CN111189938B - Quantitative detection method for adefovir dipivoxil and sulfamethoxazole in pig plasma and alveolar fluid - Google Patents
Quantitative detection method for adefovir dipivoxil and sulfamethoxazole in pig plasma and alveolar fluid Download PDFInfo
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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
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- G—PHYSICS
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Abstract
The invention discloses a quantitative detection method of adefovir dipivoxil and sulfamethoxazole in pig plasma and alveolar fluid, which comprises the steps of preparing an adefovir dipivoxil and sulfamethoxazole standard solution and a sample extracting solution to be detected; performing linear regression analysis, and drawing a standard curve; the accuracy and the precision are respectively expressed by the recovery rate and the variation coefficient; drawing a working curve and calculating a regression equation and a correlation coefficient of the adefovir dipivoxil and the sulfamethoxazole in the pig blood and the alveolar lavage fluid; actual sample measurement and result calculation; the method has the advantages of high accuracy, good precision and sensitivity and less impurities, improves the conventional HPLC method, optimizes the gradient time and conditions, increases the separation degree among all peaks, increases the sensitivity of the target drug peak and improves the recovery rate.
Description
Technical Field
The invention relates to the technical field of animal blood detection, in particular to a quantitative detection method of adefovir dipivoxil and sulfamethoxazole in pig plasma and alveolar fluid.
Background
Sulfamethoxazole belongs to sulfanilamide antibiotics, is a broad-spectrum antibiotic, has sensitive pathogenic bacteria such as streptococcus, pneumococcus, salmonella, actinomyces pyogenes, escherichia coli, haemophilus paragallinarum, staphylococcus, proteus, pasteurella, escherichia coli, clostridium perfringens, bacillus anthracis and the like in early research, and has antibacterial effect on gram-negative bacteria and gram-positive bacteria. Sulfamethoxazole is widely used and often used in combination with trimethoprim. SMZ single prescription product for animals recorded in pharmacopoeia second department of the people's republic of China comprises sulfamethoxazole tablet, and compound preparation comprises compound sulfamethoxazole oral suspension, compound sulfamethoxazole tablet and compound sulfamethoxazole tablet for children, which are composed of TMP. The SMZ has strong antibacterial effect, is an intermediate sulfanilamide for treating systemic infection, is mainly used for treating infection diseases such as hemolytic streptococcus, meningococcus, pneumococcus, pasteurella and the like, and has strong antibacterial force, lasting drug effect, low toxicity and the like;
however, the current research reports show that sulfamethoxazole gradually shows universal drug resistance to the bacteria, and the combination of the sulfonamide and the sulfonamide synergist can enhance the discovery effect and reduce the drug resistance. The adeprilin is a novel antibacterial synergist, and pharmacodynamics and pharmacokinetic researches show that the application prospect of the adeprilin is far wider than that of trimethoprim. Early studies showed that the antibacterial efficacy of ideopril in combination with sulfamethoxazole against a. pleuropneumoniae, pasteurella suis was much higher than that of ADP alone. Therefore, the research and development of a new compound preparation can reduce the dosage of a single medicine, make up the defects of the compound sulfamethoxazole, and also can effectively prevent the generation of drug resistance by formulating a reasonable administration scheme, thereby providing an alternative medicine for veterinary clinical medication, in particular to diseases caused by infection of sensitive bacteria of the idedperin such as streptococcus suis and the like;
animal-derived bacterial drug resistance is an important issue of global concern, and is of great importance in order to enhance the therapeutic level of veterinary antibacterial drugs, reduce bacterial drug resistance, ensure the health and safety of livestock and poultry and the good development of animal husbandry, and establish and perfect the drug delivery scheme of veterinary antibacterial drugs.
At present, the detection method of the ideopril and the sulfamethoxazole mainly adopts liquid chromatography single-drug detection, and the operation scheme is expensive and complex in operation, so that the invention provides a quantitative detection method of the ideopril and the sulfamethoxazole in pig plasma and alveolar fluid to overcome the defects in the prior art.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a method for quantitatively detecting adefovir dipivoxil and sulfamethoxazole in pig plasma and alveolar fluid, which has the advantages of high accuracy, good precision and sensitivity, and less impurities.
In order to realize the purpose of the invention, the invention is realized by the following technical scheme:
the quantitative detection method of the adefovir dipivoxil and the sulfamethoxazole in the pig plasma and the alveolar fluid comprises the following steps:
the method comprises the following steps: preparing an idempreglin and sulfamethoxazole standard solution and a sample extracting solution to be detected;
step two: drawing a standard curve, respectively using 3-5 parts of the standard solution of the epidophylline with different concentrations for HPLC detection, then carrying out elution treatment, recording a chromatogram and a peak area of the standard solution of the epidophylline, carrying out linear regression analysis, and drawing to obtain the standard curve;
step three: the accuracy and precision are respectively expressed by the recovery rate and the coefficient of variation, firstly, the adefovir dipivoxil and sulfamethoxazole with high, medium and low concentrations are added into blank pig blood and alveolar lavage fluid, sample purifying solution is prepared for HPLC detection according to the extraction method of a sample extracting solution to be detected, each concentration is repeated for 5 times in the same day, then an average value is taken, the relative error in the day is calculated as the variation coefficient in the day, each concentration is repeated for 5 days, then the average value and the relative error are calculated to obtain the variation coefficient in the day, and the recovery rate is calculated by adopting a single-point calibration method shown in a formula (1):
X＝AV/ASW×100％ (1)
in the formula: x: the recovery rate of the object to be detected in the sample; a: peak area of the sample; v: extracting and drying a sample, and then fixing the volume to a unit mL; as: peak area of the standard working solution; w: sample volume, in mL;
step four: drawing a working curve, taking blank pig blood and alveolar lavage fluid, adding 3-5 parts of adefovir dipivoxil and sulfamethoxazole with different concentrations, and extracting sample detection fluid for HPLC detection according to an extraction method for extracting a detected sample extracting solution; after repeating the steps for three times, performing linear regression on the obtained peak area and the corresponding concentration, drawing a working curve, and calculating a regression equation and a correlation coefficient of the ideprop and the sulfamethoxazole in the pig blood and the alveolar lavage fluid;
step five: and (3) measuring an actual sample and calculating a result, collecting blood and alveolar lavage fluid after the pig is injected with the compound injection of the ideopril and the sulfamethoxazole for a single time, extracting the blood and the alveolar lavage fluid, detecting by HPLC, recording a chromatogram and a peak area, and calculating the contents of the ideopril and the sulfamethoxazole in the collected pig blood and the alveolar lavage fluid respectively by using a linear regression equation obtained in the fourth step.
The further improvement lies in that: the preparation method of the sample extracting solution to be detected in the first step comprises the following steps: adding dichloromethane and acetonitrile into blood and alveolar lavage fluid to be detected to serve as extracting solution, taking the supernatant after vortex and centrifugal treatment, repeating the operation twice, drying the supernatant under the atmosphere of nitrogen, redissolving solid-phase residues by using a mobile phase, adding n-hexane into the redissolved solution to remove fat, and finally passing the dissolved solution through a membrane to obtain the sample extracting solution to be detected.
The further improvement lies in that: the mass ratio of the dichloromethane to the acetonitrile is 1: 1.
The further improvement lies in that: and in the first step, when the drying is carried out in the nitrogen atmosphere, the nitrogen atmosphere is 50 ℃.
The further improvement lies in that: the HPLC detection conditions are as follows: a chromatographic column: agilent ZORBAX SB-C18, 250X 4.6mm (i.d.), 5 μm; mobile phase: a-0.1% trifluoroacetic acid, C-methanol; ultraviolet detection wavelength: 270 nm; sample introduction amount: 20 mu L of the solution; flow rate: 1 mL/min; column temperature: at 30 ℃.
The further improvement lies in that: and the elution treatment in the second step is carried out in a gradient elution mode.
The invention has the beneficial effects that: the method has high accuracy, good precision and sensitivity and less impurities, is used as a method for detecting the content of the ideopril and the sulfamethoxazole in the pig plasma and the alveolar lavage fluid, and provides good guiding significance for detecting the metabolic distribution of the ideopril and the sulfamethoxazole in a pig body; meanwhile, the invention improves the prior HPLC method, optimizes the time and conditions of the gradient, increases the separation degree between the peaks, increases the sensitivity of the target drug peak, improves the recovery rate, and reduces the impurities in the sample by selecting the mixed liquid of dichloromethane and acetonitrile as the extractants of two drugs in pig plasma and alveolar lavage fluid, removing the lipid by n-hexane and then blowing nitrogen, thereby having high detection accuracy and sensitivity and the recovery rate reaching the detection requirement.
Drawings
FIG. 1 is a schematic diagram of a standard curve and a formula of an ideopril and sulfamethoxazole standard substance.
FIG. 2 is a graph showing the working curve and regression equation of adefovir dipivoxil and sulfamethoxazole after the treatment of the porcine plasma sample according to the present invention.
FIG. 3 is a graph showing the operating curve and regression equation of adefovir dipivoxil and sulfamethoxazole after sample treatment in porcine alveolar lavage fluid according to the present invention.
Fig. 4 is a schematic diagram of a blank flow phase comparison of the present invention.
FIG. 5 is a schematic diagram showing the comparison between ideopril and sulfonamide standards according to the present invention.
FIG. 6 is a schematic diagram of a porcine plasma blank sample chromatogram according to the present invention.
FIG. 7 is a chromatogram of a sample solution in plasma according to the present invention (concentration of adefovir dipivoxil and sulfamethoxazole is 0.1. mu.g/mL).
FIG. 8 is a schematic chromatogram of a blank sample of alveolar lavage fluid of the present invention.
FIG. 9 is a chromatographic schematic of a sample solution in alveolar lavage fluid (adefovir dipivoxil and sulfamethoxazole concentration 0.1. mu.g/mL).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, 2, 3, 4, 5, 6, 7, 8, and 9, the present embodiment provides a method for quantitatively detecting adefovir dipivoxil and sulfamethoxazole in pig plasma and alveolar fluid, comprising the following steps:
liquid chromatograph: waters 2695;
a detector: waters2487 uv-vis detector;
a chromatographic column: agilent ZORBAX SB-C18, 250X 4.6mm (i.d.), 5 μm;
mobile phase A: 0.1% trifluoroacetic acid;
mobile phase C: 100% methanol;
ultraviolet detection wavelength: 270 nm;
sample introduction amount: 20 mu L of the solution;
flow rate: 1 mL/min;
column temperature: at 30 ℃.
The mobile phase elution protocol is shown in table 1 below:
TABLE 1
The specific detection method is as follows:
the method comprises the following steps: preparing standard stock solutions of epidophylline and sulfamethoxazole: respectively and precisely weighing 10.00mg of ideopril and a sulfamethoxazole standard substance, dissolving the ideopril and the sulfamethoxazole standard substance in 10mL of methanol, and carrying out ultrasonic heating to dissolve the ideopril and the sulfamethoxazole standard substance to obtain a first stock solution serving as a first stock solution of the ideopril standard substance, diluting the ideopril by 5 times of the methanol to obtain a second stock solution, and diluting the sulfamethoxazole by 5 times to obtain a second stock solution; and mixing the aldeprin stock solution II with the sulfamethoxazole stock solution II. As stock solution three.
Step two: preparing an ideopril and sulfamethoxazole standard working solution: diluting the stock solution of the idemprillin by two times respectively by 10 times, 20 times, 40 times, 100 times, 200 times, 400 times, 1000 times, 2000 times and 4000 times, and preparing 9 parts of working solution with different concentrations for HPLC detection;
drawing a standard curve, opening a liquid chromatograph and a detector, balancing a base line for 20min after the parameters of the instrument meet the requirements, sequentially injecting the 9 parts of working solution, enabling each solution to be parallel for 3 times, recording a chromatogram, and performing linear regression analysis by using peak areas and corresponding concentrations to obtain the standard curve and an equation;
step three: extraction of idemprin and sulfamethoxazole in porcine plasma and alveolar lavage fluid: accurately weighing 1mL of plasma or alveolar lavage fluid into a 10mL centrifuge tube, adding 3mL of mixed solution of dichloromethane and acetonitrile, vortexing for 5min, centrifuging for 10min at 10000r/min, taking down clear liquid into the 10mL centrifuge tube, adding 3mL of dichloromethane and acetonitrile into residues, extracting once according to the method, combining the two times of clear liquid, dissolving the residues with 1mL of initial mobile phase, vortexing and uniformly mixing, adding 1mL of n-hexane, removing the n-hexane layer (upper layer) to achieve the purpose of degreasing, and then using a 0.22 mu m filter membrane to obtain a sample purified liquid;
step four: working curve: adding corresponding aldepin and sulfamethoxazole standard solutions into a blank plasma sample to enable the concentration of the aldepin drug in the sample to reach 0.05 mu g/mL, 0.1 mu g/mL, 0.2 mu g/mL, 0.5 mu g/mL, 1 mu g/mL, and sulfamethoxazole to reach 0.05 mu g/mL, 0.1 mu g/mL, 1 mu g/mL, 5 mu g/mL, 10 mu g/mL, and 20 mu g/mL; adding corresponding ideprop and sulfamethoxazole standard substance solutions into a blank alveolar lavage fluid sample to enable the concentration of an ideprop drug in the sample to reach 0.05 mu g/mL, 0.1 mu g/mL, 0.2 mu g/mL, 0.5 mu g/mL, 1 mu g/mL, sulfamethoxazole to be 0.05 mu g/mL, 0.1 mu g/mL, 1 mu g/mL, 2 mu g/mL, 5 mu g/mL to be processed according to the method steps of the step four to obtain a sample detection solution, then opening a liquid chromatograph and a detector, balancing a base line for 20min after the parameters of the instrument meet requirements, sequentially feeding the 5 samples, enabling each solution to be parallel for 3 times, and performing linear regression analysis by using a peak area and the corresponding concentration to obtain a working curve and a regression equation;
step five: respectively expressing the accuracy and precision by using the recovery rate and the variation coefficient, adding 0.1 mu g/mL, 0.5 mu g/mL and 1 mu g/mL of ideprop in blank pig plasma, and preparing a sample purifying solution for HPLC detection by using 0.1 mu g/mL, 10 mu g/mL and 20 mu g/mL of the sample extracting solution to be detected; adding adefovir dipivoxil with three concentrations of 0.1 mug/mL, 0.5 mug/mL and 1 mug/mL into the blank pig alveolar lavage fluid, preparing sample purifying fluid with three concentrations of 0.1 mug/mL, 1 mug/mL and 5 mug/mL according to the extraction method of the sample extracting solution to be detected, repeating each concentration for 5 times in the same day by HPLC detection, taking an average value, and calculating the relative error in the day to obtain the variation coefficient in the day; repeat for 5 days at each concentration; calculating the average value and the relative error to obtain a day variation coefficient;
the recovery rate is calculated by adopting a single-point calibration method, and the formula is as follows: x ═ AV/ASW × 100%, where: x: the recovery rate of the object to be detected in the sample; a: peak area of the sample; v: extracting and drying a sample, and then fixing the volume to a unit mL; as: peak area of the standard working solution; w: sample volume, in mL; the calculated recovery rate of the ideoprim in the blood plasma is 80.1-101.3%, and the diurnal variation coefficient is 2.42-6.11%. The recovery rate of sulfamethoxazole in blood plasma is 87.6-97.2%, and the diurnal variation coefficient is 4.43-6.62%. The recovery rate of the aldiprine in the alveolar lavage fluid is 84.0-94.4 percent, and the diurnal coefficient of variation is 2.15-6.38 percent. The recovery rate of sulfamethoxazole in alveolar lavage fluid is 82.8-101.5%, and the diurnal variation coefficient is 1.86-5.15%.
The method has high accuracy, good precision and sensitivity and less impurities, is used as a method for detecting the content of the ideopril and the sulfamethoxazole in the pig plasma and the alveolar lavage fluid, and provides good guiding significance for detecting the metabolic distribution of the ideopril and the sulfamethoxazole in a pig body; meanwhile, the invention improves the prior HPLC method, optimizes the time and conditions of the gradient, increases the separation degree between the peaks, increases the sensitivity of the target drug peak, improves the recovery rate, and reduces the impurities in the sample by selecting the mixed liquid of dichloromethane and acetonitrile as the extractants of two drugs in pig plasma and alveolar lavage fluid, removing the lipid by n-hexane and then blowing nitrogen, thereby having high detection accuracy and sensitivity and the recovery rate reaching the detection requirement.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (2)
1. The quantitative detection method of the adefovir dipivoxil and the sulfamethoxazole in the pig plasma and the alveolar fluid is characterized by comprising the following steps of:
the method comprises the following steps: preparing an idempreglin and sulfamethoxazole standard solution and a sample extracting solution to be detected;
step two: drawing a standard curve, respectively using 3-5 parts of the standard solution of the epidophylline with different concentrations for HPLC detection, then carrying out elution treatment, recording a chromatogram and a peak area of the standard solution of the epidophylline, carrying out linear regression analysis, and drawing to obtain the standard curve;
step three: the accuracy and precision are respectively expressed by the recovery rate and the coefficient of variation, firstly, the adefovir dipivoxil and sulfamethoxazole with high, medium and low concentrations are added into blank pig plasma and alveolar lavage fluid, sample purifying liquid is prepared for HPLC detection according to the extraction method of a sample extracting solution to be detected, each concentration is repeated for 5 times in the same day, then an average value is taken, the relative error in the day is calculated as the variation coefficient in the day, each concentration is repeated for 5 days, then the average value and the relative error are calculated to obtain the variation coefficient in the day, and the recovery rate is calculated by adopting a single-point calibration method shown in a formula (1):
X＝AV/AsW×100％ (1)
in the formula: recovering rate of the object to be detected in the X-sample; a-peak area of sample; v, extracting and drying a sample, and then fixing the volume to a unit mL; peak area of As-standard working solution; w: sample volume, in mL;
step four: drawing a working curve, taking blank pig plasma and alveolar lavage fluid, adding 3-5 parts of adefovir dipivoxil and sulfamethoxazole with different concentrations, and extracting sample detection fluid for HPLC detection according to an extraction method for extracting a detected sample extracting solution; after repeating the steps for three times, performing linear regression on the obtained peak area and the corresponding concentration, drawing a working curve, and calculating a regression equation and a correlation coefficient of the ideprop and the sulfamethoxazole in the porcine plasma and the alveolar lavage fluid;
step five: measuring actual samples and calculating results, wherein after the pigs are injected with the compound injection of the ideopril and the sulfamethoxazole for a single time, plasma and alveolar lavage fluid are collected, the plasma and alveolar lavage fluid are extracted and subjected to HPLC detection, a chromatogram and a peak area are recorded, and the contents of the ideopril and the sulfamethoxazole in the collected pig plasma and the alveolar lavage fluid are respectively calculated according to a linear regression equation obtained in the fourth step;
the preparation method of the sample extracting solution to be detected in the first step comprises the following steps: adding dichloromethane and acetonitrile into plasma and alveolar lavage fluid to be detected, performing vortex and centrifugal treatment, taking the supernatant, repeating the operation twice, drying the supernatant in a nitrogen atmosphere, redissolving solid phase residues by using a mobile phase, adding n-hexane for degreasing after redissolving, and finally passing the solution through a membrane to obtain a sample extracting solution to be detected;
the mass ratio of the dichloromethane to the acetonitrile is 1: 1;
the HPLC detection conditions are as follows: a chromatographic column: agilent ZORBAX SB-C18, 250X 4.6mm (i.d.), 5 μm; mobile phase: a-0.1% trifluoroacetic acid, C-methanol; ultraviolet detection wavelength: 270 nm; sample introduction amount: 20 mu L of the solution; flow rate: 1 mL/min; column temperature: 30 ℃;
the mobile phase elution protocol was as follows:
2. the method for quantitatively detecting adefovir dipivoxil and sulfamethoxazole in pig plasma and alveolar fluid according to claim 1, wherein the method comprises the following steps: and in the first step, when the drying is carried out in the nitrogen atmosphere, the nitrogen atmosphere is 50 ℃.
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