As a basic component of the human diet, milk is of great

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1 KIRA ET AL.:JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 1, RESIDUES AND TRACE ELEMENTS Comprison of Prtil Digestion Procedures for Determintion of C, Cr, Cu, Fe, K, Mg, Mn, N, P, nd Zn in Milk by Inductively Coupled Plsm-Opticl Emission Spectrometry CARMEN S. KIRA nd FRANCA D. MAIO Instituto Adolfo Lutz, Divis o de Bromtologi e Químic, Av. Dr. Arnldo 355, CEP , S o Pulo, SP, Brzil VERA A. MAIHARA Lbortório de Análise por Ativç o IPEN/CNEN-SP, CEP , São Pulo, SP, Brzil A fst procedure ws developed for determintion ofc,cr,cu,fe,k,mg,mn,n,p,ndzninmilk smples. This procedure consisted of prtil digestion with hydrochloric cid on hot plte. The results obtined were compred with 3 other digestion procedures (dry shing nd 2 microwve digestions). All the procedures showed similr precision levels, with coefficients of vrition <10% for most nlyzed elements. Accurcy ws evluted by using certified reference mterils, nd the vlues obtined were within the confidence intervls for these products. The results obtined were not considered sttisticlly different. The prtil digestion on hot plte with HCl cn be very prcticl for lbortories with reltively lrge numbers of smple nlyses. As bsic component of the humn diet, milk is of gret importnce. It is known tht milk is good source of clcium, proteins, nd vitmins A nd D, which re necessry for bone nd tooth development (1, 2). Milk is the min nourishment for toddlers, nd n pproprite intke is recommended for dults to retin bone mss so tht frctures nd osteoporosis cn be prevented. Mny spectroscopic techniques hve been used for the determintion of inorgnic elements in rnge of different mtrixes. However, these techniques generlly involve smple pretretment, which is the most criticl prt of the nlysis becuse it is the slowest step in the nlyticl process nd errors in the results obtined re often ssocited with this step. Trditionl methods of smple preprtion for milk smples (powder nd liquid) re well documented. These methods include wet digestion nd dry shing (3 9). Wet digestion procedures performed on hot plte or in microwve oven require the use of concentrted cids, necessry for digestion monitoring (10 12). Dry shing is time-consuming nd minly influenced by the finl temperture of the muffle furnce s well s the progrmmed temperture grdient, which Received April 21, Accepted by JS August 19, Corresponding uthor s e-mil: crmkir@il.sp.gov.br. must be low enough in order to void losses of elements. The cids used for dissolving the shes cn lso influence smple tretment (13 17). Conventionl digestion procedures with totl orgnic mtter destruction by dry or wet digestion mke the routine processing of lrge numbers of smples difficult. Therefore, with the incresing demnd of nlyses required by public helth progrms nd privte compnies, it is importnt to use digestion procedures tht require minimum smple tretment, resulting in good precision nd ccurcy. There is growing interest in smple pretretment procedures tht could both void nlyte loss nd smple contmintion leding to nlyticl costs, s well s increse productivity. In the present study, the inductively coupled plsm-opticl emission spectrometry (ICP-OES) method ws used to determinec,cr,cu,fe,k,mg,mn,n,p,ndzninmilkpowder smples. Four digestion procedures were evluted to determine inorgnic elements in milk smples, including one dry shing procedure with totl destruction of orgnic mtter, nd 3 wet digestions with prtil smple destruction one on hot plte nd the other 2 in microwve oven. Experimentl Apprtus () Muffle furnce. For dry shing of smples. (b) Focused open-vessel microwve oven. For microwve digestions (Str 2; CEM, Mtthews, NC). (c) Spectrometer. For ICP-OES spectrometric mesurements (Optim 3000 DV; Perkin Elmer Corp., Norwlk, CT). Detils on ICP-OES instrumentl operting conditions nd mesuring prmeters used re given in Tble 1. (d) Glsswre. All glsswre ws wshed with detergent nd wter. After being rinsed with wter for severl times, it ws soked in 20% HNO 3 (v/v) for 24 h. This solution ws discrded nd the glsswre ws soked gin in 20% HNO 3 (v/v) for 24 h. This procedure ws repeted. The glsswre ws then rinsed severl times with deionized wter, nd dried.

2 152 KIRA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 1, 2004 Tble 1. ICP-OES operting conditions Smple Preprtion Prmeter Regents () Deionized wter. Obtined from Milli-Q system (Millipore Corp., Bedford, MA), specific resistnce, 18 M /cm. (b) Hydrochloric cid, 37% (w/w). Anlyticl grde (Merck, Drmstdt, Germny). (c) Nitric cid, 65% (w/w). Anlyticl grde (Merck). (d) Hydrogen peroxide, 30%. Anlyticl grde (Merck). (e) C, Fe, K, Mg, N, P, nd Zn stock solutions mg/l (High Purity Stndrds). (f) Cr, Cu, Mn stndrd Titrisol solutions mg/l (Merck). The working multi-elementl stndrd solutions were mde with 10% (v/v) HCl. All working stndrd solutions were stored in Nlgene polypropylene bottles. The clibrtion curve ws mde from 5 points nd the blnk. The multi-elementl stndrd solutions rnged from 2.5 to mg/l for C, K, N, nd P; mg/l for Fe nd Zn; mg/l for Cr; mg/l for Cu; mg/l for Mg; nd mg/l for Mn. Smples Nonft milk powder contining 1% ft nd whole milk powder contining 26% ft were used in this study. The smples were purchsed in locl mrkets. Reference Mterils Opertion conditions RF power, W 1350 Smple uptke rte, ml/min 1.5 Nebuliztion gs flow rte, L/min 0.85 Plsm flow rte, L/min 15 Smple flow rte, L/min 1.5 Observtion height 15 mm bove lod coil Nebulizer Cross flow Spry chmber Scott Elements, wvelengths (nm) C ( ), Cr ( ), Cu ( ), Fe ( ), K ( ), Mg ( ), Mn ( ), N ( ), P ( ), Zn ( ) Two reference mterils from the Ntionl Institute of Stndrds nd Technology (NIST; Githersburg, MD), Stndrd Reference Mteril (SRM) 1549 Nonft Milk Powder nd RM 8435 Whole Milk Powder were used to vlidte the digestion procedure methods. These mterils were treted ccording to the different digestions (A D). Procedure A: Dry shing. About 2 g milk powder ws directly weighed in porcelin crucible. The smple ws preheted on Bunsen burner until smoke ws no longer observed. The crucible ws plced in cold muffle furnce. The temperture of the muffle ws rised from 50 C/30 min to 450 C, nd kept t this temperture for 4 h. Minerliztion ws considered complete when sh ws white or slightly colored, with no chrred prticles. If crbon ws still present, the smple ws treted with 1 ml HNO 3. The sh ws dissolved in 1 ml HCl nd trnsferred with deionized wter into 10.0 ml volumetric flsk. Procedure B: Wet digestion on hot plte. About 1 g smple ws trnsferred into 125 ml conicl flsk. A 10 ml HCl H 2 O (1 + 1) mixture ws dded to it, nd the flsk ws covered with wtch glss. The smple ws heted on hot plte t c 100 C for bout 2 h, bringing it to gentle boil. The digested solution ws filtered into 25.0 ml volumetric flsk through Whtmn No. 40, 125 mm filter pper, nd diluted with deionized wter. Procedure C: Wet digestion using focused microwve oven with HCl for whole milk smples. Step 1: The smple (1 g) ws weighed in qurtz digestion vessel. In this step, 2.5 ml concentrted HCl ws dded to the smple, heted to 80 C within 2 min, nd kept t this temperture for 5 min. Step 2: The temperture ws rised to 90 C nd kept for 10 min. The rmp used for this step ws 2 min. Step 3: The temperture wsrisedto95 C nd kept for 10 min (rmp of 1 min). Step 4: A 10 ml volume of 30% H 2 O 2 ws dded to the smple, nd temperture ws rised to 105 C nd kept for 10 min. Rmp of 1 min ws used for this step. Step 5: The temperture wsrisedto109 C with rmp of 1 min, nd kept for 5 min; 10 ml 30%. H 2 O 2 ws dded. Step 6: A 2.5 ml volume of HCl ws dded nd the temperture ws rised to 110 C within 1 min (rmp) nd kept for 3 min. The digested solution ws filtered into 25.0 ml volumetric flsk through Whtmn No. 40, 125 mm filter pper, nd diluted with deionized wter. Procedure D: Microwve digestion with HCl for nonft milk smples. Step 1: About 1 g milk powder ws weighed in qurtz digestion vessel. A 2.5 ml volume of HCl ws dded to the smple. The temperture ws rised to 70 C within2minndkeptfor5min.step2:usingrmpof 2 min, the temperture ws rised to 85 C nd kept for 5 min. A 5 ml volume of H 2 O ws dded. Step 3: The temperture wsrisedto90 C within 1 min nd kept for 10 min. In this step, 2.5 ml HCl ws dded. Step 4: The rmp used in this step ws 1 min; the temperture ws rised to 95 C nd kept for5min,nd10mlh 2 O ws dded. Step 5: The temperture wsrisedto107 C within 1 min, kept for 10 min, nd 5 ml H 2 O ws dded. The digested solution ws filtered through Whtmn No. 40, 125 mm filter pper into 25.0 ml volumetric flsk, nd diluted with deionized wter.

3 KIRA ET AL.:JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 1, Tble 2. Results of inorgnic elements in SRM 8435 Whole Milk Powder reference mteril SRM 8435 Whole Milk Powder ( ) Element Procedure A, dry shing Procedure B, wet digestion (hot plte) Procedure C, wet digestion (microwve oven) Certified vlue C, % ± ± ± ± Cu, mg/kg 0.44 ± ± ± ± 0.08 Fe, mg/kg 1.87 ± ± ± ± 1.10 K, % 1.38 ± ± ± ± 0.05 Mg, mg/kg 813 ± ± ± ± 76 Mn, mg/kg 0.17 ± ± ± ± 0.05 N, % ± ± ± ± P, % ± ± ± ± Zn, mg/kg 27.7 ± ± ± ± 3.1 Men vlue stndrd devition of 6 determintions. Results nd Discussion Both wet digestions either on hot plte or in microwve oven destroyed orgnic mtter prtilly, wheres dry shing eliminted orgnic mtter completely. Colorless smple solutions were obtined by dry shing in contrst to brown smple solutions, obtined by both wet digestions on hot plte nd in microwve oven. Smples were cutiously heted on hot plte to void ny vigorous rection. The HNO 3 /H 2 O 2 mixture successfully replced HClO 4 to digest the smples. The HNO 3 /H 2 O 2 mixture is lso efficient to oxidize orgnic mtter (10, 14). The efficiency of the microwve digestion ws verified nd compred with tht of digestion on hot plte. For the microwve oven digestion, 2 progrms with different conditions (time intervls nd initil temperture) were tested (progrms C nd D). The first ws for whole milk powder digestion, nd the other for nonft milk digestion. It ws not possible to use only one progrm in the microwve digestion for the 2 types of milk smples since intensive reflux of the smple occurred during the nonft milk digestions. Therefore, for the nonft milk smple, heting progrm ws necessry with n initil temperture lower thn tht used for the whole milk smple. Thus, miniml initil temperture ws used to void scum development nd reflux of the smple. A longer rmp of temperture ws lso chosen to void reflux of the smple. A combintion of cid(s) nd oxidnt(s) is usully used for wet digestion procedures; however, it is highly explosive. Therefore, to reduce the risk of n explosion, nonoxidnt regent, like hydrochloric cid, ws chosen to be used insted. The blnk vlue did not increse even when the digested solution obtined fter both digestions on hot plte nd in microwve oven were filtered through filter pper. Tble 3. Results of inorgnic elements in SRM 1549 Nonft Milk Powder reference mteril SRM 1549 Nonft Milk Powder ( ) Element Procedure A, drying shing Procedure B, wet digestion (hot plte) Procedure C, wet digestion (microwve oven) Certified vlue C, % 1.31 ± ± ± ± 0.05 Cu, mg/kg 0.66 ± ± ± ± 0.10 Fe, mg/kg 1.84 ± ± ± ± 0.10 K, % 1.69 ± ± ± ± 0.03 Mg, % ± ± ± ± Mn, mg/kg 0.21 ± ± ± ± 0.06 N, % ± ± ± ± P, % 1.06 ± ± ± ± 0.02 Zn, mg/kg 40.5 ± ± ± ± 2.2 Men vlue ± stndrd devition of 6 determintions.

4 154 KIRA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 1, 2004 Figure 1. Z-score for NIST 8435 Whole Milk Powder digested by different procedures. Figure 2. Z-score for NIST 1549 Nonft Milk Powder digested by different procedures. In order to ssess the ccurcy of the smple tretment procedures, certified mterils NIST SRM 1549 Nonft Milk Powder nd NIST RM 8435 Whole Milk Powder were used to nlyze C, Cu, Fe, K, Mg, Mn, N, P, nd Zn. Cr ws not nlyzed becuse its concentrtion vlue ws not certified. The element concentrtions found in the certified reference mterils by the different procedures in this study re presented in Tbles 2 nd 3. Figures 1 nd 2 show the results obtined in Whole Milk Powder (NIST RM 8435) nd Nonft Milk Powder (NIST SRM 1549) reference mterils by the different digestion procedures ccording to z-criteri, respectively. The stndrdized difference or z-vlue ws clculted ccording to Bode (18). If z < 3, it mens tht the individul result of the reference mteril should be in the 99% confidence intervl of the trget vlue. The results obtined for RM 8435 Whole Milk Powder were within the cceptble limits, wheres for SRM 1549 Nonft Milk Powder digested by focused microwve oven, only the concentrtion of Mg ws over the cceptble limits. A possible explntion for this behvior is tht Mg is strongly linked to csein micelles nd is not esily relesed, cusing lower recovery (19). Mgnesium concentrtion is higher in nonft milk thn in whole milk; tht is why the link of Mg to csein micelles is more pronounced in nonft milk smples thn in whole milk smples. The nlyticl results obtined for C, Cr, Cu, Fe, K, Mg, Mn, N, P, nd Zn determintions in whole milk nd nonft milk smples fter the different procedures of digestion re shown in Tbles 4 nd 5, respectively. The reltive stndrd devitions (RSD) obtined were <10% for most elements nlyzed in the different digestion procedures for whole milk nd nonft milk smples. It ws observed tht the RSDs were comprble between ll digestion procedures. In the wet digestion performed on hot plte, the RSDs were equl to or Tble 4. Results for minerls nd trce elements determined in whole milk powder smples purchsed loclly, digested by different procedures Digestion procedures ( ) Element Procedure A, dry shing Procedure B, wet digestion (hot plte) Procedure C, wet digestion (microwve oven) b C, % ± ± ± Cr, mg/kg ± ± ± Cu, mg/kg ± ± ± Fe, mg/kg 2.08 ± ± ± 0.18 K, % 1.18 ± ± ± 0.10 Mg, mg/kg 772 ± ± ± 13 Mn, mg/kg 0.23 ± ± ± 0.02 N, % ± ± ± P, % ± ± ± Zn, mg/kg 24.5 ± ± ± 4.1 b Men vlue ± stndrd devition of 6 determintions. Men vlue ± stndrd devition of 3 determintions.

5 KIRA ET AL.:JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 1, Tble 5. Results for elements determined in nonft milk smples purchsed loclly, digested by different procedures Digestion procedures ( ) Element Procedure A, dry shing Procedure B, wet digestion (hot plte) Procedure D, wet digestion (microwve oven) b C, % 1.18 ± ± ± 0.07 Cr, mg/kg ± ± ± Cu, mg/kg ± ± ± Fe, mg/kg 2.24 ± ± ± 0.05 K, % 1.55 ± ± ± 0.05 Mg, % ± ± ± Mn, mg/kg 0.30 ± ± ± 0.04 N, % ± ± ± P, % ± ± ± Zn, mg/kg 31.1 ± ± ± 1.1 b Men vlue ± stndrd devition of 6 determintions. Men vlue ± stndrd devition of 3 determintions. less thn the RSDs obtined in the conventionl dry shing digestion. In the wet digestion performed in microwve oven, the RSDs obtined for the elements nlyzed were greter thn those obtined in the other procedures; it probbly occurs due to smple refluxing during the digestion. To evlute the performnce of different smple pretretment procedures in determining inorgnic elements in milk smples, the conventionl procedure (dry shing) tht showed totl destruction of orgnic mtter ws compred with wet digestions on hot plte nd in microwve tht resulted in prtil destruction of orgnic mtter. The one-wy nlysis of vrince (ANOVA) ws used to compre the men vlues obtined for the different smple pretretment procedures. When the men vlues were sttisticlly different, the Tukey-Krmer test ws used. Different from ANOVA, the Tukey-Krmer procedure identifies which men vlues re significntly different from ech other. A sttisticl comprison of the results obtined for the inorgnic elements determined in milk smples is presented in Tble 6. Tble 6. Comprison mong concentrtions of elements determined in milk smples by different digestion procedures pplying the Tukey-Krmer test Digestion procedures Whole milk smples Nonft milk smples Method A B Method A C Method A B Method A D Element q vlue p vlue Result q vlue p vlue Result q vlue p vlue Result q vlue p vlue Result C > > > > Cr > > > > Cu > < > > Fe > > > > K > > > > Mg > > > > Mn > > > > N > > > > P > > > > Zn > > > > A: Dry shing; B: wet digestion on hot plte; C: wet digestion in microwve oven for whole milk smples; D: wet digestion in microwve oven for nonft milk smples; + = men concentrtions re not considered significntly different t the significnce level of 95%; = men concentrtions re considered significntly different t the significnce level of 95%.

6 156 KIRA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 1, 2004 Tble 7. Limits of detection (LOD) nd quntittion (LOQ) in mg/l Element (nm) LOD (mg/l) LOQ (mg/l) C Cr Cu Fe K Mg Mn N P Zn Tble 6 shows good greement mong the different digestion procedures pplied for the milk powder smples. The results for whole milk smples digested either by dry shing or wet digestions on hot plte or in microwve oven were not significntly different (p > 0.05), except for copper, becuse the stndrd devition obtined for the smple digested in microwve oven ws smller thn the others. The results obtined by the different smple tretment procedures for nonft milk smples were not significntly different, indicting tht the concentrtions for ll elements nlyzed yielded bsiclly the sme results. The limits of detection nd quntittion (LOD nd LOQ) of the elements were determined ccording to AOAC procedure (20) nd re presented in Tble 7. Conclusions Relible results cn be obtined by wet digestions with HCl on hot plte s well s in microwve oven. Both digestion procedures hve qulities tht mke them suitble for ICP-OES determintion of wide rnge of inorgnic elements in milk smples, but wet digestion on hot plte hs the dvntge of low-cost equipment nd little mintennce. In wet digestion on hot plte, multiple smples cn be prepred concurrently, wheres in wet digestion in microwve oven, s performed in our lbortory, only 2 smples cn be prepred simultneously. In this study, up to 16 smples could be digested t the sme time when the hot plte ws used nd, depending on the size of the hot plte, even more smples my be digested. In smple digestions performed in microwve oven, throughput is limited by the number of digestion vessels tht cn be processed simultneously, the digestion time due to cooling time, nd the smll mounts of smple tht cn be used in this system. The dvntge of wet digestion on hot plte is relted to the use of common lbortory regent, such s hydrochloric cid. Therefore the procedure proposed, with ICP-OES cpbilities, cn be successfully used for inorgnic element determintions in milk smples. References (1) Willims, S.R. (1997) Fundmentos de Nutriç oe Dietoterpi,ArtesMédics, Porto Alegre, RS, Brzil, pp (2) Neville, M.C., Zhng, P., & Allen, J.C. (1995) in Hndbook of Milk Composition, R.G. Jensen (Ed.), Acdemic Press, New York, NY, pp (3) Krushevsk, A., Brnes, R.M., & Amrsiriwrden, C. (1993) Anlyst 118, (4) Reid, H.J., Greenfield, S., & Edmonds, T.E. (1995) Anlyst 120, (5) Amrsiriwrden, D., Kotrebi, M., Krushevsk, A., & Brnes, R.M. (1997) Cn. J. Anl. Sci. Spectrosc. 42, (6) Nóbreg, J.A., Gélins, Y., Krushevsk, A., & Brnes, R.M. (1997) J. Anl. At. Spectrom. 12, (7) Borkowsk-Burneck, J., Szmigiel, E., & Zyrnicki, W. (1996) Chem.Anl.(Wrsw)41, (8) Krdjov, I., Girousi, S., Ilidou, E., & Strtis, I. (2000) Mikrochim. Act 134, (9) Sun, D.H., Wters, J.K., & Mwhinney, T.P. (2000) J. AOAC Int. 83, (10) De L Fuente, M.A., & Juárez, M. (1995) Anlyst 120, (11) Thán, J.E., Sánchez, J.M., Grndillo, V.A., Cubillán, H.S., & Romero, R.A. (1995) J. Agric. Food Chem. 43, (12) Burguer, M., & Burguer, J.L. (1998) Anl. Chim. Act 366, (13) Jorhem, L. (1995) Mikrochim. Act 119, (14) Amro-López, M.A., Moreno-Rojs, R., Sánchez-Segrr, P.J., & Zurer-Cosno, G. (1996) Alimentri 271, (15) Novozmsky, I., Lee, V.H., & Houb, V.J.G. (1995) Mikrochim. Act 119, (16) Hoening, M., & Kersbiec, A-M. (1996) Spectrochim. Act Prt B 51, (17) Subrmnin, K. (1996) Spectrochim. Act Prt B 51, (18) Bode, P. (1997) J. Rdionl. Nucl. Chem. 215, (19) De L Fuente, M.A., Crzo, B., & Juárez, M. (1997)J. Diry Sci. 80, (20) Interntionl Accredittion Criteri for Lbortories Performing Food Chemistry Testing (1999) AOAC INTERNATIONAL, Githersburg, MD, pp 1 33

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