CN115629145A - Method for simultaneously detecting content of 7 anti-new coronavirus medicines - Google Patents
Method for simultaneously detecting content of 7 anti-new coronavirus medicines Download PDFInfo
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Abstract
The invention provides a method for simultaneously detecting the content of 7 anti-new coronavirus medicines. The method comprises the following steps: preparing at least three standard solutions with different concentrations and containing 7 anti-new coronavirus drugs and internal standard substances thereof, detecting by using liquid chromatography-mass spectrometry, and establishing a standard curve equation; mixing a sample to be detected, an internal standard substance and a protein precipitator, wherein the volume of the protein precipitator is more than 50 times of that of the sample to be detected, centrifuging, and taking supernatant for dilution to obtain a sample to be detected; and detecting the sample to be detected by using liquid chromatography-mass spectrometry, and calculating the content of the 7 anti-new coronavirus medicines in the sample to be detected according to the detection result and a standard curve equation. The method provided by the invention can be used for simultaneously detecting 7 anti-new coronavirus medicines, and has the advantages of high accuracy and sensitivity, good reproducibility and short detection time.
Description
Technical Field
The invention belongs to the technical field of drug detection, and particularly relates to a method for simultaneously detecting the content of 7 anti-new coronavirus drugs.
Background
Currently, some progress has been made in the study of anti-neocoronavirus drugs, such as lopinavir, ritonavir, ribavirin, chloroquine, arbidol, darunavir, cobicistat, and the like. The anti-neocoronaviruses are generally administered by single or combined administration. Because the anti-new coronaviruses medicine may cause adverse reaction and has extremely strict requirements on the dosage, the timely detection of the blood concentration of a patient has important significance for guiding a doctor to reasonably formulate a dosing scheme so that the patient obtains the best curative effect.
CN112379029A discloses a method for detecting concentration of novel coronavirus resistant drugs in blood, the amount of blood samples used is up to 500 mu L, blood sampling, blood transportation, treatment and storage of patients are inconvenient, the analysis time is up to 12min, rapid analysis cannot be realized, and the method does not realize simultaneous detection of lopinavir, ritonavir, ribavirin, chloroquine, arbidol, darunavir and cobicistat. CN112924573A discloses an HPLC-MS/MS detection method for arbidol, ribavirin and chloroquine, only 3 anti-new coronavirus medicines can be detected, and the sample pretreatment is complex, the cost is high, and the method is not suitable for clinical practical application. In addition, the standard working solution of the method is obtained by diluting the biological matrix, so that the cost is high, and the preparation and long-term storage of the working solution have risks.
In order to assist clinical auxiliary judgment of the curative effect of antiviral drugs and ensure safe medication of patients with new coronary pneumonia, a method for simultaneously detecting the concentrations of 7 antiviral drugs (chloroquine phosphate, abidol, lopinavir, ritonavir, ribavirin, darunavir and cobicistat) in blood of patients quickly at low cost is needed to be developed.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for simultaneously detecting the content of 7 anti-new coronavirus medicines. The method can simultaneously detect 7 anti-neocoronaviruses drugs, and has the advantages of high accuracy, high sensitivity, good reproducibility and short detection time.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for simultaneously detecting the content of 7 anti-neocoronaviruses drugs, which comprises the following steps:
establishing a standard curve equation: preparing at least three standard solutions with different concentrations and containing 7 anti-new coronavirus drugs and internal standard substances thereof, wherein the concentrations of the same internal standard substance in the standard solutions with different concentrations are the same; detecting the standard solution by using liquid chromatography-mass spectrometry (LC-MS/MS), and establishing a standard curve equation of each anti-neocoronavirus drug/internal standard substance chromatographic peak area ratio-anti-neocoronavirus drug/internal standard substance concentration ratio according to the detection result;
pretreatment of a sample to be detected: mixing a sample to be detected, an internal standard substance and a protein precipitator, wherein the volume of the protein precipitator is more than 50 times of that of the sample to be detected, centrifuging, and taking supernatant for dilution to obtain a sample to be detected;
detecting a sample to be detected: detecting the sample to be detected by using a liquid chromatography-mass spectrometry, and calculating the content of 7 anti-new coronavirus medicines in the sample to be detected according to a detection result and the standard curve equation;
wherein the 7 anti-neocoronaviral drugs are Lopinavir (LPV), ritonavir (RTV), ribavirin (RBVR), chloroquine (CQ), arbidol (ABD), darunavir (DRV) and Cobicistat (CBS).
Since the blood concentrations of 7 anti-new coronavirus medicines such as lopinavir, ritonavir, ribavirin, chloroquine, arbidol, darunavir and cobicistat are very different (from hundreds to tens of thousands ng/mL), and the molecular properties are different, it is difficult to detect 7 anti-new coronavirus medicines simultaneously.
In the invention, a large amount of protein precipitant is used for treating a sample to be detected, so that the interference of a blood matrix can be sufficiently reduced, the ionization efficiency of a mass spectrum on a target object is improved, blank plasma or serum is not required to be added when a standard solution is prepared, but the concentration of the sample to be detected is reduced, the signal intensity of the target object is reduced, and the accuracy of a detection result is possibly influenced. By adopting the pretreatment method and matching with proper chromatographic conditions, the 7 new coronavirus resistant medicaments can be fully separated, the peak effect is good, the influence of blood matrix is small, the standard curve linearity of each medicament is good, the detection accuracy is high, and the reproducibility is good, so that the concentrations of the 7 new coronavirus resistant medicaments can be simultaneously detected by only once sampling.
In the invention, the structural formulas of the 7 anti-new coronavirus medicines are as follows:
if no special description is provided, the preparation process of the standard solution does not use plasma or serum, and the conditions of liquid chromatography-mass spectrometry are the same when the standard solution and the sample to be detected are detected.
It should be noted that, in the standard solutions with different concentrations, the concentration of the same anti-new coronavirus drug is different, and the concentration of the same internal standard substance is preferably the same; in the same standard solution, the concentrations of the 7 anti-new coronavirus medicines and the internal standard substance thereof are independent from each other, can be the same or different, and the invention does not make special requirements.
In the invention, the "chromatographic peak area ratio of the anti-new coronavirus drug to the internal standard substance" refers to the ratio of the chromatographic peak area of the anti-new coronavirus drug to the chromatographic peak area of the internal standard substance corresponding to the drug; the concentration of the anti-new coronavirus/internal standard substance refers to the ratio of the concentration of the anti-new coronavirus to the concentration of the internal standard substance corresponding to the anti-new coronavirus.
Since the standard solution of the present invention is prepared without using plasma or serum, the standard solution can be prepared directly with a solvent without performing a matrix treatment operation (e.g., protein precipitation). However, in order to further reduce the systematic error, the standard solution of the present invention is preferably prepared by the following method: preparing at least three target working solutions with different concentrations and containing 7 anti-new coronavirus medicines, and then treating the target working solutions according to the same pretreatment method as the sample to be detected to obtain the standard solution.
The above-mentioned "treatment is carried out by the same pretreatment method as that of the sample to be tested" means that the procedure is substantially the same (specifically, the types and the proportions of the diluents used for diluting the added internal standard substance, protein precipitant and supernatant are the same), and the other auxiliary procedures (for example, the means and parameters of mixing and centrifugation) may be different.
In some embodiments of the present invention, the solvent of the target working solution is a mixed solution of methanol and water, preferably a mixed solution of methanol and water with a volume ratio of 1:1.
In some embodiments of the invention, the internal standard of the 7 anti-neocoronavirus drugs is an isotopic internal standard.
In some embodiments of the invention, the internal standard of the 7 anti-neocoronavirus drugs is lopinavir-d 8, ritonavir-d 6, ribavirin- 13 C5, chloroquine-d 5, arbidol-d 6, darunavir-d 9 and cobicistat-d 8.
In some embodiments of the invention, the sample to be tested is plasma or serum.
In some embodiments of the invention, the internal standard is added in the form of an internal standard working fluid, which is a solution containing the 7 anti-neocoronavirus internal standard drugs.
In some embodiments of the present invention, the solvent of the internal standard working solution is a mixed solution of methanol and water, preferably a mixed solution of 1:1 in a volume ratio of methanol to water.
In some embodiments of the invention, the volume ratio of the sample to be tested to the internal standard working solution is 1; for example, 1.
In some embodiments of the invention, the protein precipitating agent is selected from one or more of methanol, ethanol, acetonitrile, and isopropanol.
In some embodiments of the invention, the volume of the protein precipitating agent is 50 to 800 times the volume of the sample to be tested; for example, the amount of the compound may be 50 times, 100 times, 120 times, 150 times, 180 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 600 times, 700 times, 800 times, or the like.
In some embodiments of the present invention, the diluent used for diluting the supernatant in the pretreatment of the sample to be tested is a mixed solution having a methanol-to-water volume ratio of 3:7-7:3 (for example, 3:7, 3.5, 4:6, 4.5.
In some embodiments of the present invention, the total dilution multiple of the sample to be tested in the pretreatment of the sample to be tested is 100-5000 times; for example, the amount may be 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 800 times, 1000 times, 1200 times, 1500 times, 1800 times, 2000 times, 2500 times, 3000 times, 3500 times, 4000 times, 4500 times, or 5000 times.
In the present invention, the total dilution factor of the sample to be tested refers to the dilution factor of the sample to be tested compared to the sample to be tested.
In some embodiments of the invention, the conditions of the liquid chromatography comprise:
the chromatographic column is a C18 chromatographic column with isobutyl side chain protection;
and/or the column temperature is 30-55 deg.C (such as 30 deg.C, 32 deg.C, 35 deg.C, 38 deg.C, 40 deg.C, 42 deg.C, 45 deg.C, 48 deg.C, 50 deg.C, 52 deg.C or 55 deg.C);
and/or the mobile phase flow rate is 0.3-0.6 mL/min (e.g., can be 0.3 mL/min, 0.35 mL/min, 0.4 mL/min, 0.45 mL/min, 0.5 mL/min, 0.55 mL/min, or 0.6 mL/min, etc.);
and/or the mobile phase consists of a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution containing 1-50 mmol/L ammonium formate and 0.1-1vol% formic acid, and the mobile phase B is acetonitrile; wherein the ammonium formate content can be 1 mmol/L, 3 mmol/L, 5 mmol/L, 8 mmol/L, 10 mmol/L, 15 mmol/L, 20 mmol/L, 25 mmol/L, 30 mmol/L, 35 mmol/L, 40 mmol/L, 45 mmol/L or 50 mmol/L, etc.; the content of formic acid may be 0.1 vol%, 0.2 vol%, 0.3 vol%, 0.5vol%, 0.8 vol%, 1vol%, or the like;
and/or the elution mode is gradient elution.
In some embodiments of the invention, the mobile phase of the gradient elution is:
0-0.5kmin: the 50% mobile phase a linearly decreased to 0%, and the 50% mobile phase B linearly increased to 100%;
0.5k -1kmin:100% mobile phase B;
1k -2.5kmin:50% mobile phase a,50% mobile phase B;
wherein the content of the first and second substances,k = 0.4/mobile phase flow rate value.
In the gradient elution procedure of the present invention, 1 iskChanges in the composition of the mobile phase before and after min, 1kThe mobile phase composition at the min time point may be 100% mobile phase B, or may be 50% mobile phase a and 50% mobile phase B.
In some embodiments of the invention, the conditions of the mass spectrum comprise:
the mass spectrum detector adopts an electrospray ion source (ESI), a positive ion mode and a multi-reaction monitoring (MRM), the electrospray voltage is 5000V, the ion source temperature is 400-600 ℃, the atomization air flow rate is 30-80L/min, and the auxiliary air flow rate is 40-70L/min.
In some preferred embodiments of the present invention, the method for simultaneously detecting the content of 7 anti-new coronavirus drugs can be performed according to the following steps:
preparation of standard solution
And (2) taking 10 mu L of target working solution, 10 mu L of internal standard working solution and 500-8000 mu L of protein precipitator, putting the target working solution, the internal standard working solution and the protein precipitator into a centrifuge tube, carrying out vortex mixing for 30s-1min at the rotating speed of 1000-2000rpm, taking the uniformly mixed solution, adding a diluent (methanol: water =3:7-7, 3, v/v) to dilute (the total dilution multiple of the target working solution is 100-5000 times), and obtaining a series of standard solutions with different concentrations.
The preparation method of the target working solution comprises the following steps:
accurately weighing 7 anti-new coronavirus drug standards, and respectively adding methanol or water for dissolving to obtain 7 target stock solutions;
the 7 target stock solutions were diluted with a diluent (methanol: water =1,v/v) to obtain 7 target intermediate solutions, respectively;
mixing the 7 target intermediate solutions, and diluting with a diluent (methanol: water =1:1, v/v) to obtain a series of target working solutions with different concentrations and containing 7 anti-new coronavirus drugs;
according to relevant authority websites, drug specifications or relevant documents of human pharmacokinetics in clinical examination, the blood concentration ranges of 7 anti-neocoronaviruses are as follows:
in order to meet the clinical requirements as much as possible, the concentration ranges of the anti-new coronavirus drugs in the target working solution are set as follows:
the concentration range basically covers the actual blood concentration range, so that the novel crown treatment application is more targeted in clinic.
The preparation method of the internal standard working solution comprises the following steps:
accurately weighing standard substances of internal standard substances corresponding to 7 anti-new coronavirus medicines, and respectively adding methanol or water for dissolving to obtain 7 internal standard stock solutions;
the 7 internal standard stock solutions were diluted with a diluent (methanol: water =1,v/v) to obtain 7 internal standard intermediate solutions, respectively;
mixing the 7 internal standard intermediate solutions, and diluting with a diluent (methanol: water =1, v/v) to obtain an internal standard substance working solution;
the types of internal standard substances and the concentrations of the internal standard substance working solution are preferably as follows:
(II) establishment of standard curve equation
And (3) taking the standard solution, detecting by using a high performance liquid chromatography-mass spectrometer to obtain a series of chromatograms of the standard solution with different concentrations, fitting to obtain a standard curve equation of each anti-new coronavirus drug by taking the ratio of the chromatographic peak area of the anti-new coronavirus drug to the chromatographic peak area of the corresponding internal standard substance as a vertical coordinate (y) and the ratio of the concentration of the anti-new coronavirus drug to the concentration of the corresponding internal standard substance as a horizontal coordinate (x).
(III) pretreatment of sample to be tested
Taking 10 mu L of a sample to be detected, 10 mu L of internal standard working solution and 500-8000 mu L of protein precipitator, placing the sample to be detected, firstly carrying out vortex mixing for 2-10 min at the rotating speed of 1000-2500 r/min, then centrifuging for 3-10 min at the rotating speed of 8000-14000 r/min, taking supernate, adding a diluent (methanol: water =3:7-7, v/v) for diluting (the total dilution multiple of the sample to be detected is 100-5000 times), and carrying out vortex mixing for 30 s-3 min at the rotating speed of 1000-2500 r/min to obtain a sample to be detected.
(IV) detection of sample to be detected
And (3) taking the sample to be detected, detecting by using a high performance liquid chromatography-mass spectrometer to obtain a chromatogram, and calculating to obtain the respective concentrations of the 7 anti-new coronavirus medicines in the sample to be detected according to the chromatographic peak areas of the anti-new coronavirus medicines, the chromatographic peak areas of the corresponding internal standard substances and the corresponding standard curve equations.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts the matching of a proper pretreatment method and chromatographic conditions, so that the provided detection method can simultaneously detect 7 anti-new coronavirus medicaments of lopinavir, ritonavir, ribavirin, chloroquine, arbidol, darunavir and cobicistat, and the method has the advantages of high accuracy and sensitivity, small required sample amount, capability of completing detection by using serum/plasma of not more than 10 mu L, good reproducibility and short detection time.
Drawings
FIG. 1 is a chromatogram of 7 anti-neocoronavirus drugs in a serum sample to be tested in example 1 of the present invention;
FIG. 2 is a mass spectrum of 7 anti-neocoronaviral drugs and their internal standards in a serum sample to be tested in example 1 of the present invention;
FIG. 3 is a chromatogram of chloroquine in comparative example 2;
FIG. 4 is a chromatogram of 7 anti-neocoronavirus drugs in a serum sample to be tested in comparative example 3;
wherein, 1-lopinavir, 2-ritonavir, 3-ribavirin, 4-chloroquine, 5-arbidol, 6-darunavir, and 7-cobicistat.
Detailed Description
The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings. It should be understood by those skilled in the art that the specific embodiments are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
The embodiment provides a method for simultaneously detecting the content of 7 anti-new coronavirus medicines, which comprises the following specific steps:
preparation of standard solution
10 μ L of the target working solution, 10 μ L of the internal standard working solution, and 1000 μ L of methanol were placed in a centrifuge tube, and after vortex mixing was performed at 1500rpm for 30 seconds, 10 μ L of the mixed solution was taken, and 200 μ L of a diluent (methanol: water =1, v/v) was added to dilute the solution, to obtain 8 standard solutions with different concentrations.
The preparation method of the target working solution comprises the following steps:
accurately weighing 7 anti-new coronavirus drug standards, and respectively adding methanol or water for dissolving to obtain 7 target stock solutions;
the 7 target stock solutions were diluted with a mixture of methanol and water (methanol: water =1,v/v) to obtain 7 target intermediate solutions;
the concentrations of the above 7 target stock solutions and 7 target intermediate solutions are shown in table 1 below:
TABLE 1
Mixing the 7 target intermediate solutions, and diluting with a mixture of methanol and water (methanol: water =1,v/v) to obtain 8 target working solutions with different concentrations containing 7 anti-neocoronavirus drugs;
the concentrations of the anti-neocoronavirus drugs in the 8 target working solutions are shown in the following table 2:
TABLE 2
The preparation method of the internal standard working solution comprises the following steps:
accurately weighing standard substances of internal standard substances corresponding to 7 anti-new coronavirus medicines, and respectively adding methanol or water for dissolving to obtain 7 internal standard stock solutions;
diluting the 7 internal standard stock solutions with a mixed solution of methanol and water (methanol: water =1, v/v) to obtain 7 internal standard intermediate solutions;
mixing the 7 internal standard intermediate solutions, and diluting with a mixed solution of methanol and water (methanol: water =1, v/v) to obtain an internal standard working solution;
the types of the internal standard substances, the concentrations of the internal standard stock solution, the internal standard intermediate solution and the internal standard working solution are shown in the following table 3:
TABLE 3
(II) establishment of standard curve equation
And (3) taking the standard solution, detecting by using a high performance liquid chromatography-mass spectrometer to obtain a series of chromatograms of the standard solution with different concentrations, fitting to obtain a standard curve equation of each anti-new coronavirus drug by taking the ratio of the chromatographic peak area of the anti-new coronavirus drug to the chromatographic peak area of the corresponding internal standard substance as a vertical coordinate (y) and the ratio of the concentration of the anti-new coronavirus drug to the concentration of the corresponding internal standard substance as a horizontal coordinate (x).
In this example, the conditions of the high performance liquid chromatography were as follows:
a chromatographic column: a C18 chromatographic column protected by isobutyl side chains (Shim-pack Velox SP-C18, filler particle size 2.7 μm, diameter 2.1mm, length 100 mm);
column temperature: at 32 ℃;
mobile phase: the method comprises the following steps of (1) preparing a liquid phase A and a liquid phase B, wherein the liquid phase A is an aqueous solution containing 10 mmol/L ammonium formate and 0.5vol% formic acid, and the liquid phase B is acetonitrile;
and (3) an elution mode: gradient elution;
sample introduction amount: 10. mu L;
analysis time: 2.5 And (5) min.
The gradient elution conditions are shown in table 4 below:
TABLE 4
The mass spectrometry conditions were as follows:
the mass spectrometer AB Sciex 4500MD adopts electrospray ionization (ESI), positive ion mode, multiple Reaction Monitoring (MRM); electrospray Voltage (ion spray Voltage): 5000V; ion source Temperature (TEM): 400 ℃; flow rate of atomizing Gas (Gas 1): 30 L/min; flow rate of assist Gas (Gas 2): 40 L/min; the ion pair parameters are shown in table 5 below:
TABLE 5
The retention times and standard curve equations for the 7 anti-neocoronavirus drugs in this example are shown in table 6 below:
TABLE 6
As can be seen from Table 6, the linear correlation coefficient R of the standard curves for the 7 anti-neocoronavirus drugs in the respective standard solution concentration ranges 2 And the Tg is more than 0.9900, and the good linear relation is shown.
(III) pretreatment of sample to be tested
Taking 10 mu L of a serum sample to be detected, 10 mu L of an internal standard working solution and 1000 mu L of methanol, placing the serum sample to be detected, 10 mu L of the internal standard working solution and 1000 mu L of methanol in a centrifuge tube, firstly carrying out vortex mixing for 5min at the rotating speed of 2500 r/min, then carrying out centrifugation for 5min at the rotating speed of 12000 r/min, taking 10 mu L of supernate, adding 200 mu L of diluent (methanol: water =1, v/v) for dilution, and carrying out vortex mixing for 1min at the rotating speed of 2500 r/min to obtain a sample to be detected.
(IV) detection of sample to be tested
And (3) taking the sample to be detected, detecting by using a high performance liquid chromatography-mass spectrometer to obtain a chromatogram, and calculating to obtain the respective concentrations of the 7 anti-new coronavirus medicines in the sample to be detected according to the chromatographic peak area of each anti-new coronavirus medicine, the chromatographic peak area of the corresponding internal standard substance and the corresponding standard curve equation.
In this embodiment, the chromatogram of 7 anti-neocoronavirus drugs in the serum sample to be tested is shown in fig. 1, and the mass spectra of 7 anti-neocoronavirus drugs and their internal standard substances in the serum sample to be tested are shown in fig. 2. As can be seen from the figure, the chromatogram obtained by the detection method provided by the invention has smooth base line and good target object peak shape, and the detection method provided by the invention can well realize the separation and detection of 7 anti-new coronavirus medicines.
Methodology analysis
(1) Recovery and in-batch precision
Taking target working solution, preparing low, medium and high 3 concentration drug-containing serum by using blank serum, and carrying out sample-adding recovery rate experiment and batch precision experiment. The above-mentioned 3 concentrations of serum containing drugs were treated and subjected to sample injection analysis in the same manner as in the "pretreatment of sample to be tested (three)" and detection of sample to be tested (four) ", and the analysis and measurement were repeated for 3 batches, and the results of recovery and precision were shown in table 7 below.
TABLE 7
As can be seen from Table 7, the average recovery rate of 7 anti-neocoronaviruses under 3 additive concentrations of low, medium and high is 98.0 to 107.9%, and the relative standard deviation RSD is 0.5 to 5.7%, which shows that the detection method provided by the invention has higher accuracy and batch precision.
(2) Daily and daytime precisions
Taking target working solution, preparing low, medium and high 3 concentrations of drug-containing serum with blank serum, and performing intra-day precision and inter-day precision experiments. The above-mentioned drug-containing sera at 3 concentrations were treated and analyzed by sample injection in the same manner as in the "pretreatment of sample to be tested (three)" and detection of sample to be tested (four) "of this example, and the results are shown in table 8 below, wherein the assay was repeated 3 batches for intraday precision and 5 batches for daytime precision.
TABLE 8
As can be seen from Table 8, the daily precision of 7 anti-neocoronavirus drugs at 3 low, medium and high addition concentrations is 1.75-7.18%, and the daytime precision is 4.93-8.23%, which indicates that the detection method provided by the invention has higher daily precision and daytime precision.
Example 2
The embodiment provides a method for simultaneously detecting the content of 7 anti-new coronavirus medicines, which is different from the method in embodiment 1 in that in the preparation of a standard solution and the pretreatment of a sample to be detected, a protein precipitator is 500 mu L of acetonitrile, 40 mu L of supernate is obtained after centrifugation, the dosage of a diluent is 160 mu L, and the total dilution multiple is 250 times.
After instrumental analysis, the standard curve equation is obtained as shown in the following table 9:
TABLE 9
Preparing high and low concentration standard blood samples, processing and sample injection analysis according to the pretreatment method of the sample to be detected in the embodiment, and performing parallel processing for 6 times to obtain the precision data shown in the following table 10:
watch 10
From the experimental results in tables 9 and 10, it can be seen that the linearity of the standard curve and the precision result are good when the sample analysis is performed within the range of the pretreatment given in the present invention.
Comparative example 1
The difference between the method and the embodiment 1 is that in the preparation of the standard solution and the pretreatment of the serum sample to be detected, the dosage of the protein precipitator, namely methanol, is 50 mu L, the dosage of the diluent is 60 mu L, and the total dilution multiple is about 50 times.
After the analysis of an instrument, fitting to obtain a standard curve equation, substituting the ratio of the chromatographic peak areas of the target substance and the internal standard substance into the standard curve equation to back calculate the concentration of the target substance, wherein the ratio of the concentration to the theoretical value is accurate. Accuracy and linear correlation coefficient R 2 The data are shown in table 11 below:
TABLE 11
From the above, when the protein was precipitated using a low-fold organic reagent and the dilution factor was low, the linearity of the plurality of substances was not qualified, and quantification was not possible.
Comparative example 2
The comparison example provides a method for simultaneously detecting the content of 7 anti-new coronavirus medicines, and the difference from the example 1 is that in the preparation of the standard solution and the pretreatment of a sample to be detected, the diluent is pure water. 1 labeled blood sample was treated using this method and the injection was repeated 6 times.
After instrumental analysis, the precision data obtained are shown in table 12 below:
TABLE 12
It can be seen from the experimental results in table 12 that when the diluent is replaced with pure water, the sample solvent is an aqueous solution containing low-concentration methanol, which results in poor detection precision of ritonavir, chloroquine, abidol, darunavir, and cobicistat, and accurate quantification cannot be guaranteed.
Further, the chromatogram of chloroquine is shown in FIG. 3. As can be seen from FIG. 3, the chloroquine chromatogram shows a change in peak shape, resulting in the appearance of a shoulder.
Comparative example 3
The comparative example provides a method for simultaneously detecting the content of 7 anti-new coronavirus medicines, and is different from the method in example 1 in that the gradient elution conditions are as follows:
the chromatogram of 7 anti-neocoronavirus drugs in the serum sample to be tested obtained in the comparative example is shown in FIG. 4.
The chromatographic conditions used in the comparative example are obtained by optimizing according to the conventional gradient elution setting idea, the target substance normally flows out in the elution and washing processes of the chromatographic column, the initial flow is balanced with the chromatographic column, and the analysis time of a single sample is long and needs 6.5min.
The gradient elution condition of the application is set according to an unconventional thought, and is continuously optimized through experiments, and finally, the chromatographic column is firstly washed by the aqueous mobile phase for a short time (0-0.5 min) so that the target substance can be kept in the fixed phase at the front section of the chromatographic column, most of blood matrix impurities directly flow out because the impurities cannot be kept, and then the target substance is gradually eluted when pure organic phase washing and balancing (0.5-2.5 min) are carried out, and when the target substance is completely eluted, the chromatographic column is completely balanced and is consistent with the initial mobile phase of the next needle. Through the special gradient setting method, the application realizes the analysis of 7 substances within 2.5min, and simultaneously realizes the accurate quantification effect under the condition that the standard solution matrix is only a reagent but not serum or plasma because the interference of the blood matrix co-flowed when the target substance is out of peak is reduced.
In conclusion, the invention adopts the proper pretreatment method and the chromatographic condition to be matched, so that the detection method has high accuracy and sensitivity and good reproducibility, the plasma/serum is directly injected after protein precipitation and dilution treatment, the pretreatment process is simple, convenient and quick, the experiment cost is reduced, the analysis time under the flow rate of 0.4 mL/min only needs 2.5min, the detection time is short, and the invention is more beneficial to the clinical large-flux sample detection. When the current treatment method or chromatographic conditions are beyond the scope of the invention, the detection effect is easily deteriorated.
The foregoing are merely exemplary embodiments of the present disclosure, which enable those skilled in the art to understand or practice the present disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the disclosure. Thus, the present disclosure is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (17)
1. A method for simultaneously detecting the content of 7 anti-new coronavirus medicines is characterized by comprising the following steps:
establishing a standard curve equation: preparing at least three standard solutions with different concentrations and containing 7 anti-new coronavirus medicines and internal standard substances thereof; detecting the standard solution by using a liquid chromatography-mass spectrum, and establishing a standard curve equation of each anti-new coronavirus drug/internal standard substance chromatographic peak area ratio-anti-new coronavirus drug/internal standard substance concentration ratio according to a detection result;
pretreatment of a sample to be detected: mixing a sample to be detected, an internal standard substance and a protein precipitator, wherein the volume of the protein precipitator is more than 50 times of that of the sample to be detected, centrifuging, and taking supernatant for dilution to obtain a sample to be detected;
detecting a sample to be detected: detecting the sample to be detected by using a liquid chromatography-mass spectrometry, and calculating the content of 7 anti-new coronavirus medicines in the sample to be detected according to a detection result and the standard curve equation;
wherein the 7 anti-neocoronavirus drugs are lopinavir, ritonavir, ribavirin, chloroquine, arbidol, darunavir and cobicistat.
2. The method of claim 1, wherein the standard solution is prepared by: preparing at least three target working solutions with different concentrations and containing 7 anti-new coronavirus medicines, and then treating the target working solutions according to the same pretreatment method as the sample to be detected to obtain the standard solution.
3. The method of claim 2, wherein the solvent of the target working fluid is a mixture of methanol and water.
4. The method according to claim 1, wherein the concentrations of the same internal standard substance in standard solutions of different concentrations are the same.
5. The method of claim 1, wherein the internal standard of the 7 anti-neocoronavirus drugs is an isotopic internal standard.
6. The method of claim 5, wherein the internal standard of the 7 anti-neocoronavirus drugs are lopinavir-d 8, ritonavir-d 6, ribavirin- 13 C5, chloroquine-d 5, arbidol-d 6, darunavir-d 9 and cobicistat-d 8.
7. The method of claim 1, wherein the sample to be tested is plasma or serum.
8. The method according to claim 1, wherein the internal standard substance is added in the form of an internal standard working fluid, which is a solution containing the 7 internal standard substances of the anti-new coronavirus drugs.
9. The method according to claim 8, wherein the solvent of the internal standard working solution is a mixture of methanol and water.
10. The method according to claim 8, wherein the volume ratio of the sample to be tested to the internal standard working solution is 1.
11. The method according to claim 1, wherein the protein precipitating agent is selected from one or more of methanol, ethanol, acetonitrile and isopropanol.
12. The method of claim 1, wherein the volume of the protein precipitating agent is 50 to 800 times the volume of the sample to be tested.
13. The method according to claim 1, wherein a diluent used for diluting the supernatant in the pretreatment of the sample to be tested is a mixed solution of methanol and water in a volume ratio of 3:7-7:3.
14. The method as claimed in claim 1, wherein the total dilution factor of the sample to be tested in the pre-treatment of the sample to be tested is 100-5000 times.
15. The method of claim 1, wherein the conditions of the liquid chromatography comprise:
the chromatographic column is a C18 chromatographic column with isobutyl side chain protection;
and/or the column temperature is 30-55 ℃;
and/or the flow velocity of the mobile phase is 0.3-0.6 mL/min;
and/or the mobile phase consists of a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution containing 1-50 mmol/L ammonium formate and 0.1-1vol% formic acid, and the mobile phase B is acetonitrile;
and/or the elution mode is gradient elution.
16. The method of claim 15, wherein the mobile phase of the gradient elution is:
0-0.5kmin: the 50% mobile phase A decreases linearly to 0%50% mobile phase B was linearly increased to 100%;
0.5k -1kmin:100% mobile phase B;
1k -2.5kmin:50% mobile phase a,50% mobile phase B;
wherein the content of the first and second substances,k = 0.4/mobile phase flow rate value.
17. The method of claim 1, wherein the conditions of mass spectrometry comprise:
the mass spectrum detector adopts an electrospray ion source, a positive ion mode and multi-reaction monitoring, the electrospray voltage is 5000V, the ion source temperature is 400-600 ℃, the atomization air flow rate is 30-80L/min, and the auxiliary air flow rate is 40-70L/min.
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