Page 85 - Modern Derivatization Methods for Separation Sciences

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has been adopted, i.e., a fluorescence product (λex360 nm, λem475 nm) is formed through a chemical
reaction of the drug with acetic anhydride which results in the formation of a conjugated dehydration
product. This dehydration produced an aromatic system which is conjugated with the 8-11 diene to
furnish the fluorophore. Being effective for end- and ectparasite, ivermectin has been used for
veterinary medicine and the wide range of residue analysis in livestock products were reported using the
pre-column HPLC method with fluorescent derivatization mentioned earlier. Samples of animal tissues
and bovine milk were extracted with aqueous acetone followed by liquid-liquid extraction with
isooctane, acetonitrile, and hexane in order [188-193]. MSPD combined with SPE (Sep-pak alumina) as
simultaneous extraction and clean-up was applied for the analysis of bovine liver [13]. The
derivatization procedure was common among the reports. A reaction with 1-methylimidazole/acetic
anhydride/N-N-dimethylformamide (2+3+9), a derivatizing agent, was carried out at 95°C for 1 hour
followed by silica gel column clean-up such as Sep-pak silica and the detection limit was 1-2ppb. The
analysis of moxidectin in cattle tissues uses the same derivatization as ivermectin [194].

Among the avermectins, avermectin B was found to be the most effective against agricultural insects
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and mites. Avermectin residues degrade rapidly by both oxidative and photochemical pathways to
various products when applied to various crops. However, the only residues of toxicological
significance are avermectin B and 8,9-Z-avermectin B which are an isomer of avermectin B
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isomerized by UV light below 280 nm. Analytical methods of these compounds were similar to those
for ivermectin, but acetic anhydride used for the derivatization reagent was replaced by trifluoroacetic
anhydride. The method included extraction with aqueous methanol or acetonitrile followed by SPE
(aminopropyl) cleanup was applied for the analysis of dried hops [38], wine [195] and apple [196] and
the detection limit was 1-2 ppb.

Malachite Green

Malachite green (MG) is a triarylmethane dye used for aquaculture as a parasiticide, commonly applied
to newly laid fish eggs to inhibit fungal and protozoal infections. Some triphenylmethane dyes,
including MG, are recognized as animal carcinogens.

Determination of MG employs detection with visible absorption at 610 nm and the reduction of
malachite green to its colorless leuco base (LMG) is a facile process, which has been reported to occur
in fish flesh. The 90% of the total malachite green species present in fish were in the leuco form. To
determine MG and LMG simultaneously, post-column reactor packed with lead dioxide (PbO ) was
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used to transform LMG to MG with detection at VIS 618 nm after HPLC separation [197]. Application
for rainbow trout or cat fish used acetonitrile-buffer extraction, liquid-liquid extraction with
dichloromethane, and SPE (alumina, propylsulfonic acid) clean-up (detection limit 1-2ppb) [198,199].
An another method for acidification of LMG to MG employs post-column electrochemical oxidation
[200]. This HPLC system consists of coulometricall efficient electrochemical cell (ESA Coulochem
model 5100A; a potential of 0.45 V). Analysis for rainbow trout included extraction with
dichloromethane/acetonitrile/perchloric acid and clean-up with SPE (Bakerbond C18). The limit of
detection for the method is 3 ppb for MG and 6 ppb for LMG.

The oxidation efficiency from MG to LMG was reported to be 100.5% for PbO column reactor [199]
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and 57% for electrochemical oxidation [200]. The use of PbO column reactor requires replenishment
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for every 30-40 injection, while the use of the electrochemical cell needs occasional generation with 5
M nitric acid.

Hormones

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