CN116577450A - Method for simultaneously detecting blood concentration of 7 antitumor drugs - Google Patents
Method for simultaneously detecting blood concentration of 7 antitumor drugs Download PDFInfo
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Classifications
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- G—PHYSICS
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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
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- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The application discloses a method for simultaneously detecting blood concentration of 7 antitumor drugs, which comprises the following steps: s1, determining a drug to be detected, wherein the drug to be detected comprises irinotecan, capecitabine, paclitaxel, docetaxel, tamoxifen, methotrexate and letrozole; s2, preparing a calibrator and a quality control product, and preparing a working curve concentration table; s3, preprocessing the calibrator and quality control product obtained in the step S2; s4, performing liquid chromatography tandem mass spectrometry detection on the pretreated liquid; s5, performing performance verification on a detection result, wherein S2 specifically comprises the following steps: s201, mixing the calibrator and the quality control intermediate solution with the blank matrix according to the ratio of 1:50. The application adopts a liquid chromatography tandem mass spectrometry method and a combination mode of high performance liquid chromatography separation and mass spectrometry, and has the advantages of strong background interference resistance, high signal to noise ratio, high sensitivity, high detection efficiency and short sample waiting time.
Description
Technical Field
The application belongs to the technical field of medicine detection, and particularly relates to a method for simultaneously detecting blood concentration of 7 antitumor drugs.
Background
The blood concentration monitoring is based on the principle of pharmacokinetics, and is used to evaluate the curative effect or determine the dosage scheme, so as to individuate the dosage scheme, raise the therapeutic level of the medicine and reach clinical safety, effective and reasonable medicine use. Blood concentration monitoring is commonly used for medicines with narrow therapeutic window, strong toxicity, long administration period and large individual difference after administration.
The traditional blood concentration monitoring method mainly uses immunology, namely, by researching the immune response of organisms to antigen substances (the reaction of organisms to antigen stimulation is also a biological process for identifying and removing the antigen substances), and the method has the advantages of high specificity, simplicity and rapidness in operation, relatively few influencing factors, easiness in control and the like. However, when the plasma concentration is monitored by immunology, there are also drawbacks that the type of the drug antibody is limited, and that it is impossible to measure a plurality of drugs or upstream and downstream metabolites of a single drug at the same time.
In addition, some institutions also use High Performance Liquid Chromatography (HPLC) to monitor blood concentration, but high performance liquid chromatography has low sensitivity, some drugs with relatively high sensitivity requirements cannot be detected, and the pretreatment methodology is relatively complex.
Therefore, in order to solve the above-mentioned problems, it is necessary to provide a method for simultaneously detecting the blood concentration of 7 antitumor drugs.
Disclosure of Invention
The application aims to provide a method for simultaneously detecting the blood concentration of 7 antitumor drugs so as to solve the problems.
In order to achieve the above object, an embodiment of the present application provides the following technical solution:
a method for simultaneously detecting blood concentration of 7 antitumor drugs, comprising the following steps:
s1, determining a drug to be detected, wherein the drug to be detected comprises irinotecan, capecitabine, paclitaxel, docetaxel, tamoxifen, methotrexate and letrozole;
s2, preparing a calibrator and a quality control product, and preparing a working curve concentration table;
s3, preprocessing the calibrator and quality control product obtained in the step S2;
s4, performing liquid chromatography tandem mass spectrometry detection on the pretreated liquid;
s5, performing performance verification on the detection result.
Further, the step S2 specifically includes the following steps:
s201, mixing the calibrator and the quality control product intermediate solution with a blank matrix according to the ratio of 1:50;
s202, vortex oscillation is carried out for 5min at 2500rpm/min to obtain standard substances J1-J7, and the concentration of each drug in the standard substances J1-J7 is drawn to obtain a working curve concentration table.
Further, the blank matrix in S201 is 20 μl of calibrator and 980 μl of blank matrix.
Further, the step S3 specifically includes the following steps:
s301, transferring 100 mu L of serum sample/calibrator/quality control product, adding the serum sample/calibrator/quality control product into a 2mL centrifuge tube, adding 20 mu L of internal standard, and adding 280 mu L of methanol-acetonitrile (1+1) solution containing 1% formic acid;
s302, vortex oscillation is carried out for 5min at 2000rpm/min, after uniform mixing, centrifugation is carried out for 5min at 14000rpm/min at the temperature of 4 ℃;
s303, transferring 200 mu L of the supernatant in the step S302, adding the supernatant into a 96-well plate, and centrifuging for 5min at the rotating speed of 4000rpm/min to obtain the liquid medicine to be detected.
Further, the step S4 includes a mass spectrum parameter setting and a liquid phase parameter setting.
Further, the liquid phase setting parameters include: chromatographic column: aglient Eclipse Plus C18,3.5 μm (100×3.0 mm), mobile phase: phase A was 2mM ammonium formate in water (0.1% formic acid); phase B was 0.1% acetonitrile formate, flow rate: sample volume of 0.6 mL/min: 20 μl, column temperature: 40 ℃.
Further, the step S4 further includes a gradient elution process.
Further, the performance verification in S5 includes a matrix curve verification and an imprecision verification.
Further, the matrix curve verification method comprises the following steps: and respectively taking 100 mu L of 7 concentration points, processing through the steps S3 and S4, drawing a standard curve by taking the concentration value of the object to be detected as an abscissa and the area ratio of the object to be detected to the internal standard as an ordinate, and carrying out regression calculation.
Further, the method for verifying the inaccuracy comprises the following steps: and respectively carrying out 6 times of parallel measurement on samples with low, medium and high concentrations, repeatedly measuring 3 batches, calculating the CV values in the batches and between the batches of each concentration by using the Area Ratio value, wherein the CV of the accuracy of the medium and high concentrations is less than 15%, and the CV of the low concentration is less than 20% so as to be acceptable.
Compared with the prior art, the application has the following advantages:
1. the application uses liquid chromatography tandem mass spectrometry and adopts a combination mode of high performance liquid chromatography separation and mass spectrometry analysis, thereby overcoming complex background interference in blood samples, improving signal to noise ratio, achieving high sensitivity for medicines with smaller in vivo concentration, including anti-tumor medicines and facilitating detection.
2. The liquid chromatography tandem mass spectrometry can be used for detecting various substances, various treatment schemes can be combined in anti-tumor treatment, various medicines can be taken simultaneously, and different active metabolites can be obtained in one medicine.
3. The pretreatment of the liquid chromatography tandem mass spectrometry is optimized, and the detection can be carried out on the machine after a few steps are processed, so that the waiting time of a sample is reduced.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to the drawings without inventive effort to those skilled in the art.
FIG. 1 is a graph showing the concentration of 7 antitumor drugs in a method for simultaneously detecting the blood concentration of the antitumor drugs according to an embodiment of the present application;
FIG. 2 is a table of mass spectrometry setup parameters for a method of simultaneously detecting blood concentrations of 7 antitumor drugs according to an embodiment of the present application;
FIG. 3 is an ion pair information table of a method for simultaneously detecting blood concentration of 7 antitumor drugs according to an embodiment of the present application;
FIG. 4 is an elution gradient table of a method for simultaneously detecting blood concentration of 7 antitumor drugs according to an embodiment of the present application;
FIG. 5 is a table of matrix curve verification data for a method of simultaneously detecting blood concentration of 7 antitumor drugs according to an embodiment of the present application;
FIG. 6 is a table of accuracy verification data for a method of simultaneously detecting blood concentrations of 7 antitumor drugs according to an embodiment of the present application;
FIG. 7 is a table of data for verifying the accuracy of a method for simultaneously detecting blood concentration of 7 antitumor drugs according to an embodiment of the present application;
FIG. 8 is a table of data for verifying the specificity of a method for simultaneously detecting blood concentration of 7 antitumor drugs according to an embodiment of the present application;
FIG. 9 is a table of residual data for a method of simultaneously detecting blood concentrations of 7 antitumor drugs according to an embodiment of the present application;
FIG. 10 is a table of repeated sample injection verification data for a method of simultaneously detecting blood concentrations of 7 antitumor drugs according to an embodiment of the present application.
Detailed Description
The present application will be described in detail below with reference to the embodiments shown in the drawings. The embodiments are not intended to limit the application, but structural, methodological, or functional modifications of the application from those skilled in the art are included within the scope of the application.
The application discloses a method for simultaneously detecting blood concentration of 7 antitumor drugs, which is shown in fig. 1-10 and comprises the following steps:
s1, determining a drug to be detected, wherein the drug to be detected comprises irinotecan, capecitabine, paclitaxel, docetaxel, tamoxifen, methotrexate and letrozole;
s2, preparing a calibrator and a quality control product, and preparing a working curve concentration table;
s3, preprocessing the calibrator and quality control product obtained in the step S2;
s4, performing liquid chromatography tandem mass spectrometry detection on the pretreated liquid;
s5, performing performance verification on the detection result.
Wherein, S2 specifically includes the following steps:
s201, mixing the calibrator and quality control product intermediate solution and a blank matrix according to a ratio of 1:50, wherein the blank matrix is 20 mu L of the calibrator and 980 mu L of the blank matrix;
s202, vortex oscillation is carried out for 5min at 2500rpm/min to obtain standard substances J1-J7, and the concentration of each drug in the standard substances J1-J7 is drawn to obtain a working curve concentration table.
In addition, S3 specifically includes the following steps:
s301, transferring 100 mu L of serum sample/calibrator/quality control product, adding the serum sample/calibrator/quality control product into a 2mL centrifuge tube, adding 20 mu L of internal standard, and adding 280 mu L of methanol-acetonitrile (1+1) solution containing 1% formic acid;
s302, vortex oscillation is carried out for 5min at 2000rpm/min, after uniform mixing, centrifugation is carried out for 5min at 14000rpm/min at the temperature of 4 ℃;
s303, transferring 200 mu L of the supernatant in the step S302, adding the supernatant into a 96-well plate, and centrifuging for 5min at the rotating speed of 4000rpm/min to obtain the liquid medicine to be detected.
Specifically, the method for detecting the liquid chromatography tandem mass spectrometry comprises the following steps: the method comprises the steps of firstly, precipitating proteins by using an organic reagent, shaking the sample by using a shaking device, centrifuging by using a high-speed refrigerated centrifuge, transferring supernatant into a 96-well plate by using a pipettor, and finally detecting mass spectrum on the 96-well plate.
Preferably, the shake requires a rotational speed: 2000rpm/min, time: 5min; high speed refrigerated centrifuge speed: 14000rpm/min, time: 5min, temperature: 4 ℃.
The sample and the organic reagent are fully and uniformly mixed by the shaking device, so that protein precipitation is promoted. The sediment in the solution is quickly precipitated to the lower layer by high-speed refrigerated centrifugation, and the solution is fully separated from the sediment at the low temperature of 4 ℃.
The mass spectrum parameter setting and the liquid phase parameter setting are included in S4.
Wherein the mass spectrum parameters are all optimized according to single compounds, including DP, CE, EP, CXP and the like, can be matched with machines and the response of related compounds is improved,
the sample injection amount is smaller through the liquid phase parameter setting, the pollution risk to the machine is reduced, the gradient time is shorter, and the mass spectrum flux is improved.
Preferably, the mass spectrometry setup parameters are as shown in fig. 2.
The liquid phase setting parameters are as follows: chromatographic column: aglient Eclipse Plus C18,3.5 μm (100×3.0 mm), mobile phase: phase A was 2mM ammonium formate in water (0.1% formic acid); phase B was 0.1% acetonitrile formate, flow rate: sample volume of 0.6 mL/min: 20 μl, column temperature: 40 ℃.
In addition, S4 also includes a gradient elution process, and the parameters of the gradient elution are shown in fig. 4.
Based on the method, the anti-tumor drugs in the blood sample are extracted by using a protein precipitation method, then the anti-tumor drugs are detected by using a liquid chromatography-tandem mass spectrometry system, mass spectrum signals are collected, and quantitative detection of the target drugs in the sample is realized by establishing a functional relation between known concentration analytes (standard substances) and target analyte response values (peak area ratios of target peak areas and corresponding isotope internal standard substances).
Furthermore, in order to verify whether the detection method is suitable for a AB SCIEX Triple Quad 4500 liquid chromatography-mass spectrometer, the clinical detection requirement of anti-tumor drug concentration monitoring is met, and performance verification needs to be carried out on detected data.
Wherein, the detection data performance verification comprises matrix curve verification and precision verification.
Specifically, the method for verifying the matrix curve comprises the following steps: and respectively taking 100 mu L of 7 concentration points, processing through the steps S3 and S4, drawing a standard curve by taking the concentration value of the object to be detected as an abscissa and the area ratio of the object to be detected to the internal standard as an ordinate, and carrying out regression calculation, wherein the calculated result is shown in the figure 5.
As can be seen from the data in fig. 5, the linear R values of the 7 antitumor drugs of irinotecan, capecitabine, paclitaxel, docetaxel, tamoxifen, methotrexate and letrozole were all greater than 0.99, indicating that the linear relationship was good across the 7 antitumor drugs.
In addition, the method for verifying the precision comprises the following steps: and respectively carrying out parallel measurement on samples with low concentration, medium concentration and high concentration for 6 times, repeatedly measuring 3 batches, calculating the CV values of the batches of each concentration by using the Area Ratio value, wherein the CV of the accuracy of the medium concentration and the high concentration is less than 15%, and the CV of the low concentration is less than 20%, so that the accuracy is acceptable, and the accuracy verification result is shown in figure 7.
As can be seen from the data in FIG. 7, the precision CV of 7 antitumor drugs was 15% or less in each of the batches and between the batches, and the performance verification requirements were satisfied.
In addition, in order to further improve the accuracy of the detection data result, accuracy verification, specificity and selectivity verification, residual verification and repeated sample injection stability verification are also required to be performed on the detection data.
The operation process of accuracy verification is as follows:
(1) The basic requirements and preparation method of the accuracy verification test sample are as follows:
selecting a conventional detection sample with proper concentration, and dividing the conventional detection sample into 3 parts with the same volume; adding different amounts of standard substances to be detected into 3 samples to prepare 3 recovered samples with different added concentrations (such as high, medium and low quality control points of a matrix), and calculating the concentration of the added substances to be detected; and adding the same amount of solvent without the tested object into the other sample to prepare a basic sample.
(2) Accuracy verification experiment procedure
The recovered sample and the basic sample are measured by a method to be evaluated, and the sample is usually subjected to 3 times of repeated analysis, and the average value is obtained for calculation.
(3) Accuracy verification data processing and result reporting
And (5) calculating recovery rate: recovery 1= (recovery sample concentration 1-base sample concentration)/addition concentration x 100%;
calculating average recovery rate:
average recovery = (recovery 1+recovery 2)/2×100%;
acceptable judgment: the average recovery rate is 85% -115%.
(4) The accuracy verification process is as follows:
quality control products with low, medium and high concentration levels of 3 are prepared, added into unknown serum to be evenly mixed, then the test is carried out, each concentration is repeatedly measured for 3 times, meanwhile, unknown serum added with the same amount of methanol is measured as a basic sample, and the verification result is shown in figure 6.
From the data in fig. 6, it can be seen that the concentration of each drug is between 85% and 115% recovered by labeling, and the accuracy performance verification requirement is satisfied.
In addition, the determination method for specificity and selectivity verification comprises the following steps: the blank (with internal standard sample matrix) samples were tested, with the following requirements: no background peak or background peak is less than 20% of the LLMI peak area.
Based on this, the results of the obtained specificity, selectivity verification are shown in fig. 8.
As can be seen from fig. 8, the blank peak area of each analyte was less than 20% of the LLMI peak area, satisfying the specificity requirements.
Specifically, the verification method of the residual verification is as follows: immediately after analyzing the high concentration sample, 1 or more blank samples are sampled, and the detection signal of the blank samples is far lower than LLMI.
Immediately after analysis of the peak S6 of the curve, the blank sample was subjected to repeated measurement 6 times, and the peak area and LLMI, i.e., the peak area of S1, were paired as shown in FIG. 9.
As can be seen from fig. 9, the residues in the double blank samples after the highest concentration sample (S7) are less than 20% of the lower limit of quantification (S1), and the performance verification requirements are satisfied.
In addition, the repeated sampling stability verification is to continuously sample the sample for 20 needles with low quality control, calculate the average value of the measurement result, require CV less than or equal to 15 percent, and record relevant data.
The results of the repeated sample injection stability verification are shown in fig. 10.
As can be seen from FIG. 10, the low-concentration repeated sampling CV of 7 antitumor drug analytes is less than or equal to 15%, and the verification requirement is met.
In summary, according to the results of the detection data of the matrix curve verification, the precision verification, the accuracy verification, the specificity, the selectivity verification, the residual verification and the repeated sample injection stability verification, the detection method disclosed by the application can accurately quantify 7 antitumor drugs based on a AB SCIEX Triple Quad 4500 liquid chromatography-mass spectrometry combined instrument, wherein the linearity can meet r >0.99, the minimum standard point deviation is within +/-20%, the rest standard point deviation is within +/-15%, the quality control accuracy meets RE less than or equal to +/-15%, and the precision in batch and between batches meets CV less than or equal to 15%. The product is suitable for AB SCIEX Triple Quad 4500 liquid chromatography-mass spectrometry, and meets the clinical detection requirement for the concentration of 7 antitumor drugs.
The technical scheme shows that the application has the following beneficial effects:
1. the application uses liquid chromatography tandem mass spectrometry and adopts a combination mode of high performance liquid chromatography separation and mass spectrometry analysis, thereby overcoming complex background interference in blood samples, improving signal to noise ratio, achieving high sensitivity for medicines with smaller in vivo concentration, including anti-tumor medicines and facilitating detection.
2. The liquid chromatography tandem mass spectrometry can be used for detecting various substances, various treatment schemes can be combined in anti-tumor treatment, various medicines can be taken simultaneously, and different active metabolites can be obtained in one medicine.
3. The pretreatment of the liquid chromatography tandem mass spectrometry is optimized, and the detection can be carried out on the machine after a few steps are processed, so that the waiting time of a sample is reduced.
It will be evident to those skilled in the art that the application is not limited to the details of the foregoing illustrative embodiments, and that the present application may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment contains only one independent technical solution, and that such description is provided for clarity only, and that the technical solutions of the embodiments may be appropriately combined to form other embodiments that will be understood by those skilled in the art.
Claims (10)
1. A method for simultaneously detecting blood concentration of 7 antitumor drugs, which is characterized by comprising the following steps:
s1, determining a drug to be detected, wherein the drug to be detected comprises irinotecan, capecitabine, paclitaxel, docetaxel, tamoxifen, methotrexate and letrozole;
s2, preparing a calibrator and a quality control product, and preparing a working curve concentration table;
s3, preprocessing the calibrator and quality control product obtained in the step S2;
s4, performing liquid chromatography tandem mass spectrometry detection on the pretreated liquid;
s5, performing performance verification on the detection result.
2. The method for simultaneously detecting the blood concentration of 7 antitumor drugs according to claim 1, wherein the step S2 specifically comprises the following steps:
s201, mixing the calibrator and the quality control product intermediate solution with a blank matrix according to the ratio of 1:50;
s202, vortex oscillation is carried out for 5min at 2500rpm/min to obtain standard substances J1-J7, and the concentration of each drug in the standard substances J1-J7 is drawn to obtain a working curve concentration table.
3. The method for simultaneously detecting blood concentration of 7 antitumor drugs according to claim 2, wherein the blank matrix in S201 is 20 μl of calibrator and 980 μl of blank matrix.
4. The method for simultaneously detecting the blood concentration of 7 antitumor drugs according to claim 1, wherein the step S3 specifically comprises the following steps:
s301, transferring 100 mu L of serum sample/calibrator/quality control product, adding the serum sample/calibrator/quality control product into a 2mL centrifuge tube, adding 20 mu L of internal standard, and adding 280 mu L of methanol-acetonitrile (1+1) solution containing 1% formic acid;
s302, vortex oscillation is carried out for 5min at 2000rpm/min, after uniform mixing, centrifugation is carried out for 5min at 14000rpm/min at the temperature of 4 ℃;
s303, transferring 200 mu L of the supernatant in the step S302, adding the supernatant into a 96-well plate, and centrifuging for 5min at the rotating speed of 4000rpm/min to obtain the liquid medicine to be detected.
5. The method for simultaneously detecting blood concentration of 7 antitumor drugs according to claim 1, wherein the step S4 comprises a mass spectrometry parameter setting and a liquid phase parameter setting.
6. The method for simultaneously detecting blood concentration of 7 antitumor drugs according to claim 5, wherein the liquid phase setting parameters comprise: chromatographic column: aglient Eclipse Plus C18,3.5 μm (100×3.0 mm), mobile phase: phase A was 2mM ammonium formate in water (0.1% formic acid); phase B was 0.1% acetonitrile formate, flow rate: sample volume of 0.6 mL/min: 20 μl, column temperature: 40 ℃.
7. The method for simultaneously detecting blood concentration of 7 antitumor drugs according to claim 5, wherein the step S4 further comprises a gradient elution process.
8. The method for simultaneously detecting blood concentration of 7 antitumor drugs according to claim 1, wherein the performance verification in S5 comprises a matrix curve verification and an imprecision verification.
9. The method for simultaneously detecting the blood concentration of 7 antitumor drugs according to claim 8, wherein the method for verifying the matrix curve is as follows: and respectively taking 100 mu L of 7 concentration points, processing through the steps S3 and S4, drawing a standard curve by taking the concentration value of the object to be detected as an abscissa and the area ratio of the object to be detected to the internal standard as an ordinate, and carrying out regression calculation.
10. The method for simultaneously detecting the blood concentration of 7 antitumor drugs according to claim 8, wherein the method for verifying the inaccuracy is as follows: and respectively carrying out 6 times of parallel measurement on samples with low, medium and high concentrations, repeatedly measuring 3 batches, calculating the CV values in the batches and between the batches of each concentration by using the Area Ratio value, wherein the CV of the accuracy of the medium and high concentrations is less than 15%, and the CV of the low concentration is less than 20% so as to be acceptable.
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