REV. CHIM. (Bucharest) 65 No. 2 2014 http://www.revistadechimie.

ro 137
Development of a Reversed-phase High Performance Liquid
Chromatographic Method for Simultaneous Determination
of Allura Red AC and Ponceau 4R in Soft Drinks
MARIA MADALINA JURCOVAN, ELENA DIACU*
University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, 1-7 Polizu Str., 011061, Bucharest,
Romania
Red Allura and Ponceau 4R are two of the most used red pigments in food and beverages. As they are
synthetic organic substances that bears azo functional group in their molecule, which, generally, is incriminated
as potential cause of malformations and tumours, these colors are perceived by consumers as harmful
substances. For this reason, European regulators have limited the content of Red Alura and Ponceau 4R in
beverages, and increased attention have been given to the development of methods of analysis for the
determination of this kind of additives permitted in soft drinks. This work presents the possibility of
simultaneous determination of Red Allura and Ponceau 4R in soft drinks by liquid chromatographic HPLC
technique. The sample preparation is quite simple, without solvent extraction, requiring before
chromatographic analysis only filtration and bringing the soft drink sample to a pH of 6.50. The values of the
analytical performances criteria (detection limit, quantification limit, and recovery, repeatability, reproducibility
and uncertainty measurement) have been determined, which shows that the described method is accurate
and appropriate to be used. for the determination at legal requirements regarding the level of concentration
(g/L) of Red Alura and Ponceau 4R in soft drinks.
Keywords: Red Allura, Red Ponceau, synthetic food dyes, HPLC, soft drinks
In order to impart or to improve the visual aspect and
flavour of food and beverages, manufacturers have used
food coloring for centuries, being known that people
associate certain colours with certain flavours [1, 2].
Among food pigments, the synthetic ones have certain
properties that recommend them to be used in food and
soft drinks, instead of the natural ones: less cost, a higher
coloring power, a better stability on time, the offset colour
loss due to exposure to light, temperature, air conditions,
moisture and long storage, a vast range of new colours, a
better distribution of color to the dyed food or drinks.
Therefore, today colour additives are recognized as an
important part of almost all processed foods and drinks;
even if they are recognized as harmful for human health in
case they are consumed excessively.
Nowadays, effects of synthetic food pigments on health
consumers are still insufficiently known, especially
regarding the effect of azo-dyes food colors over time, and
only long-term studies and experiments performed on a
number of subjects could provide accurate data.
Being able to improve overall sensory quality of foodstuff,
synthetic colors are accepted to be used in the food
industry as additives in a reduced number, only in a
maximum allowable content and they are needed to be
listed on the food labels. Therefore, all these colours were
subject to certification and they are strictly controlled by
European Union legislation and national laws [3-5]. Among
synthetic colors, Red Allura and Ponceau 4 R are two of the
most used red pigments in food and soft drinks.
Red Allura AC (RA), known as E129, is according to the
IUPAC name, disodium 6-hydroxy-5-((2-methoxy-5-
methyl-4-sulfophenyl)azo)-2-naphthalenesulfonate and is
a water soluble monoazo dye with good compatibility to
other food constituents. This dye can be used in soft drinks
either separately, or in binary combination with other red
* email:elena_diacu@yahoo.co.uk
synthetic dye, such as Ponceau 4R (P 4R), known as E124,
whose IUPAC name is trisodium (8Z)-7-oxo-8-[(4-
sulfonatonaphthalen-1-yl)hydrazinylidene]naphthalene-
1,3-disulfonate. This last dye is a suspected harmful
substance, but permitted in Europe [3].
A
B
Fig. 1. Chemical structure of Red Allura (A) and Ponceau 4R (B)
In the context of increase of consumer concern
regarding the global consumption of synthetic dyes,
considerable interest is given to development of such
analytical methods, able to separate and quantitatively
determine these dyes, whose substances show, in most
cases, quite similar physicochemical properties. In the
literature, there is a large number of references regarding
the analytical methods for synthetic food dyes
determination in different food matrices, either separately,
or in combination with other additives. Here one can
mention few of them: spectral methods [8-11], capillary
electrophoresis [12-14], electrochemical methods [15-17]
and the most numerous, chromatographic methods [18-
24]. Among all mentioned methods used for the analysis
of food dyes, HPLC methods are by far the most promising
technique regarding the accuracy, precision, repeatability
and detection limit.
This paper describes the development and in-house-
validation of a reversed-phase high-performance liquid
REV. CHIM. (Bucharest) 65 No.2 2014 http://www.revistadechimie.ro 138
chromatography method for the simultaneous
determination of two red synthetic dyes, Red Allura and
Ponceau 4R (fig 1). Both these food colors are approved by
European regulations in the maximum allowable
concentration of 100 mg/L, used either individually, or in
binary combination [3-6]. It is worth mentioning that
Ponceau 4R, due to the carcinogenic suspicion is not
allowed in USA [7].
Experimental part
Chemicals and reagents
Allura Red AC (purity 99%) and Ponceau 4R (purity
98%), both analytical standards, were purchased from
Germany. NaOH and acetonitrile were Chromasolv purity
from Germany. All aqueous solutions were prepared using
ultrapure water obtained through a Millipore Milli-Q system.
Mixed stock solution of the standard containing 1000
mg/L was prepared by weighing 0.1010 g of each colour to
100 mL and preservation at 4 8
o
C temperature (valid for
3 months). The mixed working solutions of calibration
standards were prepared on the day of determination, by
taking the appropriate volumes of stock solution after its
equilibration to room temperature and then diluted in
ultrapure water.
Apparatus
The determination of RA and P 4R content was
performed with an Agilent Liquid chromatograph-Series
1100, equipped with DAD detection, on a column with
stationary phase as Nucleosyl 5 MOS C
8
on an analytical
column length of 250 x 4.60 mm. The flow rate was set up
at 1.0 mL/min and the injection volume was 10 L. The
detection was achieved at 520 2 nm, as the optimum
wavelength for monitoring both red solutes. Peak
identification was done by the retention times of the
samples with food color standards and the quantification
by peak areas.
Samples Preparation
Soft drink samples were filtered in vacuum on a
microfiltration membrane (pore size of 0.45 m). If the
sample was carbonated, it was degassed by ultra-
sonication for 10 min. The pH sample was adjusted at 6.50
values, with a NaOH solution 10 %, using a pH-meter
equipped with a combined glass electrode. The
chromatographic separation of RA and P 4R was achieved
through a gradient-elution system consists of two
components: component A was ultrapure water, and B, a
mixture of acetonitrile and ultrapure water, in 1:4 volumes.
Results and discussions
The gradient-elution system used for mobile phase was
programmed in four steps: i) 0-1 min isocratic elution at
5% A:95% B; ii) 1-15 min linear gradient elution from 5%
A:95% B to 45% A:55% B; iii) 15-16 min linear gradient to
recover initial conditions of 5% A:95% B and iv) is the
conditioning step at 5% A:95% B for 15 min. Experiments
were performed on either standard solutions or on samples
fortified soft drinks with RA and P 4R, and a significant
difference between retention times of the two dyes was
recorded on the chromatogram (5.7 0.2 min for P 4R
and 11.4 0.2 min for RA), meaning a proper identification,
as can be seen in figure 2.
The calibration curves for RA and RP were obtained by
fortification of a juice sample with both colorants, and were
drawn based on the peak areas of different working
calibration standard solutions. From figure 3, A and B, it
Fig. 2. Typical chromatogram for
a fortified juice sample with RA
and P 4R
REV. CHIM. (Bucharest) 65 No. 2 2014 http://www.revistadechimie.ro 139
can be seen that there is a very good linearity of the
dependency of the analytical signal with both colorants
concentration in the range of 10-100 mg/L.
In the development of analytical methods care should
be taken for interferences.
Study of possible interferences (other food colorants, or
other additives usually present in beverages, such as
preservatives and sweeteners) was realized, and no
significant interference with the method here proposed
was not observed. In addition, the selectivity of the
developed HPLC method was proven by investigation of
the spectral purity of chromatogram using a fortified
beverage sample, and good values were obtained for the
purity factors of both colorants, respectively 999.929 for P
4R, and 999.920 for RA.
The HPLC method was then used to simultaneously
determine the pairs of colorants RA and P 4R in ten real
soft drink samples, with results that showed that the content
did not exceed the legal limits, respectively 100 mg/L.
Validation of the method
Next step of this study was the validation of HPLC
method for the determination of RA and P 4R in beverage
samples. The following analytical performance criteria:
linearity of calibration curve, detection limit (LOD),
quantification limit (LOQ), recover y, repeatability,
reproducibility and measurement uncertainty, (U) were
determined in accordance with the references [25- 27] for
in-house-validation procedure.
Detection limit, LOD , and quantification limit, LOQ
For the developed HPLC method, LOD was considered
as the colorant concentration giving an analytical signal
equal to the blank plus 3 times the standard deviation s
r
of
the blank. The equipment used here offers excellent
possibility to establish the LOD, the value being obtained
directly from the chromatographic software by analyzing
four different concentrations of P 4R and RA standard
solutions (100-1000 g/L). For each colorant, LOQ found
was 0.1 mg/L. Consequently, the LOQ is 0.3 mg/L,
considering LOQ = 3 LOD.
Calibration curve linearity
Linearity of calibration curve for HPLC method was
checked on the basis of the chromatographic peak areas
using red juice samples fortified with various
concentrations of mixed standard solutions RA and P 4R in
a dynamic concentration range 0-100 mg/L (as can be in
red beverages). The calibration curve was linear for all the
ranges of interest, with a good regression coefficient, of
0.99996 for RA and 0.99991 for P 4R, respectively [fig. 3].
Recovery %
Achievement of the percent recovery for RA and P 4R
determination from beverages was carried out considering
the maximum accepted level by law for these dyes, using
a set of 12 spiked soft drinks samples for each colorant, at
two concentration levels (50 and 100 mg/L), 6 samples
for each level. An unfortified sample of soft drink red, was
analyzed also here, its content being considered 0.0 mg/L,
because no analytical signal was generated. The recovery
percentage obtained is between 93- 102% for P 4R and 90-
103% for RA, which shows that the sample matrix does
not affect the determination of both colorants from soft
drinks.
Repeatability, r
Repeatability r is the measure of agreement between
results obtained under repeatability conditions. This
parameter was obtained under the requirements (i.e.,
same method, same sample, work done by one operator,
in the same laboratory, with the same apparatus, and with
short time interval) using the formula according to which
r = 2.8 x s
r
. (2.8 is the number derived from Gaussian
distribution, according with ISO 5725). Standard deviation
s
r
was calculated for a number of data n 10, with 95%
confidence. The obtained results for s
r
and r are displayed
in table 1, where it can be seen that the values for s
r
are
Fig. 3. Calibration curves for
Red Allura (A) and Ponceau 4R(B)
B
REV. CHIM. (Bucharest) 65 No.2 2014 http://www.revistadechimie.ro 140
less than 0.14, and for r are between 0.18 and 0.38, which
shows that repeatability of analytical data for both colorants
at all fortification levels is very good.
Reproducibility, R
The reproducibility R is a measure of the dispersion of
results obtained with the same method but under different
conditions, such as: work done by different persons, with
different equipment, laboratories, and times. R was
calculated after performing two sets of experiments, in
two different days, by two persons, with different
chromatographs, for both colorants, at two fortification
levels (50 and 100 mg/L) with formula R = 2.8 x s
R
, where
s
R
is the standard deviation of these results calculated for a
number of data n 8 (with 95% confidence). For both
fortification levels of P 4R and RA determination, the results
for R are less than 1 (s
R
ranges between 0.05 and 0.21 and
from 0.14 to 0.6 for R), values that gives good reproducibility
to the proposed method.
Measurement uncertainty, U
In order to evaluate the measurement uncertainty in the
determination of RA and P 4R by HPLC in soft drinks,
according with in-house-validation procedure [25-27],
the following uncertainty components have been identified
and evaluated: uncertainty on standard solutions on the
recovery, on the reproducibility, and on HPLC instrument.
Uncertainty on standard solutions
In order to obtain the associated uncertainty on the
preparation of standard solutions, two components should
be considered: the uncertainty on the preparation of the
stock solution and the uncertainty on the preparation of
the working solutions itself. Taking into account all
combined uncertainties involved here, respectively: due
to the purity of the two dyes powders (RA 99% and P 4R
98%), due to the weighing of analytical balance (calibration
certificate of Mettler Toledo AT 261), to the use of a 100
mL, 10 mL volumetric flasks Class A and of micropipette
(100-1000 L), due to variation of the temperature, the
value for the associated uncertainty on standard solutions
u
standards
was found as 0.36 mg/L.
The uncertainty due to the recovery percentage
To obtain the uncertainty due to the recover y
percentage, the relative standard deviations, RSD
P 4R
and
RSD
RA
were calculated separately for the two dyes and
were integrated for 8 series of analyses within 1 day. Table
2 presents the analytical data for both standard deviation
(SD) and relative standard deviation (RSD) of recovery
percentage. Therefore, the individual values obtained for
RSD
P 4R
= 0.098% and RSD
RA
= 0.157%, are useful for the
evaluation of associated uncertainty related to the recovery
in simultaneous determination of RA and P 4R, considered
as their sum, respectively 0.258.
The uncertainty due to the to the reproducibility
The uncertainty related to reproducibility is achieved
from the sum of the averages RSD values of eight time
analyses of a fortified soft drink sample at 100 mg/L level
of P 4R and 50 mg/L RA, performed in two different days
(RSD
P 4R
= 0.128%, RSD
RA
= 0.175%. The Associated
uncertainty related to the reproducibility in this case is
0.303%.
Table 1
DETERMINATION OF
REPEATABILITY FOR RED ALLURA
AND PONCEAU 4R DETERMINATION
Table 2
RDS VALUES FOR RED ALLURA
AND PONCEAU 4R RECOVERY
Table 3
RED ALLURA AND PONCEAU 4R
CONTENT IN DIFFERENT KINDS
OF SOFT DRINKS
REV. CHIM. (Bucharest) 65 No. 2 2014 http://www.revistadechimie.ro 141
Associated uncertainty for HPLC instrument
The associated uncertainty for liquid chromatograph,
u
HPLC
can be expressed as average between the standard
deviation for P 4R and RA, with a value of 0.2925 mg/L.
Measurement uncertainty-U calculation
In the end, the measurement uncertainty U was
calculated as expanded uncertainty by the formula U =
u k, where k, the coverage factor, has the value 2, taking
into account a normal distribution of the experimental data.
For the concentration of 100mg/L for both colorants, U =
2.62 mg/L. This U-value which defines the interval between
RA and RP content and may be acceptable to be included
in soft drinks and encompasses the conditions of food
safety and of food authenticity detection.
Developed HPLC method was applied in the
determination of RA and RP either separately and
simultaneously in 10 real soft drinks samples commercially
available, and the obtained values exhibited in table 3 show
that the content of the two studied dyes did not exceed the
legal limit.
Conclusions
An HPLC method was developed for determination of
red synthetic colors Allura Red AC and Ponceau 4R in soft
drinks. The described method was in-house validated,
and the analytical performance criteria and the critical
parameters on measurement uncertainty were determined.
The method has proven its ability to obtain reliable results,
precise and accurate, with a satisfactor y limit of
quantification of mg/L, in a relatively short time (14 min)
and with a slight sample preparation process for analysis.
All these recommend the method for use in monitoring the
legal limits of the content of synthetic dyes, Ponceau 4R
and Allura Red in soft drinks and in determining the
authenticity of the natural ones.
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Manuscript received: 1.07.2013