedure is shown in table 1.
TABLE 1 gradient elution procedure in chromatographic conditions for LC-MS/MS
(2) The mass spectrometry conditions are shown in table 2.
TABLE 2 Mass Spectrometry parameters of LC-MS/MS
Third, the detection result
And (4) carrying out data processing by using the MultiQuant MD to obtain a detection result.
In addition, the application also provides a kit for detecting the concentration of the enzyme inhibitor antibiotic drugs by using the method, and the kit comprises an internal standard compound chlorzoxazone and reagents necessary for detecting the drug concentration. The reagents necessary for detecting the concentration of the drug comprise calibrators, quality control substances, methanol, acetonitrile, formic acid and the like with different concentrations.
The application provides an application of the kit in detecting the concentration of the enzyme inhibitor antibiotic drugs. Specifically, the kit is applied to detection of the concentration of enzyme inhibitor antibiotic drugs in human serum.
The present application will be described in further detail with reference to preparation examples and examples 1 to 3.
Preparation example
The preparation example provides a screening process of an internal standard compound capable of replacing an internal standard in LC-MS/MS detection.
Among the alternative internal standard compounds are chlorzoxazone, flurbiprofen and kanamycin.
The screening process of the internal standard compound is as follows:
first, solution preparation
(1) Preparation of internal standard working solution
Precisely weighing 10.000mg of the alternative internal standard compound standard substitute into a glass sample bottle, adding 50% methanol aqueous solution, and dissolving completely to obtain an internal standard stock solution with the concentration of 1.000 mg/mL. Taking a proper amount of internal standard stock solution, diluting the internal standard stock solution with methanol, and preparing internal standard working solution with the concentration of 150 ng/mL.
(2) Standard curve sample and quality control sample processing
1. Preparation of standard substance stock solution
Weighing 10mg of tazobactam standard substance into a glass sample bottle, adding 50% methanol solution, and preparing into a standard substance stock solution with the concentration of 1 mg/mL. The stock solution of the standard was vortexed thoroughly until completely dissolved and stored at-20 ℃ as a stock solution of the linear standard.
The above operations were repeated to prepare a stock solution of standard substance having a concentration of 1 mg/mL. And fully swirling the standard substance stock solution until the standard substance stock solution is completely dissolved, and storing the standard substance stock solution at-20 ℃ to serve as a quality control sample stock solution.
2. Preparation of standard curve working solution and quality control working solution
Standard curve working solution: c, diluting the linear standard sample stock solution prepared in the step a by using methanol to prepare a series of standard curve working solutions with the concentrations of 5.80 mu g/ml, 11.60 mu g/ml, 23.20 mu g/ml, 46.40 mu g/ml, 92.80 mu g/ml, 185.60 mu g/ml, 371.20 mu g/ml and 742.60 mu g/ml;
quality control working solution: and (c) diluting the quality control sample stock solution prepared in the step (a) with methanol to prepare a series of quality control working solutions with the concentrations of 210.8 mu g/ml and 575.8 mu g/ml.
3. Preparation of standard curve sample and quality control serum sample
Standard curve sample: adding 10 μ L of each standard curve working solution into blank human serum matrix to obtain a series of standard curve samples with concentrations of 0.29 μ g/ml, 0.58 μ g/ml, 1.16 μ g/ml, 2.32 μ g/ml, 4.64 μ g/ml, 9.28 μ g/ml, 18.56 μ g/ml and 37.13 μ g/ml;
quality control of serum samples: adding 10 mu L of each quality control working solution into a blank biological matrix to obtain quality control samples, wherein the quality control samples comprise a high-value sample (28.79 mu g/ml) and a low-value sample (10.54 mu g/ml);
wherein the blank biological matrix is obtained by treating whole blood of healthy people with yellow separating gel conventional serum blood collecting tube, separating to obtain serum, storing in a refrigerator at-80 deg.C, and naturally thawing at room temperature before use.
4. Standard Curve sample and quality control sample processing
And (3) putting 50 mu L of each standard curve sample and quality control sample into a 96-hole sample plate, adding 150 mu L of internal standard working solution (150 ng/mL), whirling for 10min, centrifuging for 10min at 4000g and 4 ℃, taking 20 mu L of supernatant, adding 180 mu L of ultrapure water, mixing uniformly, and injecting a sample for LC-MS/MS analysis.
Second, detecting the conditions
Using a SCIEX Triple Quad TM 4500 detection by liquid chromatography tandem mass spectrometry (SCIEX, USA).
(1) The chromatographic conditions were as follows:
a chromatographic column: waters acquisition UPLC BEH C8, 2.1x50mm, particle size 1.7 μm;
mobile phase A: water containing 0.1% formic acid;
and (3) mobile phase B: methanol with 0.1% formic acid: the volume ratio of acetonitrile is 1: 1;
flow rate: 0.4 mL/min; sample introduction volume: 5 mu L of the solution; column temperature: 40 ℃;
the gradient elution procedure is shown in table 1.
(2) The mass spectrometry conditions were as shown in table 2, and data processing was performed using a multisant MD to obtain the detection results.
Third, screening the first step and screening results
And (3) testing the response of the alternative internal standard compound, the peak shape of a chromatographic peak and the like under the condition of a liquid chromatography method capable of simultaneously detecting tazobactam by using different alternative internal standard compounds. The results of the tests are shown in FIGS. 1-2.
According to the detection result, the alternative internal standard compound which does not meet the condition is kanamycin. Kanamycin is more easily positively charged and is not suitable for detecting compounds in a negative ion mode.
Fourth, screening the second step and screening results
And (3) simulating dilution errors caused by operation, and evaluating the corrective action of the substituted internal standard compounds of chlorzoxazone and flurbiprofen.
The way of simulating dilution errors caused by operation is:
50 mu L of the quality control samples (high value sample (28.79 mu g/mL) and low value sample (10.54 mu g/mL)) are respectively taken to be put in a 96-well sample plate, each concentration sample is divided into 4 parts in parallel, 150 mu L of internal standard working solution (150 ng/mL) is respectively added to be swirled for 10min, and the mixture is centrifuged for 10min at 4000g and 4 ℃; then, 15. mu.L (-25% variation), 18. mu.L (-10% variation), 20. mu.L (0 variation), 22. mu.L (+ 10% variation), and 25. mu.L (+ 25% variation) of the supernatant were taken, respectively, and 180. mu.L of ultrapure water was added thereto, mixed, and then, sample-fed for LC-MS/MS analysis under the conditions as described in step two. The results are shown in Table 3.
TABLE 3 corrective action of chlorzoxazone as a substitute internal standard compound
According to the detection results in table 3, chlorzoxazone can stably rectify the compound to be evaluated and can be used as a substitute internal standard compound of the compound to be evaluated.
The samples were tested by diluting 8 concentration points with commercial calibrators to generate a standard curve. Errors in sample handling operations under real assay conditions were simulated for high value samples and low value samples of known concentrations. An external standard method and a substitute internal standard method (the application) are respectively adopted to detect the sample, whether the precision CV meeting the requirements of clinical regulations and whether the accuracy RE is less than or equal to 15 percent or not is evaluated, and the detection effects among the methods are compared.
Examples
Example 1
The embodiment provides a method for detecting the concentration of enzyme inhibitor antibiotic drugs in human serum based on LC-MS/MS. Wherein, the drug detected in the embodiment is tazobactam, and the used substituted internal standard compound is chlorzoxazone.
The precision and accuracy of high value samples (28.79 ug/ml) and low value samples (10.54 ug/ml) of known concentrations of drug were analyzed by simulating the errors made during the sample handling operation.
The method for detecting the concentration of the enzyme inhibitor antibiotic drugs in human serum based on LC-MS/MS specifically comprises the following steps:
first, solution preparation
(1) Preparation of internal standard working solution
Precisely weighing 10.000mg of chlorzoxazone standard substance in a glass sample bottle, adding 50% methanol aqueous solution, and dissolving completely to obtain an internal standard stock solution with the concentration of 1.000 mg/mL. Taking a proper amount of internal standard stock solution, diluting the internal standard stock solution with methanol, and preparing internal standard working solution with the concentration of 150 ng/mL.
(2) Standard Curve sample and quality control sample processing
1. Preparation of standard substance stock solution
Weighing 10mg of a drug standard to be detected in a glass sample bottle, adding 50% methanol solution, and preparing into a standard stock solution with the concentration of 1 mg/mL. The stock solution of the standard was vortexed thoroughly until completely dissolved and stored at-20 ℃ as a stock solution of the linear standard.
The above operation was repeated to prepare a stock solution of 1mg/mL standard substance. And (4) fully swirling the standard substance stock solution until the standard substance stock solution is completely dissolved, storing at-20 ℃ and taking the standard substance stock solution as a quality control sample stock solution.
2. Preparation of standard curve working solution and quality control working solution
Standard curve working solution: diluting the linear standard sample stock solution prepared in the step a by using methanol to prepare a series of standard curve working solutions with the concentrations of 5.80 mu g/ml, 11.60 mu g/ml, 23.20 mu g/ml, 46.40 mu g/ml, 92.80 mu g/ml, 185.60 mu g/ml, 371.20 mu g/ml and 742.60 mu g/ml;
quality control working solution: and (b) diluting the quality control sample stock solution prepared in the step (a) with methanol to prepare a series of quality control working solutions with the concentrations of 210.8 mu g/ml and 575.8 mu g/ml.
3. Preparation of standard curve sample and quality control sample
Standard curve sample: adding 10 μ L of each standard curve working solution into blank human serum matrix to obtain a series of standard curve samples with concentrations of 0.29 μ g/ml, 0.58 μ g/ml, 1.16 μ g/ml, 2.32 μ g/ml, 4.64 μ g/ml, 9.28 μ g/ml, 18.56 μ g/ml and 37.13 μ g/ml;
quality control of serum samples: adding 10 mu L of each quality control working solution into a blank biological matrix to obtain quality control samples, wherein the quality control samples comprise high-value samples (28.79 mu g/ml) and low-value samples (10.54 mu g/ml);
wherein the blank biological matrix is obtained by treating whole blood of healthy people with yellow separating gel conventional serum blood collecting tube, separating to obtain serum, storing in a refrigerator at-80 deg.C, and naturally thawing at room temperature before use.
4. Standard curve samples and sample processing
And (3) putting 50 mu L of each standard curve sample into a 96-well sample plate, adding 150 mu L of internal standard working solution (150 ng/mL), vortexing for 10min, centrifuging for 10min at 4000g and 4 ℃, taking 20 mu L of supernatant, adding 180 mu L of ultrapure water, mixing uniformly, and then injecting a sample for LC-MS/MS analysis.
50 μ L of the above low value sample (10.54 μ g/mL) and high value sample (28.79 μ g/mL) were put in a 96-well sample plate, 150 μ L of internal standard working solution (150 ng/mL) was added, vortexed for 10min, and centrifuged at 4000g and 4 ℃ for 10min, and supernatants (dilution error due to simulation) of 15 μ L (-25% offset), 18 μ L (-10% offset), 20 μ L (0 offset), 22 μ L (+ 10% offset), and 25 μ L (+ 25% offset) were taken, 180 μ L of ultrapure water was added, mixed well, and then sample injection was performed for LC-MS/MS analysis.
Second, detecting the conditions
Using a SCIEX Triple Quad TM 4500 detection by liquid chromatography tandem mass spectrometry (SCIEX, USA).
(1) Chromatographic conditions are as follows:
a chromatographic column: waters acquisition UPLC BEH C8, 2.1x50mm, particle size 1.7 μm;
mobile phase A: water containing 0.1% formic acid;
mobile phase B: methanol with 0.1% formic acid: the volume ratio of acetonitrile is 1: 1;
flow rate: 0.4 mL/min; sample injection volume: 5 mu L of the solution; column temperature: at 40 ℃;
the gradient elution procedure is shown in table 1.
(2) The mass spectrometry conditions are shown in table 2.
Third, the detection result
And (4) carrying out data processing by using the MultiQuant MD to obtain a detection result.
Comparative example
Comparative example 1
The comparative example provides a method for detecting the concentration of enzyme inhibitor antibiotic drugs in human serum by an external standard method. Wherein, the drug detected in the comparative example is tazobactam.
The method for detecting the concentration of the enzyme inhibitor antibiotic drugs in human serum by the external standard method specifically comprises the following steps:
first, solution preparation
(1) Standard curve sample and quality control sample processing
1. Preparation of standard substance stock solution
In accordance with the corresponding procedure in example 1.
2. Preparation of standard curve working solution and quality control working solution
In accordance with the corresponding procedure in example 1.
3. Preparation of standard curve sample and quality control sample
In accordance with the corresponding procedure in example 1.
4. Standard curve samples and sample processing
And (3) putting 50 mu L of each standard curve sample into a 96-hole sample plate, adding 150 mu L of methanol, vortexing for 10min, centrifuging for 10min at 4000g and 4 ℃, taking 20 mu L of supernate, adding 180 mu L of ultrapure water, mixing uniformly, and then injecting a sample for LC-MS/MS analysis.
The low value sample (10.54. mu.g/ml) and the high value sample (28.79. mu.g/ml) 50. mu.L were placed in a 96-well sample plate, 150. mu.L of methanol was added, vortexed for 10min, and centrifuged at 4000g and 4 ℃ for 10min, and 15. mu.L (-25% offset), 18. mu.L (-10% offset), 20. mu.L (0 offset), 22. mu.L (+ 10% offset), and 25. mu.L (+ 25% offset) of the supernatant (dilution error due to the simulation) were taken, 180. mu.L of ultrapure water was added, mixed, and then, sample introduction was performed for LC-MS/MS analysis.
Second, detecting the conditions
In accordance with the corresponding procedure in example 1.
Third, the detection result
And (4) carrying out data processing by using the MultiQuant MD to obtain a detection result.
Detection result 1
The test results of example 1 and comparative example 1 are shown in table 4.
Among them, the results of the detection of the tazobactam concentration of 10.54. mu.g/ml (0 deviation) in example 1 are shown in FIG. 3.
TABLE 4 comparison of precision and accuracy of internal and external standard methods
As can be seen from Table 4, in the case of variation in operation, the external standard method provided in comparative example 1 all have precision greater than 10% and accuracy variation greater than + -15%, and cannot meet the requirements of clinical regulations. By adopting the substituted internal standard compound chlorzoxazone provided by the application as an internal standard, the precision of the detection result is within 10%, and the accuracy is within +/-10%. Therefore, the chlorzoxazone can be used for replacing an isotope internal standard, so that the used internal standard compound is commercialized, and the detection cost can be effectively reduced.
Example 2
Example 2 provides a method for detecting the concentration of enzyme inhibitor antibiotic drugs in human serum based on LC-MS/MS. The above embodiment is different from embodiment 1 in that: the types of the detected drugs are different, and the type of the detected drug in the embodiment is sulbactam.
Comparative example 2
Comparative example 2 provides a method for detecting blood serum concentration in human by external standard method. The above comparative example differs from comparative example 1 in that: the types of the detected medicines are different, and the type of the medicine detected in the comparative example is sulbactam.
Detection result two
The results of the tests of example 2 and comparative example 2 are shown in table 5.
Among them, the results of the detection of sulbactam concentration of 89.82. mu.g/ml (0 deviation) in example 2 are shown in FIG. 4.
TABLE 5 comparison of precision and accuracy of substituted internal standard compound (chlorzoxazone) and external standard method
As can be seen from Table 5, in the case of operation deviation, the precision of the detection result by the external standard method is greater than 10%, the deviation of the accuracy is greater than +/-15%, the precision CV which can not meet the clinical requirement is less than or equal to 15%, and the accuracy RE is less than or equal to 15%. And the chlorzoxazone is adopted as a substitute internal standard compound, the precision of the detection result is within 10%, the accuracy is within +/-10%, and the clinical requirements can be met.
Example 3
Example 3 provides a method for detecting concentration of enzyme inhibitor antibiotic drugs in human serum based on LC-MS/MS. The difference from example 1 is that: the detected medicine types comprise tazobactam and sulbactam. The results of the detection are shown in FIG. 5.
As can be seen from FIG. 5, the chlorzoxazone can be simultaneously used for detecting various drug types, and the clinical requirement of drug combination can be met.
In summary, the detection method and the kit provided by the application can be suitable for the problem that enzyme inhibitor compounds are lack or isotope internal standards are expensive, and can be used for monitoring the blood concentration of the compounds by the LC-MS/MS method. The substituted internal standard compound meets the clinical detection requirement of TDM in precision and accuracy. A substituted internal standard compound can be simultaneously used for detecting a plurality of compounds, and the requirement of clinical combined medication can be met, so that the detection cost is reduced. In addition, alternative internal standard compounds can be extended to other items of detection depending on the nature of the test compound.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (10)
1. The chlorzoxazone is applied as an internal standard compound in a method for detecting the concentration of an enzyme inhibitor antibiotic drug.
2. The use of chlorzoxazone as an internal standard compound in a method for detecting the concentration of an enzyme inhibitor antibiotic drug according to claim 1, where the enzyme inhibitor antibiotic drug is selected from at least one of tazobactam and sulbactam.
3. The use of chlorzoxazone as an internal standard compound in a method for detecting the concentration of an enzyme inhibitor antibiotic drug according to claim 2, where the chlorzoxazone can be used to detect two or more enzyme inhibitor antibiotic drugs simultaneously.
4. A method for detecting the concentration of an enzyme inhibitor antibiotic drug is characterized by comprising the steps of preparing an internal standard working solution; the internal standard compound in the internal standard working solution is chlorzoxazone.
5. The method for detecting the concentration of an enzyme inhibitor antibiotic drug according to claim 4, wherein the enzyme inhibitor antibiotic drug is at least one selected from tazobactam and sulbactam.
6. The method for detecting the concentration of an enzyme inhibitor antibiotic drug according to claim 4, wherein the detection is performed by LC-MS/MS.
7. The method for detecting the concentration of an enzyme inhibitor antibiotic drug according to claim 6,
the chromatographic conditions of the LC-MS/MS are as follows:
a chromatographic column: waters acquisition UPLC BEH C8, 2.1x50mm, particle size 1.7 μm;
mobile phase A: water containing 0.1% formic acid;
and (3) mobile phase B: methanol with 0.1% formic acid: the volume ratio of acetonitrile is 1: 1;
flow rate: 0.35-0.45 mL/min;
sample introduction volume: 4-6 μ L;
column temperature: 35-45 ℃;
the mass spectrum conditions of the LC-MS/MS are as follows:
the ion source is an ESI-ion source:
the detection mode is negative ion detection;
the scanning mode is multi-reaction monitoring MRM;
the gradient elution procedure was as follows:
8. the method for detecting the concentration of an enzyme inhibitor antibiotic drug according to claim 4, wherein the concentration of the internal standard compound in the internal standard working solution is 150 ng/mL.
9. A kit for detecting the concentration of an enzyme inhibitor antibiotic drug is characterized by comprising an internal standard compound chlorzoxazone.
10. Use of a kit according to claim 9 for the detection of the concentration of an enzyme inhibitor antibiotic drug in human serum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210928389.0A CN114994215A (en) | 2022-08-03 | 2022-08-03 | Application of chlorzoxazone, method for detecting concentration of enzyme inhibitor antibiotic drug, kit and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210928389.0A CN114994215A (en) | 2022-08-03 | 2022-08-03 | Application of chlorzoxazone, method for detecting concentration of enzyme inhibitor antibiotic drug, kit and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114994215A true CN114994215A (en) | 2022-09-02 |
Family
ID=83022633
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210928389.0A Pending CN114994215A (en) | 2022-08-03 | 2022-08-03 | Application of chlorzoxazone, method for detecting concentration of enzyme inhibitor antibiotic drug, kit and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114994215A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101093214A (en) * | 2005-10-14 | 2007-12-26 | 复旦大学附属华山医院 | Method for measuring density of anti-epileptic in blood |
| WO2015162459A1 (en) * | 2014-04-24 | 2015-10-29 | Novartis Ag | Amino pyrazine derivatives as phosphatidylinositol 3-kinase inhibitors |
| CN111537649A (en) * | 2020-05-29 | 2020-08-14 | 南京品生医学检验实验室有限公司 | Kit for detecting antibacterial drugs in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology |
-
2022
- 2022-08-03 CN CN202210928389.0A patent/CN114994215A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101093214A (en) * | 2005-10-14 | 2007-12-26 | 复旦大学附属华山医院 | Method for measuring density of anti-epileptic in blood |
| WO2015162459A1 (en) * | 2014-04-24 | 2015-10-29 | Novartis Ag | Amino pyrazine derivatives as phosphatidylinositol 3-kinase inhibitors |
| CN111537649A (en) * | 2020-05-29 | 2020-08-14 | 南京品生医学检验实验室有限公司 | Kit for detecting antibacterial drugs in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology |
Non-Patent Citations (3)
| Title |
|---|
| AN GH 等: "Aspects of matrix and analyte effects in clinical pharmacokinetic sample analyses using LC-ESI/MS/MS-Two case examples", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 * |
| 刘凤银 等: "酶抑制法快速检测牛奶中β-内酰胺酶抑制剂多残留", 《食品科学》 * |
| 王海静 等: "UPLC-MS/MS测定73例患者血中头孢哌酮与舒巴坦的浓度", 《中国医院药学杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Valen et al. | Determination of 21 drugs in oral fluid using fully automated supported liquid extraction and UHPLC‐MS/MS | |
| Alam et al. | Measurement of homocysteine: a historical perspective | |
| CN111830146B (en) | LC-MS/MS high-throughput detection method and kit for 25-hydroxyvitamin D in dried blood slices | |
| CN113917049A (en) | Biological analysis method for clinical research of chlorpromazine and metabolite concentration in plasma sample | |
| CN111458417B (en) | Method and kit for combined detection of multiple antibiotics in sample to be detected | |
| CN109900841B (en) | HPLC-MS/MS method for simultaneously determining concentration of aminoglycoside antibiotic drug in plasma | |
| CN113607854B (en) | Method and detection kit for simultaneously detecting multiple vitamins | |
| CN110927310A (en) | Method for simultaneously detecting content of 25 hydroxy-vitamin D3 and 25 hydroxy-vitamin D2 in trace blood | |
| CN113720946A (en) | Method and kit for detecting multiple steroid hormones in blood | |
| CN114544796B (en) | Method for measuring settop alcohol in plasma by liquid phase mass spectrometry | |
| CN110361485A (en) | Oxcarbazepine monitor drug concentration kit and its detection method in a kind of blood | |
| CN114994215A (en) | Application of chlorzoxazone, method for detecting concentration of enzyme inhibitor antibiotic drug, kit and application | |
| CN114397379A (en) | Method for determining concentration of ornidazole in blood plasma by liquid chromatography-mass spectrometry | |
| CN114858949A (en) | Application of clenbuterol, method for detecting drug concentration, kit and application | |
| CN114280191A (en) | Method for detecting related substances in bis-cysteine and preparation thereof | |
| CN114624362A (en) | Kit for detecting advanced glycosylation end products in serum and application thereof | |
| CN114994216A (en) | Application of ropivacaine, method for detecting concentration of antibiotic drugs, kit and application | |
| CN113820424A (en) | HPLC-MS/MS method for simultaneously determining concentration of 14 antidepressants in human plasma | |
| Jiao et al. | Development of a sensitive HPLC‐MS/MS method for 25‐hydroxyvitamin D2 and D3 measurement in capillary blood | |
| CN112213417A (en) | Kit and method for detecting concentration of mycophenolic acid medicine in dried blood spots | |
| CN112485340A (en) | Method for detecting 1, 5-sorbitan in plasma by ultra-high performance liquid chromatography tandem mass spectrometry | |
| CN114755348B (en) | Method for simultaneously detecting contents of 20 medicines and metabolites thereof | |
| Rochaeny et al. | Development and validation of an ultra performance liquid chromatography-tandem mass spectrometry method with liquid-liquid extraction for the quantification of sitagliptin in human plasma using nebivolol as internal standard | |
| CN112986452B (en) | Method for determining tandospirone concentration in human plasma | |
| CN116026971B (en) | Kit and detection method for detecting full-spectrum fat-soluble vitamins and metabolites thereof in human serum and plasma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220902 |
|
| RJ01 | Rejection of invention patent application after publication |