Food Chemistry 120 (2010) 12291237

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Improved methodology for the characterisation of transgenic Bt-11 maize

cultivars using RP-HPLC proles of albumin, globulin, prolamin, and glutelin
protein fractions and chemometric analysis
J.M. Rodriguez-Nogales a, A. Cifuentes b, M.C. Garcia c, M.L. Marina c,*
a
b
c

rea de Tecnologa de los Alimentos, Departamento de Ing. Agraria y Forestal, ETS de Ingeniera Agraria, Universidad de Valladolid, 34004 Palencia, Spain
Departamento de Anlisis de Alimentos, Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
Departamento de Qumica Analtica e Ingeniera Qumica, Facultad de Qumica, Universidad de Alcal, 28871 Alcal de Henares, Madrid, Spain

a r t i c l e

i n f o

Article history:
Received 20 November 2008
Received in revised form 26 May 2009
Accepted 28 November 2009

Keywords:
Transgenic Bt maize
Perfusion RP-HPLC
Monolithic RP-HPLC
Maize proteins
Protein fractions

a b s t r a c t
A clear differentiation between Bt-11 transgenic and isogenic non-transgenic maize cultivars has successfully achieved analysing the perfusion and monolithic RP-HPLC proles of the albumin, globulin, prolamin, and glutelin maize fractions together with a discriminant analysis. Signicant differences
between transgenic and isogenic non-transgenic cultivars were observed in the chromatograms obtained
from any of the four protein fractions. The application of linear discriminant analysis to the area percentages corresponding to every peak detected in every protein fraction was successfully employed for the
classication of transgenic Bt maize lines obtaining a global percentage of correct classication of
100%. For perfusion RP-HPLC, the variables with more discriminant power and prediction capability were
the following peaks: peaks 1 and 3 for albumins, peak 2 for globulins, peaks 3 and 6 for prolamins, and
peaks 7 and 8 for glutelins. In the case of monolithic RP-HPLC, the variables were peaks 2 and 3 for albumins, peak 5 for globulins, peaks 5, 6, and 7 of prolamins, and peak 10 for glutelins.
2009 Elsevier Ltd. All rights reserved.

1. Introduction
Genetically modied organisms (GMOs) can be dened as
organisms in which the genetic material (DNA) has been altered
in a way it would never occur naturally (Anklam, Gadani, Heinze,
Pijnenburg, & Van Den Eede, 2002). Transgenic maize is one of
the principal biotech crops grown worldwide, occupying 23% of
the total global biotech area (in 2007, 114.3 millions of ha) (James,
2007). Bt-11 transgenic maize was developed to be resistant to the
attack by European corn borer and to be tolerant to the herbicide
phosphinothricin. Bt-11 maize is grown extensively in USA (57.7
million of ha in 2007), Argentina (19.1), Brazil (15.0), Canada
(7.0), South Africa (1.8), Uruguay (0.5), and Philippines (0.3). In
Europe, the increased production of Bt-11 maize is concentrated
in Spain with 0.1 million of ha. The cultivation and commercialisation of transgenic maize is regulated in the European Union (EU)
(Regulation (EC) 1829/2003, and 1830/2003) while several other
countries are considering voluntary or mandatory labelling proposals. Therefore, the development of reliable methods to detect
transgenic events has become increasingly important for government regulators, international trade organizations, and industries
utilising these products. On the other hand, research on how the
* Corresponding author.
E-mail address: mluisa.marina@uah.es (M.L. Marina).
0308-8146/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.11.069

genetic modications can impact on the chemical composition of

these products is also of great interest (Herrero, Ibanez, MartinAlvarez, & Cifuentes, 2007; Levandi, Leon, Kaljurand, Garcia-Canas,
& Cifuentes, 2008). Hence, rapid, easy and sensitive analytical
methods must be developed to reliably determine the presence
and/or the amount of GMOs in raw agricultural materials and in
processed and rened ingredients, and also to evaluate other possible alterations in GMOs.
Current methodologies for the analysis of genetically modied
organisms are focused on either one of two targets, the transgenic
DNA inserted or the novel proteins expressed in a genetically modied product (Miraglia et al., 2004). Regarding DNA-based techniques, the most current detection methods rely on the
polymerase chain reaction (PCR) to amplify transgene sequences.
Although specic DNA sequences can be detected by hybridation,
it is PCR in its various formats (qualitative PCR, quantitative competitive PCR, and quantitative real-time PCR) which has been generally accepted by the regulatory authorities (Marmiroli et al.,
2008). However, these techniques mainly provide results on the
existence (or not) of GMOs without offering any additional information on the composition of the investigated GMOs (Levandi
et al., 2008). Protein analysis methods are another way to face
the detection of GMOs. The most usual assays employed for the
detection of proteins from GMOs are immunological methods, primarily ELISA (the enzyme-linked immunosorbent assay) and some

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J.M. Rodriguez-Nogales et al. / Food Chemistry 120 (2010) 12291237

variants of ELISA, such as lateral ow strips or dipstick kits. One of

the major drawbacks of immunological assay is that its accuracy
and precision can be adversely affected by a complex matrix, such
as those found in many processed agricultural and food products
(Anklam et al., 2002). Also, protein detection by immunoassay requires the use of antibodies raised against the protein encoded by
transgene. Due to these limitations other analytical method are
now being developed, as for instance capillary electrophoresismass spectrometry (Erny, Leon, Marina, & Cifuentes, 2008) or HPLC
to investigate GMOs. HPLC has been applied to the characterisation
and differentiation of some transgenic plants such as tomato
(Nicoletti, De Rossi, Giovinazzo, & Corradini, 2007), tobacco plants
(Halim, Verberne, & Verpoorte, 2003), sugarcane (Colombo, Lancas,
& Yariwake, 2006), and potato (Defernez et al., 2004). The combined use of reverse-phase HPLC with rapid stationary phases as
perfusion and monolithic columns could open interesting possibilities to investigate the differences in the protein proles of genetically modied and conventional plants. Recently, a rapid
analytical strategy has been proposed to analyse maize cultivars
and derived products using perfusion and monolithic RP-HPLC in
very short analysis times (<4 min with the perfusion column and
<8 min with the monolithic column) (Rodrguez-Nogales, Garca,
& Marina, 2006a, 2006b). This methodology has been applied to
the differentiation between transgenic and non-transgenic maize
cultivars based on protein proles obtained from whole protein extracts. The differences observed on the perfusion and monolithic
chromatograms of whole protein extracts from transgenic and
non-transgenic maize cultivars enabled the differentiation
amongst them (Rodrguez-Nogales, Cifuentes, Garca, & Marina,
2007) and the development of calibration models predicting the
percentage of transgenic Bt-11 maize in ours (Rodrguez-Nogales,
Cifuentes, Garca, & Marina, 2008). Moreover, protein fractions
from transgenic and non-transgenic maize lines have also been
characterised, for the rst time, by perfusion and monolithic RPHPLC being possible the assignment of the peaks appearing in
the fractions to the whole protein extract (Rodrguez-Nogales
et al., 2007).
The proposal in this work has been to investigate the potential
of the protein proles corresponding to albumins, globulins, prolamins, and glutelins obtained by perfusion and monolithic RP-HPLC
from different transgenic and isogenic non-transgenic maize cultivars for their differentiation. These results will be compared with
the previously obtained using the whole protein extract.

2. Experimental
2.1. Chemicals and samples
HPLC grade acetonitrile (ACN) (Merck, Darmstadt, Germany),
Milli-Q water (Millipore, Bredford, MA, USA), and triuoroacetic
acid (TFA) (Sigma, St. Louis, MO, USA) were used for the preparation of mobile phases. 2-mercaptoethanol, tris(hydroxymethyl)aminomethane (Tris), ethylenediamine-tetraacetic acid
(EDTA), 1-propanol (all from Merck), ammonium acetate, chloride
acid, and potassium chloride (all from Panreac, Barcelona, Spain)
were employed for the extraction of maize proteins.
Transgenic Bt-11 maize seeds with the event MON 810
(PR33P67, DKC6575, and Aristis Bt) and their non-Bt isogenic varieties (PR33P66, Tietar, and Aristis, respectively) were employed in
this study. Conventional and transgenic maize cultivars were obtained from a eld assay carried out in Estacin Experimental Agrcola Mas Bada in Tallada dEmpord (Girona, Spain) using
commercial varieties. Namely, in order to skip any inuence from
the growing conditions, Aristis maize (wild type and its Bt transgenic variety), Tietar maize (wild type and its Bt transgenic variety

DKC6575), and PR33P66 maize (wild type and its Bt transgenic

variety PR33P67) were grown under the same eld conditions
and investigated in this work. The transgenic and no transgenic
nature of all these maize samples was conrmed based on their
DNA using an analytical procedure described elsewhere (GarcaCaas, Cifuentes, & Gonzlez, 2004; Garca-Caas, Gonzlez, & Cifuentes, 2002a, 2002b, 2002c, 2004).
2.2. Protein fractionation
For every maize line, 30 kernels of transgenic and non-transgenic maize lines were ground with an analytical mill (IKA Labortechnik, Staufen, Germany) during three minutes at room
temperature. Dry matter content of maize ours was determined
by drying at 130 C to constant weight (AOAC method 925.10).
The moisture of the kernels ranged from 7% to 9%.
Osborne procedure with modications was employed for the
extraction of the maize protein fractions (Osborne, 1907). Namely,
30 mg of each milled maize line were extracted twice with Milli-Q
water (1 mL each time) and sonicated for 5 min in a bath sonicator
(150 W, 50 Hz, FS-30, Fisher Scientic, Pittsburgh, PA, USA). The
mixture was centrifuged for 5 min at 3362 g (Avanti J-25 centrifuge, Beckman Instruments Inc., Fullerton, CA, USA) at 25 C and
the supernatant (albumin fraction) was saved. The pellet was then
extracted twice with 1 mL of TrisHCl buffer (50 mM TrisHCl,
50 mM KCl, and 5 mM EDTA) at pH 7.8 for 5 min and centrifuged
as before. The supernatants corresponded to the globulin fraction.
Afterwards, the pellet was extracted twice with 1 mL of 50% (v/v)
1-propanol in water for 5 min and centrifuged. The supernatants
were decanted, mixed, and saved as prolamin fraction. Glutelins
were extracted twice from the prolamin pellet with 1 mL of 50%
(v/v) 1-propanol, and 1% (w/v) dithiothreitol in water for 5 min.
After centrifugation for 5 min the supernatants were mixed and
saved as glutelin fraction. All the protein fractions obtained by this
procedure were directly injected in the chromatographic system.
2.3. High-performance liquid chromatography
A HewlettPackard 1100 Series liquid chromatograph (HewlettPackard, Pittsburgh, PA, USA) consisting of a degassing system,
a binary pump, a thermostated compartment for the column, an
injection system, and a diode-array detector was used. Separation
of maize proteins was accomplished in a POROS R2/H perfusion
column (4.6  50 mm; 10 lm particle size) (Perseptive Biosystems,
Framingham, MA, USA) and a monolithic silica column Chromolith Performance RP-18e (4.6  100 mm) (Merck). The perfusion
chromatographic method was optimised previously by our research team (Rodrguez-Nogales et al., 2006a): mobile phase A,
0.1% (v/v) TFA in Milli-Q water; mobile phase B, 0.1% (v/v) TFA in
ACN; linear binary gradient, 5.050.2% B in 2.40 min, 50.265.4%
B in 0.98 min, and 65.45.0% in 1 min; injection volume, 20 ll;
ow-rate, 3 mL/min; temperature, 25 C; UV detection, 280 nm.
The separation conditions for the monolithic column were also
optimised previously (Rodrguez-Nogales et al., 2006b): linear binary gradient, 5.026.4% B in 5.15 min, 26.487.5% B in 2.16 min,
and 87.55.0% in 1 min; temperature, 35 C. The injection volume,
ow-rate, mobile phase composition, and wavelength detection
were as in perfusion chromatography. Data were recorded and processed with the HP-Chemstation software.
2.4. Data treatment
In all analysis, the area percentage for every peak was calculated as the average of three replicates (injected by duplicate).
The integration was performed by setting the baseline from valley
to valley. ANOVA and linear discriminant analysis were performed

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J.M. Rodriguez-Nogales et al. / Food Chemistry 120 (2010) 12291237

using the computer programme Statgraphics Plus for Windows

4.0 (Statistical Graphics Corp., Rockville, MD 20852-4999, USA).

the rst peak appeared partially defolded. Signicant differences

for the area percentages among the four maize lines were observed, mainly for peaks 1 and 2. The chromatograms obtained
using monolithic RP-HPLC were totally different from those obtained with perfusion RP-HPLC and presented a higher number of
peaks with retention times between 1 and 6 min (Fig. 1B). The
highest peak areas were found for peaks 1 and 2 for Tietar and
DKC6575 while in PR33P67 and PR33P66 the highest peak areas
were observed for peaks 1 and 3. Signicant differences in the relative area between TietarDKC6575 and PR33P6667 lines were
also observed.
Globulin proles obtained by perfusion RP-HPLC are shown in
Fig. 2A. These chromatograms exhibited mainly two pair of peaks
(peaks 23 at the beginning of the chromatograms, and peaks 6
7 at the end of them). Signicant differences for the area percentages between TietarDKC6575 and PR33P6667 were found for
peak 3 (an average of 20.3% and 6.2% for TietarDKC6575 and
PR33P6667, respectively). For the monolithic RP-HPLC (Fig. 2B),
the chromatograms for globulin fractions could be divided into
three groups of peaks: one group with retention times around
three minutes (peaks 12), a second group at the middle of the
chromatograms (peaks 34), and, nally a third group at the end

3. Results and discussion

Whole protein extracts from transgenic and non-transgenic
maize cultivars were obtained and studied using perfusion and
monolithic methodologies in a previous work being possible the
differentiation between them from their protein proles (Rodrguez-Nogales et al., 2007). In order to improve the results obtained
in this work we propose the use of the protein proles obtained by
these methodologies from the different protein fractions corresponding to albumins, globulins, prolamins, and glutelins that
were previously isolated from transgenic and non-transgenic
maize cultivars. Fig. 1 shows the chromatograms obtained for the
separation of albumins by using perfusion (A) and monolithic (B)
RP-HPLC for DKC6575 and PR33P67 maize lines and their nontransgenic isogenic varieties (Tietar and PR33P66, respectively).
Perfusion RP-HPLC chromatograms obtained for the albumin fraction exhibited two large peaks (peaks 1 and 2) and a third peak
with less intensity (peak 3), all of them migrating between 1 and
2 min. For PR33P67 and its non-transgenic maize line (PR33P66),

15

20

1
15
10

mAU

mAU

DKC6575

10

PR33P67

PR33P66
Tietar
0
0

20

25

B
3
20

mAU

DKC6575

15

mAU

7
6

10

1
2

15

PR33P67
2
4

7
6

10

Tietar
3
4

PR33P66

Time (min)

Time (min)

Fig. 1. Perfusion (A) and monolithic (B) RP-HPLC chromatograms for albumins from DKC6575 and PR33P67 and their non-transgenic isogenic varieties (Tietar and PR33P66,
respectively).

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J.M. Rodriguez-Nogales et al. / Food Chemistry 120 (2010) 12291237

10

10

A
8

DKC6575

mAU

mAU

2
4

Tietar

PR33P67

PR33P66
2

0
0

10

PR33P67

DKC6575

4
8

1
2

7
3

3
6

mAU

mAU

Tietar

PR33P66
2

0
0

Time (min)

Time (min)

Fig. 2. Perfusion (A) and monolithic (B) RP-HPLC chromatograms for globulins from DKC6575 and PR33P67 and their non-transgenic isogenic varieties (Tietar and PR33P66,
respectively).

(peaks 57). Most signicant differences between transgenic and

non-transgenic cultivars were observed for peak 4.
Perfusion RP-HPLC chromatograms obtained for the prolamin
fractions exhibited seven peaks (Fig. 3A). Peaks 3 and 8 showed
the highest values for area percentage, being peak 8 partially
defolded. The comparison of the chromatograms corresponding
to the TietarDKC6575 and PR33P6667 lines revealed signicant
differences for peak 3, showing the highest levels of area percentages for Tietar and DKC6575 (10.8% and 13.3%, respectively) and
the lowest for PR33P66 and PR33P67 (6.3% and 8.6%, respectively).
The chromatograms obtained using monolithic RP-HPLC (Fig. 3B)
showed three groups of peaks: a triplet (peaks 57) in the middle
of the chromatograms (retention times ranging from 5.2 to
5.5 min), a second triplet (peaks 810) and a big peak (peak 11)
at the end of them. Only peak 11 accounted for >79% of the total
area. Signicant differences were found for the peaks of the rst
and second triplet showing the maximum levels for the DKC6575
line.
Glutelin fraction from transgenic and isogenic lines was also
analysed using perfusion and monolithic RP-HPLC (Fig. 4). The perfusion chromatograms show two peaks (peak 7 and 8) with the
maximum signal for peak 7. The highest levels of area percentages
for peak 7 were found for PR33P66 and PR33P67 (84.1% and 89.5%,
respectively). The chromatograms obtained using monolithic RPHPLC shows a group of three peaks at the end of the chromato-

grams (peaks 8, 10, and 11), showing the highest levels of area percentages for peak 10. Peak 10 showed more intensity for PR33P66
and PR33P67 (relative area of 63.1% and 74.6%, respectively) than
for Tietar and DKC6575 (relative area of 54.9% and 56.9%,
respectively).
Showing these results, it was expected that the chromatographic proles obtained could be useful to discriminate among
non-transgenic and transgenic maize Bt maize lines. Among multivariate methods, discriminant analysis is considered to be an
important classical parametric tool for grouping samples wherein
the origin of the samples is known (Gardiner, 1997). This chemometric technique has been successfully employed by our research
team for the classication of commercial maize products (Rodrguez-Nogales et al., 2006a, 2006b) and European and North American inbred and hybrid maize lines (Rodriguez-Nogales, Garcia, &
Marina, 2006c) based on the study of the protein proles using perfusion and monolithic RP-HPLC.
Chromatographic data obtained for the perfusion and monolithic analysis of albumins, globulins, glutelins, and prolamins from
Aristis Bt, PR33P67 and DKC6575, and their non-transgenic lines
(Aristis, PR33P66, and Tietar, respectively) were analysed using
multiple linear discriminant analysis. Figs. 5 and 6 represent the
plot of the maize lines in the plane dened by the two rst discriminant functions for every protein fraction using perfusion and
monolithic RP-HPLC data, respectively.

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J.M. Rodriguez-Nogales et al. / Food Chemistry 120 (2010) 12291237

15

12

DKC6575

3
8

12

PR33P67

6
7

mAU

mAU

Tietar

3
6

2
4 5

PR33P66
0
0

15

25

11

B
11

20

9
8 10

10

9
8 10

mAU

DKC6575

15

mAU

PR33P66

10

Tietar

Time (min)

Time (min)

Fig. 3. Perfusion (A) and monolithic (B) RP-HPLC chromatograms for prolamin from DKC6575 and PR33P67 and their non-transgenic isogenic varieties (Tietar and PR33P66,
respectively).

The nal model selected for the classication of the transgenic

and non-transgenic maize lines using perfusion RP-HPLC data of
the albumin fractions showed that peaks 1 and 3 were the most
important variables for the differentiation of these samples. All
the samples were correctly classied with exception of two samples of Tietar which were non-correctly classied as Aristis lines.
A percentage of 91.6% of correct classication (72.3% using crossvalidation) was achieved. A new discriminant analysis was applied
with the area percentages obtained from protein proles corresponding to globulin fractions. For this analysis, a good discrimination between maize lines (94.5% of samples were correctly
classied and an 88.9% using cross-validation) was achieved, only
a sample of the Bt-transgenic PR33P67 lines was non-correctly
classied as its non-transgenic line (PR33P66) (Fig. 5). In this analysis, peak 2 was the most important variable for the classication
of maize lines. Then, a linear discriminant analysis was applied to
the chromatographic data obtained for the perfusion analysis of
prolamins. The results of classication matrix of the obtained model for training were worse than those obtained for albumins and
globulins, showing an average of correct classication of 86.1%.
Two samples of Tietar were non-correctly classied as Aristis Bt,
and a sample of PR33P66 was incorrectly grouped as transgenic

PR33P67 line. According to the discriminating power, peaks 3

and 6 were the main variables responsible for the observed distribution of the transgenic and non-Bt isogenic lines. A new linear
discriminant analysis was performed on the data set of glutelin
fractions analysed by perfusion RP-HPLC. In general, the plot of
the maize lines in the plane dened by the two discriminant functions showed a very good and clear separation among different
maize lines. The results of correct classication were of 94.5%
(83.4% using cross-validation), where only a sample of Aristis
was incorrectly grouped as Aristis Bt. In this case, the variables
with more discriminating power were peaks 7 and 8. Finally, a discriminant analysis was applied to the overall data set (all peak
areas from albumins, globulins, prolamins, and glutelins). All maize
lines were correctly classied into their groups showing a 100% of
correct classication and a 94.5% of prediction capacity (using
cross-validation). The results of prediction percentages were similar to those obtained in classication, which conrms the stability
of the obtained model. Taking into account that the distance between centroids is proportional to the similarity among groups,
more similarity was found between a Bt-transgenic line and its
corresponding non-Bt isogenic line than among the transgenic Bt
maize lines with the event MON 810. The rst discriminant func-

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J.M. Rodriguez-Nogales et al. / Food Chemistry 120 (2010) 12291237

DKC6575

PR33P67

7
6

4
8

mAU

mAU

8
4

Tietar

PR33P66

10

10

DKC6575

10
8

8
8

11
11

mAU

mAU

Tietar

PR33P66
4

0
3

Time (min)

Time (min)

Fig. 4. Perfusion (A) and monolithic (B) RP-HPLC chromatograms for glutelins from DKC6575 and PR33P67 and their non-transgenic isogenic varieties (Tietar and PR33P66,
respectively).

tion showed a clear separation between the samples of PR33P6667 and the other lines, while the second one achieved a notable
discriminating power between AristisAristis Bt and TietarDKC6575. The nal model selected the following seven variables:
peaks 1 and 3 of albumins, peak 2 of globulins, peaks 3 and 6 of
prolamins, and peaks 7 and 8 of glutelins. These last peaks (peaks
7 and 8 for glutelins) were the most important variables for the
classication of these maize lines.
Fig. 6 shows the plots for transgenic Bt and non-Bt isogenic
maize lines in the plane dened by the discriminant functions obtained from the analysis of the monolithic RP-HPLC chromatographic data. Initially, the discriminant analysis was applied to
the chromatographic data obtained from the analysis of the albumin fractions. All the samples were correctly classied with the
exception of Aristis and Aristis Bt, which were overlapped. An average of classication ability of 83.3% (75.0% using cross-validation)
was achieved. On the other hand, maize samples of Aristis, Tietar,
and DKC6575 were correctly classied according to the chromatographic proles of globulins. However, a sample of Aristis Bt, three
samples of PR33P66 and three samples of PR33P67 were non-correctly classied as PR33P67, PR33P67 and PR33P66 lines, respectively. The chromatographic data obtained for globulin fractions
had lower discriminating power than albumins successfully sepa-

rating 70.8% of the maize samples (37.5% using cross-validation).

The best result of discriminating ability was achieved with the
chromatographic data of prolamin fractions with a 91.7% of correct
classication (70.8% using cross-validation). All the samples of
maize lines were correctly grouped into their respective groups
with exception of two samples of Aristis Bt and two samples of
DKC6575 which were non-correctly classied as PR33P66 and Tietar lines, respectively. Peaks 5, 6, and 7 were the most important
variables for the classication of the transgenic and non-Bt isogenic maize lines. The worst results were found for the glutelin
fractions where only a 37.5% of the samples were correctly grouped
(33.3% using cross-validation), being not possible a representation
of maize samples since a unique signicant discriminant function
was obtained. Finally, a new discriminant analysis was applied to
all fractions. A global percentage of correct classication of 100%
and of global prediction of 94.5% (using cross-validation) was obtained. The nal model selected the following seven variables:
peaks 2 and 3 of albumins, peak 5 of globulins, peak 5, 6, and 7
of prolamins, and peak 10 of glutelins.
The comparison of the results obtained with the published previously (Rodrguez-Nogales et al., 2007) demonstrated that the use
of protein proles from the albumin, globulin, prolamin, and glutelin fractions enabled a better classication of different transgenic

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J.M. Rodriguez-Nogales et al. / Food Chemistry 120 (2010) 12291237

Aristis

Aristis Bt

PR33P66

Tietar

DKC6575

Centroids

PR33P67

Aristis

Aristis Bt

PR33P66

Tietar

DKC6575

Centroids

PR33P67

Albumins

Globul ins

2
2

1
0

0
-2

-1

-4

-2
-3

-6

-6

-4

-2

-15

-10

-5

10

10

Prolamins
Second discriminant function

Glutelins
8

6
1

4
0

2
-1

-2

-2
-4

-3
-10

-5

10

-10

-5

10

15

20

All protein fractions

15
10
5
0
-5
-10
-15
-40

-20

20

40

60

First discriminant function

Fig. 5. Distribution of isogenic and transgenic maize lines analysed by perfusion RP-HPLC in the plane dened by the two rst discriminant functions for albumin, globulin,
prolamins, glutelin fractions, and all protein fractions together.

and non-transgenic maize cultivars than using the whole protein

extract. In fact, the comparison of the results obtained with the
perfusion column resulted in a higher classication and prediction

capabilities when using the data corresponding to the combination

of all fractions. In the case of the monolithic column, the results obtained were signicantly improved when using most individual

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J.M. Rodriguez-Nogales et al. / Food Chemistry 120 (2010) 12291237

Aristis

Aristis Bt

PR33P66

Tietar

DKC6575

Centroids

PR33P67

15

10

Aristis

Aristis Bt

PR33P66

Tietar

DKC6575

Centroids

PR33P67

15

Globulins

Albumins
10

5
0

Second discriminant function

-5

-5

-10

-15

-10
-30

-20

-10

10

20

6
5
4

-60

-40

-20

20

40

200

400

300

Prolamins

200

All protein
fractions

100

2
1

0
-100

-1
-2

-200

-3
-4
-20

-15

-10

-5

10

-300
-600

-400

-200

First discriminant function

Fig. 6. Distribution of isogenic and transgenic maize lines analysed by monolithic RP-HPLC in the plane dened by the two rst discriminant functions for albumin, globulin,
prolamins, and all protein fractions together.

protein fractions (with the exception of glutelin) or the combination of all fractions.
4. Conclusions
Very few data were found about the differentiation between
transgenic and non-transgenic cultivars based on protein proles.
The perfusion and monolithic RP-HPLC analysis of the protein
fractions (albumin, globulin, prolamin, and glutelin), which is
possible in times close to four min and close to eight min, respectively, together with the discriminant analysis of the chromatographic proles could be an interesting alternative to the other
longer and more tedious analytical methods based on the gel
electrophoresis or conventional RP-HPLC. The perfusion and
monolithic chromatographic data were appropriate for differentiating between Bt-transgenic and non-Bt isogenic lines using linear discriminant analysis. Best results were obtained by
perfusion RP-HPLC analysis of globulin or glutelin fractions
(94.5% of discriminating power). However, the obtained results
provided evidence that Aristis-Aristis Bt, PR33P66-67, and Tietar-DKC6575 can be correctly classied according to their typology by applying discriminant analysis on the global data of the

perfusion and monolithic RP-HPLC analysis of albumin, globulin,

prolamins and glutelin fractions. This overall statistical model
showed that the variables with the highest discriminant power
were seven for both perfusion RP-HPLC (peaks 1 and 3 for albumins, peak 2 for globulins, peaks 3 and 6 for prolamins, and peaks
7 and 8 for glutelins) and monolithic RP-HPLC (peaks 2 and 3 for
albumins, peak 5 for globulins, peak 5, 6, and 7 of prolamins, and
peak 10 for glutelins). The comparison of this data with those obtained in a previous publication in which whole protein extracts
from transgenic and non-transgenic maizes were studied demonstrated that, in general, the use of maize protein fractions instead
of the whole protein extract enabled the improvement of classication and prediction capabilities.
Acknowledgements
The authors thank the Ministerio de Ciencia y Tecnologa
(Spain) for project AGL2005-05320-C02-01. M.C. Garca thanks
the Comunidad Autnoma de Madrid (Spain) for project CCG07UAH/AMB-1831. J.M. Rodrguez Nogales thanks the Ministerio de
Educacin y Ciencia (Spain) for his fellowship (SB2004-0045) in
the University of Alcal.

J.M. Rodriguez-Nogales et al. / Food Chemistry 120 (2010) 12291237

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