HILIC-QTOF-HR-MS/MS for the orthogonal (complementary) screening and identification of polar micropollutantsin environmental samples Anna Bletsou Laboratory of Analytical Chemistry Department of Chemistry University of Athens Environmental Analysis Target Suspect Non-Target Screening Screening Screening Human Technique of choice: Wide-scope Unknown metabolites – Reverse-Phase method for compounds in Environmental 2327 EPs wastewaters Liquid Chromatography TPs (RP) -MS Smart Scope of the study alternative for polar compounds HILIC wide- scope target Application of HILIC method for additional method Hydrophilic Interaction confirmation in Suspect& Non-Target LIquidChromatography Optimization- (HILIC) -MS validation- Screening application to real samples HILIC Advantages ü Retention of polar components → higher intensity ü Several different stationary phases available ü MS compatible ü Use of ACN (low viscosity solvent) → higher flow rates & better ionization ü Combination of 3 major LC techniques (NR, RP, IEX) • Complex mechanistic separation (Adsorption, Partitioning, H bonding, Ion exchange) • Great effort for the method optimization and development • More equilibration time Method development HILIC stationary phases Unmodified bare Polar chemically silica gels boned phases (c) TSKgelAmide Tosoh Bioscience (2.0 × 150 mm, 3 μm) (a) (d) Luna HILIC PHENOMENEX BEH HILIC Acquity (2.0 × 100 mm,3 (2.1 × 100 mm, 1.7 μm) μm) (b) BEH Amide Acquity (2.1 × 100 mm, 1.7 μm) (e) ZIC-pHILICSeQuant (2.1 × 150 mm, 5 μm) Method development HILIC Optimization Mobile phase •BEH Amide composition •80:20 •BEH (silica) •Luna (diol) •Ammonium Acetate •90:10 1, 5, 10 mM •ZIC-p-HILIC •95:5 •Ammonium Formate •95:5 (0.01% F.A.) •TSK-gel (amide) 1, 5, 10 mM •Formic Acid 0.01%, Stationary 0.05% Vial composition phase (ACN:H2O) M.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 MaXisImpact § bbCIDmode Ultra High Resolution Time-of-Flight Mass bbCIDmode Spectrometer Low CE (4 ev) (pass all) " MS spectra High CE (25 ev) (fragment all) " MS/MS spectra bbCIDmode † Target Analysis AutoMSmode † Suspect Screening † Non-target screening Database Chosen according to EPs, belonging to a 902 compounds environmental relevance diverse group of & compounds 601 well-retained compounds (k’>1) HILIC chromatographic behavior Sampling & Sample preparation Location: WWTPofAthens,Greece Period: 8th March2015(Sunday) Samples: 24-hcompositeflow-proportional influent&effluentwastewater SamplePreparation: ü 100 mL wastewater (GFF filtration) as performed in RP target screening method. ü IS spiking (100 ng/L) ü SPE Mixed-bed cartridges Strata-X ü Extraction: Neutral, Basic & Acidic Compounds Mixture: Strata-XCW, " 100 times Strata-XAW, pre-concentration ENVI+ *Kern et al. EST (2009) 43(18):7039 Method validation † validation dataset v 85 compounds v 10% of the compounds of the total database v Representative physicochemical properties v 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) Validation Results Linearity Amisulpride-N-oxide Standard addition Curve 5.E+05 Matrix-matched solutions Curve 6.E+05 4.E+05 y = 772353x + 12721 5.E+05 y = 933726x + 16903 R² = 0.9858 R² = 0.9837 ea 3.E+05 r 4.E+05 A ea r 2.E+05 A 3.E+05 2.E+05 1.E+05 1.E+05 0.E+00 0.E+00 0 0.125 0.25 0.375 0.5 0 0.125 0.25 0.375 0.5 Conc. (μg/L) Conc. (μg/L) Screening Detection Limits (SDL) –Limits of Identification (LOI) 35 30 SDL 25 es LOI t y l 20 a n 15 A % 10 5 0 0.0025 0.0125 0.025 0.05 0.25 0.5 C (μg/L) Validation Results % Recoveries 30 25 % Matrix Effect es t 20 y l a n 15 signal A % 10 suppression 5 >-50 0 <50 50-80 80-100 100-120 >120 -50 - 0 0-20 20-50 % Repeatability (n=6) 50-80 (RSD%) 80-100 § 0.25 μg/L: 3.4-16 % signal § 0.025 μg/L: 6.0-17 % enhancement § 0.0025 μg/L: 11-21 % Validation Results º Comparison RP –HILIC » 58 compounds Common in RP & HILIC validation (representative tR) Matrix Effect signal signal HILIC enhancement suppression RP >-50 -50 - 0 0-50 50-100 Validation Results º Comparison RP –HILIC » Sensitivity Slope (b), standard addition curve • 19% compounds → higher sensitivity in HILIC 11020 Due to tR ? 9 ? 100 Due to physicochemical properties ILIC 8 H 7 e 80 p 6 o l 60 /s 5 P 4 R 40 e 3 p o l 2 s 20 1 00 00 55 1010 1515 reternettieonnt tiiomn eti (mmei n)(min) Screening Detection Limit –Limit of Identification SDL-LOI • 38% compounds → lower SDL-LOI in HILIC …compounds different • 51% compounds → lower SDL-LOI in RP fragmentation pattern RP- • 11% compounds → equal SDL-LOI HILIC Validation Results º Comparison RP –HILIC » Different fragmentation pattern MS/MS spectra Spiked sample 0.5 μg/L Intens. -bbCID MS, 3.7-3.7min #423-425, Background Subtracted, Background Subtracted [%] 12.5 RP O 181.9896 O [M-H]- 10.0 S C7H5NO3S 7.5 N 181.9917 5.0 O 2.5 149.0478 165.0172 211.0508 66.0329 83.0230 95.9317 102.9567 112.9873 122.0302 134.0276 194.0876 203.9398 0.0 60 80 100 120 140 160 180 200 m/z Intens. -bbCID MS, 1.82-1.85min #391-397, Background Subtracted, Background Subtracted [%] HILIC 30 149.0611 181.9916 20 82.9539 - 190.9579 CNO3S 10 105.9604 126.9961 178.0501 105.9604 41.9991 92.9949 66.9590 78.9840 100.9260 135.0828 163.1140 199.0416 0 40 60 80 100 120 140 160 180 200 m/z Wastewater Results 336 compounds Criteria detected in total • Ion Intensity > 250 (+ESI) / 150 (-ESI) • Peak Area > 1000 (+ESI) / 600 (-ESI) • deltaRT ≤ 0.4 min Influent • Accuracy: Error ≤ 2.5 mDa wastewater 256 compounds • Isotopic fit: ≤ 100 mSigma Intens. x1056 2.0 Benzyl-dimethyl-dodecylammonium MeitsforlLeueuminccineine (Ile) 1.0 01.8.5 0.6 2-Nphe--ONn-Dyliedethsymlaemthineylvenlafaxine 1.0 cotinine 0.4 nicotine 0.5 1-7-dTimheeophthyylxliannethine Benzyl-dimethyl-dodecylammphoonOliedum-Dreinsem (teprat-hmyadld-evriopeolsxyhednollaLeeu-fmariucxneeciitneneihney l(aIlme)phetamine) 0.2 Climbazole N-DesmethyAlvmenphelafataxminieneO (xnoprrevennolaolfaxine) Caffeine cotinine v2en-ApheNmlph-a-ONfiasne-xuDyndillinepedirtem2ihdes-yephemltareamtznhiiyneynellevtehnylalafamxiinene 1-7-dTimheopethyhlyxliannethine phoOledp-VeDrroTAienparnphosamelma fmie(fstapernlueax-tdonehltdmipoopneyrrliad-dlin-dD6er -rveoD6eopeol l-xyhedno(D5pla --hmfriayNnexednephiotrneihoCrceyxoaTpleytatyhedihd-nemriraneipnemerihtneiheneytalammine)phetamine) BenBzenoBtzhenoiaBthzeioantlzerio a(CltzBeCraoil TH)ia(mrlCBezboff eobaaTe(ilfrBH)tneeubhzT roopae(oanR)BfCtleuhoT-ra3aR)brp-nCbaahaba-teay3mrd-bhrateoyzmxydepraozixynepe-i1pn0ire--a11p10cire-a-dt1cami1he-ytdamdihroy-d10ro--110--d11ih-yddihroyxydroxy N-DeNs-NmD-deDiNtsmehCm-smpyMeT4diV3dcmtrletr-evaoeThi mAenNtventp4sephelpaLthrCyaenoppho2mhara-AteBiyMleopTmdel4hid-alocttE3falmAitlrer-pyahBovAmspaiehfNiena ftrayeptsnlhyamphenc4opuriftLyeneedvthmosaphexamln2doharBmoneend1hlxea-enlitlop-yipCermuxopdoail-hopph-noy2irEtirneepmazArlneneBesbmrTDthliPolr aiNl-pneimaef-yrneoCtahdyimrctliD6yt6r iehrtcrioli-fnetamoaPmplpheened hofp1neopaneredlx2alD6hnNtadeep(yo-aediCermP hehila-r-zyxhenno2inohityepmzyNneo(exycltD6oberpemD5phtnThoPr epaanri-ilamilttoCnelteirhynt iit-rollamPrhnecrifnloaPpinm hinrvgneepolxNneayeoO(AnredixyCrenmPifyzzyoeabahenxednondci payNtpeophhVednemlycpetpeizor(homaepdiexcod anyrioiphyiMnenootepairtSoeglTnnelrtplioanrnehtmNPiradpneinCroerlivimyexaAiGA)hrterblyneafinrhAoamrneeinrCxhtfiacro CeytpezRnTedvnei VphedophlhmztiepnaphexiRaabaAn(tneerCmyedephnox(ocenbaenoneintodepheic-ryMadiepianBSneilmTahll -neanitoaolrodanNne)(acdnemloeityliia ZimrA)loVanetAGlli rtafhioneoli(ahamrfinot(neebrchpzPCRmnt ritineoelinRaPoxtiritfedpheA(oienexCrtinienienoda-rneeicadtBT)ianapephl)nemOildibaplanlhe) ihaitnerxol-riodne)acyoe(nomtenZneisrD7ol yzD)Vat aionnet irafi(n nein(P)athirtlaoCimcelPtoatilneeoe)leiinned pseTt-irnel)dOmhe) cyliopaNSfaneiir-olnoy(eneinesrolD7neMDzD)tnoo iphr-ir ninaneolacalel nx-smpirte(fcyNS fopaA)eidTriene(tneiiMa- iynerdeo(lonmr)aNDx m (fa)iidTrieA)ne)ineiy Bde(r)Nu a)fexBaumfeacxamac 0.0 1 1 2 2 3 3 4 4 5 5 6 6 7 7 Time [min] Effluent wastewater Intens. 257 compounds x105 Climbazole 1.0 Irbesartan 0.8 0.6 Amisulpride 1-54M-Meteh-yBl-enBeznoztoritarizaozleole 0.4 Benzyl-dimethyl-dovdeencylafalaxminemonium N-Desmethylventrlafmaxaidnole (norvenlafaxine) Ranitidine 0.2 Benzotriazole (BTR) Celiprolol 5-Methyl-Benzotriazole-D6 phendimetrazine Metformin Fluconazole piracetam 3 4-metho2yrt-phlrphenameeenadnadodeyitolaerixlft-needyhN-yN- rolia-nexmideeinethylamphetamine (MDMA) allaoll-oTT-HTHTFHFFHF ( A( T( llA(eTCotlletraoCeahtrtrtbaearyarthbarIdhmysahrydoamdrpyczorardoepcozrCcocteoiarcsnptrrrboiePtibasneirl-syo tDoiCmrls 8irlmoC aao5?rlrazi rbado5 rCe4-5bir?Pnp-a ma-F5aolirP-mneegbeoaPr-rzaerPmn4ezgran-dagnapyepnanegnA4ilne-ac-zAi3mneimn-ca-?et3?e10iaene-no-c-1110mt3o-a--1atim3?-di1?n-111a-11oait17-ido-idne-1p-1bidn1y-ha7-21itbry17ahin-ip-21nedyt1-yirTdp7or--eriyaT-21onet10r-er-i2o1net-1r-tl-0o1e--20tl1t11-er20-otd-r-ol-iod-hn20oneliy-he20dy)-