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
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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 .
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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
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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.

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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.
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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
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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.
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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

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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 .
The inancial suppo om he Join Resea ch P og amme o he Du ch wa e
companies (BTO) and om he Gene ali a Valenciana, P ojec : Collabo a i e Resea ch on
En i onmen and Food Sa e y (ISIC/2012/016) is g a e ully acknowledged. 440
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REFERENCES
[1] E. Zucca o, S. Cas iglioni, R. Bagna i, C. Chiab ando, P. G assi, R. Fanelli, Wa e Res. 445
42 (2008) 961-368.
[2] D.R. Bake , B. Kasp zyk-Ho de n, J. Ch oma og . A 1218 (2011) 8036-8059.
[3] M.R. Boleda, M. Hue a-Fon ela, F. Ven u a, M.T. Galce an, Chemosphe e 84 (2011)
1601–1607.
[4] A.L.N. an Nuijs, S. Cas iglioni, I. Ta comnicu, C. Pos igo, M. Lopez de Alda, H. 450
Neels, E. Zucca o, D. Ba celo, A. Co aci, Sci. To al En i on. 409 (2011) 3564-3577.
[5] M. Kellmann, H. Muens e , P. Zome , H. Mol, J. Am. Soc. Mass Spec om. 20 (2009)
1464-1476.
[6] M. K auss, H. Singe , J. Hollende , Anal. Bionanal. Chem. 397 (2010) 943-951.
[7] F. He nández, J.V. Sancho, M. Ibáñez, E. Abad, T. Po olés, L. Ma iolo, Anal. Bioanal. 455
Chem. 403 (2012) 1251-1264.
[8] A.C. Hogenboom, J.A. an Lee dam, P. de Voog , J. Ch oma og . A 1216 (2009) 510-
519.
[9] F. He nández, L. Bijlsma, J.V. Sancho, R. Díaz, M. Ibáñez, Anal. Chim. Ac a 684
(2011) 96-106. 460
[10] J. Nu mi, J. Pellinen, J. Ch oma og . A 1218 (2011) 6712-6719.
[11] I. González-Ma iño, J.B. Quin ana, I. Rod íguez, M. González-Díez, R. Cela, Anal.
Chem. 84 (2012) 1708-1717.
[12] J.A. an Lee dam, A.C. Hogenboom, M.M.E. an de Kooi, P. de Voog , In . J. Mass
Spec om. 282 (2009) 99-107. 465
[13] A. Kau mann, P. Bu che , K. Maden, S. Walke , M. Widme , Rapid Commun. Mass
Spec om. 25 (2011) 979-992.
[14] S. Voglia di, D. Fa e o, M. Tucci, G. S ocche o, S.D. Fe a a, Anal. Bioanal. Chem.
400 (2011) 51-67.
[15] R. Pinhancos, S. Maass, D.M. Ramana han, J. Mass Spec om. 46 (2011) 1175-1181. 470
[16] J.M. Ma ín, E. G acia-Lo , J.V. Sancho, F.J. López, F. He nández, J. Ch oma og . A
1216 (2009) 596-612.
[17] O.J. Pozo, J.V. Sancho, M. Ibáñez, F. He nández, W.M.A. Niessen, T ends Anal. Chem.
25 (2006) 1030-1042.
18/22
[18] Commission Decision 2002/657/EC o 12 Augus implemen ing Council Di ec i e 475
96/23/EC conce ning pe o mance o analy ical me hods and he in e p e a ion o
esul s. O . J. Eu . Commun. L221/8 (2002).
[19] A. Kau mann, M. Widme , K. Maden, Rapid Commun. Mass Spec om. 24 (2010)
2162-2170.
[20] P. de Voog , E. Emke, R. Helmus, P. Pan eliadis, J.A. an Lee dam, in: S. Cas iglioni, 480
E. Zucca o, R. Fanelli (Eds), Mass Spec ome ic Analysis o Illici D ugs in he
En i onmen , Wiley, New Yo k, 2011, pp. 85-114.
[21] B.K. Ma uszewski, M.L. Cons anze , C.M. Cha ez-Eng, Anal. Chem. 75 (2003) 3019-
3030.
[22] S. Cas iglioni, E. Zucca o, E. C isci, C. Chiab ando, R. Fanelli, R. Bagna i, Anal. Chem. 485
78 (2006) 8421–8429.
[23] M. S übe , T. Reem sma, Anal. Bioanal. Chem. 378 (2004) 910-916.
[24] R.J.B. Pe e s, A.A.M. S olke , J.G.J. Mol, A. Lommen, E. Ly is, Y. Angelis, A.
Vonapa i, M. S amou, C. Geo gakopoulos, M.W.F. Nielen, T ends Anal. Chem. 29
(2010) 1250-1268. 490
[25] N.G.F.M. an de Aa, E. Dijkman, L. Bijlsma, E. Emke, B.M. an de Ven, A.L.N. an
Nuijs, P. de Voog , RIVM epo 703719064, Bil ho en, (2010) pp. 1-90. A ailable
om URL: www. i m.nl (accessed Sep embe 2012)
[26] N.G.F.M. an de Aa, L. Bijlsma, E. Emke, E. Dijkman, A.L.N. an Nuijs, B.M. an de
Ven, F. He nández, J.F.M. Ve s eegh, P. de Voog , Wa e Res. unde e ision. 495
[27] F. He nández, M. Ibáñez, E. G acia-Lo , J.V. Sancho, J. Sep. Sci. 34 (2011) 3517-3526.
[28] K-C. Wang, T-S. Shih, S-G. Cheng, Fo ensic. Sci. In . 147 (2005) 81-88.
[29] L. Bijlsma, J.V. Sancho, E. Pi a ch, M. Ibáñez, F. He nández, J. Ch oma og . A 1216
(2009) 3078-3089.
500
19/22
Table 1: Exac masses o he p o ona ed a ge d ugs o abuse, nominal masses and ela i e
abundance o p oduc ions, oge he wi h hei e en ion imes and iso ope labelled in e nal
s anda ds used o quan i ica ion.
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