Validated method for the determination of perfluorinated compounds in placental tissue samples based on a simple extraction procedure followed by ultra-high performance liquid chromatography tandem mass spectrometry analysis

1 Valida ed me hod o he de e mina ion o pe luo ina ed compounds in placen al issue samples based on a simple ex ac ion p ocedu e p io o ul a–high pe o mance liquid ch oma og aphy– andem mass spec ome y analysis J. Ma ína, R. Rod íguez–Gómezb, A. Za a–Gómez*b, E. Alonsoa, J.L. Vílchezb, A. Na alónb 1 Depa men o Analy ical Chemis y, Supe io Poly echnic School, Uni e si y o Se ille, C/ Vi gen de Á ica 7, E-41011 Se ille, Spain 2 Resea ch G oup o Analy ical Chemis y and Li e Sciences, Depa men o Analy ical Chemis y, Uni e si y o G anada, Campus o Fuen enue a, E-18071 G anada, Spain ABSTRACT Exposu e o xenobio ics du ing p egnancy is ine i able. De e mina ion o pe luo ina ed compounds (PFCs), enowned en i onmen al con aminan s by Public Heal h Au ho i ies due o i s pe sis ence, bioaccumula i e e ec and oxici y, is a challenge. In he p esen wo k, a me hod based on a simpli ied sample ea men in ol ing s eps o eeze- d ying, sol en ex ac ion and dispe si e clean-up o he ex ac s wi h C18 p io o ul a-high pe o mance liquid ch oma og aphy– andem mass spec ome y (UHPLC–MS/MS) analysis o he de e mina ion o 5 pe luo ina ed ca boxylic acids ( om C4 o C8) and pe luo ooc ane sul ona e (PFOS) in placen al issue samples is de eloped and alida ed. The mos in luen ial pa ame e s a ec ing he ex ac ion me hod and he clean-up s ep we e op imized using design o expe imen s. The me hod was alida ed using ma ix-ma ched calib a ion. Found de ec ion limi s (LOD) anged om 0.03 o 2 ng g-1 and quan i ica ion limi s (LOQ) om 0.08 o 6 ng g-1, while in e - and in a-day a iabili y was unde 14% in all cases. Reco e y a es o spiked samples anged om 94 o 113%. The me hod was sa is ac o ily applied o he de e mina ion o compounds in human placen al issue samples collec ed a he momen o deli e y om 25 andomly selec ed women. Keywo ds: Pe luo ina ed compounds; Placen al issue; Dispe si e solid phase ex ac ion; UHPLC–MS/MS _________________ * Co esponding au ho 2 E–mail add ess: [email p o ec ed] (A. Za a-Gómez) 3 1. In oduc ion Exposu e o xenobio ics du ing p egnancy is ine i able. They a e likely o ac no only di ec ly on he e us bu also on he placen a i sel which may a ec i s abili y o suppo p egnancy [1]. Endoc ine dis up o s (EDs) a e ubiqui ous in he en i onmen a ound us and as a consequence in human bodies. The e is a g owing conce n abou he po en ial heal h e ec s o exposu e o a ious en i onmen al chemicals du ing p egnancy and in ancy. Mono-2- e hylhexyl ph hala e (MEHP), oc ylphenol (OP), 4-nonylphenol (4-NP) o polyb omina ed diphenyl e he s (PBDEs) ha e been de ec ed in physiologically ele an compa men s wi hin p egnan women and he de eloping e uses, such as ma e nal u ine, co d blood, b eas milk, meconium, placen a and amnio ic luid in se e al s udies [2-8], showing ha p egnan women and hei e uses a e exposed o hose chemicals. Pe luo ina ed compounds (PFCs) ha e been ecognized as an impo an class o po en ial con aminan s by Public Heal h Au ho i ies due o i s pe sis ence, bioaccumula i e e ec and oxici y [9]. Since hei i s p oduc ion in 1947, PFCs ha e been used in a wide ange o comme cial and indus ial applica ions such as polyme s, me al pla ing and cleaning, su ac an s, lub ican s, pes icides, coa ing o mula ions, inks, a nishes, i e igh ing oam, and s ain/wa e epellen s o lea he , pape and ex iles. Wo ldwide human exposu e o PFCs has been con i med [10-13]. Fo he gene al popula ion he majo sou ce o con amina ion a ises om ood and some imes d inking wa e [12, 14-16]. Human exposu e also a ises om indoo and ambien ai and house dus . Pe luo ooc ane sul ona e (PFOS) is gene ally he mos abundan PFCs ound in humans, he second usually being pe luo ooc anoic acid (PFOA) [9]. An ad anced es ima ion (2012) o die a y exposu e was ~5–10 ng/kg bw/day o PFOS, 4–7 ng/kg bw/day o PFOA, he mos impo an con ibu o s being ish and sea ood, ui and ui p oduc s [17]. Child en, especially oddle s, a e mo e exposed han adul s (2-3 imes highe ) on a body weigh basis, due o highe ela i e ood consump ion and o hand- o- mou h ans e om imp egna ed ca pe s and inges ion o dus , esul ing in highe PFCs se um le els han o adul s [14, 18, 19]. In u e o and pos na al exposu es a e o pa icula conce n. PFCs c oss he placen al ba ie , exposing neona es ia hei mo he 's blood [20-22]. In addi ion, i s scien i ic e idences sugges ing ha PFCs would be esponsible o ep oduc i e diso de s in humans ha e been ecen ly published [23-29]. Bu enho e al. [26] published a wo k on he ole o PFCs in he onse o heal h oubles a ec ing he ep oduc i e unc ion when exposu e occu s du ing c i ical s ages o he de elopmen , i.e. he pe ina al 4 pe iod. Fei e al. [25] obse ed an associa ion be ween high se um le els o PFOS and PFOA and a longe ime o p egnancy. E ec s o in u e o exposu e o PFCS on he emale ep oduc i e unc ions ha e also been epo ed. In a ecen s udy, ca ied ou by K is ensen and co-wo ke s [29], daugh e s who we e exposed in u e o o le els o PFOA ha we e highe han he e e ence g oup showed a la e age o mena che. In pa icula , a numbe o animal oxicology s udies ha e shown ha exposu e o PFOS and PFOA can al e o a ian unc ion [27] and a ec he de elopmen o mamma y gland issue [28]. In his con ex , he cha ac e iza ion o he PFCs ans e , i any, om he mo he o he e us ( h ough placen a) and/o o he newbo n ( h ough b eas eeding) is acu ely expec ed. Biomoni o ing da a e lec ing he in e nal PFCs exposu e le els in he gene al popula ion ha e al eady been p o ided in b eas milk, ma e nal and co d se um om di e en s a es/coun ies [30]. Howe e , he e is limi ed da a on he in u e o exposu e le els o newbo ns o hese PFCs so a . Mos o he esea ches on placen al ans e o PFCs we e conduc ed by animal o in i o expe imen s ins ead o human s udies. The e o e, he alida ion o analy ical me hods o de e mine PFCs in human placen al issue is o special scien i ic in e es . The p e e ed echnique o he analysis o hese compounds has been high-pe o mance liquid ch oma og aphy- andem mass spec ome y in nega i e elec osp ay ioniza ion mode (LC/ESI–MS/MS) [31-32]. The isola ion o analy es om his complex biological ma ix is a c i ical aspec e en when a ch oma og aphic echnique is employed due o selec i i y and sensi i i y issues. Some echniques such as mic owa e-assis ed ex ac ion (MAE) o p essu ized liquid ex ac ion (PLE) could be good al e na i es o maximize he ex ac ion e iciency, bu he applica ion o hese echniques in ol es d as ic condi ions, speci ic echnical esou ces and no e y clean inal ex ac s including ion supp ession, which is c i ical when mass spec ome y is employed as de ec ion echnique [33]. Sol en ex ac ion wi h o ex homogeniza ion ollowed by a clean-up o he ex ac based on dispe si e solid- phase ex ac ion (d-SPE) was chosen in ou s udy as ex ac ion echnique because i s simplici y, low cos , sho ex ac ion ime, low olume o sol en s equi ed and i s widesp ead use in mos o he labo a o ies ocused on ou ine analyses. To ou knowledge, he e is a lack o published li e a u e on PFCs de e mina ion in placen al issue. The aim o he p esen wo k was o alida e a as , simple, accu a e and sensi i e UHPLC–MS/MS me hod o he de e mina ion o PFCs in eeze-d ied human placen al issue samples. A e alida ion, he me hod was sa is ac o ily applied o de e mine he ee con en o a ge compounds in samples collec ed om 25 unknown olun ee s. 5 2. Expe imen al 2.1. Chemicals and eagen s All eagen s we e analy ical g ade unless o he wise speci ied. Wa e (18.2 MΩ cm) was pu i ied using a Milli-Q sys em om Millipo e (Bed o d, MA, USA). Pe luo obu anoic acid (PFBuA) (98%), pe luo open anoic acid (PFPeA) (97%), pe luo ohexanoic acid (PFHxA) (≥ 97%), pe lu o ohep anoic acid (PFHpA) (99%), pe lu o ooc anoic acid (PFOA) (96%), and pe luo ooc anesul onic acid (PFOS) (≥ 98%) and pe luo o-n-[1,2,3,4-13C4]oc anoic acid (MPFOA) we e pu chased om Sigma-Ald ich (Mad id, Spain). S ock solu ions o each compound, a a concen a ion o 1000 mg L-1, we e p epa ed in me hanol and s o ed a -20 ºC. Wo king solu ions we e p epa ed by dilu ing he s ock s anda d solu ions in me hanol o in he ini ial mobile phase immedia ely be o e use. These solu ions we e s o ed a 4 ºC and p epa ed esh weekly. All solu ions we e s o ed in da k glass bo les o p e en pho odeg ada ion. Anhyd ous magnesium sul a e was p o ided by Pan eac (Ba celona, Spain). PSA so ben (p ima y seconda y amine, 40–60 µm) was pu chased om Scha lab (Ba celona, Spain) and BAKERBONDs oc adecyl C18 so ben (40 µm pa icle size) was p o ided by J. T. Bake (De en e , The Ne he lands). LC–MS g ade me hanol, wa e and ace oni ile, ace ic acid and ammonia (25%, w/ ) we e pu chased om Sigma-Ald ich. 2.2. Ins umen a ion and so wa e UHPLC–MS/MS analysis was pe o med using a Wa e s Acqui y UPLCTM H-Class (Wa e s, Manches e , UK), consis ing o Acqui y UPLCTM bina y sol en manage and Acqui y UPLCTM sample manage . A Xe o TQS andem quad upole mass spec ome e (Wa e s) equipped wi h an o hogonal Z-sp ayTM elec osp ay ioniza ion (ESI) sou ce was used o PFCs de ec ion. An Acqui y UPLC® BEH C18 (50 mm × 2.1 mm i.d., 1.7 µm pa icle size) and a CORTECS UPLCTM C18 (Wa e s) column (50 mm × 2.1 mm; 1.6 µm pa icle size) om Wa e s (UK) we e assayed as ch oma og aphic columns. Placen al issue samples we e eeze-d ied using a SCANVAC CoolSa e™ eezed ye (Lynge, Denma k). A B anson digi al soni ie (Danbu y, CT, USA), a o ex-mixe (IKA, S au en, Ge many), an ul asound- HD ba h (Selec a, Ba celona, Spain), a Spec a uge™ 24D cen i uge om Labne 6 In e na ional, Inc. (New Je sey, USA) and a sample concen a o (S ua , S a o dshi e, UK) we e also used. S a g aphics Plus so wa e e sion 5.1 (S a poin Technologies Inc., Vi ginia, USA) was used o s a is ical ea men o da a. 2.3. Sample collec ion and s o age Placen a issue samples we e ob ained om heal h women li ing in G anada, Spain. Samples we e anonymized, ozen a -20 ºC and s o ed un il analysis in ou labo a o y. The s udy was pe o med in compliance wi h he E hical P inciples o Medical Resea ch In ol ing Human Subjec s issued by he Wo ld Medical Associa ion, and all olun ee s signed he in o med consen o m. Human placen al issue samples we e collec ed om olun ee s a he momen o deli e y. In o de o ensu e he homogenei y and ep esen a i eness o he whole placen a issue, each one was accu a ely examined, weighed and agmen ed. Then, hal o he placen a (including ma e nal and e al sides and cen al and pe iphe al pa s) was placed in he glass con aine o a mixe o i s homogeniza ion. Once homogenized, aliquo s o 25 g we e s o ed ozen a –86 ºC wi hin 90 min o i s collec ion. Samples we e anonymized o p ese e he con iden iali y o pa ien s. All olun ee s signed hei in o med consen o pa icipa e in he s udy. Be o e p ocessing, placen al issue aliquo s we e addi ionally homogenized using an ul asonic spindle. The con aine was placed in a glass ull o ice in o de o a oid sample hea ing and he spindle was in di ec con ac wi h he placen al issue. Ul asound se ing consis ed in pulses du y cycles o 30 s ollowed by 30 s wi hou sonica ion, un il comple e 5 min o e ec i e adia ion. Once he samples we e homogenized, hey we e ozen a −86 ºC and s o ed con iden ially and anonymously un il analysis. 2.4. Basic p ocedu e 2.4.1. P epa a ion o spiked samples Due o he absence o ce i ied ma e ials o hese compounds in his ma ix, 6 g o spiked sample we e p epa ed con aining he analy es a se en di e en concen a ion le els, om 0.025 o 62.5 ng g-1, o calib a ion s anda ds and 1.25, 6.25, 12.5 and 25 ng g-1, o quali y con ol and alida ion o he me hod. Spiked samples we e accu a ely s i ed and sligh ly hea ed (35 ºC) o homogenize. Then, aliquo s o 1 g we e weigh ed in 8 mL glass 7 ials and o i ied wi h 200 µL o a me hanolic solu ion (62.5 ng mL-1) o he su oga e MPFOA ( inal concen a ion 12.5 ng g-1 in placen al issue). A e shaking o 10 min, he spiked samples we e eady o he expe imen s. 2.4.2. Sample ea men An aliquo o placen al issue sample (1 g) was weighed in an 8 mL glass ial, o i ied wi h 200 µL o me hanol con aining 62.5 ng mL-1 o MPFOA, shaken o 5 min and eeze- d ied. The sample was homogenized wi h 5 mL o ace oni ile in o ex o 1 min. Then, he mix u e was cen i uged o 10 min a 4050 × g. In o de o dec ease he ma ix co-ex ac i es in he ex ac ha could cause he ma ix e ec , a clean-up o he ex ac based on d-SPE was ca ied ou . The ex ac was ans e ed o an 8 mL polyp opylene conical ube con aining 108 mg o C18 so ben . The mix u e was hand-shaken o 2 min and cen i uged o 5 min a 4050 4050 × g. The o ganic phase (ace oni ile) con aining he analy es was sepa a ed in o a glass ial and hen e apo a ed unde a ni ogen s eam a oom empe a u e and he inal esidue was dissol ed wi h 250 µL o he ini ial mobile phase. A e s i ing o 30 s in o ex and cen i uga ion o 5 min a 16,300 × g, he sample was eady o be injec ed in o he LC sys em. 2.4.3. Ul a-high pe o mance liquid ch oma og aphy– andem mass spec ome y condi ions Ch oma og aphic sepa a ion o compounds was pe o med using a CORTECS UPLCTM C18 (Wa e s) column (50 mm × 2.1 mm; 1.6 µm pa icle size). The compounds we e sepa a ed using a g adien mobile phase consis ing o a bu e solu ion ace ic acid/ammonium ace a e (pH 4.4) (sol en A) and me hanol (sol en B). G adien condi ions we e: 0.0-5.0 min, 20-100% B and back o 20% in 0.1 min and kep o 1.9 min o equilib a e he column. Flow a e was 0.3 mL min-1. The injec ion olume was 10 µL. The column empe a u e was main ained a 40 ºC. To al un ime was 7 min. The andem mass spec ome e was ope a ed in he selec ed eac ion moni o ing (SRM) mode and Q1 and Q3 quad upoles we e se a uni mass esolu ion. ESI was pe o med in nega i e ion mode. The ion sou ce empe a u e was main ained a 150 ºC. Ins umen pa ame e s we e as ollows: capilla y ol age, 0.60 kV; sou ce empe a u e, 150 ºC; desol a ion empe a u e, 500 ºC; cone gas low, 150 L h-1; desol a ion gas low, 500 L h-1; collision gas low, 0.15 mL min-1, and nebulize gas low, 7.0 ba . Ni ogen (99.995%) was 8 used as cone and desol a ion gas, and a gon (99.999%) was used as a collision gas. Dwell ime was 25 ms. Table 1 shows he pa ame e s and he mass ansi ions o each compound. Table 1 2.4.4. Me hod alida ion Valida ion in e ms o linea i y, selec i i y, accu acy ( ueness and p ecision) and sensi i i y, was pe o med acco ding o he US Food and D ugs Adminis a ion (FDA) guideline o bioanaly ical assay alida ion [34]. Linea i y o he calib a ion g aphs was es ed using he de e mina ion coe icien s (%, R2) and he P- alues o he lack-o - i es (%, Plo ) [35]. The speci ici y o he me hod was de e mined by compa ing he ch oma og ams o blank wi h he co esponding spiked placen al issue. Due o he absence o ce i ied ma e ials, in o de o e alua e he ueness and he ep oducibili y o he me hod, a s udy wi h spiked placen a samples, a h ee concen a ions le els (6.25, 12.5 and 25 ng g-1 o PFBuA, PFPeA and PFHxA and 1.25, 6.25 and 12.5 ng g-1 o PFHpA, PFOA and PFOS) was pe o med. P ecision, exp essed as ela i e s anda d de ia ion (%, RSD) was de e mined om iplica e spiked samples du ing he same day and in six di e en days, and he ueness was e alua ed by a eco e y assay. The eco e y o he es ed compounds in placen a samples was e alua ed by compa ing he known concen a ion in spiked samples wi h he concen a ion o each compound de e mined using he me hod p oposed. Finally, LODs and LOQs we e calcula ed by aking in o conside a ion he s anda d de ia ion o esiduals, sy/x, he slope, b, o he calib a ion g aphs and an es ima e so ob ained by ex apola ion o he s anda d de ia ion o he blank (Analy ical Me hods Commi ee). The LOD was 3·so and he LOQ was 10·s0. 2.4.54. Quali y assu ance and quali y con ol Validi y o he analy ical esul s was e i ied by some simple quali y assu ance and quali y con ol (QA/QC) measu es. P ocedu al blanks we e injec ed o moni o o backg ound con amina ion. Blanks we e p ocessed in he same way as he samples and injec ed in o he UHPLC–MS/MS sys em. No quan i iable amoun s o a ge compounds we e de ec ed. On he o he hand, in o de o e alua e possible con amina ions and he a iabili y o he ins umen al analysis, s anda ds (spiked blank samples a 0 and 25 ng g-1) and a s anda d in he ini ial mobile phase (100 ng mL-1) we e injec ed by iplica e e e y 20 samples. 9 3. Resul s and discussion 3.1. Liquid ch oma og aphic-mass spec ome ic analysis A BEH C18 UPLCTM column (50 mm × 2.1 mm i.d., 1.7 µm pa icle size) and a CORTECS UPLCTM C18 (Wa e s) column (50 mm × 2.1 mm; 1.6 µm pa icle size) we e es ed. Al hough bo h columns o e ed simila esolu ion o all he analy es in es iga ed, a he e en ion ime o he analy es elu ed CORTECS UPLCTM column p o ided be e peak shape in he sho es ime wi h, e en his las gene a ed p essu es 1000 psi lowe han BEH C18 UPLCTM. Consequen ly, his column was he one we selec ed o u he expe imen s. The e ec o he mobile phase on ch oma og aphic sepa a ion was also s udied. Ou aim was o ob ain high sensi i i y and selec i i y in a sho ime. Fi s , he pH o he mobile phase was s udied and deionized wa e wi h di e en addi i es was s udied as sol en A. Ace ic acid ( om 0% o 0.2%, / ), ammonia ( om 0% o 0.050%, w/ ) and mix u es o hem we e assayed. Highe esponses and be e peak shapes we e ob ained using a mix u e o 1:1 ( / ) o ace ic acid (0.1%, / ) and ammonia (0.025% w/ ). MeOH, ACN and mix u es o hese sol en s we e e alua ed as o ganic mobile phases (sol en B). MeOH was selec ed because o he sensi i i y, peak shapes and sepa a ion achie ed. A linea g adien , as desc ibed in he p e ious sec ion, was used. The injec ion olume was s udied in o de o enhance he analy ical signal and consequen ly he LOD o he me hod. A ange om 2.5 o 10 µL was analyzed and 10 µL was chosen as injec ion olume since a ma ked inc ease in sensi i i y wi hou loss o esolu ion was ob ained. Finally, he inc ease o empe a u e om 30 o 50 ºC did no imp o e signi ican ly he cha ac e is ics o ch oma og aphic me hod, he e o e 340 ºC was chosen as op imum. The MS/MS de ec ion me hod was se up by con inuous in usion o s anda d solu ions o each indi idual compound (1 mg L-1) o op imize he esponse o he p ecu so ion. The mass spec ome ic condi ions we e op imized o each compound. ESI and ESCI in e aces in posi i e and nega i e modes we e e alua ed. ESI in e ace in nega i e mode was selec ed because i showed highe sensi i i y o all compounds o in e es . Fo each compound wo p oduc ions ( wo eac ions) we e moni o ed: one o quan i ica ion and he o he o con i ma ion. The mos abundan ansi ion ion was selec ed o ob ain maximum sensi i i y 16 This s udy was suppo ed by he Regional Go e nmen o Andalusia (P ojec o Excellence No. P09-CTS-4470) and by he Minis y o Economy and Compe i i eness (P ojec CGL2013-44402-R). The au ho s a e g a e ul o Regional Go e nmen o Andalusia o he ellowship g an ed o R. Rod íguez-Gómez and o Uni e si y o Se ille o he mobili y g an awa ded o J. Ma ín. 17 Re e ences [1] S. Ba baux, J.J. E wich, P.O. Fa a on, S. Gil, D. Gallo , T.G. Golos, A. Gonzalez- Bulnes, J. Guibou denche, A.E. Heazell, T. Jansson, O. Lap é o e, R.M. Lewis, R.K. Mille , D. Monk, B. No ako ic, C. Oudejans, M. 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Na alón, New me hod o he de e mina ion o pa abens and bisphenol A in human milk samples using ul asound-assis ed ex ac ion and clean-up wi h dispe si e so ben s p io o UHPLC–MS/MS analysis J. Ch oma og . B 992 (2015) 47– 55. [37] A.G. González, M.A. He ado , A p ac ical guide o analy ical me hod alida ion, including measu emen unce ain y and accu acy p o iles, T ends Anal. Chem. 26 (2007) 227–238. [38] F. Pé ez, M. Llo ca, M. Fa é, D. Ba celó, Au oma ed analysis o pe luo ina ed compounds in human hai and u ine samples by u bulen low ch oma og aphy coupled o andem mass spec ome y, Anal. Bioanal. Chem. 402 (2012) 2369–2378. 22 Figu e cap ions Fig. 1. In luence o sol en on he ex ac ion p ocedu e (n = 3). Fig. 2. In luence o he ex ac ion modali y ( o ex o ul asound assis ed) on he eco e ies (n = 3). Fig. 3. S anda dized main e ec Pa e o cha s o he Box–Behnken design. (+) Posi i e e ec s on he esponse, (-) nega i e e ec s on he esponse. Ve ical line shows he limi o decision o conside he signi icance o he ac o s (based on he s anda dized e ec = es ima ed e ec /s anda d e o , a 95% o con idence le el). Fig. 4. UHPLC–MS/MS ch oma og ams o : (A) a spiked blank (25 ng g-1 o each analy e); (B) a posi i e placen al issue sample con amina ed wi h PFOA and PFOS (mo he 24). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 21 Figu e cap ions Fig. 1. In luence o sol en on he ex ac ion p ocedu e (n = 3). Fig. 2. In luence o he ex ac ion modali y ( o ex o ul asound assis ed) on he eco e ies (n = 3). Fig. 3. S anda dized main e ec Pa e o cha s o he Box–Behnken design. (+) Posi i e e ec s on he esponse, (-) nega i e e ec s on he esponse. Ve ical line shows he limi o decision o conside he signi icance o he ac o s (based on he s anda dized e ec = es ima ed e ec /s anda d e o , a 95% o con idence le el). Fig. 4. Response su ace plo s co esponding o he desi abili y unc ion when op imizing he ollowing pai o ac o s om he clean-up s ep wi h d-SPE so ben s: (A) amoun o PSA s. amoun o C18; (B) amoun o MgSO4 s. amoun o C18 and (C) amoun o MgSO4 s. amoun o C18. Resul s we e e alua ed using a 95% con idence in e al. Fig. 5. UHPLC–MS/MS ch oma og ams o : (A) a spiked blank (25 ng g-1 o each analy e); (B) a posi i e placen al issue sample con amina ed wi h PFOA and PFOS (mo he 24). Table 1 Op imized MS/MS pa ame e s o SRM analysis. Name Abb e ia ion Re en ion ime (min) P ecu so Ion (m/z) SRM 1a (m/z) SRM 2b (m/z) CV (V) CE (eV) Pe luo obu anoic acid PFPuA 1.60 213 169 51.6 36 20 Pe luo open anoic acid PFPeA 3.18 263 219 89.7 36 20 Pe luo ohexanoic acid PFHxA 3.98 313 269 119 36 20 Pe luo ohep anoic acid PFHpA 4.49 363 319 333 36 20 Pe luo ooc anoic acid PFOA 4.87 413 369 194 36 20 Pe luo ooc ane sul ona e PFOS 5.18 499 80 52 36 20 Pe luo o-n-[1,2,3,4-13C4]oc anoic acid MPFOA 4.87 417 371 168 36 20 CV: Cone ol age (V); CE: Collision ene gy (eV); a: SRM ansi ion used o quan i ica ion; b: SRM ansi ion used o con i ma ion Table Table 2 Analy ical and s a is ical pa ame e s. PFBuA PFPeA PFHxA PFHpA PFOA PFOS bMP (g ng-1) 0.066 0.12 0.24 0.19 0.19 0.59 sbMP (g ng-1) 8.5·10-4 9.4·10-4 3.1·10-3 2.2·10-3 2.2·10-3 6.9·10-3 bPT (g ng-1) 0.036 0.11 0.24 0.20 0.20 0.30 sbPT (g ng-1) 1.1·10-3 6.4·10-3 4.4·10-3 7.0·10-3 7.0·10-3 1.1·10-2 s uden 13 1 2 1 1 13 % R2 99.9 99.3 99.9 99.8 99.8 99.6 LOD (ng g-1) 1.5 2 0.5 0.03 0.03 0.03 LOQ (ng g-1) 4 6 2 0.08 0.08 0.08 LDR (ng g-1) 4.0-62.5 6.0-62.5 1.8-62.5 0.08-62.5 0.08-62.5 0.08-62.5 MP: Mobile phase; PT: Placen al issue; b: slope; sb: slope s anda d de ia ion; R2: de e mina ion coe icien ; LOD: limi o de ec ion; LOQ: limi o quan i ica ion; LDR: linea dynamic ange Table Figu e Click he e o download high esolu ion image 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 21 Figu e cap ions Fig. 1. In luence o sol en on he ex ac ion p ocedu e (n = 3). Fig. 2. In luence o he ex ac ion modali y ( o ex o ul asound assis ed) on he eco e ies (n = 3). Fig. 3. S anda dized main e ec Pa e o cha s o he Box–Behnken design. (+) Posi i e e ec s on he esponse, (-) nega i e e ec s on he esponse. Ve ical line shows he limi o decision o conside he signi icance o he ac o s (based on he s anda dized e ec = es ima ed e ec /s anda d e o , a 95% o con idence le el). Fig. 4. Response su ace plo s co esponding o he desi abili y unc ion when op imizing he ollowing pai o ac o s om he clean-up s ep wi h d-SPE so ben s: (A) amoun o PSA s. amoun o C18; (B) amoun o MgSO4 s. amoun o C18 and (C) amoun o MgSO4 s. amoun o C18. Resul s we e e alua ed using a 95% con idence in e al. Fig. 5. UHPLC–MS/MS ch oma og ams o : (A) a spiked blank (25 ng g-1 o each analy e); (B) a posi i e placen al issue sample con amina ed wi h PFOA and PFOS (mo he 24). Table 1 Op imized MS/MS pa ame e s o SRM analysis. Name Abb e ia ion Re en ion ime (min) P ecu so Ion (m/z) SRM 1a (m/z) SRM 2b (m/z) CV (V) CE (eV) Pe luo obu anoic acid PFPuA 1.60 213 169 51.6 36 20 Pe luo open anoic acid PFPeA 3.18 263 219 89.7 36 20 Pe luo ohexanoic acid PFHxA 3.98 313 269 119 36 20 Pe luo ohep anoic acid PFHpA 4.49 363 319 333 36 20 Pe luo ooc anoic acid PFOA 4.87 413 369 194 36 20 Pe luo ooc ane sul ona e PFOS 5.18 499 80 52 36 20 Pe luo o-n-[1,2,3,4-13C4]oc anoic acid MPFOA 4.87 417 371 168 36 20 CV: Cone ol age (V); CE: Collision ene gy (eV); a: SRM ansi ion used o quan i ica ion; b: SRM ansi ion used o con i ma ion Table Table 2 Analy ical and s a is ical pa ame e s. PFBuA PFPeA PFHxA PFHpA PFOA PFOS bMP (g ng-1) 0.066 0.12 0.24 0.19 0.19 0.59 sbMP (g ng-1) 8.5·10-4 9.4·10-4 3.1·10-3 2.2·10-3 2.2·10-3 6.9·10-3 bPT (g ng-1) 0.036 0.11 0.24 0.20 0.20 0.30 sbPT (g ng-1) 1.1·10-3 6.4·10-3 4.4·10-3 7.0·10-3 7.0·10-3 1.1·10-2 s uden 13 1 2 1 1 13 % R2 99.9 99.3 99.9 99.8 99.8 99.6 LOD (ng g-1) 1.5 2 0.5 0.03 0.03 0.03 LOQ (ng g-1) 4 6 2 0.08 0.08 0.08 LDR (ng g-1) 4.0-62.5 6.0-62.5 1.8-62.5 0.08-62.5 0.08-62.5 0.08-62.5 MP: Mobile phase; PT: Placen al issue; b: slope; sb: slope s anda d de ia ion; R2: de e mina ion coe icien ; LOD: limi o de ec ion; LOQ: limi o quan i ica ion; LDR: linea dynamic ange Table Table 3 Reco e y assay, p ecision and ueness o a ge compounds in placen al issue. Compound Spiked (ng g-1) Found (ng g-1) (SD) RSD (%) Reco e y (%) PFBuA 6.25 6.02 (1) 14 96 12.5 12.8 (2) 12 103 25 28 (2) 6 113 PFPeA 6.25 6.61 (1) 9 106 12.5 12.3 (2) 10 98 25 24 (3) 9 94 PFHxA 6.25 6.44 (1) 10 103 12.5 12.4 (2) 12 99 25 23 (3) 12 94 PFHpA 1.25 1.32 (0.1) 1 105 6.25 6.24 (1) 4 100 12.5 11.8 (1) 12 95 PFOA 1.25 1.30 (0.1) 1 104 6.25 6.24 (1) 4 100 12.5 11.8 (1) 12 95 PFOS 1.25 1.30 (0.1) 1 104 6.25 6.11 (1) 13 98 12.5 12.6 (2) 14 101 SD: S anda d de ia ion; RSD: Rela i e s anda d de ia ion Table Table 4 Concen a ions o PFCs de e mined in he placen a samples. Sample *Found amoun (ng g-1) (SD) PFBuA PFPeA PFHxA PFHpA PFOA PFOS Mo he 01 D ND D ND D D Mo he 02 ND ND ND ND ND ND Mo he 03 ND ND ND ND ND ND Mo he 04 ND ND D ND ND 1.1 (0.01) Mo he 05 ND ND ND ND ND D Mo he 06 ND ND ND ND ND D Mo he 07 ND ND ND ND ND 1.0 (0.01) Mo he 08 30 (2) ND 4.9 (0.5) ND D ND Mo he 09 ND ND ND ND ND 1.2 (0.01) Mo he 10 D ND D ND ND ND Mo he 11 ND ND ND ND ND ND Mo he 12 ND ND ND ND ND ND Mo he 13 ND ND ND ND ND D Mo he 14 ND ND D D D D Mo he 15 28 (1.5) ND 5.1 (0.4) ND ND D Mo he 16 ND ND D ND ND D Mo he 17 ND ND ND ND ND ND Mo he 18 ND ND D ND D 1.1 (0.01) Mo he 19 ND ND ND ND ND ND Mo he 20 D ND D ND ND D Mo he 21 D ND ND D 0.37 (0.03) 1.2 (0.01) Mo he 22 ND ND ND D ND ND Mo he 23 ND ND ND ND ND ND Mo he 24 ND ND ND D 0.37 (0.02) 0.21 (0.01) Mo he 25 ND ND ND ND ND ND ND: below limi o de ec ion; D: below he limi o quan i ica ion bu abo e he limi o de ec ion * Mean o six de e mina ions Table 0 20 40 60 80 100 120 140 PFBuA PFPeA PFHxA PFHpA PFOA PFOS Reco e y (%) Ace one Ace oni ile E hyl ace a e Figu e Click he e o download high esolu ion image Figu e Click he e o download high esolu ion image