CN111624279A - Pretreatment method and quantitative detection method for quinolone antibiotics in biological sample - Google Patents
Pretreatment method and quantitative detection method for quinolone antibiotics in biological sample Download PDFInfo
- Publication number
- CN111624279A CN111624279A CN202010443577.5A CN202010443577A CN111624279A CN 111624279 A CN111624279 A CN 111624279A CN 202010443577 A CN202010443577 A CN 202010443577A CN 111624279 A CN111624279 A CN 111624279A
- Authority
- CN
- China
- Prior art keywords
- solution
- quinolone
- minutes
- quinolone antibiotics
- biological sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000003242 anti bacterial agent Substances 0.000 title claims abstract description 55
- 229940088710 antibiotic agent Drugs 0.000 title claims abstract description 53
- LISFMEBWQUVKPJ-UHFFFAOYSA-N quinolin-2-ol Chemical compound C1=CC=C2NC(=O)C=CC2=C1 LISFMEBWQUVKPJ-UHFFFAOYSA-N 0.000 title claims abstract description 46
- 239000012472 biological sample Substances 0.000 title claims abstract description 23
- 238000001514 detection method Methods 0.000 title claims abstract description 22
- 238000002203 pretreatment Methods 0.000 title claims abstract description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 60
- 239000000243 solution Substances 0.000 claims abstract description 48
- 238000000605 extraction Methods 0.000 claims abstract description 24
- 239000000523 sample Substances 0.000 claims abstract description 21
- 239000002086 nanomaterial Substances 0.000 claims abstract description 20
- 238000002156 mixing Methods 0.000 claims abstract description 13
- 238000001914 filtration Methods 0.000 claims abstract description 11
- 239000012528 membrane Substances 0.000 claims abstract description 10
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 8
- 239000000203 mixture Substances 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 239000011521 glass Substances 0.000 claims abstract description 5
- 239000012456 homogeneous solution Substances 0.000 claims abstract description 4
- 238000002137 ultrasound extraction Methods 0.000 claims abstract description 4
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 claims description 44
- 238000000034 method Methods 0.000 claims description 27
- 229960003405 ciprofloxacin Drugs 0.000 claims description 22
- 229960003702 moxifloxacin Drugs 0.000 claims description 22
- FABPRXSRWADJSP-MEDUHNTESA-N moxifloxacin Chemical compound COC1=C(N2C[C@H]3NCCC[C@H]3C2)C(F)=CC(C(C(C(O)=O)=C2)=O)=C1N2C1CC1 FABPRXSRWADJSP-MEDUHNTESA-N 0.000 claims description 22
- OGJPXUAPXNRGGI-UHFFFAOYSA-N norfloxacin Chemical compound C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCNCC1 OGJPXUAPXNRGGI-UHFFFAOYSA-N 0.000 claims description 22
- 229960001180 norfloxacin Drugs 0.000 claims description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 17
- KYGZCKSPAKDVKC-UHFFFAOYSA-N Oxolinic acid Chemical compound C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC2=C1OCO2 KYGZCKSPAKDVKC-UHFFFAOYSA-N 0.000 claims description 16
- 239000003306 quinoline derived antiinfective agent Substances 0.000 claims description 16
- 239000012488 sample solution Substances 0.000 claims description 14
- 210000002700 urine Anatomy 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 230000014759 maintenance of location Effects 0.000 claims description 8
- 239000012086 standard solution Substances 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 7
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 7
- 210000004369 blood Anatomy 0.000 claims description 6
- 239000008280 blood Substances 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 238000010812 external standard method Methods 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 238000004811 liquid chromatography Methods 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 230000032683 aging Effects 0.000 claims description 2
- YHWCPXVTRSHPNY-UHFFFAOYSA-N butan-1-olate;titanium(4+) Chemical compound [Ti+4].CCCC[O-].CCCC[O-].CCCC[O-].CCCC[O-] YHWCPXVTRSHPNY-UHFFFAOYSA-N 0.000 claims description 2
- 238000001354 calcination Methods 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 239000008367 deionised water Substances 0.000 claims description 2
- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- 238000000227 grinding Methods 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000004570 mortar (masonry) Substances 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 239000010453 quartz Substances 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- UTCARTSNNKGRTD-UHFFFAOYSA-N terbium(3+);trinitrate;hexahydrate Chemical compound O.O.O.O.O.O.[Tb+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O UTCARTSNNKGRTD-UHFFFAOYSA-N 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 239000000706 filtrate Substances 0.000 abstract 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 abstract 1
- 239000006228 supernatant Substances 0.000 description 19
- 239000003480 eluent Substances 0.000 description 10
- 238000011084 recovery Methods 0.000 description 10
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 10
- 238000005259 measurement Methods 0.000 description 9
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 238000005070 sampling Methods 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 6
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N titanium dioxide Inorganic materials O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- 238000009210 therapy by ultrasound Methods 0.000 description 5
- 238000007792 addition Methods 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 239000012496 blank sample Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000003795 desorption Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000622 liquid--liquid extraction Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000002470 solid-phase micro-extraction Methods 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004853 microextraction Methods 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
Landscapes
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a pretreatment method and a quantitative detection method of quinolone antibiotics in biological samples, which are characterized by comprising the steps of placing biological samples to be detected in a glass centrifuge tube, adding 10 mmol/L phosphate buffer solution with the pH =6, which is 10 times of the volume of sample liquid to be detected, and uniformly mixing to obtain uniform solution; adding TiO into the mixture2Adding Tb nano material into the homogeneous solution, carrying out ultrasonic-assisted extraction for 3-8 minutes, centrifuging, removing the upper layer solution, adding an acetic acid solution with the mass concentration of 4wt% and the same volume as that of a phosphate buffer solution into the lower layer solution, carrying out ultrasonic-assisted back extraction for 12-18 minutes, centrifuging, filtering the upper layer solution through a 0.45 mu m microporous filter membrane, and then using the filtrate for high performance liquid chromatography-ultraviolet spectroscopy quantitative detection of quinolone antibiotics.
Description
Technical Field
The invention relates to a pretreatment method of antibiotics, in particular to a pretreatment method of quinolone antibiotics in a biological sample and a quantitative detection method thereof.
Background
The prior pretreatment methods of quinolone antibiotics in biological samples comprise liquid-liquid extraction, solid-phase microextraction and the like. However, the traditional liquid-liquid extraction needs a large amount of organic reagents, and the extraction can be completed only by multiple times of extraction, so that the operation is complicated; although solid phase extraction is a classical extraction method, a solid phase extraction device and a solid phase extraction column are used, the solid phase extraction column is expensive, and the accuracy of subsequent determination is influenced by the reproducibility problem; in addition, the solid phase extraction is further concentrated by a nitrogen blowing instrument, the whole extraction process takes a long time, and a large amount of organic reagents are also used for column washing and sample elution; although the solid phase micro-extraction method is a micro-extraction method, special equipment is needed, the equipment cost is high, and the service life of the extraction fiber is always a restriction factor of the wide application of the prior treatment method. In summary, the prior measurement of quinolone antibiotics in biological samples requires certain pretreatment, such as protein precipitation, before sample pretreatment, and these processes are time-consuming, labor-consuming, complex to operate, and consume a lot of organic solvents, and the increase of operation steps can reduce the recovery rate of subsequent measurement, and affect the accuracy of measurement, thereby leading to unstable and reliable rear-end detection results.
Disclosure of Invention
The invention aims to solve the technical problem of providing a simple and rapid pretreatment method and a quantitative detection method for quinolone antibiotics in a biological sample with high target recovery rate, wherein the detection method has high sensitivity and accuracy.
The technical scheme adopted by the invention for solving the technical problems is as follows:
1. a pretreatment method of quinolone antibiotics in biological samples comprises the following steps:
(1) placing a biological sample to be detected in a glass centrifuge tube, adding 10 mmol/L phosphate buffer solution with pH =6, which is 10 times of the volume of a sample solution to be detected, and uniformly mixing to obtain a uniform solution;
(2) adding TiO into the mixture2Adding Tb nanomaterial into the homogeneous solution at an addition rate of 20 mg/mL, performing ultrasonic-assisted extraction for 3-8 min, centrifuging at 4000r/min for 5min, discarding the upper layer solution, adding acetic acid solution (pH = 2.45) with a mass concentration of 4wt% and equal volume to phosphate buffer solution into the lower layer solution,and (3) centrifuging for 5 minutes at 4000r/min after carrying out ultrasonic-assisted back extraction for 12-18 minutes, and filtering the upper-layer solution by using a 0.45-micrometer microporous filter membrane to obtain a pretreatment sample for the high performance liquid chromatography detection of the quinolone antibiotics.
The TiO is2The preparation method of the-Tb nano material comprises the following steps: dissolving 20 mL of tetrabutyl titanate in 25 mL of absolute ethanol, and stirring for 30 minutes to obtain a solution A; meanwhile, 0.1335g of terbium nitrate hexahydrate is added into a mixed solution containing 10 mL of acetic acid, 20 mL of deionized water and 25 mL of absolute ethyl alcohol, and stirred for 30 minutes to obtain a solution B; slowly dripping the solution A into the solution B, reacting for 2 hours at 70 ℃, and aging for 72 hours at room temperature; drying the aged product in a drying oven at 100 ℃, transferring the dried product to a muffle furnace, calcining at 500 ℃ for 4 hours, cooling to room temperature, grinding the calcined product into powder by using a quartz mortar to obtain the TiO2-Tb nanomaterial.
The biological sample comprises human urine and rabbit whole blood.
2. A method for quantitatively detecting quinolone antibiotics by utilizing a pretreatment method of the quinolone antibiotics in the biological sample comprises the following steps:
(1) preparing a standard solution of the quinolone antibiotic by using methanol as a solvent, measuring chromatographic peak retention time and chromatographic peak area of each component in the standard solution of the quinolone antibiotic by using a high performance liquid chromatography-ultraviolet method (HPLC-UV), determining the chromatographic peak retention time by using the chromatographic peak retention time, drawing a standard curve of the quinolone antibiotic by using concentration as a horizontal coordinate and using the chromatographic peak area as a vertical coordinate, wherein the quinolone antibiotic comprises ciprofloxacin, moxifloxacin and norfloxacin, and the concentration range of each component is 10-10000 ng/mL;
(2) HPLC-UV determination conditions of quinolone antibiotics
Liquid chromatography instrument model: agilent 1260 Infinity II; a chromatographic column: the model is Pursuit 5C 18, the specification is 5 mu m particle size, 4.6 mm x 150 mm; sample introduction amount: 20 mu L of the solution; column temperature: 25 ℃; a detector: a diode array detector; detector wavelength: the ciprofloxacin and norfloxacin are 278 nm, and the moxifloxacin is 291 nm; the mobile phase A is 0.2wt% acetic acid solution, the mobile phase B is acetonitrile, and the flow rate is 1 mL/min; the gradient elution procedure is shown in the following table:
TABLE 1 HPLC gradient elution procedure
(3) And (3) quantitatively analyzing the quinolone antibiotics in the sample by adopting an external standard method, and calculating to obtain the concentrations of ciprofloxacin, moxifloxacin and norfloxacin in the biological sample to be detected.
Compared with the prior art, the invention has the advantages that: the pretreatment adopts a pH-mediated dispersed solid phase extraction method, does not need to use a large amount of organic reagents for elution, and can realize the extraction of the quinolone antibiotics in the biological sample by simply adjusting the pH value of the sample, adding nano solid particles and then eluting with a small amount of acetic acid solution. When the pH of the sample is adjusted to 6, all three quinolone antibiotics exist in a negatively charged form, and TiO2The surface of the Tb nano material is positively charged, and the antibiotics and the nano material are adsorbed by electrostatic adsorption chemical action, wherein the adsorption is strongest; when adsorbing TiO of three quinolone antibiotics2After 4wt% acetic acid solution (pH = 2.45) is added into the-Tb nano material, TiO is easily dissolved in acid solution due to three quinolone antibiotics2The antibiotics adsorbed on the surface of the Tb nano material are desorbed into the solution again. The biological sample, particularly the blood sample, has complex matrix and high protein content, so that the extraction efficiency of antibiotics can be influenced; the research determines complex sample matrix-quinolone antibiotics in urine samples and whole blood samples, does not need sample pretreatment, and HPLC determination is carried out after the samples are directly extracted by adopting dispersive solid phase extraction. The method not only can effectively remove matrix interference of protein and the like, but also can be completed by simple reagent adding and centrifugal operation, has the advantages of simplicity, rapidness, high recovery rate and easy limited connection with back-end HPLC (high performance liquid chromatography) to realize quinolone antibioticHigh-efficiency and quick determination of the element. The research can provide a certain theoretical basis for determination of antibiotics in biological samples and also provide technical support for bedside monitoring of antibiotics.
Drawings
FIG. 1 is a schematic diagram of a pH-mediated dispersed solid phase extraction process of the present invention;
FIG. 2 is a chromatogram of human urinary bladder and spiked recovery, wherein the spiked concentrations of 3 quinolone antibiotics are: 10 mu g/mL of 1 norfloxacin, 5 mu g/mL of 2 ciprofloxacin and 5 mu g/mL of 3 moxifloxacin; a is a blank sample, b is a labeled sample;
fig. 3 is a chromatogram of rabbit blood emptying and recovery by adding standard substances, wherein the standard substance concentrations of 3 quinolone antibiotics are respectively: 10 mu g/mL of 1 norfloxacin, 5 mu g/mL of 2 ciprofloxacin and 5 mu g/mL of 3 moxifloxacin; a is a blank sample, b is a labeled sample;
fig. 4 is a chromatogram of rabbit plasma air and labeled recovery, wherein the labeled concentrations of 3 quinolone antibiotics are: 10 mu g/mL of 1 norfloxacin, 5 mu g/mL of 2 ciprofloxacin and 5 mu g/mL of 3 moxifloxacin; a is a blank sample and b is a labeled sample.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
Detailed description of the preferred embodiment
Detection of quinolone antibiotics in human urine, rabbit serum and rabbit plasma samples
1. Pretreatment method
The extraction process is shown in figure 1, respectively taking 500. mu.L of human urine, rabbit serum and rabbit plasma, respectively placing the 500. mu.L of human urine, the rabbit serum and the rabbit plasma in 10 mL of glass centrifuge tubes, respectively adding 5mL of 10 mmol/L phosphate buffer solution with pH =6, and uniformly mixing to obtain uniform solution; to the direction of100 mg TiO was added to the homogeneous solution2Ultrasonic-assisted extraction of the-Tb nanomaterial for 5 minutes, centrifuging at 4000r/min for 5 minutes, removing the upper solution, and removing the lower TiO layer2Adding 5mL of 4wt% acetic acid solution (pH = 2.45) into the Tb nano material solution, performing ultrasonic-assisted back extraction for 15 minutes, centrifuging at 4000r/min for 5 minutes, filtering the upper solution through a 0.45-micron microporous membrane, and performing HPLC-UV detection;
2. HPLC-UV detection method
(1) Preparing a mixed standard solution of the quinolone antibiotics by using methanol as a solvent,
the concentration ranges of ciprofloxacin, moxifloxacin and norfloxacin are all 10-10000 ng/mL, the chromatographic peak retention time and the chromatographic peak area of each component in a standard solution of the quinolone antibiotic are measured by HPLC-UV, the chromatographic peak retention time is used for determining the nature, the concentration is used as a horizontal coordinate, the chromatographic peak area is used as a vertical coordinate, and a standard curve of the quinolone antibiotic is drawn, wherein the quinolone antibiotic comprises ciprofloxacin, moxifloxacin and norfloxacin, and the concentration ranges of all the components are all 10-10000 ng/mL;
(2) HPLC-UV conditions for determining quinolone antibiotics are
Liquid chromatography instrument model: agilent 1260 Infinity II; a chromatographic column: the model is Pursuit 5C 18, the specification is 5 mu m particle size, 4.6 mm x 150 mm; sample introduction amount: 20 mu L of the solution; column temperature: 25 ℃; a detector: a diode array detector; detector wavelength: the ciprofloxacin and norfloxacin are 278 nm, and the moxifloxacin is 291 nm; mobile phase: (A) 0.2wt% acetic acid solution (B) acetonitrile at a flow rate of 1 mL/min; the gradient elution procedure is shown in the following table:
TABLE 1 HPLC gradient elution procedure
The HPLC-UV condition is adopted to determine the 3 kinds of quinolone antibiotic mixed standard solutions, the standard sample separation effect is good, the peak shapes are symmetrical, the base line is stable, and the condition of the instrument is suitable. Under the chromatographic conditions, the retention time of the quinolone antibiotics is respectively as follows: norfloxacin 4.24 min, ciprofloxacin 4.84 min, and moxifloxacin 5.51 min, as shown in fig. 2, fig. 3, and fig. 4;
(3) and (3) quantitatively analyzing the quinolone antibiotics in the sample by adopting an external standard method.
No 3 quinolone antibiotics are detected in human urine, rabbit serum and rabbit plasma samples, and blank and labeled chromatograms are shown in fig. 2, fig. 3 and fig. 4.
Detailed description of the invention
The optimal extraction conditions are obtained by examining each experimental condition through single factor alternation.
1. Influence of the pH of the sample solution
The pH of the sample solution is 3, 4, 5, 6, 7, 8, 9 and 10, and 100 mg of TiO is accurately weighed2the-Tb nano material was added to 5mL of a mixed sample solution of 3 quinolone antibiotics (norfloxacin, ciprofloxacin, moxifloxacin). And (3) carrying out ultrasonic treatment for 5min after vortex mixing, centrifuging for 5min at 4000r/min, filtering the supernatant through a 0.45-micron filter membrane, transferring the filtered supernatant into a chromatographic sampling bottle, and carrying out machine detection. The measurement results showed that the extraction efficiency was the highest for three quinolone antibiotics when the pH of the sample solution was 6, and thus pH 6 was selected as the optimum pH of the sample solution.
2. TiO2Influence of the amount of Tb added
Accurately weighing 5, 10, 20, 30, 40, 50 and 60 mg TiO respectively2-Tb nanomaterial was added to 5mL of a sample solution of 3 quinolone antibiotics (norfloxacin, ciprofloxacin, moxifloxacin) with pH 6. And (3) carrying out ultrasonic treatment for 5min after vortex mixing, centrifuging for 5min at 4000r/min, filtering the supernatant through a 0.45-micrometer filter membrane, transferring the filtered supernatant into a chromatographic sampling bottle, and carrying out machine detection. The measurement result shows that when TiO is used2When the amount of Tb is 20 mg, the extraction efficiency of norfloxacin and ciprofloxacin is maximized; when TiO is present2When the dosage of the-Tb is 40 mg, the extraction efficiency of the moxifloxacin is highest. To extract three quinolone antibiotics simultaneously in the sample solution, we chose 100 mg as the optimal dosage.
3. Influence of eluent type and volume
Accurately weighing 100 mg TiO2the-Tb nano material was added to 5mL of a mixed sample solution of 3 quinolone antibiotics (norfloxacin, ciprofloxacin, moxifloxacin) having a pH of 6. Vortex, mix evenly, ultrasonic 5min, centrifugate 5min under 4000r/min, discard the supernatant. 5mL of a solution of methanol (MeOH) and 4wt% acetic acid in a volume ratio of 0: 10. 1: 9. 2: 8. 3: 7 and 4: 6 as eluent, fully and uniformly mixing by vortex, carrying out ultrasonic treatment for 15min, filtering the supernatant by a 0.45-micron filter membrane, transferring the filtered supernatant into a chromatographic sampling bottle, and carrying out machine detection. The results of the measurements show that the extraction efficiency is highest when the ratio of MeOH/4wt% acetic acid solution is 0:10 by volume, so we chose a 4wt% solution of acetic acid by mass as eluent.
Accurately weighing 100 mg TiO2the-Tb nano material was added to 5mL of a mixed sample solution of 3 quinolone antibiotics (norfloxacin, ciprofloxacin, moxifloxacin) having a pH of 6. Vortex, mix evenly, ultrasonic 5min, centrifugate 5min under 4000r/min, discard the supernatant. Adding 5mL of acetic acid solution with mass concentration of 1%, 2%, 3% and 4% as eluent respectively, mixing uniformly by vortexing, performing ultrasound for 15min, filtering the supernatant with 0.45 μm filter membrane, transferring to a chromatographic sampling bottle, and detecting on a machine. The measurement result shows that the extraction efficiency is highest when the mass concentration of the acetic acid solution is 4%, so that 4wt% of the acetic acid solution is selected as the eluent.
Accurately weighing 100 mg TiO2the-Tb nano material was added to 5mL of a mixed sample solution of 3 quinolone antibiotics (norfloxacin, ciprofloxacin, moxifloxacin) having a pH of 6. Vortex, mix evenly, ultrasonic 5min, centrifugate 5min under 4000r/min, discard the supernatant. Adding 1 mL, 2 mL, 3 mL, 4 mL and 5mL of 4% acetic acid solution (pH = 2.45) as eluent, mixing uniformly by vortexing, performing ultrasound for 15min, filtering the supernatant with 0.45 μm filter membrane, transferring to a chromatographic sampling bottle, and detecting on a machine. The results of the assay show that the extraction efficiency is highest when the eluent is 5mL, so the optimum eluent volume is 5 mL.
4. Influence of extraction time
Accurately weighing 100 mg TiO2-Tb nanomaterial was added to 5mL 3 quinolone antibiotics with pH 6Mixed sample solution of antibiotics (norfloxacin, ciprofloxacin and moxifloxacin). After vortex mixing, ultrasonic treatment is respectively carried out for 5min, 10min, 15min, 20min, 25min and 30min for investigation, centrifugation is carried out for 5min at 4000r/min, and supernatant is discarded. Adding 5mL of acetic acid solution (pH = 2.45) with the mass concentration of 4wt% as an eluent, fully and uniformly mixing by vortex, carrying out ultrasonic treatment for 15min, filtering the supernatant by a 0.45-micron filter membrane, transferring the filtered supernatant into a chromatographic sampling bottle, and detecting the supernatant on a machine. The measurement result shows that the extraction efficiency is highest when the ultrasonic time is 5min, so the extraction time is selected to be 5 min.
5. Effect of desorption time
Accurately weighing 100 mg TiO2the-Tb nano material was added to 5mL of a mixed sample solution of 3 quinolone antibiotics (norfloxacin, ciprofloxacin, moxifloxacin) having a pH of 6. Vortex, mix evenly, ultrasonic 5min, centrifugate 5min under 4000r/min, discard the supernatant. Adding 5mL of acetic acid solution (pH = 2.45) with the mass concentration of 4wt% as an eluent, fully and uniformly mixing by vortex, respectively carrying out ultrasonic examination for 5min, 10min, 15min, 20min, 25min and 30min, filtering the supernatant by a 0.45 mu m filter membrane, transferring the filtered supernatant into a chromatographic sampling bottle, and detecting the supernatant by an upper computer. The measurement result shows that the extraction efficiency is highest when the ultrasonic time is 15min, so that the desorption time is 15 min.
Detailed description of the preferred embodiment
The linear range, regression equation, and detection limits of one of the methods of the embodiments described above
The concentration C is used as the abscissa, the peak area A is used as the ordinate to draw a standard curve, 3 antibiotics have good linearity in the concentration range, and the correlation coefficient (R)2) All are greater than 0.990, and as shown in Table 2, the detection Limit (LOD) of each antibiotic was calculated as 3-fold signal-to-noise ratio, and the LOD of 3 quinolone antibiotics was 3.3 ng/mL.
TABLE 2 Linear Range, regression equation, correlation coefficient and detection limits for quinolone antibiotics
Detailed description of the invention
Recovery and precision of the process of the invention
Respectively taking 500 mu L of samples (namely the human urine, the rabbit serum and the rabbit plasma samples), respectively placing the samples into 10 mL glass centrifuge tubes, respectively adding 5mL phosphate buffer solution with pH =6 and 10 mmol/L, uniformly mixing to obtain uniform solutions, respectively adding 3 antibiotic mixed standard solutions with different concentration levels, wherein the addition level of the quinolone antibiotic is 0.5-2 mu g/mL, each concentration level is parallel to 3 samples, and carrying out sample pretreatment and chromatographic condition determination according to the method of the specific embodiment. The average recovery rate and the standard deviation (i.e., SD) of each antibiotic were calculated by external standard method, and the results are shown in table 3, and the sample blank and the calibration chromatogram are shown in fig. 2, fig. 3, and fig. 4.
Table 3 results of recovery of antibiotic addition in urine, rabbit serum and rabbit plasma samples (n = 3)
As can be seen from Table 3, the average recovery rates of ciprofloxacin, norfloxacin and moxifloxacin, which are 3 quinolone antibiotics, are 72-115%, 76-118% and 69-117%, and the corresponding SD rates are 3.6-9.9%, 2.4-9.3% and 4.6-9.3%, respectively, which indicates that the method has good accuracy and repeatability.
The above examples show that the dispersive solid-phase extraction-liquid chromatography-ultraviolet method can be used for detecting quinolone antibiotics in urine and rabbit whole blood samples, and the dispersive solid-phase extraction method is simple, rapid and low in consumption, and does not need a large amount of organic reagents for extraction. The liquid chromatography-ultraviolet method provides high sensitivity for the detection of such antibiotics. The result proves that the method is sensitive and rapid, and can be popularized as a method for determining quinolone antibiotics.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.
Claims (4)
1. A pretreatment method of quinolone antibiotics in biological samples is characterized by comprising the following steps:
(1) placing a biological sample to be detected in a glass centrifuge tube, adding 10 mmol/L phosphate buffer solution with pH =6, which is 10 times of the volume of a sample solution to be detected, and uniformly mixing to obtain a uniform solution;
(2) adding TiO into the mixture2Adding Tb nano material into the homogeneous solution according to the proportion of 20 mg/mL, performing ultrasonic-assisted extraction for 3-8 minutes, centrifuging at 4000r/min for 5 minutes, removing the upper layer solution, adding the lower layer solution into an acetic acid solution with the mass concentration of 4wt% and the volume equal to that of the phosphate buffer solution in the step (1), performing ultrasonic-assisted back extraction for 12-18 minutes, centrifuging at 4000r/min for 5 minutes, taking the upper layer solution, and filtering through a 0.45-micrometer microporous filter membrane to obtain a pretreatment sample for high performance liquid chromatography detection of the quinolone antibiotics.
2. The method of claim 1, wherein the TiO is selected from the group consisting of TiO, InP2The preparation method of the-Tb nano material comprises the following steps: dissolving 20 mL of tetrabutyl titanate in 25 mL of absolute ethanol, and stirring for 30 minutes to obtain a solution A; meanwhile, 0.1335g of terbium nitrate hexahydrate is added into a mixed solution containing 10 mL of acetic acid, 20 mL of deionized water and 25 mL of absolute ethyl alcohol, and stirred for 30 minutes to obtain a solution B; slowly dripping the solution A into the solution B, reacting for 2 hours at 70 ℃, and aging for 72 hours at room temperature; drying the aged product in a drying oven at 100 ℃, transferring the dried product to a muffle furnace, calcining at 500 ℃ for 4 hours, cooling to room temperature, grinding the calcined product into powder by using a quartz mortar to obtain the TiO2-Tb nanomaterial.
3. The method according to claim 1, wherein the pretreatment of the quinolone antibiotic in the biological sample comprises: the biological sample comprises human urine and rabbit whole blood.
4. A method for quantitatively detecting a quinolone antibiotic using the pretreatment method for a quinolone antibiotic in a biological sample according to any one of claims 1 to 3, comprising the steps of:
(1) preparing a standard solution of the quinolone antibiotic by using methanol as a solvent, determining chromatographic peak retention time and chromatographic peak area of each component in the standard solution of the quinolone antibiotic by using a high performance liquid chromatography-ultraviolet method, determining the nature of the chromatographic peak retention time, drawing a standard curve of the quinolone antibiotic by using concentration as a horizontal coordinate and a chromatographic peak area as a vertical coordinate, wherein the quinolone antibiotic comprises ciprofloxacin, moxifloxacin and norfloxacin, and the concentration range of each component is 10-10000 ng/mL;
(2) HPLC-UV determination conditions of quinolone antibiotics
Liquid chromatography instrument model: agilent 1260 Infinity II; a chromatographic column: the model is Pursuit 5C 18, the specification is 5 mu m particle size, 4.6 mm x 150 mm; sample introduction amount: 20 mu L of the solution; column temperature: 25 ℃; a detector: a diode array detector; detector wavelength: the ciprofloxacin and norfloxacin are 278 nm, and the moxifloxacin is 291 nm; the mobile phase A is 0.2wt% acetic acid solution, the mobile phase B is acetonitrile, and the flow rate is 1 mL/min; the gradient elution procedure is shown in the following table:
(3) and (3) quantitatively analyzing the quinolone antibiotics in the biological sample by adopting an external standard method, and calculating to obtain the concentrations of ciprofloxacin, moxifloxacin and norfloxacin in the biological sample to be detected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010443577.5A CN111624279B (en) | 2020-05-22 | 2020-05-22 | Pretreatment method and quantitative detection method for quinolone antibiotics in biological sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010443577.5A CN111624279B (en) | 2020-05-22 | 2020-05-22 | Pretreatment method and quantitative detection method for quinolone antibiotics in biological sample |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111624279A true CN111624279A (en) | 2020-09-04 |
| CN111624279B CN111624279B (en) | 2022-06-10 |
Family
ID=72273075
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010443577.5A Expired - Fee Related CN111624279B (en) | 2020-05-22 | 2020-05-22 | Pretreatment method and quantitative detection method for quinolone antibiotics in biological sample |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111624279B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113075313A (en) * | 2021-03-22 | 2021-07-06 | 武汉海关技术中心 | Method for measuring quinolone drugs in environmental water and fish |
| CN113567594A (en) * | 2021-09-02 | 2021-10-29 | 佛山市三水佛水供水有限公司 | Detection method of norfloxacin based on MOFs type molecularly imprinted polymer |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003065031A1 (en) * | 2002-01-31 | 2003-08-07 | Gl Sciences Incorporated | Method and apparatus for analyzing amino acid, peptide, protein, saccharide or lipid |
| CN106124683A (en) * | 2016-08-26 | 2016-11-16 | 中国兽医药品监察所 | A kind of method of multiple antimicrobial component in veterinary drug of detection simultaneously |
| CN108896676A (en) * | 2018-07-12 | 2018-11-27 | 吉林化工学院 | A kind of magnetic Nano microsphere and its method to five kinds of trace fluoroquinolone antibiotics extractions and analysis in water body |
-
2020
- 2020-05-22 CN CN202010443577.5A patent/CN111624279B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003065031A1 (en) * | 2002-01-31 | 2003-08-07 | Gl Sciences Incorporated | Method and apparatus for analyzing amino acid, peptide, protein, saccharide or lipid |
| CN106124683A (en) * | 2016-08-26 | 2016-11-16 | 中国兽医药品监察所 | A kind of method of multiple antimicrobial component in veterinary drug of detection simultaneously |
| CN108896676A (en) * | 2018-07-12 | 2018-11-27 | 吉林化工学院 | A kind of magnetic Nano microsphere and its method to five kinds of trace fluoroquinolone antibiotics extractions and analysis in water body |
Non-Patent Citations (7)
| Title |
|---|
| KE HE 等: "Systematic optimization of an SPE with HPLC-FLD method forfluoroquinolone detection in wastewater", 《JOURNAL OF HAZARDOUS MATERIALS》, vol. 282, 27 August 2014 (2014-08-27), pages 96 - 105 * |
| LIBIN YANG 等: "SERS investigation and detection of levofloxacin drug molecules onsemiconductor TiO2: Charge transfer contribution", 《COLLOIDS AND SURFACES A: PHYSICOCHEM. ENG. ASPECTS》, vol. 508, 28 August 2016 (2016-08-28), pages 142 - 149, XP029730333, DOI: 10.1016/j.colsurfa.2016.08.068 * |
| MITRA AMOLI-DIVA 等: "Dispersive micro-solid phase extraction using magnetic nanoparticle modified multi-walled carbon nanotubes coupled with surfactant-enhanced spectrofluorimetry for sensitive determination of lomefloxacin and ofloxacin from biological samples", 《MATERIALS SCIENCE AND ENGINEERING C》, vol. 60, 10 November 2015 (2015-11-10), pages 30 - 36, XP029365401, DOI: 10.1016/j.msec.2015.11.013 * |
| ZHENCUI WANG 等: "Rapid preparation of terbium-doped titanium dioxide nanoparticles and their enhanced photocatalytic performance", 《R. SOC. OPEN SCI.》, vol. 6, 31 December 2019 (2019-12-31), pages 1 - 14 * |
| 徐媛原 等: "原位生成TiO2·nH2O-分散微固相萃取-高效液相色谱法测定环境水样中4种酚类污染物", 《分析试验室》, vol. 37, no. 11, 30 November 2018 (2018-11-30), pages 1266 - 1270 * |
| 谈文全 等: "纳米二氧化钛/腐植酸对泰乐菌素的吸附特性", 《广东化工》, vol. 41, no. 6, 31 December 2014 (2014-12-31), pages 1 - 2 * |
| 陈文胜 等: "固相萃取-液相色谱法测定水中喹诺酮类抗生素", 《华南预防医学》, vol. 46, no. 2, 30 April 2020 (2020-04-30), pages 178 - 182 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113075313A (en) * | 2021-03-22 | 2021-07-06 | 武汉海关技术中心 | Method for measuring quinolone drugs in environmental water and fish |
| CN113567594A (en) * | 2021-09-02 | 2021-10-29 | 佛山市三水佛水供水有限公司 | Detection method of norfloxacin based on MOFs type molecularly imprinted polymer |
| CN113567594B (en) * | 2021-09-02 | 2023-06-27 | 佛山市三水佛水供水有限公司 | Detection method of norfloxacin based on MOFs type molecularly imprinted polymer |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111624279B (en) | 2022-06-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108663471B (en) | Method for determining contents of multiple endocrine disruptors in estuary sediments | |
| CN111624279B (en) | Pretreatment method and quantitative detection method for quinolone antibiotics in biological sample | |
| CN112083108B (en) | Accurate detection method and kit for folic acid in blood | |
| Namera et al. | Extraction of amphetamines and methylenedioxyamphetamines from urine using a monolithic silica disk-packed spin column and high-performance liquid chromatography–diode array detection | |
| CN110346487A (en) | ZIF-8@ SiO2Core-shell particles and its preparation method and application | |
| CN115166080B (en) | Method for detecting impurity A and impurity B in ifosfamide bulk drug | |
| CN115267017B (en) | Method for detecting organic tin by combining dispersed solid phase extraction material and GC-MS | |
| CN112198265B (en) | Pretreatment method, detection method and kit for simultaneously detecting multiple steroid hormones in blood sample | |
| CN108072712B (en) | Quantitative analysis method for blood concentration of new compound WSJ-557 in SD rat plasma | |
| CN109632997B (en) | Extraction and determination method of liliflorin B in lily medicinal material | |
| CN101614712B (en) | Method for analyzing etimicin | |
| CN114814044B (en) | Method for detecting catecholamine and metabolite content in blood | |
| CN114904492B (en) | Polymer composite microsphere for extracting and purifying diquat metabolite in blood plasma, preparation method, kit and extraction method | |
| Zhou et al. | A molecularly imprinted fiber array solid-phase microextraction strategy for simultaneous detection of multiple estrogens | |
| CN112051343B (en) | Method for determining antibiotic residues | |
| CN112903836B (en) | Method for determining isopropyl-beta-D-thiogalactopyranoside in-vitro cultured bear gall powder | |
| CN101210928B (en) | Method for analyzing carbohydrate-binding protein by functional sugar chip and flight mass spectrometer | |
| CN103048403B (en) | Method for detecting lactic acid in fermented soybean meal | |
| CN109324139A (en) | Ribosylzeatin liquid-liquid extraction-liquid chromatography-tandem mass spectrometry measuring method in a kind of tobacco leaf | |
| CN115308317B (en) | High performance liquid chromatography detection method for index components in diabetes wound healing extract | |
| CN117147738B (en) | Method for detecting aristolochic acid I in refreshment and reconstruction pill | |
| CN117491522B (en) | Method for detecting residual quantity of N-hydroxysuccinimide | |
| CN116046935A (en) | Method for detecting free cortisol in urine by utilizing liquid chromatography-tandem mass spectrometry | |
| CN107126925B (en) | Magnetic nano material and application thereof in anegliptin drug detection | |
| CN115932104A (en) | Method for determining body-strengthening and life-prolonging pills by HPLC (high performance liquid chromatography) method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20220610 |