Food Chemistry 138 (2013) Contents lists available at SciVerse ScienceDirect. Food Chemistry

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1 Food Chemistry 38 (3) 9 5 Contents lists vilble t SciVerse ScienceDirect Food Chemistry journl homepge: Anlyticl Methods Investigtion of isotope dilution mss spectrometric (ID-MS) method to determine nicin in infnt formul, brekfst cerels nd multivitmins Hyunju Shin, Byungjoo Kim b, Joonhee Lee b, Bio-engineering R&D Center, Biospce Co., Ltd., Seoul , Republic of Kore b Division of Metrology for Qulity of Life, Kore Reserch Institute of Stndrds nd Science, Yuseong, Dejeon 35-6, Republic of Kore rticle info bstrct Article history: Received 5 June Received in revised form 3 October Accepted 9 November Avilble online 7 November Keywords: Nicin ID-LC/MS Method vlidtion Infnt formul Brekfst cerels Multivitmin An isotope dilution LC/mss spectrometric (ID-LC/MS) method ws developed s cndidte reference method for the ccurte determintion of nicin in infnt formul, brekfst cerels nd multivitmin. After spiking nicotinmide-d 4 s n internl stndrd, infnt formul nd brekfst cerel smples were hydrolysed under lkline condition. Smples were then nlysed in SRM mode to detect nicotinic cid nd nicotinic cid-d 4 t m/z 4? 8 nd 7? 84, respectively. In the cse of multivitmin smple tht contins minly free nicotinmide, LC/MS monitored nicotinmide nd nicotinmide-d 4 t their SRM chnnels of m/z 3? 8 nd m/z 7? 84, respectively, fter simple extrction. The repetbility nd reproducibility were tested for the vlidtion of the developed ID/LC MS method. Additionlly, the developed nlyticl method ws pplied to determine totl nicin contents in homogenised infnt formul, homogenised multivitmin, nd commercilly vilble products including different types of infnt formul, brekfst cerels, nd multivitmin tblets. Ó Elsevier Ltd. All rights reserved.. Introduction Nicin is one of the components in vitmin B complex nd generlly includes both of nicotinic cid (pyridine 3-crboxylic cid) nd nicotinmide (pyridine 3-crboxmide). In biologicl system, nicin involved in vrious dehydrogense rections in forms of nicotinmide denine dinucleotide (NAD) nd nicotinmide denine dinucleotide phosphte (NADP) (Gregory, 996). Nicin cn be existed s free nicotinic cid, nicotinmide, NAD, or NADP in severl foods including poultry, nuts, legumes, milk, cerels, nd vegetbles. In cerel grin, nicin is lso founded s chemiclly bound form of nicotinic cid to polyscchrides nd peptides or glycopeptides. On the determintion of nicin in foods, it should be clerly specified tht the method mesure free or totl nicin. Free nicin hs been usully mesured by determining nicotinic cid nd/or nicotinmide fter extrction with cidic queous solution (Bll, 6). For the nlysis of totl nicin, it is usully required to extrct nicin fter ll forms of nicin convert to free nicin by heting the smple with lkli or cid. Colorimetric nd microbiologicl methods hve been used s n officil method to nlyse nicin in food mtrix (AOAC, 997, 997b). Colorimetric method is bsed on the König rection nd determines the totl nicin with low specificity. Microbiologicl ssy evlutes either totl nicin or free nicin depending on Corresponding uthor. Tel.: ; fx: E-mil ddress: joonhee@kriss.re.kr (J. Lee). the using microorgnisms. Free nicin is generlly determined by Lctobcillus csei or Leuconostoc mesenteroids subsp. Mesenteroides. Lctobcillus plntrum (ATCC 84) cn determine totl nicin becuse this prticulr microorgnism responds to nicotinic cid, nicotinmide, nicotinuric cid (n inctive metbolite) nd NAD (Bll, 6). However, chromtogrphicl techniques currently replced colorimetric nd microbiologicl methods. Severl liquid chromtogrphicl methods with vrious seprtion nd detection techniques hve been reported (Lng, Ygr, Eggers, & Hofmnn, 8; Perrone, Donngelo, & Frh, 8; Rose-Sllin, Blke, Genoud, & Tgliferri, ; Zfr-Go9mez, Grbllo, Morles, & Grcí-Ayuso, 6). One of those studies compred the results of totl nicin contents mesured by liquid chromtogrphy (LC) with fluorescence detector nd by microbiologicl ssy using L. plntrum (Rose-Sllin et l., ). The HPLC method showed better reproducibility (.9.7%) compred to the microbiologicl ssy ( %). Also the other method by LC UV (Zfr-Go9mez et l., 6) showed the reltive stndrd devition from.5% to 3.7% for the simultneous nlysis of nicotinmide nd seven other wter soluble vitmins in supplemented foods. Mss spectrometry (MS) coupled with LC hs been introduced to improve the efficiency of nicin nlysis in coffee ben smples, nd the repetbility of method for nicotinic cid ws mintined s.6 4.9% depending on the levels of nlytes in the smples (Perrone et l., 8). An isotope dilution mss spectrometric (IDMS) method bsed on LC ws pplied to determine nicotinic cid nd nico /$ - see front mtter Ó Elsevier Ltd. All rights reserved.

2 H. Shin et l. / Food Chemistry 38 (3) 9 5 tinmide in foods nd biologicl smples. The ID-LC/MS ws shown.% of repetbility for nicotinic cid nd 3.% for nicotinmide in coffee smples (Lng et l., 8). Adopting isotope dilution techniques elimintes the systemic bis through the ccurte correction of the recovery of the trget nlyte long the smple clenup process (De Bivère, 993; Dube, Henrion, & Richter, 997; Jung et l., 4). Therefore, IDMS method is recognised s one of the potentil primry methods in chemistry by CCQM (Comité Consulttif pour l Quntité de Mtière) (Quinn, 997) s it provide higher order metrologicl qulity for the mesurement of smples with complex mtrices. A few Ntionl Metrology Institutes (NMI), Ntionl Institute of Stndrd nd Technology (NIST) of US nd Ntionl Institute of Metrology (NIM) Chin, hve estblished reference methods by pplying the ID-LC/MS method to determine nicin in infnt formul contining nicin. (Goldschmidet & Wolf, ; Hung et l., ). These methods monitored only free nicotinmide in smple without lkline hydrolysis process, which ws required to determine totl nicin including bound forms of nicin. Current study described bout the development of n ID-LC/MS method s cndidte reference method for the nlysis of nicin in infnt formul, brekfst cerel nd multivitmin. This method ws designed to investigte totl nicin in vrious food types by dopting lkline hydrolysis step, which cn be lso pplied for the nlysis of infnt formul or milk powder regrdless of existing forms of nicin in the mtrix. The uncertinty of the ID-LC/MS ws evluted. Finlly, nicin contents in vrious commercil foods nd food supplements were determined to confirm the fesibility of the ID-LC/MS developed.. Mterils nd methods.. Chemicls nd mterils Nicotinmide ws purchsed from Chromdex. Inc. (Snt An, CA, USA) s primry reference stndrd nd its purity ws determined s (99.4 ±.3)% by the protocols mintined in this lbortory of KRISS (Kore Reserch Institute of Stndrds nd Science), the Ntionl Metrology Institute of Kore. Nicotinmide-(,4,5,6)- d 4 ws purchsed from CDN Isotopes Inc. (Quebec, Cnd). Ammonium cette ws obtined from Sigm Aldrich (Steinheim, Germny). Sodium hydroxide nd hydrochloric cid were purchsed from Smchun Chemicls (PyungTk, Kore). HPLC grde methnol nd formic cid were from Burdick nd Jckson (Muskegon, MI, USA) nd Fluk (St. Gllen, Switzerlnd), respectively. HPLC grde wter ws purified in Milli-Q system (Millipore, Beldford, MA, USA). Filter crtridges (PURDISC NYL 5 FILTER 5 mm.45 lm) were obtined from Whtmn (Clifton, NJ, USA). Certified reference mterils SRM 849 (infnt/dult nutrition formul) nd BCR 43 (Brussels sprouts powder) were purchsed from the Ntionl Institute of Stndrd nd Technology (Githersburg, MD, USA) nd Institute for Reference Mterils nd Mesurements (Geel, Belgium), respectively. Other testing mterils including severl infnt formuls, brekfst cerels nd multivitmins were purchsed from locl mrkets. Two infnt formuls were for 3 6 months bby from different producers. Brekfst cerels A nd B were products with minly corn, respectively 6% nd 89%, from different producers nd brekfst cerel C ws products contin 85% of brwn rice. All of three multivitmins were tblet types from different producers... Clibrtion stndrd solutions Stndrd solutions nd isotope rtio stndrd were prepred nd verified following the procedure mintined in this lbortory s described in our other rticles (Kim, Hwng, So, Son, & Kim, ; Lim, Kim, Ahn, & Kim, ). Briefly, weighted portion (7 mg) of nicotinmide primry reference mteril ws dissolved into weighted portion (8 g) of methnol. Four stndrd solutions were independently prepred. An isotope lbelled nicotinmide (nicotinmide-d 4 ) stndrd solution ws prepred in the sme wy. As clibrtion stndrd solution for isotope dilution method, isotope rtio stndrd solution ws prepred by grvimetriclly mixing the nicotinmide stndrd solutions nd the nicotinmided 4 stndrd solution (: isotope rtio). Two isotope rtio stndrd solutions were prepred from ech nicotinmide stndrd solution. After dilution with wter ( mg/kg in this study), the eight isotope rtio stndrds were nlysed by LC/MS in SIM (selected ion monitoring) mode to cross-check the consistencies mong them. Repetbility of prepring stndrd solutions nd isotope rtio stndrds were evluted by the LC/MS results. One isotope rtio stndrd ws selected nd used for the clibrtion stndrd on smple nlysis..3. Smple preprtion Smples were differently prepred depending on the existing forms of nicin in the smples. Alkline hydrolysis is necessry to convert into free nicotinic cid from NAD or NADP nd bound nicotinic cid in milk or brekfst cerel. Smples like multivitmins which contin minly nicotinmide were nlysed fter simple extrction without hydrolystion..3.. Preprtion for infnt formul nd brekfst cerel smples Brekfst cerel smple hs to be homogenised by grinding with lbortory mill (FRITSCH, Model No. Pulverisette 4, Germny) before smpling. SRM 849, BCR 43, nd infnt formul were homogeneous fine powder forms nd did not require grinding. Smple ( g) ws tken grvimetriclly in 6 ml mber bottle nd pproprite mount of nicotinmide-d 4 stndrd solution ws spiked in the bottle to mke the isotope rtio to. s bsed on the product informtion. Wter (3 ml) ws dded in the bottle nd mixed with smple by vortexing nd soniction. A portion ( ml) of the prepred smple solution ws trnsferred to conicl tube. To perform the lkline hydrolysis, 5 ml of NOH (5 mol/ L) ws dded to the smple solution, then they were heted in boiling wter bth for one hour. After cooling down to room temperture, ph of the smple solution ws djusted between.5 nd 3. by dding hydrochloric cid. For the solid-phse extrction, OA- SIS HLB crtridge (Wters, Milford, MA, USA) ws pre-conditioned with methnol nd wter, nd ml of smple solution ws loded. After wshing with ml of 5 mmol/l mmonium cette buffer, nicotinic cid ws eluted with 3 ml of 5 mmol/l mmonium cette buffer. The isotope rtio stndrd ( ml) ws subject to the sme smple preprtion processes including lkline hydrolysis to be used s clibrnt for smple nlysis..3.. Preprtion for multivitmin smples For the nlysis of multivitmin smple,. g of ground smple ws plced into 6 ml glss bottle nd 5 ml of wter ws dded into the bottle. The exct mount of smple nd extrction solvent were determined by weighing the bottle before nd fter ddition of ech of them. After soniction for h, ml of the smple extrct ws trnsferred to vil nd spiked with n pproprite mount of the isotope stndrd solution to mke the isotope rtio s close to :. The smple ws pssed through filter crtridge, nd then diluted with wter to level ( mg/kg) which is convenient for LC/ MS nlysis in comprison with the isotope rtio stndrd (without further tretment such s lkli hydrolysis).

3 H. Shin et l. / Food Chemistry 38 (3) Instrumenttion nd MS nlysis The LC MS nlysis ws chieved with Agilent 64 Triple Qud LC/MS (Snt Clr, CA, USA) connected with Agilent Series (Wldbronn, Germny) contining quternry pump, n utosmpler, degsser, column oven nd diode-rry detector. The chromtogrphic seprtion ws crried out on the Phenomenex Synergi Fusion RP-C8 ( mm i.d., 5 mm length, 4. lm prticle size) connected with C 8 gurd column (Lng et l., 8). Mobile phses were 5 mmol/l mmonium cette in wter (phse A) nd 5 mmol/l mmonium cette in methnol (phse B) both contining.% formic cid. Flow rte ws.3 ml/min. Grdient elution strted with % A for min, chnged linerly from % to 9% A for 4 min, from 9% to % A for 4 min, nd mintined % A for 6 min. The injection volume ws ll. MS nlysis ws conducted in the positive ion mode of electrospry ionistion (ESI). The MS conditions optimised for detecting nicotinic cid nd nicotinmide were s following; cpillry voltge, 4 V; nebulizer gs (N ) pressure, 45 psi; drying gs (N ) temperture, 35 C; drying gs flow, L/min; frgmentor voltge (pplied to the extrction skimmer), V. For the verifiction of clibrtion stndrd solution on SIM mode, [M+H] + ions of nicotinic cid nd nicotinmide were monitored t m/z 4 nd 3 nd corresponding isotope lbelled ions were detected t m/ z 8 nd 7, respectively. On the selected rection monitoring (SRM) mode, ev of collision energy ws pplied on the collision cell, nd dissocition chnnels of m/z 4? 8 nd m/z 8? 84 were chosen for nicotinic cid nd the isotope lbelled nicotinic cid, respectively, in prepred smples. SRM chnnels for nicotinmide nd nicotinmide-d 4 were m/z 3? 8 nd m/z 7? 84, respectively..5. Clcultion nd uncertinty evlution The mss frctions of nicin (equivlent to nicotinmide) in test smples (mg/kg) were determined by the isotope rtio of nicotinmide/nicotinmide-d 4 (for multivitmin smples without lkline hydrolysis) or of nicotinic cid/nicotinic cid-d 4 (for smples with lkline hydrolysis in the smple preprtion processes) in smple, in comprison with tht in the isotope rtio stndrd s following eqution (De Bivère, 993; Dube et l., 997; Jung et l., 4; Kim et l., ; Lim et l., ; Shin, Kim, Lee, & Hwng, ). All the mesurement dt obtined in this study were converted to the vlue equivlent to nicotinic cid by considering their moleculr weights. C smple ¼ M is-sol;spiked AR smple M s-sol;std C s-sol M smple AR std M is-sol;std where, C smple = concentrtion of nicotinmide in the smple; C s- sol = concentrtion of nicotinmide in the stndrd solution; M smple = mss of the smple tken for nlysis; M s-sol,std = mss of the nicotinmide stndrd solution spiked into isotope rtio stndrd solution; M is-sol,spiked = mss of the nicotinmide-d 4 solution spiked into smple solution; M is-sol,std = mss of the nicotinmide-d 4 solution spiked into isotope rtio stndrd solution; AR smple = observed re rtio of nicotinmide/nicotinmide-d 4, or nicotinic cid/nicotinic cid-d 4 for the smple solution from LC/MS mesurement; AR std = observed re rtio of nicotinmide/nicotinmide-d 4, or nicotinic cid/nicotinic cid-d 4 for the isotope rtio stndrd solution from LC/MS mesurement. The uncertinty evlution in this study ws referred to previously published ppers (Choi, Hwng, So, & Kim, 3; Lim et l., ; Prk, Lee, Lim, Hwng, & So, ) nd Guide to the Expression of Uncertinty in Mesurement (GUM) (BIPM, IEC, IFCC, ISO, IU- PAC, IUPAP & OIML, 995). The uncertinty components in ID-LC/ MS mesurement were ctegorised into systemtic nd rndom effects ccording to their influences on the vrition of multiple mesurements. Consequently, the reltive stndrd uncertinty of the mesurement vlue ws combined the ctegorised uncertinty components by following eqution, u C ¼ u sys þ u rn. The qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi observed results in this study were expressed s men nd expnded uncertinty (U), nd U were clculted by u c multiple with coverge fctor (k), which ws determined from the Student s t- distribution corresponding to the pproprite degrees of freedom nd n 95% confidence for ech men vlues. 3. Results nd discussions 3.. LC/MS performnce The LC conditions for the seprtion of nicotinic cid nd nicotinmide were dpted from prior study by Lng et l. (8) nd the mobile phse ws tested for the effects of compositions nd phs of queous phse. Nicotinic cid nd nicotinmide were hrdly retined on the selected column (Phenomenex Synergi Fusion RP- C8) with methnol, nd were properly seprted on the column when the mobile phse consists of more thn 9% of queous solution. The effects by ph of mobile phse were shown in Fig.. Mobile phse with higher ph provided better retention of nicotinmide. However, the pek of nicotinmide becomes broder nd the pek intensity becomes lower s ph increses. We note tht queous phse with ph 3.6 provide proper seprtion between the two compounds. Therefore, 5 mmol/l mmonium cette buffer with ph 3.6, which ws set by.% formic cid (Fig. b), ws chosen s n queous mobile phse in this study. Grdient elution progrm, which ws run with the selected queous phse nd methnol contining 5 mmol/l mmonium cette, crried out to properly seprte nicotinic cid nd/or nicotinmide from mtrix in current study. The optimistion of the instrumentl sensitivity nd the collection of structurl informtion on nlytes nd their isotope lbelled compounds were performed with the MS/MS experiments. As shown in Fig., nicotinic cid ([M+H] +, m/z 4) produced the frgment ions t m/z 8, which were pyridine ring ions fter losing crboxylic cid. The similr CID pttern ws observed from isotope lbelled nicotinic cid (which ws hydrolysis product of nicotinmide-d 4 ) with [M+H] + pek t m/z 8 nd [M+H COOH] + pek t m/z 84 (Fig. b). Nicotinmide ions ([M+H] + t m/z 3) were dominntly dissocited to m/z 8 s losing the crboxmide from the prent ion (Fig. c), however, nicotinmide-d 4 ([M+H] + t m/z 7) showed the frgment ions t m/z 83 s well s 84 (Fig. d). The dditionl pek t m/z 83 ws inferred to the result of H-D scrmbling in the pyridine ring during the CID process. However, the dditionl pek (m/z 83) brely ffects to the quntifiction of nicin by ID-LC/MS, becuse the scrmbling ws consistently occurred during the CID of nicotinmide-d 4 in stndrd nd smple solution. 3.. Smple preprtion The smple preprtion process ws selected between chemicl hydrolysis nd simple extrction methods depending on forms of nicin existing in smple. Infnt formul contins free nicotinic cid nd nicotinmide s well s low mount of NAD or/nd NADP. Brekfst cerel lso contins free nicotinic cid nd nicotinmide s well s bounded nicotinmide which is originted in cerel grin. Therefore infnt formul nd brekfst cerel require lkline hydrolysis, which convert nicotinmide nd other forms of nicin into free nicotinic cid to determine totl nicin. In the preliminry study, it ws confirmed tht lkline hydrolysis with

4 H. Shin et l. / Food Chemistry 38 (3) b in nicotinmide form ccording to the informtion provided by mnufcturers. This ws confirmed by tht the observed results of nicin were (9.95 ±.5) g/kg nd (9.96 ±.6) g/kg before nd fter the lkline hydrolysis, respectively. Consequently, nicotinmide in multivitmin cn be nlysed fter extrction with wter nd proper dilution without further chemicl process. However, the hydrolysis step must be included for the nlysis of totl nicin in the cse of food supplements prepred with nturl rw mterils Method vlidtion nd nlyticl performnce check Fig.. Totl ion chromtogrms of nicotinic cid (pek ) nd nicotinmide (pek ) produced with vrious phs of queous mobile phse contining 5 mmol/l mmonium cette (: ph 3., b: ph 3.6, c: ph3.9, d: ph 4.6, e: ph 6.6). phs were set by dding formic cid nd ph 3.6 ws nturl ph of.% formic cid in queous phse. The mobile phse isocrticlly rn with 9% of queous phse. sodium hydroxide in the boiling wter for h ws enough to convert ll forms of nicin into nicotinic cid in infnt formuls nd brekfst cerels. As the smple contins excess mount of slt fter ph djustment with HCl solution, SPE process ws necessry for deslting the smple to obtin reproducible dt in mss spectrometric nlysis. During the instrumentl nlysis, nicotinmide nd nicotinic cid were simultneously monitored in order to ssure tht the lkline hydrolysis procedure hd been properly conducted. All multivitmin products obtined from locl mrket were produced by blending rw mterils of vitmins nd hve nicin c d e Vlidtion by reference mterils The vlidtion of the developed ID-LC/MS method ws tested by nlysing the well-chrcterised reference mterils including SRM 849 (NIST) nd BCR 43 (IRMM). SRM 849 hs certificte vlue of (97.5 ±.3) mg/kg s expressed in nicotinmide nd this vlue ws ssigned by ID-LC/MS without ny chemicl tretment. Our observed results by the developed ID-LC/MS method were (97.5 ± 4.3) mg/kg with lkline hydrolysis nd (96.8 ± 4.4) mg/kg without lkline hydrolysis (Tble ). The results obtined by this method gree with the certified vlue within their uncertinties. BCR 43, Brussels sprouts powder, hs certificte vlue of (43 ± 3) mg/kg s expressed in nicin, which indictes totl nicin becuse the vlues were ssigned by microbiologicl ssy (L. plntrum) nd König rection ccording to their certifiction. As shown in Tble, free nicotinmide ws determined to be (9.8 ±.48) mg/kg without lkline hydrolysis. After lkline hydrolysis, however, totl nicin ws determined to be (4. ±.) mg/kg, which gree with the certified vlue within uncertinties. This greement indictes tht the pplied nlyticl method with lkline hydrolysis ws properly determined the bounded nicotinmide nd other sources of nicotinic cid in BCR 43 smple. Though the certified vlue ws produced by microbiologicl ssy which is not considered to hve higher-order metrologicl qulity, the greement of our results with the certified vlue prtly supports the vlidity of this method. All the uncertinties bove were the expnded uncertinties with 95% of level of confidence Repetbility nd reproducibility To test the repetbility of the developed ID-LC/MS method, cndidte reference mterils of infnt formul nd multivitmin powder prepred in this lbortory were used s homogenised smples. The reproducibility of the ID-LC/MS method ws lso confirmed by conducting the sme repetbility test in different dys with one month intervls. In ech time period, new set of multiple stndrd solutions were prepred nd verified by the consistency test mong them s described in experimentl section. Tble shows the observed results for infnt formul nd multivitmin smples t different time periods. In the cse of infnt formul, the men verges of observed results in three different time periods were 8.5, 8. nd 8. mg/kg, nd the reltive stndrd devitions within period were.69%,.75% nd.89%. The men verges of observed results for multivitmin smples in ech time period were 9.9,.7 nd. g/kg, nd the reltive stndrd devition within period ws between.7% nd 3.%. Observed results from the three different time periods show.568% nd.86% of reltive stndrd devition for infnt formul nd multivitmin, respectively. These observed results supported the developed method hd good repetbility nd reproducibility. Consequently, the developed ID-LC/MS method cn be pplied to ssess totl nicin in vrious types of food mtrix, nd preprtion method cn be selected between chemicl hydrolysis nd simple extrction by the forms of nicin in smples.

5 H. Shin et l. / Food Chemistry 38 (3) Fig.. Product ion spectr of collisionlly induced dissocition (CID) of the [M+H] + ions of nicotinic cid (), nicotinic cid-d 4 (b), nicotinmide (c) nd nicotinmide-d 4 (d). Dissocition mechnism of the dominnt chnnel, [M+H] + ions to [M+H COOH] +, ws identicl between two compounds Appliction of the ID-LC/MS method to vrious smples Anlysis of homogenised smples The vlidted ID-LC/MS method ws pplied to homogenised smples of infnt formul nd multivitmin, which were prepred by in-house protocol with the intention of using s cndidte reference mterils. Commercil products were purchsed from different brnds contining different levels of nicin. As shown in Tble 3, homogenised infnt formul nd multivitmin smples were determined s (8.5 ± 3.7) mg/kg nd (9.9 ±.5) g/kg, respectively, which were men vlues of multiple sub-smples nd expnded uncertinties t 95% of confidence level. These uncertinties contin the uncertinty components from both of nlyticl method nd smple inhomogeneity from mesurements of multiple subsmples (n = ). The reltive stndrd devitions (%) were obtined s.89% nd.5% for infnt formul nd multivitmin smples, respectively, nd these referred smple inhomogeneity of the distribution of nicin in smples. Excluded the

6 4 H. Shin et l. / Food Chemistry 38 (3) 9 5 Tble Comprison of observed results nd certified vlues for nicin in SRM 849 nd BCR 43 depend on the lkline hydrolysis prior to ID-LC/MS mesurement. Smples Certified vlue (mg/kg) Observed vlue (mg/kg) c With lkline hydrolysis Without lkline hydrolysis SRM ±.3 d 97.5 ± ± 4.43 BCR 43 b 43 ± 3 4. ±. 9.8 ±.48 b c d Certified vlue ws ssigned by NIST. Certified vlue for nicin ws ssigned by IRMM. Nicotinic cid equivlent. The vlues following ± re the expnded uncertinties of the preceding vlues t the 95% level of confidence. Tble Repetbility nd reproducibility results for nicin nlysis by the developed ID-LC/ MS method pplying to homogenised infnt formul nd multivitmin smples. Subsmple No. Observed results Infnt formul (mg/kg) Multivitmin (g/kg) Period # # # # Averge Stndrd devition.555 (.69 rel% b ).46 (.4 rel%) Period # 8.. # 8..7 # # Averge 8..7 Stndrd devition.4 (.75 rel%).397 (.9 rel%) Period 3 # 8..4 # # # Averge 8.. Stndrd devition.663 (.89 rel%).65 (3. rel%) Averge 8..3 Stndrd devition mong period.46 (.568 rel%).377 (.86 rel%) b Observed results were expressed s men of 4 injections. The unit rel% indictes the reltive percentge of the stndrd devition in comprison with the corresponding men vlue. uncertinty by smple homogeneity, the uncertinty of the developed nlyticl method ws expected s.8% for both of multivitmins nd infnt formul. These levels of smll uncertinties on mesurements support tht the method hs high metrologicl qulity s reference method. Additionlly, homogenised multivitmin smples were nlysed by two different testing lbortories nd reported s.5% nd 4.8% in kg/kg Appliction on the commercil products Tble 4 summrises the observed results nd the expnded uncertinties in comprison with vlues lbelled on the commercil products. The observed results for two different brnds of infnt formul were (7. ± 3.96) mg/kg nd (69.8 ± 3.8) mg/kg, which were higher thn the vlues lbelled by the mnufcturers, 6 mg/kg nd 65 mg/kg, respectively. The lbelled nicin contents of brekfst cerels rnged from 8 mg/kg to 4 mg/kg, however, the mesured vlues were between 5 mg/kg nd 5 mg/kg. The min ingredient for brekfst cerel A nd B were corn nd brekfst cerel C ws brown rice, nd nicotinmide ws dded s n dditionl source of nicin for ll three brekfst cerels. The discrepncy between the declred vlues nd the observed vlues seems to the results tht lkline hydrolysis relesed the bounded nicotinmide from corn or brown rice. The developed method is Tble 3 Observed results of nicin in homogenised smples of infnt formul nd multivitmin. Homogenised smples Observed vlues RSD b (%) Infnt formul (mg/kg) KRISS 8.5 ± Multivitmin (g/kg) KRISS 9.9 ±.5.5 Lb A c.5 Lb B c 4.8 The observed vlues by KRISS (Kore Reserch Institute of Stndrds nd Science) represented men ± expnded uncertinties t the 95% level of confidence. Smple in-homogeneity from mesurements of multiple subsmples (n = ) ws included s n uncertinty source. b Reltive stndrd devition of mesurement vlues from multiple subsmples, indicting smple in-homogeneity of the distribution of nicin in smple. c Results were collected from two different testing lbortories nd were nlysed by LC UV. Tble 4 Observed results of totl nicin contents (mg/kg) in infnt formul nd brekfst cerel products, nd free nicotinmide contents (g/kg) in multivitmin products by developed ID-LC/MS method. Smples Declred vlues Observed vlues Infnt formul A 6 (mg/kg) 7. ± 3.96 (mg/kg) Infnt formul B 65 (mg/kg) 69.8 ± 3.8 (mg/kg) Brekfst cerel A 4 (mg/kg) 5 ±. (mg/kg) Brekfst cerel B 8 (mg/kg) 5 ± 7.5 (mg/kg) Brekfst cerel C 8 (mg/kg) 7 ±.7 (mg/kg) Multivitmin A 7 (g/kg) 7.8 ±.594 (g/kg) Multivitmin B 3 (g/kg) 4.5 ±.6 (g/kg) Multivitmin C 5 (g/kg) 6.9 ±. (g/kg) The results re mens of triplicte determintion nd expnded uncertinties of the preceding vlues t the 95% level of confidence. ble to determine totl nicin including free nicotinic cid nd nicotinmide s well s bounded nicotinmide in the smple mtrix. In the cse of multivitmin, the ssessments by the developed ID- LC/MS were similr with the lbelled vlues. All three multivitmins include only nicotinmide s nicin source. 4. Conclusion An isotope dilution mss spectrometric method bsed on the LC/MS hs been estblished s cndidte reference method for the ccurte determintion of nicin in infnt formul, brekfst cerel nd multivitmin products. Depending on the existing forms of nicin in the smples, the ppliction of chemicl tretment like lkline hydrolysis helped to relese into free nicotinic cid nd to determine the totl nicin in smples. The repetbility/reproducibility studies nd the uncertinty evlution results hve proven tht the developed method hs metrologicl qulity which is dequte to be used s reference method.

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