Published online: 05 Feb 2014.

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1 This rtile ws downloded y: [Kore Institute of Siene nd Tehnology (KIST)], [Oh Min-Seok] On: 18 Ferury 2014, At: 17:41 Pulisher: Tylor & Frnis Inform Ltd Registered in Englnd nd Wles Registered Numer: Registered offie: Mortimer House, Mortimer Street, London W1T 3JH, UK Food Additives & Contminnts: Prt B: Surveillne Pulition detils, inluding instrutions for uthors nd susription informtion: Simultneous nlysis of phthltes, dipte nd polyyli romti hydrorons in edile oils using isotope dilution-gs hromtogrphy mss spetrometry Min-Seok Oh, Seon-Hw Lee, Myeong Hee Moon, Dong Soo Lee & Hyun-Mee Prk Advned Anlysis Center, Kore Institute of Siene nd Tehnology, Seoul, Repuli of Kore Deprtment of Chemistry, Yonsei University, Seoul, Repuli of Kore Pulished online: 05 Fe To ite this rtile: Min-Seok Oh, Seon-Hw Lee, Myeong Hee Moon, Dong Soo Lee & Hyun-Mee Prk, Food Additives & Contminnts: Prt B (2014): Simultneous nlysis of phthltes, dipte nd polyyli romti hydrorons in edile oils using isotope dilution-gs hromtogrphy mss spetrometry, Food Additives & Contminnts: Prt B: Surveillne, DOI: / To link to this rtile: PLEASE SCROLL DOWN FOR ARTICLE Tylor & Frnis mkes every effort to ensure the ury of ll the informtion (the Content ) ontined in the pulitions on our pltform. However, Tylor & Frnis, our gents, nd our liensors mke no representtions or wrrnties whtsoever s to the ury, ompleteness, or suitility for ny purpose of the Content. Any opinions nd views expressed in this pulition re the opinions nd views of the uthors, nd re not the views of or endorsed y Tylor & Frnis. The ury of the Content should not e relied upon nd should e independently verified with primry soures of informtion. Tylor nd Frnis shll not e lile for ny losses, tions, lims, proeedings, demnds, osts, expenses, dmges, nd other liilities whtsoever or howsoever used rising diretly or indiretly in onnetion with, in reltion to or rising out of the use of the Content. This rtile my e used for reserh, tehing, nd privte study purposes. Any sustntil or systemti reprodution, redistriution, reselling, lon, su-liensing, systemti supply, or distriution in ny form to nyone is expressly foridden. Terms & Conditions of ess nd use n e found t

2 Food Additives & Contminnts: Prt B, Downloded y [Kore Institute of Siene nd Tehnology (KIST)], [Oh Min-Seok] t 17:41 18 Ferury 2014 Simultneous nlysis of phthltes, dipte nd polyyli romti hydrorons in edile oils using isotope dilution-gs hromtogrphy mss spetrometry Min-Seok Oh,, Seon-Hw Lee,, Myeong Hee Moon, Dong Soo Lee nd Hyun-Mee Prk * Advned Anlysis Center, Kore Institute of Siene nd Tehnology, Seoul, Repuli of Kore; Deprtment of Chemistry, Yonsei University, Seoul, Repuli of Kore (Reeived 28 August 2013; epted 24 Novemer 2013) A method for simultneous determintion of 12 priority phthltes, dipte nd polyyli romti hydrorons (PAHs) in edile oils y isotope dilution-gs hromtogrphy mss spetrometry (ID-GC MS) ws developed for fst, urte nd tre nlysis. The extrtion nd len-up proedures were optimised, nd using stle isotope-lelled internl stndrds for eh nlyte, reltive stndrd devitions (RSDs) of % nd spiked smple reoveries of % were otined. Limits of detetion for PAHs were in the rnge of µg/kg nd those for phthltes were in the rnge of µg/kg. The lirtion urves exhiited good linerities with regression oeffiients of R Twelve edile oils were exmined to evlute the effiieny of this method. Among the 12 nlytes, diutyl phthltes (DBP), diethylhexyl phthltes (DEHP), diethylhexyl dipte (DEHA), enzo[]nthrene (B[]A), hrysene (Chry) nd enzo []fluornthene (B[]F) were deteted in the rnge of µg/kg. Keywords: phthltes; dipte; polyyli romti hydrorons; isotope dilution; GC MS; edile oils; simultneous determintion Introdution Endorine disrupting phthltes nd rinogeni polyyli romti hydrorons (PAHs) re present not only in the environment ut re lso migrted into foods during pkging, storge (Aurel et l. 1999) nd mnufturing (Phillips 1999). Phthltes re plstiisers tht gently soften plstis (Steiner et l. 1998) nd re widely used in industril produts, suh s toys, vinyl flooring, medil devies nd food pkging (Huser & Clft 2005; Guo et l. 2012). Beuse phthltes do not form stle onds with plsti polymers, they esily umulte in the fts of foods exposed to pkging nd proessing with plsti produts (Witorsh 2002). Crinogeni PAHs, whih hve more thn two enzene rings, re formed during the inomplete omustion of orgni sustnes, suh s wood, fuel, too nd food; thus, PAHs n e inorported into foods during prodution (Cmrgo & Toledo 2002; FAO/WHO 2005). In ddition, humns my e exposed to PAHs through umultion in soil, wter, ir, food hin nd diol epoxide. Diol epoxide is PAH intermedite tht indues ytotoxiity, muttions nd tumours y interting with proteins, DNA nd RNA (Burzynski et l. 1999; Mollerup et l. 2001). Beuse phthltes our in plsti ontiners nd PAHs in the ourse of food proessing nd ooking, suh s during rosting nd smoking, most of the exposure to these sustnes is ssoited with food (Klusk 2003). Lipophili phthltes migrte into oils from plsti pkging nd ontiners, wheres PAHs re produed y the inomplete omustion of plnt seeds during oil prodution; therefore, edile oils re ommonly ontminted with oth phthltes nd PAHs. The Sientifi Committee for Food (SCF, EU) hs designted the tolerle dily intke (TDI) of diutyl phthltes (DBP), enzylutyl phthltes (BBP), diethylhexyl phthltes (DEHP) nd diethylhexyl dipte (DEHA) s 0.1 mg/kg, 0.05 mg/kg, 0.05 mg/kg nd 0.3 mg/kg per dy, respetively (Petersen & Breindhl 2000; Kueseng et l. 2007). In vegetle oil, onentrtions of DBP nd DEHP were < mg/kg nd mg/kg (Liu et l. 2013) nd in ooking oil were μg/kg nd μg/kg (Guo et l. 2012), respetively. For PAHs, the United Sttes Environmentl Protetion Ageny (USEPA) mnges 16 speies, espeilly enzo[]pyrene (B[]P), enzo[] nthrene (B[]A), dienzo[, h]nthrene (DB[h]A) nd hrysene (Chry). The Europen Union Commission Regultion (835/2011 L215) (EPA 1984) permits mximum level of B[]P of 2 µg/kg nd the sum of B[]P, B[] A, Chry nd enzo[]fluornthene (B[]F) of 10 µg/kg for oils nd fts. Conentrtions of B[]P nd B[]A were in the rnges of µg/kg nd µg/kg in smoked food, nd B[]P, B[]A, Chry nd DB[h]A were deteted in the rnges of µg/kg, µg/kg, µg/kg nd ND 0.1 µg/kg in edile oils, respetively (Mored et l. 2001). *Corresponding uthor. Emil: phm4556@kist.re.kr 2014 Tylor & Frnis

3 Downloded y [Kore Institute of Siene nd Tehnology (KIST)], [Oh Min-Seok] t 17:41 18 Ferury M.-S. Oh et l. Extrtion nd len-up re the most importnt steps for phthltes nd PAHs nlysis in food, espeilly in ftty food smples. Currently, severl extrtion, purifition (len-up) nd detetion methods re employed for the determintion of phthltes nd PAHs. Phthlte extrtion methods inlude liquid liquid extrtion (LLE) (Wu et l. 2012; Liu et l. 2013), solid-phse extrtion (SPE) (Nnni et l. 2011; Liu et l. 2013) nd solid-phse miroextrtion (SPME) (Co 2008). Extrtion with polr or nonpolr solvents following sili nd C 18 rtridge len-up hs lso een used for phthltes nlysis, ut the limit of detetion (LOD) ws reltively poor, eing mesured in the rnge of mg/kg (Nnni et l. 2011; Wuetl.2012; Liu et l. 2013). Soxhlet (Jouen-Mdoulet et l. 2000), pressurised-liquid extrtion (PLE) (Jir 2004), mirowve-ssisted extrtion (MAE) (Sim et l. 1997; Mooiroek et l. 2002) nd LLE (Bogusz et l. 2004; Bllesteros et l. 2006) re typil PAH extrtion methods, nd len-up methods suh s gel-permetion hromtogrphy (GPC), (Liguori et l. 2006) sili gel nd florisil (Simon et l. 2006; Tfounietl.2007) reommonly used. The k extrtion of PAHs with polr solvent following nonpolr solvent is time-onsuming nd yields low reoveries (Mottier et l. 2000; Diletti et l. 2005). The detetion of phthltes is usully performed y gs hromtogrphy-flme ioniztion detetor (GC-FID) (Polo et l. 2005), gs hromtogrphy-mss spetrometery (GC-MS) (Nnni et l. 2011; Wuetl. 2012; Liu et l. 2013) nd liquid hromtogrphy-mss spetrometery (LC-MS) (Jonsson & Boren 2002), wheres PAHs re deteted y GC MS (Bogusz et l. 2004; Bllesteros et l. 2006), GC MS/MS (Helleh et l. 2005) nd high performne liquid hromtogrphy-fluoresene detetion (HPLC-FLD) (Pen et l. 2006; Tfounietl.2007). As n isotope dilution method tht uses the deuterted or C 13 isotope of nlyte s the internl stndrd, whih shows similr physil nd hemil ehviour s the nlytes, this n improve the ury y reduing reovery errors from the omplex mtrix nd interferenes during lirtion for quntifition (Boden & Reiner 2004). Usully, one or two isotopes re used s internl stndrds for the nlysis of phthltes (DBP-d4, DEHP-d 4 ; Feng et l. 2005) nd PAHs (phennthrene-d 10, nthrene-d 10, enzo()pyrene-d 12 ;Wng&Guo2010). As mentioned ove, the extrtion nd len-up methods re different for phthltes nd PAHs, nd individul ompounds re nlysed seprtely. Thus, it is diffiult to tret lrge numer of smples t the sme time. Therefore, simultneous nlysis method is needed for simple nd fst nlysis. The im of this study ws to develop pretretment nd nlytil method for edile oils with low LODs, good preision nd ury for the simultneous determintion of three phthltes (diutyl phthltes (DBP), enzylutyl phthltes (BBP), diethylhexyl phthltes (DEHP)), one dipte (diethylhexyl dipte (DEHA) nd eight PAHs (enzo[]nthrene (B[] A), hrysene (Chry), enzo[]fluornthene (B[]F), enzo [k]fluornthene (B[k]F), enzo[]pyrene (B[]P), indeno [1,2,3-d]pyrene (I[d]P), enzo(ghi)perylene (B[ghi]P) nd dienzo[, h]nthrene (DB[h]A)). To nlyse these ompounds simultneously, LLE nd solid-phse len-up were optimised, nd n isotope dilution- GC MS method, whih uses isotopes for ll nlytes, ws estlished for the improvement of the ury. The new method ws pplied to 12 smples, to demonstrte tht it is le to determine phthltes, dipte nd PAHs in edile oil. Mterils nd methods Chemils nd smples All solvents were HPLC-grde nd purhsed from J.T Bker (Phillipsurg, NJ). Anlytil stndrds of phthltes (purity > 99%) were DBP (Chem Servie, West Chester, PA), BBP nd DEHP (Wko Chemil, Osk, Jpn) nd DEHA (Sigm Aldrih, Aldrih Chemil, USA). Anlytil mixture stndrds of PAHs (2000 µg/ml), B[]A, Chry, B[]F, B[k]F, B[]P, I[d]P, B [ghi]p nd DB[h]A were otined from Supelo (Bellefonte, PA). Individul stok solutions of phthlte were prepred in methnol, nd the PAHs were prepred in dihloromethne (DCM). Clirtion stndrd mixtures of the four trget nlytes (DBP, BBP, DEHP nd DEHA, 100 µg/ml) were dissolved in methnol. Their orresponding deuterted internl stndrds, DBP-d 4, BBP-d 4, DEHA-d 8, B[]F-d 12, B[k]F-d 12, B[]A-d 12, I [d]p-d 12, DB[h]A-d 14 nd B[ghi]P-d 12 were purhsed from Chiron As (Trondheim, Norwy) nd DEHP-d 4, B[]A-d 12 nd Chry-d 12 were otined from Austndrd (New Hven, CT). Anhydrous Sodium sulphte (N 2 SO 4 ) for dehydrtion ws otined from Knto hemil (Tokyo, Jpn) nd florisil ( mesh), for SPE, ws purhsed from J.T Bker (Phillipsurg, NJ). Phthltes re widely present in the environment, suh s in soil, rivers, drinking wter, ir nd lso in plnts nd nimls. Furthermore, mny solvents, hemils, glsswre, gloves, nd vils ontin phthltes, espeilly DBP nd DEHP (Fnkhuser-Noti & Gro 2007). This is signifint soure for potentil error in the results. To minimise the lnk ontmintion, ll the glsswre ws ked for 2 h t 200 C, N 2 SO 4 ws ked for 7 8 h t 700 C nd florisil ws ked for 16ht130 Cndthenstoredinvuumdesitor until nlysis. Solvents were stored in ottles ontining lumin dsorent prior to use. Smple nlyses were

4 Food Additives & Contminnts: Prt B 3 Downloded y [Kore Institute of Siene nd Tehnology (KIST)], [Oh Min-Seok] t 17:41 18 Ferury 2014 onduted with 12 edile oils (rie rn, rown rie, nol, olive, grpe seed, orn nd soyen) tht were purhsed from generl retil mrket. Optimistion of pretretment The solvent for the LLE nd the eluent for the solidphse extrtion (SPE) used to extrt nd len-up the phthltes nd PAHs simultneously were seleted sed on following referene results. Usully, the extrtion solvents for phthltes nd PAHs re different. Methylene hloride, n-hexne nd etonitrile (Wu et l. 2012; Liu et l. 2013) were used to extrt the phthltes, wheres ylohexne, dimethylformmide (DMF):H 2 O (Diletti et l. 2005), dimethylsulfoxide (DMSO) (Mottier et l. 2000) nd mixture of n-hexne nd etonitrile were used to extrt PAHs (Bllesteros et l. 2006). Therefore, it is importnt to selet n pproprite extrtion solvent for oth sustnes. Nonpolr solvent extrtion requires onsiderle time, nd the loss of nlytes is reltively lrge with k extrtion. Conversely, the polr solvent extrtion effiieny vries depending on the polrity of the solvent. In this study, extrtions were performed using polr nd nonpolr (A) 120 Reovery (%) (C) Reovery (%) Hex:DCM = 3:1 MeOH ACN Hex/ACN Solvent Hex:DCM = 1:1 Hex solvents s follows: methnol extrtion, etonitrile extrtion, etonitrile extrtion fter dissolving in hexne nd hexne extrtion fter k extrtion with DMF:H 2 O (9:1). The extrtion effiienies re shown in Figures 1A nd B. After LLE, SPE helps to remove interferenes from the omplex mtrix. Florisil, polr sttionry phse, ws used s the SPE sorent to purify the phthltes (Wu et l. 2012) nd PAHs (Brtolome et l. 2005; Ehrenhuser et l. 2010) from interfering polr sustnes. One hundred grms of florisil, ked t 130ºC for 16 h, ws mixed with 5.7 ml of wter, nd 1 g liquot ws pked in glss syringe with methnol. Insted of ommeril plsti rtridge, glss ws used to void phthlte ontmintion from the plsti. The rtridge ws onditioned with 10 ml of DCM fter 15 ml hexne efore len up for preonditioning of the rtridge. Five solvents were ompred for effetive purifition: n-hexne, n-hexne:dcm (3:1), n-hexne: DCM (1:1), n-hexne:etone (9:1) nd n-hexne:etone (3:1). The results re shown in Figures 1C nd D. The mount of solvent used during the extrtion ws lso vried etween 5 nd 25 ml to determine the optimum volume. DBP 100 BBP 80 DEHA DEHP DMF/Hex MeOH ACN Hex/ACN DMF/Hex Solvent Hex:Aeton = 9.1 Eluent Hex:Aeton = 3.1 DBP BBP DEHA DEHP Reovery (%) (B) (D) Reovery (%) Hex:DCM = 3:1 Hex:DCM = 1:1 Hex Eluent Hex:Aeton = 9.1 Hex:Aeton = 3.1 B[]A Chry B[]F B[k]F B[]P I[d]P DB[h] A B[]A Chry B[]F B[k]F B[]P I[d]P DB[h]A B[ghi]P Figure 1. Extrtion effiienies of vrious solvents for (A) phthltes, (B) PAHs for the liquid liquid extrtion, (C) phthltes nd (D) PAHs for the solid-phse extrtion (ANOVA nlysis, P < 0.05): diutyl phthltes (DBP), enzylutyl phthltes (BBP), diethylhexyl phthltes (DEHP), diethylhexyl dipte (DEHA), enzo[]nthrene (B[]A), hrysene (Chry), enzo[]fluornthene (B []F), enzo[k]fluornthene (B[k]F), enzo[]pyrene (B[]P), indeno[1,2,3-d]pyrene (I[d]P), dienzo[, h]nthrene (DB[h]A), enzo(g,h,i)perylene (B[ghi]P), methnol (MeOH), etonitrile (ACN), n-hexne (Hex), dimethylformmide (DMF) nd dihloromethne (DCM);,, ross the group signifies tht mens with different letters differ signifintly t P < 0.05 while mens with the sme letters do not differ signifintly t P < 0.05.

5 4 M.-S. Oh et l. Downloded y [Kore Institute of Siene nd Tehnology (KIST)], [Oh Min-Seok] t 17:41 18 Ferury 2014 Isotope dilution-gc MS nlysis Aury ws ompred etween typil isotope dilution methods using DEHP-d 4 nd B[]P-d 12 s internl stndrds nd the 1:1 isotope dilution method of this study using 12 isotopes orresponding to 12 nlytes: DBP-d 4, BBP-d 4,DEHA-d 8, DEHP-d 4, B[]A-d 12,Chry-d 12, B[] F-d 12, B[k]F-d 12, B[]A-d 12, I[d]P-d 12, DB[h]A-d 14 nd B[ghi]P-d 12. Five hundred nnogrms of the isotope mixture ws dded to 4 g of the oil smple efore extrtion. Extrted smples were nlysed with gs hromtogrphy time-of-flight mss spetrometry (GC TOFMS: Agilent 6890N GC system, USA; Leo, PEGASUS IV MS system, USA). Dt proessing ws performed with LECO ChromTOF TM nd NIST MS serh 2.0. A DB- 5MS olumn (30 m 0.25 mm 0.25 μm, Agilent Tehnology, USA) ws used with He (99.999%) rrier gs t flow rte of 0.8 ml/min. The inlet ws in the split (5:1) mode t 300 C with n injetion volume of 1 μl. An oven grdient progrmme ws used for the seprtion: C with 25 C/min rmp nd durtion of 2 min, inrese to 300 C with rmp of 5 C/min nd durtion of 1 min nd finlly inrese to 315 C with rmp of 5 C/min nd durtion of 5 min. Tle 1. The operting prmeters for the MS were s follows: eletron impt (EI) ion soure temperture, 220 C; eletron energy, 70 ev; eletron multiplier detetor (EM) voltge, 1580 ev; trnsfer line temperture, 300 C; MS rnge, mu. The prmeters for the mss nlysis of the nlytes re listed in Tle 1. Smple nlysis All the 12 edile oils (rie rn, rown rie, nol, olive, grpe seed, orn nd soyen) purhsed from retil mrket were nlysed y using the optimised nlytil proedures. Four grm liquots of homogenous oil smples were weighed in 50 ml entrifuge tues, nd 500 ng of the isotope solution nd 15 ml etonitrile were dded. The tues were seled nd shken for 15 min t 200 rpm using mehnil shker (Chng-shin, Kore). This proess ws repeted twie, nd then the etonitrile extrts were olleted following dehydrtion with nhydrous N 2 SO 4. The extrt ws evported in nitrogen strem during 25 min to dryness, nd the residue ws dissolved in 1mLn-hexne. Interferenes, suh s lipids nd polr impurities, were removed from the residue using florisil. The florisil olumn ws onditioned with 10 ml DCM Prmeters for the GC MS nlysis, linerity, limits of detetion (LOD) nd quntifition (LOQ). Compound R.T (se) Q.I (m/z) Q.I (m/z) Linerity (R 2 ) LOD LOQ (µg/kg, n = 9) (µg/kg, n =9) Phthltes Diutyl phthltes Diutyl phthltes-d Denzylutyl phthltes Denzylutyl phthltes-d Diethylhexyl phthltes Diethylhexyl phthltes-d Diethylhexyl dipte Diethylhexyl dipte-d PAHs Benzo[]nthrene Benzo[]nthrene-d Chrysene Chrysene-d Benzo[]fluornthene Benzo[]fluornthene-d Benzo[k]fluornthene Benzo[k]fluornthene-d Benzo[]pyrene Benzo[]pyrene-d Indeno[1,2,3-d]pyrene Indeno[1,2,3-d]pyrene-d Dienzo[, h]nthrene Dienzo[, h]nthrene-d Benzo(g,h,i)perylene Benzo(g,h,i)perylene-d Notes: Retention time; Qulifition ion; Quntifition ion.

6 Food Additives & Contminnts: Prt B 5 Downloded y [Kore Institute of Siene nd Tehnology (KIST)], [Oh Min-Seok] t 17:41 18 Ferury 2014 following 15 ml of n-hexne. The elution solvent used for len-up ws 15 ml of 1:1 DCM nd n-hexne mixture. The elute ws dried in nitrogen strem, nd the residue ws dissolved in 250 μl ethylette. One mirolitre of this solution ws injeted into the GC MS. Sttistil nlysis Reovery results of the extrtion nd len-up proedures re presented s men vlue ± stndrd devition (SD) for four replites. Multiple omprisons of mens were performed y nlysis of vrine (ANOVA), nd the mens were seprted y Dunn s multiple rnge test onsidering signifint differenes t P < Results nd disussion Optimistion of pretretment The reoveries of the phthltes nd PAHs with methnol re shown in Figures 1A nd B. The phthlte reoveries were poor, rnging from 1.8% to 7.6%, nd PAHs were not reovered. The extrtion effiieny using DMF:H 2 O (9:1) following k extrtion with hexne lso showed poor reovery rtes: % for phthltes nd % for PAHs. Using etonitrile, ll phthltes nd PAHs were extrted with good reoveries ( % nd %) nd repetilities ( % nd %). The residue oil remining in the extrt mkes hromtogrphi seprtion poor due to the ontmintion of the olumn (olumn plugging). Therefore, it is importnt to len-up, with high reovery effiieny, oth phthltes nd PAHs in edile oil. In Figure 1C, then-hexne: Tle 2. Method vlidtion results of phthltes, dipte nd PAHs in oil smples. DCM (1:1) solvent showed the highest reovery effiieny, from 87.0% to 94.1% for phthltes, wheres the reovery effiieny ws reltively low with other eluents, rnging from 30.3% to 77.5%. As shown in Figure 1D, the extrtion effiienies for B[]A nd Chry were 94.7% nd 96.6%, respetively, with the DCM:n-hexne (1:1) solvent, wheres other solvents showed reltively low reoveries for these two PAHs, rnging from 76.0% to 89.4%. The reovery effiienies of the other PAHs (B[]F, B[k]F, B[]P, I[d]P, B[ghi]P nd DB[h]A) were good with the n-hexne:dcm (3:1) nd n-hexne:etone (9:1) solvents. However, the reoveries for these PAHs using the n-hexne:dcm (1:1) solvent were similr to the other solvents. From the sttistil nlysis of ANOVA, we n see signifint effiieny differenes etween solvents nd eluents in phthltes (Figures 1A nd C) ut no signifint differene in PAHs (Figures 1B nd D). However, euse the gol of this reserh is the simultneous determintion of PAHs nd phthltes, we hose etonitrile s the solvent nd n-hexne:dcm (1:1) s the eluent whih gve good reoveries ( % for phthltes nd % for PAHs) nd repetilities ( % for phthltes nd % for PAHs) for oth phthltes nd PAHs. Volume of 15 ml of this eluent showed mximum reoveries: phthlte ( %) nd PAHs ( %), so the florisil len-up ws onduted with 15 ml of n-hexne:dcm (1:1) eluent for ll the experiments. Insted of the typil one or two isotope internl stndrds, ll isotopes orresponding to ll nlytes were dded for the isotope dilution (1:1 isotope dilution). The ury using DEHP-d 4 nd B[]P-d 12 s internl Reovery (%) Aury (%) Preision (%) TID Interdy Intrdy Compounds (μg/kg, n =9) (μg/kg, n =9) (n =5) (n =3) Phthltes DBP BBP DEHP DEHA PHAs B[]A Chry B[]F B[k]F B[]P I[d]P DB[h]A B[ghi]P Note: Typil isotope dilution (250 μg/kg).

7 Downloded y [Kore Institute of Siene nd Tehnology (KIST)], [Oh Min-Seok] t 17:41 18 Ferury M.-S. Oh et l. stndrds ws ompred to tht of the 1:1 isotope dilution method, where 12 isotopes orresponding to ll nlytes were spiked. The results re shown in Tle 2. The ury of this method ws %, wheres tht of the typil isotope dilution ws %. As expeted, the ury ws improved using the 1:1 isotope dilution method. Therefore, we used the 1:1 isotope dilution method for ll the following experiments. Method vlidtion As mentioned, phthltes re widespred throughout the experimentl environment, suh s vils, solvents, injetor sept, gloves, glsses nd even ir; therefore, lnks hve to e mesured efore eh smple mesurement. All nlytil proedures for lnks were performed seven times, in whih the smple ws not inluded in the lnk. The kground levels of DBP nd DEHP were determined to e 41.5 ± 5.5 µg/kg nd 49.4 ± 7.5 µg/kg, respetively, nd these vlues were sutrted in the smple onentrtion lultions. To evlute the effetiveness of this new method, the preision ws mesured in inter-dy (n = 5) nd intr-dy (n = 3) intervls t low, medium nd high onentrtions (50, 250 nd 1250 µg/kg). The reltive stndrd devitions (RSDs) were s low s % for phthltes nd % for PAHs. The ury experiment ws performed y determining the reoveries of phthltes nd PAHs in the lnk oil spiked t three different onentrtion levels (n =9).Thedtin Tle 2 shows the good ury of the reoveries of % for phthltes nd % for PAHs. The extrtion reoveries were estimted y omprison of the pek re of spiked smple with the pek re of the stndrd solution. The reoveries of phthltes nd PAHs were in the rnges of % nd %, respetively. These results re not signifintly different from previous studies where reoveries rnged from 64.0% to 109.0% for phthltes (Del Crlo et l. 2008; Wuetl.2012; Liu et l. 2013) nd % for PAHs (Mottier et l. 2000; Bogusz et l. 2004; Diletti et l. 2005; Bllesteros et l. 2006). For the quntifition of phthltes nd PAHs, lirtion urves were fitted from eight points nd showed good linerity; the orreltion oeffiients (R 2 ) of the phthltes were nd those of the PAHs were (Tle 1). The limits of detetion (LOD, 3.3σ) nd limits of quntifition (LOQ, 10σ) for phthltes were µg/kg nd µg/kg, respetively, nd those of the PAHs were µg/kg nd µg/kg, respetively. The LOD results of phthlte were 3 to 10 times lower thn previous vlues in the literture ( µg/kg), (Del Crlo et l. 2008; Nnni et l. 2011; Wu et l. 2012; Liu et l. 2013) nd the vlues for the PAHs were similr (Bogusz et l. 2004; Diletti et l. 2005; Bllesteros et l. 2006). In profiieny test with sunflower oil simulnt mteril, z-sores of 1.1 nd 1.3 were hieved for DBP t 0.38 mg/kg nd 0.36 mg/kg, nd z-sores of 0.3 nd 0.1 were hieved for DEHP t 0.86 mg/kg nd 0.89 mg/kg, respetively, inditing good method performne (Food Anlysis Performne Assessment Sheme 2012) These vlidtion results show tht this new method is sensitive nd urte for the quntifition of oth phthltes nd PAHs t low onentrtion levels (µg/kg) for the studied edile oils. Applition to smples Twelve edile oils (rie rn oil, nol oil, olive oil, rown rie oil, grpe seed oil, orn oil nd soyen oil) were nlysed to determine whether this method ould e pplied to oil smples. All smples were pkged in polyethylene terephthlte (PET) exept smple 11 (polyethylene, PE). The food pkging industry hs redued its use of phthltes y employing polymers suh s polyethylene (PE) nd polypropylene (PP) insted of polyvinyl hloride (PVC). PVC ontins up to 40% phthltes, usully DEHP (Aurel et l. 1999). Although the use of phthltes s plstiisers hs deresed, they re still used in food pkging in mny pplitions, suh s dhesives, offset printing inks nd lquers (Aurel et l. 1999). Therefore, there still exists the possiility of ontmintion of phthltes from the pkging. In this study, DBP ws deteted in only two olive oil smples (16.7%) t onentrtions of 13.2 ± 2.29 nd 40.6 ± 2.30 µg/kg. Among the 12 nlysed oils, totl of 9 smples (75%) were ontminted with DEHP, whih ounts for more thn 50% of plstiisers, t slightly high onentrtions of 25.0 ± 1.77 to 806 ± 10.1 µg/kg. DEHA ws deteted only in S-6 (olive oil) t onentrtion of 5.46 ± 0.15 µg/kg. Previous results for DBP nd DEHP in Chinese vegetle oil were in the rnge of µg/kg nd µg/kg (Liu et l. 2013), respetively, whih is higher thn our results. In the se of PAHs, B[]A ws found in the rnge of 1.17 ± ± 0.42 µg/kg in seven oils (58.3%), wheres Chry ws deteted only in rie rn oil (S-1) t 2.95 ± 0.31 µg/kg nd B[]F ws found in two orn oils (Tle 3) t 2.12 ± 0.09 nd 3.76 ± 0.16 µg/kg. In Spin, B[]A, Chry nd B[]F were deteted t <0.1 97, < nd < µg/kg in edile oils, respetively (Brrno et l. 2003). Compring our results with those from Spin, the onentrtions of PAHs from our reserh re lower thn those from Spin, nd B[]A is uiquitous in edile oils. Other PAHs (B[]P, B[k]F, I[d]P, DB[h]P nd B[ghi]P) nd BBP were not deteted in the nlysed oils. B[]P, regultedt2µg/kginedileoilinsouthkore,ws

8 Food Additives & Contminnts: Prt B 7 Tle 3. Conentrtions of phthltes, dipte nd PAHs in oil smples. Compound (µg/kg, n = 4) Smple No. Pkging mteril DBP DEHP DEHA B[]A Chry B[]F Downloded y [Kore Institute of Siene nd Tehnology (KIST)], [Oh Min-Seok] t 17:41 18 Ferury 2014 Rie rn S-1 PET < ± 6.47 < ± ± 0.31 <0.55 Brown rie S-2 PET < ± 9.28 <4.56 <0.28 <0.26 <0.55 Cnol S-3 PET < ± 2.66 < ± 0.28 <0.26 <0.55 S-4 PET < ± 5.21 <4.56 <0.28 <0.26 <0.55 Olive S-5 PET 40.6 ± ± 5.98 <4.56 <0.28 <0.26 <0.55 S-6 PET 13.2 ± ± ± 0.15 <0.28 <0.26 <0.55 Grpe seed S-7 PET <9.77 <10.0 < ± 0.08 <0.26 <0.55 S-8 PET < ± 10.1 < ± 0.07 <0.26 <0.55 Corn S-9 PET <9.77 <10.0 < ± 0.20 < ± 0.16 S-10 PET < ± 1.35 < ± 0.07 < ± 0.09 Soyen S-11 PE < ± 1.77 <4.56 <0.28 <0.26 <0.55 S-12 PET <9.77 <10.0 < ± 0.08 <0.26 <0.55 Notes: Other phthltes nd PAHs (BBP, Chry, B[k]F, B[]P, I[d]P, DB[h]A nd B[ghi]P) were not deteted; Polyethylene terephthlte; Polyethylene. not deteted. The totl sum of the exmined PAHs ws µg/kg, whih is lower thn the EU ommission regultion (835/2011 L215) of 10 µg/kg for the sum of four PHAs in oils nd fts. Conlusions In this study, new isotope dilution-gs hromtogrphy mss spetrometry (ID-GC MS) method ws developed for the simultneous isoltion nd purifition of 12 nlytes from edile oils. The method showed exellent reoveries nd preisions nd limit of quntifition elow the µg/kg level for most of the phthltes, n dipte nd the PAHs. The entire proedure requires 85 min (inluding LLE, SPE nd nlysis y GC MS) nd does not require lrge volume of solvents. These results demonstrte tht this newly developed simultneous nlysis method ould e used for the nlysis of tre phthltes, dipte nd PAHs in edile oil for routine nlysis. This new method hs mny dvntges, suh s short time, lour nd solvent svings, low LOD nd good preision nd ury ompred to onventionl nlytil tehniques, nd the method n e used to ontrol food sfety nd provide si dt for the risk ssessment of phthltes nd PAHs in edile oils. Funding Finnil support from the Kore Food nd Drug Administrtion [grnt numer 10162KFDA994, 2012] nd the Kore Institute of Siene nd Tehnology [grnt numer 2VO2952, 2013] re grtefully knowledged. Referenes Aurel B, Kulml H, Soderhjelm L Phthltes in pper nd ord pkging nd their migrtion into Tenx nd sugr. Food Addit Contm. 16: Bllesteros E, Snhez AG, Mrtos NR Simultneous multidetermintion of residues of pestiides nd polyyli romti hydrorons in olive nd olive-pome oils y gs hromtogrphy/tndem mss spetrometry. J Chromtogr A. 1111: Brrno A, Alonso-Sles RM, Bkkli A, Berruet LA, Gllo B, Viente F, Sroe M Solid-phse lenup in the liquid hromtogrphi determintion of polyyli romti hydrorons in edile oils. J Chromtogr A. 988: Brtolome L, Cortzr E, Rposo JC, Usoig A, Zulog O, Etxerri N, Fernndez LA Simultneous mirowve-ssisted extrtion of polyyli romti hydrorons, polyhlorinted iphenyls, phthlte esters nd nonylphenols in sediments. J Chromtogr A. 1068: Boden AR, Reiner EJ Development of n isotope-dilution gs hromtogrphi-mss spetrometri method for the nlysis of polyyli romti ompounds in environmentl mtries. Polyyl Aromt Comp. 24: Bogusz MJ, El Hj SA, Ehide Z, Hssn H, Al-Tufil M Rpid determintion of enzo()pyrene in olive oil smples with solid-phse extrtion nd low-pressure, wide-ore gs hromtogrphy-mss spetrometry nd fst liquid hromtogrphy with fluoresene detetion. J Chromtogr A. 1026:1 7. Burzynski ME, Lin HK, Penning TM Isoform-speifi indution of humn ldo-keto redutse y polyyli romti hydrorons (PAHs), eletrophiles, nd oxidtive stress: implitions for the lterntive pthwy of PAH tivtion tlyzed y humn dihydrodiol dehydrogense. Cner Res. 59: Cmrgo MCR, Toledo MCF Polyyli romti hydroron ontmintion in different ommodity groups. Brz J Food Tehnol. 5: Co XL Determintion of phthltes nd dipte in ottled wter y hedspe solid-phse miroextrtion nd gs hromtogrphy/mss spetrometry. J Chromtogr A. 1178: Del Crlo M, Pepe A, Shetti G, Compgnone D, Mstrool D, Cihelli A Determintion of phthlte esters in wine using solid-phse extrtion nd gs hromtogrphymss spetrometry. Food Chem. 111:

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