CN113109481A - 一种固相萃取-液相色谱串联质谱法同时检测饮用水源水体中四类13种抗生素的方法 - Google Patents
一种固相萃取-液相色谱串联质谱法同时检测饮用水源水体中四类13种抗生素的方法 Download PDFInfo
- Publication number
- CN113109481A CN113109481A CN202110475591.8A CN202110475591A CN113109481A CN 113109481 A CN113109481 A CN 113109481A CN 202110475591 A CN202110475591 A CN 202110475591A CN 113109481 A CN113109481 A CN 113109481A
- Authority
- CN
- China
- Prior art keywords
- phase extraction
- antibiotics
- methanol
- formic acid
- water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 55
- 239000003242 anti bacterial agent Substances 0.000 title claims abstract description 43
- 229940088710 antibiotic agent Drugs 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 37
- 239000003651 drinking water Substances 0.000 title claims abstract description 13
- 235000020188 drinking water Nutrition 0.000 title claims abstract description 13
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 title claims abstract description 11
- 239000007790 solid phase Substances 0.000 title claims abstract description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 87
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 60
- 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 abstract description 29
- 235000019253 formic acid Nutrition 0.000 claims abstract description 29
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 28
- 238000001514 detection method Methods 0.000 claims abstract description 19
- 239000012528 membrane Substances 0.000 claims abstract description 13
- 239000007864 aqueous solution Substances 0.000 claims abstract description 10
- 238000001914 filtration Methods 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000003365 glass fiber Substances 0.000 claims abstract description 7
- 230000008569 process Effects 0.000 claims abstract description 5
- 238000001291 vacuum drying Methods 0.000 claims abstract description 5
- 238000010812 external standard method Methods 0.000 claims abstract description 4
- 239000000945 filler Substances 0.000 claims abstract description 4
- 238000011010 flushing procedure Methods 0.000 claims abstract description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 4
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 4
- 230000003213 activating effect Effects 0.000 claims abstract description 3
- 239000012491 analyte Substances 0.000 claims abstract description 3
- 238000001035 drying Methods 0.000 claims abstract description 3
- 150000002500 ions Chemical class 0.000 claims description 19
- 239000012071 phase Substances 0.000 claims description 13
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 6
- 239000005695 Ammonium acetate Substances 0.000 claims description 6
- 239000007789 gas Substances 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 5
- 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 claims description 4
- 239000004098 Tetracycline Substances 0.000 claims description 4
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 claims description 4
- 235000019364 tetracycline Nutrition 0.000 claims description 4
- 150000003522 tetracyclines Chemical class 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000001819 mass spectrum Methods 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 229930101283 tetracycline Natural products 0.000 claims description 3
- RXZBMPWDPOLZGW-XMRMVWPWSA-N (E)-roxithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=N/OCOCCOC)/[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 RXZBMPWDPOLZGW-XMRMVWPWSA-N 0.000 claims description 2
- GSDSWSVVBLHKDQ-UHFFFAOYSA-N 9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid Chemical compound FC1=CC(C(C(C(O)=O)=C2)=O)=C3N2C(C)COC3=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-UHFFFAOYSA-N 0.000 claims description 2
- SPFYMRJSYKOXGV-UHFFFAOYSA-N Baytril Chemical compound C1CN(CC)CCN1C(C(=C1)F)=CC2=C1C(=O)C(C(O)=O)=CN2C1CC1 SPFYMRJSYKOXGV-UHFFFAOYSA-N 0.000 claims description 2
- 239000004099 Chlortetracycline Substances 0.000 claims description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 2
- KIPLYOUQVMMOHB-MXWBXKMOSA-L [Ca++].CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O.CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O Chemical compound [Ca++].CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O.CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O KIPLYOUQVMMOHB-MXWBXKMOSA-L 0.000 claims description 2
- 229960004099 azithromycin Drugs 0.000 claims description 2
- MQTOSJVFKKJCRP-BICOPXKESA-N azithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)N(C)C[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 MQTOSJVFKKJCRP-BICOPXKESA-N 0.000 claims description 2
- CYDMQBQPVICBEU-UHFFFAOYSA-N chlorotetracycline Natural products C1=CC(Cl)=C2C(O)(C)C3CC4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-UHFFFAOYSA-N 0.000 claims description 2
- 229960004475 chlortetracycline Drugs 0.000 claims description 2
- CYDMQBQPVICBEU-XRNKAMNCSA-N chlortetracycline Chemical compound C1=CC(Cl)=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-XRNKAMNCSA-N 0.000 claims description 2
- 235000019365 chlortetracycline Nutrition 0.000 claims description 2
- 229960003405 ciprofloxacin Drugs 0.000 claims description 2
- NOCJXYPHIIZEHN-UHFFFAOYSA-N difloxacin Chemical compound C1CN(C)CCN1C(C(=C1)F)=CC2=C1C(=O)C(C(O)=O)=CN2C1=CC=C(F)C=C1 NOCJXYPHIIZEHN-UHFFFAOYSA-N 0.000 claims description 2
- 229950001733 difloxacin Drugs 0.000 claims description 2
- 229960001484 edetic acid Drugs 0.000 claims description 2
- 229960000740 enrofloxacin Drugs 0.000 claims description 2
- 229960003276 erythromycin Drugs 0.000 claims description 2
- 229960001699 ofloxacin Drugs 0.000 claims description 2
- 229960005224 roxithromycin Drugs 0.000 claims description 2
- SEEPANYCNGTZFQ-UHFFFAOYSA-N sulfadiazine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=NC=CC=N1 SEEPANYCNGTZFQ-UHFFFAOYSA-N 0.000 claims description 2
- 229960004306 sulfadiazine Drugs 0.000 claims description 2
- 229960002135 sulfadimidine Drugs 0.000 claims description 2
- ASWVTGNCAZCNNR-UHFFFAOYSA-N sulfamethazine Chemical compound CC1=CC(C)=NC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 ASWVTGNCAZCNNR-UHFFFAOYSA-N 0.000 claims description 2
- 229960005404 sulfamethoxazole Drugs 0.000 claims description 2
- 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 2
- 229940063650 terramycin Drugs 0.000 claims description 2
- 229960002180 tetracycline Drugs 0.000 claims description 2
- 238000011084 recovery Methods 0.000 abstract description 25
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 abstract description 4
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 238000002552 multiple reaction monitoring Methods 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 7
- 239000003480 eluent Substances 0.000 description 7
- 238000000605 extraction Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 230000003115 biocidal effect Effects 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 229940072172 tetracycline antibiotic Drugs 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 229940123317 Sulfonamide antibiotic Drugs 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000003120 macrolide antibiotic agent Substances 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000002957 persistent organic pollutant Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 229940124530 sulfonamide Drugs 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- KYGZCKSPAKDVKC-UHFFFAOYSA-N Oxolinic acid Chemical compound C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC2=C1OCO2 KYGZCKSPAKDVKC-UHFFFAOYSA-N 0.000 description 1
- 208000035896 Twin-reversed arterial perfusion sequence Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 150000007514 bases Chemical class 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 229940041033 macrolides Drugs 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 239000003306 quinoline derived antiinfective agent Substances 0.000 description 1
- 150000007660 quinolones Chemical class 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 1
- 150000003456 sulfonamides Chemical class 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 229940040944 tetracyclines Drugs 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- 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
- G01N30/08—Preparation using an enricher
-
- 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
-
- 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
-
- 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
- G01N2030/042—Standards
- G01N2030/047—Standards external
-
- 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
- G01N2030/062—Preparation extracting sample from raw material
Landscapes
- 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
本发明公开了一种固相萃取‑液相色谱串联质谱法同时检测饮用水源水体中四类13种抗生素的方法,按以下步骤进行:水样经玻璃纤维膜过滤后,加入乙二胺四乙酸二钠,用甲酸调节pH等于3;依次用甲醇和甲酸水溶液活化HLB固相萃取小柱,将步骤一中处理过的水样过HLB固相萃取小柱,整个过程始终保持液面高于HLB固相萃取小柱填料上端,待水样处理完成后,用甲酸水溶液冲淋,然后真空干燥,再用甲醇洗脱,将待测分析物收集,用氮气吹干,最后用甲醇定容,过滤膜后待测;液相色谱串联质谱法测定水环境中四类13种抗生素的含量,采用外标法进行定量分析。此方法具有准确、灵敏度高、重现性好、回收率高的优点,可适用于饮用水源中抗生素检测。
Description
技术领域
本发明属于水环境中痕量有机污染物残留检测技术领域,涉及一种水中微量有机污染物的检测方法,特别涉及一种以固相萃取预处理结合液相色谱质谱联用技术,能够高效、同时快速检测出饮用水源水体中四类13种抗生素的快速检测方法。
背景技术
抗生素是应用最广泛的药物之一,主要用于预防和治疗细菌和真菌性疾病。我国是抗生素的生产和使用大国,抗生素滥用现象严重,很多抗生素在被人或动物使用后不能完全被其机体吸收,80%以上的量会排出体外而进入水环境中,从而导致水环境受到抗生素的污染。四环素类、喹诺酮类、大环内酯类和磺胺类四大类抗生素经常在水环境中被检出。但随着抗生素残留在饮用水源水体中的频繁检出,人们对饮用水源地水体中抗生素的污染状况的关注也越来越广泛。
抗生素的检测方法主要有酶联免疫检测技术、气相色谱-质谱联用技术、液相色谱技术等。其中固相萃取-液相色谱/质谱联用法具有应用范围广、分离能力强、灵敏度高和分析速度快等特点,已成为环境水体中抗生素的重要检测分析方法,其检测限可达到ppb级水平。目前报道的测定方法涉及的抗生素种类相对较少,多集中于单一类抗生素化合物,且灵敏度和回收率不高,难以对不同种类的抗生素进行同时开展检测。因此,建立一种快速测定饮用水源水中多种抗生素残留的方法具有重要的现实意义,本发明人对此做进一步研究,研发出一种固相萃取-液相色谱串联质谱法同时检测饮用水源水体中四类13种抗生素的方法,本案由此产生。
发明内容
本发明所要解决的技术问题在于提供一种固相萃取-液相色谱串联质谱法同时检测饮用水源水体中四类13种抗生素的方法,具有准确、灵敏度高、重现性好、回收率高的优点,适用于饮用水源水中抗生素的检测,有很强的实际应用价值。
为解决上述技术问题,本发明的技术解决方案是:
一种固相萃取-液相色谱串联质谱法同时检测饮用水源水体中四类13种抗生素的方法,按以下步骤进行:步骤一、水样经玻璃纤维膜过滤后,加入乙二胺四乙酸二钠,用甲酸调节pH等于3;步骤二、依次用甲醇和甲酸水溶液活化HLB固相萃取小柱,将步骤一中处理过的水样过HLB固相萃取小柱,整个过程始终保持液面高于HLB固相萃取小柱填料上端,待水样处理完成后,用甲酸水溶液冲淋,然后真空干燥,再用甲醇洗脱,将待测分析物收集,用氮气吹干,最后用甲醇定容,过滤膜后待测;步骤三、液相色谱串联质谱法测定水环境中四类13种抗生素的含量,采用外标法进行定量分析,色谱的检测条件为:色谱柱为C18液相色谱柱,流动相A是5mmol·L-1乙酸铵的0.1%甲酸水溶液,流动相B是纯甲醇;13种抗生素分别为土霉素、四环素、金霉素、罗红霉素、阿奇霉素、红霉素、磺胺嘧啶、磺胺甲嘧啶、磺胺甲噁唑、氧氟沙星、环丙沙星、恩诺沙星、二氟沙星。
进一步,等体积的甲醇和甲酸水溶液。
进一步,在步骤二中,水样过HLB固相萃取小柱的流速为3~5ml·min-1。
进一步,在步骤一中,水样过滤所用的过滤膜是孔径为0.45μm的玻璃纤维膜。
进一步,在步骤二中,固相萃取小柱为Oasis HLB小柱。
进一步,在步骤二中,依次用6mL甲醇和6mL的0.5%甲酸水溶液活化Oasis HLB固相萃取小柱,甲醇和0.5%的甲酸水溶液各取3次,每次2mL,待水样处理完成后,用6mL的0.5%甲酸水溶液冲淋,然后真空干燥30min,以去除柱体残留的水分,再用6mL甲醇洗脱3次。
进一步,在步骤三中,色谱的检测条件还包括:流速为0.5mL·min-1,进样体积10μL,色谱柱温40℃。
进一步,在步骤三中,质谱的检测条件如下:电喷雾离子源,正离子模式,离子源Ⅰ与离子源II的气流量分别是50mL·min-1和60mL·min-1,离子源电压为5500V,辅助加热气温度为550℃。
附图说明
图1是本发明的四环素类抗生素的MRM色谱图;
图2是本发明的喹诺酮类抗生素的MRM色谱图;
图3是本发明的磺胺类抗生素的MRM色谱图;
图4是本发明的大环内酯类抗生素的MRM色谱图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步详述。首先,本实施例采用的仪器与试剂如下:
仪器:玻璃纤维滤膜(上海半岛实业有限公司净化器材厂,孔径0.45μm),酸碱pH计(pH2100,中国),固相萃取装置(Waters公司,美国),OASIS HLB固相萃取小柱(200mg,6mL,CNW),恒温水浴氮吹仪(Organomation公司,美国),真空干燥箱(DZF-6050,中国),色谱柱柱(100mm×4.6mm,2.6μm,Phenomenex),安捷伦高效液相色谱-质谱联用仪(ABI 3200Q TRAP LC-MS/MS System,Agilent,美国)。试剂:甲醇、甲酸和乙二胺四乙酸二钠(Na2EDTA)均为色谱纯(纯度≥99.0%),购自阿拉丁集团;实验室用水均为Milli-Q超纯水(≤18.2MΩ)。13种抗生素标准物质纯度均≥95%,购自生工生物工程(上海)股份有限公司。
准确量取500mL水样,经0.45μm的玻璃纤维膜过滤后,加入0.5g乙二胺四乙酸二钠;用5mol·L-1甲酸调节pH约等于3。在此步骤中选用甲酸不但可以起到调节pH,还可以在后续的液质进样过程中保护色谱柱,这是其他常选用的无机酸(例如盐酸、硫酸等)不具有的优点。乙二胺四乙酸二钠的添加主要是为了络合水中的重金属离子,提高四环素类抗生素的回收率。
依次用6mL甲醇和6mL的0.5%甲酸水溶液活化Oasis HLB固相萃取小柱(甲醇和0.5%的甲酸水溶液各取3次,每次2mL)。甲醇可以活化HLB固相萃取小柱,然后以3~5ml·min-1流速加载水样,限制流速的目的是为了延长目标物和萃取小柱里填料的接触时间,从而提高目标物的保留效果。整个过程始终保持液面高于小柱填料上端,目的是保持液体不断流,处于充盈状态。待水样处理完成后,用6mL的0.5%甲酸水溶液冲淋,然后真空干燥30min,以去除柱体残留的水分,再用6ml甲醇洗脱3次,根据目标物吸附能力来选择活化的洗脱量,甲醇的体积是可以限定的,来优化得出最佳的洗脱量,在溶剂用量最少的情况获得最高的回收率。将洗脱液收集到10mL棕色玻璃试管中,用温和的氮气吹干,最后用甲醇定容至1.0mL,过0.22μm滤膜后用HPLC-MS/MS分析测定。
色谱条件:流动相A是5mmol·L-1乙酸铵的0.1%甲酸水溶液,流动相B是纯甲醇;流速为0.5mL·min-1,进样体积10μL;色谱柱温40℃。具体的洗脱程序如表1所示。
表1梯度洗脱程序
质谱条件:电喷雾离子源(ESI),正离子模式,离子源Ⅰ(GSI)与II(GS2)的气流量分别是50mL·min-1和60mL·min-1,离子源电压为5500V,辅助加热气温度为550℃。采用多反应监测(MRM)模式扫描,对各化合物的离子源条件进行了优化,各目标物的质谱参数见表2,各目标物的MRM色谱图详见图1、图2、图3和图4。
表2 MRM模式下13种抗生素的质谱参数
良好的色谱分离效果是对目标物定量和定性的保证,为改善多种抗生素的分离效果并提高分析效率,本发明分别优化了色谱流动相、色谱柱和色谱柱柱温等色谱条件。
色谱流动相的组成除影响保留时间和峰形外,还会影响离子化效率,进而影响目标物的检测灵敏度。乙腈作为有机流动相时,峰形较差,实验改用甲醇作为有机流动相时峰形良好;同时,水相中加入0.5%的甲酸(含5mmol·L-1乙酸铵)可有效改善峰形、增加物质离子化效率和响应强度。这是因为甲酸和乙酸铵是正离子模式下增强目标化学物离子化程度的常用试剂,可有效改善峰形,使峰形更加尖锐和对称,两者具有共同作用影响。
对比了色谱柱(100mm×4.6mm,2.6μm,Phenomenex)和Kromasil 100-5 C18色谱柱(150mm×4.6mm,3.5μm,Akzo Nobel)对13种抗生素的分离效果。结果表明长度稍短的色谱柱不仅可以缩短分析时间,一定程度上实现了各目标物的离子峰的互不干扰,且分离后的抗生素峰形相对更佳。
本实施例还考察了柱温(30℃、40℃和50℃)对组分分离的影响,结果表明,40℃柱温时的峰形最好,组分的分离效果较好,这是因为适宜的柱温可以降低流动相的粘度,缩短平衡时间。
综上所述,根据目标物的性质和电离后得到的离子模式,对色谱和质谱工作条件进行优化,得到各化合物的MRM色谱图见图1至图4。
本发明中还考察了固相萃取小柱、pH、上样体积和洗脱液体积等因素对水样萃取的影响,优化了固相萃取条件。
选取Oasis HLB和C18固相萃取小柱,对比考察不同类型萃取柱对回收率的影响。结果表明,Oasis HLB对四类抗生素的萃取效率为65%~120%,而C18小柱对于磺胺类抗生素萃取效率低于50%。这可能是由于Oasis HLB固相萃取小柱填充的是亲水亲脂平衡、水可浸润的反相吸附剂,是一种可适用于酸性、中性及碱性化合物的通用型萃取柱;而C18小柱是硅胶上接十八烷基的反相键合硅胶,主要对极性偏弱的化合物保留较好。同时,HLB小柱的比表面积也大于C18小柱。因此,选择Oasis HLB固相萃取小柱对样品进行富集和净化。
样品的酸度也是影响萃取效率的重要因素,实验中采用甲酸和氨水将水样调至不同的pH值(pH=3.0,5.0,7.0),考察其对抗生素回收率的影响。结果表明,当pH值为7.0时,四环素类和磺胺类抗生素的回收率偏低,为40%~75%;当pH值为3.0和5.0时,目标化合物的回收率均高于65%,且差异不显著。综合考虑到四环素类抗生素的pKa值为2.3~3.3,为保证四类抗生素的回收率,选择在过小柱前调节水样pH值为3.0。
选用不同上样水样体积(500mL和1000mL)进行加标100ng·L-1的回收实验(详见表3)。结果表明,体积为500mL的回收率为67.5%~124.8%;而体积1000mL的回收率却降低为40.1%~87.4%,这是因为水样体积过大,会导致固相萃取小柱过载,从而影响回收率,与文献报道的研究结果一致。因此,选择上样水样体积500mL。
考察了洗脱液体积(4、6、8和10mL)对抗生素回收率的影响(见表3)。各目标化合物在洗脱液体积为4mL时,回收率为39.1%~86.1%,洗脱液体积为6、8和10mL时,各目标化合物的回收率为70.4%~117.8%,较高且变化不大。这可能是因为溶剂量少时,洗脱液与填料接触时间短,解吸不充分。考虑增加洗脱液体积可能会导致杂质被洗脱而影响净化效率,因此,洗脱液的体积确定为6mL。
表3不同上样体积和洗脱液体积下13种抗生素的回收率
按上述优化的SPE-HPLC-MS/MS方法进行了线性范围、检出限、回收率和精密度的考察。采用外标法进行定量分析,线性方程浓度由10、20、50、100、200、250μg/L的6个浓度值组成,其相关系数R2都大于0.99。按照富集倍数换算,本方法在0.02~0.5ng/L范围内线性关系良好。根据测定结果绘制标准工作曲线,得出13种抗生素的浓度(x)与峰面积(Y)所存在的线性关系,13种抗生素存在的线性关系见表4。抗生素浓度的检出限是3倍信噪比,定量限是10倍信噪比。
表4 13种抗生素的线性方程、检出限和定量限
对超纯水和水源地水样进行加标实验,考察方法的精密度和回收率。实验加标浓度为20ng/L、200ng/L和1000ng/L的低、中、高的三个水平浓度,以3次平行测得值计算回收率和相对标准偏差(RSD),结果见表5。3种不同加标浓度的回收率为60.2%~120.1%,RSD为1.1%~14.9%,表明该方法准确、可靠,可用于环境水样中13种抗生素的同时测定和分析。
表5 13种抗生素在纯水和水源水水样中加标回收率和相对标准偏差(n=3)
以上所述,仅是本发明的较佳实施例而已,并非对本发明的技术范围作任何限制,故但凡依本发明的权利要求和说明书所做的变化或修饰,皆应属于本发明专利涵盖的范围之内。
Claims (8)
1.一种固相萃取-液相色谱串联质谱法同时检测饮用水源水体中四类13种抗生素的方法,其特征在于:按以下步骤进行:步骤一、水样经玻璃纤维膜过滤后,加入乙二胺四乙酸二钠,用甲酸调节pH等于3;步骤二、依次用甲醇和甲酸水溶液活化HLB固相萃取小柱,将步骤一中处理过的水样过HLB固相萃取小柱,整个过程始终保持液面高于HLB固相萃取小柱填料上端,待水样处理完成后,用甲酸水溶液冲淋,然后真空干燥,再用甲醇洗脱,将待测分析物收集,用氮气吹干,最后用甲醇定容,过滤膜后待测;步骤三、液相色谱串联质谱法测定水环境中四类13种抗生素的含量,采用外标法进行定量分析,色谱的检测条件为:色谱柱为C18液相色谱柱,流动相A是5mmol·L-1乙酸铵的0.1%甲酸水溶液,流动相B是纯甲醇;13种抗生素分别为土霉素、四环素、金霉素、罗红霉素、阿奇霉素、红霉素、磺胺嘧啶、磺胺甲嘧啶、磺胺甲噁唑、氧氟沙星、环丙沙星、恩诺沙星、二氟沙星。
2.根据权利要求1所述的方法,其特征在于:在步骤二中,等体积的甲醇和甲酸水溶液。
3.根据权利要求1所述的方法,其特征在于:在步骤二中,水样过HLB固相萃取小柱的流速为3~5ml·min-1。
4.根据权利要求1所述的方法,其特征在于:在步骤一中,水样过滤所用的过滤膜是孔径为0 .45μm的玻璃纤维膜。
5.根据权利要求1所述的方法,其特征在于:在步骤二中,固相萃取小柱为Oasis HLB小柱。
6.根据权利要求1所述的方法,其特征在于:在步骤二中,依次用6mL甲醇和6mL的0.5%甲酸水溶液活化Oasis HLB固相萃取小柱,甲醇和0.5%的甲酸水溶液各取3次,每次2mL,待水样处理完成后,用6mL的0.5%甲酸水溶液冲淋,然后真空干燥30min,以去除柱体残留的水分,再用6mL甲醇洗脱3次。
7.根据权利要求1所述的方法,其特征在于:在步骤三中,色谱的检测条件还包括:流速为0.5mL·min-1,进样体积10μL,色谱柱温40℃。
8.根据权利要求1所述的方法,其特征在于:在步骤三中,质谱的检测条件如下:电喷雾离子源,正离子模式,离子源Ⅰ与离子源II的气流量分别是50mL·min-1和60mL·min-1,离子源电压为5500V,辅助加热气温度为550℃。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110475591.8A CN113109481A (zh) | 2021-04-29 | 2021-04-29 | 一种固相萃取-液相色谱串联质谱法同时检测饮用水源水体中四类13种抗生素的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110475591.8A CN113109481A (zh) | 2021-04-29 | 2021-04-29 | 一种固相萃取-液相色谱串联质谱法同时检测饮用水源水体中四类13种抗生素的方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113109481A true CN113109481A (zh) | 2021-07-13 |
Family
ID=76720922
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110475591.8A Pending CN113109481A (zh) | 2021-04-29 | 2021-04-29 | 一种固相萃取-液相色谱串联质谱法同时检测饮用水源水体中四类13种抗生素的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113109481A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115469027A (zh) * | 2022-08-30 | 2022-12-13 | 南开大学 | 一种水体中麻痹性贝类毒素的检测方法 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104730168A (zh) * | 2015-03-31 | 2015-06-24 | 中国地质大学(武汉) | 一种水体残留四环素类、氟喹诺酮类和磺胺类抗生素同步检测的方法 |
CN107024548A (zh) * | 2016-07-10 | 2017-08-08 | 华中农业大学 | 同时检测水环境中92种抗菌药物残留的方法 |
US20180059082A1 (en) * | 2015-10-20 | 2018-03-01 | The Florida International University Board Of Trustees | Materials and methods for the detection of trace amounts of substances in biological and environmental samples |
-
2021
- 2021-04-29 CN CN202110475591.8A patent/CN113109481A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104730168A (zh) * | 2015-03-31 | 2015-06-24 | 中国地质大学(武汉) | 一种水体残留四环素类、氟喹诺酮类和磺胺类抗生素同步检测的方法 |
US20180059082A1 (en) * | 2015-10-20 | 2018-03-01 | The Florida International University Board Of Trustees | Materials and methods for the detection of trace amounts of substances in biological and environmental samples |
CN107024548A (zh) * | 2016-07-10 | 2017-08-08 | 华中农业大学 | 同时检测水环境中92种抗菌药物残留的方法 |
Non-Patent Citations (5)
Title |
---|
中国食品药品检定研究院: "《药品检验仪器操作规程及使用指南》", 31 August 2019, 中国健康传媒集团中国医药科技出版社 * |
余胜兵: "超高效液相色谱-串联质谱同时测定饮用水中60种抗生素", 《环境卫生学杂质》 * |
吴莱文: "《治疗药物监测》", 30 June 1989, 人民卫生出版社 * |
李经纬等: "高效液相色谱-串联质谱法同时检测水中17种抗生素", 《分析科学学报》 * |
甘国强等: "固液萃取-超高效液相-串联质谱法同时测定污水处理厂进出水中14种抗生素", 《沈阳药科大学学报》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115469027A (zh) * | 2022-08-30 | 2022-12-13 | 南开大学 | 一种水体中麻痹性贝类毒素的检测方法 |
CN115469027B (zh) * | 2022-08-30 | 2023-11-03 | 南开大学 | 一种水体中麻痹性贝类毒素的检测方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Luks-Betlej et al. | Solid-phase microextraction of phthalates from water | |
Vita et al. | Development and validation of a liquid chromatography and tandem mass spectrometry method for determination of roscovitine in plasma and urine samples utilizing on-line sample preparation | |
Abdel-Rehim et al. | Microextraction in packed syringe/liquid chromatography/electrospray tandem mass spectrometry for quantification of olomoucine in human plasma samples | |
Liu et al. | Automated on-line liquid chromatography–photodiode array–mass spectrometry method with dilution line for the determination of bisphenol A and 4-octylphenol in serum | |
CN105651894B (zh) | 环境土壤中抗生素的测定方法 | |
Wu et al. | Determination of tributyltin by automated in-tube solid-phase microextraction coupled with HPLC-ES-MS | |
Kim et al. | Reversed‐phase liquid chromatographic method for the analysis of aminoglycoside antibiotics using pre‐column derivatization with phenylisocyanate | |
CN102798689A (zh) | 分离富集并检测水环境中痕量氟喹诺酮类抗生素的方法 | |
CN112326812A (zh) | 同位素稀释-OnlineSPE-HRMS同时检测地下水中五种农药的方法 | |
Kim et al. | Development of liquid chromatographic method for the analysis of kanamycin residues in varicella vaccine using phenylisocyanate as a derivatization reagent | |
CN113109481A (zh) | 一种固相萃取-液相色谱串联质谱法同时检测饮用水源水体中四类13种抗生素的方法 | |
Zhao et al. | LC-QTOF-MS method for the analysis of residual pharmaceuticals in wastewater: application to a comparative multiresidue trace analysis between spring and winter water | |
Wang et al. | A rapid and sensitive liquid chromatography/negative ion tandem mass spectrometry method for the determination of an indolocarbazole in human plasma using internal standard (IS) 96‐well diatomaceous earth plates for solid‐liquid extraction | |
CN113419013A (zh) | 一种分析环境水样中全氟烷基酸类污染物的方法及应用 | |
Gang et al. | Development of an analytical method to determine phenolic endocrine disrupting chemicals in sewage and sludge by GC/MS | |
CN210037730U (zh) | 一种质谱分析装置 | |
CN108414654B (zh) | 基于spe柱同时富集检测饮用水中喹诺酮类抗生素的方法 | |
Mekiki et al. | Application of solid-phase microextraction for the analysis of nitropolycyclic aromatic hydrocarbons in water | |
Tiwari et al. | HPLC: a modern approach of development and validation | |
Miyagi et al. | Sugar microanalysis by HPLC with benzoylation: Improvement via introduction of a C-8 cartridge and a high efficiency ODS column | |
Miyashita et al. | A simple and effective method for speciation analysis of 13 arsenic species using HPLC on a fluorocarbon stationary phase coupled to ICP-MS | |
Hsieh et al. | Determination of Dechlorane compounds in aqueous samples using ultrasound-assisted dispersive liquid–liquid microextraction and gas chromatography-electron-capture negative ion-mass spectrometry | |
CN113030300A (zh) | 一种测定农田环境水中兽用抗生素的方法 | |
CN113820421A (zh) | 一种测定水中6种酚类化合物的方法 | |
CN113433241B (zh) | 超声提取-高效液相色谱法测定土壤中联苯胺 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210713 |