MERINO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 2, 2000

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1 MERINO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 2, FOOD COMPOSITION AND ADDITIVES Liquid Chromtogrphic Determintion of Residul Nitrite/Nitrte in Foods: NMKL 1 Collbortive Study MERINO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 2, 2000 LEONARDO MERINO, ULLA EDBERG, nd GEORG FUCHS Ntionl Food Administrtion, Chemistry Division 2, PO Box 622, S Uppsl, Sweden PER ÅMAN Swedish University of Agriculturl Sciences, Box 7051, S Uppsl, Sweden Collbortors: G. Corbri; L. Dhlberg; V. D-Riz; S. Eerol; R. Gerrd; D. Grncher; S. Jensen; A. Kngro; A. Kjolby; L. Lundberg; M. Mrin; S. Melnd; B. Muriel; M. Nicols; T. Rvn; M. Tuhknen; T. Vrtil; G. Zieprth Nitrite nd nitrte re used s dditives in the food industry to provide color nd tste nd to control undesirble gs nd flvor production by nerobic bcteri by virtue of their ntimicrobil properties. The nlyticl method tht hs been widely used to determine nitrite nd nitrte involves the use of toxic cdmium. In response to request from the Nordic Committee on Food Anlysis, study ws performed to obtin n lterntive chromtogrphic method to determine residul nitrite nd nitrte in met products. The study ws done in 3 stges: (1) comprtive evlution of the performnce of 3 liquid chromtogrphic methods, (2) internl vlidtion of the selected ion chromtogrphic method, nd (3) collbortive study in which 17 lbortories from Europen countries prticipted. Furthermore, the pplicbility of the method to mtrixes other thn met nd met products ws demonstrted. The results of the collbortive study show tht the Europen Prestndrd prenv is well suited for the determintion of nitrite nd nitrte in different foods (e.g., met products, vegetbles, bby food, nd cheese). The limits of detection for nitrite nd nitrte ions re 1 nd 10 mg/kg, respectively. Recoveries of residul nitrite/nitrte rnged from 96 to 108%. Repetbility nd reproducibility were stisfctory. Received Mrch 24, Accepted by JL August 25, Nordic Committee on Food Anlysis (Secretrit Generl, c/o Veterinærinstituttet, Pb 8156 Dep., N-0033 Oslo, Norwy). Nitrite either lone or in combintion with the nitrte slt is used s curing gent for met products. The role of nitrite is to produce the chrcteristic pink color, texture, nd flvor nd, especilly, to provide protection ginst poisonous microorgnisms, such s Clostridium botulinum, which is of prticulr interest. Becuse of toxicologicl considertions, nitrite nd nitrte re the concern of gencies responsible for food sfety. Nitrite (nd nitrte s its reservoir) is precursor of the crcinogenic N-nitroso compounds. Nitrite is n unstble ion nd undergoes series of rections s soon s it is dded to food products. In n cid environment, the nitrite ion ionizes to yield nitrous cid, which further decomposes to yield nitric oxide (NO), n importnt product from the stndpoint of color fixtion in cured met. Nitric oxide rects with myoglobin to produce the red pigment nitrosomyoglobin. Time, temperture, ph, nd dditives hve n importnt effect on the depletion of nitrite in cured met. Nordin (1) reported n eqution tht reltes the rte of nitrite depletion to time, temperture, nd ph. Sen et l. (2) hd considerble losses of nitrite during nlysis of cidic smples. Fujimki et l. (3) studied the fte of nitrite in met-curing model systems tht showed the effect of sodium scorbte in the decomposition of nitrite. These fctors, besides the fct tht the nlyticl procedure itself promotes certin chemicl rections, explin why determintion of nitrite in met products is difficult. On the other hnd, Olsmn nd Cees (4) differentited between residul free nitrite nd protein-bound nitrite, nd they proposed method to estimte the protein-bound nd the residul free nitrite content of met. The principle on which the method is bsed is the relese of the bound nitrite, s described by the following rection: RS NO + HgCl 2 +H 2 O RS HgCl + HNO 2 + HCl Acording to our nlyticl objective, we were not interested in relesing nitrite from ny rection products (even though they re reservoirs for nitrosmine formtion). On the contrry, the choice of the conditions nd chemicls used in the methods we evluted ws guided by the desire to minimize the losses of or increse the numbers of free nitrite/nitrte ions present during the nlyticl procedures. Therefore, the nlyticl methods determine the residul nitrite/nitrte, i.e., the free nitrite/nitrte ions tht re present when the chemicl nlysis is initited.

2 366 MERINO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 2, 2000 Experimentl Anlyticl Methods Assessed Three ion chromtogrphic methods were ssessed: (1) Wek nionic exchnger (minopropyl bonded-phse column). This liquid chromtogrphic (LC) method is bsed on the Europen Stndrd pren Prt 2 (5). (2) Strong nionic exchnger (quternry mmonium column). This method ws proposed to the Nordic Committee on Food Anlysis (NMKL) some yers go. Erlier collbortive studies showed low recoveries of nitrites in met, nd in generl the performnce of the method ws vrible. (3) Strong nionic exchnger (quternry mmonium column). This method ws proposed to the Europen Committee for Stndrdistion (CEN). This method is ctully Europen Prestndrd (6). The methods re summrized in Tble 1. Wek Anionic Exchnger Method (Aminopropyl Bonded-Phse Column) The method hs been vlidted for determintion of nitrte in vegetbles. The liquid chromtogrm of stndrd solution contining nitrite nd nitrte, ech t 5 mg/l, is shown in Figure 1. The seprtion of the peks ws indequte, but this is not shortcoming in the nlysis of vegetbles, which usully do not contin nitrite. Although the compounds were dded t the sme concentrtion, the nitrite pek is smller thn the nitrte pek. Becuse the mobile phse is ph 3, the NO 2 forms HNO 2 (pk = 3.4), llowing the possibility of nitrite losses by the following rection in cidic solution: 3HNO 2 HNO 3 +H 2 O + 2NO To void this rection, the ph of the mobile phse ws incresed from 3 to 9. At this higher ph, the mobile phse showed high molr bsorptivity t 205 nm tht interfered with the mesurement of the nlytes. The mobile phse ws chnged to 5 mm K 2 HPO 4 in 15% cetonitrile, ph 8.6 (the mobile phse from the NMKL method). No improvement ws chieved; just 1 pek ppered. No further evlutions of this method were mde becuse of its poor performnce. Strong Anionic Exchnger Methods; Comprison of NMKL nd CEN Methods The NMKL nd the CEN methods re bsed on the sme ion chromtogrphic principle, but they use different mobile phses nd preprtion procedures. Assys were performed to ssess which mobile phses nd smple preprtions give better performnce. Evlution of Mobile Phse The extrct of met product (susge) ws used for the nlyses. The extrct ws seprted on strong nionic exchnger column (IC-PAK A mm) by using 2 different mobile phses: 5 mm K 2 HPO 4 in 15% cetonitrile, ph 8.6 (NMKL mobile phse), nd buffer (boric cid, gluconic cid, lithium hydroxide, nd glycerol) in 12.5% cetonitrile, ph 6.5 (CEN mobile phse). Figure 2 shows the Tble 1. Summrized description of the nlyticl methods ssessed Smple preprtion Chromtogrphic conditions Injection volume Extrction Clrifiction Column Mobile phse Flow Detection Method 0.7 ml/min 205 nm 20 µl 57 mm K2HPO4 in 5% cetonitrile. Adjust to ph 3 with H3PO4 Aminopropyl bonded phse LiChrosorb-NH2 (Hichrom) Filter through filter pper nd membrne filter Aminopropyl 10 g smple homogenized in hot wter (50 80 C), plced in boiling wter bth 15 min 0.6 ml/min 215 nm 20 µl 5mMK2HPO4 in 15% cetonitrile, ph 8.6 IC-PAK A mm (Wters) 2 ml Crrez I nd Crrez II solutions +5mLborx solution. Filter through filter pper nd membrne filter NMKL 10 g smple g ctivted chrcol +5mLsturted borx + 50 ml wter (80 C), plced in boiling wter bth 15 min 1 ml/min 205 nm 100 µl Buffer (boric cid, gluconic cid, lithium hydroxide, glycerol) in 12.5% cetonitrile, ph 6.5 IC-PAK A HC (High Cpcity) mm (Wters) 50 ml cetonitrile. Filter through filter pper nd membrne filter CEN 10 g smple homogenized in 50 ml wter (50 60 C)

3 MERINO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 2, Figure 1. Chromtogrm obtined with the wek nionic exchnger column for n queous stndrd solution contining nitrite nd nitrte t 5 mg/l. resolution of the peks obtined with the NMKL nd CEN mobile phses. The CEN mobile phse (buffer in 12.5% cetonitrile, ph 6.5) shows better seprtion of the peks. In ddition, this mobile phse hs prcticl dvntge, i.e., it my be left on the chromtogrphic column s storing solvent. Smple Preprtion (Extrction nd Clrifiction) Met products, purchsed from locl supermrket, were used for the comprison. Both spiked nd unspiked met products were nlyzed. One set of smples ws extrcted nd clrified exctly s described in the CEN method. The other set of smples ws treted s described in the NMKL method with modifiction: the smple ws homogenized with wter t 60 C (not t 80 C), nd it ws not plced in boiling wter bth for 15 min. Tble 2 shows the results of the evlution. Smple preprtion ffected the recoveries of nitrite nd nitrte. The smples pretreted ccording to the CEN method gve consistently higher recoveries thn did smples pretreted ccording to the modified NMKL method. Appliction of the pired t-test showed significnt difference in the results. The theoreticl vlue of t ws lower (2.73) thn the observed t vlues for nitrite nd nitrte (4.73 nd 4.10, respectively; p = 0.05; 18 degrees of freedom). Lter ssys showed tht the deproteinizing/clering Crrez regents interfere in the nlyses (see below). Smple Preprtion nd Mtrix ph One importnt difference in the smple preprtion procedures of the NMKL nd CEN methods is tht the former extrcts the nitrite/nitrte by using borx buffer, which is intended to counterct the effect of low ph on the stbility of nitrite/nitrte. In order to evlute if the ddition of borx buffer ctully enhnced the recovery of the nlytes, some ssys were performed in which the ph of the mtrix ws chnged before the smples were spiked with nitrite nd nitrte ions. () (b) Figure 2. Seprtion of nitrite nd nitrte in susge extrct on strong nionic exchnger column: () with the NMKL mobile phse nd (b) with the CEN mobile phse.

4 368 MERINO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 2, 2000 Tble 2. methods Recovery of nitrite nd nitrte from met products pretreted ccording to the CEN nd modified NMKL Nitrite Nitrte CEN NMKL CEN NMKL Smple mg/kg % mg/kg % mg/kg % mg/kg % Pork susge b NF c NF Pork susge + std b Beef b NF NF NF NF Beef + std Hm NF NF NF NF Hm + std Pte b NA d NF NA 16 Pte + std b NA NA Chorizo Chorizo + std Bcon NF NF NF NF Bcon + std Hot dog b Slmi NF NF NF NF Mortdell + std NA NA b c d Std = stndrd. Men of 2 results. NF = not found. NA = not nlyzed. A slmi smple with ph < 5 ws spiked directly nd fter ph djustment with borx, borx plus 1M NOH, nd 5M NOH. The smples were extrcted ccording to the NMKL nd CEN methods. The results re shown in Tble 3. From Tble 3 it cn be concluded tht the higher the ph of the mtrix, the higher the recovery obtined. From n nlyticl point of view, the results suggest tht the difference in the ph of the extrction procedures of the methods hs minor influence on recovery, compred with the initil ph of the mtrix. Europen Prestndrd s Horizontl Method; Anlysis of Vegetbles nd Diry Products Vegetbles (onion, crrot, potto, cucumber, spinch, nd lettuce) were nlyzed in prllel by using the wek nionic exchnger column (5) nd the strong nionic exchnger column (6). The chromtogrms (Figure 3) show tht for certin vegetbles (i.e., crrot, onion, nd potto), which normlly hve very low concentrtion of nitrte, the strong nionic exchnger column is the only one cpble of seprting this ion. The seprtion obtined by using the wek nionic exchnger column (Figure 3) did not llow clcultion of the nitrte concentrtion. However, in the cses for which both methods chieved good chromtogrphic seprtion (i.e., cucumber, lettuce, nd spinch), the concentrtions of nitrte were the sme (dt not shown). A cheese smple ws lso nlyzed. Good seprtion nd recovery were obtined. Method Vlidtion: A Simple nd Prcticl Experimentl Design Bsed on Anlysis of Vrince (ANOVA) Mny guidelines hve been written, most of them describing interesting spects of the elements of the vlidtion procedure, but the experimentl designs to estimte these prmeters re omitted or pertin to severl replicte nlyses of different smples. In generl, good experimentl design would enble the nlyst to estimte most of the prmeters with minimum of nlyses. This tsk is fcilitted by the interreltion between the elements of the vlidtion. However, the diverse pproches in the literture give the impression tht to consistently follow the vlidtion procedure proposed, n nlyst must undertke enormous nd tedious work (7), but this is not the cse. Wilson nd Chessemn (8) proposed n experimentl design using ANOVA to give n estimte of precision, bis, nd limit of detection. The design specifies tht nlyses ought to be performed in severl btches over period of 1 2 weeks. Ech btch consists of replicte nlyses of blnks (limit of detection), stndrd solutions (sensitivity, linerity, nd rnge), nd both spiked nd unspiked smples (recovery). To estimte precision over concentrtion rnge, t lest

5 MERINO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 2, Tble 3. Influence of mtrix ph on recovery of nitrite spike Nitrite CEN NMKL Smple ph mg/kg % mg/kg % Slmi (sl.) 4.57 NF NF Sl. + stndrd (std) Sl. + borx (bx) + std Sl. + bx + 1M NOH + std Sl. + 5M NOH + std 10.4 NA b b NF = not found. NA = not nlyzed. 2 stndrd solutions re recommended for the upper nd lower concentrtions of interest. The dvntge of this pproch is tht it estimtes the within-btch stndrd devition s pooled vlues from ll the btches. This pproch is of prcticl importnce becuse it provides resonble indiction of wht is chievble. In ddition, this bsic experimentl design includes the internl reproducibility (between-btches stndrd devition, S b ), which hs n importnt consequence in reltion to the distinction between the vlidtion requirement of single lbortory nd of regultory body. Actully, prerequisite for the cceptnce of method s officil by committees or regultory gencies is estimtion of its performnce through collbortive study. Becuse collbortive study is n expensive nd work-intensive tsk, it is not suitble option for ll lbortories. However, by estimting the internl reproducibility, the nlyst obtins preliminry informtion bout the reproducibility of the method, i.e., the robustness under the best conditions. One generl point is worth noting here. The vlidtion procedure should consist of the nlysis of n portions in m btches. It is recommended tht n = 2 nd m = The product m n should never be < 10; 20 is better. Such design encourges the use of sttistic criterion to estimte the number of nlyses, to help to prevent n rbitrry nd inpproprite decision. A detiled discussion of the ANOVA s pplied to method vlidtion is beyond the scope of the present rticle, but the prcticl utility of ANOVA should not be overlooked. To put it more simply, its role in the experimentl design of the vlidtion method should be similr to the importnce it hs when interlbortory studies re conducted (9). Vlidtion of the Europen Prestndrd prenv The ppliction rnge, linerity, precision, detection limit, nd percent recovery were determined for the method. The procedure ws s follows: Foods (susge, corned beef, hm, chorizo, cheese, potto, nd bby food) purchsed from locl supermrket were homogenized nd spiked (except potto nd chorizo) with different concentrtions of ni- () (b) Figure 3. Chromtogrms obtined from nlyses of crrot smples using () wek nionic exchnger column (CEN, Prt 2, 1998) nd (b) strong nionic exchnger column (CEN, Prt 4, 1998).

6 370 MERINO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 2, 2000 Tble 4. Btch Nitrite nd nitrte found in susge (mg/kg) NO - 2 NO - 3 Test 1 Test 2 Test 1 Test trite nd nitrte ions (dt not shown). Smples were stored t 4 C during the study. Six stndrd solutions of nitrite nd nitrte were used to clculte the clibrtion curve. In ddition, 2 replicte stndrd solutions of lower (0.5 mg/l) nd upper (20 mg/l) concentrtions were nlyzed s smples. The test consisted of the nlyses of replicte smples (n = 2) in ech btch (m = 5). The totl of 10 nlyses (m n = 10) gives resonble minimum number of degrees of freedom for the clcultion of the within-btch stndrd devition (S w ) nd between-btches stndrd devition (S b ) for ech smple. Results nd Discussion Linerity nd Rnge Linerity ws ddressed by prepring stndrd solutions of nitrite nd nitrte t 6 levels (0.5, 1.0, 3.0, 5.0, 10.0, nd 20.0 mg/l). Stndrds were injected in rndom order into the chromtogrph. A liner regression nlysis of nlyte concentrtion vs pek response ws performed. The coefficients of determintion (r 2 ) for nitrite nd nitrte were nd , respectively. The upper concentrtion of the rnge is equivlent to 400 mg nitrite nd/or nitrte/kg smple. Precision nd Internl Reproducibility As n exmple of the repetbility obtined on precision, only the results for the susge smple re shown in Tbles 4 nd 5. The results of the ANOVA re given in Tble 5. The criticl vlue of F (P = 0.01, n 1 =4,n 2 = 5) is 11.4, which is less thn nd 44.6, the F vlues for nitrite nd nitrte, respectively. The F-test showed tht there is significnt difference between the within-btch nd between-btches stndrd devitions; this difference is explined by the instbility of the nlytes (Figure 4). A prerequisite for the experimentl design bsed on ANOVA is the stbility of the smple. Unfortuntely, we were unble to chieve n cceptble level of control over the instbility of the nlytes through the use of borx, time, nd low temperture. Thus, the only wy to estimte the reproducibility of the method ws through n interlbortory study (see below, Collbortive Study). Limit of Detection nd Limit of Determintion The NMKL Procedure No. 4 (10) estblishes tht the detection nd determintion limits re clculted by multiplying the stndrd devition of the blnk by 3 nd 10, respectively. Becuse the studied method filed to detect n nlyticl response for the blnk smple, the stndrd devition of the stndrd solution of lowest concentrtion (0.5 mg/l) ws used to clculte these limits (11). Thus, the detection limits of nitrite nd nitrte re 1 nd 10 mg/kg, respectively. Bis nd Recovery Test The experimentl recovery is obtined from the difference between 2 mesurements (smple nd spiked smples), ccording to the following reltionship: Recovery, % = (totl nlyte found nlyte originlly present) 100/nlyte spike The recovery rnges for nitrite nd nitrte estimted immeditely fter smple preprtion (dte not reported) were % nd %, respectively. The poor recovery of the lter btches ws ttributed to the instbility of the nlytes. Identifiction of Systemtic Errors: Constnt-Type nd Reltive-Type Errors According to Linnig et l. (12), reltive-type nd constnt-type systemtic errors re ssocited with the slope nd intercept of plot of found vs dded concentrtion of the nlyte, respectively. They lso stte tht the effect of reltive-type error is generlly more importnt in the determintion of mjor components, wheres constnt-type errors re more serious in the determintion of trce components. The Tble 5. Summry of sums of squres nd degrees of freedom for susge smples Smple Source of vribility Sums of squres Degrees of freedom Men squres (MS) F Susge NO - 2 Between-btches (B) (MSB) 6852 Within-btch (W) (MSW) Susge NO - 3 Between-btches Within-btch F = (MSB)/(MSW).

7 MERINO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 2, Figure 4. Rte of depletion of nitrite nd nitrte spikes in hm. bility to use the method to nlyze other foods, which usully hve low concentrtions of nitrte nd nitrite (bby food, cheese), mde it necessry to identify these systemtic errors. An experiment ws performed. Minced met smples were spiked with different concentrtions of nitrite nd nitrte ions. The smples were nlyzed immeditely fter preprtion. Exmintion of the rtio vlues (columns 3 nd 5, Tble 6) shows tht the nitrite rtios re distributed round vlue close to unity, wheres the nitrte rtios decrese when the mount of nlyte increses. This difference suggests the presence of constnt-type systemtic error in the results of the nitrte nlyses. A correction fctor should be used, if meningful results re to be obtined. The intercept of the regression line for nitrte is 13.4 (Figure 5). This vlue could be n dequte correction fctor for the constnt-type error in the nlyses for residul nitrte in minced met. On the other hnd, there is no definitive criterion for judging whether the intercept is n dequte correction fctor for the nlyses for nitrite, becuse the observed intercept flls into the confidence intervl of the clculted intercept (dt not shown). The existence of reltive-type systemtic error (error proportionl to the mount of smple nlyzed) is relted to slope different from unity. Nitrite nd nitrte stright lines show slopes of 1.10 nd 1.09, respectively, which indicte reltive error of bout 10%. Comprison of Ion Chromtogrphic (CEN-Prt 4) nd Spectrophotometric (CEN-Prt 3) Methods for Determintion of Nitrite in Minced Met Spiked minced met smples were nlyzed for nitrite by both methods. The spectrophotometric method is bsed on Tble 6. Results of nlyses of spiked minced met smples for nitrite nd nitrte Nitrite Nitrte NO - + NO- 2 3 dded, mg/kg Found, mg/kg Rtio, found/dded Found, mg/kg Rtio, found/dded Ech vlue is the men of 2 results.

8 372 MERINO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 2, 2000 Figure 5. Effect of experimentl errors in nitrte determintion. dizotiztion of sulfnilmide by nitrite, followed by coupling with N-(1-nphthyl)ethylenedimine dichloride to form n zo dye, which is mesured spectrophotometriclly t 540 nm (13). The pired t-test did not give significnt difference (columns 2 nd 3, Tble 7). Furthermore, nother portion ws nlyzed simultneously by the sme ion chromtogrphic method using cetonitrile nd the Crrez solution, which re the clrifiction solutions of the CEN nd NMKL methods, respectively (columns 3 nd 4, Tble 7). The results show the disturbing effect of Crrez solution when the ion chromtogrphic method is used. These results gree very well with those of previous nlyses performed during the evlution of the methods tht led to the choice of the Europen Prestndrd (Tble 2). Therefore, the Crrez solution is not recommended s clrifying solution for the ion chromtogrphic method. Collbortive Study Prticipting Lbortories, Test Mterils, nd Sttisticl Procedure A totl of 17 lbortories prticipted in the collbortive studies I nd II. The prticipting lbortories were in Denmrk, Estoni, Finlnd, Frnce, Germny, Itly, Norwy, Spin, nd Sweden. The procedure estblished by AOAC INTERNATIONAL to conduct collbortive study ws followed (14). If cidic, the test mterils were neutrlized with solution of 5M NOH nd spiked with sturted solution of borx to increse the ph. The test mterils were sent by courier in freeze-box. Recommendtions were given to the prticipnts to store the test mterils in refrigertor (4ºC) nd strt the nlysis in the morning on the sme dy. Ech lbortory ws provided well in dvnce with the written version of the method, the schedule, instructions, etc. Tble 7. Comprison of residul nitrite levels in minced met s determined by 2 different methods Found, mg/kg Spectrophotometry Liquid chromtogrphy NO NO- 3 dded, mg/kg Crrez solution Acetonitrile Crrez solution ND b b Ech vlue is the men of 2 results. ND = not detected.

9 MERINO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 2, Sttisticl Anlysis The bsis of the outliers test is to determine if the cndidte outlier vlues re consistent with the Gussin distribution ssumed priori. The outliers exclusion ws done with the Cochrn test nd the single nd double Grubbs tests t the 2.5% rejection level. The Cochrn test is for the removl of lbortories showing significntly greter vribility mong replictes (blind duplictes). The Grubbs test is for the removl of lbortories with extreme vlues. The Horrt vlues were used to judge the relibility of the method. The Horrt vlue for reproducibility is the observed (RSD R ) vlue divided by the RSD R vlue clculted from the Horwitz eqution t the concentrtion of interest (RSD R =2 (1 0.5logC) ). If the Horrt vlue is 2, then the method my be ssumed to hve stisfctory reproducibility (15). This is the cceptbility criterion. Collbortive Study I Results were received from 13 lbortories (Tble 8). Dt for the test mterils were rounded without decimls. The test mterils were the following: A = susge smple spiked with low concentrtion of nitrite nd nitrte; B, D = susge smple spiked with high concentrtion of nitrite nd nitrte; C = bby food smple spiked with nitrite nd nitrte; E = cbbge smple; nd Std = 10 mg NO - 2 /L nd 10 mg NO- 3 /L. The results from lbortories 9 nd 11 were considered s invlid dt.when the outliers tests were pplied to ll lbortories, lbortories 9 nd 11 hd the most outliers. Furthermore, the results of the check stndrd solution were significntly different from those of other lbortories. Becuse the effect of retining rel outlier is usully more serious thn the effect of rejecting vlid point, the decision to eliminte ll results of these lbortories s invlid dt is correct. Vlid dt re the vlues tht the collbortor hs no reson to suspect s being wrong (14). The outliers tests were pplied to the results of the remining lbortories. Lbortory 2 hd Grubbs outliers in 4 of 5 nlyses for nitrte. Lbortory 4 hd Grubbs outlier for the susge smple (test mteril A). In ll nlyses of the preliminry collbortive study, the precision (repetbility nd reproducibility) ws stisfctory. Tble 8. Reported vlues of nitrite nd nitrte from collbortive study I NO - 2, mg/kg NO - 3, mg/kg Lbortory code Std, mg/l A B C D E Std, mg/l A B C D E b 210 b 173 b 210 b b c c b Accepted results No. of outliers Men d SD r e SD R RSD R, % f Horrt vlue b c d e f Std = stndrd. Grubbs outlier. Invlid dt. SD r = repetbility stndrd devition (clculted for the blind duplicte test mterils). SD R = reproducibility stndrd devition. RSD R, % = reproducibility reltive stndrd devition.

10 374 MERINO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 2, 2000 Collbortive Study II Results were received from 15 lbortories (Tble 9). The test mterils were the following: F, K = slmi smple spiked with low concentrtion of nitrite nd nitrte; G = lettuce smple; H = pté smple spiked with nitrite nd nitrte; I = slmi smple spiked with nitrite nd nitrte; J = cheese smple; nd Std = 5 mg NO - /L nd 5 mg NO- 2 3 /L. Lbortories 9 nd 11 hd Cochrn outliers. Lbortories 3, 4, 8, 9, 11, 13, nd 14 hd Grubbs outliers. Test mterils G nd J contined only nitrte. However, some lbortories reported very low concentrtion of nitrite. When the retention times were checked, most of them showed significnt difference from retention times reported by the other lbortories. These results (which could indicte the presence of interfering substnces) were considered s invlid dt. On the bsis of the Horrt vlues, 3 out of 10 did not fulfill the cceptnce criterion (see Tble 9, Horrt vlues; vlues re not given for test mterils G nd J, which hd nitrite concentrtion below the limit of detection). Two comments should be mde. First, test mterils F nd K were the sme (blind duplictes). In one cse the cceptnce criterion ws fulfilled, nd in the other cse it ws not. Second, the concentrtion of the nitrite spike in the test mterils ws very close to the limit of determintion (5 nd 25 mg/kg for nitrite nd nitrte ions, respectively, vlues obtined in the vlidtion performed by our lbortory), nd obviously the uncertinty is higher t this level. Tble 9. Reported vlues of nitrite nd nitrte from collbortive study II NO - 2, mg/kg NO - 3, mg/kg Lbortory code Std, mg/l F G H I J K Std, mg/l F G H I J K b c c c c b c c c c d b c c c c c c c c Accepted results No. of outliers Men <LOD e <LOD f SD r g SD R RSD R, % h Horrt vlue b c d e f g h Std = stndrd. Test mterils F nd K gve Cochrn outliers (nitrite). Grubbs outlier. Excess dt reported. LOD = limit of detection. SD r = repetbility stndrd devition. SD R = reproducibility stndrd devition. RSD R,% = reproducibility reltive stndrd devition.

11 MERINO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 2, Conclusions Nitrite determintion with the wek nionic exchnger column, CEN-Prt 2 (5), is not suitble. This method is not relible for the nlysis of certin vegetbles with low concentrtions of nitrte. The ph of the mtrix t the time of the ddition of nitrite/nitrte hs the gretest influence on the stbility of these ions; the higher the ph, the better the recovery. Smple extrction with buffer hs minor influence, compred with the ph of the mtrix. Recoveries of nitrite from met products, vegetbles, cheese, nd bby food rnged from 96 to 108% with CEN-Prt 4 (6). The corresponding rnge for nitrte recovery ws %. The detection limits for nitrite nd nitrte ions were 1 nd 10 mg/kg, respectively. The limits of determintion were 5 nd 25 mg/kg for nitrite nd nitrte ions, respectively. Constnt-type nd reltive-type systemtic errors were found in the nlysis for nitrite nd nitrte in minced met. To obtin ccurte nlyticl results, correction of this systemtic error is necessry. Reltive-type errors were pproximtely 10% for both nlytes. In generl, the performnce of the method differs when nitrite nd nitrte re determined. Better performnce is obtined for nitrite determintions. The study shows tht the method cn be pplied to the nlysis of foods other thn met nd met products, e.g., bby food, vegetbles, nd cheese. In nlyses for nitrite, no sttisticlly significnt difference ws found between the methods studied, CEN-Prt 4 (6) nd the spectrophotometric method, CEN-Prt 3 (13). The precision of the method fulfills the criteri recommended by AOAC INTERNATIONAL (14) nd the Europen Committee for Stndrdiztion (15); therefore, CEN-Prt 4 (6) is suitble for the determintion of nitrte nd/or nitrte in vrious kinds of mtrixes. Acknowledgments We thnk the following lbortories for their prticiption: Anstlten veterinärmedicin och livsmedel, Helsinki, Finlnd Chemisches Untersuchungsmt Minz, Minz, Germny Ministere de L Agriculture (CNEVA), Misons, Frnce Centre Technique de l Slison (CTSCCV), Misons, Frnce ENVL-Lbo Chrles FLACHT, Mrcy L Etoile, Frnce Institute of Food Reserch nd Nutrition, Ringsted, Denmrk Helsingin ympäristölbortorio, Helsinki, Finlnd Joensuun kupungin eli.-j ympäristölb, Joensuun, Finlnd Lbortorio Arbitrl-Agrolimentrio, Mdrid, Spin Lb. Interrégionl de l Concurrence (DGCCRF), Illkirch Grffenstden, Frnce Næringsmiddeltillsyn for Midt-Roglnd, Forus, Norwy Ntionl Food Administrtion, Uppsl, Sweden Dnish Met Reserch Institute, Roskilde, Denmrk Sttens Lbortorium A/S, Holstebro, Denmrk Stzione Sperim. Ind. Conserve Alimentri, Prm, Itly Swedish Met Reserch Institute, K vlinge, Sweden Trtu Public Helth Service, Trtu, Estoni References (1) Nordin, H.R. (1969) J. Technol. Aliment. 2, (2) Sen, N.P., Young, C.L., & McPherson, M. (1979) J. Assoc. Off. Anl. Chem. 62, (3) Fujimki, M., Emi, M., & Okitni, A. (1975) Agric. Biol. Chem. 39, (4) Olsmn, W., & Cees, M. (1977) Z. Lebensm. Unters. Forsch. 164, (5) Europen Committee for Stndrdiztion (CEN) (1998) Europen Stndrd pr ENV Prt 2, HPLC/IC Method for the Determintion of Nitrte Content of Vegetbles nd Vegetble Products, Brussels, Belgium (6) Europen Committee for Stndrdiztion (CEN) (1998) Europen Prestndrd pr ENV Prt 4, IC Method for the Determintion of Nitrte nd Nitrite Content of Met Products, Brussels, Belgium (7) EURACHEM Drft 1.0 (1996) Method Vlidtion, A Lbortory Guide, LGC, with the support of the UK DTI VAM inititive, Middlesex, UK (8) Wilson, A.L., & Chessemn, R.V. (1978) Mnul on Anlyticl Qulity Control for the Wter Industry, Technicl Report Tr66, Wter Reserch Center, Bucks, UK (9) Thompson, M., & Wood, R. (1993) J. AOAC Int. 76, (10) Nordic Committee on Food Anlysis (1996) NMKL Procedure No. 4, Vlidtion of Chemicl Anlyticl Methods, NMKL, Espoo, Finlnd (11) Kirchmer, C.J. (1988) in Estimtion of Detection Limits for Environmentl Anlyticl Procedures, Ch. 4, Americn Chemicl Society, Wshington, DC, pp (12) Linnig, F., Mndel, J., & Peterson, J.M. (1954) Anl. Chem. 26, (13) Europen Committee for Stndrdiztion (CEN) (1998) Europen Prestndrd pr ENV Prt 3, Spectrometric Determintion of Nitrte nd Nitrite Content of Met Products fter Enzymtic Reduction of Nitrte to Nitrite, Brussels, Belgium (14) AOAC INTERNATIONAL (1995) J. AOAC Int. 78, 143A 160A (15) CEN/TC 275/WG 7 N 103 (1997) Report of the 6th Meeting of the CEN/TC 275/WG 7 N 103, Annex B, Secretrit DIN, Berlin, Germny

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