Lawrence H. Pacquette, Andre Szabo, and Joseph J. Thompson Abbott Nutrition, Global Research Services, 3300 Stelzer Rd, Columbus, OH 43219

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1 Pcquette et l.: Journl of AOAC Interntionl Vol. 95, No. 3, INFANT FORMULA AND ADULT NUTRITIONALS Appliction of Inductively Coupled Plsm/Mss Spectrometry for the Mesurement of Chromium, Selenium, nd Molydenum in Infnt Formul nd Adult Nutritionl Products: First Action Lwrence H. Pcquette, Andre Szo, nd Joseph J. Thompson Aott Nutrition, Glol Reserch Services, 3300 Stelzer Rd, Columus, OH Steve Bugh AOAC INTERNATIONAL, 481 N. Frederick Ave, Suite 500, Githersurg, MD An inductively coupled plsm/ms method ws developed for the simultneous determintion of Cr, Se, nd Mo in infnt formul nd other nutritionl products. All smples were digested using closed vessel microwve oven system, together with Ni nd Te internl stndrds. The prcticl quntittion limits for Cr, Se, nd Mo were 0.4, 0.2, nd 0.4 ng/ml, respectively; dilution fctors were 250 for powders nd 50 for liquids. The Cr, Se, nd Mo concentrtions in 10 nutritionl products were within specifiction limits; within-dy nd dy-to-dy (6 independent dys) precision vlues were <5% RSD. For two control smples, the oserved precision ws 2% RSD over 10 independent dys. Cr, Se, nd Mo results were within the certified limits in three Ntionl Institute of Stndrds nd Technology stndrd reference mterils. The verge smple spike recoveries for 10 nutritionl products rnged from 93 to 107%. Roustness studies showed miniml effect from concomitnt esily ionized element concentrtions. However, the choice of internl stndrd nd mtrix-mtching cron content were criticl to otining ccurte Se results. All indictions re tht this method would e suitle cndidte s glol reference method for the determintion of these trce elements in infnt formul, dult nutritionls, nd other nutritionl products. On Mrch 28, 2011, the AOAC Bord of Directors pproved n lterntive pth to chieve Officil First Action sttus for methods selected nd reviewed using Sumitted for puliction Jnury 23, The method ws pproved y the Expert Review Pnel on Infnt Formul nd Adult Nutritionls s First Action. See Stndrds News, (2011) Inside Lortory Mngement, Septemer/Octoer issue. The AOAC Stkeholder Pnel on Infnt Formul nd Adult Nutritionls (SPIFAN) invites method users to provide feedck on the First Action methods. Feedck from method users will help verify tht the methods re fit for purpose nd re criticl to gining glol recognition nd cceptnce of the methods. Comments cn e sent directly to the corresponding uthor. Corresponding uthor s e-mil: lwrence.pcquette@ott.com DOI: /jocint.CS2011_19 AOAC s voluntry consensus stndrds process. Following this process, selected methods re reviewed nd pproved y n Expert Review Pnel (ERP) for AOAC Officil First Action sttus. This process follows selected methods for period of pproximtely 2 yers s First Action methods. This llows n opportunity for methods to e used in lortories nd to generte dditionl informtion. The ERPs will monitor the method s performnce; fter out 2 yers, the ERP will recommend the method to the Officil Methods Bord for Finl Action if the method is found to e suitle (1). An ERP reviewed the method, Simultneous Determintion of Chromium, Selenium, nd Molydenum in Nutritionl Products y Inductively Coupled Plsm/Mss Spectrometry: Single Lortory Vlidtion, during the Stndrds Development nd Interntionl Hrmoniztion: AOAC INTERNATIONAL Mid-Yer Meeting on June 29, 2011 (2). After evlution of the vlidtion dt ville, n ERP greed tht the method meets the stndrd method performnce requirements (SMPRs), s rticulted y the Stkeholder Pnel on Infnt Formul nd Adult Nutritionls. The ERP grnted the method First Action sttus, pplicle to infnt formul nd dult nutritionl products. The SMPR nlyticl rnge is ng/g for Cr, ng/g for Se, nd ng/g for Mo. This ICP-MS method provides sensitive nd ccurte method for the nlysis of Cr, Se, nd Mo in infnt formul nd dult nutritionl products. Mny infnt formuls re fortified with Se; mny up-ge peditric nd dult medicl nutritionl products re lso fortified with Cr nd Mo (3, 4). Together, these three trce nutrients offer huge chllenge to ny lortory involved in testing these kinds of mtrixes ecuse they usully cnnot e determined with dequte speed, ccurcy, or precision. In ddition, different kinds of instrumenttion nd smple preprtion my e required versus methods in common use for mjor minerls. For exmple, inductively coupled plsm-tomic emission spectrometry (ICP-AES) techniques, used routinely to determine nine minerls simultneously (5), might need to e fitted with ultrsonic neulizers to determine Cr nd Mo, or with hydride genertors to determine Se (6 8). Alterntive techniques, such s grphite furnce-tomic sorption spectrometry, my require complicted extrction procedures to otin the requisite sensitivity, nd they cn only determine one element t time, which precludes the use of the internl stndrd technique (9). ICP-AES instruments usully require ultrsonic neulizers to

2 2 Pcquette et l.: Journl of AOAC Interntionl Vol. 95, No. 3, 2012 Tle 1. Typicl polytomic interferences for Cr, Mo, nd Se Element m/z Aundnce, % Interferences Cr Mo Se Se Se Ar 12 C +, 37 Cl 14 N 1 H +, 36 Ar 16 O +, 35 Cl 16 O 1 H +, 26 Mg 26 Mg + 79 Br 16 O + 38 Ar 40 Ar + 40 Ar 40 Ar + 40 Ar 40 Ar 1 H +1 H +, 12 C 35 Cl 35 Cl determine Cr nd Mo, or hydride genertors to determine Se. The ICP-MS technique offers quntittion limits t prts-pertrillion levels using internl stndrds (10 12), nd the newest genertion of instruments hve collision rection cells (CRCs) tht cn reduce or eliminte interferences from low-mss moleculr ions generted y the plsm gs, orgnic mtrix components, nd solvent-cids (13, 14). For exmple, using CRC, 52 Cr cn e used to determine Cr in the presence of polytomic interferences, such s 40 Ar 12 C, nd 78 Se cn e used to determine Se in the presence of 40 Ar 38 Ar (15, 16). In the former exmple, the CRC is pressurized with He gs, which collides with the lrger polytomic interferences more efficiently, cusing them to lose kinetic energy reltive to the smller nlyte ion ( 52 Cr); potentil rrier is pplied tht lets only the lighter, fster 52 Cr enter the qudrupole y kinetic energy discrimintion (17). In the second exmple, the rection mode of the CRC is used: the cell is pressurized with H 2 gs (produced y hydrogen genertor), which neutrlizes ionized rgon dimer Ar(Ar + ) to reduce the formtion of 40 Ar 38 Ar nd other interfering species (17, 18). Exmples of polytomic interferences in ICP-MS re listed in Tle 1. The isotope 95 Mo hs very few interferences nd does not require collision gs, ut it is determined with Cr in the He gs mode in order to sve time. The determintion is not truly simultneous, ut cn e ccomplished in 2 3 min, nlogous to using oth xil nd rdil modes in ICP-AES determintions. Another kind of interference encountered ws the enhncement of Se signls y cron (ioniztion energy, ev; 15, 16, 19). It is suggested tht the incresed popultion of C + in the plsm increses the degree of ioniztion of Se y improving the trnsfer of electrons from Se (ioniztion energy, 9.75 ev) to C + (15). The interference is overcome y dding methnol to oth stndrds nd smples. Smple preprtion is esily nd rpidly ccomplished y closed vessel microwve digestion in Teflon vessels. Digestion tkes pproximtely 1.5 h (including cool down), is utomted, nd cn totlly digest the smple without dngerous perchloric cid (20). Volumetric wre is unnecessry ecuse the internl stndrd is dded to the vessels t the strt; the digested solution is merely poured into the utosmpler tue for nlysis. The uthors elieve tht this comintion of closed vessel microwve digestion with ICP-AES or ICP-MS nlysis nd internl stndrdiztion is so compelling tht it should merit first considertion for virtully ny minerl nlysis (21). This report descries single-lortory vlidtion (SLV) for the determintion of Cr, Se, nd Mo in infnt formul nd dult nutritionl products y ICP-MS (see Tle 2). Given tht the whole rnge of nutrient minerls cn now e tested y similr methodology tht is widely ville nd cost-effective (microwve-icp-aes, ICP-MS), these methods re strong cndidtes to e dvnced s next-genertion stndrd reference methods for the AOAC nd Interntionl Formul Council inititive recently descried (22). AOAC Officil Method Chromium, Selenium, nd Molydenum in Infnt Formul nd Adult Nutritionl Products Inductively Coupled Plsm/Mss Spectrometry First Action 2011 A. Principle Test portion is heted with nitric cid in closed vessel microwve digestion system t 200 C. Digested test solution, or n pproprite dilution, is presented to the ICP-MS instrument stndrdized with cid mtched stndrd clirnt solutions. An ioniztion uffer (potssium) is used to minimize esily ionizle element (EIE) effects, methnol is dded to normlize Tle 2. Nutritionl products evluted during the SLV Concn, % Product Type of product Protein Ft Crohydrte LC Infnt/dult nutritionl - powder A Infnt/dult nutritionl - powder B Medicl nutritionl - liquid C Adult nutritionl - liquid D Medicl nutritionl - liquid E Medicl nutritionl - liquid F Adult nutritionl - powder G Peditric nutritionl - liquid H Medicl nutritionl - powder I Medicl nutritionl - powder J Medicl nutritionl - liquid

3 Pcquette et l.: Journl of AOAC Interntionl Vol. 95, No. 3, Tle A. ICP-MS prmeters Typicl operting conditions RF power, W 1600 RF mtching, V 1.8 Smpling depth, mm 9 Extrct 1 lens, V 0 Crrier gs, L/min 0.9 Mke-up gs, L/min 0.2 Neulizer (glss concentric) MicroMist Spry chmer temperture, C 2 Interfce cones Ni He cell gs flow rte, ml/min 4.5 H2 cell gs flow rte, ml/min 4.2 Neulizer pump rte, rps 0.1 (0.5 ml/min) Anlyte/internl stndrd/ 52 Cr, 95 Mo/ 60 Ni,/He 78 Se/ 130 Te/H2 gs mode the cron content, nd nickel nd tellurium re used s internl stndrds. B. Apprtus () Microwve. Commercil microwve designed for lortory use t C, with closed vessel system nd controlled temperture rmping cpility. It is recommended tht the vessel design e selected tht will withstnd the mximum possile pressure, since orgnic mteril, nd lso crontes if not given sufficient time to predigest, will generte significnt pressure during digestion. (Vessels cn rech 700 psi or more on occsion.) Vessels must e designed to operte with only 6 ml solution volume, or the volume must e djusted ccordingly. Vent ccording to mnufcturer s recommendtion. (Cution: Microwve opertion involves hot pressurized cid solution. Use pproprite fce protection nd lortory clothing.) Additionl instrument prmeters re summrized in Tle A. () ICP-mss spectrometer. With CRCs. (c) Vrious plsticwre nd pipets. C. Chemicls nd Regents Employ norml lortory sfety precutions (lortory cots nd sfety glsses with side shields) when hndling concentrted cids, ses, nd orgnic solvents. Additionl protections such s fce shields, neoprene gloves, nd prons should e used where splshing my occur. Avoid rething vpors y working in pproved hoods. () Lortory wter. Use 18 MΩ wter throughout for dilution. () Concentrted nitric cid (HNO 3 ) % trce metl-grde HNO 3 throughout. (c) Hydrogen peroxide. 30% ACS regent grde. (d) Methnol % nlyticl regent grde for mtrix mtching. (e) Potssium mg/l in nitric cid for mtrix mtching. Tle B. Operting prmeters Stge 1 smple digestion 1 Power 100% (1600 W) 2 Rmp to temperture 20 min 3 Hold time 20 min 4 Temperture 180 C 5 Cool down 20 min D. Stndrds Stge 2 smple digestion 1 Power 100% (1600 W) 2 Rmp to temperture 20 min 3 Hold time 20 min 4 Temperture 200 C 5 Cool down 20 min () 2 mg/l Cr nd Mo nd 1 mg/l Se multielement stock stndrd solution in nitric cid. High-Purity Stndrds (Chrleston, SC) or equivlent. () 5 mg/l Ni nd Te multielement stock stndrd solution in nitric cid. High-Purity Stndrds, or equivlent. E. Procedure () Stndrd preprtion. Prepre intermedite stndrds from commercil stock stndrds t 40 ng/ml Cr nd Mo, nd 20 ng/ml Se. Custom-lended multielement stock stndrd in HNO 3 is cceptle. Prepre minimum of three multielement working stndrds contining 0.8, 4.0, nd 20 ng/ml Cr nd Mo nd 0.4, 2.0, nd 10 ng/ml Se, plus lnk, with oth Ni nd Te internl stndrd, in HNO 3. Ni is used s the internl stndrd for oth Cr nd Mo, nd Te must e used for Se. () Smple preprtion. Prepre powder smples y reconstituting pproximtely 25 g smple in 200 ml wrm lortory wter (60 C). Accurtely weigh pproximtely 1.8 g reconstituted test portion into the digestion vessel. This represents pproximtely 0.2 g originl powder smple. Fluid smples my e prepred y ccurtely weighing pproximtely 1 g test portion weighed directly into the digestion vessel fter mixing. For one-step digestion (two stges in microwve progrm) dd 0.5 ml 5000 ng/ml Ni nd Te internl stndrd solution nd 5 ml trce metl-grde HNO 3 followed y 2 ml H 2 O 2, to the microwve digestion vessels. Sel vessels ccording to mnufcturer s directions nd plce in microwve. Rmp temperture from mient to 180 C in 20 min, nd hold for 20 min in stge one. In stge two, the microwve will utomticlly rmp to 200 C in 20 min, nd hold for 20 min (See Tle B). For microwve ovens without the two stge progrm nd where it is more convenient use the two-step digestion. Add 0.5 ml 5000 ng/ml Ni nd Te internl stndrd solution nd 5 ml trce metl-grde HNO 3. With power settings pproprite to microwve model nd numer of vessels, rmp temperture from mient to 200 C in 20 min. Hold t 200 C for 20 min. Cool vessels ccording to mnufcturer s directions, pproximtely 20 min. Slowly open the microwve vessels, venting the rownish nitrogen dioxide gses. (Cution: Venting

4 4 Pcquette et l.: Journl of AOAC Interntionl Vol. 95, No. 3, 2012 must e performed in hood ecuse NO 2 is very toxic.) Add 1 ml H 2 O 2 nd redigest smples y rmping the temperture from mient to 180 C in 15 min. Hold t 180 C for 15 min nd cool for 20 min. (c) Preprtion of test solution. Add pproximtely 20 ml lortory wter to the contents of the vessel with the digested smples nd trnsfer to 50 ml smple vil. Rinse the vessel nd trnsfer the rinste into the smple vil. Add 0.5 ml methnol to the smple vil nd dilute to pproximtely 50 ml with lortory wter. F. Determintion Tle summrizes typicl instrument prmeters for nlysis. Anlyze test solutions using n ICP-MS instrument stndrdized with stndrd solutions. Ni is used s the internl stndrd for oth Cr nd Mo (helium mode), nd Te must e used for Se (hydrogen mode). Anlyze 4 ng/ml Cr nd Mo, nd 2 ng/ml Se working stndrd or other suitle qulity control solution every 10 test portions to monitor for instrument drift nd linerity (result 100 ± within 5% of nominl). The inclusion of method lnk (run s smple), duplicte smple [reltive percent difference (RPD) within 10%], nd known reference mterils serving s control smples (recovery check within control limits) re considered mndtory for good method performnce. If ny of these QC checks fils, results should e considered invlid. G. Clcultions Smple concentrtions were utomticlly clculted y the ChemSttion softwre using nonweighted lest-squres liner regression clirtion nlysis to produce est-fit line: Y = x + lnk The nlyte concentrtion in the smple ws then clculted: x = y lnk DF where x = nlyte concentrtion (ng/g); y = smple response rtio (ng/ml), which is the mesured count of ech nlyte s stndrd solution dt point in the clirtion curve divided y the rtio of the counts/concentrtion of the internl stndrd t the sme level; lnk = lnk stndrd solution (ng/ml), which is the mesured count of the lnk stndrd solution dt point in the clirtion curve divided y the rtio of the counts/concentrtion of the internl stndrd t the sme level s the lnk stndrd solution; = slope of the clirtion curve; nd DF = dilution fctor of the smple solution divided y smple weight (ml/g). H. Method Vlidtion () Linerity. All clirtion curves were prepred using nonweighted lest-squres liner regression nlysis, nd correltion coefficient (r) vlues were clculted with ech clirtion curve. Ech clirtion curve ws prepred with four multielement stndrd solutions, including the lnk stndrd solution. It should e noted tht ll nlyte concentrtions in smples were within liner rnge of the clirtion curve nd ove the estlished lower linerity limit. () LOQ. The LOQ is the lowest concentrtion of the nlyte in the smple tht cn e relily quntitted y the instrument. The method LOQ is typiclly determined y multiplying the verge SD of 10 digested lnks y fctor of 10, nd the instrument LOQ y multiplying the instrument LOD y 3 (23). However, in this method the useful LOQ, or prcticl LOQ (PLOQ), ws determined to e the lower liner limit vlue of the clirtion curve ecuse the ccurcy nd precision of smple mesurements elow tht vlue would e uncertin. Almost ll minerl-fortified nutritionl products cn e prepred with DF such tht Cr, Se, nd Mo will e present in the nlyticl solution ove the PLOQ. (c) Mtrix mtching with methnol. The presence of cron (orgnic compounds) in nlyticl solutions cuses signl enhncement of Se during nlysis y ICP-MS (15, 16, 18). To determine the optimum concentrtion of methnol (source of cron) needed to compenste for Se signl enhncement, vrious concentrtions of methnol were dded to oth clirtion stndrds nd digested smples. (d) Effects of EIEs. Mny nutritionl products contin significnt levels of EIEs, such s C, N, K, nd Mg. Therefore, lnk solutions nd solutions contining 4 ng/ml Cr nd Mo nd 2 ng/ml Se were nlyzed oth with nd without EIEs to determine ny chnges in concentrtions of the nlytes. (e) Specificity. Specificity of the method is its ility to ccurtely mesure the nlyte in the presence of other components in the smple mtrix tht might cuse spectrl interferences. To demonstrte the specificity of the method, undigested lnk solutions were spiked with multielement solutions t concentrtions tht re representtive of nutritionl products in smples for ICP-MS nlysis. The typicl H 2 gs mode for Se, nd He gs mode for Cr nd Mo, were used. (f) Accurcy. Accurcy ws demonstrted y nlyzing three Ntionl Institute of Stndrds nd Technology (NIST) stndrd reference mterils (SRMs) on 2 independent dys, mesuring spike recoveries in 10 nutritionl products on 3 different dys, nd compring results for 10 nutritionl products otined y this method to results otined y other in-house vlidted ICP-AES nd tomic fluorescence spectrometry (AFS) methods. The spike levels of the nlytes dded to the products were etween 50 nd 200% of the nlyte concentrtions in ech product. (g) Precision. Both within- nd etween-dy RSD vlues were determined y nlyzing two in-house lortory control smples. Within-dy precision ws determined y nlyzing the lortory control smples in duplicte on ech dy, nd etweendy precision ws mesured y using the men results of the duplicte smples nlyzed on ech dy on 10 different dys. (h) Ruggedness nd roustness. To determine the ruggedness of the method, lortory control smples were nlyzed y two nlysts on 10 different dys. Also, NIST SRM 1849 ws nlyzed in triplicte with vrying smple weights nd with different internl stndrds. References: J. AOAC Int. xxxx (2012) Results nd Discussion System Suitility All system suitility cceptnce criteri used in this vlidtion were met. Results for the r-vlue nd preprtion lnk were etter thn nd less thn the method criteri of 0.4 ng/ml Cr nd Mo, nd 0.2 ng/ml Se (Tle 3). The RSD vlues for duplicte mesurement for ll 10 products were <4% for Cr, <3% for Se, nd <3% for Mo (dt not shown). Tle 4

5 Pcquette et l.: Journl of AOAC Interntionl Vol. 95, No. 3, Tle 3. System suitility dt for r-vlue nd preprtion lnk r Preprtion lnk, ng/ml Anlysis Cr Se Mo Cr Se Mo A clirtion curve nd preprtion lnk were prepred for ech nlysis on 10 seprte dys. The preprtion lnks nd clirtion lnk re nerly identicl. The only difference is the ddition of hydrogen peroxide to the former. Both solutions were prepred dily. Thus, these dt should e very close to zero. lso shows the duplicte RSD vlues for Cr, Se, nd Mo in oth lortory controls used. The RSD vlues were <3%. Initil control chrt vlues for SRM 1849 (lortory control 1) nd lortory control 2 for 10 seprte dys re shown in Tle 5. Linerity The clirtion curve of ll three nlytes produced r-vlues of or etter (Tle 3). In ddition to the r-vlues, the linerity of the clirtion curve ws determined y nlyzing multielement solutions contining 0.4, 2, 8, nd 16 ng/ml Cr nd Mo concentrtions, nd 0.2, 2, 4, nd 8 ng/ml Se concentrtions. The nlysis ws crried out in duplicte on 3 seprte dys. The overll verge recovery for ech concentrtion of ll nlytes ws etween 95 nd 103%, with precision vlues rnging from 0.3 to 2.7% RSD (Tle 6). These results demonstrte tht good linerity from 0.4 to 20 ng/ml for Cr nd Mo, nd from 0.2 to 10 ng/ml for Se clirtion curves ws chieved. Below 0.4 ng/ml for Cr nd Mo, or elow 0.2 ng/ml for Se, the mesured recoveries were outside the rnge of % (dt not shown). The method requires ll smple nlyticl solution Tle 4. Precision of the method demonstrted y nlyzing two lortory controls Within-dy RSD, % (n = 2) Lortory control 1 (SRM 1849) Lortory control 2 (LC) Dy Cr Se Mo c Cr d Se e Mo f c d e f Overll concentrtion = ng/g. Overll concentrtion = ng/g. Overll concentrtion = ng/g. Overll concentrtion = ng/g. Overll concentrtion = ng/g. Overll concentrtion = ng/g.

6 6 Pcquette et l.: Journl of AOAC Interntionl Vol. 95, No. 3, 2012 Tle 5. Demonstrtion of ruggedness for two nlysts on 10 seprte dys Lortory control 1: SRM 1849-Infnt/Adult Nutritionl Formul, ng/g Anlyte Dy 1 Dy 2 Dy 3 Dy 4 Dy 5 Dy 6 Dy 7 Dy 8 Dy 9 Dy 10 Averge RSD, % Cr Se Mo Lortory control 2, ng/g Cr Se Mo Reference vlues, mg/kg: Cr = 1.09 ± 0.21; Se = ± 0.057; Mo = 1.62 ± concentrtions to e ove this lower linerity limit to prevent excessive is (>5%) due to nonlinerity. The lower linerity limit, thus, represents the PLOQ for this method. Note tht the lower linerity limits re well ove the instrumentl LOQ for these elements. LOQ Tle 7 shows tht the instrument LOQs otined for Cr, Se, nd Mo were 0.03, 0.03, nd 0.01 ng/ml, respectively. These vlues re pproximtely one order of mgnitude smller thn the PLOQ vlues. The smple PLOQs, clculted from the PLOQs for Cr, Se, nd Mo in pproximtely 0.2 g powder nd 1.0 g liquid smples, diluted to 50 ml solutions, re lso shown in Tle 7. DFs for powder nd liquid products were 250 nd 50, Tle 6. Mesurement of stndrd solutions to determine linerity of Cr, Se, nd Mo using clirtion stndrds 0, 0.8, 4.0, nd 20.0 ng/ml Cr nd Mo, nd 0, 0.4, 2.0, nd 10.0 ng/ml Se Anlyte Concn, ng/ml Recovery, % RSD, % c Cr Se Mo c Expected concentrtion. Overll verge for 3 independent dys nd duplicte mesurements ech dy. Precision for 3 seprte independent dys. respectively. If the nlyte concentrtions in the products re lower thn the PLOQs, lrger smple weights should e used. Powder nd liquid smples should e less thn or equl to 0.5 g nd 3 g, respectively. Mtrix-Mtching with Methnol Initilly, 2% methnol ws dded to oth stndrds nd digested smples (SRMs 1849 nd 8415), ut sustntil ccumultion of cron deposits on the cones nd ion lenses resulted in signl drift nd loss of sensitivity fter nlyzing few smples. Therefore, the concentrtion of methnol in the clirtion stndrds nd SRM smples ws reduced to 1.5, 1.0, or 0%. The concentrtions of Cr, Se, nd Mo in the SRMs with the reduced methnol concentrtions re shown in Tle 8. In the sence of methnol, the concentrtions of Se in oth SRMs were high, outside the reference vlue rnges, wheres Se concentrtions in the SRMs contining 1 nd 1.5% methnol were comprle nd within the reference vlue rnges. Also, the Se spike recovery in SRM 1849 ws 113.5% in the sence of methnol, ut nd 102.2% in 1 nd 1.5% methnol, respectively. The spike recovery vlues for Cr nd Mo rnged from 96.4 to 105.7%. It is importnt to note tht the vrious Tle 7. Instrument LOQ nd smple PLOQ clculted from their corresponding PLOQ Anlyte Instrument LOQ, ng/ml Lower liner limit PLOQ, ng/ml Liquid, ng/g c Smple PLOQ Powder, ng/g d Cr Se Mo c d Clculted y multiplying the instrument LOD y 3 (LOD clculted y the instrument softwre). PLOQ is equivlent to the lower liner limit of the clirtion curve. Clculted y multiplying the PLOQ y dilution fctor of 50. Clculted y multiplying the PLOQ y dilution fctor of 250.

7 Pcquette et l.: Journl of AOAC Interntionl Vol. 95, No. 3, Tle 8. Effects of different methnol concentrtions on Cr, Se, nd Mo in SRMs 1849 nd 8415 SRM 1849-Infnt/Adult Nutritionl Formul concn, mg/kg (n = 3), spike recovery, % SRM 8415-Whole Egg Powder concn, mg/kg (n = 3) Anlyte Reference vlue MeOH, 0% MeOH, 1% MeOH, 1.5% Reference vlue MeOH, 0% MeOH, 1% MeOH, 1.5% Cr 1.09 ± ± 0.01 [105.7] 1.04 ± 0.01 [101.9] 1.04 ± 0.01 [102.5] 0.37 ± ± ± ± 0.03 Se ± ± 0.01 [113.5] ± [101.3] ± [102.2] 1.39 ± ± ± ± 0.01 Mo 1.62 ± ± 0.01 [101.0] 1.70 ± 0.01 [99.1] 1.68 ± [96.4] ± ± ± ± levels were etween 50 nd 200%. MeOH = Methnol. concentrtions of methnol hd negligile effects on Cr nd Mo in oth SRMs, nd cron ccumultions on the cones nd ion lenses were not sustntil enough to ffect signl stility. Effects of EIEs Tle 9 shows the recovery results for 4 ng/ml Cr nd Mo, nd 2.0 ng/ml Se in solutions contining vrious comintions of EIEs. A significnt Cr concentrtion (pproximtely 0.7 ng/ml) ws produced y Mg due to contmintion of our Mg stock solution; therefore, Mg ws not used in ny EIE experiments. The clirtion stndrds for this experiment were prepred with 50 mg/l concomitnt K. Recoveries etween 95 nd 105% of the expected vlues were otined, nd stndrd with no EIEs dded yielded close to 100% recovery of its nominl concentrtion. Thus, lthough there ws reltively little effect of EIE dditions on the results, we thought it prudent to dd some K to the stndrds in cse other instruments show more sensitivity to EIE effects. Specificity Tle 10 shows the verge results of n experiment using two groups of 10 lnk solutions to determine the specificity of the method. One group of lnk solutions ws spiked with multielement solution, nd the other ws left unspiked (i.e., PB). The concentrtions of the individul elements dded to ech lnk solution (concentrtions shown t the ottom of Tle 10) re representtive of the element solution concentrtions in nutritionl products. The smll increse in Cr (0.000 to ng/ml) nd Mo (0.000 to ng/ml) concentrtions ws likely due to contmintion from the multielement stndrds. The Cr nd Mo concentrtions in the spiked PB were significntly elow the PLOQ vlues (0.4 ng/ml for oth Cr nd Mo). The method PLOQ for Se, 0.2 ng/ml, ws lso much greter thn the lnk concentrtions of Se in the presence of the multielement solution. These dt (otined in the H 2 gs mode for Se, nd the He gs mode for Cr nd Mo) demonstrte tht the method is specific for Cr, Se, nd Mo ecuse only negligile nlyte signl is produced y ckground moleculr gses or concomitnt smple elements. Accurcy First, to verify the ccurcy of the method, three SRMs, Infnt/Adult Nutritionl Powder (SRM 1849), Whole Egg Powder (SRM 8415), nd Whole Milk Powder (SRM 8435), were nlyzed (Tle 11). The overll verge concentrtions of Cr, Se, nd Mo were within the reference vlue specifiction limits of the SRMs (24 26). The second verifiction of ccurcy ws crried out y nlyzing 10 spiked nutritionl products on 3 seprte dys. The spike levels of the nlytes in the products rnged from 71 to 214%. The 3 dy verge spike recoveries for Cr, Se, nd Mo were etween 93 nd 107%, with precision vlues rnging from 0.2 to 5.7% RSD (Tle 12). It is lso importnt to note tht the concentrtions of Cr, Se, nd Mo mesured in ll unspiked product smples were within product specifiction limits (Tle 13). The results in Tle 12 show tht the method is ccurte for the determintion of Cr, Se, nd Mo in nutritionl products. A third ccurcy verifiction ws crried out y compring the results of this ICP-MS method to results otined y in-house ICP-AES nd AFS methods for 11 nutritionl products, including NIST SRM 1849 (Tle 14). The ICP-AES method determined Cr nd Mo using internl stndrds Y nd Co, respectively. The AFS method, using only one Se hollow cthode lmp, determined Se without n internl stndrd. The results in Tle 14 show tht except for Cr in nutritionl product J (10.7%) nd Se nd Mo in nutritionl product A (16.1 nd 15.2%, respectively), the difference etween the ICP-MS nd the in-house methods ws less thn 10%. It should e noted tht the ICP-MS results were the verge of 6 seprte dys (smples nlyzed in duplicte on ech dy) nd otined y two nlysts, while oth the ICP-AES nd AFS results were the verge of duplicte smples on 1 dy nd otined y one Tle 9. Effects of vrious comintions of EIEs on 4 ng/ml Cr nd Mo, nd 2 ng/ml Se using clirtion stndrds spiked with 50 mg/l K (n = 3) Anlyte Expected concn, ng/ml 50 mg/l K, N; 20 mg/l C 50 mg/l N; 20 mg/l C 50 mg/l K; 20 mg/l C 50 mg/l K, N 50 mg/l K No EIEs Cr ± ± ± ± ± ± 0.00 Se ± ± ± ± ± ± 0.01 Mo ± ± ± ± ± ± 0.01

8 8 Pcquette et l.: Journl of AOAC Interntionl Vol. 95, No. 3, 2012 Tle 10. Investigtion of specificity y determining the effect of multielement solution on Cr, Mo, nd Se mesurements in lnk solutions (n = 10) Anlyte Blnk verge concn, ng/ml Blnk + multielement verge concn, ng/ml PLOQ concn, ng/ml Cr ± ± Se ± ± Mo ± ± Averge of 10 solutions nlyzed on different dys. Multielement concentrtion in lnk, mg/l: C = 22.5, Mg = 6, K = 30, Cu = 12, Mn = 0.1, N = 22.5, Fe = 7.5, P = 15, Zn = 7.5. Tle 11. Results from nlysis of NIST SRMs for Cr, Se, nd Mo Anlyte Reference vlue, mg/kg d NIST SRM 1849 NIST SRM 8415 NIST SRM 8435 c Overll vg. concn, mg/kg (n = 10) Reference vlue, mg/kg d Overll vg. concn, mg/kg (n = 2) Reference vlue, mg/kg d Overll vg. concn mg/kg (n = 2) Cr 1.09 ± ± ± ± e 0.4 ± 0.02 Se ± ± ± ± ± ± Mo 1.62 ± ± ± ± ± ± 0.01 c d e Infnt/Adult Nutritionl Powder NIST SRM (lso used s the lortory control). Whole Egg Powder NIST SRM. Whole Milk Powder NIST SRM. The uncertinty in the reference vlues is clculted for 95% confidence intervl, nd the uncertinty in the ICP-MS results is sed on one-sigm confidence intervl. This vlue ws sed on results of limited determintions; therefore, no uncertinty vlue ws provided (ref. 20). Tle 12. Averge recoveries of Cr, Se, nd Mo in 10 spiked nutritionl products on 3 seprte dys (n = 3) Product level, % Cr Se Mo recovery, % RSD, % level, % recovery, % RSD, % level, % recovery, % RSD, % A B C D E F G H I J Anlyte concentrtions in nlyticl solutions of products re etween 0.6 nd 7.0 ng/ml.

9 Pcquette et l.: Journl of AOAC Interntionl Vol. 95, No. 3, Tle 13. Overll verge concentrtion nd precision for 10 nutritionl products evluted y two nlysts on 6 seprte dys (n = 6) Product Specifiction limits, g/g Cr Se Mo Concn, Specifiction ng/g RSD, % limits, ng/g Concn, Specifiction ng/g RSD, % limits, ng/g Concn, ng/g RSD, % A B C D E F G H H J The 95% confidence intervl (mesurement uncertinty) is pproximtely equl to the intervl represented y these RSDs, since the t fctor for 5 degrees of freedom (2.57) is pproximtely equl to the squre root of six mesurements (2.45). nlyst. Therefore, t these low concentrtions, the greement etween ICP-MS nd the in-house methods is resonle. The dt in Tle 14 lso show tht there my e is mong the methods. The ICP-MS results re usully lower thn the ICP-AES results for oth Cr nd Mo, nd higher thn the AFS results for Se. Given the good overspike recovery dt (Tle 12), the superior sensitivity of ICP-MS versus ICP-AES, nd the etter precision over hydride genertion techniques, especilly without n internl stndrd, the ICP-MS method my e more ccurte, ut this cnnot e proven conclusively. Difficulty in exct mtrix-mtching my cuse is of 1 5% with this method (see EIE discussion). Precision Tle 13 shows the intermedite precision vlues for 10 nutritionl products nlyzed y two nlysts on 6 seprte dys (totl n = 6). Precision vlues for ll 10 nutritionl products were <5% RSD for concentrtion vlues rnging from to , 50.9 to 819.3, nd 46.9 to ng/g for Cr, Se, Tle 14. Comprison of ICP-MS results to ICP-AES (Cr nd Mo) nd AFS (Se) for 11 nutritionl products Product ICP-AES concn, ng/g (n = 1) Cr Se Mo ICP-MS concn, ng/g (n = 6) Difference, % AFS concn, ng/g (n = 1) ICP-MS concn, ng/g Difference, (n = 6) % ICP-AES concn, ng/g (n = 1) ICP-MS concn, ng/g (n = 6) Difference, % A B C D E F G H I J SRM c d 1.02 d d d 1.70 d 1.2 c d ICP-AES method uses ultrsonic neuliztion for improved sensitivity. AFS method uses prereduction nd hydride genertion. Infnt/Adult Nutritionl Formul SRM certified vlues, mg/kg: Cr = 1.09 ± 0.21; Se = ± 0.057; Mo = 1.62 ± mg/kg.

10 10 Pcquette et l.: Journl of AOAC Interntionl Vol. 95, No. 3, 2012 Tle 15. Vrying lortory control smple (SRM 1849) weights to determine roustness Dy 1 Dy 2 Concn, ng/g (n = 2) Concn, ng/g (n = 2) Smple weight, g (n = 2) Cr Se Mo Smple weight, g (n = 2) Cr Se Mo Averge, ng/g RSD, % Tle 16. Anlysis of NIST SRM 1849 with different internl stndrds to determine roustness of the method Anlyte concn, mg/kg (n = 3) Internl stndrd Anlyte Reference vlue Co Ni Ge Y Te Cr 1.09 ± ± ± ± ± ± 0.02 Se ± ± ± ± ± Mo 1.62 ± ± ± ± ± ± 0.01 nd Mo, respectively. Tle 14 lso shows tht ll nutritionl product concentrtions were within the product specifiction limits. Therefore, these results demonstrte tht the method is very precise, nd is well suited to routine nlysis. Ruggedness nd Roustness The results in Tle 13 demonstrte tht the method is very rugged. Excellent precision (<5% RSD) ws otined dy-to-dy, with results otined y two nlysts. The roustness of the method ws demonstrted erlier in Tle 8 for Cr nd Mo. The concentrtions of Cr nd Mo were comprle in 0, 1, nd 1.5% methnol solutions. The roustness of the method ws lso evluted y vrying smple weights of SRM 1849 from 0.17 to 0.23 g on 2 seprte dys (Tle 15). Duplicte smple weights were prepred t low, middle, nd high levels so tht the smple weights were pproximtely ±10% of the nominl smple weight. The results show tht vrying the smple weights hd little effect on the results for Cr, Mo, nd Se in the smples. The overll verge precision on ech dy, cross ll smple weights, ws <1% RSD. Also, the difference etween the verge result of ech nlyte on ech dy ws =5%. Another roustness study showed tht when SRM 1849 ws nlyzed with different internl stndrds (Co, Ni, Ge, Y, nd Te), the results were within the certified reference vlue concentrtion rnges for Cr nd Mo, regrdless of the choice of internl stndrd (Tle 16). However, Se concentrtions were significntly higher when Ni nd Ge were used s internl stndrds, which is why Te ws chosen s the internl stndrd. Presumly this ws ecuse Te (similr first ioniztion energy compred to Se) ws etter t correcting for cron mtrix effects on Se (27). It should e noted tht Co levels in nutritionl products re negligile, nd Co hs proven to e n excellent internl stndrd in ICP-AES methods. Also, no Ni contmintion from the cones ws oserved, ut the fct tht Ni is not used s n internl stndrd in ny of our in-house methods provides us with the cpility of diluting smples prepred for ICP-AES nlysis nd then dding Ni for nlysis of Cr nd Mo y this ICP-MS method. Hence, one smple preprtion is used for oth ICP-sed methods. References (1) Sullivn, D. (2012) J. AOAC Int. 95, org/ /jocint.sullivn_intro (2) Pcquette, L.H., Szo, A., & Thompson, J.J. (2011) J. AOAC Int. 94, (3) Cudd, F., Rggi, A., Testoni, A., & Znsi, F. (2002) J. AOAC Int. 85, (4) Shrpless, K.E., Thoms, J.B., Christopher, S.J., Greenerg, R.R., Snder, L.C., Shntz, M.M., Welch, M.J., & Wise, S.A. (2007) Anl. Bionl. Chem. 389, s y (5) Poitevin, E., Nicols, M., Grvelleu, L., Richoz, J., Andrey, D., & Monrd, F. (2009) J. AOAC Int. 92, (6) Cudd, F., & Rggi, A. (2005) Microchem. J. 79, dx.doi.org/ /j.microc (7) Dedin, J., & Tslev, D. (1995) Hydride Genertion nd Atomic Asorption Spectrometry, Vol. 130, Wiley nd Sons, New York, NY (8) Stiilj, V., Mzej, D., & Flnog, I. (2003) Anl. Bionl. Chem. 377, (9) Nrdi, E.P., Evngelist, F.S., Tormen, L., SintPierre, T.D., Curtuis, A.J., Sous, S.S., & Bros, F. (2009) Food Chem. 112, (10) Lozk, A., Soltyk, K., Ostpczuk, P., & Fijlek, Z. (2004) Phrmzie 59, (11) Phifer, E.C. (1995) J. AOAC Int. 78, (12) Ginnens, I., Nisinkis, P., Grvriil, A., Kontopidis, G., & Kyrizkis, I. (2009) Food Chem. 114, org/ /j.foodchem (13) Wilur, S. (2007) Performnce Chrcteristics of the Agilent 7500cx: Evluting Helium Collision Mode for Simpler, Fster, More Accurte ICP-MS, (14) Pechey, E., Hern, R., & Elhi, S. (2008) Multi-Element Determintion of Hevy Metls in Dietry Supplements Using

11 Pcquette et l.: Journl of AOAC Interntionl Vol. 95, No. 3, Collision/Rection Cell ICP-MS, LGC, Teddington, Middlesex, UK (15) Krlj, P., & Verer, M. (2003) Act Chim. Slov. 50, (16) Gmmelgrd, B., & Jons, O. (1999) J. Anl. Atom. Spectrom. 14, (17) Agilent Technologies (2005) Inductively Coupled Plsm: A Primer, (18) Feldmnn, I., Jkuowski, N., Thoms, C., & Stuewer, D. (1999) Fresenius J. Anl. Chem. 365, (19) Olivs, R.M., Quétel, C.R., & Donrd, O.F.X. (1995) J. Anl. Atom. Spectrom. 10, j (20) Dlpozzo, R., Brtoli, G., Smri, L., & Melchiorre, P. (2010) Chem. Rev. 110, (21) Zinden, P., & Andrey, D. (1998) Atom. Spectrosc. 19, (22) Inside Lortory Mngement (My/June 2010) AOAC INTERNATIONAL, Githersurg, MD, (23) IUPAC (1996) Pure Appl. Chem. 68, org/ /pc (24) Certificte of Anlysis (July 9, 2009) Stndrd Reference Mteril 1849 Infnt/Adult Nutritionl Formul, NIST, Githersurg, MD, (25) Certificte of Anlysis (Jnury 25, 2008) Stndrd Reference Mteril 8415 Whole Egg Powder, NIST, Githersurg, MD, (26) Certificte of Anlysis (Mrch 17, 2008) Stndrd Reference Mteril 8435 Whole Milk Powder, NIST, Githersurg, MD, (27) Lst, W.K., Cornelius, V., Delves, T., Sieniwsk, C., Fitzgion, J., Norton, A., Amess, J., Wilson, A., Rohtiner, S.Z.A., & Lister, A.T. (2003) J. Clin. Oncol. 21,