Multiresidue analytical method for pharmaceuticals and personal care products in sewage and sewage sludge by online direct immersion SPME on-fiber derivatization – GCMS

1 Mul i esidue analy ical me hod o pha maceu icals and pe sonal ca e p oduc s in 1 sewage and sewage sludge by online di ec imme sion SPME on- ibe de i a iza ion – 2 GCMS 3 4 Rebeca López-Se na () • Da id Ma ín-de-Jesús • Rubén I us a-Ma a • Ped o An onio 5 Ga cía-Encina • Raquel Leb e o • Ma ía Fdez-Polanco • Raúl Muñoz 6 7 Depa men o Chemical Enginee ing and En i onmen al Technology, Uni e si y o 8 Valladolid, C/ D . Me gelina, s/n, 47011 Valladolid, Spain 9 10 () Co esponding au ho : 11 Rebeca López-Se na 12 e-mail: ebeca.lopezs[email p o ec ed]a.es 13 14 Abs ac 15 The wo k he e p esen ed aimed a de eloping an analy ical me hod o he 16 simul aneous de e mina ion o 22 pha maceu icals and pe sonal ca e p oduc s, 17 including 3 ans o ma ion p oduc s, in sewage and sludge. A me iculous me hod 18 op imiza ion, in ol ing an expe imen al design, was ca ied ou . The de eloped 19 me hod was ully au oma ed and consis ed o he online ex ac ion o 17 mL o wa e 20 sample by Di ec Imme sion Solid Phase Mic oEx ac ion ollowed by On- ibe 21 De i a iza ion coupled o Gas Ch oma og aphy - Mass Spec ome y (DI-SPME – On-22 ibe De i a iza ion – GC - MS). This me hodology was alida ed o 12 o he ini ial 23 compounds as a eliable ( ela i e eco e ies abo e 90% o sewage and 70% o 24 sludge; epea abili y as %RSD below 10% in all cases), sensi i e (LODs below 20 ng L-1 25 in sewage and 10 ng g-1 in sludge), e sa ile (sewage and sewage-sludge samples up o 26 15,000 ng L-1 and 900 ng g-1, espec i ely) and g een analy ical al e na i e o many 27 medium- ech ou ine labo a o ies a ound he wo ld o keep up wi h bo h cu en and 28 o ecas en i onmen al egula ions equi emen s. The emaining 10 analy es ini ially 29 conside ed showed insu icien sui abili y o be included in he inal me hod. The 30 me hodology was success ully applied o eal samples gene a ed in a pilo scale 31 sewage ea men eac o . 32 2 33 Keywo ds: DI-SPME • GC-MS • On-Fibe De i a iza ion • PPCPs • Sewage sludge • 34 Was ewa e 35 36 1 In oduc ion 37 The de elopmen o analy ical me hodologies o he de e mina ion o 38 pha maceu icals and pe sonal ca e p oduc s (PPCPs) in en i onmen al ma ices has 39 boomed in he pas yea s. In his con ex , Zwiene and F immel [1] epo ed ha he 40 analysis o PPCPs has been adi ionally domina ed by Liquid Ch oma og aphy 41 de ec ed by andem Mass Spec ome ic (LC-MS/MS) echniques. Fische e al. [2] 42 ecen ly obse ed majo ends in he use o Ul a High Pe o mance Liquid 43 Ch oma og aphy (UHPLC) [3] and High Resolu ion Mass Spec ome y (HRMS) [4-6] like 44 Time O Fligh (TOF) and O bi ap [7] analyze s. Howe e , hese echniques equi e 45 cos ly ins umen a ion no a o dable by many labo a o ies wo ldwide. In con as , 46 Gas Ch oma og aphy coupled o single quad upole Mass Spec ome y (GC-MS) is an 47 analy ical con igu a ion a mo e common in ou ine analysis labo a o ies a ound he 48 wo ld, including de eloping coun ies. Despi e PPCPs a e mainly pola compounds and 49 no eadily analyzable by GC, Lopez-Se na e al. [8] ecen ly showed how GC-MS is a 50 alid ins umen al echnique o he analysis o eme ging con aminan s in 51 en i onmen al ma ices like sewage, when a de i a iza ion s ep is included in he 52 me hod. In e ms o sample p epa a ion, Solid-Phase Ex ac ion (SPE) ep esen s 53 nowadays he mos popula echnique o he ex ac ion o pollu an s om 54 en i onmen al aqueous samples, and ecen de elopmen s in his ield ha e mainly 55 ocused on SPE au oma ion [9]. In addi ion, a g ea e o has been la ely made o 56 de elop new analy ical me hodologies able o pe o m di ec analyses using 57 minia u ized equipmen , he eby achie ing high en ichmen ac o s, minimizing 58 sol en consump ion and educing was e [7, 10] in acco dance o he equi emen s o 59 g een analy ical chemis y. Solid-Phase Mic oEx ac ion (SPME) was i s ly de eloped 60 in he 1990s by Pawliszyn and cowo ke s [11]. Since hen many con igu a ions ha e 61 been success ully implemen ed, which can be classi ied in o s a ic and dynamic 62 echniques [12]. S a ic p ocedu es a e ypically ca ied ou in s i ed samples, including 63 ibe SPME, and cons i u e he mos common o ma o his echnique. Fibe SPME 64 3 u ilizes a so ben coa ing on he ou e su ace o a used silica ibe o ex ac he 65 analy e(s) om he sample ma ix in a p ocess ha occu s h ough di ec imme sion 66 (DI-SPME) o om he sample headspace in a closed con aine (HS-SPME) [10]. Thus, 67 analy es ha exhibi a high apo p essu e can be ex ac ed ei he by imme sing he 68 ibe in o he aqueous sample o by sampling i s headspace. In con as , analy es ha 69 exhibi a low apo p essu e could only be ex ac ed by imme sion. Fibe SPME has 70 become a e y popula echnique, especially o ola ile compounds, due o i s 71 simplici y, ela i ely sho ex ac ion ime, sol en - ee na u e, ull au oma ion 72 po en ial and easy coupling wi h ch oma og aphy [12]. These ad an ages e en ually 73 educe he con amina ion o he o iginal sample and he loss o analy es. In addi ion, 74 SPME can also be used o onsi e sample ex ac ion and is able o ob ain good esul s 75 e en o ace analy es in complex ma ices [12]. Howe e , i s applica ion o he 76 en i onmen al analysis o pola compounds has been poo ly explo ed, especially when 77 his sample p e ea men is coupled o GC. This applica ion implies he addi ion o a 78 de i a iza ion s ep, which is essen ial o he analysis o non- ola ile and/o 79 he molabile compounds by GC. Today, wo app oaches a e commonly used o ca y 80 ou de i a iza ion when SPME is he p e ea men echnique. The i s one, namely in-81 si u de i a iza ion, is based on he addi ion o he de i a izing agen di ec ly o he 82 sample and he collec ion o he de i ed ola ile analy es by SPME in he headspace o 83 a closed ial. In he second app oach, namely on- ibe de i a iza ion, analy e 84 ex ac ion occu s ia di ec ibe imme sion in he sample combined wi h a headspace 85 de i a iza ion by exposing he analy es-loaded ibe o he apo s o he de i a izing 86 agen . This second app oach is en i onmen ally and economically p e e ed, because 87 he de i a izing agen can be eused o a la ge numbe o analyses (wi h he 88 subsequen dec ease o eagen consump ion). 89 This s udy aimed a de eloping and op imizing a ully au oma ed me hod 90 consis ing o Online DI-SPME - On-Fibe De i a iza ion - GC-MS o he analysis o 19 91 PPCPs and 3 o hei T ans o ma ion P oduc s (TPs) in sewage (SW) and sludge (SS) 92 using s a is ical expe imen al design. To he au ho s’ knowledge, he e a e only wo 93 o he publica ions [13, 14] p oposing he use o his echnique o he analysis o 94 PPCPs in sewage and none o sludge. Howe e , none o hem included he le el o 95 4 au oma ion he e p esen ed. Finally, he analy ical limi a ions encoun e ed du ing he 96 applica ion o his inno a i e me hodology we e also discussed. 97 98 2 Ma e ial and me hods 99 2.1 Chemicals 100 The s anda ds o all PPCPs and hei TPs, p o ided in Table S1 as 101 supplemen a y da a, we e o high pu i y g ade (>95%). They we e pu chased om 102 Sigma-Ald ich (T es Can os, Mad id, Spain) as neu al non-sol a ed molecules, excep 103 o amoxicillin (acqui ed as ihyd a e), a o as a in (acqui ed as calcium sal ) and 104 diclo enac (acqui ed as sodium sal ). The iso opically labelled compounds Diclo enac-105 d4, Ibup o en-d3, Salicylic acid-d4, Nap oxen-d3, P opylpa aben-d7 and T iclosan-d3 106 we e ob ained om TRC Canada (To on o, ON, Canada). 107 Indi idual s ock solu ions a 1 g L-1 o bo h PPCPs s anda ds and iso opically-108 labelled-in e nal-s anda ds we e p epa ed on a weigh basis in me hanol (MeOH), 109 excep o he lu oquinolones (cip o loxacin, le o loxacin and no loxacin), which 110 we e dissol ed in a wa e -me hanol (H2O/MeOH) mix u e (1:1) con aining 0.2% / 111 hyd ochlo ic acid (HCl) due o hei low solubili y in pu e MeOH [15]. F om hem, a 112 s ock solu ion wi h all he analy es was hen p epa ed in MeOH a 20 mg L-1. Se ial 113 aqueous dilu ions we e subsequen ly p epa ed om i . A sepa a e mix u e o 114 iso opically labelled in e nal s anda ds and u he dilu ions we e also p epa ed. A e 115 p epa a ion, all s ock solu ions we e s o ed a -20 °C in da kness. 116 High pu i y sol en s, i.e., Sup aSol ® GC-MS g ade MeOH by Me ck Millipo e 117 (Mad id, Spain), LC-MS Ch omasol ® g ade E hyl Ace a e (EA) by Fluka (Mad id, Spain), 118 Sodium chlo ide (NaCl) and 37% HCl we e supplied by Pan eac (Ba celona, Spain). 119 Ace one, 99% pu e, was supplied by Co a cas (Bu gos, Spain). N- e -120 Bu yldime hylsilyl-N-me hyl i luo oace amide, wi h a pu i y >99%, (MTBSTFA), was 121 ob ained om Regis Technologies Inc. (Mo on G o e, IL, USA). SPME ibe s we e 122 pu chased om Supelco (T es Can os, Mad id, Spain). Milli-Q® g ade wa e was in-123 house p oduced. Helium 99.999% (He) was pu chased om Abelló Linde S.A. (Alcalá de 124 Hena es, Mad id, Spain). 125 5 126 2.2 Sewage analy ical me hodology 127 The de elopmen o he analy ical me hod, u he explained in Sec ions SD.1.1 128 and SD.1.2 wi hin he Supplemen a y da a (SD), was ca ied ou in Milli-Q® wa e and 129 alida ed o sewage as de ailed in Sec ion 3.2.1. In addi ion, he op imized me hod 130 based on Online DI-SPME – On-Fibe De i a iza ion – GC – MS was applied o he 131 analysis o aw and ea ed was ewa e om a pilo scale ac i a ed sludge eac o , and 132 he esul s a e p esen ed in Sec ion 3.2.2. 133 134 2.2.1 Online DI-SPME – On-Fibe De i a iza ion 135 Wa e samples (100 mL) we e supplemen ed wi h NaCl a 30 % (w ./ ol.). A e 136 s i ing o 20 min o assu e comple e dissolu ion, he esul ing wa e sample pH was 137 adjus ed o 3 by adding as ew d ops o dilu ed solu ions o HCl (1%, 0.1% and/o 138 0.01%) as needed. A olume o 17 mL o he esul ing solu ion was placed in a 20-mL 139 SPME ial along wi h 200 µL o an aqueous mix u e o he iso opically labelled in e nal 140 s anda ds a 0.5 mg L-1. 141 The esul ing ial was placed in he sample ack o a CTC PAL RSI au osample . A 142 SPME ool held a 2-cm long 50/30-µm hick 143 Di inylbenzene/Ca boxen/Polydime hylsiloxane (DVB/CAR/PDMS) S ableFlex/SS ibe 144 ha was p o ec ed inside a 23 Ga needle. The ully au oma ed DI-SPME me hod 145 included a ibe p e-condi ioning o 15 min a 270 °C in he spa e GC inle , ollowed by 146 120 min sample ex ac ion a a pene a ion dep h o 60 mm, which en ailed ha he 147 ibe was ully imme sed in he sample (DI-SPME). On- ibe de i a iza ion o he 148 analy es abso bed on o he ibe was hen ca ied ou by in oducing he ibe in 149 ano he 20-mL SPME ial con aining 1 mL o he de i a izing agen MTBSTFA o 48 150 min a a pene a ion dep h o 60 mm. Thus, he ibe was exposed o he apo s o he 151 MTBSTFA in he headspace o he ial. Bo h he DI-SPME and On-Fibe De i a iza ion 152 we e ca ied ou a a cons an empe a u e o 50 °C unde o bi al agi a ion a 500 pm 153 wi h a s i ing egime o 6s on / 30 s o . The ibe , loaded wi h he de i a ized 154 analy es, was hen aken o he GC inle connec ed o he GC column o deso p ion a 155 250 °C o 3 min. Finally, he ibe was pos -condi ioned o 15 min a 270 °C in he 156 spa e GC inle p io o he nex analysis. 157 6 158 2.2.2 GC – MS 159 Ch oma og aphic uns s a ed concomi an ly wi h ibe deso p ion in a pulsed 160 spli less mode a 250 °C in he spli /spli less back inle . A SPME injec ion slee e, 0.75 161 mm i.d., was used as a line . The es s we e pe o med in an Agilen 7890B GC Sys em 162 coupled o a 5977A MSD. A capilla y HP-5MS GC column (30 m leng h, 0.25 mm i.d., 163 0.25 μm ilm hickness) was used o he ch oma og aphic sepa a ion wi h He as 164 ca ie gas a a cons an low a e o 1.2 mL min-1. Injec o empe a u e was se a 250 165 °C, while he GC o en empe a u e inc eased om 70 °C (held o 3 min du ing ibe 166 deso p ion) o 120 °C a 20 °C min-1, hen o 250 °C a 10 °C min-1 and inally o 300 °C 167 (held o 5 min) a 5 °C min-1. The o al analysis ime o each GC un was 33.5 min. The 168 mul imode on GC inle was se a 270 °C in spli mode o acili a e he elimina ion o 169 esidual compounds du ing ibe p e- and pos -condi ioning. 170 Mass de ec ion was ob ained in elec on impac ioniza ion mode (70 eV) wi h 171 selec ed ion moni o ing (SIM) and a ilamen delay o 12 min. The GC–MS in e ace, 172 ion sou ce and quad upole empe a u es we e se a 280, 230 and 150 °C, 173 espec i ely. Quad upole esolu ion was se a low. Ta ge compounds we e eco ded 174 in i e acquisi ion windows along he un ime. Table 1 shows he p ima y (in i alics) 175 and he wo seconda y ions moni o ed pe compound. Acquisi ion s opped a min 26. 176 Ins umen con ol and da a acquisi ion we e pe o med by Agilen Technology Mass 177 Hun e B.07.03.2129 so wa e. 178 179 2.3 Sewage sludge analy ical me hodology 180 Ae obic sludge was used o de elop and alida e he me hodology u he 181 discussed in Sec ions SD.1.3 and 3.2.1, espec i ely. The sewage sludge analy ical 182 me hod was designed as ollows: 1) One hund ed millili e s o esh sludge sample 183 we e eeze-d ied. 2) A known amoun o d ied sludge (~800 mg) was weighed in o a 184 20-mL glass ial, along wi h 200 µL o a mix u e o he iso opically labelled in e nal 185 s anda ds a 20 mg L-1 in ace one. 3) The mix u e was ho oughly o ex-s i ed and 186 emained o e nigh o allow sol en e apo a ion and in e nal s anda d ixa ion. 4) A 187 olume o 12 mL o MilliQ® wa e a pH 9 was hen added o he ial, which was hen 188 igo ously o ex-s i ed o ob ain a homogenous suspension. 5) Then, he ial 189 7 unde wen Ul asound Assis ed Ex ac ion (UAE) o 30 min a oom empe a u e in a 190 JP Selec a Uni eba ul asonic ba h o 50 W and 60 Hz (Ba celona, Spain). 6) 191 Subsequen ly, he suspension was cen i uged o 5 min a 2,655 x g in a Fishe 192 Bioblock Scien i ic Cen i uge 2-16P (Mad id, Spain). 7) The esul ing supe na an was 193 hen collec ed wi h a glass pipe e and ans e ed o a 20-mL glass ial. 8) S eps 4-7 194 we e epea ed once mo e and he supe na an s we e pooled oge he . 9) The esul ing 195 solu ion was analyzed by Online DI-SPME – On- ibe de i a iza ion – GC-MS using he 196 op imized me hod desc ibed in Sec ion 2.2, excep o he addi ion o in e nal 197 s anda ds as hey we e al eady added in s ep 2. 198 199 2.4 Expe imen al design 200 As a i s app oach, a sc eening design was ca ied ou . Hence, he key 201 pa ame e s in luencing he pe o mance o he Online DI-SPME – On-Fibe 202 De i a iza ion me hodology we e iden i ied o he de elopmen o he ins umen al 203 leg o bo h sewage and sludge me hods. As a esul , a o al o 18 pa ame e s we e 204 so ed ou in ou ca ego ies, depending on he a ge o hei in luence, i.e., DI-SPME 205 ex ac ion, On-Fibe De i a iza ion, Fibe Deso p ion and Ca y-O e a oidance (Table 206 S2). A e wa ds, echnical limi a ions o his inno a i e me hodology we e poin ed ou , 207 which na owed down o 6 he numbe o pa ame e s admi ing u he op imiza ion. 208 None heless, 4 o hem, i.e., ibe coa ing, sample Ionic s eng h, sample pH and 209 de i a iza ion empe a u e could easily be op imized by a one- ac o -a -a- ime 210 app oach as hey a e disc e e a iables o o he wise consolida ed e e ences exis in 211 he scien i ic li e a u e which d as ically delimi s he ange o a ia ion. E en ually, 212 only wo pa ame e s emained as signi ican , ex ac ion and de i a iza ion imes, and 213 in need o u he op imiza ion. Thus, a esponse su ace me hodology (RSM), 214 consis ing o a ull ac o ial 22 wi h a cen al poin epea ed i e imes and ex ended 215 wi h 4 s a poin s, was applied o hem. Thus, a se o 13 expe imen s was andomly 216 pe o med. A e wa ds, he so wa e S a g aphics Cen u ion XVII was used o p ocess 217 he acqui ed expe imen al da a and ma hema ically i i o a second o de polynomial 218 model h ough he leas squa es me hod. 219 220 3 Resul s and discussion 221 8 3.1 Analy ical me hod de elopmen and op imiza ion o sewage and sludge 222 A selec ion o 22 PPCPs, in pa icula , 5 pha maceu icals and 2 o hei TPs as 223 well as 14 pe sonal ca e p oduc s and 1 o hei TPs, we e ini ially chosen as a ge 224 analy es. 225 The p o ocol ollowed o de elop and op imize he analy ical me hod, including 226 an expe imen al design, is desc ibed in he supplemen a y da a SD ile. In b ie , a e 227 he GC-MS leg was de eloped, he sample p e ea men pa o he me hodology was 228 op imized. Hence all he pa ame e s wi h a ole du ing he Online DI-SPME – On-Fibe 229 De i a iza ion we e iden i ied and some echnical limi s we e se . A e wa ds, some 230 p elimina y expe imen s we e ca ied ou in a one- ac o -a -a- ime app oach o 231 op imize he Type o Fibe Coa ing, Sample Ionic S eng h, Sample pH and 232 De i a iza ion empe a u e. Finally, as he ex ac ion and de i a iza ion ime could 233 in e e e wi h each o he , a esponse su ace me hod was designed based on a ull 234 ac o ial 22 wi h a cen al poin epea ed i e imes and ex ended wi h 4 s a poin s. 235 TS/N was selec ed as he esponse a iable du ing he op imiza ion, in o de o ge a 236 comp omise among he pe o mance o all he compounds. As a esul , he op imum 237 alue o he esponse a iable ob ained co esponded o an ex ac ion ime o 120 238 min and a de i a iza ion ime o 48 min. Tha is g aphically shown in Figu e 1. 239 A e he op imiza ion, en o he ini ial a ge PPCPs, including he analgesics 240 ace aminophen and ace ylsalicylic acid, he lipid egula o a o as a in, and he 241 an ibio ics amoxicillin, cip o loxacin, le o loxacin, no loxacin, sul ame hoxazole, 242 e y h omycin and cla i h omycin u ned ou o be unsui able o hei analysis by 243 Online DI-SPME – On-Fibe De i a iza ion – GC-MS, as hey exhibi ed a e y weak o 244 e en no esponse wha soe e . The e o e, hey we e uled ou and no included in he 245 me hod. 246 The inal me hods, which allowed o he analysis o 12 PPCPs including 3 TPs, 247 a e summa ized in Sec ions 2.2 and 2.3. Rep esen a i e SIM ch oma og ams, ob ained 248 om MilliQ® wa e and sewage sludge samples spiked wi h he a ge PPCPs a 4 µg L-1 249 and 400 ng g-1, espec i ely, using he op imized me hod condi ions, a e illus a ed in 250 Figu e 2. 251 252 253 9 3.2 Me hod alida ion and applica ion 254 3.2.1 Me hod alida ion 255 Se e al egula o y bodies (like he Uni ed S a es Food and D ug Adminis a ion 256 (FDA) [17] o Eu achem [18]), s anda diza ion agencies (like he In e na ional 257 Associa ion o O icial Analy ical Chemis s (AOAC In e na ional) [19]), and wo king 258 g oups and commi ees (like he Food and Ag icul u al O ganiza ion/Wo ld Heal h 259 (FAO/WHO) [20]) ha e published guidelines and equi emen s o me hod alida ion. 260 In addi ion, he Eu opean Union adop ed a decision [21] implemen ing a di ec i e 261 conce ning he pe o mance o analy ical me hods and he in e p e a ion o esul s. I 262 e e s o animal p oduc s. Howe e , i has been widely used as an illus a i e e e ence 263 in he design o cus omized alida ion p o ocols o en i onmen al analysis like in [22-264 25], as well as in he p esen s udy because o he lack o speci ic guidelines. 265 Hence, i e alida ion pa ame e s, i.e., accu acy, ME, p ecision, sensi i i y and 266 dynamic ange we e de e mined o all 12 a ge analy es included in he me hod 267 (clo ib a e, 1,4-benzoquinone, me hylpa aben, e hylpa aben, clo ib ic acid, ibup o en, 268 p opylpa aben, salicylic acid, p-hyd oxybenzoic acid, nap oxen, iclosan, diclo enac) in 269 sewage and sludge. In addi ion, a ca yo e es was also pe o med o ensu e he 270 absence o con amina ion be ween samples du ing he ins umen al leg o he 271 analysis. Two meaning ul le els o concen a ion pe ma ix −100 and 1000 ng L-1, and 272 100 and 400 ng g-1− ypical o he a ge compounds in eal sewage and sludge 273 samples, espec i ely, we e es ed o he ou i s pa ame e s, as ecommended by 274 [23, 24]. Each es was un in iplica e wi h he op imized me hod. The esul s, 275 a e age o bo h concen a ion le els, which a e discussed below, a e shown in Table 276 S4. 277 278 1) Accu acy: Absolu e eco e ies (%) we e de e mined by compa ing he 279 peak a eas ob ained om spiked samples analyzed using he op imized me hods wi h 280 he a eas ob ained om di ec injec ions (2 µL) o equi alen amoun s o s anda ds in 281 EA solu ions. As bo h sewage and sludge can con ain some o he a ge compounds, 282 non-spiked samples we e also analyzed and he peak a eas we e a e wa ds 283 sub ac ed om he spiked samples in o de o calcula e he absolu e eco e y. Table 284 S4A shows e y a iable absolu e eco e ies o sewage. Hence, SPME suppo ed good 285 16 [9] A. Balles e os-Gómez, S. Rubio, Recen ad ances in en i onmen al analysis, 474 Analy ical Chemis y 83(12) (2011) 4579-4613. 475 [10] M.E.T. Pad ón, C. A onso-Oli a es, Z. Sosa-Fe e a, J.J. 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Bayona, E. 549 Béca es, Tempo al e olu ion in PPCP emo al om u ban was ewa e by cons uc ed 550 we lands o di e en con igu a ion: A medium- e m s udy, Chemosphe e 88(2) (2012) 551 161-167. 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 19 Figu e 1: Response su ace a e applying an expe imen al design 22 + s a + 5 cen al 568 poin s 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 20 Figu e 2: Ch oma og ams ob ained om A) 4000 ng L-1 MilliQ wa e and B) 400 ng g-1 598 sludge samples a e he op imized me hods we e applied 599 A) 600 601 602 B) 603 604 21 Table 1: MS pa ame e s o he inal a ge compounds and in e nal s anda ds 1IS Analy e Chemical Name Acquisi ion window # 2 R (min) 3SIM ions, m/z 1 Clo ib a e 1 13.17 128 130 169 2 1,4-Benzoquinone 13.70 281 338 282 3 Me hylpa aben 2 15.23 209 210 135 4 Ace ylsalicylic acid 15.28 195 237 135 5 E hylpa aben 16.02 223 224 151 6 Clo ib ic acid 16.02 143 271 185 7 Ibup o en 16.38 263 264 117 1 Ibup o en-d3 16.34 266 267 164 8 P opylpa aben 17.04 237 238 151 2 P opylpa aben-d7 16.96 244 245 152 9 Salicylic acid 17.50 309 310 195 3 Salicylic acid-d4 17.45 313 314 312 10 Ace aminophen 3 18.24 208 265 166 11 P-hyd oxybenzoic acid 18.91 309 265 310 12 Nap oxen 4 21.07 287 185 288 4 Nap oxen-d3 20.95 290 188 207 13 T iclosan 21.66 347 345 200 5 T iclosan-d3 21.54 350 348 200 14 Diclo enac 5 23.73 352 214 354 6 Diclo enac-d4 23.73 356 218 358 1IS: In e nal S anda d; 2 R: Re en ion Time; 3SIM: Selec ed Ion Moni o ing 22 1