CN116699020A - Method and kit for detecting antiarrhythmic drugs in blood plasma by HPLC-MS - Google Patents
Method and kit for detecting antiarrhythmic drugs in blood plasma by HPLC-MS Download PDFInfo
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- CN116699020A CN116699020A CN202310630547.9A CN202310630547A CN116699020A CN 116699020 A CN116699020 A CN 116699020A CN 202310630547 A CN202310630547 A CN 202310630547A CN 116699020 A CN116699020 A CN 116699020A
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- 239000003416 antiarrhythmic agent Substances 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 35
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 title claims abstract description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 45
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 40
- 239000003085 diluting agent Substances 0.000 claims abstract description 34
- 238000001514 detection method Methods 0.000 claims abstract description 26
- LOUPRKONTZGTKE-LHHVKLHASA-N quinidine Chemical compound C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@H]2[C@@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-LHHVKLHASA-N 0.000 claims abstract description 24
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Classifications
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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
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- G—PHYSICS
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- 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
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a method and a kit for detecting antiarrhythmic drugs in blood plasma by HPLC-MS, comprising the following steps: adding methanol into the plasma, mixing the plasma and the methanol in a volume ratio of 1:1.5-3, shaking uniformly, carrying out protein precipitation, and centrifuging to obtain a supernatant; mixing and diluting the supernatant with 0.05-0.3% formic acid aqueous solution according to the volume ratio of 1:4-10 to obtain a diluent; and detecting the obtained diluent by adopting a liquid chromatography tandem mass spectrometry method, and simultaneously detecting quinidine, lidocaine, procainamide and N-acetylprocainamide in blood plasma. The supernatant after precipitation is diluted by a proper diluent, so that peak saturation phenomenon of procainamide and N-acetylprocainamide caused by overhigh concentration is effectively avoided, peak bifurcation phenomenon of procainamide caused by short retention time and influence of solvent effect is avoided, different antiarrhythmic drugs can be detected simultaneously, and the method is a detection method which is simple in sample processing, reliable in result, small in matrix effect and friendly to detection equipment.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a method and a kit for detecting an antiarrhythmic drug in blood plasma by HPLC-MS.
Background
The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.
Arrhythmia is an important group of cardiovascular diseases, has become one of global public health problems, seriously affects more than 3000 tens of thousands of people worldwide, can be independently developed, and can also be accompanied with other cardiovascular diseases. Sudden death due to sudden onset of arrhythmia may also continue to involve the heart and cause its failure, placing a heavy burden on the healthcare system in many countries. At present, the arrhythmia diseases are clinically controlled by using anti-arrhythmia drugs quinidine, lidocaine, procainamide (N-acetylprocainamide) and the like, but aiming at different drug-taking individuals, the differences of metabolic degrees of drugs in bodies are obvious due to the influences of factors such as individual metabolic enzyme differences, combined drug effects, pathological factors, living and eating habits and the like, and the in-vivo stable drug concentrations of different individuals can be different by more than 20 times. The therapeutic concentration range of the antiarrhythmic drugs is generally narrow, the poisoning concentration and the therapeutic concentration are very close, for patients with faster in vivo metabolism of the drugs, the in vivo drug concentration is lower than the therapeutic concentration, and an effective therapeutic effect cannot be achieved, while for patients with slower in vivo metabolism of the drugs, the in vivo drug concentration is higher than the therapeutic concentration and even reaches the poisoning concentration due to drug accumulation. Through the rapid monitoring of the free blood concentration after administration of the arrhythmia patient, a powerful technical support is provided for a clinician aiming at the optimal administration dosage and administration time of the arrhythmia patient, blindness of administration (comprising dosage, decrement, medicine changing, medicine stopping and the like) is reduced to a great extent, the medicine can exert the optimal curative effect in the patient, reduce adverse reaction of the medicine and save treatment cost.
Current methods for monitoring the blood concentration of quinidine, lidocaine, procainamide and N-acetylprocainamide include enzyme-linked immunoassay and High Performance Liquid Chromatography (HPLC). However, the enzyme-linked immunoassay has poor specificity and sensitivity, because the medicines have similar chemical structures and similar antigen surface areas, antigen-antibody reactions can occur, and the detected medicine concentration is difficult to completely distinguish by adopting an immunoassay, so that deviation exists between the detected medicine concentration and the actual situation, the curative effect cannot be well predicted and the adverse reaction cannot be well estimated, and the enzyme-linked immunoassay has specificity and can only detect one compound. The high performance liquid chromatography has high background signal and poor specificity, has very high requirements on the pretreatment of samples, and needs to remove impurities as much as possible to eliminate the interference of co-outflow compounds on detection results.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a method and a kit for detecting antiarrhythmic drugs in blood plasma by HPLC-MS.
In order to achieve the above object, the present invention is realized by the following technical scheme:
in a first aspect, the present invention provides a method for detecting an antiarrhythmic agent in plasma by HPLC-MS, comprising the steps of:
adding methanol into the plasma, mixing the plasma and the methanol in a volume ratio of 1:1.5-3, shaking uniformly, carrying out protein precipitation, and centrifuging to obtain a supernatant;
mixing and diluting the supernatant with 0.05-0.3% formic acid aqueous solution according to the volume ratio of 1:4-10 to obtain a diluent;
and detecting the obtained diluent by adopting a liquid chromatography tandem mass spectrometry method, and simultaneously detecting quinidine, lidocaine, procainamide and N-acetylprocainamide in blood plasma.
In some embodiments, the aqueous formic acid solution has a concentration of 0.05 to 0.15%.
In some embodiments, the liquid chromatography conditions are: chromatographic column model: YS LCC 104MD,2.1 mm. Times.50 mm,2.6 μm;
mobile phase a: wherein the concentration of formic acid is 0.05% -3%, and the percentage is mass percent; the concentration of ammonium formate is 5-10mmol/L;
mobile phase B: methanol;
flow rate: 0.1-0.4mL/min;
column temperature: 25-50 ℃;
sample injection volume: 1-10 mu L;
gradient elution procedure: 0min:5% B,3min:98% b,4min:98% b,4.1min:5% B,5.5min:5% B.
Preferably, the mobile phase A is 0.05% -1% formic acid, 5-8mmol/L ammonium formate aqueous solution.
Further preferably, the mobile phase A is 0.05% -0.5% formic acid, 5-6mmol/L ammonium formate aqueous solution.
Still more preferably, the mobile phase A is 0.1% formic acid, 5mmol/L ammonium formate in water.
In some embodiments, the mass spectrometry conditions are: ion source: electrospray ion source (ESI), positive ion mode; ion spray voltage: 3500V; sheath gas: 5.58L/min; auxiliary gas: 8L/min; sweep gas: 1.5L/min; ion transport tube temperature: 325 deg.c; atomization temperature: 350 ℃; scanning mode: multiple reaction monitoring.
In some embodiments, mass spectrometry detection ion pairs for four species to be tested are as follows:
in a second aspect, the invention provides a kit for detecting antiarrhythmic drugs in plasma by HPLC-MS, which at least comprises a calibrator, low, medium and high level quality control substances, a calibrator diluent (blank plasma), a protein precipitant, a sample diluent, a mobile phase additive, a chromatographic column and a sample injection plate;
the antiarrhythmic drugs are quinidine, lidocaine, procainamide and N-acetylprocainamide.
The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:
according to the invention, an organic solvent is established for precipitating protein aiming at complex biological matrixes, and a proper diluent (0.05% -0.3% formic acid aqueous solution) is selected for dilution of the supernatant after precipitation, so that peak saturation phenomenon of procainamide and N-acetylprocainamide caused by overhigh concentration is effectively avoided, peak bifurcation phenomenon of procainamide caused by influence of solvent effect due to short retention time is avoided, and different antiarrhythmic drugs can be detected simultaneously.
According to the invention, a liquid chromatography-tandem mass spectrometry method of quinidine, lidocaine, procainamide and N-acetylprocainamide is established simultaneously, the compound peaks within 3min, the analysis time is short, the simultaneous monitoring of the concentration of four antiarrhythmic drugs can be realized by one-needle sample injection, and the trouble that the inspector needs to monitor different drugs by adopting different monitoring means is solved.
The invention applies the accuracy and the effectiveness of three-level quality control product investigation methods to avoid the distortion of detection results.
According to the invention, three pairs of parent ions/child ions are collected for each compound, and besides quantitative ion pairs are used for quantification, two pairs of qualitative ions assist qualitative determination, so that false positive results in detection results can be effectively avoided.
The invention adopts the external standard method to quantitatively detect the concentration of the antiarrhythmic drugs, avoids using expensive internal standard substances, and greatly reduces the detection cost.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a sample preprocessing flow chart of an embodiment of the present invention;
FIG. 2 shows peak shape comparisons of different amounts of procainamide using 0.1% formic acid aqueous solution as diluent, wherein a is 120. Mu.L and b is 170. Mu.L;
FIG. 3 is a total ion flow chromatogram (TIC) in an embodiment of the invention;
FIG. 4 is an ion-extracting chromatogram (EIC) of procainamide in an embodiment of the invention;
FIG. 5 is an Extraction Ion Chromatogram (EIC) of N-acetylprocainamide in an embodiment of the invention;
FIG. 6 is an ion-extracting chromatogram (EIC) of lidocaine in an embodiment of the invention;
FIG. 7 is a quinic Ding Diqu ion chromatogram (EIC) of an embodiment of the invention;
FIG. 8 is a standard working curve of procainamide in an embodiment of the present invention;
FIG. 9 is a standard operating curve for N-acetylprocainamide in an embodiment of the present invention;
FIG. 10 is a standard working curve of lidocaine in an embodiment of the present invention;
FIG. 11 is a quinic Ding Biaozhun operating curve in an embodiment of the invention;
FIG. 12 is a lyophilization curve in an example of the invention.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The Chinese and English names and CAS numbers of the 4 antiarrhythmic drugs are shown in Table 1:
information such as Chinese and English names and CAS numbers of 14 antiarrhythmic drugs
The invention is further illustrated below with reference to examples.
Examples
The invention provides a method for detecting the concentration of an antiarrhythmic drug in blood plasma by combining a precipitated protein method with a high performance liquid chromatography-tandem mass spectrometry, which fully verifies the performance of the method in the aspects of linearity, carrying pollution, accuracy, precision, matrix effect and the like. The technological formula of the kit is illustrated, and the performance of the kit is verified in terms of bottle opening/redissolution stability, validity period stability and the like.
Instrument for measuring and controlling the intensity of light
YSEXACT 9050MD liquid chromatography (shandong flourishing biotechnology limited); electronic balance (radtag, germany); a 24-bit vortex mixer (eppendorf, germany); centrifuge (eppendorf, germany); ultrapure water machines (millipore, usa).
Reagent consumable
Quinidine, lidocaine, procainamide standard (Tianjin allta technologies limited); n-acetylprocainamide (Sigma-Aldrich); methanol was chromatographically pure (Sigma-Aldrich); formic acid, ammonium formate (LC-MS grade, fisher); drohent distilled water (purified by Milli-Q ultra-pure water); chromatographic column (Shandong Yingsheng Biotechnology Co., ltd.).
Preparation of standard mother liquor
Accurately weighing 50 mg+/-0.01 mg quinine Ding Biaozhun products in a 10mL brown volumetric flask, dissolving with methanol, and fixing the volume to a scale to obtain 5mg/mL standard mother liquor. The preparation process of the mother liquor of other standard substances is the same as that of the mother liquor of other standard substances, and the standard mother liquor corresponding to 4 antiarrhythmic drugs is obtained.
Preparation of standard Mixed stock solutions
Preparation of standard mixed stock solution: respectively transferring 0.6mL, 1.2mL and 2.4mL of quinidine, lidocaine, procainamide and N-acetylprocainamide standard mother liquor into a 10mL brown volumetric flask, and fixing the volume to the scale by using methanol. A mixed stock solution was obtained.
Preparation of calibrator solution and preparation of standard working solution (plasma matrix)
The high-concentration calibrator is prepared by adding a certain volume of solvent mixed standard solution of anti-arrhythmia drugs quinidine, lidocaine, procainamide and N-acetylprocainamide into a calibrator diluent (blank plasma).
Specifically, 100. Mu.L of a standard mixed stock solution (2.4.4) was removed, 2mL of a calibrator dilution (plasma) was added, and the mixture was vortexed and homogenized to obtain a high concentration calibrator solution S8.
The standard working solution is prepared by gradually diluting high-concentration calibrator by calibrator diluent (blank plasma) at S1-S8 8 concentration points. The specific formulation process and concentration points are shown in Table 2:
TABLE 2 working fluid concentration points and formulation procedure for standard
Low, medium and high level quality control product preparation (plasma matrix)
The high-level quality control product is prepared by adding a certain volume of calibrator dilution (blank plasma) into a calibrator solution (2.4.5), the medium-concentration quality control product is prepared by adding a certain volume of calibrator dilution (blank plasma) into the high-level quality control product, and the low-concentration quality control product is prepared by adding a certain volume of calibrator dilution (blank plasma) into the medium-level quality control product.
Specifically, mixing a calibrator solution (2.4.5) and a calibrator diluent (blank plasma) according to a ratio of 2:1, transferring 1mL of the calibrator solution (2.4.5), adding 0.5mL of the calibrator diluent (blank plasma), and uniformly mixing by vortex oscillation to obtain a high-level quality control product solution; mixing the high-level quality control product solution with calibrator dilution (blank plasma) according to a ratio of 1:1, transferring 500 mu L of the high-level quality control product solution, adding 500 mu L of calibrator dilution (blank plasma), and mixing by vortex oscillation to obtain a medium-level quality control product solution; mixing the medium-level quality control product solution with the calibrator dilution (blank plasma) according to the ratio of 1:4, transferring 250 mu L of the medium-level quality control product solution, adding 1mL of the calibrator dilution (blank plasma), and uniformly mixing by vortex oscillation to obtain the low-level quality control product solution.
The concentration of the quality control product is shown in table 3:
TABLE 3 concentration points for low, medium and high quality control products
Pretreatment method
Precisely removing standard solution (plasma matrix), quality control sample, or human plasma 50 μl into a clean centrifuge tube; adding 100 μl of methanol, stirring at 2000rpm, mixing for 2min, and centrifuging at 10000rpm at room temperature for 10min; and (3) sucking 30 mu L of supernatant, transferring to a 1mL 96-well plate, adding 170 mu L of 0.1% formic acid aqueous solution for dilution, repeatedly sucking and beating for several times by using a 200 mu L row gun, and uniformly mixing to obtain the sample. The specific pretreatment flow is shown in fig. 1:
detection method
Chromatographic conditions
Chromatographic column model: YS LCC 104MD (2.1 mm. Times.50 mm,2.6 μm);
mobile phase a:0.1% formic acid, 5mmol/L ammonium formate aqueous solution;
mobile phase B: methanol;
flow rate: 0.3mL/min;
column temperature: 35 ℃;
sample injection volume: 1 μl;
gradient elution procedure is shown in table 4:
TABLE 4 gradient elution procedure
Mass spectrometry conditions:
ion source: electrospray ion source (ESI), positive ion mode;
ion spray voltage: 3500V;
sheath gas: 5.58L/min;
auxiliary gas: 8L/min;
sweep gas: 1.5L/min;
ion transport tube temperature: 325 deg.c;
atomization temperature: 350 ℃;
scanning mode: multiple reaction monitoring
Mass spectral parameters for each target are shown in table 5:
TABLE 5 Mass Spectrometry parameters for each target
Experimental results
Precipitant type and amount optimization
According to the invention, three precipitants of methanol, acetonitrile and methanol-acetonitrile=1:1 (v/v) are respectively examined, the examined proportions are respectively samples of the precipitants=1:2, 1:3 and 1:4, as shown in table 6, and the results show that the three precipitants have no obvious change in the response of the four medicaments under the conditions of different extraction proportions. From the comprehensive consideration of saving cost, reducing toxicity and dosage of the organic solvent, finally, methanol is selected as a precipitant, and the precipitation ratio is sample, namely precipitant=1:2.
TABLE 6 response of antiarrhythmic drugs at different precipitants and ratios
Diluent type and amount optimization
After protein precipitation, because the therapeutic concentration of the four medicines is higher, especially procainamide and N-acetylprocainamide, the high concentration point chromatographic peak is easy to generate saturation phenomenon, and proper diluents are needed to be selected to dilute the supernatant, and then the supernatant is detected by an upper machine, so that the types and the dosage of the diluents are examined. Pure water and 0.1% formic acid aqueous solution are respectively examined as diluents, and the volumes of the diluents added are 70 mu L, 120 mu L and 170 mu L, as shown in figure 2, the procainamide has stronger solvent effect and peak bifurcation (as shown in figure 2) due to the fact that the retention time of the procainamide is relatively short and the type of the diluents is not selected or the volume is too small, the solvent effect is eliminated, and the peak bifurcation phenomenon disappears. Thus, 0.1% formic acid water was finally selected as the diluent, the volume of which was 170. Mu.L.
Total ion flow chromatography (TIC) and Extracted Ion Chromatography (EIC) under optimal chromatography and mass spectrometry conditions
The MRM mode is adopted to monitor the ion pairs of 4 antiarrhythmic drugs, the quantitative ion pairs are used for result quantification, and the 2 pairs of qualitative ion pairs assist in qualitative confirmation. The obtained TIC diagram and EIC diagram of the lowest point of the standard curve are shown in fig. 3-7.
Quantitative determination of external standard
An external standard quantitative method is adopted, S1-S8 8 concentrations of standard working solution (plasma matrix) are taken as X axis, peak area of the standard working solution is taken as Y axis, an external standard curve is established, linear fitting equations of quinidine, lidocaine, procainamine and N-acetylprocainamine in respective concentration ranges are established, linearity is good, correlation coefficients are basically above 0.99, and a Quantic fitting mode is adopted for the N-acetylprocainamine. The specific linear regression equation and the linear correlation coefficient are shown in Table 7.
TABLE 7 Linear regression equation and Linear correlation coefficient for antiarrhythmic drugs
The standard operating curves are shown in fig. 8-11.
Portable pollution verification
In the process of detecting target analytes in samples by utilizing high performance liquid chromatography-tandem mass spectrometry, the problem of carrying pollution is often encountered, and the accuracy and reliability of detection results are affected. The invention verifies the carrying pollution in two ways.
One way of verification is to repeatedly sample the standard curve low concentration sample for 5 needles, then alternately sample the standard curve high concentration sample for 1 needle and the low concentration sample for 2 continuous needles, repeat for 5 times, and compare the deviation of the high-low concentration value conversion sample mean (H-L) and the low-low concentration value conversion sample mean (L-L). The second verification mode is to alternately sample the standard curve high-concentration sample and the blank sample 5 times, and compare the average value of the peak areas of the sample analytes sampled 5 times for the blank sample with the average value of the peak areas sampled 5 times for the standard curve low-concentration sample of the first verification mode.
The result of the first verification mode shows that the deviation between the average value of the detection results of the standard curve low-concentration samples sampled repeatedly for 5 times and the average value of the detection results of the standard curve low-concentration samples sampled alternately with the H-L concentration is lower than 20%; and the difference between the average value of the H-L concentration value conversion samples and the average value of the L-L concentration value conversion samples is smaller than 3SD of the L-L concentration value conversion samples. The second validation mode shows that the average value of the area of the analyte peak in the blank sample after the high concentration sample is lower than 20% of the average value of the area of the analyte peak in the standard curve low concentration sample. Details are shown in Table 8.
Table 8 carries contamination verification data
Accuracy verification
And (5) evaluating the accuracy of the method by adopting a standard adding recovery rate test. According to the preparation method of the quality control product with the low level, the medium level and the high level of 2.4.6, 3 samples to be tested with different concentrations of low, medium and high are prepared, 5 samples with each concentration are treated in parallel, and the result shows that the average recovery rate of 4 antiarrhythmic drugs is between 91.62% and 111.73%, and the requirement of 85% to 115% is met. The CV of 5 replicates was between 1.97% -5.96%. See in particular table 9.
Table 9 recovery of four antiarrhythmic drugs at low, medium and high levels
Precision verification
According to the preparation method of the quality control product with the low level, the medium level and the high level of 2.4.6, 3 samples to be tested with different concentrations are prepared, 5 samples with each concentration are treated in parallel in one day, and the continuous treatment is carried out for three days, and the results show that the intra-batch precision of 4 antiarrhythmic drugs is expressed by a variation coefficient CV, the inter-batch precision CV value is between 1.31 and 8.59 percent, and the inter-batch precision CV value is between 2.82 and 7.22 percent, and is specifically shown in Table 10.
Table 10 four antiarrhythmic drugs low, medium, high three levels of intra-and inter-lot precision
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Matrix effect validation
The matrix effect is verified in two ways, wherein one verification way is to prepare low, medium and high 3 different concentrations according to a preparation method of a 2.4.6 low, medium and high three-level quality control product, each concentration sample is processed in parallel for 5 times, and each sample is repeatedly measured for 3 times; according to the same concentration setting, preparing a pure solvent standard solution, detecting the pure solvent standard solution on the same machine as a matrix sample, and verifying a matrix effect. The results show that the average value of matrix effector (MF) at low, medium, and high 3 concentration levels for the 4 antiarrhythmic drugs is between 0.87-1.22, and the CV value of matrix effector (MF) is between 3.9% -10.0%. See in particular table 11.
Another way of verification is to select a calibrator dilution to add the target analyte (analyte concentration is the standard curve midpoint concentration) as a matrix sample, a biological matrix sample, and 1:1, respectively carrying out pretreatment on the mixed solution, and then carrying out machine-on detection. Comparing the average value of the peak areas of the matrix sample added with the target analyte and the biological matrix sample with 1:1, and the result shows that the deviation is between 7.26 and 12.31. See table 12.
Table 11 matrix effector at low, medium and high three levels for four antiarrhythmic drugs
Table 12 results of matrix effect of mixing experiments
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From the results of the two verification modes, the method can effectively avoid the influence of the matrix effect on the detection result.
In a word, the detection method of the embodiment is used for detecting 4 antiarrhythmic drugs, has good reproducibility, stability and accuracy, and in a word, the method has high sensitivity, strong specificity, high accuracy and simpler pretreatment process, can rapidly complete the detection of the antiarrhythmic drugs, and provides a reliable detection method for monitoring the blood concentration of patients with clinical arrhythmia after taking the drugs.
Kit composition for detecting antiarrhythmic drugs in blood plasma by liquid chromatography-tandem mass spectrometry
The composition, formulation, specification and loading of the kit are shown in Table 13.
Table 13: kit composition of antiarrhythmic drugs
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Preparation of calibration Material
According to the preparation method of the standard mixed stock solution of 2.4.4, the standard mixed stock solution is prepared, then 5mL of the standard mixed stock solution is removed, added into 100mL of calibrator diluent (blank plasma) matrix, and fully mixed. Packaging into 3mL brown freeze-dried bottles according to 200 μl/bottle, freeze-drying the packaged calibrator solution in a freeze dryer to obtain dry powder, and storing at-18deg.C or below. When in use, the mixture is taken out, and 200 mu L of water is added for re-dissolution. Preparation of low, medium and high level quality control product
The quality control product is prepared by using a calibrator solution (2.4.9.1), and the quality control product is specifically prepared as follows:
high level quality control solution: the calibrator solution and calibrator diluent (blank plasma) are mixed according to the proportion of 2:1, 100mL of calibrator solution is removed by taking 150mL as a unit, 50mL of calibrator diluent (blank plasma) is added, and vortex oscillation and uniform mixing are carried out.
Medium level quality control product solution: mixing the high-level quality control product solution with the calibrator dilution (blank plasma) according to the ratio of 1:1, taking 75mL of the high-level quality control product solution with 150mL as a unit, adding 75mL of the calibrator dilution (blank plasma), and mixing by vortex oscillation.
Low level quality control solution: mixing the calibrator solution and calibrator diluent (blank plasma) according to a ratio of 1:4, taking 150mL as a unit, transferring 30mL of the middle-level quality control solution, adding 120mL of calibrator diluent (blank plasma), and mixing by vortex oscillation.
And sub-packaging the low, medium and high level quality control product solution into 3mL brown freeze-dried bottles according to 200 mu L/bottle, and freeze-drying the sub-packaged solution into dry powder in a freeze dryer, and preserving at-18 ℃ or below. When in use, the mixture is taken out, and 200 mu L of water is added for re-dissolution.
Preparation of calibrator dilution
Taking blank plasma, sub-packaging into 3mL brown freeze-dried bottles according to 1.5 mL/bottle, placing into a freeze dryer for freeze-drying to obtain dry powder, and preserving at-18 ℃ or below. When in use, the mixture is taken out, and 1.5mL of water is added for re-dissolution.
Preparation of the Diluent
Taking 1000mL as a unit, transferring 1mL of formic acid, adding ultrapure water to 1000mL, and carrying out ultrasonic dissolution and uniform mixing. Split charging is carried out according to 25 mL/bottle, and the product is preserved at room temperature.
Preparation of mobile phase A additives
15.765g of ammonium formate is precisely weighed by taking 100mL as a unit, ultrapure water is added to 100mL, and the mixture is dissolved and mixed uniformly by ultrasonic. Split charging is carried out according to 2 mL/bottle, and the storage is carried out at room temperature.
Lyophilization conditions
Freezing at-30 to-50 deg.c for 3-6 hr, sublimating at-30 to 0 deg.c for 12-18 hr, and resolving and drying at 0 to 20 deg.c for 6-10 hr.
Specifically, the calibrator, calibrator diluent and quality control solution to be freeze-dried are packaged into 3mL brown freeze-dried bottles, half plugs are added, the bottles are placed in a vacuum freeze dryer, frozen for 5 hours at the temperature of minus 40 ℃, and the samples are frozen. Then sublimating for 5h at-20 ℃ and sublimating for 10h at 0 ℃, wherein the sublimating can directly remove about 90% of water from the frozen semi-finished solid product, and meanwhile, the original solid framework structure is reserved, the product is spongy, and the product has excellent re-solubility. Finally, resolving and drying for 8 hours at 20 ℃, removing the bound water, improving the storage stability of the freeze-dried product and prolonging the protection period of the freeze-dried product. The lyophilization curve is shown in fig. 12.
Calibration material, calibrator diluent, low, medium and high three-level quality control material freeze-dried product moisture content and state verification
3 lyophilized products of the same batch of calibrator, calibrator diluent and low, medium and high level quality control products are randomly extracted, stored in a dark place at room temperature, and the water content of the lyophilized products is respectively measured by a volume titration method by using a Karl Fischer water content meter, and the appearance and the re-dissolved state of the lyophilized products are examined, and the specific results are shown in Table 14.
TABLE 14 moisture content, appearance and reconstitution status of calibrator, calibrator dilutions and low, medium and high three-level quality control
As can be seen from Table 14, the moisture content of the calibrator, calibrator dilutions and the low, medium and high level quality control lyophilized products is not more than 3%, and the lyophilized products are light yellow dry powder and light yellow clear liquid after reconstitution, so that the calibrator, calibrator dilutions and low, medium and high level quality control lyophilized products in the kit have good moisture and appearance.
Calibrator accuracy verification
Randomly taking 3 calibration products, re-dissolving the calibration products to be used as samples to be tested, performing sample treatment according to the pretreatment method described in 2.4.7.1, calibrating the system by using a selected higher-level standard product, detecting the samples to be tested according to the detection method described in 2.4.7.2, testing each sample for 1 time, calculating a measured value, comparing the measured value with a target value, and calculating relative deviation. The results are shown in Table 15.
Table 15 calibrator accuracy validation
As can be seen from table 15, the calibration material was calibrated at a higher level, and the relative deviation between the measured value and the target value was not more than 10%, so that the accuracy of the calibration material was very reliable.
Bottle opening/reconstitution stability verification of calibrator and low, medium and high three-level quality control product
And (3) opening bottles/re-dissolving the calibrator and the low, medium and high three-level quality control product freeze-dried products, covering, placing under the expected storage condition, placing 0d, 3d, 6d, 9d, 12d and 14d from 0h, taking out each time, placing below-70 ℃, finally performing machine detection according to a detection method, measuring three parallel samples at each time point, calculating the concentration of the antiarrhythmic medicament obtained by detection according to the average value, comparing with the concentration of 0h, and calculating the relative deviation. The results are shown in Table 16.
Table 16 calibrator and low, medium and high three level quality control vial opening/reconstitution stability
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As can be seen from Table 16, after the calibrator and the low, medium and high level quality control materials are subjected to bottle opening and reconstitution, the calibrator and the low, medium and high level quality control materials are placed at the temperature of-70 ℃ and stored for 14 days, and the relative deviation between the concentrations of the 4 antiarrhythmic drugs and the 0h detection concentration is not more than 10%, so that the calibrator and the low, medium and high level quality control materials have better stability within 14 days after bottle opening/reconstitution.
Validity period stability verification of calibrator and low, medium and high three-level quality control product
Storing the calibrator and the low, medium and high three-level quality control product freeze-dried products at the temperature of-18 ℃ or below under the condition of light-shielding sealing, detecting the calibrator, the low, medium and high three-level quality control product freeze-dried products at different time periods of 0 month, 1 month, 3 months, 6 months, 9 months and 12 months respectively, measuring 3 parallel samples at each time point, comparing the concentrations of the antiarrhythmic drugs obtained by mean calculation detection with target value concentrations, and calculating relative deviation, wherein the results are shown in table 17.
Table 17 calibrator and low, medium and high three level quality control shelf life stability
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As shown in Table 17, after the calibrator and the low, medium and high level quality control products are re-dissolved after being opened, the calibrator and the low, medium and high level quality control products are stored at-18 ℃ for 12 months, and the relative deviation between the concentration of the 4 antiarrhythmic drugs and the target value is not more than 10%, so that the calibrator and the low, medium and high level quality control products have better stability.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for detecting antiarrhythmic drugs in plasma by HPLC-MS, characterized in that: the method comprises the following steps:
adding methanol into the plasma, mixing the plasma and the methanol in a volume ratio of 1:1.5-3, shaking uniformly, carrying out protein precipitation, and centrifuging to obtain a supernatant;
mixing and diluting the supernatant with 0.05-0.3% formic acid aqueous solution according to the volume ratio of 1:4-10 to obtain a diluent;
and detecting the obtained diluent by adopting a liquid chromatography tandem mass spectrometry method, and simultaneously detecting quinidine, lidocaine, procainamide and N-acetylprocainamide in blood plasma.
2. The method for detecting antiarrhythmic drugs in plasma by HPLC-MS according to claim 1, characterized in that: the concentration of the aqueous solution of formic acid is 0.05-0.15%.
3. The method for detecting antiarrhythmic drugs in plasma by HPLC-MS according to claim 1, characterized in that: the liquid chromatography conditions were: chromatographic column model: YS LCC 104MD,2.1 mm. Times.50 mm,2.6 μm;
mobile phase a: 0.05-3% formic acid, wherein the percentage is 5-10mmol/L ammonium formate aqueous solution;
mobile phase B: methanol;
flow rate: 0.1-0.4mL/min;
column temperature: 25-50 ℃;
sample injection volume: 1-10 mu L;
gradient elution procedure: 0min:5% B,3min:98% b,4min:98% b,4.1min:5% B,5.5min:5% B.
4. A method for HPLC-MS detection of antiarrhythmic drugs in plasma according to claim 3, characterized in that: the mobile phase A is 0.05% -1% formic acid, 5-8mmol/L ammonium formate aqueous solution.
5. The method for detecting antiarrhythmic drugs in plasma by HPLC-MS according to claim 4, wherein: the mobile phase A is 0.05% -0.5% formic acid, 5-6mmol/L ammonium formate aqueous solution.
6. The method for detecting an antiarrhythmic drug in plasma by HPLC-MS according to claim 5, wherein: the mobile phase A is 0.1% formic acid and 5mmol/L ammonium formate aqueous solution.
7. The method for detecting antiarrhythmic drugs in plasma by HPLC-MS according to claim 1, characterized in that: the mass spectrum conditions are as follows: ion source: electrospray ion source (ESI), positive ion mode; ion spray voltage: 3500V; sheath gas: 5.58L/min; auxiliary gas: 8L/min; sweep gas: 1.5L/min;
ion transport tube temperature: 325 deg.c; atomization temperature: 350 ℃; scanning mode: multiple reaction monitoring.
8. The method for detecting an antiarrhythmic agent in plasma by HPLC-MS according to claim 7, wherein: mass spectrometry detection ion pairs of four substances to be tested are shown in the following table:
9. a kit for detecting an antiarrhythmic drug in plasma by HPLC-MS, characterized in that: the device at least comprises a calibrator, low, medium and high three-level quality control products, a calibrator diluent, a protein precipitant, a sample diluent, a mobile phase additive, a chromatographic column and a sample injection plate;
the antiarrhythmic drugs are quinidine, lidocaine, procainamide and N-acetylprocainamide.
10. A kit for HPLC-MS detection of antiarrhythmic drugs in plasma according to claim 9, characterized in that: the sample diluent is 0.05-0.3% formic acid water solution.
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| JP2020051960A (en) * | 2018-09-28 | 2020-04-02 | 株式会社日立ハイテクサイエンス | Liquid chromatograph analysis method and liquid chromatograph analyzer |
| CN110596272A (en) * | 2019-09-26 | 2019-12-20 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | Method for detecting 16 local anesthetic components in external adult product |
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