HILIC-QTOF-HR-MS/MS for the Orthogonal (Complementary) Screening and Identification of Polar Micropollutantsin Environmental Samples

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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 3.E+05 4.E+05 A r ea

2.E+05 A r ea 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 LOI 20 15

% A n a l y t es 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 20

15 signal

% A n a l y t es 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 ? 8 807 6 605 4 40 3 2

s l o p e R P /s H ILIC 20 1 00 00 55 1010 1515 reternettieonnt tiiomn et i(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 MeistfolrLeueuminccineine (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-evripoesolhedxynollLeaeu-fmrauicnexeciinetneihney (laIle)mphetamine) 0.2 Climbazole N-DesmethyAlmvenphelaftaaxminieneO (xnoprrevnoenlolafaxine) Caffeine cotinine v2en-ANphemlph--aONifsane-Duxyndlilipneedirtme2ihdes-yephemltareamtznhiiyneneyllevtehnylalafamxiinene 1-7-dTimheopethhyylxliannethine phoOledp-VeDrTorAeinrpanphoasmelmaf mie(fstaeprlnueaxdt-onelhtdmipoopneyrrliad-ildndD6-er -vreoD6epoeol l-hedxyo(nD5pl a--hfmriayNnexednephoitrneihoCcreyxoaTpleytatyihedhd-nemriraneipnemerihtneiheneytalammine)phetamine) BenBzenoBtzhenoiaBtzheoinatlzerio a(CtlzBCeralio TH)iam(rlCzeBffboebao aTe(ilrfBH)tneeubhz Troeopao(anR)BfCtleuhoTr-aa3R)brpCn-baahaba-ate3ymr-dbhrateyozmdxyepraozixynepe-i1pn0ire--a11p10cire-a-dt1cami1he-ytdamdihroy-d10ro--110--d11ih-yddihroyxydroxy N-DeNsN-mD-dDeiNstmehCm-smpMyTe4diV3dcmrtelrt-eoavTeih AenmNvttpen4sepaphelLCrthenayphoop2marhaA-tBeMiyTelop4idemlhdc-oaltE3tfmlariAltp-aeryBAohviesapmNihf enatfrpayehlsntya4phenmcLtopifureneyedvtmhosa2rhadoamxlphenBendmone1-eahlenxiltopl-yCedormopuixp-ailphophE-tnoiy2rieprnemaAzrneelnBeTtbshlirmDPolNari pnel-maei-frynoteCayimcrdhlD6ityteir 6rctrihoftamol-neiPmpalephehonedf 12oparednpalenexhD6lnNtadeo-epy(CeraiedPhe mhial--henzyrnohi2xnotepiymzyNneeo(xtycD6lnTtbephmopeD5rhoParp eri-anmalitlitneoCyielrht itntr-iPchmnelolarrfinlPaomhpiin rgennevpoxlNneOoyea(AnedriCPrmxyenizyfhenabadzynoedxenociNtpay hVophnepeedetlycpmpeoizrho(emxepaidodricyan ophiyneiMnoepotSoaegritTnenltplrhonelanirtNPmidarCrepinonerliviyxemAGrhaA)ieytlrbneahAnirforamneeCrhfnitxaciro CeytRTednpezvVhophphedilne mzeptinaRAxabaai(phenCmredenetnoephycbao(enxtodeenopheneinyc-riMiepadiSanBTailhmnell -neantoiaolNrodand(aclnemne)eotiyliioari lZmA)AGtValinelhtar foineolraamhi(oniferhpneb(ctCtmnRzP iotrineeilnRxtaoiPtrifAedphe(Croeinextienineno-rdianeiceadtBTipephaan)l)ilmneObapldianl he)hiatiolnerxodir-acne)yeot(nomenZsinerol D7yzaVatD) otinne iraf(inein (nP)ahtirtlaomCiclePtoatielneoe)lieinen dpseTtine-rl)dOmhe)cyli aopanNSfeiiolrno-yene(inesrolD7znetD)MDnoo iphri-rin naneloacalel nxpsm-rtie(fcyNS fopaA)eTidriene(tneiiMai y-nerde(olonmr)aND xm (fa)iidrieTA)ne)inei yBde(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 (ll(AeTCotllertaoCahetrrttabaeryrathbarIdmhysahrydomadrpyczorardoepcozrCcocteioarcnsptrrrbioPeitbasneir-lsyo tDoiCrmls i8rlmoCa a5?orlraiz bard5oCr e4-ib5?rnPp-a ma-F5aolirPnem-beegoaPr-rzaerPm4nezgr-daanpgnayepnanegn4Ail-neacz-Ai3mneimn-ca-?et3?10eieane-no-c11-10tm3o-a-1-atmi3?-di1?n1-11a-11oait-17ido-neid-1p-1ibnd1yh-a721-tibryh17ani-ip21-nedyt1-yirdTp7or-re-iyTa-neo2110tre--ri2ne1ot-r1-0l-to1e--20lt111t-er20-otd-r-ol-iod-hn20oneliy-he20dy)- ord()o-K)n ronexy(oeK)xy) ()K) (K) MdiemNtmhe3-La-DadOtNep h2e4fm2-ye-op-DeAh-mlnpnepheeraoteeniTmende3atmtefrher fyiteoit(4onypghs3eMnpd-xyohelheoiym ry-tnero4mENarlilenneexre-eptmTe-ASoi-atptonth1H)xhhemihoxeenatne3 yByanedti tCiadelanlhlinevlaeoOle3yeolnmnxdtn--l zadeneedD3yDriliaineaneeGmBefiiPanesoCda xirmxyphed(ophinlibaBipoAtinendezner-xaeatthap1Neymnhtmaamymen-no-zyyobenmdNphadepliCrGA-rdte-llyvoiniomapeinapetolnehcxzleno4tiebaphed npeehedinyaya- ltPi-ma(lcnehmpMrarfilayiperiidoznenpeDzlxneen laapet-(iiA)rthD7Mnem riin(dayeMepizl rnat(heoneiDopBcnemA)aZtprarP) obhm(lNoeoixta)lna aaemmc (iiMDMidne)d) A) 0.0 1 2 3 4 5 6 7 Time [min] Wastewater Results

HILIC ü Complex mechanism of Comparison of retention of selected analytes separation Intens. ü Complementarity x105 2.0 with RP Benzyl-dimethyl dodecylammonium HILIC

1.5

1.0 Cotinine

Clarithromycin 0.5 Ephedrine irbersartan Amisulpride 0.0 2 3 4 5 6 Time [min]

Intens. x105 Clarithromycin RP 2.5 Cotinine

2.0

1.5

1.0

EpheAdmrineisulpride 0.5 irbersartan Benzyl-dimethyl-dodecylammonium 0.0 4 5 6 7 8 9 10 11 Time [min] Wastewater Results

Comparison of retention of isomers

Intens. x104 O-desmethyl venlafaxine HILIC 3

N-desmethyl venlafaxine

0 4 5 6 7 8 9 10 Time [min] Intens. x104 O-desmethyl venlafaxine RP

N-desmethyl venlafaxine

0 2 4 6 8 10 12 Time [min] Wastewater Results

Intens. x105 HILIC 6.48 min 1.0 HILIC ü Better 0.8 retention 0.6

0.4 ü Better peak 0.2 shape

Inte0n.0s. ü Higher 4000 RP 3.66 min intensity 3000 2-phenethyamine 2000 C8H11N 1000

0 0 2 4 6 8 (-) ESI

Intens. INFLUENT _7_3_15In_teRAn5s._02_10805.d: EIC 136.1121±0.005 +All MS, Masses excluded x104 6000 6.38 min HILIC 2.00 min 3 HILIC

4000 2 Ethyl sulfate 1 2000 C2H6O4S 0 0 Intens. Sample_15_3_inf_RE4_01_3616.d: EICIn 1te36n.s.1121±0.005 +All MS, Masses excluded, Smoothed (1.00,1,GA) x104 600 4.01 min RP 1.25 Amphetamine RP 500 1.00 C9H13N 400 1.53 min 0.75 300

0.50 200

0.25 100

0.00 0 0 2 4 6 8 10 0 1 2 3 Wastewater Results HILIC ü Better retention Guanylurea Intens. ü Better peak EFFLUENT_7_3_15_RA4_02_10803.d: EIC 103.0614±0.005 +All MS, Masses excluded x104 C H N O 2 6 4 6.77 min 4 shape HILIC

0 Intens. eff_8_3_2015_pos_RC3_01_8665.d: EIC 103.0614±0.005 +All MS, Masses excluded x105 1.43 min 3 RP

0 0 1 2 3 4 5 6 7 8 9 Time [min]

Intens. INFLUENT _7_3_15_RA5_02_10805.d: Benzyl-dimethyl-dodecylammonium, 304.2999±0.005, C 21 H 38 N 1, 5.8min x105 2.0 5.83 min Benzyl dimethyl HILIC 1.5 dodecylammonium 1.0 + C21H38N 0.5

0.0 Intens. inf_8_3_2015_pos_RC4_01_8666.d: Benzyl-dimethyl-dodecylammonium, 304.2999±0.005, C 21 H 38 N 1, 11.2min x104 1.0 11.27 min RP 0.8

0.6

0.4

0.2

0.0 4 5 6 7 8 9 10 11 12 13 Time [min] Wastewater Results HILIC ü Compounds HILIC Intens. NOTeluted in INFLUENT _7_3_15_RA5_02_10805.d: dexamethasone, 393.2072±0.005, C 22 H 29 F 1 O 5, 2.4min 1500 RP mode Dexamethasone 2.45 min 2.5 mDa, 53 mSigma 1000

500

0 Intens. INFLUENT _7_3_15_RA5_02_10805.d: Oxprenolol, 266.1751±0.005, C 15 H 23 N 1 O 3, 6.5min x104 Oxprenolol 3 0.2 mDa, 32 mSigma 6.66 min 2

Intens.0 Pyrimidinol INFLUENT _7_3_15_RA5_02_10805.d: Pyrimidinol, 153.1022±0.005, C 8 H 12 N 2 O 1, 2.0min 2.03 min 2000 0.4 mDa, 35 mSigma

1000

0 Intens. INFLUENT _7_3_15_RA5_02_10805.d: Tyramine, 138.0913±0.005, C 8 H 11 N 1 O 1, 6.7min x104 Tyramine 0.8 0.1 mDa, 5 mSigma 6.79 min 0.6 0.4 0.2 0.0 0 1 2 3 4 5 6 7 8 Time [min] Comparison HILIC-RP

HILIC RP

902 compounds database 2327 compounds

336 compounds detected 371 compounds

203 compounds in common

133 compounds 168 compounds only in HILIC only in RP Further evaluation 104/133 present in Why not detect some 95/168 present compounds in RP or RP db in HILIC db HILIC mode? Conclusions

v Development of HILIC wide-scopetarget method

v Optimization& validationof the HILIC method

v In-house database with information for 902 compounds

v Application in influent & effluent wastewater samples

v Comparison with RP target screening method

ü Complementary technique for target screening ü Use in suspect & non-target screening for additional information Acknowledgments to.. AlexandrosMarkatis NikolaosThomaidis

& our collaborators from

Thank you for your attention!

Laboratory of Analytical Chemistry Department of Chemistry University of Athens