CN108414654B - Method for simultaneously enriching and detecting quinolone antibiotics in drinking water based on SPE (solid phase extraction) column - Google Patents

Method for simultaneously enriching and detecting quinolone antibiotics in drinking water based on SPE (solid phase extraction) column Download PDF

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CN108414654B
CN108414654B CN201810035741.1A CN201810035741A CN108414654B CN 108414654 B CN108414654 B CN 108414654B CN 201810035741 A CN201810035741 A CN 201810035741A CN 108414654 B CN108414654 B CN 108414654B
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CN108414654A (en
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崔长征
张天阳
金郁
韩琪
胡亚茹
曹赞
雷丹丹
姚诗杰
奚桢浩
王漫莉
任静
徐晓庆
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East China University of Science and Technology
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Abstract

The invention relates to a method for simultaneously enriching and detecting quinolone antibiotics in drinking water based on SPE (solid phase extraction) columns. Compared with the prior art, the method adopts SPE-HPLC-MS/MS to realize simultaneous enrichment detection of trace (micro) amount typical quinolone antibiotics in the drinking water, has reliable detection result precision, low detection limit, small relative standard deviation and the like, and can be effectively applied to detection and quantitative analysis of trace (micro) amount quinolone in excrement, soil, surface water and drinking water.

Description

Method for simultaneously enriching and detecting quinolone antibiotics in drinking water based on SPE (solid phase extraction) column
Technical Field
The invention relates to a detection and analysis technology of trace (micro) organic pollutants in a water environment, in particular to a method for simultaneously enriching and detecting quinolone antibiotics in drinking water by using a novel SPE (solid phase extraction) column based on reversed phase/ion exchange mixed mode chromatographic packing.
Background
Quinolone (Quinolones, QNs) antibiotics are used as a kind of artificially synthesized antibiotics with broad-spectrum resistance, strong antibacterial power, strong penetrating power, high efficiency, low toxicity and long half-life, and are widely applied to prevention and treatment of bacterial diseases existing in human beings, livestock breeding and the like. However, since QNs antibiotics are only partially degraded by biological absorption in organisms, most of the antibiotics are discharged from the bodies in a maternal form, and migrate into the drinking water environment through surface runoff in nature to directly affect human health, and meanwhile, the accumulation of quinolones in the environment can induce drug resistance of environmental bacteria to cause partial drug 'failure'. Therefore, QNs antibiotic residues in drinking water sources are receiving more and more attention from human beings.
At present, QNs antibiotics are commonly detected in surface water and water plants, wherein NOR, OFL, ENR, CIP, LOM and the like have high detection frequency and large detection concentration in water environment, and part of water plants also have part of residues, even part of veterinary quinolone is detected in human urine, such as NOR, OFL, CIP, ENR and the like (Wang H, Wang N, Wang B., et al, antibiotics detected in genes and adipogenesis in school children [ J ]. environmental International,2016,89-90: 204-. Therefore, establishing an accurate detection and analysis method for QNs antibiotics in drinking water sources is the basis and precondition for effective control.
Although many studies have been carried out on QNs antibiotic detection and analysis methods at home and abroad, most detection methods only can be used for analyzing animal-derived QNs antibiotic, and the detection limit is relatively high. For example, chinese patent No. CN101315351A discloses a method for simultaneously detecting 19 QNs drugs. The method adopts acetonitrile to extract animal tissue samples, and norfloxacin-D is used5As an internal sample standard, the treated sample was analyzed for sample concentration by HPLC-ESI-MS/MS. However, the method has the advantages that samples are directly subjected to sample injection determination without enrichment after being extracted, the applicable concentration is high, and the method is not suitable for analyzing trace QNs antibiotics in drinking water sources.
Chinese patent No. CN104730168A discloses a method for synchronously detecting tetracycline, fluoroquinolone and sulfonamide antibiotics remained in a water environment. The method relates to a technology for enriching and detecting residual antibiotics in a water environment, in particular to a method for simultaneously detecting 21 antibiotics such as residual tetracyclines, fluoroquinolones, sulfanilamide antibiotics and the like in a water body by adopting HLB (hydrophile-lipophile balance) solid-phase extraction and high performance liquid chromatography tandem mass spectrometry. Although the method can detect the residual situation of trace antibiotics in the environmental water body, the method has the advantages of considering more types of antibiotics and interference among different types of antibiotics, and is not sensitive enough to QNs antibiotics.
Chinese patent No. CN101696964A discloses a solid-phase extraction and HPLC-fluorescence detection method of fluoroquinolone antibiotics. The method relates to a fluoroquinolone antibiotic detection technology, in particular to a method for detecting 4 typical QNs antibiotics such as OFL, CIP, NOR, ENR and the like in a water environment sample through solid phase extraction and HPLC-fluorescence detection. According to the method, a C18 bonded silica gel solid phase extraction column is adopted to enrich antibiotics in a water sample, concentrated ammonia methanol solution with the volume ratio of concentrated ammonia being 6% is used for elution for three times, the volume is fixed to 1mL by nitrogen blowing, and the concentration of quinolone in the sample is analyzed by HPLC-fluorescence detection. Compared with the previous method, the method is simple to operate, low in cost and strong in pertinence to the quinolone antibiotics, but only aims at 4 quinolone antibiotics and few detection types of quinolone substances possibly remaining in the water environment at present.
The method disclosed by the invention fills the blank just by taking wide attention to the situation of residual antibiotics in the drinking water environment at present, but has the advantages of higher detection limit, fewer research types and relatively lower recovery rate aiming at the QNs antibiotic detection method, and can realize simultaneous detection of trace quinolone in the drinking water through one-time quantitative analysis by analyzing and detecting QNs antibiotics in the water environment, thereby greatly improving the detection efficiency.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for simultaneously enriching and detecting QNs antibiotics in drinking water based on an SPE column.
The purpose of the invention can be realized by the following technical scheme:
a method for simultaneously enriching and detecting QNs antibiotics in drinking water based on SPE columns comprises the following steps:
(1) sample pretreatment:
taking a test water sample, filtering to remove suspended matters, adjusting pH, adding a complexing agent, and adding an internal standard substance to complete pretreatment;
(2) concentrating and enriching the target quinolone:
activating the SPE cartridge, extracting and enriching the pretreated water sample obtained in the step (1) through the cartridge, leaching the SPE cartridge with a leacheate, drying in vacuum to remove water, continuously eluting a target with an elution solvent, collecting the eluent, drying the eluent under nitrogen flow, metering volume with an organic solution, and transferring the eluent to a brown sample injection bottle to be tested after purification;
(3) and (3) standard working curve preparation:
respectively drawing a standard working curve of each QNs antibiotic by taking the concentration ratio of each quinolone to the corresponding internal standard substance as a horizontal coordinate and the peak area ratio of each quinolone quantifier ion to the corresponding internal standard substance as a vertical coordinate;
(4) analyzing and detecting:
analyzing the concentrated and enriched sample in the step (2) by adopting HPLC-MS/MS, determining the peak area ratio of the quantitative daughter ions of each target object in the sample to the corresponding internal standard object, substituting the peak area ratio into the standard working curve obtained in the step (3) to obtain the concentration ratio of each target object to the corresponding internal standard object, and finally multiplying the obtained concentration ratio by the concentration of each internal standard object added in the step (1) to obtain the content of each quinolone antibiotic in the test water sample.
Preferably, the quinolone antibiotics as the target are Norfloxacin (Norfloxacin, NOR), Ofloxacin (Ofloxacin, OFL), Enrofloxacin (Enrofloxacin, ENR), Ciprofloxacin (CIP), Danofloxacin (Danofloxacin, DOF), Lomefloxacin (Lomefloxacin, LOM), Sarafloxacin (Sarafloxacin, SAR), Flumequine (fluequinine, FJK) and Oxolinic acid (OXO acid, OXO) which are 9 typical QNs antibiotics, and the selected 9 target antibiotics are currently used in large amounts and are detected frequently in water sources and tap water plants, wherein the Flumequine and the Oxolinic acid belong to typical acidic quinolones, and the other 7 quinolones belong to typical piperazine quinolones and have good representativeness.
More preferably, in the HPLC-MS/MS analysis in the step (3),
the liquid chromatography operating parameters were:
the organic phase is 0.05-0.3% formic acid/acetonitrile solution (v/v), the aqueous phase is 0.05-0.3% formic acid/pure water solution (v/v), and the gradient elution procedure is as follows: the volume ratio of the aqueous phase/organic phase is linearly reduced from 85%/15% to 75%/25% at 0-5min, to 55%/45% at 13min, to 15%/85% at 14min and maintained for 1.5min, returned to 65%/35% at 17min and continuously increased, and returned to 85%/15% at 18.5min and maintained for 0.5 min; the sample injection flow rate is 0.4mL/min, and the sample injection volume is 5-10 μ L.
More preferably, in the HPLC-MS/MS analysis in the step (3),
the operating parameters of the mass spectrum were:
adopting positive electric spray ionization source (ESI +), drying gas temperature is 350 ℃; the gas flow was 11L/min; positive ionization mode capillary voltage 4000 v; negative ionization mode capillary voltage 3000 v; the atomizer pressure was 15 psi.
More preferably, when the mass spectrometer is operated, in a Multiple Reaction Monitoring (MRM) mode, the target quinolone is qualitatively and quantitatively analyzed through three characteristic ions, namely a parent ion and two daughter ions, wherein the fragmentation voltage (V)/the ion one (daughter ion two) collision energy (eV) of NOR, CIP, OFL, ENR, DOF, LOM, SAR, FJK, OXO are 125/18(24), 125/16(12), 115/18(26), 110/16(24), 135/22(22), 115/22(24), 130/20(25), 110/30(15), 100/16(30), respectively.
Even more preferably, during the liquid chromatography run:
the organic mobile phase is formic acid/acetonitrile solution with the volume ratio of 0.1%/99.9%;
the aqueous phase was a 0.1%/99.9% formic acid/water solution.
Preferably, in the pretreatment process in the step (1):
filtering with 0.45 μm glass fiber membrane as filter membrane, adjusting pH to 5.0-9.5, and selecting Na as complexing agent2EDTA, internal standard substance of 9 target QNs antibiotics is 100 mug/L ciprofloxacin-D8。
Preferably, the reagent used for adjusting the pH value of the test water sample in the step (1) is 0.1mol/L sodium hydroxide solution.
Preferably, the SPE cartridge in step (2) is a reverse phase/ion exchange mixed mode chromatography packing solid phase extraction cartridge (HCE-C18). The filler is prepared by bonding alkyl chains such as C18 and C8 and positive charges on the surface of silica gel (Guo Z, WangC, Liang T, et al polar-polymerized aproach base on cationic polymerization on silica surface for preparation of polar-modified stable peptides [ J ]. Journal of Chromatography A,2010,1217(27): 4555-4560), and is available from China Hua spectral New science and technology Co., Ltd. (with the product number SC18 HCE-0506A).
Preferably, the target quinolone concentration enrichment of step (2):
SPE cartridge activation was performed sequentially using 5mL of methanol and ultrapure water in equal volumes through the cartridge.
Preferably, the target quinolone concentration enrichment of step (2):
the leacheate used was a 1% -8% methanol/water solution (v/v).
Preferably, the target quinolone concentration enrichment of step (2):
the vacuum drying time is 15-30 min.
Preferably, the target quinolone concentration enrichment of step (2):
the elution solvent is 0.05-3% formic acid/methanol solution (v/v).
Preferably, the target quinolone concentration enrichment of step (2):
the temperature of the eluent is controlled to be 35 ℃ when the nitrogen flow is dried.
Preferably, the target quinolone concentration enrichment of step (2):
the organic solution for constant volume is 40-80% methanol/water solution (v/v).
Preferably, the target quinolone concentration enrichment of step (2):
after the volume is fixed, a 0.22 mu m nylon needle filter is adopted for filtration and purification.
Preferably, the drawing process of the standard working curve in the step (3) is specifically as follows:
(a) the method comprises the following steps Mixing different quinolone antibiotics by using methanol to prepare a single mixed standard stock solution with known and same quinolone antibiotic concentration, and placing the mixed standard stock solution into a brown reagent bottle;
(b) the method comprises the following steps Diluting the mixed standard stock solution obtained in the step (a) into a plurality of groups of samples with different concentration gradients by using 40-80% methanol/water solution (v/v), simultaneously adding corresponding internal standard substances into each group of samples with the concentration gradients respectively, and ensuring the concentration of the internal standard substances in the samples with the different concentration gradients to be consistent;
(c) the method comprises the following steps And analyzing each concentration gradient sample by adopting LC-MS/MS, and drawing standard working curves of different quinolones by taking the concentration ratio of each quinolone to the corresponding internal standard substance as a horizontal coordinate and taking the peak area ratio of the quantitive daughter ion of each quinolone to the corresponding internal standard substance as a vertical coordinate.
Preferably, when the sample is analyzed by LC-MS/MS, the method for determining the peak area of the quantitive ion comprises the following steps: 1) determining target parent ions (m/z) with a full SCAN monitoring mode (SCAN); 2) optimizing the fragmentation voltage and the acceleration voltage of the parent ions by using a selective ion monitoring mode (SIM) to ensure that the target parent ions enter a collision chamber in the optimal state (namely the maximum target peak area); 3) screening two characteristic ion ions (m/z) with the maximum responsivity after the parent ions are cracked by collision through preset collision energy in a Product Scan mode; 4) further optimizing collision energy by using a multi-reaction monitoring mode (MRM) to ensure that the peak area of a target object taking the parent ions and the characteristic daughter ions as ion pairs is maximum; 5) and finally, recording the peak appearance area corresponding to the ion pair by taking the parent ion and the characteristic ion of the target quinolone as the ion pair under the MRM mode and the optimal condition, namely the peak area of the quantitative daughter ion corresponding to the quinolone.
More preferably, the samples with different concentration gradients in step (b) include samples with concentrations of the corresponding quinolones of 1, 2, 5, 10, 20, 50, 80, 100 and 200. mu.g/L, respectively.
The invention realizes simultaneous enrichment detection of trace (micro) amount typical QNs in drinking water by sample pretreatment concentration enrichment and exploration and optimization of detection conditions and parameters of an LC-MS/MS instrument, greatly saves sample measurement time and reduces detection cost. In the pretreatment process, the recovery rate of QNs is comprehensively improved and the peak appearance conditions of different QNs and internal standard substances in subsequent detection are improved by selecting and optimizing the adding amount of the internal standard substance, the filler of the solid-phase extraction column, the pH of a water sample and an elution solvent; in the process of analyzing and detecting the instrument, the peak shapes and the separation degrees of 9 QNs and three internal standard substances are ensured by optimizing the sample introduction condition of the liquid chromatogram and the detection parameters of the mass spectrum, so that a lower detection limit and a smaller relative standard deviation are obtained.
Compared with the prior art, the invention has the following advantages:
(1) the reversed phase/ion exchange mixed mode chromatographic packing (HCE-C18) solid phase extraction column is selected to simultaneously enrich and purify 9 types of QNs, the recovery rate is 80-120%, the effect is stable, the selectivity is strong, the adsorption capacity is large, the aim of effectively separating and enriching the target is fulfilled, and the simultaneous enrichment detection of multiple types of QNs is realized.
(2) The LC-MS/MS is adopted for quantitative analysis and detection, the sensitivity is high, the detection limit of QNs is lower than 2ng/L, the quantitative limit is lower than 5ng/L, and the detection requirement of QNs in trace amount in drinking water can be met. In addition, the tandem triple quadrupole mass spectrometer carries out quantitative analysis according to characteristic fragment ions generated by the collision of corresponding parent ions, has strong selectivity, and eliminates the interference of other impurity signals in a sample.
(3) The concentration of QNs is measured by an internal standard curve method, the linear relation is good, the relative deviation is small, and the precision of analysis is improved.
(4) The pretreatment process is simple to operate and environment-friendly.
Drawings
FIG. 1 is a schematic analysis flow chart of the present invention;
FIG. 2 is a graph of the effect of different SPE packings on target recovery in accordance with the present invention;
FIG. 3 is a liquid phase permeation curve of norfloxacin in reversed phase/ion exchange mixed mode chromatography packing under different pH conditions in accordance with the present invention;
FIG. 4 is a graph showing the effect of pH values of different water samples on the recovery rate of a target substance in the present invention;
FIG. 5 is a graph showing the effect of different elution solvents on the recovery of a target in the present invention;
FIG. 6 is a graph showing the recovery of the target under the optimum conditions in the present invention;
FIG. 7 is a total ion flow diagram of a mixed standard solution (100. mu.g/L) of object QNs in accordance with the present invention;
fig. 8, 9 and 10 are extraction chromatograms of each target QNs and corresponding internal standard in the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
The analytical method flow shown in figure 1 is adopted to simultaneously detect quinolones in drinking water, and comprises the following steps:
(1) sample pretreatment:
taking a test water sample, filtering to remove suspended matters, adjusting pH, adding a complexing agent, and adding an internal standard substance to complete pretreatment;
(2) concentrating and enriching the target quinolone:
activating the SPE cartridge, extracting and enriching the pretreated water sample obtained in the step (1) through the cartridge, leaching the SPE cartridge with a leacheate, drying in vacuum to remove water, continuously eluting a target with an elution solvent, collecting the eluent, drying the eluent under nitrogen flow, metering volume with an organic solution, and transferring the eluent to a brown sample injection bottle to be tested after purification;
(3) and (3) standard working curve preparation:
respectively drawing a standard working curve of each quinolone antibiotic by taking the concentration ratio of each quinolone to the corresponding internal standard substance as a horizontal coordinate and taking the peak area ratio of each quinolone quantifier ion to the corresponding internal standard substance as a vertical coordinate;
(4) analyzing and detecting:
analyzing the concentrated and enriched sample in the step (2) by adopting HPLC-MS/MS, determining the peak area ratio of the quantitative daughter ions of each target object in the sample to the corresponding internal standard object, substituting the peak area ratio into the standard working curve obtained in the step (3) to obtain the concentration ratio of each target object to the corresponding internal standard object, and finally multiplying the obtained concentration ratio by the concentration of each internal standard object added in the step (1) to obtain the content of each quinolone antibiotic in the test water sample.
The detection method further comprises sample pretreatment optimization and detection method optimization, wherein the sample pretreatment optimization comprises determination of SPE filler, optimal pH and elution solvent. And optimizing various conditions in the detection method, including optimizing liquid chromatography conditions and optimizing mass spectrum operating parameters.
Wherein, the optimization comprises the following specific steps:
1. sample pretreatment:
1.1 determination of SPE pillar Filler
The choice of solid phase extraction column depends on the interaction of the column packing with the functional groups on the target, and to maximize the enrichment efficiency of each QNs, the present invention selects the extraction efficiency of three different column packings (Isolute C18, reversed phase/ion exchange mixed mode chromatography packing (HCE-C18), and Oasis HLB) according to empirical summary, and the results are shown in fig. 2. The experimental results show that: in the three SPE columns, when the solid phase extraction column is enriched by the reversed phase/ion exchange mixed mode chromatographic packing, the extraction recovery rate of most of the target QNs is in or close to the range of 50% -70%, so that QNs effective enrichment can be realized, and the control limit requirement of the environmental sample standard addition recovery rate is met.
The penetration volume of the reversed-phase/ion exchange mixed-mode chromatographic packing of the extraction column to the quinolone antibiotics is predicted and optimized rapidly through a liquid phase, and then the enrichment multiple of the reversed-phase/ion exchange mixed-mode chromatographic packing material to the quinolone antibiotics is predicted. The method specifically comprises the following steps: filling the reversed phase/ion exchange mixed mode chromatographic packing with determined mass in a hollow chromatographic column, and compacting and activating for later use. And (3) taking the target sample with determined concentration as a water phase and methanol as an organic phase, and drawing the adsorption effect of the reversed phase/ion exchange mixed mode chromatographic packing on the target substance. The preferable type of the extraction column can ensure good enrichment degree and recovery rate of the target QNs, the preferable activation mode can improve the adsorption and elution effect of the filler in the extraction column on the target QNs to the maximum extent and improve the recovery rate, the preferable elution mode can remove impurities in the filler of the extraction column and reduce the interference of subsequent detection while reducing the loss of the target QNs to the maximum extent, the preferable elution mode can ensure that the target QNs enriched on the extraction column is eluted to the maximum extent and improve the recovery rate of the target, the preferable nitrogen blowing mode can ensure that the loss of the target QNs is reduced to the maximum extent while the elution solvent is rapidly volatilized, and the preferable organic solution can ensure that the target QNs attached to the wall of the container after nitrogen is blown dry is completely dissolved and mixed and is convenient for the sample analysis of a subsequent instrument.
Meanwhile, the method takes a target substance as a chromatographic column packing material with a water phase reverse phase/ion exchange mixed mode as a chromatographic column packing material, and draws a penetration curve as shown in fig. 3, and experimental results show that the chromatographic column packing material with the reverse phase/ion exchange mixed mode has different penetration curves under different pH conditions, the chromatographic column packing material with the reverse phase/ion exchange mixed mode is difficult to retain the target quinolone antibiotics under the condition of pH 3, and a sample starts to penetrate after 0 moment; the sample can be well preserved in the reversed phase/ion exchange mixed mode chromatographic packing under the conditions of pH 5, pH 7 and pH 8, the sample starts to penetrate after 15min, the retention time is long, the enrichment amount of the reversed phase/ion exchange mixed mode chromatographic packing material on the quinolone is estimated to be about 53 mu g/g through the volume of the reversed phase/ion exchange mixed mode chromatographic packing, the sampling flow rate and the penetration time, and trace (nanogram) antibiotics remained in the drinking water environment can be effectively adsorbed and cannot penetrate. Therefore, the reversed phase/ion exchange mixed mode chromatographic packing can be determined to be suitable for detecting trace quinolone antibiotics in drinking water.
1.2 determination of optimum pH
Since the target QNs is mostly a weakly acidic compound containing phenolic hydroxyl, the pH of the water sample is selected to be optimized at 3.0, 5.0, 7.0, 8.0, 9.0 and 11.0, which respectively represent a strongly acidic condition, a weakly acidic condition and a neutral condition, and the result is shown in FIG. 4. The experimental results show that: when the pH value is 8.0, more target quinolones are extracted and recovered within the range of 60-80%, and the experimental result is the same as the liquid phase prediction experimental result, so that the water sample condition with the pH value of 8.0 is selected to complete the extraction and enrichment of the target QNs.
1.3 determination of elution solvent
In order to ensure that the target substance can be eluted to the maximum extent, the invention inspects the influence of different elution solvents on the recovery rate of the target substance. The elution solvents are methanol, acetonitrile, formic acid: methanol (0.1%: 99.9%, v/v). Weak acid can well separate quinolone substances adsorbed by the reversed phase/ion exchange mixed mode chromatographic packing material, and the result is shown in figure 5. The experimental results show that: when the elution solvent is acetonitrile target, the recovery rate is the lowest, and the recovery rate of partial substances is lower than 40%; when the elution solvent is formic acid/methanol (the mixing ratio of the two is most preferably 0.1%: 99.9%, v/v), the recovery rate of the target object is the highest, and basically reaches between 80% and 120%, and meets the quantitative requirement; when the elution solvent is methanol, the elution time and the target recovery rate lie between the first two. The most preferred elution solvent of the present invention is therefore formic acid: methanol (0.1%: 99.9%, v/v).
The recovery of pure water normalized under the optimum conditions is shown in FIG. 6.
2. Detection method optimization
2.1 optimization of liquid chromatography conditions
In order to realize chromatographic peak separation and improve signal intensity, the invention respectively optimizes key factors influencing liquid phase separation, such as mobile phase, flow velocity, sample injection quantity, gradient elution program and the like. The liquid chromatography detection conditions adopted by the invention are as follows:
mobile phase a (i.e. organic phase): acetonitrile solution containing 0.05% to 0.3% formic acid (formic acid/acetonitrile: 0.1%/99.9%, v/v), mobile phase B (i.e. aqueous phase): pure water containing 0.05% to 0.3% formic acid (formic acid/water: 0.1%/99.9%, v/v); flow rate: 0.4 mL/min; sample introduction amount: 5-10 μ L; column temperature: 35 ℃; gradient elution procedure: 0-5min, 85% of the B mobile phase is linearly reduced to 75%, 5-13min is linearly reduced to 55% and continuously reduced, 14min is reduced to 15% and kept for 1.5min, 17min is returned to 65% and continuously increased, 18.5min is increased to 85% and continuously increased for 0.5 min.
The sample introduction flow rate is 0.4mL/min, and the sample introduction volume is 5-10 mu L;
2.2 Mass Spectrometry operating parameter optimization
The liquid chromatogram-tandem mass spectrometer can accurately quantify the characteristic fragment ions, and for this reason, the fragmentation voltage and the collision energy need to be optimized to obtain the optimal molecular ion-fragment ion pair, so that the subsequent detection and analysis of the actual water sample are more accurate. The mass spectrum operating parameters adopted by the invention are as follows:
an ionization mode: ESI +; temperature of the drying gas: 350 ℃; gas flow rate: 11L/min; capillary voltage: 4000v (ESI +), 3000v (ESI-); atomizer pressure: 15 psi.
The invention selects CIP-D8As an internal standard substance of QNs, quantification was performed by an internal standard method. The internal standard and target were eluted at the same chromatographic time period and responded well in electrospray ionization source mode without significant matrix interference. Therefore, the above-mentioned internal standard substance is a highly desirable internal standard substance in the present invention. On the basis of this, the total ion flux chromatogram of 9 quinolone antibiotics represented by 100. mu.g/L in ESI positive ion mode is shown in FIG. 7. Although the total ion flow graph only has 8 chromatographic peaks, the total ion flow chromatographic peaks of individual substances overlap due to the fact that the relation between the extraction chromatographic peak area and the concentration of the quantitative daughter ions is used for quantification in the tandem mass spectrum, and the analysis and the determination of the target substances are not influenced. HPLC-MS/MS extraction chromatograms of 8 quinolone antibiotics represented by 100. mu.g/L and an internal standard are shown in FIG. 8, FIG. 9 and FIG. 10. Therefore, the peak shape of the extracted chromatographic peak of each target object under the optimized condition is complete, the response value is high, and reliable guarantee is provided for the subsequent quantitative analysis work of antibiotics in the water source water.
In addition, the tandem mass spectrometer uses the characteristic fragment ion having the largest signal response intensity as a quantitative ion, except for using an internal standard curve, and the quantitative ion of each substance is shown in table 1.
TABLE 1 target quinolone antibiotic profile parameters
Figure BDA0001547870620000101
Example 1
A method for simultaneously enriching and detecting typical quinolone antibiotics in drinking water based on a novel reversed-phase/ion-exchange mixed-mode chromatographic packing SPE column comprises the following specific steps of:
(a) preparing a mixed standard stock solution: mixing 9 kinds of QNs with known concentration by using 40-80% methanol/water solution (v/v) to prepare a single mixed standard stock solution with QNs concentration which is respectively the same, and placing the mixed standard stock solution into a brown reagent bottle;
(b) diluting the mixed standard stock solution in the step (a) into different concentration gradients by using 40% -80% methanol/water solution (v/v): 1.2, 5, 10, 20, 50, 80, 100 and 200. mu.g/L, while adding CIP-D to each concentration gradient sample8Preparing 100 mug/L internal standard working solution;
(c) analyzing each concentration gradient sample by adopting LC-MS/MS, operating the LC-MS/MS by adopting the optimized liquid chromatogram operating parameters and mass spectrum operating parameters, taking the concentration ratio of each QNs and the corresponding internal standard substance as a horizontal coordinate, taking the peak area ratio of each QNs quantitive ion and the internal standard substance obtained by analysis as a vertical coordinate, and respectively drawing three types of QNs standard working curves.
The method detection Limit and the method quantitative Limit are calculated by a Signal-to-noise (S/N), with 3 times the Signal-to-noise ratio as the method detection Limit (LOD) and 10 times the Signal-to-noise ratio as the method quantitative Limit (LOQ). The linear range, correlation coefficient, detection limit and quantitation limit results for this method are listed in table 2.
The result shows that the linear correlation of the target substance under the optimized instrument condition is good, and the linear correlation coefficient R2>0.98. The method detection limit range of the target substance is 0.01-1.36ng/L, the method quantification limit range is 0.03-4.53ng/L, and the detection limit and the quantification limit of the low concentration level ensure the possibility of detecting the trace QNs in the water environment by the analysis method.
TABLE 2 detection and quantitation limits for target quinolone antibiotics
Figure BDA0001547870620000111
Example 2
A method for simultaneously enriching and detecting typical 9 quinolone similar antibiotics in drinking water based on a novel reverse phase/ion exchange mixed mode chromatographic packing SPE column comprises the following steps:
(1) sample pretreatment:
selecting water sample of water source in east China, filtering with 0.45 μm glass fiber membrane as filter membrane to remove suspended substances, adjusting pH to 5.0-9.5, and adding complexing agent Na2EDTA, adding an internal standard with a known amount of 100 mug/L to complete the pretreatment;
(2) concentration and enrichment of target QNs:
firstly, 5-10mL of methanol and ultrapure water with the same volume are sequentially used for activation through an SPE small column (the embodiment selects a reversed phase/ion exchange mixed mode chromatographic packing solid phase extraction small column), then 500mL of water sample pretreated in the step (1) is taken to pass through the column for extraction and enrichment, then taking 4% methanol/water solution (v/v) as eluent to drip the small column, vacuum drying for 15-30min to remove water, continuously eluting the target object by using 8mL formic acid/methanol solution (0.1%/99.9%, v/v) as eluting solvent, collecting eluent, controlling the water bath temperature to 35 ℃, blow-drying under nitrogen flow, dissolving the residue with 50% methanol/water solution (v/v) and fixing the volume, finally, filtering the solution by adopting a 0.22 mu m nylon needle filter, and transferring the solution to a brown sample injection bottle to be tested;
(3) analyzing and detecting by an instrument:
analyzing the concentrated and enriched sample in the step (2) by adopting LC-MS/MS, and setting the operation parameters of liquid chromatography as follows: the organic phase was an acetonitrile solution of formic acid (formic acid/acetonitrile 0.1%/99.9%, v/v), and the aqueous phase was pure water containing formic acid (formic acid/water 0.1%/99.9%, v/v in this example); gradient elution procedure: from 0 to 5min, the 85% aqueous phase mobile phase is linearly reduced to 75%, at 13min to 55%, at 14min to 15% and kept for 1.5 min; returning to 85% at 16min, and introducing sample at flow rate of 0.4mL/min and volume of 5-10 μ L; the operating parameters of the mass spectrum were: adopting positive electric spray ionization source (ESI +) and negative electric spray ionization source (ESI-), drying gas temperature is 350 ℃; the gas flow was 11L/min; positive ionization mode capillary voltage 4000 v; negative ionization mode capillary voltage 3000 v; atomizer pressure 15 psi; and simultaneously recording by adopting a full-SCAN SCAN mode, a Selective Ion Monitoring (SIM) mode, a Product Ion scanning mode and a multi-reaction monitoring MRM mode.
Determining peak area ratios of the quantitive daughter ions of each target QNs and the corresponding internal standard substance in the sample according to the LC-MS/MS analysis result, obtaining concentration ratios of each QNs and the corresponding internal standard substance in the sample by combining the standard working curve drawn in the example 1, and determining the content of each QNs in the sample by combining the known concentrations of each internal standard substance added in the step (1).
Finally, the analysis and detection results and the recovery rate of the obtained drinking water sample in east China under the detection operation conditions of the embodiment are shown in Table 3.
TABLE 3 residue and recovery of QNs from a source of water in east China
Figure BDA0001547870620000131
The experimental results show that: the established method is successfully applied to the analysis and detection of QNs residues in a water source water in east China, all targets QNs are detected in the water source, and the detected concentrations of NOR, CIP, ENR, DOF and the like are higher than those of other antibiotics.
Example 3
A method for simultaneously enriching and detecting typical 9 quinolone similar antibiotics in drinking water based on a novel reverse phase/ion exchange mixed mode chromatographic packing SPE column comprises the following steps:
(1) sample pretreatment:
selecting water sample of water source in east China, filtering with 0.45 μm glass fiber membrane as filter membrane to remove suspended substances, adjusting pH to 5.0-9.5, and adding complexing agent Na2EDTA, adding an internal standard with a known amount of 100 mug/L to complete the pretreatment;
(2) concentration and enrichment of target QNs:
firstly, 5-10mL of methanol and ultrapure water with the same volume are sequentially used for activation through an SPE small column (the embodiment selects a reversed phase/ion exchange mixed mode chromatographic packing solid phase extraction small column), then 500mL of water sample pretreated in the step (1) is taken to pass through the column for extraction and enrichment, then taking 1% methanol/water solution (v/v) as eluent to drip the small column, vacuum drying for 15-30min to remove water, continuously eluting the target object by using 5mL formic acid/methanol solution (0.1%/99.9%, v/v) as eluting solvent, collecting eluent, controlling the water bath temperature to 35 ℃, blow-drying under nitrogen flow, dissolving the residue with 50% methanol/water solution (v/v) and fixing the volume, finally, filtering the solution by adopting a 0.22 mu m nylon needle filter, and transferring the solution to a brown sample injection bottle to be tested;
(3) analyzing and detecting by an instrument:
analyzing the concentrated and enriched sample in the step (2) by adopting LC-MS/MS, and setting the operation parameters of liquid chromatography as follows: the organic phase is an acetonitrile solution containing 0.05% (v/v) formic acid, and the aqueous phase is a pure water solution containing 0.05% (v/v) formic acid; gradient elution procedure: from 0 to 5min, the 85% aqueous phase mobile phase is linearly reduced to 75%, at 13min to 55%, at 14min to 15% and kept for 1.5 min; returning to 85% at 16min, and introducing sample at flow rate of 0.4mL/min and volume of 5-10 μ L; the operating parameters of the mass spectrum were: adopting positive electric spray ionization source (ESI +) and negative electric spray ionization source (ESI-), drying gas temperature is 350 ℃; the gas flow was 11L/min; positive ionization mode capillary voltage 4000 v; negative ionization mode capillary voltage 3000 v; atomizer pressure 15 psi; and simultaneously recording by adopting a full-SCAN SCAN mode, a Selective Ion Monitoring (SIM) mode, a Product Ion scanning mode and a multi-reaction monitoring MRM mode.
Determining peak area ratios of the quantitive daughter ions of each target QNs and the corresponding internal standard substance in the sample according to the LC-MS/MS analysis result, obtaining concentration ratios of each QNs and the corresponding internal standard substance in the sample by combining the standard working curve drawn in the example 1, and determining the content of each QNs in the sample by combining the known concentrations of each internal standard substance added in the step (1).
Example 4
A method for simultaneously enriching and detecting typical 9 quinolone similar antibiotics in drinking water based on a novel reverse phase/ion exchange mixed mode chromatographic packing SPE column comprises the following steps:
(1) sample pretreatment:
selecting water sample of water source in east China, filtering with 0.45 μm glass fiber membrane as filter membrane to remove suspended substances, adjusting pH to 5.0-9.5, and adding complexing agent Na2EDTA, adding an internal standard with a known amount of 100 mug/L to complete the pretreatment;
(2) concentration and enrichment of target QNs:
firstly, 5-10mL of methanol and ultrapure water with the same volume are sequentially used for activation through an SPE small column (the embodiment selects a reversed phase/ion exchange mixed mode chromatographic packing solid phase extraction small column), then 500mL of water sample pretreated in the step (1) is taken to pass through the column for extraction and enrichment, then, 8 percent methanol/water solution (v/v) is taken as eluent to drip the small column, the small column is dried in vacuum for 15 to 30min to remove water, then 5mL formic acid/methanol solution (0.1 percent/99.9 percent, v/v) is continuously used as an elution solvent to elute the target object, the eluent is collected, the water bath temperature of the eluent is controlled to be 35 ℃, blow-drying under nitrogen flow, dissolving the residue with 50% methanol/water solution (v/v) and fixing the volume, finally, filtering the solution by adopting a 0.22 mu m nylon needle filter, and transferring the solution to a brown sample injection bottle to be tested;
(3) analyzing and detecting by an instrument:
analyzing the concentrated and enriched sample in the step (2) by adopting LC-MS/MS, and setting the operation parameters of liquid chromatography as follows: the organic phase is an acetonitrile solution containing 0.3% (v/v) formic acid, and the aqueous phase is a pure water solution containing 0.3% (v/v) formic acid; gradient elution procedure: from 0 to 5min, the 85% aqueous phase mobile phase is linearly reduced to 75%, at 13min to 55%, at 14min to 15% and kept for 1.5 min; returning to 85% at 16min, and introducing sample at flow rate of 0.4mL/min and volume of 5-10 μ L; the operating parameters of the mass spectrum were: adopting positive electric spray ionization source (ESI +) and negative electric spray ionization source (ESI-), drying gas temperature is 350 ℃; the gas flow was 11L/min; positive ionization mode capillary voltage 4000 v; negative ionization mode capillary voltage 3000 v; atomizer pressure 15 psi; and simultaneously recording by adopting a full-SCAN SCAN mode, a Selective Ion Monitoring (SIM) mode, a Product Ion scanning mode and a multi-reaction monitoring MRM mode.
Determining peak area ratios of the quantitive daughter ions of each target QNs and the corresponding internal standard substance in the sample according to the LC-MS/MS analysis result, obtaining concentration ratios of each QNs and the corresponding internal standard substance in the sample by combining the standard working curve drawn in the example 1, and determining the content of each QNs in the sample by combining the known concentrations of each internal standard substance added in the step (1).
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims (3)

1. A method for simultaneously enriching and detecting quinolone antibiotics in drinking water based on SPE (solid phase extraction) columns is characterized by comprising the following steps:
(1) sample pretreatment:
taking a test water sample, filtering to remove suspended matters, adjusting pH, adding a complexing agent, and adding an internal standard substance to complete pretreatment;
(2) concentrating and enriching the target quinolone:
activating the SPE cartridge, extracting and enriching the pretreated water sample obtained in the step (1) through the cartridge, leaching the SPE cartridge with a leacheate, drying in vacuum to remove water, continuously eluting a target with an elution solvent, collecting the eluent, drying the eluent under nitrogen flow, metering volume with an organic solution, and transferring the eluent to a brown sample injection bottle to be tested after purification;
(3) and (3) standard working curve preparation:
respectively drawing a standard working curve of each quinolone antibiotic by taking the concentration ratio of each quinolone to the corresponding internal standard substance as a horizontal coordinate and taking the peak area ratio of each quinolone quantifier ion to the corresponding internal standard substance as a vertical coordinate;
(4) analyzing and detecting:
analyzing the concentrated and enriched sample obtained in the step (2) by adopting HPLC-MS/MS, determining the peak area ratio of the quantitative daughter ions of each target object in the sample to the corresponding internal standard object, substituting the peak area ratio into the standard working curve obtained in the step (3) to obtain the concentration ratio of each target object to the corresponding internal standard object, and finally multiplying the obtained concentration ratio by the concentration of each internal standard object added in the step (1) to obtain the content of each quinolone antibiotic in the test water sample;
in the pretreatment process in the step (1):
filtering with 0.45 μm glass fiber membrane as filter membrane, adjusting pH to 5.0-9.5, and selecting Na as complexing agent2EDTA, the internal standard substance is ciprofloxacin-D with the concentration of 100 mug/L8；
In the step (1), the reagent for adjusting the pH value of the test water sample is sodium hydroxide and/or phosphate buffer solution;
the SPE small column in the step (2) is a reversed phase/ion exchange mixed mode chromatographic packing solid phase extraction column HCE-C18;
concentrating and enriching the target quinolone in the step (2):
the SPE small column activation is sequentially activated by passing 5mL of methanol and ultrapure water with the same volume through the column;
the leacheate is 1-8% methanol/water solution, v/v;
vacuum drying for 15-30 min;
the elution solvent is 0.05-3% formic acid/methanol solution, v/v;
controlling the temperature of the eluent to be 35 ℃ when blowing dry by nitrogen flow;
the organic solution for constant volume adopts 40-80% methanol/water solution, v/v;
after constant volume, filtering and purifying by adopting a 0.22 mu m nylon needle filter;
the target quinolone antibiotics are norfloxacin, ofloxacin, enrofloxacin, ciprofloxacin, danofloxacin, lomefloxacin, sarafloxacin, flumequine and oxolinic acid;
when the HPLC-MS/MS analysis is carried out in the step (3),
the liquid chromatography operating parameters were:
the organic mobile phase is 0.1% by volume: 99.9% formic acid/acetonitrile solution;
the water phase is 0.1% by volume: 99.9% formic acid/water solution, gradient elution procedure: the volume ratio of the aqueous phase/organic phase is linearly reduced from 85%/15% to 75%/25% at 0-5min, to 55%/45% at 13min, to 15%/85% at 14min and maintained for 1.5min, returned to 65%/35% at 17min and continuously increased, and returned to 85%/15% at 18.5min and maintained for 0.5 min; the sample introduction flow rate is 0.4mL/min, and the sample introduction volume is 5-10 mu L;
the operating parameters of the mass spectrum were:
adopting a positive electric spray ionization source, wherein the temperature of the drying gas is 350 ℃; the gas flow was 11L/min; positive ionization mode capillary voltage 4000 v; negative ionization mode capillary voltage 3000 v; atomizer pressure 15 psi; in a multi-reaction monitoring mode, the target substance quinolone is qualitatively and quantitatively analyzed through three characteristic ions of a parent ion and two daughter ions, wherein the fragmentation voltages of norfloxacin, ciprofloxacin, ofloxacin, enrofloxacin, danofloxacin, lomefloxacin, sarafloxacin, flumequine and oxolinic acid/the collision energies of the daughter ion I/daughter ion II are respectively 125V/18eV/24eV, 125V/16eV/12eV, 115V/18eV/26eV, 110V/16eV/24eV, 135V/22eV/22eV, 115V/22eV/24eV, 130V/20eV/25eV, 110V/30eV/15eV and 100V/16eV/30 eV.
2. The method for simultaneously enriching and detecting the quinolone antibiotics in the drinking water based on the SPE column as claimed in claim 1, wherein the drawing process of the standard working curve in step (3) is specifically as follows:
(a) the method comprises the following steps Mixing different quinolone antibiotics by using methanol to prepare a single mixed standard stock solution with known and same quinolone antibiotic concentration, and placing the mixed standard stock solution into a brown reagent bottle;
(b) the method comprises the following steps Diluting the mixed standard stock solution obtained in the step (a) into a plurality of groups of samples with different concentration gradients by using 40-80% methanol/water solution v/v, simultaneously adding corresponding internal standard substances into each group of samples with different concentration gradients respectively, and ensuring the concentration consistency of the internal standard substances in the samples with different concentration gradients;
(c) the method comprises the following steps And analyzing each concentration gradient sample by adopting LC-MS/MS, and drawing standard working curves of different quinolones by taking the concentration ratio of each quinolone to the corresponding internal standard substance as a horizontal coordinate and taking the peak area ratio of the quantitive daughter ion of each quinolone to the corresponding internal standard substance as a vertical coordinate.
3. The SPE column based method for simultaneous enrichment and detection of quinolone antibiotics in drinking water according to claim 2, wherein the samples with different concentration gradients in step (b) comprise samples with corresponding quinolone concentrations of 1, 2, 5, 10, 20, 50, 80, 100 and 200 μ g/L.
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