Performance of the linear ion trap Orbitrap mass analyzer for qualitative and quantitative analysis of drugs of abuse and relevant metabolites in sewage water

Tí ulo a ículo / Tí ol a icle: Pe o mance o he LTQ-O bi ap mass analyze o quali a i e and quan i a i e analysis o d ugs o abuse and ele an me aboli es in sewage wa e Au o es / Au o s Lube us Bijlsmaa, E ik Emke, Félix He nández, Pim de Voog Re is a: Analy ica Chimica Ac a, . 768 (2013) Ve sión / Ve sió: P ep in de l’au o Ci a bibliog á ica / Ci a bibliog à ica (ISO 690): BIJLSMA, L.; EMKE, E.; HERNÁNDEZ, F.; DE VOOGT, P. Pe o mance o he LTQ-O bi ap mass analyze o quali a i e and quan i a i e analysis o d ugs o abuse and ele an me aboli es in sewage wa e . Analy ical Chimica Ac a, . 768 (2103), p. 102-110 u l Reposi o i UJI: h p:// eposi o i.uji.es/xmlui/handle/10234/94472 1/22 Pe o mance o he LTQ-O bi ap mass analyze o quali a i e and quan i a i e analysis o d ugs o abuse and ele an me aboli es in sewage wa e Lube us Bijlsmaa,§, E ik Emkeb, Félix He nándeza, Pim de Voog b,c,* 5 a Resea ch Ins i u e o Pes icides and Wa e , Uni e si y Jaume I, A da. Sos Bayna s/n, E- 12071 Cas ellón, Spain. b KWR Wa e cycle Resea ch Ins i u e, Chemical Wa e Quali y and Heal h, P.O. Box 1072, 3430 BB Nieuwegein, he Ne he lands. c Ins i u e o Biodi e si y and Ecosys em Dynamics, Uni e si y o Ams e dam, P.O. Box 10 94248, 1090 GE Ams e dam, he Ne he lands §Visi ing scien is a KWR * Co esponding au ho : [email p o ec ed], Tel +31 20 5256565, Fax +31 20 5257431. 15 2/22 ABSTRACT This wo k illus a es he po en ial o liquid ch oma og aphy coupled o a hyb id linea ion ap Fou ie T ans o m O bi ap mass spec ome e o he simul aneous iden i ica ion and quan i ica ion o 24 d ugs o abuse and ele an me aboli es in sewage wa e . The de eloped 20 me hodology consis ed o au oma ic solid-phase ex ac ion using Oasis HLB ca idges, ch oma og aphic sepa a ion o he a ge ed d ugs, ull-scan accu a e mass da a acquisi ion unde posi i e elec osp ay ioniza ion mode o e an m/z ange o 50 - 600 Da a a esolu ion o 30,000 FWHM and simul aneous MSn measu emen s o ob ain in o ma ion o agmen ions gene a ed in he linea ion ap. Accu a e mass o he p o ona ed molecule, oge he wi h 25 a leas one nominal mass p oduc ion and e en ion ime allowed he con iden iden i ica ion o he compounds de ec ed in hese complex ma ices. In addi ion o he highly eliable quali a i e analysis, O bi ap analyze also p o ed o ha e sa is ac o y po en ial o quan i ica ion a sub-ppb analy e le els, a possibili y ha has been e y li le explo ed in he li e a u e un il now. The limi s o quan i ica ion anged om 4 o 68 ng L-1 in in luen sewage 30 wa e , and om 2 o 35 ng L-1 in e luen , wi h he excep ion o MDA, mo phine and THC ha p esen ed highe alues as a consequence o he high ioniza ion supp ession in his ype o samples. Sa is ac o y eco e ies (70 – 120%) and p ecision (< 30%) o he o e all p ocedu e we e ob ained o all compounds wi h he excep ion o me a- chlo ophenylpipe azine, me hylphenida e and ke amine. Iso ope-labelled in e nal s anda ds 35 we e added o sewage samples as su oga es in o de o co ec o ma ix e ec s and also o possible losses du ing sample ea men . The me hodology de eloped was applied o sewage wa e samples om he Ne he lands (in luen and e luen ), and he esul s compa ed wi h hose ob ained by LC-MS/MS wi h iple quad upole. Se e al d ugs o abuse could be iden i ied and quan i ied, mainly MDMA, benzoylecgonine, codeine, oxazepam and 40 emazepam. O bi ap also showed po en ial o e ospec i e in es iga ion o ke amine me aboli es in he samples wi hou he need o addi ional analysis. Keywo ds 45 D ugs o abuse, accu a e mass, O bi ap analyze , high esolu ion mass spec ome y, quan i a i e analysis, sewage wa e . 3/22 1. INTRODUCTION 50 The p esence o d ugs o abuse, unal e ed o as me aboli es, in he wa e cycle has spu ed esea che s on o in es iga e hei occu ence in sewage wa e , su ace wa e and d inking wa e [1-3]. Al hough concen a ions ound a e gene ally low (sub µg L-1 le el), da a ob ained om analysis o u ban was ewa e can be used o s udy consump ion and usage ends in communi ies [4]. Fu he mo e, en i onmen al loads can be calcula ed, as hei 55 po en ial impac on aqua ic o ganisms, human heal h and he en i onmen may no be uled ou [1]. Mos o he exis ing me hods o de e mina ion o d ugs o abuse in wa e a e based on liquid ch oma og aphy coupled o andem mass spec ome y (LC-MS/MS), using iple quad upole (QqQ) analyze s. Despi e i s excellen sensi i i y and selec i i y , his app oach 60 also has some limi a ions [5, 6], he main being ha o he d ugs, di e en om hose included in he scope o he me hod, may be igno ed in analyses, as analy e speci ic in o ma ion is acqui ed and only he a ge analy es a e no mally de ec ed and quan i ied. The inc easing in e es o using accu a e mass High Resolu ion Mass Spec ome e s (HRMS), e.g. O bi ap and ime-o - ligh (TOF) ins umen s, in en i onmen al sciences elies on i s capabili y o 65 pe o m bo h a ge ed as well as non- a ge ed analysis, based on ull-spec um accu a e-mass acquisi ion a good sensi i i y [7]. E icien sc eening s a egies using HRMS ha e allowed he de ec ion and iden i ica ion o a ious d ugs o abuse in en i onmen al and was ewa e samples, in some cases e en wi hou he need o e e ence s anda ds, bu wi h high con idence due o he high mass accu acy measu emen s [8, 9]. 70 Ad an ages o HRMS a e widely ecognized in quali a i e analysis; howe e HRMS- based quan i a i e analyses ha e ha dly been explo ed in he scien i ic li e a u e un il now. One o he classical c i icisms conce ns he ela i e low sensi i i y and low linea dynamic ange. This limi a ion was mo e e iden in he i s -gene a ion ins umen s, e.g. i s LC-TOF MS. Howe e , he imp o ed echnology o he la es TOF ins umen s, i.e. highe sensi i i y 75 and esol ing powe , and wide linea dynamic ange, p o ides be e quan i a i e capabili ies. This has allowed quan i ica ion o pes icides, pha maceu icals and illici d ugs in was ewa e by using LC-TOF MS [10, 11]. As o O bi ap ins umen s, good quan i a i e pe o mances, i.e. high sensi i i y and selec i i y, ha e been demons a ed in some applied ields [5, 12-15]. 80 Ne e heless, o he bes o ou knowledge, he quan i a i e po en ial o O bi ap has no been p e iously demons a ed o d ugs o abuse in sewage wa e samples. The 4/22 de e mina ion o hese compounds is complica ed due o he complexi y o he samples and low analy e concen a ions. Sample p e-concen a ion is no mally equi ed, mos ly based on solid phase ex ac ion (SPE), bu he key poin is he quan i ica ion o analy es, which is 85 p oblema ic in LC-MS based me hods due o he s ong ma ix e ec s commonly obse ed o his ype o sample ma ices. The use o iso ope-labeled in e nal s anda ds (ILIS) is he app oach mos equen ly applied o sol e his p oblem, al hough i s applica ion is di icul in mul i- esidue mul iclass me hods whe e a la ge numbe o ILIS would be equi ed. Typically he own analy e ILIS is used, as he use o analogue compounds as in e nal s anda ds does no 90 always ensu e an app op ia e co ec ion [10, 16]. In he p esen wo k, analy ical me hodology based on he use o SPE ollowed by LC coupled o a hyb id linea ion ap (LTQ) Fou ie T ans o m (FT) O bi ap MS, has been de eloped o he de e mina ion o 24 d ugs o abuse and me aboli es in u ban was ewa e . The acquisi ion o ull-scan accu a e-mass da a by O bi ap oge he wi h he simul aneous 95 MS/MS measu emen s pe mi ed by LTQ is a powe ul combina ion o con iden iden i ica ion and con i ma ion. As he excellen quali a i e po en ial o O bi ap analyze is widely accep ed in he ecen li e a u e, an addi ional and ele an objec i e was o demons a e he quan i a i e capabili ies o his HRMS, a ea u e ha has been li le explo ed un il now. To he bes o ou knowledge, he quan i a i e po en ial o O bi ap has no been 100 p e iously demons a ed o d ugs o abuse in complex sewage wa e samples. 5/22 2. MATERIALS AND METHODS 2.1. Reagen s 105 D ugs o abuse and me aboli es e e ence s anda ds: amphe amine, me hamphe amine, 3,4-me hylenedioxyamphe amine (MDA), 3,4-me hylene-dioxyme hamphe amine (MDMA, o ecs asy), 3,4-me hylenedioxye hylamphe amine (MDEA), cocaine, benzoylecgonine, he oin, mo phine, 6-monoace yl mo phine (6-MAM), me hadone, codeine, -9- e ahyd ocannabinol (THC), 11-no -9-ca boxy--9- e ahyd ocannabinol (THC-COOH), 11-110 hyd oxy--9- e ahyd ocannabinol (OH-THC), ke amine, me hylphenida e, oxazepam, diazepam, emazepam, no dazepam, desalkyl- lu azepam, me a-chlo ophenylpipe azine (mCPP), and en anyl we e ob ained om Lipomed AG (A lesheim, Swi ze land) as solu ions in me hanol (MeOH), e hanol (E OH) o ace oni ile (ACN) a a concen a ion o 1 g L-1. S anda d solu ions o each compound we e p epa ed a 36 mg L-1 in MeOH. A inal mix 115 solu ion was made by dilu ing aliquo s om e e y compound indi idually o a concen a ion o 3.6 mg L-1. Wo king mix solu ions o calib a ion cu es we e made in MeOH. Be o e each analy ical un, he calib a ion s anda ds we e dilu ed 10 imes wi h ul apu e wa e esul ing in a mix o wa e : MeOH (90:10 / ) and we e injec ed in o he O bi ap sys em. Final concen a ions o s anda ds anged om: 0.7 o 288 µg L-1. 120 Deu e a ed compounds we e pu chased om Lipomed AG as solu ions in MeOH, E OH o ACN a a concen a ion o 1 g L-1 and we e used as su oga e iso ope labelled in e nal s anda ds (ILIS) o quan i ica ion: amphe amine-d11, me hamphe amine-d5, 3,4- me hylenedioxyamphe amine-d2 (MDA-d2), 3,4-me hylenedioxyme hamphe amine-d5 (MDMA-d5), 3,4-me hylenedioxye hyl-amphe amine-d5 (MDEA-d5), cocaine-d3, 125 benzoylecgonine-d3, mo phine-d3, 6-monoace yl mo phine-d3 (6-MAM-d3), 9- e ahyd ocannabinol-d3 (THC-d3), 11-no -9-ca boxy-9- e ahyd ocannabinol-d3 (THC- COOH-d3), 11-hyd oxy-9- e ahyd ocannabinol-d3 (OH-THC-d3), oxazepam-d5, diazepam- d5, no dazepam-d5. A mixed ILIS wo king solu ion was p epa ed in MeOH and added o all calib a ion s anda ds o ge a inal ILIS concen a ion o 72 µg L-1, as well as o he in luen 130 and e luen sewage wa e samples p io o sample ea men ( inal ILIS concen a ion in sample o 360 ng L-1 and 180 ng L-1, espec i ely). All s anda d and wo king solu ions we e s o ed in ambe glass bo les a -18 °C. 6/22 The ul apu e wa e was ob ained by pu i ying demine alized wa e in a Milli-Q sys em om Millipo e (Bed o d, MA, USA). Fo mic acid (98 – 100%), HPLC-g ade MeOH, 135 E OH and ACN we e acqui ed om Mallinck od Bake (De en e , The Ne he lands). Glass ib e il e s (1 µm, ype A/E) we e pu chased om Pall Co po a ion (Po Washing on, NY, USA). Polye he sul one il e s (0.45 µm) wi h disposable se up we e acqui ed om Nalgene (Roches e , NY, USA). SPE ca idges, buil o a hyd ophilic and a lipophilic monome (Oasis-HLB; 6 mL, 140 150 mg) we e pu chased om Wa e s (Mil o d, MA, USA). Poly y osine-1,3,6 s anda d used o mass axis calib a ion was pu chased om Cs Bio Co. (Menlo Pa k, CA, USA). 2.2. Wa e samples 145 24-hou s low dependen in luen and e luen composi e-samples om di e en sewage ea men plan s (STPs) loca ed in he Ne he lands we e aken on he same weekend day, wi hou accoun ing o lag- ime. Samples we e collec ed in ambe glass bo les, and s o ed in he da k a 4 °C. Upon ecep ion in he labo a o y, he samples we e immedia ely analysed. 150 2.3. Ex ac ion p ocedu e P io o SPE, samples we e acuum il e ed h ough 1 µm ype A/E glass ib e il e s, ollowed by 0.45 µm polye he sul one (PES) il e s wi h disposable se up. Subsequen ly, 200 mL o e luen sewage wa e , o 100 mL o in luen sewage wa e sample, we e spiked wi h a 155 mixed in e nal s anda d solu ion o gi e a concen a ion o each compound in sample o 180 ng L-1 and 360 ng L-1, espec i ely. SPE was pe o med au oma ically using a GX-274 ASPEC (Gilson). Oasis HLB ca idges we e condi ioned by washing and insing wi h 8 mL o ACN, 8 mL o MeOH and 8 mL o Milli-Q wa e . Samples we e loaded on o he ca idges a 5 mL min-1, and hen ca idges we e washed wi h 8 mL o Milli-Q wa e and d ied wi h 160 ni ogen o 15 min a a p essu e o 1 ba . Analy es we e elu ed using 8 mL o MeOH a a low o 0.5 mL min-1. The SPE elua es (MeOH) we e e apo a ed o 200 µL a 35°C unde a gen le s eam o ni ogen. Then, 250 µL o Milli-Q wa e was added and he emaining MeOH (200 µL) e apo a ed. E apo a ion o he ex ac s was pe o med au oma ically using Ba key 165 op ocon ol (Ge many). The inal ex ac was hen made up, by weigh , o exac ly 250 µL wi h Milli-Q wa e . As a inal s ep, he olume was adjus ed o 500 µL, by weigh , wi h 7/22 wa e :MeOH (80:20 / ) o achie e a inal pe cen age o 10% MeOH. An aliquo o he sample ex ac (20 µL) was injec ed di ec ly in o he LC-LTQ FT O bi ap sys em. 170 2.4. Liquid Ch oma og aphy A hyb id linea ion ap Fou ie T ans o m (LTQ FT) O bi ap mass spec ome e was in e aced o a Su eyo HPLC sys em, consis ing o a Su eyo au o sample model Plus and a Su eyo qua e na y g adien HPLC-pump (The mo Fishe Scien i ic, B eda, The Ne he lands). Ch oma og aphic sepa a ion o he compounds was made using an XB idge C18 175 column (150 mm x 2.1 mm I.D., pa icle size 3.5 µm) (Wa e s). The p e-column used was a 4.0 x 2.0 mm I.D. Phenomenex Secu i y Gua d column (Bes e , Ams e dam, he Ne he lands). The analy ical column and he gua d column we e main ained a a empe a u e o 21 °C in a column he mos a . An op imized g adien was used a a cons an low a e o 0.3 mL min-1 using Milli-Q wa e (Sol en A) and MeOH (Sol en B) bo h wi h 0.05% 180 o mic acid. The pe cen age o o ganic modi ie (B) was changed linea ly as ollows: 0 min, 5%; 20 min, 100%; 30 min, 100%; 32 min, 5%. Be ween consecu i e uns, he analy ical column was e-equilib a ed o 10 min. 2.5. LTQ FT O bi ap mass spec ome y 185 An LTQ FT O bi ap mass spec ome e (The mo Elec on, B emen, Ge many) was used. The LTQ pa o his sys em was equipped wi h an Ion Max Elec osp ay Ioniza ion (ESI) p obe and ope a ed in he posi i e ion mode. The condi ions in ESI posi i e mode we e: sou ce ol age 4.0 kV, hea ed capilla y empe a u e 300 °C, capilla y ol age 30 V and ube lens 45 V. In he LTQ componen o he ins umen , he empe a u e was se o 26 °C and 190 helium was used as damping gas. All measu emen s we e done using he au oma ic gain con ol (AGC) o he LTQ o adjus he numbe o ions en e ing he ap. P oduc s ions we e gene a ed in he LTQ ap a a no malized collision ene gy se ing o 40% and using an isola ion wid h o 2 Da. Full-scan accu a e mass spec a (mass ange om 50 o 600 Da) we e ob ained a a 195 mass esolu ion o 30,000 FWHM (m/z 400). The o al cycle ime depends upon he esolu ion; a he selec ed esolu ion he o al cycle ime is abou 0.55 s. The mass spec ome e ope a ed unde da a-dependen -acquisi ion (DDA) mode du ing he comple e ch oma og aphic un, in which bo h MS and MSn spec a we e acqui ed simul aneously. The ins umen was ini ially se o ope a e in ull-scan (‘su ey’) mode wi h accu a e mass 200 measu emen s. When an ion exceeded a p ese h eshold and co esponded o he a ge mass 8/22 lis speci ied by he use , he ins umen swi ched o p oduc -ion scan mode (MSn) in he ion- ap pa wi h nominal mass measu emen s. In his way, ele an in o ma ion o iden i ica ion and con i ma ion, e.g. e en ion ime, molecula weigh and agmen a ion, was ob ained in a single analysis. All da a we e acqui ed and p ocessed using Xcalibu e sion 2.1 so wa e. 205 Mass calib a ion was pe o med wi h e e y ba ch un jus p io o s a ing he ba ch by using low injec ion o a Poly y osine-1,3,6 solu ion ([M+H]+ 182.01170 / 508.20783 and 997.39781) a a low a e o 10 µL min-1. Iden i ica ion and quan i ica ion o a ge compounds was pe o med using he accu a e mass o he p o ona ed molecule wi hin a mass window o 5 ppm. Fo con i ma ion 210 o he iden i y o he compounds, in addi ion o he accu a e mass o he p ecu so ion, a leas one nominal mass p oduc ion was used oge he wi h e en ion ime, which was compa ed wi h ha o he e e ence s anda ds (wi hin 2.5%) [17, 18]. 2.6. Me hod alida ion 215 The pe o mance o he me hod was e alua ed in e ms o linea i y, limi s o quan i ica ion, ueness and p ecision. The o e all eco e y (including sample ea men and po en ial ma ix e ec s) was s udied and e alua ed. Ins umen al linea i y was es ima ed by analyzing s anda d solu ions in iplica e. Sa is ac o y linea i y was assumed when he coe icien o de e mina ion ( 2) was > 0.99, 220 based on analy e/in e nal s anda d peak a eas, excep o hose compounds ha we e quan i ied wi hou ILIS (absolu e esponse). Limi o quan i ica ion (LOQ): To acili a e he ou ie ans o ma ion o he acqui ed equency da a and con e sion o m/z in he o bi ap, noise is il e ed ou . This is why he common app oaches o e alua e he limi s o quan i ica ion do no apply [19]. The e o e, a 225 di e en app oach was applied as p e iously epo ed by de Voog e al. [20]. I is based on he ma ix supp ession o he deu e a ed analogue and he iden i ica ion c i e ia [18] o each enough iden i ica ion poin s. The ma ix e ec (exp essed as de ined by Ma uszewski e al. [21]) is calcula ed by using he a ea o he accu a e mass signal o he deu e a ed s anda d, spiked be o e ex ac ion (in ma ix), di ided by he a e age a ea o he deu e a ed s anda d in 230 he calib a ion cu e (in sol en ). By using he lowes s anda d isible in he calib a ion cu e which mee s all he iden i ica ion c i e ia ( ypically he absence/p esence o he con i ma ion p oduc ion is he c i ical pa ame e ) and co ec ed o he ma ix supp ession and he concen a ion ac o , he LOQ can be de e mined. 15/22 4. CONCLUSIONS Analy ical me hodology based on ull-spec um accu a e-mass and MS/MS acquisi ion p o ided by LC-LTQ FT O bi ap MS has been de eloped o he simul aneous quan i ica ion and con i ma ion o 24 a ge d ugs o abuse a ng L-1 le els in sewage wa e . Al hough O bi ap is ecognized as an excellen analyze o quali a i e pu poses, i s sui abili y o 420 pe o m quan i a i e analysis has no been much explo ed. In his wo k, O bi ap has been applied o he i s ime o he quan i a i e analysis o d ugs o abuse in sewage wa e . Ou da a showed ha his analyze can be used o he eliable quan i ica ion wi h almos he same sensi i i y han he mos commonly used me hodologies based on LC-MS/MS wi h iple quad upole. The quan i a i e applicabili y has been demons a ed by me hod alida ion and 425 he analysis o quali y con ol samples included in each sample sequence, and also ia a compa ison wi h da a epo ed by iple quad upole analysis. In addi ion, MS da a p o ided by O bi ap ha e allowed e ospec i e analysis leading o an indica ion o he p esence o wo ke amine me aboli es. In conclusion, his unique ea u e o high- esolu ion accu a e-mass spec ome y demons a es ha ke amine is likely o be p esen in se e al samples. 430 16/22 ACKNOWLEDGEMENTS The au ho s wish o hank Juan V. Sancho o Resea ch Ins i u e o Pes icides and Wa e (IUPA) o his use ul commen s. 435 L. Bijlsma is e y g a e ul o he KWR Wa e cycle Resea ch Ins i u e o allowing him o pe o m an in e nship as isi ing scien is . 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Compound R P ecu so ion [M+H]+ P oduc ion 1 P oduc ion 2 In e nal s anda d (min) m/z m/z m/z RA (%) Amphe amine 10.28 136.11208 119.1 91.1 0.5 Amphe amine-d11 Me hamphe amine 10.64 150.12773 119.0 91.1 9.0 Me hamphe amine-d5 MDA 10.75 180.10191 163.2 MDA-d2 MDMA 10.90 194.11755 163.1 58.0 1.0 MDMA-d5 MDEA 11.66 208.13321 163.1 72.0 2.7 MDEA-d5 Cocaine 13.42 304.15433 182.1 150.2 2.6 Cocaine-d3 Benzoylecgonine 12.51 290.13868 168.2 272.2 4.8 Benzoylecgonine-d3 He oin 13.07 370.16490 328.2 268.2 99.1 n/a Mo phine 4.71 286.14334 201.1 229.1 51.9 Mo phine-d3 6-MAM 10.33 328.15433 211.2 268.2 73.7 6-MAM-d3 Me hadone 18.80 310.21654 265.1 247.2 0.1 n/a Codeine 9.10 300.15942 215.2 243.1 47.7 6-MAM-d3 THC 26.26 315.23186 259.2 193.2 76.7 THC-d3 THC-COOH 24.84 345.20604 327.2 299.3 6.1 THC-COOH-d3 OH-THC 24.48 331.22677 313.3 OH-THC-d3 Ke amine 12.43 238.09932 220.1 207.1 23.9 n/a Me hylphenida e 13.61 234.14886 84.0 174.2 0.3 n/a Oxazepam 19.53 287.05818 269.1 241.1 3.9 Oxazepam-d5 Diazepam 20.62 285.07892 257.1 222.2 30.4 Diazepam-d5 Temazepam 19.85 301.07383 283.0 255.2 9.2 No dazepam-d5 No dazepam 20.13 271.06327 243.1 208.1 37.7 No dazepam-d5 Desalkyl- lu azepam 19.67 289.05385 261.1 140.0 44.5 No dazepam-d5 mCCP 13.62 197.08400 154.0 119.1 6.9 n/a Fen anyl 15.66 337.22744 188.2 216.3 5.6 No dazepam-d5 RA: ela i e abundance o p oduc ions. n/a: adequa e in e nal s anda d was no a ailable. 505 20/22 Table 2: Me hod alida ion in in luen (n = 4) and e luen (n = 4) sewage wa e Compound In luen E luen Linea i y Ra (%) CVb (%) LOQ (ng L-1) Ra (%) CVb (%) LOQ (ng L-1) 2 Ins umen al LOQ (pg) Amphe amine 104 8 40 105 6 4 0.9960 58 Me hamphe amine 98 4 15 92 7 5 0.9994 28 MDA 113 13 360c 92 4 158 0.9996 720 MDMA 102 4 12 97 7 4 0.9999 14 MDEA 101 4 17 98 5 4 0.9999 14 Cocaine 70 6 40 93 6 6 0.9999 14 Benzoylecgonine 111 8 10 93 7 2 0.9999 14 He oin 70 20 19 72 21 7 0.9943 28 Mo phine 102 5 360c 98 12 125 0.9996 144 6-MAM 117 11 19 119 15 7 0.9995 14 Me hadone 73 19 45 76 5 6 0.9917 14 Codeine 90 15 19 120 26 7 0.9988 28 THC 109 11 360c 94 12 180c 0.9995 720 THC-COOH 102 7 33 90 5 7 0.9995 28 OH-THC 82 4 68 85 10 35 0.9988 58 Ke amine 48 21 10 81 7 2 0.9956 14 Me hylphenida e 45 20 20 65 27 2 0.9969 14 Oxazepam 97 6 14 91 6 4 0.9994 28 Diazepam 100 4 18 94 7 6 0.9998 20 Temazepam 105 6 4 90 7 2 0.9980 28 No dazepam 96 5 4 91 8 2 0.9995 28 Desalkyl- lu azepam 88 25 4 109 15 2 0.9994 28 mCCP 52 28 20 62 18 6 0.9911 14 Fen anyl 73 16 4 74 14 2 0.9964 14 a T ueness, es ima ed by means o eco e y expe imen s. 510 b P ecision, exp essed as epea abili y in e ms o coe icien s o a ia ion. c These alues we e de i ed om alida ion expe imen s ( o de ailed explana ion see ex ). 515 21/22 Table 3: Compa ison o concen a ions o d ugs o abuse de ec ed in in luen and e luen sewage wa e s analyzed by LC-MS/MS wi h iple quad upole ( wo di e en labo a o ies) and by he O bi ap me hod p esen ed in his wo k. Each numbe is he esul o a single measu emen o he pe aining sample using a speci ic MS de ec ion echnique Compound In luen sewage wa e (ng L-1) E luen sewage wa e (ng L-1) Sample 1 Sample 2 Sample 3 Sample 4 QqQ 1a QqQ 2b O bi ap QqQ 1 QqQ 2 O bi ap QqQ 1 QqQ 2 O bi ap QqQ 1 QqQ 2 O bi ap Amphe amine 95 117 123 282 249 245 - - - - - - MDMA 21 96 144 - 56 86 84 88 137 50 54 76 Cocaine 439 296 dc 179 114 dc 2 - - 14 - 8 Benzoylecgonine 1178 1136 1637 528 645 615 26 19 45 85 77 99 THC-COOH 378 n/ad dc - n/a - - n/a - - n/a - Ke amine n/a -e - n/a - - n/a - 2 n/a 16 6 a P e- ea men : cen i uga ion; P e-concen a ion by SPE (Oasis MCX, 150 mg); p e-concen a ion ac o : in luen 10x, e luen , 50x; [29] b P e- ea men : none; P e-concen a ion by SPE (Oasis HLB, 200 mg); p e-concen a ion ac o : in luen 50x, e luen 250x; [25] 520 c d: de ec ed d n/a: no da a a ailable e - : no de ec ed 22/22 FIGURE CAPTIONS 525 Figu e 1: The in luence o in ake olume (100, 300, 600 and 900 mL) on ma ix e ec in a ypical in luen Figu e 2: A e age ma ix e ec s [21] obse ed in bo h in luen s (100 mL) and e luen s (200 530 mL) o 5 di e en STPs. Figu e 3: The mass de ia ion (mass d i ) o he p o ona ed ions o selec ed deu e a ed analy es in ela ion o he heo e ical mass o e a pe iod o 55 hou s. 535 Figu e 4: LC-MS (ESI + mode) ex ac ed-ion ch oma og ams (le ) and MS/MS spec a ( igh ) o d ugs o abuse de ec ed in in luen (A) and e luen (B) sewage wa e om he sewage ea men plan o Ams e dam. Concen a ions ound in hese samples we e he ollowing (in luen and e luen , espec i ely); MDMA: 136 and 190 ng L-1; benzoylecgonine: 3701 and 155 ng L-1; THC-COOH: 431 and 22 ng L- 540 1; Oxazepam: 430 and 422 ng L-1. A ows indica e ch oma og aphic peak o MDMA. Figu e 5: LC-MS (ESI+ mode) ex ac ed-ion ch oma og ams o ke amine, no ke amine and dehyd ono ke amine in an in luen sewage wa e sample om Eindho en 545 ( e ospec i e sea ch). 0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 160.0 0.0 100.0 200.0 300.0 400.0 500.0 600.0 700.0 800.0 900.0 Ma ix e ec Mo phine-d3 6-MAM-d3 MDMA-d5 MDA-d2 Amphe amine-d11 Me hamphe amine-d5 MDEA-d5 Cocaine-d3 Benzoylecgonine-d3 Oxazepam-d5 Diazepam-d5 OH-THC-d3 THC-COOH-d3 THC-d3 Figu e 1 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% THC-d3 Mo phine-d3 Cocaine-d3 MDEA-d5 OH-THC-d3 THC-COOH-d3 Me amphe amine-d5 MDMA-d5 Amphe amine-d11 Benzoylecgonine-d3 6-MAM-d3 Diazepam-d5 MDA-d2 No dazepam-d5 Oxazepam-d5 ma ix e ec In luen E luen Figu e 2