CN106596780A - Method for detecting contents of various antibiotics in water by combined use of high pressure liquid chromatography and mass spectrum - Google Patents
Method for detecting contents of various antibiotics in water by combined use of high pressure liquid chromatography and mass spectrum Download PDFInfo
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 239000003242 anti bacterial agent Substances 0.000 title claims abstract description 18
- 229940088710 antibiotic agent Drugs 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 9
- 238000001819 mass spectrum Methods 0.000 title abstract 4
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 39
- 238000001514 detection method Methods 0.000 claims abstract description 28
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- 150000002500 ions Chemical class 0.000 claims description 30
- 230000003115 biocidal effect Effects 0.000 claims description 28
- 239000000523 sample Substances 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 19
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 16
- 239000003480 eluent Substances 0.000 claims description 14
- 239000012488 sample solution Substances 0.000 claims description 12
- 239000012224 working solution Substances 0.000 claims description 12
- 239000008367 deionised water Substances 0.000 claims description 11
- 229910021641 deionized water Inorganic materials 0.000 claims description 11
- 239000003643 water by type Substances 0.000 claims description 9
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 8
- 235000019253 formic acid Nutrition 0.000 claims description 8
- 238000004949 mass spectrometry Methods 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims description 6
- XUFXOAAUWZOOIT-SXARVLRPSA-N (2R,3R,4R,5S,6R)-5-[[(2R,3R,4R,5S,6R)-5-[[(2R,3R,4S,5S,6R)-3,4-dihydroxy-6-methyl-5-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)-1-cyclohex-2-enyl]amino]-2-oxanyl]oxy]-3,4-dihydroxy-6-(hydroxymethyl)-2-oxanyl]oxy]-6-(hydroxymethyl)oxane-2,3,4-triol Chemical compound O([C@H]1O[C@H](CO)[C@H]([C@@H]([C@H]1O)O)O[C@H]1O[C@@H]([C@H]([C@H](O)[C@H]1O)N[C@@H]1[C@@H]([C@@H](O)[C@H](O)C(CO)=C1)O)C)[C@@H]1[C@@H](CO)O[C@@H](O)[C@H](O)[C@H]1O XUFXOAAUWZOOIT-SXARVLRPSA-N 0.000 claims description 5
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 claims description 5
- PCZOHLXUXFIOCF-UHFFFAOYSA-N Monacolin X Natural products C12C(OC(=O)C(C)CC)CC(C)C=C2C=CC(C)C1CCC1CC(O)CC(=O)O1 PCZOHLXUXFIOCF-UHFFFAOYSA-N 0.000 claims description 5
- 229930182555 Penicillin Natural products 0.000 claims description 5
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 5
- 229960002632 acarbose Drugs 0.000 claims description 5
- XUFXOAAUWZOOIT-UHFFFAOYSA-N acarviostatin I01 Natural products OC1C(O)C(NC2C(C(O)C(O)C(CO)=C2)O)C(C)OC1OC(C(C1O)O)C(CO)OC1OC1C(CO)OC(O)C(O)C1O XUFXOAAUWZOOIT-UHFFFAOYSA-N 0.000 claims description 5
- PCZOHLXUXFIOCF-BXMDZJJMSA-N lovastatin Chemical compound C([C@H]1[C@@H](C)C=CC2=C[C@H](C)C[C@@H]([C@H]12)OC(=O)[C@@H](C)CC)C[C@@H]1C[C@@H](O)CC(=O)O1 PCZOHLXUXFIOCF-BXMDZJJMSA-N 0.000 claims description 5
- 229960004844 lovastatin Drugs 0.000 claims description 5
- QLJODMDSTUBWDW-UHFFFAOYSA-N lovastatin hydroxy acid Natural products C1=CC(C)C(CCC(O)CC(O)CC(O)=O)C2C(OC(=O)C(C)CC)CC(C)C=C21 QLJODMDSTUBWDW-UHFFFAOYSA-N 0.000 claims description 5
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 5
- 229940049954 penicillin Drugs 0.000 claims description 5
- SEEPANYCNGTZFQ-UHFFFAOYSA-N sulfadiazine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=NC=CC=N1 SEEPANYCNGTZFQ-UHFFFAOYSA-N 0.000 claims description 5
- 229960004306 sulfadiazine Drugs 0.000 claims description 5
- 229960005404 sulfamethoxazole Drugs 0.000 claims description 5
- JLKIGFTWXXRPMT-UHFFFAOYSA-N sulphamethoxazole Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 JLKIGFTWXXRPMT-UHFFFAOYSA-N 0.000 claims description 5
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 4
- 229930182566 Gentamicin Natural products 0.000 claims description 4
- 229930194936 Tylosin Natural products 0.000 claims description 4
- 239000004182 Tylosin Substances 0.000 claims description 4
- 229960002518 gentamicin Drugs 0.000 claims description 4
- 239000007791 liquid phase Substances 0.000 claims description 4
- 229960004059 tylosin Drugs 0.000 claims description 4
- WBPYTXDJUQJLPQ-VMXQISHHSA-N tylosin Chemical compound O([C@@H]1[C@@H](C)O[C@H]([C@@H]([C@H]1N(C)C)O)O[C@@H]1[C@@H](C)[C@H](O)CC(=O)O[C@@H]([C@H](/C=C(\C)/C=C/C(=O)[C@H](C)C[C@@H]1CC=O)CO[C@H]1[C@@H]([C@H](OC)[C@H](O)[C@@H](C)O1)OC)CC)[C@H]1C[C@@](C)(O)[C@@H](O)[C@H](C)O1 WBPYTXDJUQJLPQ-VMXQISHHSA-N 0.000 claims description 4
- 235000019375 tylosin Nutrition 0.000 claims description 4
- 238000007405 data analysis Methods 0.000 claims description 3
- 238000010812 external standard method Methods 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 230000014759 maintenance of location Effects 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000004044 response Effects 0.000 claims description 3
- 239000011550 stock solution Substances 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract 1
- 238000011084 recovery Methods 0.000 abstract 1
- 230000029219 regulation of pH Effects 0.000 abstract 1
- 230000001105 regulatory effect Effects 0.000 abstract 1
- 239000013077 target material Substances 0.000 abstract 1
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 8
- 229960003276 erythromycin Drugs 0.000 description 4
- 238000004817 gas chromatography Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 239000007921 spray Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 239000004809 Teflon Substances 0.000 description 2
- 229920006362 Teflon® Polymers 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000008485 antagonism Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000003859 hyphenated technique Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
Classifications
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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
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a method for detecting the contents of various antibiotics in water by combined use of high pressure liquid chromatography and mass spectrum. The method comprises the following steps of (1) regulating pH of a water sample; (2) performing solid-phase extraction, enriching and concentration on the water sample subjected to pH regulation; (3) performing detection by a high pressure liquid chromatography and mass spectrum instrument; (4) performing result analysis. The method for detecting the contents of various antibiotics in water by combined use of high pressure liquid chromatography and mass spectrum has the characteristics of short detection time and high sensitivity, and belongs to a novel method for detecting the contents of various kinds of antibiotics in the water environment sample. The method has the advantages that the interference on the detection of a target material is greatly reduced; the high-sensitivity detection can be performed on trace antibiotics and various kinds of antibiotics. When the detection method provided by the invention is used, the mass accuracy on 9 kinds of antibiotics is less than 1 ppm; the detection limit is 0.1 to 5 ug/L; the addition recovery rate is 70 to 100 percent; the relevant deviation difference is smaller than 10 percent.
Description
Technical field
The present invention relates to technical field of environmental detection, is specifically related to many in high pressure liquid chromatography-mass spectrometry detection water
The method for planting antibiotic content.
Background technology
Increasingly improve and the newtype drug of the general level of the health constantly puts into clinical practice makes the dependence of the raw element of people's antagonism more next
Stronger, all in the trend for increasing year by year, a large amount of use of antibiotic is inevitably led for the species or quantity of antibiotic
Cause it to pass through number of ways and enter environment.It is certain that increasing research finds that the antibiotic in environment has to environmental organism
Toxicity, the antibiotic in environment has been increasingly becoming generally acknowledged emerging pollutant, and the detection to antibiotic in environment has been current
Home and abroad environment detection, the important content administered and evaluate.
At present, the detection of antibiotic mainly has gas chromatography, liquid chromatography, GC-MS, liquid phase
Chromatograph-mass spectrometer coupling method.Gas-chromatography and liquid chromatogram rely primarily on chromatogram post separation and detector to antibiotic responsiveness, right
Antibiotic detection in one pack system or simple system has a preferably qualitatively and quantitatively effect, and due to water environment matrix it is more multiple
Miscellaneous, comprising gas chromatography matter or interfering ion, there is certain difficulty in chromatogram on antibiotic is qualitative and quantitative.Gas-chromatography-
Mass spectrometry and liquid chromatograph mass spectrography mainly utilize chromatogram post separation, mass analyzer according to determine molecular weight and
The intensity of the Middle Molecular Substance carries out qualitative and quantitative, and than gas-chromatography and liquid chromatogram, chromatogram and mass spectrometric hyphenated technique can
The qualitative and quantitative of complex sample is preferably completed, yet with there is substantial amounts of organic matter in environment, ambient interferences are more serious,
The substance classes of identical molecular weight are still a lot, so easily causing the false positive of measurement result.
Triple level Four pole technologies are broken into pieces compound molecule by applying energy, and every kind of compound is in certain condition
Under have the fragments characteristic of oneself, compound is carried out by determining compound molecular weight itself and fragments characteristic molecular weight simultaneously
Qualitative and quantitative not only can reduction reduces false positive and judges to the erroneous judgement of compound, and greatly improves detection sensitivity.At present
It is domestic less to antibiotic research in high pressure liquid chromatography-mass spectrograph combination method measure water, reducing to qualitative and quantitative interference
Difficult problem aspect is also no to cause concern in environment measuring industry.
The content of the invention
Present invention solves the technical problem that being to provide a kind of quick, easy, sensitivity height, and effectively reduce or eliminate
Multiple Classes of Antibiotics contains in the high pressure liquid chromatography of the complex matrices interference antibiotic content from water environment-mass spectrometry detection water
The method of amount.
The technical scheme is that：The method of Multiple Classes of Antibiotics content in high pressure liquid chromatography-mass spectrometry detection water,
Comprise the following steps：
1) pH of water sample is adjusted：A 250mL water samples are measured, appropriate formic acid is added, pH to 2.0 is adjusted, portion is separately measured
250mL water samples, add appropriate ammoniacal liquor, adjust pH to 12.0；
2) the solid phase extraction concentration concentration of water sample after pH is adjusted：Respectively with 5mL methyl alcohol and 10mL deionized water activated solids
Extraction column 1, flow velocity is that the 250mL water samples that pH is adjusted to 2.0 are crossed into solid-phase extraction column after 5-10mL/min activation, and flow velocity is 3-5mL/
Min, after the excessively complete solid-phase extraction column of water sample, with 5mL deionized waters solid-phase extraction column is got express developed, and flow velocity is 5-10mL/min, so
After be dried solid-phase extraction column 30min, finally with 5mL methyl alcohol with 3mL/min flow velocitys elute solid-phase extraction column, collect eluent, mark
It is designated as eluent 1；Respectively with 5mL methyl alcohol and 10mL deionized water activated solids extraction column 2, flow velocity is after 5-10mL/min activation
The 250mL water samples that pH is adjusted to 12.0 are crossed into solid-phase extraction column, flow velocity is 3-5mL/min, after the excessively complete solid-phase extraction column of water sample, used
5mL deionized waters get solid-phase extraction column express developed, and flow velocity is 5-10mL/min, is then dried solid-phase extraction column 30min, is finally used
5mL methyl alcohol elutes solid-phase extraction column with 3mL/min flow velocitys, collects eluent, is labeled as eluent 2；Merge eluent 1 and 2,
In 40 DEG C of water-baths, dried up with gentle nitrogen, be finally settled to 1mL with water, after 0.45um teflon membrane filters, that is, obtained
Sample solution；
3) using high pressure liquid chromatography-mass spectrograph detection：
A. the preparation of standard liquid：Standard items antibiotic is accurately weighed respectively, using deionized water dissolving, is made into respectively
The Standard Stock solutions of 0.2mg/mL, according to the response of each antibiotic when using, deionized water is diluted to standard working solution；
B. using high pressure liquid chromatography-mass spectrograph examination criteria working solution and sample solution：Inhaled respectively by automatic sampler
Standard working solution and sample solution are taken, in injection high pressure liquid chromatography-mass spectrograph, the antibiotic group lease making liquid chromatogram in sample
Post separation, the detection of mass spectrograph multiple-reaction monitoring pattern；
4) data analysis：With the parent ion and daughter ion of compound as quota ion and qualitative ion；With standard working solution
Mass concentration X be abscissa, the peak area Y of quantitative and qualitative ion is ordinate, draws standard working curve, is worked with standard
Curve carries out single ion quantified by external standard method to sample；With in sample solution occur chromatographic peak retention time and parent ion/son from
Son is qualitative to carrying out.
Further, in such scheme, formic acid used and ammoniacal liquor are top pure grade in the step 1.
Further, in such scheme, solid-phase extraction column used is solid for Waters Oasis HLB in the step 2
Phase extraction column, specification is 6cc/500mg, 60um.
Further, in such scheme, the antibiotic standard items in the step 3 be respectively hygromycin, Lovastatin,
Acarbose, tylosin, erythromycin, sulphadiazine, gentamicin, penicillin, sulfamethoxazole.
Further, in such scheme, liquid phase chromatogram condition in the step 3：Chromatographic column：SB-C18,3.5um, 2.1
×150mm；Mobile phase：A is 0.1% formic acid, and B is acetonitrile；Gradient elution：0-2min, 10%B；2-7min, 10-40%B, 7-
9min, 40-90%B, 9-10min, 90-100%B；Column temperature：Room temperature.
Further, in such scheme, Mass Spectrometry Conditions in the step 3：Mass analyzer：Triple level Four bars, scanning
Pattern：Cation-reaction monitoring pattern, ion gun：Electric spray ion source, spray voltage：4.5kV, removes solvent temperature：500 DEG C,
Remove solvent gas flow velocity：600L/h.
The invention has the beneficial effects as follows：Multiple Classes of Antibiotics contains in the high pressure liquid chromatography of the present invention-mass spectrometry detection water
The method of amount has the characteristics of detection time is short, sensitivity is high, is a kind of new for Multiple Classes of Antibiotics in environmental water sample
The assay method of content, the method greatly reduces the interference of the detection to object, can be same to trace and Multiple Classes of Antibiotics
The highly sensitive detections of Shi Jinhang.Using the detection method of the present invention for the mass accuracy of 9 kinds of antibiotic is less than 1ppm, inspection
Survey is limited to 0.1-5ug/L, and TIANZHU XINGNAO Capsul is 70-100%, and relative standard deviation is less than 10%.
Description of the drawings
Fig. 1 is corresponding multiple-reaction monitoring (MRM) chromatogram of 9 kinds of antibiotic (100 μ g/L).
Specific embodiment
Below in conjunction with the accompanying drawings to the side of Multiple Classes of Antibiotics content in the high pressure liquid chromatography-mass spectrometry detection water of the present invention
Method is described further：
As shown in figure 1, selecting hygromycin, Lovastatin, acarbose, tylosin, erythromycin, sulphadiazine, celebrating big
Mycin, penicillin, sulfamethoxazole are carried out various in high pressure liquid chromatography-mass spectrometry detection water as antibiotic standard items
Antibiotic content, concrete grammar is comprised the following steps：
1) pH of water sample is adjusted：A 250mL water samples are measured, appropriate formic acid is added, pH to 2.0 is adjusted, portion is separately measured
250mL water samples, add appropriate ammoniacal liquor, adjust pH to 12.0；Formic acid and ammoniacal liquor are top pure grade；
2) the solid phase extraction concentration concentration of water sample after pH is adjusted：Respectively with 5mL methyl alcohol and 10mL deionized water activated solids
Extraction column 1, flow velocity is that the 250mL water samples that pH is adjusted to 2.0 are crossed into solid-phase extraction column after 7mL/min activation, and flow velocity is 4mL/min,
After the excessively complete solid-phase extraction column of water sample, with 5mL deionized waters solid-phase extraction column is got express developed, flow velocity is 7mL/min, be then dried solid
Phase extraction column 30min, finally elutes solid-phase extraction column with 5mL methyl alcohol with 3mL/min flow velocitys, collects eluent, is labeled as wash-out
Liquid 1；Respectively with 5mL methyl alcohol and 10mL deionized water activated solids extraction column 2, flow velocity is to be adjusted to pH after 7mL/min activation
12.0 250mL water samples cross solid-phase extraction column, and flow velocity is 4mL/min, after the excessively complete solid-phase extraction column of water sample, uses 5mL deionized waters
Solid-phase extraction column is got express developed, flow velocity is 7mL/min, is then dried solid-phase extraction column 30min, finally with 5mL methyl alcohol with 3mL/
Min flow velocitys elute solid-phase extraction column, collect eluent, are labeled as eluent 2；Above-mentioned solid-phase extraction column used is Waters
Oasis HLB solid-phase extraction columns, specification is 6cc/500mg, 60um；Merge eluent 1 and 2, in 40 DEG C of water-baths, with gentle
Nitrogen is dried up, and is finally settled to 1mL with water, after 0.45um teflon membrane filters, that is, obtains sample solution；
3) using high pressure liquid chromatography-mass spectrograph detection：
A. the preparation of standard liquid：Standard items antibiotic, hygromycin, Lovastatin, acarbose, Thailand are accurately weighed respectively
Happy rhzomorph, erythromycin, sulphadiazine, gentamicin, penicillin, sulfamethoxazole, using deionized water dissolving, are made into respectively
The Standard Stock solutions of 0.2mg/mL, according to the response of each antibiotic when using, deionized water is diluted to standard working solution；
B. using high pressure liquid chromatography-mass spectrograph examination criteria working solution and sample solution：Inhaled respectively by automatic sampler
Standard working solution and sample solution are taken, in injection high pressure liquid chromatography-mass spectrograph, the antibiotic group lease making liquid chromatogram in sample
Post separation, liquid phase chromatogram condition：Chromatographic column：SB-C18,3.5um, 2.1 × 150mm；Mobile phase：A is 0.1% formic acid, and B is second
Nitrile；Gradient elution：0-2min, 10%B；2-7min, 10-40%B, 7-9min, 40-90%B, 9-10min, 90-100%B；Post
Temperature：Room temperature；The detection of mass spectrograph multiple-reaction monitoring pattern, Mass Spectrometry Conditions：Mass analyzer：Triple level Four bars, scan pattern：Just from
Son-reaction monitoring pattern, ion gun：Electric spray ion source, spray voltage：4.5kV, removes solvent temperature：500 DEG C, remove solvent gas
Rate of flow of fluid：600L/h；
4) data analysis：As shown in table 1, with the parent ion and daughter ion of compound as quota ion and qualitative ion；With
Mass concentration X of standard working solution is abscissa, and the peak area Y of quantitative and qualitative ion is ordinate, draws standard working curve,
Single ion quantified by external standard method is carried out to sample with standard working curve；With the chromatographic peak retention time and mother that occur in sample solution
Ion/daughter ion is qualitative to carrying out, and the chromatogram for obtaining is shown in Fig. 1.
Table 1：The mass spectrometry parameters of 9 kinds of antibiotic
| Title | Parent ion/(m/z) | Daughter ion/(m/z) | Taper hole voltage/V | Collision energy/V |
| Gentamicin | 478.28 | 157.16 | 26 | 20 |
| Lovastatin | 405.16 | 131.35 | 52 | 38 |
| Penicillin | 335.07 | 217.10 | 56 | 16 |
| Sulfamethoxazole | 254.02 | 156.00 | 28 | 16 |
| Sulphadiazine | 251.02 | 155.99 | 24 | 16 |
| Tylosin | 916.55 | 174.12 | 90 | 36 |
| Erythromycin | 734.49 | 158.12 | 44 | 38 |
| Acarbose | 646.28 | 304.19 | 40 | 26 |
| Hygromycin | 528.26 | 177.11 | 52 | 32 |
Finally it should be noted that：Above example only to illustrate technical scheme, rather than a limitation；Although
The present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those within the art that：It still may be used
To modify to the technical scheme described in previous embodiment, or equivalent is carried out to which part technical characteristic；And
These modifications are replaced, and do not make the spirit and model of the essence disengaging embodiment of the present invention technical scheme of appropriate technical solution
Enclose.
Claims (4)
1. the method that high pressure liquid chromatography-mass spectrometry detects Multiple Classes of Antibiotics content in water, it is characterised in that including following step
Suddenly：
1) pH of water sample is adjusted：A 250mL water samples are measured, appropriate formic acid is added, pH to 2.0 is adjusted, a 250mL is separately measured
Water sample, adds appropriate ammoniacal liquor, adjusts pH to 12.0；
2) the solid phase extraction concentration concentration of water sample after pH is adjusted：Extracted with 5mL methyl alcohol and 10mL deionized waters activated solid respectively
Post 1, flow velocity is that the 250mL water samples that pH is adjusted to 2.0 are crossed into solid-phase extraction column after 5-10mL/min activation, and flow velocity is 3-5mL/min,
After the excessively complete solid-phase extraction column of water sample, with 5mL deionized waters solid-phase extraction column is got express developed, flow velocity is 5-10mL/min, Ran Hougan
Dry solid-phase extraction column 30min, finally elutes solid-phase extraction column with 5mL methyl alcohol with 3mL/min flow velocitys, collects eluent, is labeled as
Eluent 1；Respectively with 5mL methyl alcohol and 10mL deionized water activated solids extraction column 2, flow velocity is by pH after 5-10mL/min activation
The 250mL water samples for being adjusted to 12.0 cross solid-phase extraction column, and flow velocity is 3-5mL/min, after the excessively complete solid-phase extraction column of water sample, is gone with 5mL
Ionized water gets solid-phase extraction column express developed, and flow velocity is 5-10mL/min, is then dried solid-phase extraction column 30min, finally uses 5mL first
Alcohol elutes solid-phase extraction column with 3mL/min flow velocitys, collects eluent, is labeled as eluent 2；Merge eluent 1 and 2, at 40 DEG C
In water-bath, dried up with gentle nitrogen, be finally settled to 1mL with water, that is, obtain sample solution.
3) using high pressure liquid chromatography-mass spectrograph detection：
A. the preparation of standard liquid：Standard items antibiotic is accurately weighed respectively, using deionized water dissolving, 0.2mg/ is made into respectively
The Standard Stock solutions of mL, according to the response of each antibiotic when using, deionized water is diluted to standard working solution；
B. using high pressure liquid chromatography-mass spectrograph examination criteria working solution and sample solution：Mark is drawn respectively by automatic sampler
Quasi- working solution and sample solution, in injection high pressure liquid chromatography-mass spectrograph, the antibiotic group lease making liquid-phase chromatographic column in sample point
From the detection of mass spectrograph multiple-reaction monitoring pattern.
4) data analysis：With the parent ion and daughter ion of compound as quota ion and qualitative ion；With the matter of standard working solution
Amount concentration X is abscissa, and the peak area Y of quantitative and qualitative ion is ordinate, draws standard working curve, uses standard working curve
Single ion quantified by external standard method is carried out to sample；With the chromatographic peak retention time and parent ion/daughter ion pair that occur in sample solution
Carry out qualitative.
2. the method that high pressure liquid chromatography as claimed in claim 1-mass spectrometry detects Multiple Classes of Antibiotics content in water, it is special
Levy and be, formic acid used and ammoniacal liquor are top pure grade in the step 1.
3. the method that high pressure liquid chromatography as claimed in claim 2-mass spectrometry detects Multiple Classes of Antibiotics content in water, it is special
Levy and be, solid-phase extraction column used is Waters Oasis HLB solid-phase extraction columns in the step 2, and specification is 6cc/
500mg, 60um.
4. the method that high pressure liquid chromatography as claimed in claim 1-mass spectrometry detects Multiple Classes of Antibiotics content in water, it is special
Levy and be, the antibiotic standard items in the step 3 are respectively hygromycin, Lovastatin, acarbose, tylosin, red mould
Element, sulphadiazine, gentamicin, penicillin, sulfamethoxazole.
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