CN109270192B - Pretreatment device, method and application for detecting aquatic product sulfonamide antibiotics - Google Patents
Pretreatment device, method and application for detecting aquatic product sulfonamide antibiotics Download PDFInfo
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Abstract
The invention relates to the field of aquatic product quality monitoring, in particular to the field of monitoring of antibiotics in aquatic products, and more particularly relates to a pretreatment device, a pretreatment method and application for detecting aquatic product sulfonamide antibiotics. By establishing an on-line solid phase extraction elution pretreatment device for on-line enrichment and purification of sulfonamide antibiotics and an on-line enrichment and purification pretreatment method based on the device, high automation of pretreatment and low loss of sulfonamide antibiotics in the pretreatment process can be realized. The method can realize automatic online purification of complex substrate samples, has the characteristics of high analysis efficiency, less acetonitrile solvent consumption, good purification and impurity removal effects, good accuracy and reproducibility of measurement results and the like, and provides a more concise, convenient, sensitive and reliable analysis basis and an analysis method for the measurement of sulfonamides in aquatic products.
Description
Technical Field
The invention relates to the field of aquatic product quality monitoring, in particular to the field of monitoring of antibiotics in aquatic products, and more particularly relates to a pretreatment device, a pretreatment method and application for detecting aquatic product sulfonamide antibiotics.
Background
Aquatic products are the general name of aquatic animal and plant products and processed products thereof produced in marine and freshwater fishery. In the aquaculture process of aquatic products, prevention and control drugs such as antibiotics are needed for preventing and controlling aquaculture diseases. Sulfonamides are common control antibiotics for fish drugs. At present, the abuse phenomenon of antibiotics frequently occurs, and sulfonamide antibiotics can be accumulated and stored in aquatic products, which threatens the aquatic ecosystem.
In order to prevent abuse of antibiotics, in particular of sulphonamides, detection standards have been established by the national relevant sector. The existing detection method of the sulfonamide antibiotics mainly comprises the detection steps of leaching, purifying, concentrating, on-machine detection and the like. Wherein leaching, purifying and concentrating are pretreatment steps of detection, and the accuracy of the detection result is closely related to the pretreatment steps, so that improving the effectiveness of pretreatment is an important factor for improving the accuracy of the detection result.
The purification is used as a ring in the pretreatment process, namely the technical difficulty of sample pretreatment, and is also a key link for improving the effectiveness of pretreatment, and the quality of the purification effect is a key factor for influencing the accuracy of the final detection result.
In the prior art, the interference substances such as nonpolar lipids and the like in the leaching solution are removed by utilizing n-hexane, so that the interference degree of a matrix is reduced. The method has the defects that the main function of purifying the normal hexane is to carry out degreasing treatment on the sample, but the sample also contains partial substances such as polar phospholipids and/or glycoprotein, which can not be removed under the action of the normal hexane, and the polar phospholipids and/or glycoprotein remained in the sample can also generate matrix effect to influence the accuracy of the detection result. In addition, n-hexane is not ideal as a degreasing agent, and after the degreasing operation of n-hexane, the leaching solution still contains a small amount of nonpolar lipids, and the unremoved lipids also generate matrix effect, so that the accuracy of the detection result is affected.
Meanwhile, the protein, lipid and other substances remained in the sample not only can influence the accuracy of the detection result, but also can increase the detection difficulty of aquatic product antibiotics, and because the sample contains the protein and lipid substances, the pressure of the chromatographic column is easy to rise, the ion source needle of the mass spectrometer is blocked, and the taper hole, the pre-quaternary rod pollution and other adverse results are caused, so that the service life of the detection equipment is greatly shortened.
In order to reduce the residues of substances such as protein, lipid and the like and improve the purification effect, the existing purification operation is complex in steps, a large amount of acetonitrile and other toxic reagents are needed to be used, the consumption of solid phase extraction consumable materials in the purification process is large, the nitrogen blowing concentration is long, and the manual control difficulty is large. Not only is time and labor wasted, but also the target compound is not easy to be blown to dry excessively in the pretreatment process, so that the purification effect is poor, the repeatability of samples among batches is poor, and the accuracy of the quantitative result is unstable.
Therefore, for those skilled in the art, a new purification technical method is established, so that the purification effect is improved, the matrix effect in the sample detection process is reduced, and the improvement of the sample detection accuracy is the key point of improving the detection efficiency of the aquatic product sulfonamide antibiotics, and is also the research key point in the field.
Disclosure of Invention
One of the technical problems to be solved by the invention is to provide a pretreatment device for detecting aquatic product sulfonamide antibiotics, so that the on-line purification of complex substrate samples can be realized, the pretreatment flow of the samples is simplified, the pretreatment time of the samples is shortened, the pretreatment efficiency of the samples is improved, and the rapid and efficient completion of the pretreatment flow of detecting aquatic product sulfonamide antibiotics is realized.
Meanwhile, the second technical problem to be solved by the invention is to provide a pretreatment method based on the pretreatment device for detecting the aquatic product sulfonamide antibiotics, so that the consumption of organic reagents such as acetonitrile can be reduced, and the purification effect of pretreatment can be effectively improved.
Finally, the third technical problem to be solved by the invention is to further provide a method for detecting the sulfonamide antibiotics in the aquatic products based on the pretreatment device and the method for detecting the sulfonamide antibiotics in the aquatic products.
In order to solve the technical problems, the invention discloses a pretreatment device for detecting aquatic product sulfonamide antibiotics, which comprises four six-way valves, namely a first valve, a second valve, a third valve and a fourth valve, wherein each six-way valve is provided with six interfaces, and the connection state of adjacent interfaces in the valves is regulated by the rotation of an inner rotor of the six-way valve. The fifth port of the first valve is connected with the first port of the second valve through a pipeline, the fourth port of the first valve is connected with the second port of the fourth valve through a pipeline, and the third port of the third valve is connected with the third port of the fourth valve through a pipeline; the mixer is provided with two inlets and an outlet, the first inlet of the mixer is connected with the third port of the second valve through a pipeline, the second inlet of the mixer is connected with the sixth interface of the fourth valve through a pipeline, and the outlet of the mixer is connected with the first interface of the third valve through a pipeline; a quantitative ring is arranged between a third interface and a sixth interface of the first valve, a solid-phase extraction column is arranged between a second interface and the sixth interface of the second valve, and a solid-phase extraction column is also arranged between the second interface and the sixth interface of the third valve; the first port of the first valve is a sample inlet, the fifth port of the second valve and the fifth port of the third valve are waste discharge ports, the fourth port of the second valve is plugged by a solid plug, the first port of the fourth valve is an on-line solid phase extraction mobile phase liquid inlet, the fifth port of the fourth valve is a liquid phase pump mobile phase liquid inlet, and the fourth port of the fourth valve is a sample outlet.
Preferably, the on-line solid phase extraction column comprises a mixed cation exchange on-line solid phase extraction column and a hydrophilic and lipophilic on-line solid phase extraction column.
More preferably, a mixed cation exchange on-line solid phase extraction column (SPE 1) is arranged between the second interface and the sixth interface of the second valve, and a hydrophilic and lipophilic on-line solid phase extraction column (SPE 2) is also arranged between the second interface and the sixth interface of the third valve.
By utilizing the device, the repeated cyclic operation of the processes such as activation, sample introduction, solid phase extraction and the like can be realized by switching the valves of different six-way valves, and meanwhile, the processes are seamlessly switched through the switching of the six-way valves, so that the automation degree is high, the automatic online pretreatment of complex matrix samples is effectively realized, the loss of the samples in the pretreatment process is small, the purifying and impurity removing effects are good, and a foundation is provided for the follow-up efficient and accurate detection.
For example, six different connection states are further disclosed in the present invention, thereby forming six different pipe connection structures:
state one: the valve I is communicated in a mode of 1-6,2-3,4-5, and the valve II and the valve IV are communicated in a mode of 1-2,3-4, 5-6;
state two: the first valve is switched to a communication mode of 1-2,3-4,5-6, and the second valve and the fourth valve maintain the communication mode of 1-2,3-4, 5-6;
state three: the valve I is communicated in a mode of 1-6,2-3,4-5, and the valve II and the valve IV are communicated in a mode of 1-2,3-4, 5-6;
state four: the states of the first valve and the fourth valve are switched to be in a communication mode of 1-2,3-4 and 5-6, and the second valve and the third valve maintain the communication mode of 1-2,3-4 and 5-6;
state five: the state of the first valve and the state of the second valve are switched into a communication mode of 1-6,2-3,4-5, and the third valve and the fourth valve maintain the communication mode of 1-2,3-4, 5-6;
state six: the state of the first valve and the state of the third valve are switched to be in a communication mode of 1-6,2-3,4-5, and the second valve and the fourth valve maintain the communication mode of 1-2,3-4, 5-6;
wherein "1", "2", "3", "4", "5", "6" respectively represent a first interface, a second interface, a third interface, a fourth interface, a fifth interface, and a sixth interface.
Meanwhile, the invention also discloses a pretreatment method based on a pretreatment device for detecting the aquatic product sulfonamide antibiotics, which comprises the following steps:
s1: transferring the pretreated sample to be detected into a quantitative ring through an on-line solid phase extraction system sample inlet, then enriching sulfonamide antibiotics into a mixed cation exchange on-line solid phase extraction column (SPE 1) along with a mobile phase, and removing impurities such as protein, phospholipid and the like in the on-line solid phase extraction column through leaching and waste discharge;
s2: switching a six-way valve, reversely eluting the mixed cation exchange on-line solid phase extraction column (SPE 1) by an alkaline mobile phase, and converging a sample to be detected into a mixer along with alkaline eluent;
s3: the other path of acidic low organic phase mobile phase is converged into the mixer while SPE1 is reversely eluted; after neutralization by acid and alkali, the to-be-detected substance flows into a hydrophilic and lipophilic online solid phase extraction column (SPE 2) along with the uniformly mixed low-organic phase mobile phase;
s4: and (3) reversely eluting SPE2 by using a liquid phase gradient to obtain a purified sample to be detected.
The pretreatment method specifically comprises the following steps of:
(1) the valve I is communicated in a mode of 1-6,2-3,4-5, and the valve II and the valve IV are communicated in a mode of 1-2,3-4, 5-6; and the mobile phase I' are sequentially introduced through an online solid-phase extraction mobile phase liquid inlet; then injecting a sample solution to be treated through a sample inlet;
(2) the first valve is switched to a communication mode of 1-2,3-4,5-6, and the second valve and the fourth valve maintain the communication mode of 1-2,3-4, 5-6; injecting the mobile phase II into the device from the sample inlet;
(3) the valve I is communicated in a mode of 1-6,2-3,4-5, and the valve II and the valve IV are communicated in a mode of 1-2,3-4, 5-6; and the third mobile phase is introduced through an on-line solid phase extraction mobile phase liquid inlet;
(4) the states of the first valve and the fourth valve are switched to be in a communication mode of 1-2,3-4 and 5-6, and the second valve and the third valve maintain the communication mode of 1-2,3-4 and 5-6; and the mobile phase IV' are respectively and sequentially introduced through an on-line solid phase extraction mobile phase liquid inlet;
(5) the state of the first valve and the state of the second valve are switched into a communication mode of 1-6,2-3,4-5, and the third valve and the fourth valve maintain the communication mode of 1-2,3-4, 5-6; introducing the mobile phase five into the mobile phase inlet through the online solid-phase extraction mobile phase liquid inlet, introducing the mobile phase five 'into the mobile phase inlet through the liquid phase pump after introducing the mobile phase five into the mobile phase mixer for a period of time, and mixing the mobile phase five and the mobile phase five' in a mixer according to the proportion;
(6) the state of the first valve and the state of the third valve are switched to be in a communication mode of 1-6,2-3,4-5, and the second valve and the fourth valve maintain the communication mode of 1-2,3-4, 5-6; introducing a mobile phase six through a mobile phase liquid inlet of the liquid phase pump;
wherein "1", "2", "3", "4", "5", "6" respectively represent a first interface, a second interface, a third interface, a fourth interface, a fifth interface, and a sixth interface.
Preferably, the mobile phase I is methanol, and the mobile phase I' is pure water. And it is further preferred that the flow rates of mobile phase one and mobile phase one' be 1mL/min. More preferably, the mobile phase A is 1.5mL of methanol, and the mobile phase A' is 1.0mL of pure water.
Preferably, the second mobile phase is pure water. And it is further preferred that the flow rate of mobile phase two is 1.0mL/min.
Preferably, the mobile phase III is a methanol-water mixed solution, wherein the volume ratio of methanol to water is 5:95. And a further preferred flow rate of mobile phase three is 1.0mL/min.
Preferably, the mobile phase four is methanol, the mobile phase four 'is pure water, and further preferably, the flow rates of the mobile phase four and the mobile phase four' are 1.0mL/min. More preferably, the mobile phase IV is 1.5mL of methanol, and the mobile phase IV' is 1.0mL of pure water.
Preferably, the mobile phase five is a mixed solution of methanol-acetonitrile-ammonia water-water, wherein the methanol: acetonitrile: ammonia water: the volume ratio of water is 10:10:1:79; the mobile phase five' is formic acid-water solution, wherein the volume ratio of formic acid to water is 1:1000. More preferably, the flow rate of the mobile phase five is 0.15ml/min, and the flow rate of the mobile phase five' is 0.45ml/min.
It is further preferred that the mixing ratio of the mobile phase five to the mobile phase five' is 1:3 (volume ratio).
Preferably, the mobile phase six is a formic acid-water solution, wherein the volume ratio of formic acid to water is 1:1000, the gradient elution procedure is as follows: wherein the volume ratio of formic acid to water is 1:1000; the gradient elution procedure was as follows: 0.1% (volume fraction) formic acid/water solution (a): acetonitrile (B) =95:5, 0.4ml/min starting reverse elution SPE2, hold 0.5min, rise to 25% (B) at 1.0min, rise to 95% (B) immediately within 3.5min, hold 6.1min, drop 5% (B) at 6.5min, end at 8.0 min.
More preferably, the method further comprises (7) the system regeneration cycle is performed by setting reasonable online transfer time and adjusting online flow path composition, so that the original state is switched after the online transfer is completed, and the regeneration activation state is entered, so that the sample injection and solid phase extraction of the next cycle are ensured.
Finally, the invention also discloses a detection device of the sulfonamide antibiotics in the aquatic products, which comprises a pretreatment device based on detection of the aquatic product sulfonamide antibiotics, and the detection device comprises a pretreatment device based on detection of the aquatic product sulfonamide antibiotics, wherein a sample outlet is connected to a liquid chromatograph mass spectrometer through a pipeline.
Meanwhile, the invention also discloses a method for detecting the sulfonamide antibiotics in the aquatic products based on the pretreatment method of the pretreatment device for detecting the aquatic product sulfonamide antibiotics, and the purified sample enters a liquid chromatograph mass spectrometer along with a mobile phase six through a pipeline for analysis and detection.
The invention provides an online solid-phase extraction elution pretreatment device for online enrichment and purification of sulfonamide antibiotics, further establishes an online enrichment and purification pretreatment method based on the device, and further combines the device with high performance liquid chromatography mass spectrometry, so that the device can be used for simultaneously measuring sulfonamide antibiotics in aquatic products. The method disclosed by the invention has high automation degree and less loss of the sulfonamide antibiotics in the pretreatment process. The method can realize automatic online purification of complex substrate samples, has the characteristics of high analysis efficiency, less acetonitrile solvent consumption, good purification and impurity removal effects, good accuracy and reproducibility of measurement results and the like, and provides a more concise, convenient, sensitive and reliable analysis basis and an analysis method for the measurement of sulfonamides in aquatic products.
Drawings
FIG. 1 is a schematic diagram of a different state flow.
FIG. 2 is a liquid chromatography mass spectrometry spectrum of a conventional method (n-hexane liquid-liquid extraction purification).
FIG. 3 is a liquid chromatograph mass spectrum of the method.
FIGS. 4-21 show mass spectra of 18 sulfonamides.
FIG. 22 is a schematic diagram of 18 sulfonamide standard curves detected by the method, wherein the abscissa represents the concentration (ng/L) of 18 sulfonamide, and the ordinate represents the area of 18 sulfonamide peaks.
Detailed Description
For a better understanding of the present invention, we will further describe the present invention with reference to specific examples and drawings, but it should be noted that the practice of the present invention is not limited thereto.
Example 1
Firstly, assembling a pretreatment device for detecting aquatic product sulfonamide antibiotics in the following manner, wherein the pretreatment device comprises four six-way valves, namely a first valve 11, a second valve 22, a third valve 33 and a fourth valve 44, each six-way valve is provided with six interfaces, a fifth interface 11 (5) of the first valve 11 is connected with a first interface 22 (1) of the second valve 22 through a pipeline, a fourth interface 11 (4) of the first valve 11 is connected with a second interface 44 (2) of the fourth valve 44 through a pipeline, and a third interface 33 (3) of the third valve 33 is connected with a third interface 44 (3) of the fourth valve 44 through a pipeline; the mixer 55 is provided with two inlets and one outlet, the first inlet 55 (1) of the mixer 55 is connected with the third port 22 (3) of the second valve 22 through a pipeline, the second inlet 55-2 of the mixer is connected with the sixth port 44 (6) of the fourth valve 44 through a pipeline, and the outlet 55 (3) of the mixer 55 is connected with the first port 33 (1) of the third valve 33 through a pipeline; a quantitative ring is arranged between the third port 11 (3) and the sixth port 11 (6) of the first valve 11, a solid-phase extraction column 66 is arranged between the second port 22 (2) and the sixth port 22 (6) of the second valve 22, and a solid-phase extraction column 77 is also arranged between the second port 33 (2) and the sixth port 33 (6) of the third valve 33; the first port 11 (1) of the first valve 11 is a sample inlet, the fifth port 22 (5) of the second valve 22 and the fifth port 33 (5) of the third valve 33 are waste discharge ports, the first port 44 (1) of the fourth valve 44 is an on-line solid phase extraction mobile phase liquid inlet, the fifth port 44 (5) of the fourth valve 44 is a liquid phase pump mobile phase liquid inlet, and the fourth port 44 (4) of the fourth valve 44 is a sample outlet.
Preferably, the solid phase extraction column comprises a mixed cation exchange on-line solid phase extraction column and a hydrophilic lipophilic on-line solid phase extraction column.
More preferably, a mixed cation exchange on-line solid phase extraction column (SPE 1) is arranged between the second port 22-1 and the sixth port 22-6 of the second valve 22, and a hydrophilic and lipophilic on-line solid phase extraction column (SPE 2) is also arranged between the second port and the sixth port of the third valve.
Then, the pretreatment of the sample was performed according to the following steps:
(1) the valve I is communicated in a mode of 1-6,2-3,4-5, and the valve II and the valve IV are communicated in a mode of 1-2,3-4, 5-6; and the mobile phase I' are sequentially introduced through an online solid-phase extraction mobile phase liquid inlet; then injecting a sample solution to be treated through a sample inlet, as shown in fig. 1;
(2) the first valve is switched to a communication mode of 1-2,3-4,5-6, and the second valve and the fourth valve maintain the communication mode of 1-2,3-4, 5-6; injecting the second mobile phase into the device from the sample inlet as shown in fig. 1;
(3) the valve I is communicated in a mode of 1-6,2-3,4-5, and the valve II and the valve IV are communicated in a mode of 1-2,3-4, 5-6; and the third mobile phase is introduced through an on-line solid phase extraction mobile phase liquid inlet, as shown in figure 3;
(4) the states of the first valve and the fourth valve are switched to be in a communication mode of 1-2,3-4 and 5-6, and the second valve and the third valve maintain the communication mode of 1-2,3-4 and 5-6; and the mobile phase IV' are respectively introduced into the online solid phase extraction mobile phase liquid inlet in sequence as shown in figure 1;
(5) the state of the first valve and the state of the second valve are switched into a communication mode of 1-6,2-3,4-5, and the third valve and the fourth valve maintain the communication mode of 1-2,3-4, 5-6; and the mobile phase five is introduced into the mobile phase five through the on-line solid phase extraction mobile phase liquid inlet, after a period of time, the mobile phase five ' is introduced into the mobile phase five ' through the mobile phase liquid inlet of the liquid phase pump, and the mobile phase five ' are mixed in a mixer according to the proportion, as shown in fig. 1;
(6) the state of the first valve and the state of the third valve are switched to be in a communication mode of 1-6,2-3,4-5, and the second valve and the fourth valve maintain the communication mode of 1-2,3-4, 5-6; introducing a mobile phase six through a mobile phase liquid inlet of the liquid phase pump;
wherein "1", "2", "3", "4", "5", "6" respectively represent a first interface, a second interface, a third interface, a fourth interface, a fifth interface, and a sixth interface, as shown in fig. 1;
preferably, the mobile phase I is methanol, and the mobile phase I' is pure water. And it is further preferred that the flow rates of mobile phase one and mobile phase one' be 1mL/min. More preferably, the mobile phase I is 1.5mL of methanol, and the mobile phase I' is 1mL of pure water.
Preferably, the second mobile phase is pure water. And it is further preferred that the flow rate of mobile phase two is 1mL/min.
Preferably, the mobile phase III is a methanol-water mixed solution, wherein the volume ratio of methanol to water is 5:95. And a further preferred flow rate of mobile phase three is 1mL/min.
Preferably, the mobile phase four is methanol, the mobile phase four 'is pure water, and further preferably, the flow rates of the mobile phase four and the mobile phase four' are 1mL/min. More preferably, the mobile phase IV is 1.5mL of methanol, and the mobile phase IV' is 1mL of pure water.
Preferably, the mobile phase five is a mixed solution of methanol-acetonitrile-ammonia water-water, wherein the methanol: acetonitrile: ammonia water: the volume ratio of water is 10:10:1:79; the mobile phase five' is formic acid-water solution, wherein the volume ratio of formic acid to water is 1:1000. More preferably, the flow rate of the mobile phase five is 0.15ml/min, and the flow rate of the mobile phase five' is 0.45ml/min.
It is further preferred that the mixing ratio of the mobile phase five to the mobile phase five' is 1:3 (volume ratio).
Preferably, the mobile phase six is a formic acid-water solution, wherein the volume ratio gradient of formic acid to water is 1:1000, and the liquid phase mobile phase is 0.1% (volume fraction) of formic acid/water solution (A): acetonitrile (B) =95:5, 0.4ml/min starting reverse elution SPE2, hold 0.5min, rise to 25% (B) at 1.0min, rise to 95% (B) immediately within 3.5min, hold 6.1min, drop 5% (B) at 6.5min, end at 8.0 min.
More preferably, the method further comprises (7) the system regeneration cycle is performed by setting reasonable online transfer time and adjusting online flow path composition, so that the original state is switched after the online transfer is completed, and the regeneration activation state is entered, so that the sample injection and solid phase extraction of the next cycle are ensured.
Further, the following means is adopted as an optimal technical scheme:
(1) the valve 1 is communicated in a mode of 1-6,2-3,4-5, and the valve 2-4 is communicated in a mode of 1-2,3-4, 5-6. The flow rate of the mobile phase of the online solid phase extraction flow path is set to be 1mL/min, and 1.5mL of methanol and 1mL of pure water are used for activating and balancing SPE1. At this time, the automatic sampler moves 1mL of the liquid to be measured, injects the liquid to be measured into the No. 1 position of the No. 1 valve, and stores the liquid to be measured in the 1mL quantitative ring of the No. 6-3 position. See fig. 1.
(2) The valve 1 is switched to a communication mode of 1-2,3-4,5-6, and the valve 2-4 maintains the communication mode of 1-2,3-4, 5-6. The flow rate of 1mL/min is set in the online solid-phase extraction flow path, and pure water pushes the liquid to be detected in the quantitative ring in the valve No. 1 into SPE1. See fig. 1.
(3) Valve status 1-4 is the same as (1) SPE1 activation balance. The flow rate of the online solid-phase extraction flow path was set at 1mL/min, and 2.0mL of an aqueous methanol solution (methanol: water=5:95) was injected, see FIG. 1.
(4) The state of the valve No. 1 and the state of the valve No. 4 are switched into a communication mode of 1-2,3-4 and 5-6, and the valve No. 2 and the valve No. 3 maintain the communication mode of 1-2,3-4 and 5-6. The flow rate of 1mL/min was set for the online solid phase extraction flow path, followed by 1.5mL methanol and 1mL pure water, activating balance SPE2. See fig. 1.
(5) The state of the valve No. 1 and the state of the valve No. 2 are switched into a communication mode of 1-6,2-3,4-5, and the valve No. 3 and the valve No. 4 maintain the communication mode of 1-2,3-4, 5-6. Setting a flow rate of 0.15mL/min in an online solid-phase extraction flow path, wherein a mobile phase is methanol acetonitrile ammonia water mixed solution (methanol: acetonitrile: ammonia water: water=10:10:1:79), and reversely eluting SPE (valve No. 2, 6-2 position); the flow rate of the liquid phase pump flow path was set to 0.45mL/min, and the mobile phase was formic acid aqueous solution (formic acid: water=1:1000). The on-line solid-phase extraction flow path and the liquid-phase pump flow path are combined with the mixer according to the ratio of 1:3, so that the organic phase proportion of the mobile phase carrying the object to be detected is reduced to below 5%. Mixing the mobile phases, entering the No. 1 position of the No. 3 valve, and enriching the No. 2-6 positions on line through an SPE2HLB solid phase extraction column. See fig. 1.
(6) The state of the valve 1 and the state of the valve 3 are switched into a communication mode of 1-6,2-3,4-5, and the valve 2 and the valve 4 maintain the communication mode of 1-2,3-4, 5-6.
And (3) carrying out gradient elution on the mobile phase of the liquid phase pump, and stopping the online solid phase extraction flow path. Starting a liquid phase pump, and enabling the liquid phase pump to perform gradient reverse elution SPE2HLB on-line solid phase extraction column, and enabling the liquid phase pump to enter a liquid chromatograph mass spectrometer along with a flowing phase of a liquid phase pump flow path for detection and analysis. See fig. 1.
Comparative example 1
(1) Instrument and reagent
UPLC-5500Trap high performance liquid chromatography tandem quadrupole mass spectrometer (AB, USA), ACE C18-PFP column (100 mm.3.0 mm,2.1 μm, ACE, UK), Q-700DE ultrasonic cleaner (Xinzhi, ningbo), 5810R centrifuge (Eppendorf Centrifuge, germany), 24-position nitrogen blower (Hanno, shanghai), vortex Cenie 2 Vortex mixer (Scientifie Industries, USA).
(2) Chromatographic and mass spectral conditions
Mobile phase 0.1% (V: V, hereinafter the same) formic acid/water solution (A), acetonitrile (B); the flow rate is 0.4mL/min; the initial mobile phase ratio was 5% (B), 1min was maintained, 1.1min was increased to 15% (B), 9.5min was increased to 75% (B), 9.6min was increased to 95% (B), 11.5min was maintained, 11.6min was decreased to 5% (B), and 13.5min was completed. Sample injection amount is 10 mu L, column temperature is 40 ℃, electrospray ionization source (ESI), atomizer pressure is 60psi, capillary voltage is 4000V, drying gas temperature is 400 ℃, flow rate is 9L/min, and detection mode is multi-reaction monitoring (MRM) positive ion mode.
(3) Sample pretreatment
After the aquatic products are crushed, 2g of the aquatic products are taken, 10mL of acetonitrile is added, ultrasonic leaching is carried out for 15min, the leaching solution is transferred into a nitrogen blowing pipe after centrifugation, and soft nitrogen is blown to near dryness. Adding 0.1moL/L hydrochloric acid aqueous solution, vortex redissolving, adding n-hexane liquid-liquid extraction, vortex discarding, repeating for 2-3 times until the leaching solution is clear.
Example 2
The samples treated in example 1 and comparative example 1 were tested for sulfonamide antibiotic drugs, respectively.
In this example, we detected a total of 18 common sulfonamide drugs, see in table 1:
table 1:
standard solution preparation
1.0ng/L, 5.0ng/L, 10.0ng/L, 20.0ng/L, 50.0ng/L of standard solution is prepared by using 0.1moL/L of hydrochloric acid aqueous solution, 1mL of each concentration is sampled, a standard curve is drawn by using the peak area as an abscissa and the concentration as an ordinate, and the detection Limit (LOD) is calculated by using a 3-time signal to noise ratio (S/N) (see Table 2). The linear correlation coefficient is not lower than 0.998.
The linear curve is shown in fig. 22, wherein,
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the experimental results are shown in fig. 2 and 3. As can be seen from the comparison of fig. 2 and fig. 3, the liquid chromatograph obtained by the method has few impurity peaks, less noise, good peak shape, high sample purity and clear detection result data.
Example 3
Determination of sample recovery of the methods disclosed herein
One water sample was prepared and 7 portions were measured, 10mL each. Reference numerals 0-6. Sample No. 0 was set as blank, and 5.0ng/L standard was added to each sample No. 1-3; no. 3-6, 50.0ng/L standard was added to each sample. Sample recovery experiments of low and high concentrations are carried out, and the sample recovery is calculated by subtracting the ratio of the concentration of the analyte in the blank sample to the concentration of the added analyte from the concentration of the analyte in the standard added sample, and the results are shown in Table 2.
Determination of sample precision by the methods disclosed herein
One water sample was prepared and 8 portions were measured, 10mL each. Sample numbers 0-7 and 0 are set as blank, and sample numbers 1-7 are added with 10.0ng/L standard substance; the relative standard deviation of the samples was calculated from the concentration of the analyte in the standard addition sample subtracted from the concentration of the analyte in the blank sample, and the results are shown in table 2.
Table 2:
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what has been described above is a specific embodiment of the present invention. It should be noted that modifications and adaptations to the invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.
Claims (10)
1. The utility model provides an aquatic products sulfonamide antibiotic detection device, includes high performance liquid chromatography detection device, its characterized in that, aquatic products sulfonamide antibiotic detection device is still including the preprocessing device who is used for aquatic products sulfonamide antibiotic to detect, the preprocessing device who is used for aquatic products sulfonamide antibiotic to detect includes:
the four six-way valves are respectively a first valve, a second valve, a third valve and a fourth valve, wherein each six-way valve is provided with six interfaces, the connection state of the adjacent interfaces in the valve is adjusted through the rotation of the inner rotor of the six-way valve, the fifth interface of the first valve is connected with the first interface of the second valve through a liquid phase pipeline, the fourth interface of the first valve is connected with the second interface of the fourth valve through a liquid phase pipeline, and the third interface of the third valve is connected with the third interface of the fourth valve through a pipeline; the mixer is provided with two inlets and an outlet, the first inlet of the mixer is connected with the third port of the second valve through a pipeline, the second inlet of the mixer is connected with the sixth interface of the fourth valve through a pipeline, and the outlet of the mixer is connected with the first interface of the third valve through a pipeline; a quantitative ring is arranged between a third interface and a sixth interface of the first valve, an online solid-phase extraction column is arranged between a second interface and the sixth interface of the second valve, and an online solid-phase extraction column is also arranged between the second interface and the sixth interface of the third valve; the first port of the first valve is a sample inlet, the fifth port of the second valve and the fifth port of the third valve are waste discharge ports, the fourth port of the second valve is plugged by a solid plug, the first port of the fourth valve is an on-line solid phase extraction mobile phase liquid inlet, the fifth port of the fourth valve is a liquid phase pump mobile phase liquid inlet, and the fourth port of the fourth valve is a sample outlet;
the on-line solid phase extraction column comprises a mixed cation exchange on-line solid phase extraction column and a hydrophilic lipophilic on-line solid phase extraction column; a mixed cation exchange on-line solid phase extraction column is arranged between the second port and the sixth port of the second valve and is marked as SPE1, and a hydrophilic and lipophilic on-line solid phase extraction column is also arranged between the second port and the sixth port of the third valve and is marked as SPE2.
2. A pretreatment method for detecting aquatic product sulfonamide antibiotics is characterized by comprising the following steps of: the pretreatment method for detecting the aquatic product sulfonamide antibiotics is based on the pretreatment device for detecting the aquatic product sulfonamide antibiotics according to claim 1, and comprises the following steps:
(1) the valve I is communicated in a mode of 1-6,2-3,4-5, and the valve II and the valve IV are communicated in a mode of 1-2,3-4, 5-6; and the mobile phase I' are sequentially introduced through an online solid-phase extraction mobile phase liquid inlet; then injecting a sample solution to be treated through a sample inlet;
(2) the first valve is switched to a communication mode of 1-2,3-4,5-6, and the second valve and the fourth valve maintain the communication mode of 1-2,3-4, 5-6; injecting the mobile phase II into the device from the sample inlet;
(3) the valve I is communicated in a mode of 1-6,2-3,4-5, and the valve II and the valve IV are communicated in a mode of 1-2,3-4, 5-6; and the third mobile phase is introduced through an on-line solid phase extraction mobile phase liquid inlet;
(4) the states of the first valve and the fourth valve are switched to be in a communication mode of 1-2,3-4 and 5-6, and the second valve and the third valve maintain the communication mode of 1-2,3-4 and 5-6; and the mobile phase IV' are respectively and sequentially introduced through an on-line solid phase extraction mobile phase liquid inlet;
(5) the state of the first valve and the state of the second valve are switched into a communication mode of 1-6,2-3,4-5, and the third valve and the fourth valve maintain the communication mode of 1-2,3-4, 5-6; introducing the mobile phase five into the mobile phase inlet through the online solid-phase extraction mobile phase liquid inlet, introducing the mobile phase five 'into the mobile phase inlet through the liquid phase pump after introducing the mobile phase five into the mobile phase mixer for a period of time, and mixing the mobile phase five and the mobile phase five' in a mixer according to the proportion;
(6) the state of the first valve and the state of the third valve are switched to be in a communication mode of 1-6,2-3,4-5, and the second valve and the fourth valve maintain the communication mode of 1-2,3-4, 5-6; introducing a mobile phase six through a mobile phase liquid inlet of the liquid phase pump;
wherein "1", "2", "3", "4", "5", "6" respectively represent a first interface, a second interface, a third interface, a fourth interface, a fifth interface, and a sixth interface;
the mobile phase I is methanol, and the mobile phase I' is pure water; the first mobile phase is 1.5mL of methanol, and the first mobile phase' is 1.0mL of pure water;
the second mobile phase is pure water;
the mobile phase III is a methanol-water mixed solution, wherein the volume ratio of methanol to water is 5:95;
the mobile phase IV is methanol, and the mobile phase IV' is pure water; the mobile phase IV is 1.5mL of methanol, and the mobile phase IV' is 1mL of pure water;
the mobile phase five is a mixed solution of methanol-acetonitrile-ammonia water-water, wherein the methanol is as follows: acetonitrile: ammonia water: the volume ratio of water is 10:10:1:79; the mobile phase five' is formic acid-water solution, wherein the volume ratio of formic acid to water is 1:1000; the mixing volume ratio of the mobile phase five to the mobile phase five' is 1:3;
the mobile phase six of the on-line solid phase extraction is formic acid-water solution, wherein the volume ratio of formic acid to water is 1:1000.
3. The pretreatment method for detecting aquatic product sulfonamide antibiotics according to claim 2, wherein: the flow rates of mobile phase one and mobile phase one' were 1.0mL/min.
4. The pretreatment method for detecting aquatic product sulfonamide antibiotics according to claim 2, wherein: the flow rate of mobile phase two was 1.0mL/min.
5. The pretreatment method for detecting aquatic product sulfonamide antibiotics according to claim 2, wherein: the flow rate of mobile phase three was 1.0mL/min.
6. The pretreatment method for detecting aquatic product sulfonamide antibiotics according to claim 2, wherein: the flow rate of mobile phase four and mobile phase four' was 1mL/min.
7. The pretreatment method for detecting aquatic product sulfonamide antibiotics according to claim 2, wherein: the flow rate of the mobile phase five is 0.15ml/min, and the flow rate of the mobile phase five' is 0.45ml/min.
8. The pretreatment method for detecting aquatic product sulfonamide antibiotics according to claim 2, wherein: the gradient elution procedure was as follows: formic acid/water solution with volume fraction of 0.1% is used as phase A, acetonitrile is used as phase B, and phase A: b=95:5, 0.4mL/min starts the hydrophilic lipophilic online solid phase extraction column SPE2, starts the reverse elution, keeps 0.5min, increases the B phase to 25% at 1.0min, increases the B phase to 95% immediately after 3.5min, keeps to 6.1min, decreases the B phase to 5% at 6.5min, and ends at 8.0 min.
9. The pretreatment method for detecting aquatic product sulfonamide antibiotics according to claim 2, wherein: and (7) the regeneration circulation of the system is realized by setting reasonable online transfer time and adjusting online flow path composition, so that the original state is switched after the online transfer is finished, the regeneration activation state is entered, and the sample injection and solid phase extraction of the next period are ensured to be circularly carried out.
10. A method for detecting aquatic product sulfonamide antibiotics, which is characterized by comprising the pretreatment method for detecting aquatic product sulfonamide antibiotics according to any one of claims 2-9.
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