Else ie Edi o ial Sys em( m) o Talan a
Manusc ip D a
Manusc ip Numbe : TAL-D-11-00529R1
Ti le: Simul aneous analysis o chlo ophenols, alkylphenols, ni ophenols and c esols in was ewa e
e luen s, using solid phase ex ac ion and u he de e mina ion by gas ch oma og aphy- andem mass
spec ome y
A icle Type: Full Leng h A icle
Keywo ds: Phenols; Was ewa e ; Solid-phase ex ac ion (SPE); Gas ch oma og aphy-mass
spec ome y (GC-MS); S anda d addi ion calib a ion; Ma ix-ma ched calib a ion.
Co esponding Au ho : D . A. Ga ido F enich, Ph.D
Co esponding Au ho 's Ins i u ion: Resea ch G oup Analy ical Chemis y o Con aminan s
Fi s Au ho : Juan An onio Padilla-Sánchez
O de o Au ho s: Juan An onio Padilla-Sánchez; Pa icia Plaza-Bolaños; Robe o Rome o-González;
Nie es Ba co-Bonilla; José Luis Ma ínez-Vidal; A. Ga ido F enich, Ph.D
Abs ac : An analy ical me hodology has been de eloped o he simul aneous ex ac ion o 13
phenolic compounds, including chlo ophenols (CPs), ni ophenols (NTPs), c esols and alkylphenols
(APs) in di e en ypes o was ewa e (WW) e luen s. A solid-phase ex ac ion (SPE) me hod has been
op imized p io o he de e mina ion by gas ch oma og aphy coupled o iple quad upole andem
mass spec ome y (GC-QqQ-MS/MS). Due o he complexi y o he ma ix, a compa ison s udy o
ma ix-ma ched-calib a ion (MMC) and s anda d addi ion calib a ion (SAC) was ca ied ou o
quan i ica ion pu poses. The op imized p ocedu e was alida ed using he SAC app oach since i
p o ided he mos adequa e quan i ica ion esul s (in e ms o eco e y and p ecision alues).
Reco e ies we e in he ange 60- - and 78-
wi h p ecision alues (exp essed as ela i e s anda d de ia ion, RSD) ≤ 30% (excep o 2-ni ophenol)
in ol ing in a-day and in e -day p ecision s udies. Limi s o de ec ion (LODs) and quan i ica ion
(LOQs) we e also e alua ed, an - -1. The p oposed me hod
was applied o he analysis o 8 eal WW e luen samples, inding some phenolic compounds (e.g. 2-
chlo ophenol, 2,4,6- ichlo ophenol and 4- e -oc ylphenol) a concen a ions highe han he
es ablished LOQs.
1
Simul aneous analysis o chlo ophenols, alkylphenols, ni ophenols and c esols 1
in was ewa e e luen s, using solid phase ex ac ion and u he de e mina ion 2
by gas ch oma og aphy– andem mass spec ome y 3
4
J. A. Padilla-Sáncheza,1, P. Plaza-Bolañosa,b, R. Rome o-Gonzáleza, N. Ba co-5
Bonillaa, J. L. Ma ínez-Vidala, N. Ba co-Bonillaa,1, A. Ga ido-F enicha*
6
7
aDepa men o Analy ical Chemis y, Uni e si y o Alme ia, Ca e e a Sac amen o 8
s/n, E-04071 Alme ia, Spain 9
bDepa men o Analy ical Chemis y, Uni e si y o G anada, E-18071 G anada, 10
Spain 11
12
13
* Co espondence o: An onia Ga ido F enich, Depa men o Analy ical Chemis y, 14
Alme ia Uni e si y o Alme íia, 04120, Alme iía, Spain. 15
Tel: +34950015985; Fax: +34950015483; e-mail: [email p o ec ed] 16
17
1 Bo h au ho s con ibu ed equally o his wo k. 18
19
Ma ked-up manusc ip
2
20
Abs ac 21
An analy ical me hodology has been de eloped o he simul aneous ex ac ion o 22
13 phenolic compounds, including chlo ophenols (CPs), ni ophenols (NTPs), c esols 23
and alkylphenols (APs) in di e en ypes o was ewa e (WW) e luen s. A solid-24
phase ex ac ion (SPE) me hod has been op imized p io o he de e mina ion by gas 25
ch oma og aphy coupled o iple quad upole andem mass spec ome y (GC-QqQ-26
MS/MS). Due o he complexi y o he ma ix, a compa ison s udy o ma ix-ma ched-27
calib a ion (MMC) and s anda d addi ion calib a ion (SAC) was ca ied ou o 28
quan i ica ion pu poses. The op imized p ocedu e was alida ed using he SAC 29
app oach since i p o ided he mos adequa e quan i ica ion esul s (in e ms o 30
eco e y and p ecision alues). Reco e ies we e in he ange 60–135% (0.5 g L−1), 31
70–115% (1 g L−1), and 78–120% (5 g L−1), wi h p ecision alues (exp essed as 32
ela i e s anda d de ia ion, RSD) ≤ 30% (excep o 2-ni ophenol) in ol ing in a-33
day and in e -day p ecision s udies we e ob ained. Limi s o de ec ion (LODs) and 34
quan i ica ion (LOQs) we e also e alua ed, and LOQs anged om 0.03 g L-1 o 2.5 35
g L-1. The p oposed me hod was applied o o he analysis o 8 eal WW e luen 36
samples, inding some phenolic compounds (e.g. 2-chlo ophenol, 2,4,6-37
ichlo ophenol and 4- e -oc ylphenol) a concen a ions highe han he es ablished 38
LOQs es ablished du ing he me hod alida ion. 39
Keywo ds: Phenols, was ewa e , solid-phase ex ac ion (SPE), gas ch oma og aphy-40
mass spec ome y (GC-MS), s anda d addi ion calib a ion, ma ix-ma ched 41
calib a ion. 42
43
Fo ma ed: None
3
44
1. In oduc ion 45
Phenolic compounds can be ound in was ewa e (WW) e luen s ia di e en 46
sou ces. They can be de ec ed in his ype o samples because o hei use in plas ics 47
[1], d ug manu ac u ing, phy osani a y p oduc s o lea he colo ing [2], by 48
an h opogenic emission [2] o and by he use o ea men s wi h ae obic o anae obic 49
mic oo ganisms [4]. Some phenols show high oxici y, es ogenic [5] and an i-50
and ogenic ac i i y [6], andas well as hey can ac as endoc ine dis up e s [7]. 51
Phenols can be classi ied in a wide ange o amilies. The mos s udied analy es in 52
wa e a e chlo ophenols (CPs) [1] and alkylphenols (APs) [8]. Howe e , he Uni ed 53
S a es En i onmen al P o ec ion Agency (US EPA) classi ies CPs, ni ophenols and 54
APs as p io i y pollu an s [9] and i has es ablished a maximum con amina ion le el 55
(MCL) o pen achlo ophenol (PCP) o 1 µg L-1 in d inking wa e s [10]. On he o he 56
hand, he Eu opean Union (EU) has adop ed a lis o p io i y subs ances in he ield o 57
wa e policy, including 4-n-nonylphenol (4-n-NP), 4- e -oc ylphenol (4- e OP) and 58
PCP [11]. Fu he mo e, maximum allowable concen a ions (MAC) ha e been 59
es ablished o NP (2 g L-1) and PCP (1 g L-1) in inland and o he su ace wa e s 60
[12]. Howe e , i mus be poin ed ou ha legisla ion o WWs is s ill e y sca ce, and 61
he alues es ablished in d inking wa e a e usually used as guide in WWs. Bea ing in 62
mind hese ac s, he de elopmen o sensi i e analy ical me hodologies o he 63
simul aneous de e mina ion o phenols belonging o di e en g oups, such as CPs, 64
APs, ni ophenols (NTPs) and c esols (also known as me hyl-phenols) wi h di e en 65
pola i y ange (log Kow 1.77-5.01) is needed in o de o p o ide a comple e o e iew 66
o he occu ence o phenolic compounds in WW e luen s. 67
4
Se e al ex ac ion echniques ha e been applied o he ex ac ion o phenols om 68
aqueous samples, such as solid-phase ex ac ion (SPE) [8,13-15] and liquid-liquid 69
ex ac ion (LLE) [16]. Recen ly, mic oex ac ion echniques, such as solid-phase 70
mic oex ac ion (SPME) [17-19], s i ba so p i e ex ac ion (SBSE) [19-21], liquid 71
phase mic oex ac ion (LPME) [22] o dispe si e liquid-liquid mic oex ac ion 72
(DLLME) [23] ha e been appliedused. Howe e , mos o hem ha e only been used 73
o he simul aneous analysis o only one o ew phenols belonging o he same amily 74
such as APs [18,20] and CPs [17]. I is well-known ha SPE is he mos used 75
echnique in wa e analysis [24] due o he less educed exposi ionu e and 76
con amina ion by o ganic sol en s, he high p e-concen a ion ac o s ha make 77
possible o a oididing e apo a ion s eps, he semi-au oma ion o he ex ac ion 78
p ocess, educing he sample handling, and i allows he ex ac ion o compounds wi h 79
di e en physico-chemical p ope ies. The applica ion o mic oex ac ion echniques 80
is inc easing bu se e al disad an ages, such as cos and li e ime o ibe s and ba s, o 81
he limi ed scope o a wide pola i y ange can hinde hei u iliza ion. 82
83
Fo he de e mina ion o phenolic compounds, gas ch oma og aphy (GC) [13,25] o 84
liquid ch oma og aphy (LC) [26,27] a e he p edominan he mos used echniques, 85
mainly coupled o andem mass spec ome y (MS/MS) [28-31]. When GC is used, a 86
de i a iza ion s ep is equi ed in o de o imp o e he ch oma og aphic pe o mance 87
and sensi i i y o he selec ed compounds, and se e al de i a iza ing eagen s can be 88
applied [32,33]. 89
A well-known c i ical poin in he analysis o WW is he ma ix e ec [34]. In o de 90
o minimize i , di e en calib a ion me hods such as ma ix-ma ched calib a ion 91
5
(MMC) [33,35], s anda d addi ion calib a ion (SAC) [34] and he use o iso ope-92
labeled in e nal s anda ds [36,37] ha e been employed o complex ma ixes. 93
Quan i ica ion based on iso ope-labeled in e nal s anda ds has disad an ages due o 94
he expensi eness o hese s anda ds and hei limi ed a ailabili y. MMC is o en he 95
mos used quan i ica ion me hod in ace analysis. Howe e , he lack o blank 96
ma ixes and he need o s o ing hem can make his app oach logis ically one ous 97
and no necessa ily accu a e. SAC is he mos adequa e echnique o use when i is 98
di icul o ind a blank samples o he s udied ma ix s udied, bu a calib a ion se is 99
equi ed o each sample, inc easing he o al numbe o injec ions and he ime spen 100
in da a p ocessing. 101
Ano he p oblem ela ed o he de e mina ion o phenols in WW is ha depending 102
on he ype o WW ea men , WW e luen s can ha e di e en amoun s o suspended 103
pa icula e ma e (SPM). This SPM is no mally disca ded du ing he ex ac ion 104
p ocess by il a ion in mos o he analy ical me hods epo ed in li e a u e [38]. 105
Howe e , a ecen s udy [35] has demons a ed ha ce ain analy es can be e ained in 106
he SPM, depending on i s pola i y. The e o e, i should be necessa y o e alua e he 107
p esence o phenols in bo h phases in o de o de e mine whe he he SPM mus be 108
disca ded o no . 109
Fu he mo e, i mus be poin ed ou ha many a icles epo ing simul aneous 110
ex ac ion and de e mina ion o di e en classes o phenols (including APs, CPs and, 111
NTPs) in wa e [39,40] can be ound. Howe e , hey ha e been de eloped o he 112
analysis o his ype o compounds in su ace wa e , and hey a e no alid o he 113
analysis in WW samples, due o hey a e mo e complex ma ices wi h di e en 114
physico-chemical cha ac e is ics (SPM le els, o ganic ma e , e c.). 115
6
The e o e, Iin his s udy, a simul aneous SPE ex ac ion by SPE and de e mina ion 116
o di e en phenolic amilies (, namely CPs, NTPs, c esols and APs), has been 117
de eloped o WW e luen samples. In addi ion wo no el aspec s o his wo k 118
mus be poin ed ou : (i) a s udy o he p esence o phenolic compounds in he 119
SPM acco ding o he s a egy ecen ly p oposed by Ba co-Bonilla e al [35], and 120
(ii) a compa ison o MMC and SAC in o de o e alua e he bes quan i ica ion 121
s a egy o phenolic compounds in complex ma ices such as WWs. Fo ha , A 122
s udy o he p esence o phenolic compounds in he SPM has been ca ied ou 123
acco ding o he s a egy ecen ly p oposed by Ba co-Bonilla e al [35]. Due o he 124
complexi y o he ma ix and he di icul y o ob ain blank samples, a s udy o 125
quan i ica ion s udy using MMC and SAC was de eloped in o de o e alua e he bes 126
quan i ica ion s a egy. T wo di e en WW e luen s we e s udied indi idually: 127
memb ane bio eac o (MBR, low SPM con en ) and anae obic pond (ANAP, high 128
SPM con en ). The op imized SPE and quan i ica ion me hod was alida ed in bo h 129
ypes o WWs e luen samples. 130
131
2. Expe imen al 132
2.1. Chemicals and ma e ials 133
Phenolic compounds s anda ds, 2-ni ophenol (2-NTP), 4-ni ophenol (4-NTP), 2,4-134
dime hylphenol (2,4-DMP), 2-CP, 4-chlo ophenol (4-CP), 2,4-dichlo ophenol (2,4-135
diCP), 2,4,5- ichlo ophenol (2,4,5- iCP), 2,4,6- ichlo ophenol (2,4,6- iCP) and 4-136
n-NP we e ob ained om Fluka (Buchs, Swi ze land). On he o he hand, 3-137
ni ophenol (3-NTP), 4-chlo o-3-me hylphenol (4-C-3-MP), 4- e OP and PCP we e 138
supplied by Supelco (Belle on e, PA, USA). Pu i ies we e always >97%. A s anda d 139
7
solu ion (100 mg L−1) o iso opically labeled PCP ([13C6]-PCP) was used as in e nal 140
s anda d (IS) and i was ob ained om D . E hens o e (Augsbu g, Ge many). S ock 141
s anda d solu ions o indi idual compounds (wi h concen a ions anging om 200 o 142
450 mg L-1) we e p epa ed by exac weighing o he powde o liquid and dissolu ion 143
in 50 mL o ace one. These solu ions we e hen s o ed unde e ige a ion (T <5 ºC). 144
A wo king s anda d solu ion o he 13 phenolic compounds (2 mg L-1 o each 145
compound) was p epa ed by app op ia e dilu ion o he s ock solu ions wi h ace one, 146
and i was s o ed unde e ige a ion (T <5 ºC). A wo king s anda d solu ion o [13C6]-147
PCP (22 mg L-1) was p epa ed by app op ia e dilu ion o he s anda d solu ion wi h 148
ace one and s o ed unde he a o emen ioned condi ions. HPLC-g ade me hanol 149
(MeOH), anhyd ide ace ic acid (AAA) (99.9%), and py idine (Py) (99.8%) we e 150
pu chased om Sigma-Ald ich (Mad id, Spain). Ace one and hyd ochlo ic acid (HCl) 151
we e ob ained om J.T. Bake (De en e , Ne he lands). Dichlo ome hane (DCM) was 152
pu chased om Riedel-de Haën (Seelze-Hanno e , Ge many). Ul apu e wa e was 153
ob ained om a Milli-Q G adien wa e sys em (Millipo e, Bed o d, MA, USA). 154
Thi y mm cellulose il e s and 47-mm glass mic o ibe il e s om Wha man 155
(Maids one, England, UK) and 0.45-µm HNWP nylon memb ane il e s om 156
Millipo e (Ca ig wohill, Coun y Co k, I eland) we e also a ailable o il a ion 157
s ages. Fo SPE, Oasis HLB (200 mg, 6 cm3) ca idges we e ob ained om Wa e s 158
(Mil o d, MA, USA). 159
160
2.2. Appa a us 161
A GC sys em Va ian 3800 (Va ian Ins umen s, Sunny ale, CA, USA) equipped wi h 162
elec onic low con ol was in e aced o a 1200L iple quad upole (QqQ) mass 163
8
spec ome e . Samples we e injec ed in o an SPI/1079 spli /spli less p og ammed-164
empe a u e injec o using a Combi Pal (CTC Analy ics AG, Zwingen, Swi ze land) 165
wi h a 100-µL sy inge. A used-silica un ea ed capilla y column (2 m × 0.25 mm i.d.) 166
om Supelco was used as p e-column connec ed o a VF-5 ms Fac o Fou capilla y 167
column (30 m × 0.25 mm i.d. × 0.25 µm ilm hickness) pu chased om Va ian. 168
Helium was used as ca ie gas (99.9999%) a a cons an low a e o 1 mL min−1, and 169
a gon (99.999%) was used as collision gas. The mass spec ome e was ope a ed in 170
elec on ioniza ion (EI) mode a 70 eV. The mass spec ome e was calib a ed e e y 171
ou days wi h pe luo o ibu ylamine. Va ian Wo ks a ion so wa e was used o 172
ins umen con ol and da a analysis. 173
A Reax-2 o a y agi a o om Heidolph (Schwabach, Ge many) was used o 174
agi a ion o he de i a iza ion mix u e. An analy ical balance AB204-S om Me le 175
Toledo (G ei ensee, Swi ze land) and a o a y e apo a o R-114 (Büchi, Flawil, 176
Swi ze land) we e used du ing ex ac ion and s anda d p epa a ion. The ho izon al 177
shake used in he dis ibu ion s udy was ob ained om P-Selec a (Selec a, Ba celona, 178
Spain). 179
180
2.3. Sampling 181
WW u ban e luen s om wo di e en ea men s, namely, MBR and ANAP, wi h 182
low and high SPM con en espec i ely, we e collec ed om WW ea men plan 183
(WWTP) o he ounda ion Cen e o New Wa e Technologies (“Cen o de las 184
Nue as Tecnologías del Agua”, CENTA, Se ille, Spain). This WWTP has 41,000 m2 185
and i cu en ly holds mo e han 20 sys ems wi h di e en echnologies. Addi ional 186
physicochemical da a ela ed o he ea men s e alua ed in his s udy can be ound in 187
15
Reco e y and p ecision we e e alua ed using bo h quan i ica ion app oaches. I can 329
be obse ed ha in WW e luen s wi h high SPM, such as ANAP, MMC did no 330
p o ided adequa e esul s o he lowe spiked concen a ions (0.5 and 1 g L-1). 331
Reco e ies and in a and in e -day p ecision o mos o compounds we e below 60% 332
and o e 30%, espec i ely o hese wo concen a ion le els. On he con a y, o 5 333
g L-1, eco e y alues we e in he ange 60-120%, excep o 4-n-NP (51%) and 334
in a and in e -day p ecision we e <12%. These esul s (Table 2) sugges ed ha MMC 335
is no a sui able op ion o he adequa e quan i ica ion o a e y low concen a ions o 336
phenols in WWs e luen s wi h high SPM. On he o he hand, when SAC was used, 337
eco e ies o all compounds we e in ange 60-125%, excep o 4- e OP (135%) a 338
he lowes o i ica ion le el (0.5 g L-1). In a and in e -day p ecision alues we e 339
<27% and <31% o all compounds, espec i ely. As i is shown in Table 2, he SAC 340
app oach is mo e app op ia e o WW e luen s wi h high SPM con en . Linea i y was 341
s udied in he ange 10-150 g L-1 (excep o NTPs which was 100-300 g L-1) and 342
he ob ained de e mina ion coe icien s (R2) we e in he ange 0.9912 (3-NTP)-0.9999 343
(2-CP, 2,4,5- T iCP, PCP and 4-n-NP a ious compounds) o ANAP (Table 4). 344
Fo WW e luen s wi h low SPM, such as MBR (Table 3), he eco e ies ob ained 345
when MMC was used o he h ee le els assayed anged om 62-119%, excep o 4-346
n-NP, wi h eco e ies lowe han 56%. Despi e he adequa e eco e y esul s p o ided 347
by MMC o all he s udied o i ica ion le els, in gene al, RSD alues we e <30% 348
only o he highes spiked le el s udied (5 g L-1), as i can be obse ed in Table 3, 349
whe eas a he lowes concen a ion le els e alua ed (0.5 and 1 g L-1), in a and 350
in e -day p ecision anged om 22 o 113%. On he o he hand, he applica ion o 351
SAC on MBR WW samples yielded eco e y alues in he ange 70-120%, excep o 352
4-CP (125%) a 0.5 g L-1. Besides, RSD alues we e always <28% o in a-day 353
16
p ecision and <27% o in e -day p ecision in all cases, excep o 2-NTP, which was 354
41% a 0.5 g L-1. In consequence, i can be concluded ha o MBR ea ed WW 355
e luen s, SAC was also he mos sui able me hod o an adequa e quan i ica ion o 356
WW e luen s wi h low SPM con en , such as MBR WW samples (Table 3). 357
Fu he mo e, linea i y was also e alua ed o MBR and R2 alues anged om 0.9943 358
(4-NTP) o 0.9999 (2-CP, 4-CP, 2,4,6- iCP and 4-n-NP). 359
Conside ing hese esul s, he SAC me hod should be applied o a eliable 360
quan i ica ion o phenols in WW e luen s samples o compensa e ma ix e ec s on 361
he signal a ia ion du ing de ec ion and his does no depend on he SPM con en o 362
he WW. The SAC me hodology was he e o e applied o he quan i ica ion o 363
phenols in eal samples. 364
365
3.4. Es ima ion o he lowe limi s o he me hodology 366
Despi e o he es ima ion o he ueness and p ecision ca ied ou in he p e ious 367
sec ion, o he pe o mance cha ac e is ics o he me hod, such as limi s o de ec ion 368
(LODs) and quan i ica ion (LOQs) we e s udied. LODs and LOQs we e de e mined 369
as he lowes concen a ion le el ha yielded a signal- o-noise (S/N) a io o 3 and 10, 370
and hey a e shown in Table 4. LODs and LOQs we e de e mined in WW sample 371
blanks o each phenolic compound s udied. LODs we e om 0.01 o 1 g L-1 and 372
LOQs anged om 0.03 g L-1 o 2.5 g L-1 o ANAP and MBR (Table 4). I mus 373
be no iced ha simila alues we e ob ained o bo h ypes o WW e luen s, excep 374
o 2-NTP and 4-C-3-MP, which showed highe LOD and LOQ alues in ANAP han 375
in MBR. This could be explained aking in o accoun ha he SPM con en is highe 376
17
in ANAP, inc easing he amoun o co-ex ac ed ma e ial and a ec ing he es ima ion 377
o he lowe limi s o he me hod. 378
379
3.5. Applica ion o he analysis o eal WW e luen samples 380
The de eloped me hodology was applied o he analysis o 8 WW e luen samples 381
om he CENTA, ob ained a e he applica ion o di e en WW ea men s 382
employed in his WWTP. To assu e he quali y o he esul s and a oid e o s, he 383
quan i ica ion o he phenolic compounds was achie ed using he SAC app oach. An 384
in e nal quali y con ol (IQC) was pe o med consis ing o he analysis o spiked 385
blank WW samples a 1 g L-1 (excep o 3-NTP and 4-NTP a 5 g L-1), which we e 386
used o assess he ex ac ion e iciency and a SAC calib a ion cu e o check linea i y 387
and sensi i i y. Se e al phenolic compounds we e ound o e he LOQs es ablished 388
by he me hod, showing he ob ained esul s in Table 5. 2-CP and 2,4,6- iCP we e 389
ound in six and i e samples, espec i ely, wi h concen a ions anging om 0.04 o 390
0.20 g L-1 o 2-CP and om 0.05 o 0.10 g L-1 o 2,4,6- iCP. 4-CP and 4- e OP 391
we e ound in ou samples, and he concen a ions anged om 0.04 o 0.08 g L-1 392
and 0.04 o 0.16 g L-1 espec i ely. 2-CP, 2,4-DiCP, 4- e OP, PCP and 4-n-NP we e 393
ound simul aneously in one o he samples (Table 5). I mus be highligh ed ha 394
phenolic compounds we e no ound o e he MCLs and MACs es ablished by he 395
EPA and he EU o hese compounds [10,12]. Finally, Figu e 4 shows a posi i e 396
sample o 4- e OP de ec ed in a WW e luen sample a 0.12 g L-1. 397
398
5. Conclusions 399
18
A single ex ac ion me hod o he simul aneous ex ac ion o CPs, APs, NTPs and 400
c esols in WW e luen samples has been de eloped using SPE. A dis ibu ion s udy 401
o he phenolic compounds be ween he aqueous phase and he SPM was ca ied ou , 402
e i ying ha he SPM could be in ac disca ded du ing he ex ac ion since only 403
phenolic compounds wi h high log Kow we e ound in he SPM a a negligible 404
pe cen age. Due o he di icul y o ind WW blank samples and o ha e good 405
accu acy in he quan i ica ion, a s udy using MMC e sus SAC was pe o med in wo 406
di e en ea ed WW e luen samples (ANAP and MBR) showing ha SAC is he 407
mos sui able quan i ica ion app oach. The me hod was alida ed s udying eco e y, 408
in a and in e -day p ecision, lowe limi s (LODs and LOQs) and linea i y. 409
De e mina ion o he analy es was ca ied ou using GC-QqQ-MS/MS ope a ing in 410
SRM mode. The me hod was applied o WW e luen samples wi h sa is ac o y 411
esul s, obse ing ha phenols o se e al amilies we e simul aneously de ec ed in 412
WW e luen s, highligh ing he po en ial o analy ical me hods ha allows he 413
simul aneous de e mina ion o se e al classes o phenolic compounds. 414
415
Acknowledgmen s 416
The au ho s g a e ully acknowledge he Andalusian Regional Go e nmen (Regional 417
Minis y o Inno a ion, Science, and En e p ise-FEDER) o inancial suppo 418
(P ojec Re . P08-RNM-03892). PPB acknowledges o pe sonal unding h ough 419
Juan de la Cie a P og am (Spanish Minis y o Science and Inno a ion-Eu opean 420
Social Fund). RRG is also g a e ul o pe sonal unding h ough Ramón y Cajal 421
P og am (Spanish Minis y o Science and Inno a ion-Eu opean Social Fund). NBB 422
is g a e ul o he p e-doc o al g an om he a o emen ioned p ojec . CENTA is 423
g a e ully acknowledged o p o iding WWs e luen s samples. 424
19
425
426
20
Re e ences 427
[1] S. Eke , K.J. Fik e , En i on. Manage. 90 (2009) 692-698 428
[2] M. Conce a-Tomei, M. C is ina-Annesini, En i on. Sci. Technol. 39 (2005) 5059-429
5065. 430
[3] C. Li, S. Taneda, A.K. Suzuki, C. Fu u a, G. Wa anabe, K. Taya, Toxicol. Appl. 431
Pha m. 217 (2006) 1–6. 432
[4] D. Li, J. Pa k, J.R. Oh, Anal. Chem. 73 (2001) 3089-3095. 433
[5] M. Solé, M.J. López-De-Alda, M. Cas illo, C. Po e, K. Ladegaa d-Pede sen, D. 434
Ba celó, En i on. Sci. Technol. 34 (2000) 5076-5083. 435
[6] M.A.J. Ha ison, S. Ba a, D. Bo ghesi, D. Vione, C. A sene, R.I. Ola iu, A mos. 436
En i on. 39 (2005) 231–248. 437
[7] J. Wang, H. Pan, Z. Liu, F. Ge, J. Ch oma og . A 1216 (2009) 2499–2503. 438
[8] P. Po hi ou, D. Vou sa, Chemosphe e 73 (2008) 1716–1723. 439
[9] Lis o he 129 p io i y pollu an s es ablished by he Uni ed S a es En i onmen al 440
P o ec ion Agency (U.S. EPA). Appendix A o pa 423. 441
(h p://www.epa.go /wa e science/me hods/pollu an s.h m, las accessed Feb ua y 442
2011). 443
[10] h p://www.epa.go /sa ewa e /con aminan s/index.h ml (las access Feb ua y 444
2011). 445
[11] Decision No 2455/2001/EC o he Eu opean Pa liamen and o he Council o 20 446
No embe 2001, es ablishing he lis o p io i y subs ances in he ield o wa e policy 447
and amending Di ec i e 2000/60/EC. O . J. Eu . Communi ies L 331/1, 16.12.2001. 448
[12] Di ec i e 2008/105/ECec o he Eu opean Pa liamen and o he Council o 16 449
Decembe 2008 on en i onmen al quali y s anda ds in he ield o wa e policy, 450
amending and subsequen ly epealing Council Di ec i es 82/176/EEC, 83/513/EEC, 451
21
84/156/EEC, 84/491/EEC, 86/280/EEC and amending Di ec i e 2000/60/EC o he 452
Eu opean Pa liamen and o he Council. 453
[13] G. Ga idou, M.S. Tomaidis, A.S. S asinakis, T.D. Lekkas, J. Ch oma og . A 1138 454
(2007) 32–41. 455
[14] J. Pa sias, E. Papadopoulou-Mo kidou, J. Ch oma og . A 904 (2000) 171-188. 456
[15] C. Mahugo-San ana, Z. Sosa-Fe e a, M.E. To es-Pad ón, J.J. San ana-457
Rod íguez, Molecules 14 (2009) 298-320. 458
[16] H. Fa aji, M. Sabe -Teh ani, F. Waqi -Hussain, J. Ch oma og . A 1216 (2009) 459
8569–8574. 460
[17] J. Reguei o, E. Bece il, E. Ga cía-Ja es, M. Llompa , J. Ch oma og . A 1216 461
(2009) 4693–4702. 462
[18] P. Yi-Ping, T. Shigh-Wei, Anal. Chim. Ac a 624 (2008) 247–252. 463
[19] J.B. Quin ana, I. Rod íguez, Anal. Bioanal. Chem. 384 (2006) 1447-1461. 464
[20] M. Kawaguchi, K. Inoue, M. Yoshimu a, R. I o, N. Sakui, H. Nakazagua, Anal. 465
Chim. Ac a, 505 (2004) 217–222. 466
[21] F. Sánchez-Rojas, C. Bosch-Ojeda, J.M. Cano-Pa ón, Ch oma og aphia 69 467
(2009) S79-S94. 468
[22] X. Chen, T. Zhang, P. Liang, Y. Li, Mic ochim. Ac a 155 (2006) 415–420. 469
[23] M. Sa aji, M. Ma zbam, Anal. Bioanal. Chem. 396 (2010) 2685–2693. 470
[24] I. Rod iguez, M.P. Llompa , R. Cela, J. Ch oma og . A 885 (2000) 291–304. 471
[25] L. Donghao, J. Pa k, Anal. Chem. 73 (2001) 3089-3095. 472
[26] J.B. Baug os, C. C en-Oli é, B. Gi oud, J.Y. Gau i , P. Lan e i, M.F. G enie -473
Lous alo , J. Ch oma og . A 1216 (2009) 4941–4949. 474
[27] J.L. Ma ínez Vidal, A. Belmon e-Vega, A. Ga ido F enich, F.J. Egea-González, 475
F.J. A ebola-Liébanas, Anal. Bioanal. Chem. 379 (2004) 125–130. 476
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[28] M. Pe o ic, D. Ba celó, A. Díaz, F.J. Ven u a, Am. Soc. Mass Spec om. 14 477
(2003) 516–527. 478
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(2002) 335–344. 480
[30] R. Loos, G. Hanke, G. Umlau , S.J. Eisen eich, Chemosphe e 66 (2007) 690–481
699. 482
[31] C. Sánchez B une e, E. Miguel, J.L. Tadeo, J. Ch oma og . A 1216 (2009) 5497–483
5503. 484
[32] J.L. Pé ez-Pa ón, A.M. Casas-Fe ei a, M.E. Fe nández-Laespada, B. Mo eno-485
Co de o, J. Ch oma og . A 1216 (2009) 1192–1199. 486
[33] J.A. Padilla-Sánchez, P. Plaza-Bolaños, R. Rome o-González, A. Ga ido 487
F enich, J.L. Ma ínez Vidal, J. Ch oma og . A 1217 (2010) 5724-5731. 488
[34] A. Ga ido F enich, J.L. Ma ínez Vidal, J.L. Fe nández-Mo eno, R. Rome o-489
González, J. Ch oma og . A 1216 (2009) 4798-4808. 490
[35] M.N. Ba co-Bonilla, R. Rome o-González, P. Plaza-Bolaños, A. Ga ido 491
F enich, J.L. Ma ínez Vidal, J.Ch oma og . A 1217 (2010) 7817–7825. 492
[36] M. Bade , W. Rosenbe g, F.M. Gu zki, D. Tsikas, J. Ch oma og . B 877 (2009) 493
1402–1415. 494
[37] P. Ho mann, M.F. Ha mann, T. Reme , K.P. Zimme , S.A. Wudy, S e oids 75 495
(2010) 1067–1074. 496
[38] S. Te zic, I. Sen a, M. Ahel, M. G os, M. Pe o ic, , J. Mulle , T. 497
Kneppe , I. , F. Ven u a, , D. Jabuca , Sci.To al En i on. 399 (2008) 498
66-77. 499
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23
[40] M.E. To es-Pad ón, C. Mahugo-San ana, Z. Sosa-Fe e a, J.J. San ana-501
Rod iguez, J. Ch oma og . Sci. 46 (2008) 325-331. 502
503
504
24
505
Figu e Cap ions 506
Fig. 1. Compa ison o he eco e y alues ob ained applying di e en elu ion sol en s 507
o he ex ac ion o spiked WW samples a 0.5 g L-1. Abb e ia ions: DCM: 508
dichlo ome hane; Sequen ial: sequen ial elu ion. 509
Fig. 2. To al ion ch oma og am (TIC) o an ex ac ed spiked WW sample (5 g L-1) 510
ob ained by GC-QqQ-MS/MS. Fo compound ab e ia ions, see Table 1. 511
Fig. 3. Calib a ion cu es in he ange 10-150 g L-1 o 4- e OP when SAC and 512
MMC we e used: a) ANAP; b) MBR. Abb e ia ions: ANAP: anae obic pond; MBR: 513
memb ane bio eac o ; MMC: ma ix-ma ched calib a ion; SAC: s anda d addi ion 514
calib a ion; 4- e OP: 4- e oc ylphenol 515
Fig. 4. Selec ed- eac ion moni o ing (SRM) (a) ch oma og am and (b) MS/MS 516
spec um o 4- e OP (0.12 g L-1) ound in a eal WW sample and (c) SRM 517
ch oma og am and (d) MS/MS spec um o a SAC s anda d (50 g L-1). 518
519
520
521
3
1. In oduc ion
39
Phenolic compounds can be ound in was ewa e (WW) e luen s ia di e en
40
sou ces. They can be de ec ed in his ype o samples because o hei use in plas ics
41
[1], d ug manu ac u ing, phy osani a y p oduc s o lea he colo ing [2], by
42
an h opogenic emission [2] and by he use o ea men s wi h ae obic o anae obic
43
mic oo ganisms [4]. Some phenols show high oxici y, es ogenic [5] and an i-
44
and ogenic ac i i y [6], and hey can ac as endoc ine dis up e s [7].
45
Phenols can be classi ied in a wide ange o amilies. The mos s udied analy es in
46
wa e a e chlo ophenols (CPs) [1] and alkylphenols (APs) [8]. Howe e , he Uni ed
47
S a es En i onmen al P o ec ion Agency (US EPA) classi ies CPs, ni ophenols and
48
APs as p io i y pollu an s [9] and i has es ablished a maximum con amina ion le el
49
(MCL) o pen achlo ophenol (PCP) o 1 µg L-1 in d inking wa e s [10]. On he o he
50
hand, he Eu opean Union (EU) has adop ed a lis o p io i y subs ances in he ield o
51
wa e policy, including 4-n-nonylphenol (4-n-NP), 4- e -oc ylphenol (4- e OP) and
52
PCP [11]. Fu he mo e, maximum allowable concen a ions (MAC) ha e been
53
es ablished o NP (2 g L-1) and PCP (1 g L-1) in inland and o he su ace wa e s
54
[12]. Howe e , i mus be poin ed ou ha legisla ion o WWs is s ill e y sca ce, and
55
he alues es ablished in d inking wa e a e usually used as guide in WWs. Bea ing in
56
mind hese ac s, he de elopmen o sensi i e analy ical me hodologies o he
57
simul aneous de e mina ion o phenols belonging o di e en g oups, such as CPs,
58
APs, ni ophenols (NTPs) and c esols (also known as me hyl-phenols) wi h di e en
59
pola i y ange (log Kow 1.77-5.01) is needed in o de o p o ide a comple e o e iew
60
o he occu ence o phenolic compounds in WW e luen s.
61
4
Se e al ex ac ion echniques ha e been applied o he ex ac ion o phenols om
62
aqueous samples, such as solid-phase ex ac ion (SPE) [8,13-15] and liquid-liquid
63
ex ac ion (LLE) [16]. Recen ly, mic oex ac ion echniques, such as solid-phase
64
mic oex ac ion (SPME) [17-19], s i ba so p i e ex ac ion (SBSE) [19-21], liquid
65
phase mic oex ac ion (LPME) [22] o dispe si e liquid-liquid mic oex ac ion
66
(DLLME) [23] ha e been applied. Howe e , mos o hem ha e been used o he
67
simul aneous analysis o only one o ew phenols belonging o he same amily such
68
as APs [18,20] and CPs [17]. I is well-known ha SPE is he mos used echnique in
69
wa e analysis [24] due o he educed exposi ion and con amina ion by o ganic
70
sol en s, he high p e-concen a ion ac o s a oiding e apo a ion s eps, he semi-
71
au oma ion o he p ocess, and i allows he ex ac ion o compounds wi h di e en
72
physico-chemical p ope ies. The applica ion o mic oex ac ion echniques is
73
inc easing bu se e al disad an ages, such as cos and li e ime o ibe s and ba s, o
74
he limi ed scope o a wide pola i y ange can hinde hei u iliza ion.
75
Fo he de e mina ion o phenolic compounds, gas ch oma og aphy (GC) [13,25] o
76
liquid ch oma og aphy (LC) [26,27] a e he p edominan echniques, mainly coupled
77
o andem mass spec ome y (MS/MS) [28-31]. When GC is used, a de i a iza ion
78
s ep is equi ed in o de o imp o e he ch oma og aphic pe o mance and sensi i i y
79
o he selec ed compounds, and se e al de i a iza ing eagen s can be applied [32,33].
80
A well-known c i ical poin in he analysis o WW is ma ix e ec [34]. In o de o
81
minimize i , di e en calib a ion me hods such as ma ix-ma ched calib a ion (MMC)
82
[33,35], s anda d addi ion calib a ion (SAC) [34] and he use o iso ope-labeled
83
in e nal s anda ds [36,37] ha e been employed o complex ma ixes. Quan i ica ion
84
based on iso ope-labeled in e nal s anda ds has disad an ages due o he
85
expensi eness o hese s anda ds and hei limi ed a ailabili y. MMC is o en used
86
5
quan i ica ion me hod in ace analysis. Howe e , he lack o blank ma ixes and he
87
need o s o ing hem can make his app oach logis ically one ous and no necessa ily
88
accu a e. SAC is he mos adequa e echnique o use when i is di icul o ind blank
89
samples o he s udied ma ix, bu a calib a ion se is equi ed o each sample,
90
inc easing he o al numbe o injec ions and he ime spen in da a p ocessing.
91
Ano he p oblem ela ed o he de e mina ion o phenols in WW is ha depending
92
on he ype o WW ea men , WW e luen s can ha e di e en amoun s o suspended
93
pa icula e ma e (SPM). This SPM is no mally disca ded du ing he ex ac ion
94
p ocess by il a ion in mos o he analy ical me hods epo ed in li e a u e [38].
95
Howe e , a ecen s udy [35] has demons a ed ha ce ain analy es can be e ained in
96
he SPM, depending on i s pola i y. The e o e, i should be necessa y o e alua e he
97
p esence o phenols in bo h phases in o de o de e mine whe he he SPM mus be
98
disca ded o no .
99
Fu he mo e, i mus be poin ed ou ha many a icles epo ing simul aneous
100
ex ac ion and de e mina ion o di e en classes o phenols (including APs, CPs and,
101
NTPs) in wa e [39,40] can be ound. Howe e , hey ha e been de eloped o he
102
analysis o his ype o compounds in su ace wa e , and hey a e no alid o he
103
analysis in WW samples, due o hey a e mo e complex ma ices wi h di e en
104
physico-chemical cha ac e is ics (SPM le els, o ganic ma e , e c.).
105
The e o e, in his s udy, a simul aneous SPE ex ac ion and de e mina ion o
106
di e en phenolic amilies (CPs, NTPs, c esols and APs) has been de eloped o WW
107
e luen samples. In addi ion wo no el aspec s o his wo k mus be poin ed ou :
108
(i) a s udy o he p esence o phenolic compounds in he SPM acco ding o he
109
s a egy ecen ly p oposed by Ba co-Bonilla e al [35], and (ii) a compa ison o
110
6
MMC and SAC in o de o e alua e he bes quan i ica ion s a egy o phenolic
111
compounds in complex ma ices such as WWs. Fo ha , wo di e en WW
112
e luen s we e s udied indi idually: memb ane bio eac o (MBR, low SPM con en )
113
and anae obic pond (ANAP, high SPM con en ). The op imized SPE and
114
quan i ica ion me hod was alida ed in bo h ypes o WWs e luen samples.
115
116
2. Expe imen al
117
2.1. Chemicals and ma e ials
118
Phenolic compounds s anda ds, 2-ni ophenol (2-NTP), 4-ni ophenol (4-NTP), 2,4-
119
dime hylphenol (2,4-DMP), 2-CP, 4-chlo ophenol (4-CP), 2,4-dichlo ophenol (2,4-
120
diCP), 2,4,5- ichlo ophenol (2,4,5- iCP), 2,4,6- ichlo ophenol (2,4,6- iCP) and 4-
121
n-NP we e ob ained om Fluka (Buchs, Swi ze land). On he o he hand, 3-
122
ni ophenol (3-NTP), 4-chlo o-3-me hylphenol (4-C-3-MP), 4- e OP and PCP we e
123
supplied by Supelco (Belle on e, PA, USA). Pu i ies we e always >97%. A s anda d
124
solu ion (100 mg L−1) o iso opically labeled PCP ([13C6]-PCP) was used as in e nal
125
s anda d (IS) and i was ob ained om D . E hens o e (Augsbu g, Ge many). S ock
126
s anda d solu ions o indi idual compounds (wi h concen a ions anging om 200 o
127
450 mg L-1) we e p epa ed by exac weighing o he powde o liquid and dissolu ion
128
in 50 mL o ace one. These solu ions we e hen s o ed unde e ige a ion (T <5 ºC).
129
A wo king s anda d solu ion o he 13 phenolic compounds (2 mg L-1 o each
130
compound) was p epa ed by app op ia e dilu ion o he s ock solu ions wi h ace one,
131
and i was s o ed unde e ige a ion (T <5 ºC). A wo king s anda d solu ion o [13C6]-
132
PCP (22 mg L-1) was p epa ed by app op ia e dilu ion o he s anda d solu ion wi h
133
ace one and s o ed unde he a o emen ioned condi ions. HPLC-g ade me hanol
134
7
(MeOH), anhyd ide ace ic acid (AAA) (99.9%), and py idine (Py) (99.8%) we e
135
pu chased om Sigma-Ald ich (Mad id, Spain). Ace one and hyd ochlo ic acid (HCl)
136
we e ob ained om J.T. Bake (De en e , Ne he lands). Dichlo ome hane (DCM) was
137
pu chased om Riedel-de Haën (Seelze-Hanno e , Ge many). Ul apu e wa e was
138
ob ained om a Milli-Q G adien wa e sys em (Millipo e, Bed o d, MA, USA).
139
Thi y mm cellulose il e s and 47-mm glass mic o ibe il e s om Wha man
140
(Maids one, England, UK) and 0.45-µm HNWP nylon memb ane il e s om
141
Millipo e (Ca ig wohill, Coun y Co k, I eland) we e also a ailable o il a ion
142
s ages. Fo SPE, Oasis HLB (200 mg, 6 cm3) ca idges we e ob ained om Wa e s
143
(Mil o d, MA, USA).
144
145
2.2. Appa a us
146
A GC sys em Va ian 3800 (Va ian Ins umen s, Sunny ale, CA, USA) equipped wi h
147
elec onic low con ol was in e aced o a 1200L iple quad upole (QqQ) mass
148
spec ome e . Samples we e injec ed in o an SPI/1079 spli /spli less p og ammed-
149
empe a u e injec o using a Combi Pal (CTC Analy ics AG, Zwingen, Swi ze land)
150
wi h a 100-µL sy inge. A used-silica un ea ed capilla y column (2 m × 0.25 mm i.d.)
151
om Supelco was used as p e-column connec ed o a VF-5 ms Fac o Fou capilla y
152
column (30 m × 0.25 mm i.d. × 0.25 µm ilm hickness) pu chased om Va ian.
153
Helium was used as ca ie gas (99.9999%) a a cons an low a e o 1 mL min−1, and
154
a gon (99.999%) was used as collision gas. The mass spec ome e was ope a ed in
155
elec on ioniza ion (EI) mode a 70 eV. The mass spec ome e was calib a ed e e y
156
ou days wi h pe luo o ibu ylamine. Va ian Wo ks a ion so wa e was used o
157
ins umen con ol and da a analysis.
158
8
A Reax-2 o a y agi a o om Heidolph (Schwabach, Ge many) was used o
159
agi a ion o he de i a iza ion mix u e. An analy ical balance AB204-S om Me le
160
Toledo (G ei ensee, Swi ze land) and a o a y e apo a o R-114 (Büchi, Flawil,
161
Swi ze land) we e used du ing ex ac ion and s anda d p epa a ion. The ho izon al
162
shake used in he dis ibu ion s udy was ob ained om P-Selec a (Selec a, Ba celona,
163
Spain).
164
165
2.3. Sampling
166
WW u ban e luen s om wo di e en ea men s, namely, MBR and ANAP, wi h
167
low and high SPM con en espec i ely, we e collec ed om WW ea men plan
168
(WWTP) o he ounda ion Cen e o New Wa e Technologies (“Cen o de las
169
Nue as Tecnologías del Agua”, CENTA, Se ille, Spain). This WWTP has 41000 m2
170
and i cu en ly holds mo e han 20 sys ems wi h di e en echnologies. Addi ional
171
physicochemical da a ela ed o he ea men s e alua ed in his s udy can be ound in
172
[35]. WW e luen samples we e s o ed a 4 ºC and p ocessed wi hin 5 days a e
173
collec ion. In he MMC expe imen s, and due o he di icul y o inding WW e luen
174
blank samples, he co esponding signal o he blank was emo ed om he MMC
175
plo in hose samples whe e analy e signal was obse ed.
176
177
2.4. Dis ibu ion s udy
178
Non- il e ed WW e luen samples we e spiked wi h 0.5 µg L-1 o he s udied
179
phenolic compounds, and hen, hey we e agi a ed o e nigh a a a e o 100
180
oscilla ions pe min o allow a ho oughly in e ac ion be ween he analy es and bo h
181
9
phases o WW (aqueous phase and SPM). A e his, samples we e il e ed o sepa a e
182
and analyze bo h phases. The aqueous phase was ex ac ed by SPE, whe eas o he
183
analysis o he SPM, a me hod de eloped by Padilla-Sánchez e al. [33] o he
184
ex ac ion o phenolic compounds in ag icul u al soils was employed. The dis ibu ion
185
o he compounds be ween bo h phases was de e mined as he pe cen age o hem
186
p esen in each phase.
187
188
2.5. GC-QqQ-MS/MS
189
Aliquo s o 10 µL we e injec ed in o he GC sys em ope a ing a a sy inge injec ion
190
low a e o 10 µL s−1. The injec o empe a u e p og am was as ollows: 70 ºC (hold
191
o 0.5 min) → 310 ºC (100 ºC min−1, hold o 10 min). The injec o spli a io was
192
ini ially se a 10:1. Spli less mode was swi ched on a 0.5 min un il 3.5 min. A 3.5
193
min, he spli a io was 100:1 and a 10 min, 20:1. The column o en p og am was as
194
ollows: 70 ºC (hold o 3.5 min) → 300 ºC (20 ºC min−1) → 300 ºC (hold 4 min).
195
C yogenic cooling wi h CO2 was applied when he injec o empe a u e was 170 ºC.
196
The o al unning ime was 19 min.
197
The QqQ mass spec ome e was mainly ope a ed in he selec ed eac ion
198
moni o ing (SRM) mode, al hough selec ing ion moni o ing (SIM) mode was also
199
used o con i ma ion pu poses. The elec on mul iplie was se +200 V abo e he
200
op imal alue indica ed by he so wa e ins umen . The empe a u es o he ans e
201
line, mani old and ioniza ion sou ce we e se a 300, 40 and 265 °C, espec i ely. The
202
op imal alues o he scan ime anged om 0.132 o 0.240 s. Peak wid hs o m/z 2.0
203
and 1.5 we e se in he i s (Q1) and hi d quad upole (Q3), espec i ely. The
204
op imized MS/MS pa ame e s a e indica ed in Table 1.
205
10
2.6. SPE ex ac ion and de i a iza ion p ocedu e
206
WW e luen samples we e il e ed consecu i ely (250 mL) using wo di e en po e-
207
size il e s (47-mm glass mic o ibe il e s and 0.45-µm nylon memb ane il e s). The
208
il e ed WW e luen s showed pH alues be ween 7.7 and 8.3. Then, pH was adjus ed
209
o 2.5-2.7 wi h HCl (2 M) o ensu e he p o ona ed o m o he phenolic compounds,
210
acili a ing he abso p ion in o he solid phase, and an adequa e p ese a ion o he
211
samples. The Oasis HLB ca idges we e condi ioned wi h 5 mL o ace one ollowed
212
by 5 mL o MeOH and 3 x 5 mL o ul apu e wa e wi hou allowing he ca idges o
213
d y ou . Then, he il e ed WW sample (250 mL) was passed h ough he ca idges
214
unde acuum a a low a e o 10 mL min-1. The ca idges we e d ied o 2 h and he
215
phenolic compounds we e elu ed sequen ially wi h 3 mL o ace one and 2 mL o
216
DCM. The ex ac s we e collec ed in o 5-mL olume ic lasks, adjus ing he o al
217
olume wi h DCM, wi hou any e apo a ion s ep. Then, he de i a iza ion s age was
218
pe o med acco ding o he p ocedu e desc ibed by Padilla-Sánchez e al. [33].
219
B ie ly, 860 µL o he ex ac we e ans e ed o a 2-mL ial and 20 µL o [13C6]-
220
PCP (IS), 20 µL o Py and 100 µL o AAA we e added o ca y ou he de i a iza ion
221
eac ion. The mix u e was shaking in a o a y agi a o o 2 min and hen injec ed
222
di ec ly o he GC-QqQ-MS/MS sys em.
223
224
3. Resul s and discussion
225
WWs can be submi ed o di e en ea men s, ob aining e luen s wi h a a ie y o
226
SPM con en s, and hus, WW e luen s can p esen di e en physico-chemical
227
p ope ies. When an analy ical me hod is de eloped o his ype o samples, his
228
di e si y should be aken in o accoun . In o de o co e a wide ange o WW
229
11
e luen s, wo ypes o hem we e e alua ed, MBR and ANAP, which ha e low and
230
high SPM con en [35], espec i ely. The op imiza ion o he ex ac ion p ocedu e as
231
well as he quan i ica ion me hods, we e e alua ed in bo h ypes o WW e luen s. Fo
232
ha pu pose, a GC-QqQ-MS/MS me hod ecen ly de eloped [33] was applied.
233
234
3.1. Ex ac ion me hod
235
Fo he op imiza ion o he SPE p ocedu e, a me hodology epo ed by Po hi ou e al.
236
[8] was i s conside ed. This s udy epo ed he de e mina ion o only one amily o
237
phenolic compounds, APs, using Oasis HLB ca idges and ace one as elu ion sol en .
238
Besides, ce ain p oblems ega ding he e apo a ion s ages ha e been p e iously
239
epo ed [33], and he e o e, he ex ac ion me hod was designed wi hou any
240
e apo a ion s ep. Since he amilies o phenolic compounds included in his s udy
241
showed a wide pola i y ange, se e al elu ion sol en s we e es ed o achie e a
242
simul aneous ex ac ion [8]. Ace one (5 mL), DCM (5 mL) and a sequen ial elu ion
243
wi h ace one (3 mL) and DCM (2 mL) we e es ed. Bea ing in mind ha e apo a ion
244
s eps we e no included in he ex ac ion p ocedu e and aliquo s o he ex ac s a e
245
di ec ly injec ed in he ch oma og aphic sys em, he elu ion sol en could be pa ially
246
e ained in he solid phase o e apo a ed du ing he elu ion s ep. This can p o oke an
247
o e es ima ion o he inal concen a ion in ela ion o he heo e ical alue, ob aining
248
high eco e y alues. In o de o a oid his, 5-mL olume ic lasks we e used o
249
collec he ex ac s and he inal olume was adjus ed o 5 mL wi h he co esponding
250
sol en used du ing he elu ion s ep. The ob ained esul s a e shown in Figu e 1 and i
251
can be obse ed ha ace one p o ided adequa e esul s o all compounds, excep o
252
2,4-dMP and 4-n-NP. When DCM was used, eco e ies highe han 120% we e
253
12
ob ained o 2-CP, 2-NTP and 4-CP, al hough eco e y o 4-n-NP was imp o ed.
254
Consequen ly, in o de o ob ain good eco e ies o all he compounds, a sequen ial
255
elu ion wi h ace one and DCM was es ed. In gene al, his elu ion imp o ed he
256
eco e y a es, especially o 2,4-dMP, 2-CP, 2-NTP and 4-CP. None heless,
257
eco e ies be ween 50-60% may be accep ed ex ao dina ily in en i onmen al
258
analysis whene e he p ecision alues a e adequa e (<30%). The e o e, u he
259
expe imen s we e ca ied ou using he sequen ial elu ion wi h ace one (3 mL) and
260
DCM (2 mL) as elu ion sol en s. Finally, a o al ion ch oma og am (TIC) o an
261
ex ac ed spiked WW sample a 50 g L-1 is showed in Figu e 2.
262
263
3.2. Dis ibu ion s udy
264
Once he ex ac ion me hod was op imized o he analysis o he aqueous phase o
265
WW e luen samples, a dis ibu ion s udy is needed o e i y whe he he phenolic
266
compounds a e also p esen in he SPM. I phenolic compounds a e p esen
267
quan i a i ely in he SPM, he analysis o WW e luen s should no be limi ed o he
268
aqueous phase. The dis ibu ion s udy was he e o e ca ied ou , applying he
269
app oach desc ibed in Sec ion 2.4 o bo h ype o samples. I was obse ed ha only
270
he phenolic compounds wi h high log Kow we e ound in he SPM, bu a negligible
271
pe cen ages (<5%). On he con a y, phenolic compounds wi h lowe log Kow whe e
272
no ound in he SPM (da a no shown). Taking in o accoun his esul , u he
273
expe imen s we e limi ed o he analysis o he a ge analy es in he aqueous phase,
274
disca ding he SPM phase. These esul s a e in acco dance wi h a p e ious s udy [35]
275
epo ing ha pola compounds we e no e ained in he SPM.
276
277
19
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405
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419
(h p://www.epa.go /wa e science/me hods/pollu an s.h m, las accessed Feb ua y
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428
amending and subsequen ly epealing Council Di ec i es 82/176/EEC, 83/513/EEC,
429
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84/156/EEC, 84/491/EEC, 86/280/EEC and amending Di ec i e 2000/60/EC o he
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480
481
482
23
Figu e Cap ions
483
Fig. 1. Compa ison o he eco e y alues ob ained applying di e en elu ion sol en s
484
o he ex ac ion o spiked WW samples a 0.5 g L-1. Abb e ia ions: DCM:
485
dichlo ome hane; Sequen ial: sequen ial elu ion.
486
Fig. 2. To al ion ch oma og am (TIC) o an ex ac ed spiked WW sample (5 g L-1)
487
ob ained by GC-QqQ-MS/MS. Fo compound ab e ia ions, see Table 1.
488
Fig. 3. Calib a ion cu es in he ange 10-150 g L-1 o 4- e OP when SAC and
489
MMC we e used: a) ANAP; b) MBR. Abb e ia ions: ANAP: anae obic pond; MBR:
490
memb ane bio eac o ; MMC: ma ix-ma ched calib a ion; SAC: s anda d addi ion
491
calib a ion; 4- e OP: 4- e oc ylphenol
492
Fig. 4. Selec ed- eac ion moni o ing (SRM) (a) ch oma og am and (b) MS/MS
493
spec um o 4- e OP (0.12 g L-1) ound in a eal WW sample and (c) SRM
494
ch oma og am and (d) MS/MS spec um o a SAC s anda d (50 g L-1).
495
496
497
498
Depa amen o de Hid ogeología y Química Analí ica
C a . S a c a m e n o L a C a ñ a d a d e S a n U b a n o 0 4 1 2 0 A l m e í a ( E s p a ñ a ) T e l . : 9 5 0 0 1 5 4 8 3 F A X : 9 5 0 0 1 5 4 8 3 w w w . u a l . e s
Alme ia, 12 July 2011
Dea Edi o :
Please, ind enclosed he e ised e sion o he manusc ip en i led “Simul aneous analysis
o chlo ophenols, alkylphenols, ni ophenols and c esols in was ewa e e luen s, using
solid phase ex ac ion and u he de e mina ion by gas ch oma og aphy– andem mass
spec ome y”, Manusc ip No. TAL-D-11-00529.
We e ised ou manusc ip aking in o accoun he e iewe ’s commen s (changes a e
indica ed by using “T ack Changes”). Al hough we ied o cla i y he e iewe ’s
commen s, we ha e conside ed ha i was also necessa y o indica e some aspec s, which
can be seen in he documen uploaded as “Response o Re iewe s”.
I hope ha he e iewing p ocess inds he e ised manusc ip accep able o publica ion in
he jou nal.
You s Since ely,
P o . An onia Ga ido F enich
Co e Le e
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