For the beverages (Table 3), repeatability and reproducibility
values were related to 10 mg l1 spiking level. RSD% values for
drinks are generally lower than those resulting from the solid
matrices. This could be due to a poor sample handling. All the recoveries
were higher than 75% except for azocarmine G and metanil
yellow. However, their recoveries were higher than 60%. This could
be explained considering the filtration step before HPLC analysis:
perhaps the filter partly retained the dyes. The repeatability and
reproducibility were quite low.
Optimized gradients for separation of dyes are achieved
considering the formation of neutral species from the ionized
analytes. A gradient elution prepared by a mixture of sodium acetate
buffer (0.1 M and pH 7) with acetonitrile was used for the
successful separation of 14 synthetic food colorants in standard
solutions [26]. At pH 7 all dyes are suitable for reverse phase
chromatography since they are neutral. The colorants were eluted
from the column according to increasing polarity. The first colorant
eluted from the column was the more polar dye tartrazine, while
the last was metanil yellow which presents a single sulfonated
group and no alcoholic substituents. Quinoline yellow (E104) consists
essentially of sodium salts of a mixture of disulfonates (principally),
monosulfonates and trisulfonates [34]. It shows three
peaks corresponding to relative isomers. The sum of the three
peaks was used for determination of recoveries and precision
validation data. Figs. 1e3 show chromatograms related to three
samples spiked with red (group AeB together), yellow (group C)
and blue dyes (group D).
DAD detector higher signal was obtained by setting pertinent
wavelengths for each group of dyes. For red colorants, wavelength
for quantificationwas fixed at 515 nm as a reasonable average of the
maximum absorptions within the red colorants. For yellow dyes 2
different wavelengths were set: E102, E104 and metanil yellow
were monitored at 420 nm, while E110 and orange II were observed
at 480 nm. For blue dyes DAD wavelength was set at 620 nm.
Fortification levels for allowed food colors in foodstuffs were
chosen in accordance to legislation limits. For unauthorized dyes in
solid matrices, spiking levels was slightly lower. It was estimated
that significant quantities of colorant should be added to foodstuffs
for changing color appearance. For this reason, validation of illegal
red dye in solid matrices was carried out at the ppm order of
magnitude.