Characterization and quantitation of soybean proteins in commercial soybean products by capillary electrophoresis

Cha ac e iza ion and quan i a ion o soybean p o eins in comme cial soybean p oduc s by capilla y elec opho esis Capilla y elec opho esis was applied o he i s ime o de e mine soybean p o eins in comme cial soybean p oduc s. The mos sui able condi ions o he analysis o hese p oduc s in less han 7 min we e 0.05 Mphospha e bu e (pH 8) wi h 1 Mu ea; de ec- ion wa eleng h, 254 nm; applied ol age, 20 kV; and empe a u e, 30 o C. Quan i a ion o soybean p o eins was achie ed using e e enced condi ions by means o he me h- od o s anda d addi ions, using as s anda d a soybean p o ein isola e. This me hod was alida ed and applied o he quan i a ion o soybean p o eins in comme cial p od- uc s de i ed om soybean p o ein isola e and soybean seeds. Keywo ds: Capilla y elec opho esis / Soybean p o eins / Quan i a ion EL 3514 Ca men Ga cía-Ruiz 1 M. Concepción Ga cía 2 Me cedes To e 2 M. Luisa Ma ina 1,2 1 Cen o de Tecnología de Alimen os y Se icios Biosani a ios, Uni e sidad de Alcalµ, C a. Mad id- Ba celona, Alcalµ de Hena es (Mad id), Spain 2 Depa amen o de Química Analí ica, Facul ad de Ciencias, Uni e sidad de Alcalµ, C a. Mad id- Ba celona, Alcalµ de Hena es (Mad id), Spain 1 In oduc ion In ecen yea s, he consump ion o soybean-de i ed p oduc s has inc eased eno mously due o he high nu i- ional alue, low cos , and abundance o his leguminous plan . Mo eo e , echnological ad ances, such as bio- echnology and o he indus ial p ocesses, ha e imp o ed he quali y and unc ional p ope ies o soybean and soy- bean-de i ed p oduc s, enabling he use o soybean p o- eins (which cons i u e 48±50% o he soybean) in he manu ac u e o a wide a ie y o oods u s. App oxi- ma ely 80±90% o he soybean p o eins a e s o age p o- eins; hese majo p o eins a e globulins ha can be ac- iona ed based on hei sedimen a ion coe icien s in he 11S globulin (isoelec ic poin 6.4) and 7S globulin (iso- elec ic poin 4.8) [1]. A g ea a ie y o soybean-de i ed p oduc s can be ound in ma ke s. Some a e di ec ly de- i ed om soybean seeds (as is he case o soybean lou and ex u ed soybean) while o he s a e p epa ed om soybean p o ein isola es. These p o ein isola es a e ob- ained om soybean lou o whi e lakes by emo ing mos o he nonp o ein componen s. Among some o he p oduc s made om soybean p o ein isola es a e soy- bean dai y-like de i a i es [1]. These p oduc s (powde ed o liquid milk and in an o mulas) a e an al e na i e o dai y p oduc s o indi iduals alle gic o animal milk p o- eins [2]. Fu he uses o soybean ha e been in he manu- ac u e o o he p oduc s such as mea , ish, bake y goods, e c. [1]. The e sa ili y, inc easing use, and in e es ing p ope ies o soybean p o eins ha e p omo ed he de elopmen o di e en me hods o hei analysis. In ecen yea s, among he analy ical echniques mos o en used o he cha ac e iza ion o soybean p o eins in comme cial p od- uc s a e high pe o mance liquid ch oma og aphy (HPLC) echniques, speci ically e e sed-phase HPLC (RP- HPLC) [3±8]. Pe usion RP-HPLC has la ely been used o he ul a apid de e mina ion o hese ege able p o- eins [9] and sepa a ion o soybean p o eins and bo ine whey p o eins [10]. Al hough capilla y elec opho esis (CE) has been widely used o sepa a e biopolyme s such as p o eins, his echnique has a ely been employed o analyze soybean p o eins. Thus, Wong e al. [11] em- ployed a CE me hod o moni o ing soybean p o ein hyd olysis du ing p ocessing and o inge p in ing a ious ypes o p o ein p oduc s. On he o he hand, Kanning e al. [12] employed capilla y zone elec opho esis (CZE) and hyd ophilically coa ed used-silica capilla y o analyze soybean p o eins, esol ing soybean p o eins and hei isola ed ac ions in a g ea numbe o peaks in abou 35 min. The pu pose o his wo k was o de elop a apid CE me hod o he sepa a ion o soybean p o eins and i s applica ion o he quan i a ion o soybean p o eins in p od- uc s de i ed om soybean p o ein isola es (powde ed o liquid soybean milk o soybean in an o mulas) and om soybean seeds (soybean lou , ex u ed soybean, and liq- uid soybean milk). 2 Ma e ials and me hods 2.1 Chemicals and samples All eagen s we e o analy ical g ade. Bo ic acid, disodium phospha e anhyd ous, and ci ic acid we e ob ained om Scha lau (Ba celona, Spain); 2-[N-cyclohexylamino]e h- anesul onic acid (CHES) was pu chased om Sigma (S . Co espondence: D . M. Luisa Ma ina, Depa amen o de Quími- ca Analí ica, Facul ad de Ciencias, Uni e sidad de Alcalµ, C a. Mad id-Ba celona Km. 33.600, 28871 Alcalµ de Hena es (Ma- d id), Spain E-mail: [email p o ec ed]s Fax: +34-91-8854971 Abb e ia ions: MHEC, me hylhyd oxye hylcellulose; SPI, soy- bean p o ein isola e Elec opho esis 1999, 20, 2003±2012 2003  WILEY-VCH Ve lag GmbH, 69451 Weinheim, 1999 0173-0835/99/1010-2003 $17.50+.50/0 CE and CEC Louis, MO, USA); sodium hyd oxide, sodium sul a e anhyd ous, and u ea we e supplied om Pan eac (Ba ce- lona, Spain); me hylhyd oxye hylcellulose (MHEC) was u nished om Ald ich (Milwaukee, WI, USA). All solu- ions we e p epa ed wi h HPLC-g ade wa e (Milli-Q sys- em; Millipo e, Bed o d, MA, USA). The soybean p o ein isola e (SPI), aken as s anda d, was om ICN (Au o a, OH, USA); soybean milk, soybean in an o mulas, soy- bean lou , and ex u ed soybean we e pu chased om local ma ke s in Alcalµ de Hena es and Mad id (Spain). 2.2 Appa a us The capilla y elec opho esis ins umen was a 279A-HT model om Applied Biosys ems (No walk, CT, USA), equipped wi h a UV de ec o , a empe a u e-con olled capilla y compa men , and an au osample . Da a ea - men was pe o med wi h a Tu boch om acquisi ion sys- em (Pe kin Elme , No walk, CT, USA). The used-silica column, 60 cm (40 cm o he de ec o ) ´50 mmID (360 mm OD), was ob ained om Polymic o Technologies (Phoenix, AZ, USA). The empe a u e was kep cons an and equal o 30 o C. Injec ion was pe o med by acuum (67.73 kPa o 1 s). 2.3 P ocedu e Bu e s we e p epa ed as ollows: (i) sal s, acids ( iso- dium ci a e, CHES, bo ic acid and disodium phospha e), and addi i es (sodium sul a e, MHEC and u ea) we e weighed and dissol ed in HPLC-g ade wa e ; (ii) solu ions we e sonica ed o a ew minu es; (iii) pH was adjus ed o he selec ed alue wi h a concen a ed solu ion o sodium hyd oxide (bo a e and CHES bu e s) o wi h he app op i- a e acid (ci ic acid o phospho ic acid) in he case o ci - a e and phospha e bu e s; (i ) once p epa ed, hese sepa a ion media we e sonica ed o 5 min and il e ed be o e injec ion. Sample solu ions we e p epa ed by weighing and dissol ing he adequa e amoun o soybean p oduc in each sepa a ion medium; sonica ion was o 5 min, and cen i uga ion a 3000 pm o 5 min. The clea sample solu ion ob ained was injec ed in he elec opho- e ic sys em. Typically, analyses we e pe o med au o- ma ically wi h he equipmen desc ibed be o e, using a un sequence in o de o imp o e ep oducibili y o e en- ion imes. A he beginning o he day, he column was i s insed wi h HPLC-g ade wa e (5 min), ollowed by 0.1 Msodium hyd oxide (5 min), and inally HPLC-g ade wa e (5 min). The p o ocol used o pe o m each analysis was: (i) 2 min inse wi h HPLC-g ade wa e ; (ii) 2 min inse wi h 0.1 Msodium hyd oxide; (iii) 2 min inse wi h HPLC-g ade wa e ; (i ) 4 min inse wi h he sepa a ion medium (bu e and addi i es); ( ) hyd odynamic sample injec ion om he sample ial; ( i) sample sepa a ion un o 7±10 min wi h he sepa a ion medium in inle ial. All s eps (i± i) we e made wi h he sepa a ion medium in he ou le ial. Du ing he sepa a ion s ep ( i), inle and ou le bu e s we e enewed o each sample; howe e , o p e- limina y expe imen s hey we e eu ilized o ou injec- ions. 2.4 Quan i a i e analysis Fo quan i a i e analysis o eal samples, condi ions we e: 0.05 Mphospha e bu e (pH 8)/1 Mu ea; de ec ion wa e- leng h, 254 nm, and applied ol age, 20 kV. Quan i a i e analysis o eal samples de i ed om SPI and soybean seeds was achie ed wi h he me hod o s anda d addi- ions. Fo his pu pose, ou solu ions (excep o in an o mulas o which, due o he complexi y o he ma ix, i e solu ions we e conside ed) we e p epa ed by aking he same amoun o soybean p oduc and inc easing amoun s o SPI ( anging om 1.5 o 4.5 mg/mL). Peak a eas we e in eg a ed by se ing he baseline om alley o alley. 2.5 Da a ea men The linea i y in he calib a ion plo s was es ed by leas - squa es eg ession analysis ca ied ou wi h a Uni a ia e Linea Calib a ion P og am [13] and by he p ocedu e de- sc ibed by Do schel e al. (5% ole ance) [14]. 3 Resul s and discussion Some p elimina y expe imen s we e pe o med in o de o selec he expe imen al condi ions enabling he analysis o soybean p o eins. The SPI s anda d was injec ed unde di e en expe imen al condi ions in which he na u e and concen a ion o he sepa a ion bu e and i s pH, de ec- ion wa eleng h, and he applied ol age we e modi ied. Two bu e s o ino ganic na u e (bo a e and phospha e) and wo o he s o o ganic na u e (ci a e and CHES) we e employed wi h and wi hou addi i es such as sodium sul- a e, MHEC, and u ea. Sodium sul a e was used as modi- ie o he elec oosmo ic low (EOF) because he inc ease o ionic s eng h o he bu e dec eases he EOF and imp o es esolu ion [15]. MHEC was used o p e en adso p ion o p o eins o he inne wall o he cap- illa y [12, 15]. U ea, a dena u ing agen o p o eins, was used a low concen a ion (1 M; excep o ci a e bu e o which a 6 Mu ea concen a ion was added) o inc ease p o ein solubiliza ion [15, 16]. Ci a e bu e was used wi h u ea and MHEC as addi i es a di e en pH anging om 2.6 o 3.5. In he condi ions 2004 C. Ga cía-Ruiz e al. Elec opho esis 1999, 20, 2003±2012 speci ied in Table 1, he injec ion o he SPI s anda d a wo di e en de ec ion wa eleng hs was pe o med. A 214 nm (co esponding o he maximum abso p ion o pep ide bonds) no signal was de ec ed and a 264 nm (co esponding o he maximum in he abso p ion spec a o he SPI s anda d in ci a e bu e agains he same ci - a e bu e as blank) one b oad peak was ob ained a abou 25 min. Such bad esul s could be expec ed since a acid pH, soybean p o eins would be posi i ely cha ged and hey could be adso bed in o he inne wall o he cap- illa y (nega i ely cha ged). When bo a e, CHES, and phospha e bu e s we e used a basic pH (pH ~ 8) wi h di e en addi i es (Table 1), wo peaks we e ob ained when he SPI s anda d was injec ed. In his case, sho analysis imes we e ob ained (Fig. 1). Mig a ion imes o peaks 1 and 2 o SPI we e abou 4 and 6 min o bo a e and phospha e bu e s, espec i ely, and e en sho e o he CHES bu e (abou 3 and 4 min). These esul s we e ob ained using a de ec ion wa eleng h o 254 nm; howe e , o he de ec ion wa e- leng hs we e also in es iga ed o hese bu e s. In ac , in addi ion o 214 nm and he maximum abso p ion wa e- leng h o SPI in each bu e (202 nm o bo a e bu e , 230 nm o CHES bu e and 233 nm o phospha e bu - e ), wa eleng hs co esponding o he maximum abso p- ion o ce ain amino acids we e in es iga ed: 245 nm (cys ine), 254 nm (phenylalanine), and 280 nm (a oma ic amino acids such as y osine and yp ophan). As an example, Fig. 2 shows he e ec o he de ec ion wa e- leng h on he analysis o SPI wi h a bo a e bu e . As in he case o phospha e and CHES, 254 nm was ound o be he bes wa eleng h in o de o ob ain maximum signal and minimum baseline noise oge he wi h good esolu- ion. A compa ison o he esul s ob ained a acid pH alues wi h ci a e bu e and a basic pH wi h bo a e, phospha e, and CHES bu e s showed he con enience o using basic pH. A hese pH alues, soybean p o eins a e nega- i ely cha ged (isoelec ic poin s close o 4.8 and 6.4) and he adso p ion o he nega i ely cha ged inne wall o he capilla y is minimum. Howe e , when pH alues highe han 8 we e in es iga ed, esul s did no imp o e. In ac , when he pH was inc eased o e 8, he signal ob ained o SPI de e io a ed and cu en in ensi y inc eased. The e o e, pH 8 was conside ed app op ia e o analyze soybean p o eins. Finally, al hough di e en alues o applied ol age anging om 15 o 25 kV we e es ed (see Table 1), a alue o 20 kV was chosen in o de o dec ease analysis ime wi hou excessi ely inc easing he cu en in ensi y. Elec opho esis 1999, 20, 2003±2012 CE o soybean p o eins 2005 Table 1. Resul s o some p elimina y expe imen s in which SPI was injec ed in a CE sys em Bu e Addi i es l(nm) pH V (kV) I (mA) Numbe Sepa a ion Peaks o peaks ime (min) shape 0.01 M Ci a e a) 6 M U ea + 0.7 mg/mL 214 3.5 25 27 ± ± ± MHEC 264 3.5 20 17 1 25 B oad 264 2.6 25 40 1 25 B oad 0.2 M Bo a e a), b) 0.03 M Na 2 SO 4 214 8.2 25 98 3 9 2 ou o 3 we e na ow peaks 245 8.2 25 98 2 9 Na ow 280 8.2 25 98 2 9 Na ow and small peaks 254 8.2 25 98 2 9 Na ow and la ge peaks 254 8.2 20 67 2 13 Na ow 254 8.2 15 42 2 22 Na ow 0.1 M Bo a e c) 0.03 M Na 2 SO 4 254 8.2 20 77 2 6 Na ow 0.1 M Bo a e c) 0.03 M Na 2 SO 4 + 0.2 mg/mL 254 8.2 20 80 2 6 Na ow MHEC 0.05 M CHES c) 1 M U ea 230 8.6 20 2 1 (wi h 5 B oad o he signals) 0.1 M CHES c) 1 M U ea 230 8.0 20 2 2 4.5 B oad 230 9.0 20 11 1 5 B oad 230 10.0 20 41 1 6.5 B oad 254 8.0 20 2 2 4 Na ow 0.05 M Phospha e c) None 254 8.0 20 85 2 5 Na ow 0.03 M Na 2 SO 4 254 8.0 20 190 2 5 Na ow 1 M U ea 254 8.0 20 88 2 6 Na ow a) Sample injec ion, 67.73 kPa o 2 s b) Capilla y, 75 cm (50 cm o he de ec o ) ´50 mm ID (360 mm OD) c) Sample injec ion, 67.73 kPa o 1 s 3.1 Cha ac e iza ion o comme cial soybean p oduc s All o he abo e-men ioned p elimina y esul s enabled us o selec he bes expe imen al condi ions o analyze soy- bean p o eins: bo a e, CHES o phospha e bu e s a pH close o 8 using a de ec ion wa eleng h o 254 nm and an applied ol age o 20 kV. These ini ial condi ions we e applied o cha ac e ize comme cial soybean p oduc s. Howe e , o he condi ions such as bu e concen a ion o he addi i e©s na u e we e o be selec ed. In o de o cha - ac e ize comme cial soybean p oduc s by capilla y elec- opho esis, some o he expe imen al condi ions used in he p elimina y s udy and included in Table 1 we e chosen: a 0.1 Mbo a e bu e wi h 0.03 Msodium sul a e and 0.2 mg/mL MHEC, a 0.1 MCHES bu e wi h 1 Mu ea and a 0.05 Mphospha e bu e wi h 1 Mu ea. These bu - e s we e used o injec in o he capilla y elec opho esis sys em he SPI s anda d and comme cial soybean de i a- i es such as soybean milk (liquid and powde ed), in an o mulas, soybean lou , and ex u ed soybean. Simila esul s we e ob ained in he case o SPI wi h bo a e, CHES and phospha e bu e s (as shown in Fig. 1). Howe e , when comme cial soybean p oduc s we e injec ed, elec ophe og ams ob ained wi h bo a e and CHES bu e s p esen ed a highe complexi y han hose ob ained wi h phospha e bu e due o he inc ease in he baseline noise and he numbe o signals om he sample ma ix, especially o in an o mulas, he composi ion o which is mo e complex han ha o he o he samples. Figu e 3 shows he elec ophe og ams ob ained wi h phospha e bu e o ou di e en kinds o soybean milks ( wo powde ed and wo liquid) made om a soybean p o- ein isola e. In hese samples, he wo peaks o soybean p o eins can be iden i ied, ob aining a highe numbe o signals o he powde ed han o he liquid soybean milk acco ding o hei mo e complex composi ion. Especially o he powde ed soybean milk, hese elec ophe og ams a e simila o ha ob ained o SPI (Fig. 1), which is he second majo peak. Howe e , he con a y is obse ed o hose p oduc s di ec ly de i ed om soybean seeds and no om a soybean p o ein isola e (soybean lou , 2006 C. Ga cía-Ruiz e al. Elec opho esis 1999, 20, 2003±2012 Figu e 1. Elec ophe og ams co esponding o he injec ion o SPI wi h di e en sepa a ion media. Condi ions: de ec ion wa eleng h, 254 nm; applied ol age, 20 kV; injec ion by acuum, 67.73 kPa o 1 s; empe a u e, 30 o C; concen a ion o SPI, as basis, 6 mg/mL; capilla y, 60 cm (40 cm o he de ec- o ) ´50 mm ID (360 mm OD). Sepa a ion media: (a) 0.1 Mbo a e bu e , 0.03 Msodium sul a e, 0.2 mg/mL MHEC (pH 8.2); (b) 0.1 MCHES bu e , 1 Mu ea (pH 8); (c) 0.05 Mphospha e bu e , 1 M u ea (pH 8). ex u ed soybean, and milk om soybean seeds). Figu e 4 shows he elec ophe og ams ob ained wi h phospha e bu e o h ee p oduc s di ec ly de i ed om soybean seeds. These elec ophe og ams a e cha ac e ized by hei simplici y and by he ac ha he i s peak is he majo one, and no he second as o SPI. Finally, elec o- phe og ams co esponding o in an o mulas we e he mos complex acco ding o he composi ion o he sam- ple. Since phospha e bu e showed g ea e possibili ies o s udy comme cial soybean samples, his bu e was chosen o ca y ou quan i a i e analysis o soybean p o- eins in his kind o p oduc s. 3.2 Quan i a i e analysis In o de o achie e he quan i a i e analysis o eal sam- ples, he me hod o ex e nal s anda d was i s employed using he SPI as s anda d. Howe e , quan i a ion o soy- bean p o eins in hese p oduc s using he addi ion o he a eas o wo peaks as well as he a ea o one o he wo peaks ob ained o each sample, and in e pola ing hese alues in he calib a ion plo , ga e ise o a highe soy- bean p o ein con en han ha indica ed on he label o each p oduc . In o de o in es iga e he exis ence o ma ix e ec s, calib a ion was pe o med by he me hod Elec opho esis 1999, 20, 2003±2012 CE o soybean p o eins 2007 Figu e 2. Elec ophe og ams showing he e ec o he de ec ion wa eleng h on he analysis o he SPI wi h a bo a e bu e . Condi ions: 0.2 Mbo a e bu e , 0.03 Msodium sul a e (pH 8.2); applied ol - age, 25 kV; injec ion by acuum, 67.73 kPa du ing 2 s; empe a u e, 30 o C; concen a ion o SPI, as basis, 2 mg/mL; capilla y, 75 cm (50 cm o he de ec o ) ´50 mm ID (360 mm OD). De ec ion wa e- leng h: (a) 214 nm; (b) 245 nm; (c) 254 nm; (d) 280 nm. o s anda d addi ions and he slopes o he wo calib a ion plo s (me hods o ex e nal s anda d and s anda d addi- ions) we e compa ed. Resul s showed ha he wo slopes we e signi ican ly di e en (P<0.05), indica ing he exis ence o ma ix e ec s. Consequen ly, he me hod o s anda d addi ions was chosen o ca y ou quan i a i e analysis o soybean p oduc s. This me hod was employed using he o al a ea ( he addi ion o he a eas o wo peaks) as well as he a ea o he second peak. To e alua e he p ecision o his me hod, epea abili y and ep oducibili y we e s udied. The epea abili y in mig a ion ime and peak a ea was de e mined (as ela i e s anda d de ia ion, RSD) o i e consecu i e injec ions o some ep esen a i e samples (powde ed soybean milk 2, liquid soybean milk 1, soybean in an o mula 3, and soy- bean lou ). The esul s (g ouped in Table 2) show ha he RSD was less han 2.4% o he mig a ion ime and less han 3.2% o he peak a ea. On he o he hand, 2008 C. Ga cía-Ruiz e al. Elec opho esis 1999, 20, 2003±2012 Figu e 3. Elec ophe og ams co esponding o ou ypes o soybean milk manu ac u ed om SPI. Condi ions: 0.05 Mphospha e bu e , 1 Mu ea (pH 8); de ec ion wa eleng h, 254 nm; applied ol age, 20 kV; injec ion by acuum, 67.73 kPa o 1 s; empe a u e, 30 o C; capilla y, 60 cm (40 cm o he de ec o ) ´50 mm ID (360 mm OD). Concen a ion, as basis: (a) powde ed soybean milk 1, 8.4 mg/ mL; (b) powde ed soybean milk 2, 12.2 mg/mL; (c) liquid soybean milk 1, 99.6 mg/mL; (d) liquid soy- bean milk 2, 102 mg/mL. Elec opho esis 1999, 20, 2003±2012 CE o soybean p o eins 2009 Figu e 4. Elec ophe og ams co esponding o h ee p oduc s di ec ly de i ed om soybean seeds. Concen a ion, as basis: (a) soybean lou , 7.5 mg/mL; (b) ex u ed soybean, 7.5 mg/mL; (c) liquid soybean milk 3, 95.5 mg/mL; Expe imen al condi ions as in Fig. 3. Table 2. E alua ion o epea abili y o mig a ion imes and peak a eas employing he calib a ion by he me hod o s anda d addi ions (5 de e mina ions) Samples C M +C SPI Peak 1 Peak 2 (mg/mL) 1 RSD (%) A 1 RSD (%) 2 RSD (%) A 2 RSD (%) Powde ed soybean milk 2 a) 11.37 + 1.28 4.24 0.48 10554 1.14 6.32 0.81 26833 2.55 Liquid soybean milk 1 a) 80.00 + 1.28 4.08 0.81 10667 1.39 6.27 1.11 12254 1.93 Soybean in an o mula 3 a) 19.29 + 2.58 4.06 0.46 6654 1.74 5.80 0.84 19258 2.42 Soybean lou b) 6.94 + 1.28 4.23 1.53 53671 0.57 6.19 2.43 43993 3.21 a) De i ed om SPI b) De i ed om soybean seeds C M , sample concen a ion exp essed as d y basis C SPI , SPI concen a ion exp essed as d y basis and co ec ed by he pu i y o he s anda d ep oducibili y o he same samples was measu ed as he RSD ob ained o he i e alues o concen a ion ob- ained when i e indi idual analyses we e pe o med o a gi en sample. Resul s ob ained measu ing he o al a ea (adding he a eas o peaks 1 and 2) and he a ea o he second peak a e shown in Table 3. The RSD was less han 6.6% in he i s case and less han 4.2% in he sec- ond. Since he cha ac e is ics o he analy ical me hod de el- oped we e accep able, quan i a i e analysis o soybean p o eins in di e en comme cial samples was achie ed. Table 4 g oups he esul s ob ained conce ning he soy- bean p o ein con en o di e en comme cial p oduc s using o al a ea ( he addi ion o he a eas o peaks 1 and 2) and he a ea o he second peak and he calib a ion by means o he me hod o s anda d addi ions. Two g oups o p oduc s we e analyzed: a g oup o in an o mulas and powde ed and liquid milk manu ac u ed om a soybean p o ein isola e and a g oup o p oduc s di ec ly de i ed om soybean seeds (soybean lou , ex u ed soybean, and di e en ypes o liquid milk). Table 4 shows how he a ea o he second peak, co esponding o soybean p o- eins, enables hei co ec quan i a ion in he comme cial p oduc s s udied, especially o hose samples de i ed om soybean seeds. Gene ally, he con en ob ained o hese p o eins is in good ag eemen wi h he alue indi- ca ed on he label o he p oduc and wi h he p o ein con- en measu ed by he Kjeldahl me hod (a e age o six de e mina ions) [9]. The g ea es di e ences be ween he p o ein con en ob ained by he CE me hod and he Kjel- dahl me hod co espond o hose samples wi h a highe 2010 C. Ga cía-Ruiz e al. Elec opho esis 1999, 20, 2003±2012 Table 3. E alua ion o ep oducibili y employing he calib a ion by he me hod o s anda d addi ions (5 indi idual de e mina ions) Samples C M (mg/mL) P(A ) (%) RSD (%) P(A2) (%) RSD (%) Powde ed soybean milk 2 a) 11.37 25.88 4.13 21.46 2.44 Liquid soybean milk 1 a) 80.00 3.19 4.86 2.04 3.43 Soybean in an o mula 3 a) 19.29 10.78 6.64 11.06 4.25 Soybean lou b) 6.94 91.31 0.76 50.16 1.69 a) De i ed om SPI b) De i ed om soybean seeds C M , sample concen a ion exp essed as d y basis P(A ) (%), soybean p o ein con en de e mined om he o al peak a ea P(A2) (%), soybean p o ein con en de e mined om he a ea o peak 2 Table 4. Quan i a i e analysis o soybean p o eins in di e en comme cial soybean p oduc s Sample C M (mg/mL) P(A ) (%) RSD (%) P(A2) (%) RSD (%) P.L.(%) P.Kj.(%) Powde ed soybean milk 1 a) 7.45 35.44 1.37 34.19 0.35 35 34.81 Powde ed soybean milk 2 a) 11.37 23.14 1.22 20.86 0.59 21.3 22.31 Liquid soybean milk 1 a) 99.60 3.51 7.97 2.26 7.08 3.3 2.89 Liquid soybean milk 2 a) 102.00 3.24 0.01 2.16 0.46 3.1 2.82 Soybean in an o mula 1 a) 19.25 17.44 3.09 12.25 3.42 14.7 14.70 Soybean in an o mula 2 a) 19.04 15.67 11.10 12.52 14.85 15 14.30 Soybean in an o mula 3 a) 19.26 11.19 1.70 11.38 1.74 14 14.00 Soybean in an o mula 4 a) 19.34 8.72 4.81 9.58 3.54 14.3 14.30 Soybean lou b) 6.94 95.33 4.24 47.81 1.46 38 51.65 Tex u ed soybean b) 6.91 95.51 0.48 54.60 3.08 54 55.61 Liquid soybean milk 3 b) 95.80 11.25 7.85 4.63 9.31 3.8 ± Liquid soybean milk 4 b) 103.00 11.47 0.43 3.21 1.87 3.7 ± Liquid soybean milk 5 b) 110.10 5.43 6.64 3.02 9.37 3.6 3.70 a) De i ed om SPI b) De i ed om soybean seeds C M , sample concen a ion exp essed as d y basis P(A ) (%), soybean p o ein con en de e mined om he o al peak a ea P(A2) (%), soybean p o ein con en de e mined om he a ea o peak 2 P.L. (%), p o ein con en indica ed on he label o he p oduc P.Kj (%), p o ein con en de e mined by he Kjeldahl me hod (a e age o six de e mina ions) [9] amoun o addi i es such as i amins o amino acids which inc ease he alue o p o ein con en ob ained om o al ni ogen (Kjeldahl me hod). The e o e, he p oposed CE me hod is an in e es ing possibili y o e alua ing he eal soybean p o ein con en o a sample. Quan i a ion wi h o al a ea is no ecommended, especially no o hose p oduc s de i ed di ec ly om soybean seeds. As men ioned abo e, in such a case, a g ea inc ease in he a ea o he i s peak was ob ained, p obably due o a coe- lu ion o o he componen s exis ing in hese p oduc s wi h he i s peak o soybean p o eins. In o de o e alua e he obus ness o he me hod, he slopes o he calib a ion lines ob ained wi h he me hod o s anda d addi ions (by peak 2) we e compa ed. Table 5 shows he cha ac e is ics o hese calib a ion lines and he RSDs ob ained o he slopes, which in all cases we e less han 8.8%. Accu acy was e alua ed calcula ing he eco e y (%) o soybean p o eins ound when a known quan i y o he SPI was added o a eal sample o a soy- bean p oduc . As ep esen a i e samples, soybean pow- de ed milk 2 (de i ed om SPI) and soybean lou (de- i ed om soybean seeds) we e conside ed. Table 6 Elec opho esis 1999, 20, 2003±2012 CE o soybean p o eins 2011 Table 5. Slopes, in e cep s, co ela ion coe icien s, and obus ness (exp essed as RSD o slopes alues) co esponding o he calib a ion s aigh lines ob ained by he me hod o s anda d addi ions a) Sample Slope In e cep Co ela ion Sample Slope In e cep Co ela ion coe icien coe icien Powde ed soybean 7770 18400 0.9931 Soybean in an 4281 9284 0.9991 milk 2 b) 6672 16440 0.9985 o mula 3 b) 4093 9049 0.9993 6300 15060 0.9992 4271 9404 0.9997 6340 15940 0.9999 4318 8849 0.9927 6855 16688 0.9965 4336 8772 0.9989 RSD = 8.78% RSD = 2.28% Liquid soybean 4204 6838 0.9999 Soybean lou c) 9765 34430 0.9948 milk 1 b) 4383 7277 0.9988 11240 39210 0.9934 4316 7393 0.9971 10610 35920 0.9980 4283 6670 0.9999 9814 31480 0.9904 4135 6651 0.9994 10060 34910 0.9982 RSD = 2.67% RSD = 6.06% a) Based on he measu emen o he a ea o peak 2 in he elec ophe og am o SPI and samples b) De i ed om SPI c) De i ed om soybean seeds Table 6. Reco e y o he powde ed soybean milk 2 (de i ed om SPI) and soybean lou (de i ed om soybean seeds) by he CE me hod a) Sample C M Amoun o To al p o ein P o ein concen a ion Reco e y (mg/mL) SPI added o concen a ion ound by he CE me hod (%) he sample (mg) b) (mg/mL) (mg/mL) c) Powde ed soybean milk 2 11.37 0.86 3.30 3.22 (9.63) e) 97.58 (21.46 mg/100 mg) d) 1.72 4.16 3.84 (1.75) 92.31 2.58 5.02 4.76 (5.04) 94.82 Mean alue: 94.90 (2.64) Soybean lou 6.94 0.86 4.34 4.44 (0.56) 102.30 (50.16 mg/100 mg) 1.72 5.20 5.22 (2.59) 100.38 2.58 6.06 5.63 (2.52) 92.90 Mean alue: 98.53 (5.04) a) Expe imen al condi ions as in Fig. 3 and calib a ion by he me hod o s anda d addi ions using he a ea o peak 2 b) Exp essed as d y basis and co ec ed by he pu i y o he s anda d c) Mean alue co esponding o wo indi idual injec ions d) Mean alue o soybean p o ein concen a ion, exp essed as d y basis, ound by analyzing i e indi idual samples by he CE me hod e) RSD in % C M , sample concen a ion exp essed as d y basis