Because PAH metabolites are subject to thermal degradation
and photodegradation, antioxidant addition should prevent or
at least reduce these processes. Some authors recommended
their use in the preparation of the mobile phase and standard
solutions to avoid degradation and increase the stability of the
analytes. The addition of ascorbic acid at 1 mg/mL to the
mobile phase was tried; nonetheless, because no improvement
in the standards’ stability was observed, this procedure was
abandoned in further studies.9,33 The addition of tertbutylhydroquinone
(TBHQ), a hydroquinone derivative
substituted with a tert-butyl group, in the preparation of
standard solutions has also been reported to avoid standards’
degradation.17 However, this compound shows fluorescence at
the same wavelength as 2-hydroxyfluorene, causing an overload
of the photomultiplier tube (PMT) of the detector. Because the
aim of this study was to quantitate simultaneously 1-
hydroxynaphthalene, 2-hydroxyfluorene, and 1-hydroxypyrene,
TBHQ usage was also excluded. To ensure standard stability,
degradation studies on working standard solutions were
performed during five consecutive days, and it has been
concluded that the standard solutions were stable until the third
day without antioxidant addition. Representative LC-FD
chromatograms of a mixed standard solution and a spiked
squid sample are shown in Figure 1.