Bletsou, Anna

Digital Repository of Hellenic Managing Authority of the Operational Programme &quot;Education and Lifelong Learning&quot; (EDULLL)

HILIC-QTOF-HR-MS/MS FOR WIDE-SCOPE SCREENING OF POLAR MICROPOLLUTANTS IN ENVIRONMENTAL SAMPLESAnna BletsouLaboratory of Analytical ChemistryDepartment of ChemistryUniversity of AthensTarget screeningSuspect screeningNon-target screeningEnvironmental AnalysisData Processing IdentificationConfirmationSample& Sample preparationLC-QTOFTechnique of choice:Reverse-Phase Liquid ChromatographySmart alternative for polar compounds Hydrophilic Interaction LIquidChromatography(HILIC)Why HILIC ? Retention of polar components → higher  intensity  Several different stationary phases available MS compatible Use of ACN (low viscosity solvent) → higher flow rates & better ionization Complex mechanistic separation Great effort for the method optimization and development• Stationary phase• Mobile phase (solvents, buffer, pH)• Gradient elution program & flow rate• Column temperature• Vial compositionWide-scope screening of emerging pollutantsMethod development• BEH Amide• BEH (silica)• Luna (diol)• ZIC-p-HILIC• TSK-gel (amide)Stationary phase• Ammonium Acetate  1, 5, 10 mM• Ammonium Formate1, 5, 10 mM• Formic Acid 0.01%, 0.05%Mobile phase composition •80:20•90:10•95:5•95:5 (0.01% F.A.)Vial composition(ACN:H2O)HILIC OptimizationM.P.(+) ESI: (A) H2O, 1mM Amm. Form. 0.01% F.A.(B) ACN:H2O (95:5), 1mM Amm. Form. 0.01% F.A.(-) ESI: (A) H2O, 10mM Amm. Form.(B) ACN:H2O (95:5), 10mM Amm. Form.  Flow rate: 200 μL/min Column T: 40 °C Chromatogram: 20 min (+5 min re-equilibration)Method development Positive & Negative ESI bbCID modebbCID modeLow CE (4 ev) (pass all)MS spectraHigh CE (25 ev) (fragment all)MS/MS spectraMaXis ImpactUltra High ResolutionTime-of-Flight Mass SpectrometerDatabase902 compounds- 601 well-retained compounds (k’>1) -EPs, belonging to a diverse group of compoundsChosen according to environmental relevance & HILIC chromatographic behaviorMethod validationvalidation dataset  85 compounds 10% of the compounds of the total database Representative physicochemical properties Compounds from every class of EPs Calibration curves (solvent, matrix & spiked samples) (6 levels of concentration) Repeatability, recoveries and matrix effect The screening detection limit (SDL) and the limit of identification (LOI):• SDL: the lowest concentration level tested for which a compound was detected in all samples; tR + Precursor ion= 2 Identification Points (2 IPs)• LOI: the lowest concentration tested for which a compound was satisfactorily identified in all spiked samples; tR + Precursor ion + fragment = 4 Identification Points (4 IPs) 100 mL wastewater (GFF filtration) IS spiking (100 ng/L) SPE           Mixed-bed cartridges Extraction: Neutral, Basic & Acidic CompoundsStrata-XMixture:Strata-XCW,Strata-XAW,ENVI+ 100 times pre-concentrationLocation: WWTP of Athens, GreecePeriod: 8th March 2015 (Sunday)Samples: 24-h composite flow-proportionalinfluent & effluent wastewaterSample Preparation:*Kern et al. EST (2009) 43(18):7039Sample & Sample Preparationas performed in RP target screening method.Validation ResultsLinearityAmisulpride-N-oxidey = 772353x + 12721R² = 0.98580.E+001.E+052.E+053.E+054.E+055.E+050 0.125 0.25 0.375 0.5AreaConc. (μg/L)Standard addition Curvey = 933726x + 16903R² = 0.98370.E+001.E+052.E+053.E+054.E+055.E+056.E+050 0.125 0.25 0.375 0.5AreaConc. (μg/L)Matrix-matched solutions CurveScreening Detection Limits (SDL) – Limits of Identification (LOI)051015202530350.0025 0.0125 0.025 0.05 0.25 0.5% AnalytesC (μg/L)SDLLOIValidation Results051015202530<50 50-80 80-100 100-120 >120% Analytes% Recoveries% Matrix Effect>-50-50 - 00-2020-5050-8080-100signalsuppressionsignalenhancement% Repeatability (n=6)(RSD%) 0.25 μg/L: 3.4-16 % 0.025 μg/L: 6.0-17 % 0.0025 μg/L: 11- 21 %Wastewater ResultsInfluent wastewaterEffluent wastewater256 compounds257 compounds336 compounds detected in totalCriteria• Ion Intensity > 250 (+ESI) / 150 (-ESI)• Peak Area > 1000 (+ESI) / 600 (-ESI)• deltaRT ≤ 0.4 min• Accuracy: Error ≤ 2.5 mDa• Isotopic fit: ≤ 100 mSigmaWastewater Results2-phenethyamineC8H11NHILICRPAmphetamineC9H13N HILICRPHILIC Better retention Better peak shape Higher intensityWastewater ResultsGuanylureaC2H6N4OHILICRPEthyl sulfateC2H6O4S HILICRP(-) ESIWastewater ResultsHILIC Compounds NOT eluted in RP mode Dexamethasone2.5 mDa, 53 mSigmaOxprenolol0.2 mDa, 32 mSigmaPyrimidinol0.4 mDa, 35 mSigmaTyramine0.1 mDa, 5 mSigmaHILIC Vs RP2327 compoundsdatabase902 compoundsHILIC RP371 compoundsdetected336 compounds203 compoundsin common133 compoundsonly in HILIC168 compoundsonly in RPFurther evaluation Why not detect some compounds in RP or HILIC mode?104/133 present in RP db95/168 present in HILIC dbConclusions Development of HILIC wide-scope target method Optimization & validation of the HILIC method In-house database with information for 902 compounds Application in influent & effluent wastewater samples Comparison with RP target screening method Complementary technique for target screening Use in suspect & non-target screening for additional informationAcknowledgments to..Alexandros MarkatisNikolaos Thomaidis& our collaborators fromThank you for your attention!This research has been co-financed by the European Union and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) ARISTEIA 624 (TREMEPOL project).

HILIC-QTOF-HR-MS/MS for wide-scope screening of polar micropollutants in environmental samples

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HILIC-QTOF-HR-MS/MS for wide-scope screening of polar micropollutants in environmental samples

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Digital Repository of Hellenic Managing Authority of the Operational Programme "Education and Lifelong Learning" (EDULLL)

HILIC-QTOF-HR-MS/MS for wide-scope screening of polar micropollutants in environmental samples

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Digital Repository of Hellenic Managing Authority of the Operational Programme &quot;Education and Lifelong Learning&quot; (EDULLL)

Digital Repository of Hellenic Managing Authority of the Operational Programme "Education and Lifelong Learning" (EDULLL)