CN109060997A - A kind of detection method and application of sulfa antibiotics - Google Patents

A kind of detection method and application of sulfa antibiotics Download PDF

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Publication number
CN109060997A
CN109060997A CN201811060807.9A CN201811060807A CN109060997A CN 109060997 A CN109060997 A CN 109060997A CN 201811060807 A CN201811060807 A CN 201811060807A CN 109060997 A CN109060997 A CN 109060997A
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sample
solution
water
filter membrane
detection method
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于志浩
张新波
温海涛
祁丽
张丹
李超灿
刘阳
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Tianjin Chengjian University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G01N30/724—Nebulising, aerosol formation or ionisation
    • G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography

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Abstract

The invention discloses a kind of detection method of sulfa antibiotics and applications, the detection includes the steps that sample pre-treatments and sample analysis, the sample pre-treatments at least include the following steps: (1) water sampling filter membrane cleans, adjusted the pH of water sample after film, and sodium ethylene diamine tetracetate is added, internal standard compound is added, solution A is obtained；(2) Solid Phase Extraction that solution A is carried out using HLB solid-phase extraction column, obtains solution B, after drying, constant volume, filtering solution B, obtains sample to be tested and be placed in sample injection bottle to save.The method of the present invention significantly reduces the influence of chelation, biotransformation and hydrolysis to sulfa antibiotics content detection in municipal wastewater, in the case where guaranteeing the higher situation of the rate of recovery, reduces the time required for pre-treatment, improves detection efficiency.And the waste of methanol and filter membrane is avoided, reduce testing cost.The method of the present invention good test effect and easy to operate, as a result accurately the detection of Successful utilization sulfa antibiotics content in municipal wastewater improves detection efficiency.

Description

A kind of detection method and application of sulfa antibiotics
Technical field
The invention belongs to water pollutant detection technique field, in particular to a kind of detection method of sulfa antibiotics and Using more particularly to a kind of municipal wastewater in sulfa antibiotics detection method and application.
Background technique
Antibiotic is one of most widely used drug, is mainly used for preventing and treating bacterium and fungal disease.In recent years Come, water pollution caused by abuse of antibiotics causes concern in American-European some countries, and sulfa antibiotics are a kind of typical Widely used antibiotics, sulfa antibiotics are often used as antibacterial agent and treatment bacterium infection.
China is the production of antibiotic and uses big country, annual to produce 210,000 tons of antibiotic raw material, wherein domestic use 18 Ten thousand tons, annual consumption is 10 times of the U.S. per capita.Antibiotic will pass through body discharges, the medical treatment of the sewage that pharmaceutical factory is discharged and hospital Waste water enters in municipal water body.In recent years, the event of antibiotic pollution is frequently reported in news and network in water body, but I The regulation of the temporarily anhydrous middle antibiotic limitation of state.The antibacterial and antibacterial characteristics that antibiotic medicine itself has persistently exist in environment Antibiotic not only some environmental microorganisms are killed in the property of can choose suppression, but also can induce the production of some drug resistance floras or resistant gene It is raw, microorganism ecological environment in water environment is influenced, and easily lead to the wide-scale distribution of resistant gene, causes its special ecology poison Effect is managed, to generate to human health and aquatic ecosystem balance potentially hazardous.Water is the indispensable necessity of human body Therefore resource is monitored antibiotic in water extremely urgent.
The analytical technology means of existing antibiotic mainly have (super) high performance liquid chromatography series connection ultraviolet corona detector, efficiently Liquid chromatography tandem detection technique of fluorescence, enzyme-linked immunization, capillary electrophoresis, gas-chromatography tandem mass spectrometry, liquid chromatogram Method and liquid chromatography tandem mass spectrometry etc..Based on liquid chromatography tandem mass spectrometry by the high separability energy of chromatography and mass spectrographic Gao Jian Other feature combines, with analyst coverage is wider, sensitivity is higher, detection limit is low, qualitative framework is more reliable, it is more to detect simultaneously The advantages that component pollutant, has great importance to qualitative and quantitative analysis micro and trace components in complex compound, It is most widely used in Analysis of antibiotic.However, current detection method handles generally existing process complexity, and it is difficult to do To the rate of recovery is guaranteed, low efficiency, accuracy rate are poor, stability is lower.
Summary of the invention
The object of the present invention is to provide a kind of detection method of sulfa antibiotics and application, detection method of the invention is aobvious Work reduces chelation, biotransformation and hydrolysis to the shadow of sulfa antibiotics content detection in municipal wastewater It rings, as a result accurately, detector efficiency is high.
For this purpose, technical solution of the present invention is as follows:
In a first aspect, the present invention provides a kind of detection method of sulfa antibiotics, the detection includes sample pre-treatments And the step of sample analysis, the sample pre-treatments at least include the following steps:
(1) water sampling filter membrane cleans, and adjusted the pH of water sample after film, and sodium ethylene diamine tetracetate is added, and added Internal standard compound obtains solution A；
(2) Solid Phase Extraction that solution A is carried out using HLB solid-phase extraction column, obtains solution B, drying, constant volume, filtering solution B Afterwards, it obtains sample to be tested and is placed in sample injection bottle to save.
Internal standard compound is to be added by a known quality and in the sample without containing the pure material of impurity to testing sample solution In, using the amount of this pure material as standard, the content of comparative determination target to be measured；In the present invention, internal standard compound and object to be measured Physical property matter is similar, such as can choose13C3- caffeine and/or13C3-trimethylxanthine。
Preferably, filter membrane described in step (1) is glass fiber filter, to remove impurity in water sample.Glass fibre material Filter membrane belong to may only filtering solution filter membrane, advantage of lower cost；Nylon leaching film, which belongs to, can both filter organic solution Again can be with the filter membrane of filtering solution, but higher cost.Therefore glass fiber filter is chosen in the present invention as step (1) In filter membrane, preferred nylon leaching film in step (2).In addition, aperture is the important parameter of filter membrane selection, it is related to two aspects and asks Topic: 1. either with or without the cleared particulate matter that will cause blocking；2. particulate matter has been filtered, it can be faster on the time.In the present invention, If aperture is too small, when filtering, waste time and filter membrane；Aperture is too big, and when filtering, falling flat (i.e. cannot be effective Remove impurity).
Preferably, the aperture of the filter membrane is 0.1-0.8 μm, such as can be 0.1 μm, 0.15 μm, 0.22 μm, 0.25 μ m、0.3μm、0.35μm、0.4μm、0.45μm、0.5μm、0.55μm、0.6μm、0.65μm、0.65μm、0.7μm、0.75μm、0.8 μm and the numberical range in all point values, since the limitation of length is not listed, preferably 0.4-0.6 μm；
Preferably, adjusting pH described in step (1) be adjusted to pH to 2.0-5.0, such as can be 2.0,2.1,2.2, 2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、 4.2,4.3,4.4,4.5,4.6,4.7,4.8,4.9,5.0 and the numberical range in all point values, due to the limitation of length It is not listed, preferably 3.5-4.0；
Preferably, pH is adjusted with strong acid and/or highly basic, preferably adjusts pH with HCl and/or NaOH；
Preferably, the concentration of sodium ethylene diamine tetracetate described in step (1) is 2-8g/L, such as can be 2g/L, 2.5g/ L, 3g/L, 3.5g/L, 4g/L, 4.5g/L, 5g/L, 5.5g/L, 6g/L, 6.5g/L, 7g/L, 7.5g/L, 8g/L and the numerical value All point values in range, since the limitation of length is not listed, preferably 4-5g/L；
Preferably, the additional amount of the sodium ethylene diamine tetracetate be 5-20mL, such as can be 5mL, 6mL, 7mL, 8mL, 9mL、10mL、11mL、11.2mL、11.4mL、11.6mL、11.8mL、12mL、13mL、14mL、15mL、16mL、17mL、18mL、 All point values in 19mL, 20mL and the numberical range, since the limitation of length is not listed, preferably 10mL.
Wherein, the purpose for adjusting pH and sodium ethylene diamine tetracetate being added is to eliminate the influence of antibiotic chelating metal ion. The present invention passes through a large number of experiments, it is determined that suitable pH value, the additional amount of sodium ethylene diamine tetracetate and concentration, if it exceeds this hair The numerical value of bright restriction, the then effect that Ao closes can decline to a great extent.
Preferably, described in step (2) using HLB solid-phase extraction column carry out Solid Phase Extraction specifically includes the following steps:
Solution A is eluted after draining in air with methanol by extraction column later with ultrapure Water warfare first, is obtained molten Liquid B；
Preferably, specifically includes the following steps:
By solution A by HLB solid-phase extraction column, the column flow rate of crossing of solution A is that 2-3mL/min (such as can be 2mL/ min、2.1mL/min、2.2mL/min、2.3mL/min、2.4mL/min、2.5mL/min、2.6mL/min、2.7mL/min、 All point values in 2.8mL/min, 2.9mL/min, 3mL/min and the numberical range, since the limitation of length no longer arranges It lifts)；The ultrapure Water warfare of 6mL is used later, drains 1h in air, is finally eluted with the methanol of 12mL, is obtained solution B.
Solid-phase extraction column is a kind of material for carrying out Solid Phase Extraction, and the filler difference in extraction column is divided into HLB；C18； ProElut PXL etc..Different object ingredients is extracted, the extraction column of use is different, and the present invention is detection sulfamido antibiosis Otherwise it is anti-can not to detect sulfamido so cannot be substituted for other extraction columns using the extraction column of Oasis (HLB) for element Raw element.
Preferably, the nitrogen stripping instrument that use device water-bath is dried up described in step (2) carries out, and eluent is dried up；
Preferably, the temperature of the water-bath be 35-45 DEG C, such as can be 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C, All point values in 40 DEG C, 41 DEG C, 42 DEG C, 43 DEG C, 44 DEG C, 45 DEG C and the numberical range, since the limitation of length no longer arranges It lifts；
Preferably, in the water-bath nitrogen flow rate be 2-8mL/min, such as can be 2mL/min, 2.5mL/min, 3mL/min、3.5mL/min、4mL/min、4.5mL/min、5mL/min、5.5mL/min、6mL/min、6.5mL/min、7mL/ All point values in min, 7.5mL/min, 8mL/min and the numberical range, since the limitation of length is not listed；Preferably 5-6mL/min。
Preferably, constant volume described in step (2) is to be carried out with ammonium formate formic acid mixed aqueous solution and methanol；
Preferably, in ammonium formate formic acid mixed aqueous solution, formic acid ammonium concentration is 1mol/L, the volume ratio of formic acid and aqueous solution For 1:1000；
Preferably, the methanol is chromatographic grade；
Preferably, the volume ratio of ammonium formate formic acid mixed aqueous solution and methanol is 4:1；
Preferably, it is settled to 1mL；
Preferably, filtering filter membrane used described in step (2) is organic filter membrane, preferably nylon leaching film, to be measured to remove Impurity in sample；
Preferably, the aperture of the filter membrane is 0.1-0.3 μm, such as can be 0.1 μm, 0.12 μm, 0.14 μm, 0.16 μ M, all point values in 0.18 μm, 0.2 μm, 0.22 μm, 0.24 μm, 0.26 μm, 0.28 μm, 0.3 μm and the numberical range, by It is not listed in the limitation of length；Preferably 0.2-0.3 μm；
Preferably, it is saved as described in step (2) and saves sample to be tested at -20 DEG C.
Preferably, the method is limited to 0.05 μ g/L to the detection of sulfa antibiotics.
Preferably, the analysis of the sample to be tested is carried out using Liquid Chromatography-Tandem Mass Spectrometry；
Preferably, it is carried out using the electro-spray ionization source holotype of Liquid Chromatography-Tandem Mass Spectrometry；
Preferably, the Liquid Chromatography-Tandem Mass Spectrometry is equipped with C18 liquid-phase chromatographic column.
Preferably, the detection method at least includes the following steps:
(1) water sampling filter membrane cleans, the pH to 3.0-5.0 of water sample after adjusting film with HCl and/or NaOH, and adds Enter the sodium ethylene diamine tetracetate 9-12mL that concentration is 2-8g/L, adds internal standard compound, obtain solution A；The filter membrane is glass fibre Filter membrane, aperture are 0.4-0.6 μm；
(2) by solution A by HLB solid-phase extraction column, the column flow rate of crossing of solution A is 2-3mL/min；It is ultrapure with 6mL later Water warfare drains 1h in air, is finally eluted with the methanol of 12mL, obtains solution B, the nitrogen stripping instrument of use device water-bath It is dried up, the temperature of the water-bath is 35-45 DEG C, and nitrogen flow rate is 2-8mL/min in water-bath；It is with formic acid ammonium concentration The ammonium formate formic acid mixed aqueous solution and hplc grade methanol of 1mol/L carries out being settled to 1mL；The nylon for being 0.2-0.3 μm with aperture After membrane filtration solution B, obtain sample to be tested and be placed in sample injection bottle to save sample to be tested at -20 DEG C；
(3) analysis of the sample to be tested is carried out using the electro-spray ionization source holotype of Liquid Chromatography-Tandem Mass Spectrometry, The Liquid Chromatography-Tandem Mass Spectrometry is equipped with C18 liquid-phase chromatographic column；The method is limited to 0.05 μ g/ to the detection of sulfa antibiotics L。
Second aspect, the present invention provide detection method as described in relation to the first aspect in sulfa antibiotics residue detection Using.
The third aspect, it is residual that the present invention provides the sulfa antibiotics in municipal wastewater of detection method as described in relation to the first aspect Stay the application in detection.
Compared with prior art, sulfa antibiotics method for detecting residue provided by the invention at least has below beneficial to effect Fruit:
(1) the method for the present invention adjusts water sample pH to 3.5- because suitable sodium ethylene diamine tetracetate is added in the sample 4.0,35-45 DEG C of suitable bath temperature equal measures reduce chelation, biotransformation and hydrolysis pair significantly The influence of sulfa antibiotics content detection in municipal wastewater, improves the rate of recovery of sulfa antibiotics, recycles it averagely Rate reaches 80% or more, in the case that sulfa antibiotics concentration is micro in water sample, still recovery efficiency with higher.
(2) the method for the present invention is because using suitable meoh eluate, the measures such as suitable filter membrane, with guaranteeing high-recovery Simultaneously, it is possible to reduce the waste of meoh eluate and filter membrane reduces testing cost.
(3) the method for the present invention because using suitable sample cross column flow rate 2-3mL/min and nitrogen stripping condition (water-bath Temperature is 35-45 DEG C, and nitrogen flow rate is the measures such as 2-8mL/min) in water-bath, with guaranteeing high-recovery simultaneously, Ke Yijie About pre-treatment time about 1h, improves detection efficiency.
(4) the method for the present invention the method can achieve 0.05 μ g/L to the detection limit of sulfa antibiotics, for 0.05 μ The sample of g/L concentration, the separating effect of sulfa antibiotics is obvious in the test map of Liquid Chromatography-Tandem Mass Spectrometry, and peak shape is complete Beauty, response is high, is not influenced, is can satisfy in municipal wastewater to the sulfa antibiotics content of low concentration by impurity Testing requirements.
(5) the method for the present invention good test effect and easy to operate, at low cost, Successful utilization sulfamido in municipal wastewater is anti- The detection of raw cellulose content, as a result accurately, detection efficiency is high, it is a kind of relatively rapid, accurate, stable test method.
Detailed description of the invention
Fig. 1 is the MRM mass spectrogram of the Liquid Chromatography-Tandem Mass Spectrometry of embodiment 1.
Specific embodiment
The present invention is described further combined with specific embodiments below, but following embodiments absolutely not to the present invention have appoint What is limited.
In all embodiments of the invention, using the extraction column of Oasis (HLB) (500mg, 6cc, Waters, the U.S.) For Solid Phase Extraction；Sample is tested and analyzed, chromatographic column using Liquid Chromatography-Tandem Mass Spectrometry (1200 series of Agilent) For Agilent EclipseC18 column (diameter, length and aperture are respectively 2.1mm, 150mm, 3.5 μm), mobile phase is ammonium formate Formic acid mixed aqueous solution (solvent A)/acetonitrile (solvent B), volume ratio 20:80, constant gradient, sample injections volume are 5 μ L, stream Speed is 0.4mL/min, and column oven temperature is 35 DEG C, and the total run time of system is 3min, before inject next time, balance 1min.And nitrogen is used as spray gas (gas flows 10L/min, nebulizer pressure 35psi).Impact energy, fragmentation voltage, mother Other Mass Spectrometry Conditions such as ion and daughter ion selection then use software optimization, and antibiotic is finally carried out under multiple-reaction monitoring pattern Measurement.
Experimental water is artificial distribution, simulates moderate strength municipal wastewater, i.e., dissolves grape in the deionized water of 500mL Sugar, (NH4)2SO4、KH2PO4, yeast powder and microelement, be made water quality be TOC (Total Organic Carbon, referred to as TOC, total organic carbon), NH4 +- N, TP (Total Phosphorus, total phosphorus content), TN (Total Nitrogen, nitrogen pool), divide Not Wei 90-110mg/L, 16-20mg/L, 2.5-3.5mg/L, 16-20mg/L, MgSO in Trace Elements4·7H2O、 CaCl2·2H2O、MnCl2·7H2O、ZnSO4·7H2O、CoCl2·6H2O、CuSO4·5H2O、FeCl3、Na2MoO4·2H2O's Concentration be respectively 5.07mg/L, 0.368mg/L, 0.275mg/L, 0.44mg/L, 0.42mg/L, 0.391mg/L, 1.45mg/L, 1.26mg/L adds the sulphadiazine (SDZ) and sulfamethoxazole of equivalent as several pieces sample respectively in every a sample (SMZ), making its concentration is respectively 0.05,0.1,0.2,0.5 μ g/L.Five aliquots number consecutivelies, are denoted as embodiment 1-4.Separately The sulphadiazine (SDZ) and sulfamethoxazole (SMZ) and concentration of preparation several pieces addition equivalent are respectively 0.05 μ g/L sample, note For embodiment 1-15.
Embodiment 1
(1) the water sample filter membrane that concentration is 0.05 μ g/L is taken to clean, the pH of water sample after adjusting film with HCl and/or NaOH To 3.0, and the sodium ethylene diamine tetracetate 9mL that concentration is 2g/L is added, add internal standard compound (13C3- caffeine), obtain solution A；The filter membrane is glass fiber filter, and aperture is 0.4 μm；
(2) by solution A by HLB solid-phase extraction column, the column flow rate of crossing of solution A is 2mL/min；6mL ultrapure water is used later Purification, drains 1h in air, is finally eluted with the methanol of 12mL, obtains solution B, the nitrogen stripping instrument of use device water-bath into Row drying, the temperature of the water-bath are 35 DEG C, and nitrogen flow rate is 2mL/min in water-bath；It is mixed with ammonium formate formic acid water-soluble Liquid (formic acid ammonium concentration is 1mol/L, and the volume ratio of formic acid and aqueous solution is 1:1000) and methanol (chromatographic grade) are settled to 1mL；After being 0.2 μm of nylon leaching film filtering solution B with aperture, obtain sample to be tested and be placed in sample injection bottle to protect at -20 DEG C Deposit sample to be tested；
(3) analysis of the sample to be tested is carried out using the electro-spray ionization source holotype of Liquid Chromatography-Tandem Mass Spectrometry, The Liquid Chromatography-Tandem Mass Spectrometry is equipped with Agilent EclipseC18 column.
Embodiment 2
(1) take concentration be 0.1 μ g/L water sample filter membrane clean, after adjusting film with HCl and/or NaOH the pH of water sample to 5.0, and the sodium ethylene diamine tetracetate 12mL that concentration is 8g/L is added, internal standard compound is added, solution A is obtained；The filter membrane is glass Glass fibrous filter membrane, aperture are 0.6 μm；
(2) by solution A by HLB solid-phase extraction column, the column flow rate of crossing of solution A is 3mL/min；6mL ultrapure water is used later Purification, drains 1h in air, is finally eluted with the methanol of 12mL, obtains solution B, the nitrogen stripping instrument of use device water-bath into Row drying, the temperature of the water-bath are 45 DEG C, and nitrogen flow rate is 8mL/min in water-bath；It is mixed with ammonium formate formic acid water-soluble Liquid (formic acid ammonium concentration is 1mol/L, and the volume ratio of formic acid and aqueous solution is 1:1000) and methanol (chromatographic grade) are settled to 1mL；After being 0.3 μm of nylon leaching film filtering solution B with aperture, obtain sample to be tested and be placed in sample injection bottle to protect at -20 DEG C Deposit sample to be tested；
(3) analysis of the sample to be tested is carried out using the electro-spray ionization source holotype of Liquid Chromatography-Tandem Mass Spectrometry, The Liquid Chromatography-Tandem Mass Spectrometry is equipped with Agilent EclipseC18 column.
Embodiment 3
(1) take concentration be 0.2 μ g/L water sample filter membrane clean, after adjusting film with HCl and/or NaOH the pH of water sample to 4.0, and the sodium ethylene diamine tetracetate 10mL that concentration is 5g/L is added, internal standard compound is added, solution A is obtained；The filter membrane is glass Glass fibrous filter membrane, aperture are 0.5 μm；
(2) by solution A by HLB solid-phase extraction column, the column flow rate of crossing of solution A is 2.5mL/min；It is ultrapure with 6mL later Water warfare drains 1h in air, is finally eluted with the methanol of 12mL, obtains solution B, the nitrogen stripping instrument of use device water-bath It is dried up, the temperature of the water-bath is 40 DEG C, and nitrogen flow rate is 5mL/min in water-bath；With ammonium formate formic acid mixing water Solution (formic acid ammonium concentration is 1mol/L, and the volume ratio of formic acid and aqueous solution is 1:1000) and methanol (chromatographic grade) are settled to 1mL；After being 0.25 μm of nylon leaching film filtering solution B with aperture, obtain sample to be tested and be placed in sample injection bottle to protect at -20 DEG C Deposit sample to be tested；
(3) analysis of the sample to be tested is carried out using the electro-spray ionization source holotype of Liquid Chromatography-Tandem Mass Spectrometry, The Liquid Chromatography-Tandem Mass Spectrometry is equipped with Agilent EclipseC18 column.
Embodiment 4
(1) take concentration be 0.5 μ g/L water sample filter membrane clean, after adjusting film with HCl and/or NaOH the pH of water sample to 3.5, and the sodium ethylene diamine tetracetate 10mL that concentration is 5g/L is added, internal standard compound is added, solution A is obtained；The filter membrane is glass Glass fibrous filter membrane, aperture are 0.55 μm；
(2) by solution A by HLB solid-phase extraction column, the column flow rate of crossing of solution A is 2.8mL/min；It is ultrapure with 6mL later Water warfare drains 1h in air, is finally eluted with the methanol of 12mL, obtains solution B, the nitrogen stripping instrument of use device water-bath It is dried up, the temperature of the water-bath is 43 DEG C, and nitrogen flow rate is 7mL/min in water-bath；With ammonium formate formic acid mixing water Solution (formic acid ammonium concentration is 1mol/L, and the volume ratio of formic acid and aqueous solution is 1:1000) and methanol (chromatographic grade) are settled to 1mL；After being 0.25 μm of nylon leaching film filtering solution B with aperture, obtain sample to be tested and be placed in sample injection bottle to protect at -20 DEG C Deposit sample to be tested；
(3) analysis of the sample to be tested is carried out using the electro-spray ionization source holotype of Liquid Chromatography-Tandem Mass Spectrometry, The Liquid Chromatography-Tandem Mass Spectrometry is equipped with Agilent EclipseC18 column.
Embodiment 5
(1) take concentration be 1 μ g/L water sample filter membrane clean, after adjusting film with HCl and/or NaOH the pH of water sample to 3.7, and the sodium ethylene diamine tetracetate 10mL that concentration is 5g/L is added, internal standard compound is added, solution A is obtained；The filter membrane is glass Glass fibrous filter membrane, aperture are 0.45 μm；
(2) by solution A by HLB solid-phase extraction column, the column flow rate of crossing of solution A is 2.2mL/min；It is ultrapure with 6mL later Water warfare drains 1h in air, is finally eluted with the methanol of 12mL, obtains solution B, the nitrogen stripping instrument of use device water-bath It is dried up, the temperature of the water-bath is 40 DEG C, and nitrogen flow rate is 5mL/min in water-bath；With ammonium formate formic acid mixing water Solution (formic acid ammonium concentration is 1mol/L, and the volume ratio of formic acid and aqueous solution is 1:1000) and methanol (chromatographic grade) are settled to 1mL；After being 0.22 μm of nylon leaching film filtering solution B with aperture, obtain sample to be tested and be placed in sample injection bottle to protect at -20 DEG C Deposit sample to be tested；
(3) analysis of the sample to be tested is carried out using the electro-spray ionization source holotype of Liquid Chromatography-Tandem Mass Spectrometry, The Liquid Chromatography-Tandem Mass Spectrometry is equipped with Agilent EclipseC18 column.
Comparative example 1
Difference with embodiment 1 is only that the aperture of glass fiber filter is 0.22 μm, remaining is all the same.
Comparative example 2
Difference with embodiment 1 is only that the aperture of glass fiber filter is 0.8 μm, remaining is all the same.
Comparative example 3
Difference with embodiment 1 was only that the whole pH of water sample after film was 7.6, remaining is all the same.
Comparative example 4
Difference with embodiment 1 was only that the whole pH of water sample after film was 1.0, remaining is all the same.
Comparative example 5
Difference with embodiment 1 is only that the volume that sodium ethylene diamine tetracetate is added is 5g/L, remaining is all the same.
Comparative example 6
Difference with embodiment 1 is only that the volume that sodium ethylene diamine tetracetate is added is 20g/L, remaining is all the same.
Comparative example 7
Difference with embodiment 1 is only that the aperture of nylon leaching film is 0.45 μm, remaining is all the same.
Comparative example 8
Difference with embodiment 1 is only that effluent volume is 6mL, remaining is all the same.
Comparative example 9
Difference with embodiment 1 is only that effluent volume is 18mL, remaining is all the same.
Comparative example 10
Difference with embodiment 1 was only that column flow rate was 1mL/min, remaining is all the same.
Comparative example 11
Difference with embodiment 1 was only that column flow rate was 10mL/min, remaining is all the same.
Comparative example 12
Difference with embodiment 1 is only that draining the time in air is 0.5h, remaining is all the same.
Comparative example 13
Difference with embodiment 1 is only that draining the time in air is 2h, remaining is all the same.
Comparative example 14
Difference with embodiment 1 is only that in water-bath that nitrogen flow rate is 15mL/min, remaining is all the same.
Comparative example 15
Difference with embodiment 1 is only that sample to be tested saves at room temperature, remaining is all the same.
Test result:
The testing result of embodiment 1-4 and comparative example 1-15 are tested, test result is as shown in table 1.It can be with from table 1 Find out, the method for the present invention detects sulfa antibiotics, and recycling is greater than 85%.In comparative example 1-15, in addition to comparative example 9,10, 15, the rate of recovery is below embodiment 1-4, but the time of 9,10,15 pre-treatment of comparative example, is more than embodiment 1-4 about 1h.It can Sample recovery rate to illustrate embodiment 1-4 is higher, and testing result is accurate and efficient.
Table 1
Fig. 1 is the MRM mass spectrogram of the Liquid Chromatography-Tandem Mass Spectrometry of embodiment 1, as shown in Figure 1, sulphadiazine (SDZ), sulphur Amine first oxazole (SMZ) and internal standard compound (13C3- caffeine is denoted as13C3) appearance time be respectively 3.12min, 1.08min and 1.76min, the separating effect of sulfa antibiotics is obvious in the mass spectrogram of Liquid Chromatography-Tandem Mass Spectrometry, and peak shape is perfect, response Height, not by the interference of impurity.The MRM mass spectrogram result of embodiment 2-5 is similar to Example 1, will not enumerate herein With repeat.These results suggest that municipal wastewater sulfamido antibiosis of the method for 1-5 of the embodiment of the present invention for 0.05 μ g/L concentration Plain sample detection effect is good, can satisfy in municipal wastewater to the testing requirements of the sulfa antibiotics content of low concentration.
In conclusion method of the invention significantly reduces chelation, biotransformation and hydrolysis to municipal administration The influence of sulfa antibiotics content detection in sewage, for micro sulfa antibiotics (0.05 μ g/L).Guaranteeing to recycle In the higher situation of rate, reduces the time required for pre-treatment, improve detection efficiency.Using suitable meoh eluate, And correct filter membrane has been selected, in the case where guaranteeing the higher situation of the rate of recovery, the waste of methanol and filter membrane is avoided, detection is reduced Cost.In short, the method for the present invention good test effect and easy to operate, at low cost, Successful utilization sulfamido in municipal wastewater The detection of antibiotic content, as a result accurately, detection efficiency are high.
It should be noted that and understand, in the feelings for not departing from the spirit and scope of the present invention required by appended claims Under condition, various modifications and improvements can be made to the present invention of foregoing detailed description.It is therefore desirable to the model of the technical solution of protection It encloses and is not limited by given any specific exemplary teachings.
The Applicant declares that the above content is combine specific preferred embodiment made for the present invention further specifically It is bright, and it cannot be said that specific implementation of the invention is only limited to these instructions.For the ordinary skill of the technical field of the invention For personnel, without departing from the inventive concept of the premise, a number of simple deductions or replacements can also be made, all should be considered as belonging to In protection scope of the present invention.

Claims (10)

1. a kind of detection method of sulfa antibiotics, includes the steps that sample pre-treatments and sample analysis, which is characterized in that institute Sample pre-treatments are stated at least include the following steps:
(1) water sampling filter membrane cleans, and adjusted the pH of water sample after film, and sodium ethylene diamine tetracetate is added, and added internal standard Object obtains solution A；
(2) Solid Phase Extraction that solution A is carried out using HLB solid-phase extraction column, obtains solution B, after drying, constant volume, filtering solution B, obtains It is placed in sample injection bottle and saves to sample to be tested.
2. detection method according to claim 1, which is characterized in that filter membrane described in step (1) is glass fiber filter；
Preferably, the aperture of the filter membrane is 0.1-0.8 μm, preferably 0.4-0.6 μm；
Preferably, adjusting pH described in step (1) is to be adjusted to pH to 2.0-5.0, preferably 3.5-4.0；
Preferably, pH is adjusted with strong acid and/or highly basic, preferably adjusts pH with HCl and/or NaOH；
Preferably, the concentration of sodium ethylene diamine tetracetate described in step (1) is 2-8g/L, preferably 4-5g/L；
Preferably, the additional amount of the sodium ethylene diamine tetracetate is 5-20mL, preferably 10mL.
3. detection method according to claim 1 or 2, which is characterized in that use HLB Solid Phase Extraction described in step (2) Column carry out Solid Phase Extraction specifically includes the following steps:
Solution A is eluted with methanol after draining in air later with ultrapure Water warfare by extraction column, obtains solution B first；
Preferably, specifically includes the following steps:
By solution A by HLB solid-phase extraction column, the column flow rate of crossing of solution A is 2-3mL/min；The ultrapure Water warfare of 6mL is used later, 1-2h is drained in air, is finally eluted with the methanol of 12mL, is obtained solution B.
4. detection method according to any one of claim 1-3, which is characterized in that dry up and use described in step (2) The nitrogen stripping instrument of device water-bath carries out；
Preferably, the temperature of the water-bath is 35-45 DEG C；
Preferably, nitrogen flow rate is 2-8mL/min, preferably 5-6mL/min in the water-bath.
5. detection method described in any one of -4 according to claim 1, which is characterized in that constant volume described in step (2) is to use Ammonium formate formic acid mixed aqueous solution and methanol carry out；
Preferably, in ammonium formate formic acid mixed aqueous solution, formic acid ammonium concentration is 1mol/L, and the volume ratio of formic acid and aqueous solution is 1: 1000；
Preferably, the methanol is chromatographic grade；
Preferably, the volume ratio of ammonium formate formic acid mixed aqueous solution and methanol is 4:1；
Preferably, it is settled to 1mL；
Preferably, the filter membrane used of filtering described in step (2) is organic filter membrane, preferably nylon leaching film；
Preferably, the aperture of the filter membrane is 0.1-0.3 μm, preferably 0.2-0.3 μm；
Preferably, it is saved as described in step (2) and saves sample to be tested at -20 DEG C.
6. detection method according to any one of claims 1-5, which is characterized in that the method is to sulfa antibiotics Detection be limited to 0.05 μ g/L.
7. detection method according to claim 1 to 6, which is characterized in that the analysis of the sample to be tested uses Liquid Chromatography-Tandem Mass Spectrometry carries out；
Preferably, it is carried out using the electro-spray ionization source holotype of Liquid Chromatography-Tandem Mass Spectrometry；
Preferably, the Liquid Chromatography-Tandem Mass Spectrometry is equipped with C18 liquid-phase chromatographic column.
8. detection method described in any one of -7 according to claim 1, which is characterized in that the detection method include at least with Lower step:
(1) water sampling filter membrane cleans, the pH to 3.0-5.0 of water sample after adjusting film with HCl and/or NaOH, and is added dense Degree is the sodium ethylene diamine tetracetate 9-12mL of 2-8g/L, adds internal standard compound, obtains solution A；The filter membrane is glass fiber filter, Aperture is 0.4-0.6 μm；
(2) by solution A by HLB solid-phase extraction column, the column flow rate of crossing of solution A is 2-3mL/min；It is net with 6mL ultrapure water later Change, drain 1-2h in air, finally eluted with the methanol of 12mL, obtain solution B, the nitrogen stripping instrument of use device water-bath into Row drying, the temperature of the water-bath are 35-45 DEG C, and nitrogen flow rate is 2-8mL/min in water-bath；It is with formic acid ammonium concentration The ammonium formate formic acid mixed aqueous solution and hplc grade methanol of 1mol/L carries out being settled to 1mL；The nylon for being 0.2-0.3 μm with aperture After membrane filtration solution B, obtain sample to be tested and be placed in sample injection bottle to save sample to be tested at -20 DEG C；
(3) analysis of the sample to be tested is carried out using the electro-spray ionization source holotype of Liquid Chromatography-Tandem Mass Spectrometry, it is described Liquid Chromatography-Tandem Mass Spectrometry is equipped with C18 liquid-phase chromatographic column；The method is limited to 0.05 μ g/L to the detection of sulfa antibiotics.
9. application of the detection method according to claim 1 to 8 in sulfa antibiotics residue detection.
10. detection method according to claim 1 to 8 sulfa antibiotics residue detection in municipal wastewater In application.
CN201811060807.9A 2018-09-12 2018-09-12 A kind of detection method and application of sulfa antibiotics Pending CN109060997A (en)

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