Ca men Ga cía-Ruiz1
MaConcepción Ga cía1
Alejand o Ci uen es2
MaLuisa Ma ina1
1Depa men o Analy ical
Chemis y,
Facul y o Chemis y,
Uni e si y o Alcalá,
C a. Mad id-Ba celona,
Alcalá de Hena es,
Mad id, Spain
2Depa men o Food Analysis,
Ins i u e o Indus ial
Fe men a ions, CSIC
Juan de la Cie a,
Mad id, Spain
Recei ed Decembe 7, 2006
Re ised Ma ch 9, 2007
Accep ed Ma ch 9, 2007
Resea ch A icle
Cha ac e iza ion and di e en ia ion o
di e se ansgenic and non ansgenic
soybean a ie ies om CE p o ein p o iles
Nowadays, soybeans a e comme cialized in a wide a ie y o colo s and ones. Mo eo e ,
some pigmen ed seeds a e being comme cialized as soybeans while, on o he occasions,
hese seeds a e labeled as mung beans, azuki beans o soybean ijoles gene a ing con u-
sion on hei iden i y. In his wo k, CE has been applied o he i s ime o he cha ac e -
iza ion and di e en ia ion o di e en pigmen ed beans comme cialized as soybeans.
O he seeds comme cialized as azuki, mung g een soybeans o soybean ijoles we e also
analyzed. Bo a e bu e (a pH 8.5) con aining 20% / ACN was used as he sepa a ion
media and solu ion con aining ACN/wa e (75:25 / ) wi h 0.3% / ace ic acid was used o
solubilize he p o eins om he samples. A 50 cm ba e used-silica capilla y was employed
o ob aining adequa e sepa a ions in abou 12 min. The CE p o ein pa e n obse ed o
yellow soybeans was di e en om ha co esponding o g een and ed soybeans. The
seeds comme cialized as black soybean p esen ed elec ophe og ams iden ical o simila o
hose yielded by he yellow seeds wi h he excep ion o he sample labeled as black soybeans
ijoles ha p esen ed a o ally di e en pa e n. In addi ion, CE p o ein p o iles ob ained
o azuki and mung g een soybeans we e e y simila o hose co esponding o ed soy-
beans and g een soybeans, espec i ely. Finally, he CE me hod was also applied o di e -
en ia e ansgenic and non ansgenic soybean a ie ies. Disc iminan analysis, using se -
e al p o ein peak a eas as a iable, was used o success ully classi y hese samples.
Keywo ds:
CE / Pigmen ed soybean / P o ein p o iles / T ansgenic soybean
DOI 10.1002/elps.200600799
2314 Elec opho esis 2007, 28, 2314–2323
1 In oduc ion
Soybean is comme cialized in a wide a ie y o colo s and
ones: om yellow o black passing h ough g een, b own o
ed [1–6]. The mos comme cial and abundan soybean is he
yellow pigmen ed one. Al hough, in p inciple, he o he
colo ed seeds a e also conside ed as soybean, i is no e y
clea whe he hey a e di e en a ie ies o soybean o di -
e en legumes. Fo example, g een-coa soybeans could no
be eal soybeans bu a di e en legume o iginally om
China called mung bean (Vigna adia a (L) R. Wilezek).
Ano he example is ed-coa soybeans which could be mixed
up wi h he azuki bean (Vigna angula is (Willd.) Ohwi and
Ohashi), a swee la o bean e y much used in con ec ione y
[7, 8]. Simila ly, black soybeans a e comme cially a ailable as
black soybeans o as black soybean ijoles. The e o e, he
use o he wo d ‘soybean’ o he designa ion o di e en
legumes can esul in misuse gene a ing some consume
con usion. In ac , soybean is a e y aluable bean due o i s
nu i ional and unc ional p ope ies and hese p ope ies
can be di e en om hose o o he legumes. In addi ion,
gene ic enginee ing has also yielded a u he numbe o
soybean a ie ies gene a ed by he inco po a ion o ce ain
genes in o he o iginal soybean genome. The gene ic mod-
i ica ion o soybean c ops esul ed in ansgenic soybean. Fo
ins ance, he ansgenic glyphosa e- ole an soybean (called
Roundup Ready) is a gene ically modi ied soybean esis an
o glyphosa e he bicide used o kill nea by weeds wi hou
killing he gene ically modi ied c ops, comme cialized i s
by Monsan o in 1998. To da e, al hough ansgenic soybean
is allowed o human consump ion in he Eu opean Union
(EU), he cul i a ion o he beans is no app o ed ye (h p://
www.monsan o.com). De elopmen , g ow h and elease o
new gene ically modi ied seeds and hei labelling when
used as oods a e egula ed by he EU [9].
Co espondence: P o esso MaLuisa Ma ina, Dep . o Analy ical
Chemis y, Facul y o Chemis y, Uni e si y o Alcalá, E-28871
Alcalá de Hena es, Mad id, Spain
E-mail: [email p o ec ed]
Fax: 134-91-8854971
Abb e ia ions: IE-HPLC, ion-exchange ch oma og aphy; GMO,
gene ically modi ied o ganism
©2007 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com
Elec opho esis 2007, 28, 2314–2323 CE and CEC 2315
Soybean cul i a di e en ia ion has adi ionally been pe -
o med based on he pigmen a ion and mo phological ai s.
Mo e accu a e me hods we e based on he s udy o DNA and
p o eins p esen in soybean. The iden i ica ion o soybean cul-
i a s based on he analysis o soybean p o eins (mainly glyci-
nin and conglycinin) has mainly been ca ied ou by using gel
elec opho esis [10–15] and ch oma og aphic [16, 17] ech-
niques. RP [16, 17] and size-exclusion [17] ch oma og aphies
ha ebeen applied o he cha ac e iza ion o soybean cul i a s.
The ch oma og aphic p o iles enabled he di e en ia ion
among e y di e en cul i a s bu i was e y di icul o di e -
en ia e among close cul i a s. Mo eo e , he analysis imes
employed o he sepa a ion o soybean p o eins anged om
20 o90 min. Mo e ecen ly, ou esea ch g oup hasapplied RP
ch oma og aphy (RP-HPLC) [18] and ion-exchange ch oma-
og aphy (IE-HPLC) [19] o soybean cul i a cha ac e iza ion.
Fo ha pu pose, en beans wi h di e en coa pigmen a ion
(yellow, g een, ed and black) and ma ke ed as soybeans we e
analyzed by RP-HPLC and IE-HPLC. In bo h cases, analysis
imes needed o he sepa a ion o soybean p o eins (3 min by
RP-HPLC and 9 min by IE-HPLC) we e signi ican ly lowe
han hose p e iously employed by he o he au ho s [16, 17].
Mo eo e , all seeds p esen ing he same coa colo showed he
same p o eins p o ile by RP-HPLC wi h he excep ion o black
soybeans. The applica ion o IE-HPLC o he same pu pose
did no enable he clea di e en ia ion among soybean cul i-
a s because simila ch oma og ams we e obse ed o all
seeds. Rega ding he de ec iono Roundup Ready soybean, he
mos equen p ocedu e is o apply PCR me hods whe eDNA
agmen s a e ampli ied and hen usually de e mined by
s anda d gel elec opho esis [20]. Ve y ecen ly, o he p oce-
du escombiningPCRandCGE oanalyseDNA agmen s[21,
22]o 2-DE os udy he p o ein p o ilesha ebeen usedasgood
app oaches o assess he sa e y o ood and eed de i ed om
gene ically modi ied c ops [23–25].
CE is a powe ul sepa a ion echnique use ul o he
analysis o ag icul u al p oduc s and oods [26]. Thus, he
iden i ica ion o cul i a s, quali y con ol and de ec ion o
ood adul e a ions a e impo an issues ha ha e al eady
been ca ied ou by CE o p o eins [27, 28]. The goal o his
wo k was o in es iga e o he i s ime he po en ial o CE
o cha ac e ize soybeans comme cialized as yellow, ed,
g een o black soybeans based on hei di e en p o ein
p o iles. Mo eo e , a s udy is ca ied ou o in es iga e he
use ulness o CE o di e en ia e be ween some ansgenic
and non ansgenic soybeans.
2 Ma e ials and me hods
2.1 Chemicals and samples
All eagen s employed o he p epa a ion o he sepa a ion
bu e s we e o analy ical g ade. Bo ic acid was om Fluka
(Buchs, Swi ze land), sodium hyd oxide and glacial ace ic
acids we e supplied om Pan eac (Ba celona, Spain) and
HPLC-g ade ACN was om Me ck (Da ms ad , Ge many).
All solu ions we e p epa ed wi h ul apu e wa e om a
Milli-Q sys em (Millipo e, Bed o d, MA, USA).
Six een beans comme cialized as soybeans (see Table 1)
and wi h di e en coa pigmen a ion (six yellow soybeans
(S1, S2, S3, S4, S5 and S6), i e g een soybeans (S7, S8, S9,
Table 1. Seed mo phological ai s o he s udied beans
Comme cial name Pigmen a ion App oxima e
size (cm)
Shape
Whi e soybean (S1)a) Yellow coa /yellow inside ,0.7 Round
Yellow soybean (S2)a) Yellow coa /yellow inside ,0.7 Round
Soybean (S3) Yellow coa /yellow inside ,0.7 Round
Soybean (S4) Yellow coa /yellow inside ,0.7 Round
Soybean (S5) Yellow coa /yellow inside ,0.7 Round
Soybean (S6) Yellow coa /yellow inside ,0.7 Round
G een soybean (S7)a) G een coa /yellow inside ,0.5 O al
G een soybean (S8) G een coa /yellow inside ,0.5 O al
Mung g een soybean (S9)a) G een coa /yellow inside ,0.5 O al
G een soybean (S10)a) G een coa /yellow inside ,0.5 O al
G een soybean (S11)a) G een coa /yellow inside ,0.5 O al
Azuki (S12)a) Red coa /yellow inside 0.5–0.7 O al
Red soybean (S13) Red coa /yellow inside 0.5–0.6 O al
Azuki (S14)a) Red coa /yellow inside 0.6–0.8 O al
Red soybean (S15) Red coa /yellow inside 0.6–0.8 O al
Azuki (S16) Red coa /yellow inside 0.6–0.7 O al
Black soybean ijoles (S17)a) Black coa /whi e inside ,1.5 Kidney
Black soybean (S18) Black coa /yellow inside ,0.8 Round/o al
Black soybean (S19) Black coa /g een inside ,0.8 Round/o al
a) Ecological cul i e.
©2007 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com
2316 C. Ga cía-Ruiz e al. Elec opho esis 2007, 28, 2314–2323
S10 and S11), wo ed soybeans (S13 and S15) and h ee
black soybeans (S17, S18 and S19)) we e pu chased om
local ma ke s in Mad id (Spain). O he seeds comme cia-
lized as azuki we e om local ma ke s o Mad id (Spain)
(S12 and S14) and Wien (Aus ia) (S16). Seed mo phological
ai s o he s udied samples a e summa ized in Table 1. The
non ansgenic soybean lou was acqui ed om El G ane o
(Mad id, Spain). In addi ion, wo soybean lou s p epa ed
om ansgenic soybean we e kindly p o ided by INIA
(Mad id, Spain) (h p://www.inia.es/) and 34 di e en yellow
soybeans we e gi en by a Soybean Ge mplasm Bank (Cen o
de Recu sos Fi ogené icos, CRF, Mad id, Spain).
2.2 Appa a us
All expe imen s we e pe o med on an HP3DCE sys em
(Agilen Technologies, Waldb onn, Ge many) equipped wi h
an on-column DAD. Ins umen con ol and da a acquisi ion
we e pe o med by he HP3DCE ChemS a ion so wa e.
Sepa a ions we e pe o med on un ea ed used-silica capil-
la ies o 75 mm id and 375 mm od wi h a o al leng h o
58.5 cm and an e ec i e leng h o 50 cm om Composi e
Me al Se ices (Wo ces e , England, UK). The selec ed
ins umen al condi ions we e: capilla y empe a u e 257C,
injec ion by p essu e, 50 mba o 5 s, applied ol age 20 kV
and UV de ec ion a 210, 254 and 280 nm wi h a bandwid h
o 5 nm in all cases and esponse ime o 0.1 s. A 744 pH
Me e om Me ohm (He isau, Swi ze land) was used o
adjus he pH o he sepa a ion bu e s.
2.3 P ocedu e
Condi ioning o capilla y consis ed o lushing wi h 1 M
sodium hyd oxide (1 ba ) o 30 min and wi h wa e (1 ba )
o 10 min be o e i s use. Be ween sample injec ions,
capilla ies we e condi ioned wi h 0.1 M sodium hyd oxide
(1 ba ) o 2 min, Milli-Q wa e (1 ba ) o 2 min and, inally,
wi h he sepa a ion bu e (1 ba ) o 2 min.
A 100 mM bo a e bu e a pH 8.5 was p epa ed by dis-
sol ing ,1.5 g o bo ic acid in Milli-Q wa e , adjus ing he
pH a 8.5 wi h 1 M sodium hyd oxide and adjus ing he inal
olume o he solu ion a 250 mL. This bu e solu ion
(80 mL) was mixed wi h 20 mL o ACN o ob ain he sepa a-
ion bu e solu ion: 80 mM bo a e bu e wi h 20% / ACN
(appa en pH 9.2). Bu e solu ions we e il e ed p io o use
h ough 0.45 mm po e size disposable nylon il e s om
Ti an (Ea on own, NJ, USA).
All beans we e g ound wi h an au oma ic mille . P o ein
ex ac s we e p epa ed by dissol ing he app op ia e amoun
o he g ound sample in ACN/wa e (25:75 / ) con aining
0.3% / ace ic acid, he solubiliza ion solu ion ha p o ided
success ul esul s o soybean p o eins ex ac ion in he p e-
ious wo k o he au ho s [29]. Di e en sample concen a-
ions we e used o di e en soybeans s udied o ob ain
simila de ec ion signals since di e en pigmen ed soybeans
con ain di e en p o ein pe cen ages m/m on d y basis (yel-
low soybeans ,40–50%, and ed, g een and black soybeans
,20–30% [18]). Final concen a ions o 60 mg/mL o yellow
beans (,0.3 g o sample in ,5 g o solubiliza ion solu ion),
80 mg/mL o g een and ed beans (,0.3 g o sample in
,3.7 g o solubiliza ion solu ion) and 100 mg/mL o black
beans (,0.3 g o sample in ,3 g o solubiliza ion solu ion)
we e employed. Soybean lou solu ions we e 40 mg/mL o
non ansgenic soybean (,0.3 g o sample in ,7.5 g o solu-
biliza ion solu ion) and 60 mg/mL o ansgenic soybeans
(,0.3 g o sample in ,5 g o solubiliza ion solu ion). Finally,
hese solu ions we e manually shaken and cen i uged
(33626g o 10 min, 257C) be o e hei injec ion in o he CE
sys em.
2.4 Da a ea men
Peak a eas we e in eg a ed by se ing he baseline om al-
ley- o- alley. The a ea pe cen age o e e y peak was calcu-
la ed as he a e age o wo eplica es (injec ed by duplica e)
using Excel Mic oso XP®. Box-and-whiske plo s and dis-
c iminan analyses we e pe o med using he compu e
p og am S a g aphics Plus o Windows 5.0. G aphs wi h
di e en elec ophe og ams we e composed in O igin® e -
sion 7.0 so wa e.
3 Resul s and discussion
3.1 Cha ac e iza ion o di e en pigmen ed soybeans
A CE me hod p e iously de eloped by ou esea ch g oup o
achie e he sepa a ion o soybean and ice p o eins [29] was
applied in his wo k o he cha ac e iza ion o soybeans.
Bo a ebu e (80 mM)a pH 8.5con aining20% / ACNwas
employed as he sepa a ion media. The solubiliza ion medi-
um o he p epa a ion o p o ein ex ac s consis ed o ACN/
wa e (75:25 / ) con aining 0.3% / ace ic acid. Figu es 1
and 2 show he elec ophe og ams co esponding o ou di -
e en soybeans as examples o e e y pigmen a ion (yellow,
g een, ed and black) a h ee di e en wa eleng hs: 210 nm
(abso p ion o pep ide bonds), 254 nm (abso p ion o phenyl-
alanine esidues) and 280 nm (abso p ion o y osine and
yp ophan esidues). The elec ophe og am co esponding
o one azuki sample (S12) was also included in Fig. 2 oge he
wi h ha ob ained o a ed soybean sample (S13). Fo yellow,
g een and black soybeans, he wo elec ophe og ams o e -
lapped in e e y igu e ( h ee in he case o black beans) co e-
sponded o hose soybeans wi h he same coa colo p esen -
ing he mos di e en pa e ns. In all elec ophe og ams, he
i s peak co esponded o EOF while he las peak appea ed
due o he p esence o ace ic acid in he solubiliza ion medi-
um used o he p epa a ion o all sample solu ions.
Clea di e ences be ween he elec opho e ic pa e ns
ob ained o yellow soybeans and hose co esponding o ed,
g een o black soybeans could be obse ed a any o he h ee
©2007 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com
Elec opho esis 2007, 28, 2314–2323 CE and CEC 2317
Figu e 1. Elec ophe og ams co esponding o he injec ion o p o ein ex ac s ob ained om yellow soybeans
(,60 mg/mL) and g een soybeans (,80 mg/mL) using a solu ion o wa e /ACN (75:25 / ) wi h 0.3% / ace ic acid.
Sepa a ion condi ions: 80 mM bo a e bu e a pH 8.5 con aining 20% / ACN; uncoa ed capilla y wi h 75 mmid
and 50 cm e ec i e leng h (58.5 cm o al leng h); injec ion by p essu e, 50 mba o 5 s; capilla y empe a u e,
257C; applied ol age, 20 kV; UV de ec ion a 210, 254 and 280 nm.
©2007 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com
2318 C. Ga cía-Ruiz e al. Elec opho esis 2007, 28, 2314–2323
Figu e 2. Elec ophe og ams co esponding o he injec ion o p o ein ex ac s ob ained om ed beans ( ed
soybeans and azuki) (,80 mg/mL) and black soybeans (,100 mg/mL) using a solu ion o wa e /ACN (75:25 / )
wi h 0.3% / ace ic acid. O he expe imen al condi ions as in Fig. 1.
©2007 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com

Elec opho esis 2007, 28, 2314–2323 CE and CEC 2319
wa eleng hs used. In ac , when UV-abso p ion de ec ion a
210 nm was used, nine peaks we e obse ed o yellow soy-
beans, en peaks o g een soybeans, se en peaks o ed
soybeans and azuki and nine peaks o black beans. As
expec ed, when using UV de ec ion a 254 and 280 nm,
selec i i y inc eased while sensi i i y and he numbe o
peaks dec eased (see Figs. 1 and 2).
Despi e 210 nm was he wa eleng h ha enabled o ob-
ain he highe numbe o peaks, 254 and 280 nm a e he
wa eleng hs co esponding o he maximum abso p ion o
ce ain common amino acids p esen in p o eins. Since he
aim o his wo k was he cha ac e iza ion and di e en ia ion
o soybean a ie ies om CE p o ein p o iles, 254 and
280 nm we e selec ed as de ec ion wa eleng hs o u he
wo k.
In spi e o he ac ha p o eins a e he main compo-
nen s in soybean ( om 40 o 50% m/m on d y basis in yel-
low soybeans and om 20 o 30% m/m o he o he s udied
soybeans [18]), he e a e o he mino componen s p esen in
soybean ha can also abso b a hese wa eleng hs such as
iso la ones. Al hough soybean is he mos signi ican die a y
sou ce o iso la ones [30, 31], hei con en in soybean is
abou 0.4% m/m o he p o ein con en [32]. In addi ion, he
ex ac ion o iso la ones is usually pe o med wi h e y high
pe cen ages o o ganic sol en s like ACN o me hanol and
we use only a 25% / ACN in ou ex ac ion medium o
soybean p o eins. As a consequence, he signals obse ed a
254 and 280 nm in Figs. 1 and 2 could be a ibu ed o p o-
eins.
UV-abso bance de ec ion a 254 nm enabled he sepa a-
ion o p o eins in h ee main peaks o yellow soybeans and
black soybeans S18 and S19 (co esponding o peaks 1, 4 and
5 ma ked a 210 nm) al hough o black soybeans S18 an
addi ional big peak a abou 7.5 min appea ed ha did no
appea o he sample S19. Mo eo e , ou main peaks o
g een soybeans (co esponding o peaks 4g, 5g, 6g and 7g
ma ked a 210 nm) and ed soybeans (co esponding o
peaks 2 , 3 , 5 and 6 ma ked a 210 nm) we e obse ed. A
di e en nomencla u e o design elec opho e ic peaks was
adop ed o g een and ed soybeans wi h espec o yellow
and black soybeans because he peaks obse ed had di e en
mig a ion imes om hose ob ained o yellow and black
soybeans. Finally, when UV de ec ion was pe o med a
280 nm, he main peaks obse ed o yellow soybean and
black soybeans (samples S18 and S19) we e peaks 1, 2 and 4
al hough o black soybean S19 an addi ional peak was
obse ed as o 254 nm. The main peaks obse ed o g een
soybeans a 280 mn we e peaks 5g, 6g and 7g and in he case
o ed soybeans he main peaks we e 2 , 5 and 6 . The com-
pa ison o peak a ea a ios a 254 and 280 nm o he selec ed
peaks ob ained o he di e en beans wi h he same pig-
men a ion (yellow, ed and g een), showed ha he de ec o
esponse a io could be conside ed cons an (RSD ,13% o
majo peaks; peak 5 o ed beans p esen ing he highes
a iabili y, RSD ,25%). In addi ion, om elec ophe og ams
o Figs. 1 and 2, i can be obse ed ha mig a ion imes o he
di e en samples a ied. In ac , he majo a ia ion o
mig a ion imes was om sample o sample (,1–4%),
whe eas mig a ion imes epea abili y o he same sample
was easonable (,1%, n= 4). Rega ding peak a ea epea -
abili y, he injec ion o sample ou imes consecu i ely, yiel-
ded an RSD o abou 5% while peak a ea ep oducibili y in he
injec ion o wo indi idually ex ac ed samples was abou 3%.
Figu es 1 and 2 also show ha in gene al, elec opho e ic
pa e ns we e cha ac e is ic o e e y pigmen a ion. In ac ,
he main di e ence among pa e ns co esponding o soy-
beans wi h he same coa colo was he peak size. Thus, yel-
low soybeans could be cha ac e ized by a big peak a abou
6.3 min (peak 4), g een soybeans pa e ns could be cha -
ac e ized by wo big peaks appea ing be ween 7 and 8 min
(peaks 6g and 7g) and ed soybeans could be cha ac e ized by
he p esence o wo o e lapped peaks a app oxima ely 7 min
(peaks 2 and 3 a 254 nm) om which only peak 2 is
obse ed a 280 nm. I is in e es ing o ema k ha al hough
ed beans we e ma ke ed as ed soybeans o azuki (see Table
1), he elec opho e ic pa e ns o he i e ed beans analyzed
we e qui e simila . Howe e , as s a ed be o e, in he case o
black soybeans, wo o he h ee black soybeans s udied
(samples S18 and S19) p esen ed simila pa e ns ha , a he
same ime, esul ed simila o he pa e ns ob ained om
yellow soybeans, while he o he sample (S17) showed a di -
e en p o ile. This sample was comme cialized as black
soybean ijoles and p esen ed a kidney shape while he
o he black soybeans p esen ed a ound/o al shape. Simila
esul s we e obse ed o hese samples by RP-HPLC [18].
F om hese esul s, i is possible o s a e ha yellow and black
soybeans seemed o be di e en a ie ies o soybean while
g een and ed soybeans seemed o be e y di e en soybean
a ie ies o e en o he legumes since no signi ican di e -
ences we e ound among p o ein p o iles ob ained o azuki
and ed soybean samples o o g een soybean and mung
g een soybean samples.
Di e ences among peak a ea pe cen ages ob ained o
e e y peak and o he di e en beans we e analyzed using
he box-and-whiske plo . Al hough he di e en ia ion
among he pigmen ed beans s udied in his wo k could be
obse ed a any o he h ee wa eleng hs used, as s a ed
be o e, 254 and 280 nm we e selec ed since he di e ences
we e mo e clea ly displayed due o he simplici y o he elec-
ophe og ams in compa ison wi h hose ob ained a
210 nm. Figu e 3 shows he box-and-whiske plo s ob ained
o yellow (six samples), g een ( i e samples) and ed ( i e
samples) beans a 254 and 280 nm. I can be obse ed ha
e y simila plo s we e ob ained o yellow soybeans a he
wo wa eleng hs conside ed, he g ea es dispe sion in peak
a ea pe cen age being ob ained o peaks 1 and 4. Mo eo e ,
he highes a iabili y in peak a ea pe cen age o g een
beans co esponded o peak 5 (a 254 nm) and peak 6 (a
280 nm) and o ed beans o peaks 3, 5 and 6 (a 254 nm)
and 5 and 6 (a 280 nm), peak 6 a 254 nm o ed beans
being he one accoun ing o he maximum a iabili y in
peak a ea pe cen age.
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2320 C. Ga cía-Ruiz e al. Elec opho esis 2007, 28, 2314–2323
Figu e 3. Box-and-whiske plo s ob ained om peak a ea pe cen ages o six yellow beans, i e g een beans and
i e ed beans.
The me hod was modi ied in o de o use i as a as
sc eening me hod [33]. In ac , he elec opho e ic pa e n
was ob ained in a 8.5 cm capilla y (injec ion by he ou le
posi ion) ins ead o using he 50 cm capilla y (injec ion by
he inle posi ion) used p e iously. Al hough, as expec ed,
wi h an 8.5 cm capilla y he sepa a ions we e wo se han
hose ob ained wi h he longes capilla y (see Fig. 4); he
elec opho e ic pa e ns ob ained enabled he clea and as
di e en ia ion among he di e en pigmen ed beans, hus
making possible a signi ican educ ion in he analysis ime.
Using a capilla y ol age o 20 kV enabled ob aining p o ein
p o iles in only 70 s (12 imes sho e han he analysis ime
employed using he ini ial CE me hod).
3.2 Disc imina ion be ween ansgenic and
non ansgenic soybeans
The same analy ical me hod used o he cha ac e iza ion o
he di e en pigmen ed soybeans was also applied o dis-
c imina e be ween ansgenic and non ansgenic yellow
soybeans. Figu e 5 shows he elec opho e ic p o iles
ob ained o a comme cial soybean lou , a yellow soybean
and a ansgenic soybean lou a 254 and 280 nm. When
compa ing he elec ophe og ams co esponding o he
comme cial soybean lou and he ansgenic soybean lou
wi h ha ob ained o soybeans (S2), a signi ican inc ease in
he a ea o peak 1 wi h espec o ha o peak 4 was obse ed.
©2007 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com
Elec opho esis 2007, 28, 2314–2323 CE and CEC 2321
Figu e 4. Elec ophe og ams ob ained o yellow, g een, black
and ed beans using he 8.5 cm sepa a ion capilla y (58.5 o al
leng h). O he expe imen al condi ions as in Fig. 1.
Since his inc ease was cha ac e is ic o soybean lou s
( ansgenic and non ansgenic), i could no be a ibu ed o
he gene ic modi ica ion o he ansgenic soybean. As a
consequence, peaks 2, 4 and 5 we e selec ed in o de o s udy
i i was possible o cha ac e ize ansgenic ma e ial using
chemome ic ools. Disc iminan analysis was applied o he
a ea pe cen age ob ained o he selec ed peaks in nine sam-
ples ha we e dis ibu ed in he ollowing ca ego ies: ans-
genic samples (gene ically modi ied o ganism (GMO)), non-
ansgenic samples (nonGMO) and mix u es o ansgenic
and non ansgenic samples (mix u es) (see Fig. 6A). A 100%
o cases co ec ly classi ied we e ob ained using he abo e-
men ioned h ee peaks a 254 and 280 nm. Mo eo e , a 100%
o co ec classi ica ion was also ob ained when he lea e-
one-ou c oss alida ion p ocedu e was used. A 280 nm,
e en wi h only wo a iables (peaks 2 and 4), i was possible
o co ec ly classi y he nine samples o soybeans analyzed.
Mo eo e , hi y- ou new samples analyzed by he CE
Figu e 5. Elec ophe og ams co esponding o he injec ion o
he p o ein ex ac s ob ained om a comme cial soybean lou
(,40 mg/mL), a yellow soybean (,60 mg/mL) and a ansgenic
soybean lou (,60 mg/mL) using a solu ion o wa e /ACN (75:25
/ ) wi h 0.3% / ace ic acid. O he expe imen al condi ions as in
Fig. 1.
me hod we e used o p o e he alidi y o he disc iminan
model (see Fig. 6B whe e a o al o o y- h ee samples we e
conside ed). A 93% o he cases we e co ec ly classi ied as
non ansgenic samples. Only h ee samples we e inco ec ly
classi ied as a mix u e o ansgenic and non ansgenic soy-
beans al hough hey we e nonGMO samples.
4 Concluding ema ks
This is he i s ime ha CE has success ully been applied
o he cha ac e iza ion o di e en pigmen ed soybeans as
well as o he di e en ia ion o ansgenic and non-
ansgenic soybeans based on hei p o ein p o iles.
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2322 C. Ga cía-Ruiz e al. Elec opho esis 2007, 28, 2314–2323
Figu e 6. Disc iminan plo
ob ained o he classi ica ion o
nine soybean samples (A) and
o y- h ee soybean samples (B).
Elec opho e ic pa e ns obse ed o he di e en pig-
men ed soybeans we e ob ained in 12 min and enabled he
clea di e en ia ion be ween yellow soybeans and he o he
colou ed soybeans. Yellow soybeans p esen ed an elec o-
pho e ic pa e n cha ac e ized by a huge peak a ,6.5 min
ha was simila o he pa e n ob ained o wo o he black
soybeans. Samples ma ke ed as g een soybeans, ed soy-
beans and black soybean ijoles showed elec opho e ic
p o iles cha ac e is ic o each pigmen a ion and di e en
om hose ob ained o yellow soybean. These di e ences
could be obse ed a 210, 254 and 280 nm, al hough we e
mo e clea ly displayed a he wo la e wa eleng hs due o
he simplici y o he elec ophe og ams. Howe e , p o ein
p o iles ob ained o mung g een soybean and azuki we e
e y simila o hose ob ained o g een and ed soybeans,
espec i ely.
The modi ica ion o he me hod by injec ing samples
by he ou le posi ion enabled a signi ican educ ion in
he analysis ime o 70 s making his me hod use ul as
a as sc eening ool. This me hod would enable an
ini ial classi ica ion o he samples ha , a e wa ds, could
be cha ac e ized wi h mo e de ail by he ini ial CE
me hod.
A ea pe cen ages o peaks 2, 4 and 5 and he use o dis-
c iminan analysis we e also success ully applied o he di -
e en ia ion be ween ansgenic and non ansgenic soy-
beans.
The au ho s hank he Comunidad Au ónoma de Mad id
(Spain) (p ojec S-0505/AGR/0312) and he Minis e io de
Ciencia y Tecnología (Spain) (p ojec AGL2005-05320-C02-01/
©2007 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.elec opho esis-jou nal.com