National Institute of Research & Development for Food Bioresources, IBA Bucharest, 6 Dinu Vintila, Bucharest, Romania

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1 J. Brz. Chem. Soc., Vol. 27, No. 10, , Printed in Brzil Sociedde Brsileir de Químic $ Article Appliction of n Accurte nd Vlidted Method for Identifiction nd Quntifiction of Acrylmide in Bred, Biscuits nd Other Bkery Products Using GC-MS/MS System Mior Negoiță nd Alin Culețu* Ntionl Institute of Reserch & Development for Food Bioresources, IBA Buchrest, 6 Dinu Vintil, Buchrest, Romni A gs chromtogrphy tndem mss spectrometry hs been developed nd vlidted for the seprtion, detection, identifiction nd quntifiction of crylmide in bred, biscuits nd similr products. The method showed good precision with vlues lower thn 6%. A good sensitivity ws chieved for bred with 2.41 nd 7.23 µg kg -1 limit of detection (LOD) nd limit of quntifiction (LOQ), respectively, while for biscuits, LOD nd LOQ were 4.63 nd µg kg -1, respectively. Accurcy obtined through the bis of 2 certified reference mterils ( crisp bred - ERM - BD272 nd rusk - ERM -BD274 ) gve vlue below %. The method ws pplied by nlyzing 49 types of bred, biscuits nd other similr products. The results showed different levels of crylmide in bred (vlues rnged between 7.6 nd µg kg -1 ), biscuits (between LOD nd µg kg -1 ), sndwich biscuits with crem ( µg kg -1 ), biscuits for infnts nd young children (between LOD nd µg kg -1 ), gingerbred ( µg kg -1 ) nd crckers ( µg kg -1 ). Keywords: crylmide (AA), vlidtion, bred, biscuits, GC-MS/MS Introduction Acrylmide (AA) is chemicl tht cn be formed in some foods during certin types of high-temperture cooking. The neurotoxicity of AA in humns is well estblished from occuptionl nd ccidentl exposures, nd experimentl studies hve shown reproductive, genotoxic nd crcinogenic effects in nimls. AA hs been clssified s probbly crcinogenic to humns by the Interntionl Agency for Reserch on Cncer (IARC). 1 The possible public helth risks from dietry exposure to AA ttrcted concern by public uthorities ll over the world. 2 Bsed on the expert committee evlution of FAO/ WHO, 1 μg kg -1 body weight (bw) dy -1 is considered s n verge exposure to AA while 4 μg kg -1 bw dy -1 is considered s high exposure to AA. 3 The men dietry exposure rnge to AA is μg kg -1 bw dy -1 for the generl dult popultion while 95th-percentile rnge is μg kg -1 bw dy In 2013, the U.S. Food nd Drug Administrtion (FDA) 5 issued drft guidnce to help the food industry to reduce the mount of AA in certin foods. *e-mil: linculetu@gmil.com Commission Recommendtion 2013/647/EU estblished, bsed on the monitoring results received from the Member Sttes during the period , the indictive vlues for AA in different foods. The indictive vlues re not sfety thresholds, but their im is to indicte the need for n investigtion in the cse the vlues re exceeded nd to tke pproprite mesures to control the formtion of AA. 6 Tking into ccount tht the presence of AA in food products could increse the risk of cncer in consumers of ll ges, 7,8 n interntionlly priority is to develop relible nlysis method to quntify low concentrtions of AA in foods (dozens of ppb) nd to find solutions to reduce this contminnt. Worldwide, in recent yers severl methods hve been developed for the determintion of AA from food products Although in the lst decde were published lrge number of nlyticl methods bsed on gs chromtogrphy (GC) nd liquid chromtogrphy (LC) coupled with mss spectrometry (MS; MS/MS), the methods re minly used by privte lbortories nd officil control uthority for routine nlysis of AA in food products. Lst publictions pprently confirm the vlidity of LC nd GC techniques, which enbles to gurntee the determintion

2 Vol. 27, No. 10, 2016 Negoiță nd Culețu 1783 of AA with quntifiction limits from 30 to 50 μg kg -1 for HPLC-MS, 4 to 30 μg kg -1 for GC-MS nd LC MS/MS. Commission recommendtion of 2 June 2010, sttes tht for ensuring comprbility of nlyticl results, methods tht cn chieve limit of quntifiction (LOQ) of 30 µg kg -1 for bred nd foods for infnts nd young children nd 50 µg kg -1 for potto products, other cerel products, coffee nd other products should be chosen. 23 The im of this study is to present the results obtined on internl vlidtion of GC-MS/MS method for determintion of AA in bred, biscuits nd other similr products with LOQ below the bove recommended vlues. Moreover, ssessing the level of AA in some bkery products from the Romnin mrket ws performed. Experimentl Smpling Seventeen types of bred nd similr products, 18 types of biscuits, 5 types of biscuits for infnts nd young children, 4 types of sndwich biscuits, 2 crckers nd 3 gingerbred were studied. Smples were either purchsed in locl mrket (vrious types produced in different countries) or produced on pilot plnt of the Ntionl Reserch & Development Institute for Food Bioresources (Romni). Chemicls nd regents Ntive crylmide (min. 99% purity, concentrtion 1000 mg L -1 in methnol) (AA) ws purchsed from Ultr Scientific (N. Kingstown, Rhode Islnd, USA), internl stndrd of lbeled crylmide (1,2,3-13 C lbelled AA) (IS), min. 99% purity (+100 ppm hydroquinone) of concentrtion of 1000 mg L -1 in methnol ws from Cmbridge Isotope Lbortories, Inc. (Andover, MA, USA). All other regents used during the vlidtion of the method for determintion of AA were of chromtogrphic purity from Merck (Drmstdt, Germny), LGC Promochem GmbH (Wesel, Germny) nd Schrlu (Sentment, Spin). Ultrpure wter ws obtined through PURELAB Option-S7 nd PURELAB Ultr Ionic system (Elg Lbwter, High Wycombe, UK). Crrez I nd II solutions were prepred by dissolving 28.8 g of potssium hexcynoferrte nd 57.6 g of zinc sulfte in 100 ml of wter, respectively. Helium s mobile phse (min %) nd rgon (min %) s collision gs were used. Smples concentrtion ws chieved under strem of nitrogen (min %). Reference test mterils ERM -BD272 crispbred (AA content of 980 ± 90 µg kg -1 ) nd ERM -BD274 rusk (AA content of 74 ± 7 µg kg -1 ) were certified t the BAM Federl Institute for Mterils Reserch nd Testing (Berlin, Germny) to verify the ccurcy of the method. Preprtion of stock nd working solutions Stock solution of AA (100 mg L -1 ) nd IS (100 mg L -1 ) were prepred in mber vils by dissolving in ultrpure wter. Working solutions I, II nd III (10, 1 nd 0.1 mg L -1 of AA, respectively) nd working solution I (10 mg L -1 ) of IS were prepred by diluting the stock solution with ultrpure wter. All stock nd working solutions were kept in refrigertor t 4 C. Smple preprtion The procedure of smple preprtion for AA quntifiction in bred, biscuits nd other bkery products ws ccording to previous study with some modifictions 24,25 regrding smple preprtion (smple preprtion for soft bred, smple weight tken for nlysis, quntities nd volumes for regents) nd AA nlysis by GC-MS/MS in selected rection monitoring (SRM) cquisition mode, respectively. Briefly, the steps were s follow: Drying, milling nd weighting Only fresh bred smples were dried in n oven t 90 C, for 120 min, while biscuits nd other similr products were used s they were. The smples were fine milled using Retch GM 200 mill (Germny) nd n ultr centrifugl ZM 200 mill (Germny). In centrifuge vil of 50 ml, 3 g of bred nd other similr products nd 1.5 g from biscuits nd other similr products ctegory were weighed for AA nlysis, respectively. AA extrction in wter Over the smple weighed, working solution I of the IS of 10 mg L -1 (110 µl) nd 30 ml ultrpure wter t 60 C were dded. Extrction in wter t ph 4-5 ws chieved by ddition of µl of glcil cetic cid, followed by deproteiniztion with 400 µl Crrez I nd II solutions nd centrifugtion (6000 g) t 5 C, for 30 min. AA derivtiztion The superntnt obtined ws derivtized with 7.5 g KBr, µl HBr (ph 1-3), 10 ml sturted bromine-wter solution (round 1.6%) on shking wter bth below 4 C, for t lest 2 h. After the end of the derivtiztion rection, bromine in excess ws removed by dding round ml of 1 mol L -1 sodium thiosulfte, until the yellow color disppered.

3 1784 Appliction of n Accurte nd Vlidted Method for Identifiction nd Quntifiction of Acrylmide J. Brz. Chem. Soc. Extrction of dibromo derivtive of AA 2,3-dibromopropnl (2,3-DBPA) It ws chieved with 70 ml mixture of ethyl cette nd hexne (4:1, v/v). 2,3-DBPA The concentrtion ws chieved in first step using vcuum evportion system (Rotovpor R-210, BUCHI Lbortechnik AG, Germny) till 2 ml, followed in second step by dryness under strem of nitrogen. 2,3-DBPA purifiction nd concentrtion The residue dissolved in 50 ml hexne ws purified on glss column filled with ctivted Florisil nd clcinted sodium sulfte, previously conditioned with 20 ml hexne. The 2,3-DBPA derivtive ws eluted with cetone, then concentrted till dryness nd the residue ws redissolved in 400 µl ethyl cette nd 40 µl triethylmine. The finl solution ws filtered through 0.2 μm regenerted cellulose microfilter (17 mm dimeter, Sprtn 13RC, Whtmn GmbH, Dssel, Germny) directly in vil nd nlyzed by GC-MS/MS in SRM mode. Preprtion of the clibrtion solutions A blnk nd 7 clibrtion levels in the rnge mg L -1 were prepred in flsks of 250 ml. In ech flsk, the following were dded: 100 ml ultrpure wter, 110 µl IS of 10 mg L -1 nd working solution II/I of AA (1 mg L -1 per 10 mg L -1 ) ccording to the clibrtion level. These solutions were derivtized in the sme mnner s the derivtiztion steps described for bred, biscuits nd other similr smples, without purifiction nd the finl residue ws redissolved in 1000 µl ethyl cette nd 100 µl triethylmine. GC-MS/MS-SRM nlysis The clibrtion solutions nd the derivtized smple extrcts were nlyzed using gs chromtogrph (TRACE GC ULTRA) coupled with triple qudrupole mss spectrometer (TSQ Quntum XLS) from Thermo Fisher Scientific (USA). The nlysis ws performed in the electron impct positive ioniztion mode (EI + ); cquisition mode: selected rection monitoring-srm nd ion scnning mode: product. The determintion ws crried out with cpillry column bsed on polyethylene-glycol (30 m 0.25 mm internl dimeter; 0.25 µm) (TrceGOLD TG-WxMS, Thermo Fisher Scientific, USA). The mobile phse ws helium with constnt flow rte of 1.6 ml min µl smple ws injected in Right PTV type injector using TriPlus AS utosmpler (Thermo Fisher Scientific, USA), in split mode with split rtio of 1:10 nd injector temperture of 220 C. Under these conditions, the retention time of AA nd of the IS ws of ± 0.5 min. AA nd IS were identified by the ion frgments corresponding to the derivtized ions, 2-BPA (2-bromopropenmide) nd 2-BP( 13 C 3 )A. The frgmenttion of the precursor ions with m/z 151 nd 154 ws chieved with rgon (1 mtorr), leding to the formtion of product ions (dughter) with m/z 70 (2-BPA) nd 73 (2-BP( 13 C 3 )A), being used for quntifiction. The clcultion of the AA concentrtion in the test smples ws bsed on the rtio of the pek re corresponding to the product ions, with m/z 70 nd 73 for 2-BPA nd 2-BP( 12 C 3 )A, respectively. For bred smples nd other similr products, AA concentrtion ws clculted bsed on eqution 1, while for biscuits nd other similr products using eqution 2. C clc. = [440 C (100 U 1 )]/[w (100 U 2 )] µg kg -1 (1) C clc. = C 440/w µg kg -1 (2) where C is the 2-BPA concentrtion mesured by the instrument in the food smple (mg L -1 ), C clc. is the 2-BPA concentrtion clculted for the food smple (µg kg -1 ), U 1 is the moisture content of the bred smple before drying (%), U 2 is the moisture content of the bred smple fter drying (%), w is the weight of the smple (g), 440 is the finl volume of the smple extrct (µl). Vlidtion procedure The method ws vlidted ccording to the guides nd recommendtions for methods vlidtion The following performnce prmeters were ssessed: Clibrtion curve nd linerity Clibrtion curve nd linerity were verified by the method of lest squres in the rnge mg L -1, choosing the method of the internl stndrd. The clibrtion curve ws obtined bsed on derivtized stndrd solutions nd plotting the rtio of the pek re of 2-BPA nd 2-BP( 13 C)A ginst the concentrtion of 2-BPA. The clibrtion model ws considered correct if reltive stndrd devition (RSD) in repetbility conditions ws within the limits of ± 15% for ll the levels investigted. The correltion ws ssessed to be liner for vlue greter thn 0.99 for the correltion coefficient (R). Linerity rnge In order to estblish higher vlues for the linerity rnge, the smples were spiked with working solution I of

4 Vol. 27, No. 10, 2016 Negoiță nd Culețu 1785 ntive AA (10 mg L -1 ) t different levels of concentrtion: 40; 75; 80; 150; 225 nd 300 µg kg -1 (bred nd other similr products) nd 250; 500; 750; 1000 nd 2000 µg kg -1 (biscuits nd other similr products). The rtio of the pek re of 2-BPA nd 2-BP( 13 C)A ginst the concentrtion of 2-BPA from the food smples studied ws plotted. The correltion ws ssessed to be liner for vlue greter thn 0.99 for the correltion coefficient (R). Sensitivity Sensitivity ws chrcterized by limit of detection (LOD) nd limit of quntifiction (LOQ). Assessment of LOD nd LOQ for AA nlysis in bred nd similr products by GC-MS/MS were performed pplying IUPAC pproch, while ICH pproch ws used for biscuits nd other similr products. The criteri imposed for LOQ were: precision (expressed s reltive stndrd devition, RSD 20%) nd ccurcy (expressed s recovery in the rnge %). Selectivity Selectivity ws verified by observing the possible interference given by AA in bred, biscuits nd other similr products. 32 Specificity ws chieved by SRM detection. Precision Precision ws determined by repetbility (injection nd nlysis repetbility), inter-lbortory reproducibility nd intermedite precision. Injection repetbility ws chieved by crrying out between 9 nd 10 consecutive injections in short period of time (the sme dy). Anlysis repetbility (intr-dy) ws determined by 5-6 repeted nlyses of the sme smple, in the sme dy by the sme nlyst nd under the sme experimentl conditions. Inter-lbortory reproducibility (inter-dy) ws chieved through Food Anlysis Performnce Assessment Scheme (FAPAS) 33 of the Centrl Science Lbortory (FERA). Intermedite precision (inter-dy) ws crried out by repeted nlyses of the sme smple in 3 different dys by the sme nlyst nd under the sme experimentl conditions. Repetbility nd intermedite precision ws expressed s RSD (%), while interlbortory reproducibility s z-score. Accurcy The ccurcy of the method ws evluted with recovery nd bis mesurements for 2 reference test mterils. The recovery percentge (R%) of the method ws estblished from minimum 6 repeted nlysis of the sme smple fortified with working solution II of AA t different concentrtions. Robustness To ssess the robustness of the method developed in the lbortory, the following prmeters were vried: volume of hexne used in the purifiction of the extrcts, derivtiztion time nd mobile phse flow. It ws considered tht the method is robust for the prmeters investigted, whether obtined under repetbility conditions RSD or reproducibility ws below 2%. Mesurement uncertinty The uncertinty sources were identified nd nlyzed nd the uncertinty budget ws estimted, ccording to the reference documents. 34,35 Expnded uncertinty (U ) ws clculted by multiplying the combined stndrd uncertinty (U c ) with coverge fctor (k = 2) for confidence level of 95%. Method ppliction The vlidted nd developed method ws pplied by nlyzing different products presented in Tbles 1-8. The AA level ws lso studied in different btches of the sme brnd (3 btches 2 types of breds; 2 btches 5 types of biscuits). Sttisticl nlysis AA content ws expressed s men ± stndrd devition. The differences mong smple groups were nlyzed by one-wy ANOVA (nlysis of vrince) followed by Tukey s test, p < 0.05 ws considered sttisticlly significnt. Regrding vlidtion prmeters, verge for concentrtion vlues, C clc (µg kg -1 ), stndrd devition in repetbility SD(r) nd reproducibility conditions SD(R) in µg kg -1 s well s reltive stndrd devition in repetbility RSD(r) nd reproducibility conditions RSD(R) in % were clculted using Microsoft Excel. The repetbility nd reproducibility limits in µg kg -1 were lso considered, where the rtio between repetbility nd reproducibility is 2.8 SD(r)/SD(R) for confidence level of 95%. Results nd Discussion An overview of the test smples together with their coding nd AA content is given in Tbles 1-8. Clibrtion curve nd linerity Linerity ws investigted with the id of regression line with 7 clibrtion levels by the method of lest squres.

5 1786 Appliction of n Accurte nd Vlidted Method for Identifiction nd Quntifiction of Acrylmide J. Brz. Chem. Soc. Tble 1. Bred nd similr products produced in the pilot plnt Min ingredients AA / (μg kg -1 ) Pn bred white whet flour type 550, type ± 0.5 White bred white whet flour type ± 0.4 b Bred stick white whet flour, drk whet flour 26.2 ± 1.1 c Bred stick with olive white whet flour 47.9 ± 1.5 d Bred stick with nut white whet flour 41.6 ± 1.6 e Bred stick with onion white whet flour 87.3 ± 1.8 f AA: crylmide; dt shown s men ± SD (n = 3) for the mount of AA; mens followed by different letters re significntly different (p < 0.05). The clibrtion curve for the determintion of AA in bred, biscuits nd other similr products by GC-MS/MS nlysis (y = x ) ws liner over the rnge of mg L -1. R ws between , which proves high linerity degree. The results showed tht RSD(r) for ll the levels of concentrtion studied rnged between 0.1 nd 7.8%, being within the limits of ± 15%. The linerity rnge ws verified ccording to ISO 36 for bred, biscuits nd other similr products, respectively. The linerity rnge of the method for AA nlysis ws in the rnge of: μg kg -1 (R > 0.998) for bred nd similr products (n = 23) nd μg kg -1 (R > 0.999) for biscuits nd similr products (n = 42), respectively, n denotes the number of the nlyzed smples. The results obtined re in line with recent study, 37 which reported n LC-MS/MS method for AA determintion in different types of breds, with liner rnge of up to 750 μg kg -1 food nd determintion coefficient of Sensitivity For bred nd similr products, LOD nd LOQ obtined were 2.41 nd 7.23 µg kg -1, respectively. For biscuits nd similr products, LOD nd LOQ were: 4.63 nd µg kg -1, respectively. LOQ vlues fulfilled the two criteri (RSD < 10% nd recovery between nd %). LOQ vlue for the present method ws below 30 μg kg -1 which fulfils the criteri defined in Europen Commission recommendtion on the monitoring of AA levels in food. 23 Also, the results obtined re in ccordnce with previous GC-MS study, which reported LOD nd LOQ vlues of 2 nd 5 μg kg -1, respectively for bred smples. 14 The working rnge reched from µg kg -1 AA in food smple. Selectivity Selectivity ws demonstrted by chromtogrphic seprtion of AA in the presence of other components from the food mtrix (bred, biscuits nd similr products). For specificity, SRM detection nd the use of IS method led to specific nlysis. The retention time of AA within lbortory condition for the smples ws lmost identicl with the retention time of the IS, which fits in the Tble 2. Bred nd similr products (origin: retil store, produced in Romni) Smple, producer code Min ingredients AA / (μg kg -1 ) White bred 60/R white whet flour 7.6 ± 0.3 Round sliced bred 31/M whole whet flour, drk whet flour 61.3 ± 0.7 b Whole tosted bred 32/M whole whet flour, drk whet flour, mlt flour ± 0.3 c 33/M different btches 49.7 ± 0.3 d 34/M 57.2 ± 0.4 e White bred with sge seeds 35/M white whet flour 28.0 ± 0.5 f Hypoglucidic sliced bred 36/M white whet flour, brn, drk whet flour ± 0.3 g Whole whet tosted bred 37/T whole whet flour, fermented whet flour, soy flour ± 1.3 h Whole whet bred 38/T whole whet flour, fermented whet flour, soy flour 56.9 ± 0.3 i 39/T different btches 62.2 ± 0.1 b 47/T 79.8 ± 0.2 j Germn bred with seeds 40/T drk whet flour, rye flour, whet brn, whet mlt flour 52.2 ± 0.4 k Trnsylvnin drk bred 41/U drk whet flour 21.3 ± 0.1 l Bred with drk flour nd brn 42/V drk nd white whet flour, whet brn, mlt flour 37.4 ± 0.1 m Whole tosted bred 43/S drk nd white whet flour, rye flour, soy flour, mlt flour, rice flour 46.0 ± 0.3 n AA: crylmide; dt shown s men ± SD (n = 3) for the mount of AA; mens followed by different letters re significntly different (p < 0.05).

6 Vol. 27, No. 10, 2016 Negoiță nd Culețu 1787 tolernce of ± 0.5% imposed by Commission Decision (2002/657/CE). 30 For AA quntifiction, ion m/z 70 (2 BPA) nd ion m/z 73 for IS (2-BP( 12 C 3 )A) were used. The vlues obtined for injection nd nlysis repetbility, intermedite precision re presented in Tble 9. RSD(r) ws between 0.71 nd 3.30% for injection repetbility nd between 0.44 nd 4.90% for repetbility nlysis, respectively. The RSD(R) intermedite precision ws between 0.35 nd 5.83%. The pplied method showed good precision, RSD < 10% for AA nlysis in bred, biscuits nd similr products nd the results were within the limits set by the following regultions: VICH GL49, 28 CAC/GL 16, 29 EC. 30 The AA result for crispbred smple from the FAPAS proficiency test (lbortory No. 24) showed vlue of µg kg -1 (z-score of 0.2), which fulfilled the cceptbility criteri from the orgniztion. 33 Method ccurcy The bis obtined for the two reference mterils were: 1.68% (n = 39) for ERM -BD272 crispbred nd 2.52% (n = 30) for ERM -BD274 rusk, respectively. The described method demonstrted stisfctory ccurcy, between nd % for bred nd similr products nd between nd % for biscuits nd similr products. Tble 3. Biscuits smples produced in the pilot plnt Min ingredients AA / (μg kg -1 ) Biscuit from rice flour RF b, ft, sugr, eggs 31.3 ± 0.6 Biscuit from white whet flour WF c, ft, sugr, eggs ± 2.9 b Biscuit from white whet nd ot flour WF c 75%, OF 25%, ft, sugr, eggs ± 2.7 b Biscuit from white whet nd ot flour WF c 50%, OF 50%, ft, sugr, eggs ± 4.2 c Biscuit from whole ot flour OF d 100%, ft, sugr, eggs ± 2.3 d Biscuit from ot nd brn flour OF d 70%, ot brn 30%, ft, sugr, eggs < LOQ e e AA: crylmide; dt shown s men ± SD (n = 3) for the mount of AA; mens followed by different letters re significntly different (p < 0.05); b RF: rice flour; c WF: whet flour; d OF: ot flour; e LOQ: limit of quntifiction. Tble 4. Biscuits smples (origin: retil store, produced in different countries) Smple, producer code, country Min ingredients AA / (μg kg -1 ) Biscuit from ot 50/E/Turkey OF b, WF c, WWF d, ft, sugr, eggs ± 0.7 Biscuit with whet brn 51/B/Romni WF c, whet brn, sugr, ft, brley mlt extrct, whet fiber ± 2.7 b Digestive biscuit with cinnmon 52/M/Romni WWF b, fts, sugr, whet brn, mlt extrct 52b/M/Romni different btches Digestive biscuit 53/E/Turkey WWF d, fts, sugr, egg, brley mlt extrct, whet brn, milk powder ± 6.7 c ± 17.9 d ± 3.2 e Biscuit with slt 56/H/Romni WF c flour, oil, brley mlt extrct, egg, slt ± 0.7 f Biscuit from ot flour nd fruit jm (fsting) Biscuit from whole ot nd fruits, without gluten (fsting) 57/C/Moldvi WF c, OF b, sugr, ft, fruit jm ± 2.8 g 69/A/Scotlnd OF b, plm oil, risins, sugr, Tpioc strch ± 13.1 h Biscuit from BIO whole ot 70/P/Germny OF b, WF c, sugr, ft, egg powder, mize strch 46.8 ± 1.2 i Biscuit with ot 71/Z/Romni WF c, OF b, sugr, plm oil, whet strch ± 17.7 j Digestive biscuit with cerels, risins, ornge nd lemon Biscuit with whole cerels nd crnberry red, vitmins nd minerls Biscuit with whole cerels, nuts nd honey, vitmins nd minerls 72/M/Romni 72b/M/Romni 73/H/Romni 73b/H/Romni 74/H/Romni 74b/H/Romni cerels, risins, ornge nd lemon different btches whole cerels nd crnberry red, vitmins, minerls different btches whole cerels, nuts nd honey, vitmins, minerls different btches ± 2.7 e k ± 1.4 e l ± ± 6.8 m ± 2.5 n ± 2.7 o AA: crylmide; dt shown s men ± SD (n = 3) for the mount of AA; mens followed by different letters re significntly different (p < 0.05); b OF: ot flour; c WF: whet flour; d WWF: whole whet flour; e the AA levels were higher thn the mximum vlue of the working rnge, but in order to quntify it, the smples weight tken for nlysis ws lower (0.75 g insted of 1.5 g).

7 1788 Appliction of n Accurte nd Vlidted Method for Identifiction nd Quntifiction of Acrylmide J. Brz. Chem. Soc. Tble 5. Biscuits for infnts nd young children (origin: retil store, produced in different countries) Smple, producer code, country Min ingredient AA / (μg kg -1 ) Biscuit with vitmins nd minerls 89/I/Romni WF b, fts, sugr, mize strch, egg powder, milk powder, vitmins Biscuit with pple for children 91/J/Switzerlnd WF b, WWF c, pple juice, pple extrct First biscuit of the child 92/J/Switzerlnd WF b, sugr, whet strch, milk powder, vitmin B1 Biscuit enriched with 5 vitmins, clcium nd iron Biscuit enriched with 5 vitmins, clcium nd iron for young children 93/K/Itly 94/K/Itly WF b, sugr, milk ft, whet strch, honey, vitmins, minerls WF b, sugr, oil, milk powder, rice mlt extrct, vitmins, minerls ± ± 1.9 b < LOQ d c ± 2.8 d 37.8 ± 0.3 e AA: crylmide; dt shown s men ± SD (n = 6) for the mount of AA; mens followed by different letters re significntly different (p < 0.05); b WF: whet flour; c WWF: whole whet flour; d LOQ: limit of quntifiction. Tble 6. Sndwich biscuits with crem filling (origin: retil store) Smple, producer code, country Min ingredients AA / (μg kg -1 ) Biscuit with coco crem 80/L/Romni WF b, ft, sugr ± 5.8 Biscuit with honey nd milk crem 81/M/Romni WF b, ft, sugr, honey, powder milk, strch ± 7.2 b Biscuit with sour crem 82/B/Romni WF b, sugr, ft, coco powder, milk powder, crem powder Vnill biscuit with coco crem 76/I/Romni WF b, sugr, plm oil, mize strch, coco 76b/I/Romni different btches ± 3.1 c ± 5.8 d ± 6.0 e AA: crylmide; dt shown s men ± SD (n = 5) for the mount of AA; mens followed by different letters re significntly different (p < 0.05); b WF: whet flour. Tble 7. Crckers smples (origin: retil store) Smple, producer code, country Min ingredients AA / (μg kg -1 ) Cheese crcker 3437/O/Romni WF b, oil, cheese powder, pe flour, corn strch ± 4.3 Crwy crcker 3438/O/Romni WF b, oil, crwy, cheese powder, pe flour ± 21.3 AA: crylmide; dt shown s men ± SD (n = 6) for the mount of AA; vlues with the sme letter denote no significnt difference (p > 0.05) between smples; b WF: whet flour. Tble 8. Gingerbred smples (origin: retil store) Smple, producer code, country Min ingredients AA / (μg kg -1 ) Gingerbred 58/N/Romni WF b, rye flour, sugr, honey ± 2.9 Gingerbred 59/C/Moldvi WF b, sugr, sorbitol ± 16.8 b Gingerbred 90/C/Moldvi WF b, sugr, sorbitol ± 3.9 c AA: crylmide; dt shown s men ± SD (n = 3) for the mount of AA; mens followed by different letters re significntly different (p < 0.05); b WF: whet flour. Robustness The smll vritions in the volume of hexne used in the purifiction of the extrcts, derivtiztion time nd mobile phse flow re not susceptible to vritions in the re corrected. Stndrd devition ws between 1.79 nd 2.07%. Mesurement uncertinty The uncertinty of results for AA determintion in bred, biscuits nd similr products by GC-MS/MS ws ± 21% s estimted by budget uncertinty.

8 Vol. 27, No. 10, 2016 Negoiță nd Culețu 1789 Tble 9. Performnce prmeters of the method Performnce prmeter Injection repetbility Anlysis repetbility (intr-dy) Intermedite precision (inter-dy) AA / (mg L -1 ) Bred nd similr products (n = 9) Smple Biscuit Sndwich biscuit Crcker Gingerbred Precision (n = 10) (n = 9-10) RSD(r) / % NC NC AA / (µg kg -1 ) (n = 5-6) (n = 6) (n = 5-6) NC (n = 6) NC (n = 5-6) RSD(r) / % AA / (µg kg -1 ) (n = 3-4) (n = 3) (n = 2-3) NC (n = 2-3) RSD(R) / % NC AA: crylmide; NC: not clculted; RSD(r): reltive stndrd devition in repetbility; RSD(R): reltive stndrd devition reproducibility conditions. Method ppliction As it cn be noticed from Tbles 1-8, the results for the AA content in the different food smples studied, vried gretly not only from one type of product to nother one, but lso in the sme type of product coming from different brnds or the sme product with different btches. From the 49 types of bred, biscuits, crckers nd gingerbred nlyzed coming from 20 different suppliers, 16 of them (4 bred smples, 11 biscuits nd sndwich biscuits nd 1 biscuit for infnts nd young children) hd n AA level higher thn the indictive vlues provided in the Commission Recommendtion (EC 2013). 6 The indictive vlues were for soft bred obtined from whet flour were set t vlue of 80 µg kg -1, biscuits for infnts nd young children t 200 µg kg -1, biscuits nd crckers t 500 µg kg -1 nd gingerbred t 1000 µg kg -1, respectively. Bred products mnufctured in the pilot plnt under controlled conditions (Tble 1) do not exceed the indictive vlues, except the bred smple prepred with onion. This could be explined by the fct tht onion contributes to AA formtion. It ws shown 38 tht onion mong other ingredients used for sesoning incresed the AA content. Anlysis of the AA content in the sme type of food from the mrket (the sme receipt nd mnufcturing process ccording to the producer declrtion) with different btches showed differences in the AA content. For exmple, for 3 btches of bred (code 32, 33 nd 34) coming from the sme producer, M, the AA level showed significnt differences, while for nother 3 btches of bred (code 38, 39 nd 47, from producer T), the AA levels showed similr vlues (Tble 2). This could be explined by the technologicl conditions (temperture, time) which could vry or by vrition in the qulity of the rw mterils. Adult chronic dietry exposure estimted by EFSA 39 ws between 0.4 nd 0.9 µg kg -1 bw per dy. Considering thn in Romni, the bred intke for n dult weighing 80 kg is 300 g per dy nd bsed on the verge level of AA obtined in this study for the breds from the mrket (62.99 µg kg -1, Tble 2), it cn be concluded tht bred contributes to chronic dietry exposure of 0.9 µg kg -1 bw per dy with bout 26%. The sme procedure ws pplied for biscuits nd similr products. Thus, for dily intke of 5.8 g nd n verge for AA obtined for these products ( µg kg 1, Tbles 3-8), the biscuits contributes to chronic dietry exposure of 0.9 µg kg -1 bw per dy with bout 3.7%. The AA content obtined for bkery products re in ccordnce with the dt presented in literture (Tble 10). Conclusions The proposed GC-MS/MS method corresponds to the vlidtion prmeters imposed nd it ws pplied with good results to determine AA content in bred, biscuits nd other similr products. The LOD nd LOQ vlues were 2.41 nd 7.23 µg kg -1 for bred nd similr products, while for biscuits nd similr products were 4.63 nd µg kg 1, respectively. This study ssesses the presence of AA in foods found on the Romnin mrket. Tking into ccount the consumption of bkery products mong dults nd children, estblishment of mesures is necessry in order to protect consumer helth. In this respect, n importnt mesure is to control in order to void the sle of foods with high levels of AA. Considering ll of the bove dt for method performnce nd proficiency test, the GC-MS/MS method nd smple pretretment employed in the present work cn be regrded s being sensible, precise nd robust.

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