Detection and quantitation of additions of soybean proteins in cured-meat products by perfusion reversed-phase high-performance liquid chromatography

J. Sep. Sci. 2005, 28, 987–995 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim O iginal Pape C iado, Cas o-Rubio, Ga ca-Ruiz, Ga ca, Ma ina 987 Mnica C iado Flo en ina Cas o-Rubio Ca men Ga ca-Ruiz Ma a Concepcin Ga ca Ma a Luisa Ma ina Depa men o Analy ical Chemis y, Facul y o Chemis y, Uni e si y o Alcal, C a. Mad id- Ba celona Km. 33.600, 28871 Alcal de Hena es (Mad id), Spain De ec ion and quan i a ion o addi ions o soybean p o eins in cu ed-mea p oduc s by pe usion e e sed-phase high-pe o mance liquid ch oma og aphy Pe usion liquid ch oma og aphy has been applied in his wo k o he de e mina ion o soybean p o eins in comme cially a ailable cu ed mea p oduc s, enabling he de ec- ion o addi ions o soybean p o eins in cu ed mea p oduc s o which he addi ion o hese ege able p o eins is o bidden and he quan i a ion o soybean p o eins in cu ed mea p oduc s o which he addi ion o hese p o eins is allowed up o a ce ain limi . The analy ical me hodology is based on a sample ea men ( a ex ac ion and soybean p o ein solubiliza ion) p io o ch oma og aphic analysis. Fa ex ac ion wi h ace one and soybean p o ein solubiliza ion wi h a bu e solu ion a basic pH (pH 10 o 9) we e necessa y o ob ain selec i e and sensi i e condi ions. Use o wa e -ace o- ni ile- i luo oace ic acid o wa e - e ahyd o u an- i luo oace ic acid linea bina y g adien s a a low a e o 3 mL/min, a empe a u e o 508C, and UV de ec ion a 280 nm enabled ch oma og aphic analysis o soybean p o eins in cu ed mea p od- uc s in less han 3 min. Key Wo ds: Soybean p o eins; Cu ed-mea p oduc s; Pe usion e e sed-phase high-pe o m- ance liquid ch oma og aphy; Recei ed: Janua y 10, 2005; e ised: Ma ch 18, 2005; accep ed: Ma ch 26, 2005 DOI 10.1002/jssc.200500011 1 In oduc ion The use o soybean p o eins as mea ex ende s has sig- ni ican ly inc eased in ecen yea s due o he in e es ing unc ional and nu i ional p ope ies o hese ege able p o eins oge he wi h hei low cos [1–4]. In ac , soy- bean p o eins help o imp o e echnological p ocesses used in he manu ac u e o mea p oduc s, educe hei o mula ion cos , and can also be used as a eplace s enabling he manu ac u e o mea p oduc s wi h low a con en . Howe e , maximum le els a which hese p o- eins can be added o mea p oduc s a e es ablished by egula ions which also con ol he accu a e labelling o hese p oduc s. Thus, he addi ion o soybean p o eins is o bidden o allowed only up o a ce ain ex en depending on he kind o mea p oduc [5]. In he case o cu ed-mea p oduc s, he maximum allowance anges om 1 o 5% (w/w) ( ela i e o d y p oduc ) o non-mea p o eins (milk p o eins and/o ege able p o eins) depending on he quali y o he p oduc . In addi ion o his, he Eu opean Commission has amended he Eu opean Food Labelling Di ec i e 2000/13/EC (Di ec i e 2003/89/EC) o ensu e ha consume s a e in o med o he comple e composi ion o oods u s [6]. Consequen ly, analy ical me hods o he de ec ion o addi ions o soybean p o eins in mea p o- duc s a e u gen ly equi ed. Howe e , he de ec ion o soybean p o eins in mea p oduc s is no an easy ask since hese ood sys ems p esen a complex o mula ion. The main di icul ies ound in he de e mina ion o hese ege able p o eins in mea p oduc s a e ela ed o he composi ion and he p ocessing o mea p oduc s. In ac , he kind o mea used, i s quali y, he soybean p o ein sou ce added, he p esence o o he non-mea p o eins, o he p ocessing condi ions employed du ing he p oduc- ion o mea p oduc s a e some o he main p oblems ha can make he analysis o soybean p o eins in hese p od- uc s e y di icul . Despi e his, di e en a emp s ha e been made o de e mine hese p o eins in mea p oduc s. Mos o he p oposed me hods, being based on elec o- pho e ic (SDS-PAGE) [7] o immunochemical ech- niques [7–9], a e gene ally edious and ime consuming. Mo eo e , in e e ences om o he mea componen s made he measu emen o soybean bands in elec o- pho e ic me hods qui e di icul while he p epa a ion o a sui able an ibody agains soybean p o eins was he main limi a ion in immunochemical me hods due o he di e en echnological p ocessing s eps o which mea p oduc s a e submi ed. In spi e o hese d awbacks and due o he absence o mo e app op ia e me hodologies, he AOAC Co espondence: M. Luisa Ma ina, Depa men o Analy ical Chemis y, Facul y o Chemis y, Uni e si y o Alcal, C a. Ma- d id-Ba celona Km. 33.600, 28871 Alcal de Hena es (Mad id), Spain. Phone: +34 91 8854935. Fax: +34 91 8854971. E-mail: [email p o ec ed]. 988 C iado, Cas o-Rubio, Ga ca-Ruiz, Ga ca, Ma ina o icial me hod based on an Enzyme-Linked Immunoso - ben Assay (ELISA) is nowadays he me hod mos e- quen ly employed [9]. Ch oma og aphic me hods ha e also been applied o he de ec ion o soybean p o eins in mea p oduc s [7, 10–12]. Howe e , he me hods p o- posed in he li e a u e we e used wi h esh mea s and no applica ion was ound o p ocessed mea p oduc s. To his end, ou esea ch g oup has de eloped a pe usion high pe o mance liquid ch oma og aphy (HPLC) me hod o he de e mina ion o soybean p o eins in hea -p o- cessed mea p oduc s [13]. The use o pe usi e s a ion- a y phases in HPLC pe mi ed he ch oma og aphic anal- ysis o soybean p o eins in hese mea p oduc s in anal- ysis imes sho e han 3 min, p o iding a simple and inex- pensi e al e na i e o he ELISA me hod. While he p od- uc s s udied in ou p e ious esea ch wo k a e submi ed o he mal ea men du ing hei p oduc ion, he e is also ano he kind o p ocessed mea p oduc which is no sub- mi ed o he mal ea men bu o a ipening p ocess (cu ed mea p oduc s). In addi ion o he complexi y o hese p oduc s, he ipening p ocess may subs an ially al e he p o eins p esen in he ood sys em [14–17], making hei de e mina ion e en mo e di icul . Depending on he ype o cu ed mea p oduc , he addi ion o soybean p o eins is o bidden o allowed up o a ce ain legal limi depending on he ype o p oduc . The main goal o his wo k was o de elop an analy ical pe usion HPLC me hodology enabling he de ec ion o soybean p o eins in cu ed mea p oduc s o which he addi ion o soybean p o eins is o bidden and he quan i a- ion o soybean p o eins in cu ed mea p oduc s o which he addi ion o soybean p o eins is allowed up o a ce ain limi . Because he elu ion o mea componen s om he ch oma og aphic columns can be di icul , a compa a i e s udy o wo o ganic modi ie s in he mobile phase wi h di e en elu ion s eng h (ace oni ile and e ahyd o u an) has been ca ied ou in his wo k. 2 Ma e ials and me hods 2.1 Chemicals and samples Ace oni ile (ACN) (Me ck, Ge many), e ahyd o u an (THF) (Scha lab, Ba celona, Spain), i luo oace ic acid 99.5 a om% (TFA) (Sigma, S . Louis, MO, USA), and HPLC g ade wa e (Milli-Q sys em, Millipo e, Bed o d, MA, USA) we e used o he p epa a ion o he mobile phases. Sodium bica bona e, is-(hyd oxyme hyl)aminome hane (T is), 2-me cap oe hanol, and u ea (all o hem om Me ck, Da ms ad , Ge many), di hio h ei ol (ICN, Au o a, OH, USA), and sodium dodecylsul a e (SDS) (Fluka, Ba - celona, Spain) we e used o he solubiliza ion o soybean p o eins. Ace one (Me ck, Da ms ad , Ge many) was used o emo e he a o he mea p oduc s. Hyd ochlo ic acid o sodium hyd oxide (bo h om Pan eac, Ba celona, Spain) we e used o adjus he bu e pH. A soybean p o ein isola e (SPI) wi h a p o ein con en o 89.07% (s anda d de ia ion, s= 0.17, n= 7) was ob ained om ICN (Au o a, OH, USA). Th ee di e en ypes o cu ed mea p oduc s we e used in his wo k: (i) a cu ed mea sp ead elabo a ed wi h po k mea and po k a , pap ika, sal , suga , species, and addi- i es, (ii) a d y- e men ed sausage (Spanish cho izo) con- aining soybean p o eins (po k mea , bacon, pap ika, sal , lac ose, dex in, suga , ga lic, soybean p o eins, and addi- i es), and (iii) a high-quali y d y- e men ed sausage (Spanish cho izo) wi hou soybean p o eins (po k mea , sal , species, and addi i es). These cu ed mea p oduc s we e subjec ed o a echnological manu ac u ing p ocess consis ing o mixing o mea , a , and addi i es, homogeni- za ion, s u ing in o he casing, e men a ion, and d ying (17–30 days a 10 o 258C) [18]. All mea p oduc s we e pu chased a local ma ke s a Alcal de Hena es (Mad id, Spain). De e mina ion o he con en o soybean p o eins in he d y- e men ed sausage (Spanish cho izo) by he ELISA me hod [9] was pe o med a he Labo a o io A bi al o he Minis e io de Ag icul u a, Pesca y Alimen acin (Spain). 2.2 P epa a ion o samples, s anda ds, and solu ions Cu ed mea p oduc s we e de a ed be o e soybean p o- ein solubiliza ion. Fa ex ac ion om 10 g o p e iously g ound mea p oduc was achie ed by homogeniza ion wi h ace one in an Ul a u ax mixe , mechanical s i ing (13000 pm) o 20 min, and cen i uga ion (3362 g o 60 min a 258C). Fa ex ac ion om cu ed mea p oduc s (10 g) was achie ed wi h a 50 mL olume o ace one (one ex ac ion) o h ee consecu i e ex ac ions wi h 25 mL o ace one, depending on he p oduc . The esul an pelle was inally d ied o e nigh a 608C o gi e a de a ed and d ied esidue. Soybean p o ein solubiliza ion was pe o med using a bu - e a basic pH (0.05 M NaHCO3a pH 10.0 o he cu ed mea sp ead and 0.05 M T is-HCl a pH 9.0 o he d y- e - men ed sausages) by homogenizing abou 0.8 g o he de a ed and d ied esidue ob ained om he cu ed mea p oduc in 25 mL o he bu e (single ope a ion) o 10 mL o he bu e ( h ee consecu i e ope a ions), s i ing o 10 min a 50 8C, and sonica ing o 5 min. A e cen i uga- ion (3362 g o 45 min a 258C) he supe na an s ob ained we e il e ed (Wha man No. 1) p io o injec ion in he ch oma og aphic sys em. P epa a ion o SPI solu ions was pe o med by weighing he equi ed amoun o SPI, dissol ing i in he bu e solu- ion, sonica ing o 5 min, and cen i uging (3362 g o J. Sep. Sci. 2005, 28, 987–995 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim De ec ion and quan i a ion o addi ions o soybean p o eins 989 45 min a 258C). The esul an supe na an was di ec ly injec ed in he ch oma og aphic sys em. Mobile phases used o he ch oma og aphic sepa a ion con ained 0.05 o 0.1% ( / ) TFA in Milli-Q wa e (mobile phase A) and in ACN o THF (mobile phase B). Mobile phase B was il e ed in a sol en il a ion sys em h ough a 0.45 lm memb ane il e o PVDF. 2.3 Ins umen a ion A Hewle -Packa d liquid ch oma og aph (Hewle -Pack- a d, Pi sbu gh, PA, USA) equipped wi h a UV de ec o , an injec ion sys em (bo h o he se ies 1050), a degassing sys em, a qua e na y pump, and a he mos a ed compa - men o he column (all o he se ies 1100) we e employed. Con ol and da a acquisi ion we e accom- plished wi h HP-Chems a ion so wa e. A Hewle -Pack- a d 1100 Se ies liquid ch oma og aph (Hewle -Packa d, Pi sbu gh, PA, USA) equipped wi h a diode a ay de ec o was used in o de o ob ain UV spec a o peaks. The injec ion olume was 20 lL and de ec ion was ca ied ou a 280 nm. The sepa a ion was accomplished wi h a e e sed-phase pe usion column POROS R2/H (5064.6 mm ID) om Pe sep i e Biosys ems (F aming- ham, MA, USA), packed wi h polys y ene di inylbenzene beads (10 lm pa icle diame e ). The ch oma og aphic sepa a ions we e achie ed by pe - usion RP-HPLC using he ollowing linea bina y g adi- en s (i) 5–25% B in 0.8 min, 25–40% B in 0.8 min, 40– 50% B in 1.1 min, and 50–5% B in 0.5 min whe e he com- posi ion o he mobile phases was 0.05% ( / ) TFA in wa e (mobile phase A) and 0.05% ( / ) TFA in ACN (mobile phase B), and (ii) 5–25% B in 0.8 min, 25–42% B in 0.8 min, 42–50% B in 0.6 min, and 50–5% B in 0.5 min whe e he mobile phase A con ained 0.05% ( / ) TFA in wa e and he mobile phase B con ained 0.05% ( / ) TFA in THF. A low a e o 3 mL/min, a empe a u e o 508C, and UV de ec ion a 280 nm we e employed. 2.4 Calib a ion Calib a ion was pe o med by he ex e nal s anda d and by he s anda d addi ions me hods o in es iga e he exis- ence o ma ix in e e ences. Calib a ion by he ex e nal s anda d me hod was pe o med by injec ing SPI solu- ions o e he ange 0.1–2.2 mg/mL. Calib a ion by he s anda d addi ions me hod was pe o med by adding known and inc easing amoun s o SPI o a cu ed mea p oduc (d y- e men ed sausage). Peak in eg a ion was pe o med by se ing he baseline om alley o alley. 2.5 Da a ea men Calib a ion cu es ob ained by leas -squa es eg ession analysis we e alida ed as linea models by ANOVA (a= 5%). Limi s o de ec ion (LOD, 3 Sa/b) and quan i a- ion (LOQ, 10 Sa/b) we e de e mined om he s anda d e o o he in e cep (Sa) and he slope (b) o he calib a- ion lines [19]. All he s a is ical analyses we e ca ied ou using S a g aphics Plus 5.0 (S a is ical G aphics Co p.). Expe imen al da a we e ea ed using Excel 7.0 (Mic o- so ) and g aphs we e plo ed wi h O igin 7.0 (O iginLab Co po a ion) so wa e. 3 Resul s and discussion In o de o de elop an analy ical me hod o de e mine soy- bean p o eins in cu ed mea p oduc s, h ee di e en mea p oduc s we e employed: wo mea p oduc s o which he addi ion o soybean p o eins is o bidden (a cu ed mea sp ead and a high quali y d y- e men ed sausage) and a mea p oduc o which he addi ion o soybean p o eins is allowed o a ce ain limi (1%) (a d y- e men ed sausage). In all cases, he analysis o he mea p oduc s in ol ed he ollowing s eps: (i) a ex ac ion om he mea p oduc ; (ii) solubiliza ion o soybean p o eins om he de a ed and d ied p oduc ; and (iii) ch oma og aphic analysis. Fa ex ac ion was achie ed wi h ace one because his sol- en was shown o ha e ad an ages o e se e al o he sol en s in es iga ed o ex ac ing he a om hea -p o- cessed mea p oduc s [13]. Using his sol en , he numbe o ex ac ions needed o emo e he high a con en o he p oduc s as well as he highly UV-abso ben componen s (i.e. pap ika) [20] p esen in he cu ed mea p oduc s was s udied. Fo ha pu pose, he a o one cu ed mea sp ead and one d y- e men ed sausage was ex ac ed wi h ace one in a single s ep, in wo successi e s eps, and in h ee successi e s eps. Th ee successi e ace one ex ac ions o he g ound samples we e needed o ob ain a de a ed, d y, and manageable powde om he cu ed mea sp ead due o i s high a and pap ika con en s. How- e e , in he case o he d y- e men ed sausages one ex ac ion was enough o emo e he a . Fo soybean p o ein solubiliza ion, di e en bu e s a basic pHs wi h and wi hou added dena u ing o educing agen s we e in es iga ed in o de o ind he mos adequa e condi ions o he solubiliza ion o soybean p o eins om cu ed mea p oduc s. Wa e as well as 0.05 M T is-HCl bu e a pH 8.0 o 9.0 and 0.05 M NaHCO3bu e a pH 10.0 o 11.0 we e used. 3.1 De ec ion and quan i a ion o soybean p o eins in cu ed mea p oduc s using ace oni ile as o ganic modi ie in he mobile phase Ini ially, he ch oma og aphic condi ions p e iously op i- mized o he analysis o hea -p ocessed mea p od- uc s [13] we e employed. These condi ions consis ed o a wa e -ace oni ile linea bina y g adien om 5 o 25% B in J. Sep. Sci. 2005, 28, 987–995 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim 990 C iado, Cas o-Rubio, Ga ca-Ruiz, Ga ca, Ma ina 0.8 min, om 25 o 42% B in 0.8 min, and om 42 o 50% B in 0.6 min ollowed by a e e sed g adien om 50 o 5% B in 0.5 min o e u n o he ini ial condi ions a a low a e o 3 mL/min, 508C as wo king empe a u e, and UV de ec- ion a 280 nm (mobile phase A: 0.05% TFA in wa e , mobile phase B: 0.05% TFA in ace oni ile). The ch oma- og ams ob ained unde hese condi ions o a cu ed mea sp ead spiked wi h SPI when wa e and bu e solu ions a ou di e en pHs we e used o solubilize soybean p o- eins a e shown in Figu e 1. The e was a ch oma o- g aphic peak a 1.7 min which had he same e en ion ime as one o he peaks co esponding o SPI and ha did no appea in he ch oma og am ob ained o he same cu ed p oduc wi hou added SPI. In addi ion, he UV spec a and i s and second de i a i es ob ained o his peak in he SPI and he cu ed mea p oduc spiked wi h SPI we e iden ical. This peak (1.7 min) was chosen o cal- cula e he eco e y ob ained o soybean p o eins when using he abo e-men ioned solu ions as solubilizing media o hese p o eins. This eco e y was calcula ed by di iding he peak a ea measu ed o he selec ed ch oma- og aphic peak in he ch oma og am ob ained o he cu ed mea p oduc by he peak a ea measu ed o he same peak in he ch oma og am ob ained o SPI (unde he same expe imen al condi ions and a he same con- cen a ion as in he mea p oduc ), and exp essed as a pe cen age, ha is, Ap oduc /ASPI6100. Resul s (a e age o h ee de e mina ions) indica e ha he use o a 0.05 M NaHCO3bu e a pH 10.0 ga e he highes eco e y o soybean p o eins (L91%) (see Figu e 2) om he cu ed mea sp ead. Then, his bu e solu ion was selec ed o in es iga e he e ec o he addi ion o dena u ing o educing agen s on he eco e y o soybean p o eins. Howe e , he addi ion o hese agen s led o eco e ies highe han 100%, sugges ing ha solubiliza ion o mea componen s occu s oge he wi h ha o he soybean p o- eins. As a consequence, a 0.05 M NaHCO3bu e a pH 10.0 was employed and successi e ex ac ions we e pe o med o inc ease he solubiliza ion o soybean p o- eins. The highes eco e y (L96%) was ob ained when h ee consecu i e ex ac ions we e pe o med. Howe e , in he case o d y- e men ed sausages, only one s ep was necessa y o solubiliza ion o soybean p o eins and he highes eco e ies oge he wi h he bes selec i i y (peak pu i y) we e ob ained o a 0.05 M T is-HCl bu e a pH 9.0. Al hough hese ini ial ch oma og aphic condi ions enabled op imiza ion o he solubiliza ion media o soybean p o- eins, as obse ed in Figu e 1 he e was a pa ial o e lap- ping o he peak o in e es (1.7 min) and peaks co e- sponding o mea componen s. Fo his eason, he ch o- ma og aphic sepa a ion condi ions used o analyze cu ed mea p oduc s we e u he op imized in o de o inc ease he esolu ion. Thus, six di e en g adien s wi h di e en slopes we e es ed. F om he esul s ob ained on using hese g adien s, a g adien sligh ly longe and wi h lowe slope han he ini ial g adien was chosen: om 5 o 25% J. Sep. Sci. 2005, 28, 987–995 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim Figu e 1. Ch oma og am co esponding o 46 mg/mL o a cu ed-mea p oduc o sp ead (L1.5 mg/mL o he de a ed and d ied mea p oduc ) spiked wi h a 0.4% (w/w) o SPI e e ed o ini ial p oduc (10 g o he mea p oduc was spiked wi h 40 mg o soybean p o eins). The peak chosen o de ec soybean p o eins is indica ed by an a ow. The media employed o soybean p o ein solubiliza ion we e he ollow- ing: (a) wa e , (b) 0.05 M T is-HCl bu e a pH 8.0 (c) 0.05 M T is-HCl bu e a pH 9.0, (d) 0.05 M NaHCO3bu e a pH 10.0, and (e) 0.05 M NaHCO3bu e a pH 11.0. Ch oma- og aphic condi ions: empe a u e 508C; low a e, 3 mL/min; g adien : 5–25% B in 0.8 min, 25–42% B in 0.8 min, and 42–50% B in 0.6 min, ollowed by 50–5% B in 0.5 min. Mobile phases: A, wa e wi h 0.05% TFA and B, ACN wi h 0.05% TFA. Figu e 2. Reco e y (%) o soybean p o eins om a solu ion o 46 mg/mL o a cu ed-mea sp ead (L1.5 mg/mL o he de a ed and d ied mea p oduc ) spiked wi h a 0.4% (w/w)o SPI e e ed o ini ial p oduc (10 g o mea p oduc was spiked wi h 40 mg o SPI), using: wa e , 0.05 M T is-HCl bu - e a pH 8.0 o 9.0, and 0.05 M NaHCO3bu e a pH 10.0 o 11.0 o soybean p o ein solubiliza ion. Ch oma og aphic condi ions as in Figu e 1. De ec ion and quan i a ion o addi ions o soybean p o eins 991 B in 0.8 min, om 25 o 40% B in 0.8 min, and om 40 o 50% B in 1.1 min ollowed by a e e sed g adien om 50 o 5% B in 0.5 min o e u n o he ini ial condi ions (mobile phase A: 0.05% TFA in wa e , mobile phase B: 0.05% TFA in ace oni ile). In addi ion, a s udy o he in luence o he pe cen age o TFA in he mobile phase on he sepa a- ion o soybean p o eins and mea componen s e ealed ha a 0.05% concen a ion o TFA was he mos app op i- a e alue o his pa ame e . Figu e 3 shows he ch oma- og ams co esponding o SPI, he cu ed mea sp ead, his same p oduc spiked wi h SPI, and a d y- e men ed sausage p oduced wi h SPI and hen subjec ed o he cu - ing p ocess. I is shown ha he peak chosen o de e mine soybean p o eins (peak a 1.7 min) pe mi ed he de ec- ion o soybean p o eins in cu ed mea p oduc s o which he addi ion o soybean p o eins is o bidden e en a e hey ha e been subjec ed o he p oduc ion p ocess (case o he d y- e men ed sausage). Once we had demons a ed he applicabili y o he de el- oped me hod o de ec po en ial adul e a ions o cu ed mea p oduc s by audulen addi ion o soybean p o eins o mea p oduc s o which he addi ion o soybean p o eins is o bidden, he cha ac e is ics o he me hod o quan i a- i e analysis o soybean p o eins in cu ed p oduc s we e s udied. The quan i a ion o soybean p o eins in cu ed mea p oduc s would enable he de e mina ion o he le el o adul e a ion in cu ed mea p oduc s in which he addi- ion o soybean p o eins is o bidden o he quali y con ol o hose p oduc s o which he addi ion o soybean p o- eins is allowed up o a ce ain limi . Linea i y, limi s o de ec ion and quan i a ion, p ecision, and accu acy we e de e mined. Table 1 shows he esul s ob ained o a d y- e men ed sausage. The linea wo king concen a ion ange used was om 0.2 o 2.2 mg/mL o soybean p o eins, wi h a linea ela ionship be ween sig- nal and soybean p o ein concen a ion being obse ed up o 6 mg/mL. The limi o de ec ion (calcula ed as he con- cen a ion co esponding o a signal equal o he in e cep plus h ee imes he s anda d e o o he calib a ion plo ) was 0.03 mg/mL o soybean p o eins, which means ha i is possible o de ec up o 0.04 g o soybean p o ein pe 100 g o p oduc . The limi o quan i a ion (calcula ed as he concen a ion co esponding o a signal equal o he in e cep plus en imes he s anda d e o o he calib a- ion plo ) was 0.10 mg/mL o soybean p o eins, ha is, 0.13 g o soybean p o ein pe 100 g o p oduc may be quan i ied. In o de o in es iga e he exis ence o ma ix in e e - ences, he slope o he calib a ion lines ob ained by he ex e nal s anda d me hod and he s anda d addi ions me hod we e compa ed ( - es ). Resul s ob ained o he compa ison o he slopes e ealed ha he de eloped me hod did no su e om ma ix in e e ences. The p ecision o he me hod was e alua ed by de e min- ing he epea abili y, ep oducibili y, and in e media e p e- cision (see esul s in Table 1). Repea abili y was calcu- la ed by injec ing en consecu i e imes a solu ion o L2.8 mg/mL o he de a ed and d ied esidue ob ained om he d y- e men ed sausage s udied, ob aining a RSD alue o he e en ion ime o 0.1% and o peak a ea o 3.4%. Rep oducibili y was de e mined by injec ing six solu ions o L1.5 mg/mL o he de a ed and d ied esidue ob ained om he d y- e men ed sausage on di e en days. The RSD alue o e en ion ime was 0.4% and o peak a ea was 10%. In e media e p ecision was de e - mined as he RSD o he slopes o he s aigh lines ob ained by he ex e nal s anda d me hod on ou di e en days. Good in e media e p ecision was ob ained (RSD alues abou 1.6%). Finally, he accu acy o he me hod was e alua ed in wo ways: (i) by spiking he d y- e men ed sausage wi h known amoun s o SPI (absolu e eco e y) o (ii) by com- pa ison o he soybean con en de e mined by his me hod wi h he con en ob ained by he ELISA AOAC o icial me hod [9]. The absolu e eco e y o soybean p o eins when he g ound cu ed mea p oduc was spiked wi h inc easing amoun s o SPI anging om 95 o 99% (see J. Sep. Sci. 2005, 28, 987–995 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim Figu e 3. Ch oma og ams co esponding o: (a) a solu ion o 1.5 mg/mL o SPI, (b) a solu ion o 46 mg/mL o a cu ed mea p oduc o sp ead (L1.5 mg/mL o he de a ed and d ied mea p oduc ) spiked wi h a 0.34% (w/w) o SPI (10 g o mea p oduc was spiked wi h 34 mg o SPI), (c) a solu ion o 46 mg/mL o a cu ed mea p oduc o sp ead (L1.5 mg/mL o he de a ed and d ied mea p oduc ) wi hou soybean p o- eins, and (d) a solu ion o 46 mg/mL o a cu ed mea p oduc (d y- e men ed sausage) (L0.8 mg/mL o he de a ed and d ied mea p oduc ) wi h soybean p o eins in i s composi ion. Ch oma og aphic condi ions: empe a u e, 508C; low a e, 3 mL/min; g adien : 5–25% B in 0.8 min, 25–40% B in 0.8 min, and 40–50% B in 1.1 min, ollowed wi h 50–5% B in 0.5 min. Mobile phases: A, wa e wi h 0.05% TFA and B, ACN wi h 0.05% TFA. 992 C iado, Cas o-Rubio, Ga ca-Ruiz, Ga ca, Ma ina Table 1). On he o he hand, he soybean p o ein con en de e mined by he HPLC me hod in he d y- e men ed sausage con aining soybean p o eins (whose ch oma o- g am appea s in Figu e 3.d) was 0.79 l0.03% (w/w) ela- i e o he d ied p oduc (ini ial p oduc co ec ed wi h i s mois u e). This soybean p o ein con en was in good ag eemen wi h he con en de e mined by he ELISA AOAC o icial me hod [9]: 0.74% (w/w) ela i e o he d ied p oduc . In addi ion, hese alues we e wi hin he limi s au ho ized by law o his p oduc , a 1% (w/w) o non-mea p o eins (milk p o eins and/o ege al p o eins) ela i e o d ied p oduc . F om he esul s ob ained in his wo k, i can be s a ed ha he analy ical me hod de eloped is eliable o de ec ing and quan i ying soybean p o eins in cu ed mea p oduc s. The only d awback o he de eloped me hod was ha pe - iodic washing o he column was necessa y in o de o elu e mea componen s e ained on he s a iona y phase when using a mobile phase con aining ace oni ile as o ganic modi ie . Fo his eason, a di e en o ganic modi- ie wi h a highe elu ion s eng h such as e ahyd o u an has also been in es iga ed in his wo k o achie e he de e mina ion o soybean p o eins in cu ed mea p od- uc s. 3.2 De ec ion and quan i a ion o soybean p o eins in cu ed mea p oduc s using e ahyd o u an as o ganic modi ie in he mobile phase Mobile phases consis ing o e ahyd o u an-wa e -0.05% TFA we e also employed in his wo k. In o de o op imize he elu ion g adien , di e en linea bina y g adien s wi h di e en g adien slopes (% o mobile phase B/min) and di e en g adien imes we e es ed. F om hese expe i- men s, a linea bina y g adien om 5 o 25% B in 0.8 min, 25 o 42% B in 0.8 min, and 42 o 50% B in 0.6 min ol- lowed by a e e sed g adien om 50 o 5% B in 0.5 min o e u n o he ini ial condi ions was chosen (mobile phase A: 0.05% TFA in wa e , mobile phase B: 0.05% TFA in e - ahyd o u an). A low a e o 3 mL/min, a wo king em- pe a u e o 508C, and UV de ec ion a 280 nm we e used as be o e. J. Sep. Sci. 2005, 28, 987–995 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim Table 1. Cha ac e is ics o he pe usion RP-HPLC me hod using ACN as o ganic modi ie o he analysis o soybean p o eins in cu ed-mea p oduc s con aining soybean p o eins. Linea i ya) Up o 6 mg/mL De ec ion limi a) 0.03 mg/mL (0.04% (w/w))b) Quan i a ion limi a) 0.10 mg/mL (0.13% (w/w))b) Exis ence o ma ix in e e encesc) – Slope by he ex e nal s anda d me hod 3.65 l0.24 (n=5) – Slope by he s anda d addi ions me hod 3.41 l0.11 (n=5) P ecision Repea abili y (RSD,%) (n= 10)d) – Re en ion ime 0.1 – Peak a ea 3.4 In e nal ep oducibili y (RSD,%) (n=6) e) – Re en ion ime 0.4 – Peak a ea 10.0 In e media e p ecision (RSD,%) ) – Slope (ex e nal s anda d me hod, n= 5) 6.6 Accu acy – Absolu e eco e yg) 97 l2 a) Linea i y and de ec ion and quan i a ion limi s ela i e o mg/mL o soybean p o eins. b) Limi s o de ec ion and quan i a ion exp essed as w/w uni s we e de e mined ela ed o 1 g o sample. c) An F- es o he compa ison o a iances and an - es o he compa ison o slopes we e employed. d) Numbe o injec ions o a solu ion o 2.8 mg/mL o a de a ed and d ied cu ed-mea p oduc . e) Analysis o six solu ions o 1.5 mg/mL o a de a ed and d ied cu ed-mea p oduc on di e en days. ) Analysis pe o med on six di e en days. g) Calcula ed om he soybean p o ein con en p edic ed by he HPLC me hod and he heo e ical con en conside ing he SPI added ( om 0.14 o 0.23 mg/mL) and he soybean p o eins con ained in he p oduc (0.79% (w/w) ela i e o d ied p oduc ). De ec ion and quan i a ion o addi ions o soybean p o eins 993 Figu e 4 shows he ch oma og ams ob ained o he SPI and o he d y- e men ed sausages wi h and wi hou soy- bean p o eins when solubiliza ion o he p o eins was pe - o med wi h a 0.05 M T is-HCl bu e a pH 9.0. I can be obse ed ha he e is a ch oma og aphic peak co e- sponding o SPI elu ing a 1.0 min which appea s in he ch oma og am co esponding o he d y- e men ed sau- sage con aining soybean p o eins bu does no appea in he d y- e men ed sausage wi hou soybean p o eins (a high quali y d y- e men ed sausage o which he addi ion o soybean p o eins is o bidden). In addi ion, he UV spec a and he i s and second de i a i es ob ained o his peak in he ch oma og am o SPI and in ha o he d y- e men ed sausage con aining soybean p o eins we e e y simila , indica ing ha his peak could be used o he de ec ion and quan i a ion o soybean p o eins in cu ed mea p oduc s when employing e ahyd o u an as o ganic modi ie in he mobile phase. In o de o show he possibili ies o he de eloped me hod o quan i a ion o soybean p o eins in cu ed mea p od- uc s, he analy ical cha ac e is ics o he me hod we e s udied. Table 2 shows he linea i y, limi s o de ec ion and quan i a ion, p ecision, and accu acy de e mined o J. Sep. Sci. 2005, 28, 987–995 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim Table 2. Cha ac e is ics o he pe usion RP-HPLC me hod using THF as o ganic modi ie o he analysis o soybean p o eins in cu ed-mea p oduc s con aining soybean p o eins. Linea i ya) Up o 6 mg/mL De ec ion limi a) 0.05 mg/mL (0.06% (w/w))b) Quan i a ion limi a) 0.17 mg/mL (0.22% (w/w))b) Exis ence o ma ix in e e encesc) – Slope by he ex e nal s anda d me hod 7.01 l0.16 (n=5) – Slope by he s anda d addi ions me hod 7.63 l1.10 (n=3) P ecision Repea abili y (RSD,%) (n= 10)d) – Re en ion ime 0.1 – Peak a ea 1.5 In e nal ep oducibili y (RSD,%) (n=4) e) – Re en ion ime 0.9 – Peak a ea 3.3 In e media e p ecision (RSD,%) ) – Slope (ex e nal s anda d me hod, n= 5) 2.3 Accu acy – Absolu e eco e yg) 99 l3 a) Linea i y and de ec ion and quan i a ion limi s ela i e o mg/mL o soybean p o eins. b) Limi s o de ec ion and quan i a ion exp essed as w/w uni s we e de e mined ela i e o 1 g o sample. c) An F- es o he compa ison o a iances and an - es o he compa ison o slopes we e employed. d) Numbe o injec ions o a solu ion o 0.8 mg/mL o a de a ed and d ied cu ed-mea p oduc . e) Analysis o six solu ions o 2.3 mg/mL o a de a ed and d ied cu ed-mea p oduc on di e en days. ) Analysis pe o med on i e di e en days. g) Calcula ed om he soybean p o ein con en p edic ed by he HPLC me hod and he heo e ical con en conside ing he SPI added ( om 0.14 o 0.22 mg/mL) and he soybean p o eins con ained in he p oduc (0.56% (w/w) ela i e o d ied p oduc ). Figu e 4. Ch oma og ams co esponding o: (a) a solu ion o 6 mg/mL o SPI, (b) a solu ion o 46 mg/mL (L0.8 mg/mL o he de a ed and d ied mea p oduc ) o a d y- e men ed sau- sage wi h soybean p o eins in i s composi ion, and (c) a solu- ion o 46 mg/mL (L0.8 mg/mL o he de a ed and d ied mea p oduc ) o a d y- e men ed sausage wi hou soybean p o eins in i s composi ion. Ch oma og aphic condi ions: empe a u e, 508C; low a e, 3 L/min; g adien : 5–25% B in 0.8 min, 25–42% B in 0.8 min, and 42–50% B in 0.6 min, ollowed by 50–5% B in 0.5 min. Mobile phases: A, wa e wi h 0.05% TFA and B, THF wi h 0.05% TFA. 994 C iado, Cas o-Rubio, Ga ca-Ruiz, Ga ca, Ma ina he d y- e men ed sausage con aining soybean p o eins unde he abo e ch oma og aphic condi ions using e a- hyd o u an as o ganic modi ie in he mobile phase. A lin- ea ela ionship was obse ed be ween he signal and he soybean p o ein concen a ion up o 6 mg/mL, wi h he wo king concen a ion ange lying be ween 0.1 and 2.2 mg/mL o soybean p o eins. The me hod enabled us o de ec up o 0.05 mg/mL o soybean p o eins (0.06 g o soybean p o eins pe 100 g o cu ed mea p oduc ) and o quan i a e up o 0.17 mg/mL o soybean p o eins (0.22 g o soybean p o eins pe 100 g o cu ed mea p oduc ). Compa ison o he alues o he slope o he calib a ion lines ob ained by he ex e nal s anda d me hod and he s anda d addi ions me hod ( - es ) o he d y- e men ed sausage e ealed he absence o ma ix in e e ences. As i can be seen in Table 2, he p ecision o he me hod was e alua ed by de e mining he epea abili y, ep oduci- bili y, and in e media e p ecision. Repea abili y was calcu- la ed by injec ing en consecu i e imes a solu ion o L0.8 mg/mL o he de a ed and d ied esidue ob ained om he d y- e men ed sausage, gi ing a RSD alue o 0.1% o e en ion ime and o 1.5% o peak a ea. In e nal ep oducibili y was de e mined by injec ing ou solu ions o L2.3 mg/mL o he de a ed and d ied esidue ob ained om he d y- e men ed sausage on di e en days. The RSD alue was 0.9% o e en ion ime and 3.3% o peak a ea. In e media e p ecision de e mined as he RSD o he slopes o he s aigh lines ob ained by he ex e nal s anda d me hod on i e di e en days was 2.3%. The accu acy o he me hod was calcula ed by de e min- ing he absolu e eco e y ob ained o soybean p o eins when spiking he d y- e men ed sausage wi h known amoun s o SPI. This eco e y anged om 96 o 102% (see Table 2). In addi ion, he soybean p o ein con en de e mined in he d y- e men ed sausage (whose ch o- ma og am appea s in Figu e 4.b) was 0.56 l0.04% (w/w) ela i e o he d ied p oduc (ini ial p oduc co ec ed by i s mois u e) which can be conside ed close o he con en de e mined by he ELISA me hod o a di e en ba ch o he same p oduc (0.74% (w/w) ela i e o he d ied p od- uc ). Again he soybean p o ein con en was wi hin he limi au ho ized by law o his p oduc , i.e. 1% (w/w)o non-mea p o eins based on d ied p oduc . The e o e, his al e na i e analy ical me hod is also able o de e mine he amoun o soybean p o eins con ained in cu ed mea p oduc s such as he d y- e men ed sausage s udied. Al hough his analy ical me hodology ga e sa is ac o y esul s a ou ing he elu ion o p o eins om he s a iona y phase and a oiding he need o pe iodic washing, he use o e ahyd o u an as o ganic modi ie dec eased he li e- ime o he ch oma og aphic columns due o i s highe elu- ion s eng h, which can be conside ed he only d awback o his me hodology compa ed o he use o ace oni ile as o ganic modi ie in he mobile phase. 4 Concluding ema ks An ini ial app oach enabling he de ec ion and quan i a ion by pe usion HPLC o soybean p o eins in comme cial cu ed mea p oduc s whe e he addi ion o hese ege a- ble p o eins is o bidden o allowed up o a ce ain limi has been achie ed in his wo k. The analy ical me hodol- ogy de eloped ( a ex ac ion wi h ace one and p o ein solubiliza ion wi h a bu e a basic pH be o e he ch oma- og aphic analysis) has enabled he de ec ion o soybean p o eins no only in spiked cu ed mea p oduc s (a cu ed mea sp ead o which he addi ion o soybean p o eins is o bidden), bu also in p oduc s con aining soybean p o- eins, a d y- e men ed sausage (Spanish cho izo) whe e he ege able p o eins we e added be o e he ipening p ocess showing ha he echnological p ocessing o he mea p oduc did no a ec he analy ical me hodology. Wa e -ace oni ile- i luo oace ic acid o wa e - e ahyd o- u an- i luo oace ic acid linea bina y g adien s a a low a e o 3 mL/min, a empe a u e o 508C, and UV de ec ion a 280 nm could be used in o de o achie e he ch oma o- g aphic analysis o soybean p o eins wi h e e sed-phase pe usi e s a iona y phases in less han 3 min. The use o e ahyd o u an as o ganic modi ie in he mobile phase a oided he need o a pe iodic washing o he column due o he di icul elu ion om he s a iona y phase o some mea componen s al hough i dec eased he li e ime o he column due o i s highe elu ion s eng h. Good pe o m- ance o he de eloped analy ical me hods was obse ed, pe mi ing he quan i a ion o soybean p o eins in a cu ed mea p oduc which p o ed o ha e a soybean con en below he legal limi o his p oduc (1% (w/w) ela i e o d y p oduc o non-mea p o eins (milk p o eins and/o ege able p o eins)). Acknowledgmen s The au ho s hank he Comunidad Au onma de Mad id (Spain) o p ojec 07G/0025/2003. D . C. Ga ca-Ruiz also hanks he Minis e io de Ciencia y Tecnologa o he con ac om he Ramn y Cajal p og am (RYC-2003- 001). F. Cas o-Rubio hanks he Minis e io de Educacin y Ciencia (Spain) o he p edoc o al g an . Re e ences [1] J.T. Kee on, in 44 h Recip ocal Mea Con e ence P oceed- ings, Ame ican Mea Science Associa ion, 1991, p. 79. [2] J.T. Kee on, Food, Fa s and Heal h, Council o Ag icul u al Science and Technology (CAST). Task Fo ce Repo No. 118, 1991, p. 42. [3] J.G. End es, Soy P o ein P oduc s. Cha ac e is ics, Nu i- ional Aspec s and U iliza ion, AOCS P ess, 2001, Cham- paign, IL. J. Sep. Sci. 2005, 28, 987–995 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim De ec ion and quan i a ion o addi ions o soybean p o eins 995 [4] M.C. Ga ca, M. To e, M.L. Ma ina, F. Labo da, CRC C i . Re . Food Sci. Nu . 1997,37, 361– 391. [5] Legislacin Alimen a ia de Aplicacin en Espaa (Clasi ica- cin po Alimen os). Ca ne, a es, caza y de i ados: no ma gen ica de calidad pa a p oduc os c nicos a ados po el calo , Eypasa: Mad id, 2002. [6] Eu opean Commission, 2003, amending Di ec i e 2000/13/ EC as ega ds indica ion o he ing edien s p esen in ood- s u s, O icial Jou nal o he Eu opean Union. 10 No embe 2003. [7] J. Belloque, M.C. Ga ca, M. To e, M.L. Ma ina, CRC C i . Re . Food Sci. Nu . 2002,42, 507– 532. [8] C.H.S. Hi chcock, F.J. Bailey, A.A. C imes, D.A.G. Dean, P.J. Da is, J. Am. Oil Chem. Soc. 1979,56, 285–287. [9] AOAC O icial Me hod 998.10, Soy p o ein in aw and hea p ocessed mea p oduc s, Enzyme-Linked Inmunoso ben Assay, O icial Me hods o Analysis, 16 h edi ion, Re . 1996, 1997, 1998. AOAC In e na ional, Gai he sbu g, MD. [10] B. Flahe y, Chem. Ind. 1975,12, 495–497. [11] N. Pa is, P.J. Gillespie, in: Me hods o P o ein Analysis, J.P. Chemy, R.A. Ba o d (Eds.). Am. Oil Chem. Soc., Champaign 1988, pp. 142– 155. [12] S.H. Ashoo , P.G. S iles, J. Ch oma og .1987,393, 321– 328. [13] F. Cas o, M.C. Ga ca, R. Rod guez, M.L. Ma ina, J. Ag ic. Food Chem., in p ess. [14] M. Flo es, M.A. Du , A. Ma co, F. Told , Mea Sci. 2004, 68, 439–446. [15] A. Ma n, M.A. Asensio, M.E. Be mffldez, M.G. C doba, E. A anda, J.J. C doba, Mea Sci. 2002,62, 129–137. [16] Y. Sanz, S. Fadda, G. Vignolo, M.C. A is oy, G. Oli e , F. Told , In . J. Food Mic obiol. 1999,53, 115–125. [17] Y. Sanz, S. Fadda, G. Vignolo, M.C. A is oy, G. Oli e , F. Told , J. Ag ic. Food Chem. 1999,47, 3441– 3448. [18] A.M. Pea son, T.A. Gille , in: P ocessed Mea s, Champman & Hall, New Yo k, 1996, Chap e 9, pp. 210–241. [19] J.N. Mille , J.C. Mille , S a is ics and Chemome ics o Ana- ly ical Chemis y. P en ice Hall, Ha low, UK, 2000. [20] T. Cse h i, E. Fo gcs, J. Ch oma og . 2001,936, 119– 137. J. Sep. Sci. 2005, 28, 987–995 www.jss-jou nal.de i2005 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim