Agriculture Reference
In-Depth Information
components nevertheless, it produces a cleaner extract [20, 42, 116]. This
approach is used to confirm the presence of nitrofuran residues in samples
because total residues are now widely considered to be insufficiently specific
to identify illegal use of nitrofurans, especially in the case of nitrofurazone
abuse (monitoring of the SEM metabolite). Samples are disrupted in the
presence of methanol: water followed by subsequent washings with ice-cold
methanol, ethanol and diethyl ether. Diethyl ether is allowed to evaporate
overnight and the sample pellet is hydrolysed, derivatised and neutralised prior
to extraction with ethyl acetate. A disadvantage of this protocol is that an extra
day is required to allow evaporation of the diethyl ether, which increases
sample turnaround time. An alternative approach for the determination of
bound residues has been developed, based on two methanol:water (50:50 and
75:25, v/v) washes followed by a pure methanol and a pure water wash [119].
The advantage of this approach is that the sample pellet can proceed to the
hydrolytic derivatisation step on the same day as washing, reducing assay time
by 1 day.
One of the major obstacles for nitrofuran analysis is the identification of a
suitable residue marker for nitrofurazone abuse. The suitability of SEM as a
definitive marker for nitrofurazone misuse has been questioned in light of the
finding that, in food, SEM may arise from sources (azodicarbonamide and
carrageenan) other than this illegal veterinary antibiotic. In response to this
problem the retina was investigated as an alternative matrix for the verification
of nitrofuran abuse [83]. This group found that total antibiotic metabolites
could be detected at mg kg
−1
levels in the retina of pigs due to the
accumulation of drug residues in the eye. It was proposed that retinal analysis
may allow detection of nitrofuran abuse in animals at any point from birth to
slaughter. The metabolism of nitrofurans in chicken has also been investigated
and it was found that the intact nitrofuran parent compounds could be detected
in the eyes of treated birds [120]. A major advantage of retinal analysis comes
from the high concentrations of nitrofurans that can occur in the retina which
allows samples to be analysed by HPLC-UV rather than LC-MS/MS. Retinal
analysis is clearly a promising technique for future applications.
For nitrofuran parent compounds detection in biological samples,
extraction with ethyl acetate is used after homogenization of the sample.
Organic solvent is evaporated to dryness with N
2
, and acetonitrile first and n-
hexane next are added to the dry residue while vortexing. The n-hexane layer
is discarded and the dry residue of the acetonitrile phase is used for analysis by
LC-MS/MS [54].
