Reversed-phase high-performance liquid chromatography applied to the determination of soybean proteins in commercial heat-processed meat products

Analy ica Chimica Ac a 559 (2006) 215–220 Re e sed-phase high-pe o mance liquid ch oma og aphy applied o he de e mina ion o soybean p o eins in comme cial hea -p ocessed mea p oduc s MaConcepci´ on Ga c´ ıa, Ma Dom´ ınguez, Ca men Ga c´ ıa-Ruiz, MaLuisa Ma ina∗ Depa amen o de Qu´ımica Anal´ı ica, Facul ad de Qu´ımica, Uni e sidad de Alcal´a, 28871 Alcal´a de Hena es, Mad id, Spain Recei ed 14 July 2005; ecei ed in e ised o m 21 No embe 2005; accep ed 25 No embe 2005 A ailable online 6 Janua y 2006 Abs ac A e e sed-phase ch oma og aphic me hod has been de eloped and op imised in o de o de ec and quan i a e soybean p o eins in comme cial hea -p ocessed mea p oduc s. The op imised condi ions consis ed o a linea bina y g adien e ahyd o u ane–wa e –0.05% i luo oace ic acid a a low a e o 1mL/min. Mea p oduc s we e de a ed wi h ace one and soybean p o eins we e ex ac ed wi h a bu e ed solu ion a pH 9.60. The injec iono his ex ac in o he ch oma og aphic sys em enabled hede ec ion o soybeanp o eins in hea -p ocessed mea p oduc sin abou 12min. The me hod enabled he de ec ion and quan i a ion o addi ions o 0.38% (w/w) and 0.63% (w/w), espec i ely, o soybean p o eins ( ela ed o 10g o ini ial p oduc ). The me hod has been p o en o be p ecise wi h ela i e s anda d de ia ions (R.S.D.) o epea abili y, in e media e p ecision, and in e nal ep oducibili y lowe o 7.0%. Reco e ies ob ained o spiked mea p oduc s we e close o 100% and no ma ix in e e ences we e obse ed. The applica ion o he me hod o comme cial hea -p ocessed mea p oduc s in whose o mula ion soybean p o eins we e p esen yielded soybean p o ein con en s anging om 0.90% o 1.54%, below he maximum le els es ablished by egula ions. © 2005 Else ie B.V. All igh s ese ed. Keywo ds: Soybean p o eins; Hea -p ocessed mea p oduc s; High-pe o mance liquid ch oma og aphy 1. In oduc ion The addi ion o non-mea p o eins o mea p oduc s is a com- mon p ac ice. Among he di e en p o eins ha can be added, soybean p o eins a e one o he mos used due o di e en ea- sons: (i) Soybean p o eins can ac as emulsi ying agen s p e en ing he coalescence o a du ing mea hea ing when he lean mea con en is low and, consequen ly, he con en in mea p o eins oo [1]. (ii) The addi ion o soybean p o eins o mea sys ems has also enabled he o mula ion o mea p oduc s wi h a lowe a con en esponding, hus, o he socie y demand o low a p oduc s [2,3]. ∗Co esponding au ho . Tel.: +34 918 854 935; ax: +34 918 854 971. E-mail add ess: [email p o ec ed] (M.L. Ma ina). (iii) Soybean p o eins can also be added as mea ex ende s enabling he educ ion o he cos o mea p oduc s [4,5]. As a consequence o he addi ion o o eign p o eins o mea p oduc s, legal limi a ions es ic ing he quan i y o non-mea p o eins o ce ain maximum le els as well as con olling he labelling o hese mea p oduc s ha e appea ed [6]. In o de o a oidanypo en ial audandensu e hequali yo mea p oduc s, analy ical p ocedu es enabling he quan i a i e analysis o non- mea p o eins in hese mea sys ems a e needed. De ec ion o soybean p o eins in mea p oduc s has been pe o med by di e en echniques such as polyac ylamide gel elec opho esis and immunochemical echniques [7–9]. In ac , an enzyme-linked immunoso ben assay (ELISA) is p oposed as AOAC O icial Me hod o he de e mina ion o soybean p o- einsin awandhea -p ocessedmea p oduc s[8].Ne e heless, noneo heseme hodsa ecomple elysa is ac o ybeing,inmos cases, e y edious and ime consuming o e en no enabling he quan i a i e analysis o soybean p o eins. 0003-2670/$ – see on ma e © 2005 Else ie B.V. All igh s ese ed. doi:10.1016/j.aca.2005.11.074 216 M.C. Ga c´ıa e al. / Analy ica Chimica Ac a 559 (2006) 215–220 Ch oma og aphic echniques ha e also been ied wi h his pu pose. In some cases, soybean p o eins we e de ec ed om ce ain cha ac e is ic pep ides o soybean p o eins in ypsin hyd olysa es [10–12]. In o he cases, o al hyd olysis and s udy o he amino acid pa e n ob ained by high-pe o mance liq- uid ch oma og aphy (HPLC) was employed [13–16]. Some ew a emp s we e also ound in li e a u e in which soybean p o eins we edi ec ly de e mined using HPLC. Ne e heless,in allcases aw mea s we e used and in no case he di ec de e mina ion o soybean p o eins in comme cial hea -p ocessed mea p oduc s was ied [17,18]. Recen ly, ou esea ch eam has de eloped di e en ch o- ma og aphicme hodsusingpe usioncolumns ha ha eenabled he de e mina ion o soybean p o eins in comme cial hea - p ocessed mea p oduc s p epa ed wi h po k, u key, chicken, and/o bee mea s [19–21]. The soybean p o ein con en s de e mined in hese p oduc s ha e been compa ed wi h hose ob ained by he AOAC O icial Me hod obse ing simila esul s. Ne e heless, despi e he ad an ages associa ed o he use o pe usion ch oma og aphy in e ms o sepa a ion ime, pe - usion ch oma og aphy is no always an al e na i e o non- pe usion ch oma og aphy. In ac , he s uc u e o po es o a pe usi e column and a non-pe usi e one is o ally di e en and, hus, he selec i i y, sensi i i y and o he cha ac e is ics (e.g. exis ence o ma ix in e e ences) o a me hod. Since con- en ional HPLC has ne e been applied o he quan i a ion o soybean p o eins in comme cial hea -p ocessed mea p oduc s, he aim o his wo k was o in es iga e he po en ial o e e sed- phase HPLC o he de e mina ion o soybean p o eins in hese p oduc s. 2. Expe imen al 2.1. Chemicals and samples HPLC g ade ace oni ile, isop opanol, and e ahyd o u ane (THF) (Scha lau, Ba celona, Spain) as well as i luo oace ic acid (TFA) (Sigma, S . Louis, MO) ha e been used o he p epa a ion o mobile phases. HPLC g ade wa e ob ained om a Milli-Q sys em (Millipo e, Bed o d, MA) has also been employed. T is(hyd oxyme hyl)aminome hane (T is), 2- me cap oe hanol(MER),andu ea omMe ck(Da ms ad ,Ge - many), di io h ei ol (DTT) om ICN (Au o a, OH), sodium dodecyl sulpha e (SDS) om Fluka (Ba celona, Spain), and sodium hyd ogen ca bona e om Pan eac (Ba celona, Spain) we e employed o he op imiza ion o he sample p epa a ion. Ace one (Me ck) was employed o a ex ac ion. Soybean p o- ein isola e (SPI), ob ained om ICN, was used as s anda d o soybean p o eins. The p o ein con en o SPI, de e mined by Kjeldahl analysis, was 93.0% (calcula ed by mul iplying he ni ogen con en in pe cen age by 6.25) and i s d y ma - e con en was de e mined by d ying a 130◦C o cons an weigh . One comme cial hea -p ocessed mea p oduc no con- aining soybean p o eins and ou di e en hea -p ocessed mea p oduc s (mea p oduc s 1–4) con aining soybean p o eins we e pu chased in local ma ke s in Mad id (Spain). Hea -p ocessed mea p oduc s1–4p esen edasimila composi ionandwe e ep- esen a i e o he mos common hea -p ocessed mea p oduc s comme cially a ailable. The composi ion consis ed o u key and po k mea s, po a o s a ch, sal , wa e , and soybean p o eins om soybean p o ein isola e. Mea p oduc s 3 and 4 also con- ained oli es and in mea p oduc s 2–4 a was also added. Mea p oduc s we e de a ed be o e hei analysis by he ollowing p ocedu e: 10g o mea p oduc we e g ound in an au oma ic mille , homogenised wi h 50mL o ace one in an Ul a u ax mixe , and cen i uged (3362g, 30min); he supe na an was emo ed and he pelle was ex ac ed again wi h ano he 50mL o ace one and cen i uged du ing 1h (3620g); inally, he pel- le ob ained om his second a ex ac ion was d ied o e nigh a 60◦C o emo e he emaining ace one. Mea ex ac s we e p epa ed by weighing 2.3g o ha de a ed mea p oduc , sus- pending in 50mL o he app op ia e solu ion (0.05M ca bona e bu e (pH 9.60)), shaking o 30min in a wa e ba h a 60◦C, and cen i uging (3362g, 30min, 40◦C). The supe na an was injec ed in o he ch oma og aphic sys em. The p o ocol used o he p epa a ion o he SPI consis ed o weighing and dissol - ing he s anda d in he app op ia e solu ion (0.05M ca bona e bu e (pH 9.60)),sonica ing o 3min,andcen i uging(1450g, 5min) o collec he supe na an ha was injec ed in o he ch o- ma og aphic sys em. 2.2. High-pe o mance liquid ch oma og aphy A Hewle -Packa d 1100 Se ies liquid ch oma og aph (Hewle -Packa d, Pi sbu gh, PA) equipped wi h a diode a ay de ec o was used. The sepa a ion was accomplished wi h a PLRP-S column (150mm×4.6mm i.d.) om Polyme Labo- a o ies LTD (Chu ch S u on, UK), packed wi h polys y ene di inylbenzene beads (300 ˚ A, 8␮m pa icle size). The RP- HPLC sepa a ion was pe o med a a low a e o 1mL/min using a linea bina y g adien wa e –TFA–o ganic modi ie , an injec ion olumeo 20␮L,andUVde ec iona 254nm.Theg a- dien , column empe a u e, and mobile phase composi ion we e op imised. The o ganic modi ie s used we e il e ed h ough a 0.45-␮m nylon il e be o e use. 2.3. Calib a ion Calib a ionwaspe o medby heex e nals anda dandby he s anda daddi ionscalib a ionme hods.Calib a ionby heex e - nal s anda d me hod was ca ied ou by injec ing SPI solu ions o e he ange 2–12mg/mL. In eg a ion o he peak selec ed o he de e mina ion o soybean p o eins in mea p oduc s was achie ed by se ing he baseline om alley o alley. The a ea (a e age o wo consecu i e injec ions) o ha peak was plo - ed agains he co esponding SPI concen a ion (co ec ed o he pu i y and mois u e) o ob ain he calib a ion cu e. The con en o soybean p o eins in he mea p oduc s was de e - mined by in e pola ion o he a ea o his peak in he mea ch oma og ams in he calib a ion cu e. Calib a ion by he s an- da daddi ionsme hod waspe o medbyinjec ing mea ex ac s (47mg/mL) spiked wi h known and inc easing amoun s o SPI (0–3.75mg/mL). M.C. Ga c´ıa e al. / Analy ica Chimica Ac a 559 (2006) 215–220 217 2.4. Da a ea men The linea i y o he calib a ion cu es was ob ained by leas - squa es eg ession analysis and he linea model was alida ed bymeanso heanalysiso esidualsand heanalysiso a iance (ANOVA) (α=5%). 3. Resul s and discussion 3.1. Ch oma og aphic sepa a ion The de e mina ion o soybean p o eins in hea -p ocessed mea p oduc s has been pe o med by ou esea ch eam using pe usion e e sed-phase HPLC [19–21]. In he p esen wo k, we ied o explo e he po en ial o con en ional e e sed- phase HPLC o he de e mina ion o soybean p o eins in hea - p ocessed mea p oduc s. RP-HPLC has also been used by ou esea ch eam o he de e mina ion o soybean p o eins in comme cial soybean p od- uc ssuchassoybeanin an o mulasandsoybeanmilks [22,23]. Thus, heini ialexpe imen alcondi ionschosen o hede elop- men o ame hodenabling hede e mina iono soybeanp o eins in hea -p ocessed mea p oduc s by RP-HPLC we e he p e i- ously employed o he sepa a ion o soybean p o eins in soy- bean p oduc s: a linea bina y g adien ace oni ile–wa e –0.1% ( / ) TFA, a empe a u e o 45◦C, and a low a e o 1mL/min [22]. Using hese condi ions, a hea -p ocessed mea p oduc no con aining soybean p o eins and a hea -p ocessed mea p od- uc wi h soybean p o eins we e analysed. P io o hei injec- ion, hese mea p oduc s we e de a ed wi h ace one and he p o eins we e ex ac ed in wa e . Ne e heless, he analysis o hese samples unde hose ch oma og aphic condi ions esul ed unsuccess ul since some componen s o he mea ex ac s we e s ongly e ained on he s a iona y phase and we e no easily elu ed om he column. In o de o o e come his p oblem, o he o ganic modi ie s wi h highe elu ion s eng hs we e ied: isop opanol and e ahyd o u an (THF). When isop opanol was used, s ong adso p ion o mea componen s on he s a iona y phase su ace was also obse ed being e y di icul hei elu- ion. THF had p e iously been used by ou esea ch eam o he elu ion o much e ained componen s in he analysis o soybean p o eins in cu ed mea p oduc s using pe usion ch oma og a- phy [24]. The use o THF as o ganic modi ie (wi h a highe elu ion s eng h han ace oni ile and isop opanol) oge he wi h alowe concen a iono TFA(0.05%, / ),ino de o educe he in e ac ions o mea p o eins wi h he s a iona y phase, esul ed in he comple e elu ion o mea componen s om he column being possible he analysis o mea ex ac s wi hou any u he wash o he column be ween uns. Thus, using mobile phases consis ing o THF–wa e –0.05% ( / ) TFA he elu ion g adien was op imised and he e ec o he a ia ion o he empe a- u e on he sepa a ion was s udied. Fo he op imisa ion o he elu ion g adien , 12 linea bina y g adien s wi h di e en g a- dien slopes (% o mobile phase B/min) and di e en g adien imes we e ied. F om hese expe imen s, a linea bina y g adi- en om 1 o 42% B in 12min and om 42 o 100% B in 1min ollowed by a e e sed g adien om 100 o 1% B in 1min o Fig. 1. Ch oma og ams co esponding o an ex ac o a hea -p ocessed mea p oduc no con aining soybean p o eins (20.75mg/mL) (a) and o a solu ion o soybean p o ein isola e (4.08mg/mL) (b). Expe imen al condi ions: column, PLRP-S (150mm×4.6mm i.d.); empe a u e, 45◦C; low a e, 1mL/min; g a- dien :1–42%Bin12min and 42–100% in 1min; mobile phases: A, 0.05% ( / ) TFA in wa e ; B, 0.05% TFA ( / ) in THF; injec ion olume, 20␮L; de ec ion, 254nm. e u n o he ini ial condi ions and an equilib a ion ime o 1min was chosen. Using his g adien , empe a u e was a ied om 25 o 50◦C. Re en ion imes dec eased and e iciency enhanced wheninc easing he empe a u e.F om hese esul s, he highes empe a u e ha he column could s and wi hou comp omising i s li e ime was selec ed, 45◦C. The ch oma og ams ob ained wi h he op imised condi ions and co esponding o a solu ion o SPI and o an aqueous ex ac o he hea -p ocessed mea p oduc no con aining soybean p o- eins a e shown in Fig. 1. Soybean p o eins we e sepa a ed in se en peaks and he hea -p ocessed mea p oduc no con ain- ing soybean p o eins yielded one big band a he beginning o he ch oma og am and wo small peaks a app oxima ely 9 and 11min. The op imised me hod was also applied o he analysis o soybean p o eins in a hea -p ocessed mea p oduc con ain- ing soybean p o eins. The gene al aspec o he ch oma og am ob ained was e y simila o ha obse ed o he mea p oduc wi hou soybeanp o eins.Only azoom e ealed heexis enceo a inypeak a 12.30min ha could be a ibu ed opeak7 o SPI. In o de o imp o e he sensi i i y o his peak, he p ocedu e o he p epa a ion o mea ex ac s was op imised. 3.2. Op imisa ion o he sample p epa a ion Mos o heme hodsused o hep epa a iono mea samples o hei injec ion in a sepa a ion sys em consis ed o de a ing hemea p oduc inace oneandex ac ingsoybeanp o einswi h solu ionsbu e eda basic pHs. Ou esea ch eam,in a p e ious wo k, ied di e en sol en s (hexane, ace one, pe oleum e he , and e hanol) o he a ex ac ion in mea p oduc s obse ing hebes esul s whenace one was employed[19]. The e o e, he op imisa ion o he sample p epa a ion in his wo k was mainly 218 M.C. Ga c´ıa e al. / Analy ica Chimica Ac a 559 (2006) 215–220 Fig. 2. Ch oma og ams co esponding o he hea -p ocessed mea p oduc 1 when using di e en bu e ed solu ions o he ex ac ion o p o eins: (a) wa e , (b) 0.05M T is–HCl bu e a pH 8.30, (c) 0.05M ca bona e bu e a 9.60, (d) 0.05M ca bona e bu e a pH 10.00, and (e) 0.05M ca bona e bu e a 11.00. O he condi ions as in Fig. 1. ocused on he ex ac ion o soybean p o eins om he mea p oduc s, which had been he s ep wi h mo e signi ican di - e ences among he me hods ound in he li e a u e. Fo ha pu pose, he hea -p ocessed mea p oduc 1 was de a ed wi h ace one, as desc ibed in Sec ion 2, and soybean p o eins we e ex ac ed using di e en media: wa e , 0.05M T is–HCl bu e (pH8.30),0.05Mca bona ebu e a di e en pHs( om9.60 o 11.00),anddi e sedena u ingand educingagen s(MER,DTT, SDS, and u ea). Fig. 2 shows he ch oma og ams co esponding o he ex ac s o he hea -p ocessed mea p oduc 1 ob ained wi h he di e en media. The injec ion o he ob ained ex ac s in o he ch oma og aphic sys em esul ed in ch oma og ams wi h a peak a he same e en ion ime (∼12.30min) ha he peak de ec ed when he ex ac ion was pe o med in wa e . This peak could co espond wi h peak 7 o SPI. The size o his peak wasno hesameinallsolu ions iedand anged om helowes alue ob ained wi h wa e o he highes wi h ca bona e bu e a pH 9.60. A his pH, he e ec o he addi ion o di e en dena u ing and educing agen s was s udied. In all cases, he a ea ob ained was lowe han ha ob ained wi hou he addi ion o hese agen s. Fu he mo e, when adding SDS o he ca bon- a e bu e a pH 9.60, ano he peak a 13.30min was obse ed ins ead o he peak a 12.30min. Consequen ly, a 0.05M ca - bona e bu e a pH 9.60 was chosen as op imum ex ac ing sol en . The ch oma og ams ob ained when using his bu e o he ex ac ion o soybean p o eins om he hea -p ocessed mea p oduc 1 and he SPI a e shown in Fig. 3. Mo eo e , he spec a and i s and second de i a i es o his peak in he SPI we e compa ed wi h hose o he same peak in he mea p oduc obse ing ha hey we e iden ical (Fig. 4). 3.3. Analy ical cha ac e is ics o he me hod The s udy o he analy ical cha ac e is ics o he me hod was pe o med by he e alua ion o di e en pa ame e s: de ec- ion and quan i a ion limi s, p esence o ma ix in e e ences, p ecision, and accu acy. Table 1 g oups he esul s ob ained. Good linea co ela ion ( 2>0.99) was obse ed be ween he signal and he concen a ion o soybean p o eins in he wo king concen a ion ange (2.00–12.00mg/mL). Mo eo e , he in e - Fig. 3. Ch oma og ams ob ained om he hea -p ocessed mea p oduc 1 (47.40mg/mL) and he soybean p o ein isola e (4.08mg/mL) ex ac ed wi h 0.05M ca bona e bu e (pH 9.60). O he condi ions as in Fig. 1. cep alues in all calib a ion plo s did no signi ican ly di e om ze o ( - es , P<0.05) and he ep oducibili y (exp essed as R.S.D. (%)) in he calib a ion slopes was 4.60% (calcula ed om six calib a ion plo s ob ained in a pe iod o wo mon hs). The lowes concen a ion o soybean p o eins de ec ed by his me hod was 0.38mg/mL (calcula ed as he concen a ion co - esponding o a signal equal o he in e cep plus h ee imes Fig. 4. UV spec a and i s and second de i a i es o he peak appea ing a 12.30minin hehea -p ocessedmea p oduc 1andin hesoybeanp o einisola e. M.C. Ga c´ıa e al. / Analy ica Chimica Ac a 559 (2006) 215–220 219 Table 1 Cha ac e is ics o he de eloped RP-HPLC me hod o he de e mina ion o soybean p o eins in hea -p ocessed mea p oduc s De ec ion limi 0.38mg/mL (0.19% (w/w))a Quan i a ion limi 1.26mg/mL (0.63% (w/w))a Exis ence o ma ix in e e encesb Slope by he ex e nal s anda d me hod 30.23±1.39 (n=6) Slope by he s anda d addi ions me hod 31.33±0.34 (n=4) Repea abili y (R.S.D., %) (n=10)c0.55 In e media e p ecision (R.S.D., %) (n=7)d7.05 (SPI solu ion o 1.86mg/mL), 1.67 (SPI solu ion o 11.27mg/mL) In e nal ep oducibili y (R.S.D., %) (n=5)e0.28 Reco e y (%) 3.05mg/mL soybean p o eins 99 6.65mg/mL soybean p o eins 102 9.20mg/mL soybean p o eins 102 12.00mg/mL soybean p o eins 101 Absolu e eco e yg(%) P ocessed mea spiked wi h 5.90mg SPI 96 P ocessed mea spiked wi h 8.75mg SPI 100 P ocessed mea spiked wi h 11.80mg SPI 102 P ocessed mea spiked wi h 13.50mg SPI 95 aLimi s o de ec ion and quan i a ion e e ed o soybean p o eins we e de e - mined ela i e o 10g o ini ial mea p oduc . bThecompa isono slopeswaspe o medbyusinga - es and hecompa ison o a iances by using a F- es . cRepea abili y in peak a ea when injec ing 10 consecu i e imes a solu ion o 7.53mg/mL o soybean p o ein isola e. dIn e media e p ecision in peak a ea ob ained in he analysis o wo SPI solu ions co esponding o he lowes and he highes concen a ions o he cal- ib a ion plo (maximum e o ) in ou di e en days. eRep oducibili y in peak a ea ob ained in he analysis o i e indi idually p epa ed ex ac s o he hea -p ocessed mea p oduc 1 (46.3±1.08mg/mL). Reco e y o soybean p o eins when di e en amoun s o SPI we e added o he ex ac ob ained om he hea -p ocessed mea p oduc 1. E e y de e mina- ion was pe o med by duplica e. gReco e y o soybean p o eins when di e en amoun s o he SPI we e ini- ially added o he hea -p ocessed mea p oduc 1. E e y de e mina ion was pe o med by duplica e. he s anda d e o o he calib a ion plo ), which means ha he me hod can de ec an addi ion o 0.19% (w/w) o soybean p o eins( ela ed o10go ini ial mea p oduc ).Thelowes con- cen a ion o soybean p o eins de e mined by his me hod was 1.26mg/mL (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 ), which means ha i was possible o quan- i a e an addi ion o soybean p o eins o 0.63% (w/w) ( ela ed o 10g o ini ial mea p oduc ). Bo h de ec ion and quan i a ion limi s we e a bi highe han hose ob ained o simila mea p oduc s using pe usion ch oma og aphy [20]. The e alua ion o he p esence o ma ix in e e ences was pe o med by compa ing he slopes o he calib a ion lines ob ained by he ex e nal s anda d calib a ion me hod wi h he slopesob ainedwhenapplying hes anda daddi ionscalib a ion me hod. The use o - and F- es s (P<0.05) o make his com- pa ison e ealed ha he p oposed me hod did no su e om ma ixin e e ences.This ac cons i u edoneo hemainad an- ageso hep esen me hodsince he analysis o simila samples by pe usion ch oma og aphy esul ed in ma ix in e e ences, hus, being necessa y he use o s anda d addi ions [20]. The p ecision o he me hod was de e mined by he e alua- ion o epea abili y, in e media e p ecision, and in e nal ep o- ducibili y. The epea abili y, exp essed as R.S.D. (%) in peak a ea, was be e han 0.6%. In e media e p ecision was de e - mined by injec ing wo s anda d solu ions o SPI whose p o- ein concen a ions co esponded o he highes and he lowes concen a ions o he wo king concen a ion ange (maximum e o ). The R.S.D. alues obse ed we e close o 7.0% a low concen a ion le els and lowe han 1.7% a high concen a ion le els. Rep oducibili y in he same day was be e han 0.3%. The p ecision in e ms o e en ion ime was always excellen wi h e y low R.S.D. alues. The eco e y o soybean p o eins when di e en mea ex ac s we e spiked wi h di e en amoun s o SPI was also de e mined obse ing alues e y close o 100%. Mo eo e , he accu acyo heme hodwasalsoe alua edwhendi ec lyspiking he mea p oduc s obse ing eco e ies as good as he p e ious ones. 3.4. Quan i a i e analysis o soybean p o eins in comme cial hea -p ocessed mea p oduc s Thede elopedme hodwasapplied o hequan i a iono soy- bean p o eins in ou di e en hea -p ocessed mea p oduc s. Fo e e y mea p oduc , a leas , h ee indi idual de e mina ions we e pe o med and e e y eplica e was injec ed wice in o he ch oma og aphicsys em.Theconcen a iono soybeanp o eins de e mined in e e y sample by he ex e nal s anda d calib a ion me hod is shown in Table 2. The concen a ion o soybean p o- eins anged om 0.90% o he hea -p ocessed mea p oduc 4 o 1.54% o he hea -p ocessed mea p oduc 1 and he R.S.D. ob ained in all cases was always lowe han 5%. Fu he mo e, heconcen a iono soybeanp o einsin hehea -p ocessedmea p oduc 1 was also de e mined by using he s anda d addi ions calib a ion me hod and he alue ob ained was 1.60%, which was e y close o he alue de e mined by he ex e nal s anda d me hod o calib a ion (1.54%). The soybean p o ein con en s obse ed o hese samples we e compa ed wi h hose ob ained o p oduc s wi h he same composi ion by using pe usion ch oma og aphy [20]. Soybean p o ein con en s ob ained by he pe usion me hod (soybean p o ein con en s anged om 0.60% o 1.31% when 18 di e en mea p oduc s we e analysed) we e e y simila o hose ob ained by he p oposed me hod and we e Table 2 Soybean p o ein con en de e mined in di e en comme cial hea -p ocessed mea p oduc s by RP-HPLCa Hea -p ocessed mea p oduc Soybean p o ein con en (mg/100mg o sample) R.S.D. (%) 1 1.54 0.28 2 1.27 4.39 3 1.48 2.50 4 0.90 2.53 aE e y esul is he a e age alue ob ained om h ee indi idual de e mina- ionsexcep o he hea -p ocessed mea p oduc 1 in whose case, i eindi idual de e mina ions we e pe o med. All esul s a e exp essed as is basis. 220 M.C. Ga c´ıa e al. / Analy ica Chimica Ac a 559 (2006) 215–220 always lowe han he maximum allowance by Spanish egula- ions, 3% soybean p o eins in he mea p oduc as is basis. 4. Conclusions De ec ion and quan i a ion o soybean p o eins in hea - p ocessed mea p oduc s has been possible by e e sed-phase HPLC. The me hod used a linea bina y g adien o he sepa- a ion o soybean p o eins om mea componen s. Due o he ac ha mea componen s we e e y s ongly e ained on he s a iona y phase, a mobile phase con aining an o ganic modi ie wi h high elu ion s eng h was needed. The ex ac ion o soy- beanp o eins om mea p oduc swasbes pe o medbyusinga ca bona e bu e a pH 9.60. The ch oma og ams ob ained om hea -p ocessed mea p oduc s we e e y simila and all showed a peak a he same e en ion ime (∼12.30min) close o one o he peaks appea ing in he soybean p o ein isola e (used as s an- da d o soybean p o eins) ch oma og am. Using his peak, he me hod enabled he de ec ion and quan i a ion o up o 0.19% (w/w) and 0.63% (w/w), espec i ely, o soybean p o eins. No in e e ences om he ma ix o he sample we e de ec ed o he mea p oduc s analysed. Resul s ob ained in he e alua ion o he p ecision and eco e y o he me hod we e sa is ac o y. The quan i a i e analysis o ou di e en hea -p ocessed mea p oduc s comme cially a ailable e ealed ha he concen a ion o soybeanp o einsin hesep oduc swasalwaysbelow hemax- imum allowance by Spanish egula ions, 3% soybean p o eins in he mea p oduc as is basis. Acknowledgmen s The au ho s hank he Comunidad Au on´ oma de Mad id (Spain) o p ojec 07G/0025/2003. D . C. Ga c´ ıa-Ruiz also hanks he Minis y o Science and Technology (Spain) o he con ac om he Ram´on y Cajal p og am (RYC-2003-001). Re e ences [1] S.C.M. Yuso , A.S. Babji, In . J. Food Sci. Nu . 47 (1996) 323. [2] J.T. Kee on, Mea Sci. 36 (1994) 261. [3] P.J. Shand, P oduc ion and P ocessing o Heal hy Mea , Poul y and Fish P oduc s, Chapman & Hall, London, 1997, pp. 191– 209. [4] G. del Campo, B. Gallego, I. Be egi, A. Casado, Food Chem. 36 (1998) 187. [5] S.A. Abdel-Aziz, S.A. Esmail, L. Hussein, F. Janssen, Food Chem. 60 (1997) 389. [6] Soy P o ein Council, Soy P o ein P oduc s. Cha ac e is ics, Nu i ional Aspec s and U iliza ion, Washing on, DC, 1987. [7] J. Belloque, M.C. Ga c´ ıa, M. To e, M.L. Ma ina, CRC C i . Re . Food Sci. Nu . 42 (2002) 507. [8] AOAC O icial Me hod 998.10. Soy p o ein in aw and hea -p ocessed mea p oduc s, Enzyme-Linked Immunoso ben Assay. In O icial Me h- ods o Analysis, 16 h ed., Re . 1996, 1997. 1998; AOAC In e na ional, Gai he sbu g, MD. [9] S.J. Koppelman, C.M.M. Lakemond, R. Vlooswijk, S.L. He le, De ec- ion o soy p o eins in p ocessed oods: li e a u e o e iew and new expe imen al wo k, J. AOAC In . 87 (2004) 1398. [10] F.J. Bailey, J. Sci. Food. Ag ic. 27 (1976) 827. [11] J.W. Llewellyn, A.C. Dean, R. Sawye , F.J. Bailey, C.H.S. Hi chcock, J. Food. Technol. 13 (1978) 249. [12] I.B. Aga e , K.J. B ian , J.W. Llewellyn, R. Sawye , F.J. Bailey, C.H.S. Hi chcock, J. Sci. Food Ag ic. 37 (1986) 317. [13] B. Lindq is , J. ¨ Os g en, I. Lindbe g, Z. Lebensm. Un e s. Fo sch 159 (1975) 15. [14] W. Linsbe g, J. ¨ Ohman, S. Wold, Anal. Chim. Ac a 171 (1985) 1. [15] C.G. Za kadas, C.N. Ka a zas, S. Khanizadeh, J. Ag ic. Food Chem. 41 (1993) 642. [16] M. Zhi-Ling, W. Yan-Ping, W. Chun-Xu, M. Fen-Zhi, Am. Lab. 29 (1997) 27. [17] N. Pa is, P.J. Gillespie, HPLC sepa a ion o soy and bee p o ein isola es. In: J.P., Chemy, R.A., Ba o d (Eds.), Me hods o P o- ein Analysis, Champaign: Am. Oil Chem. Soc., 1988, pp. 142– 155. [18] S.H. Ashoo , P.G. S iles, J. Ch oma og . 393 (1987) 321. [19] F. Cas o-Rubio, M.C. Ga c´ ıa, R. Rod ´ ıguez, M.L. Ma ina, J. Ag ic. Food Chem. 53 (2005) 220. [20] F. Cas o, M.L. Ma ina, J. Rod ´ ıguez, M.C. Ga c´ ıa, Food Addi . Con am. 22 (2005) 1209. [21] F. Cas o-Rubio, M.C. Ga c´ ıa, R. Rod ´ ıguez, J. Rod ´ ıguez, M.L. Ma ina, Food Chem., in p ess. [22] M.C. Ga c´ ıa, M. To e, F. Labo da, M.L. Ma ina, J. Ch oma og . A 758 (1997) 75. [23] M.C. Ga c´ ıa, M.L. Ma ina, M. To e, J. Liq. Ch om. Rel. Technol. 23 (2000) 3165. [24] M. C iado, F. Cas o-Rubio, C. Ga c´ ıa-Ruiz, M.C. Ga c´ ıa, M.L. Ma ina, J. Sep. Sci. 28 (2005) 987.