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The use of the post-column method with OPA/2-ME for the analysis of fradiomycin (FM) and DM in
beef and neomycin (NM) in milk was reported [173,174]. The method employs extraction and clean-up
procedure described in KM analysis followed by separation with reversed-phase HPLC and post-
column derivatization with fluorescent detection (λex340 nm, λem450 nm). The detection limits were
0.1 ppm for DM and 0.2 ppm for FM.

For the analysis of spectinomycin (SPCM), one of other aminoglycoside antibiotics, the use of o-
nitrophenylhydrazine, a pre-column derivatizing agent, which reacts with carbonyl group of SPCM
followed by detection at VIS 420 nm was reported [175]. Sample reparation for chicken and pork
tissues employs deproteinization with trichloroacetic acid and SPE (C18) clean-up. The detection limit
was 0.05 ppm.

Polyether Antibiotics
Monensin, salinomycin, naracin, and lasalocid are used primarily as coccidiostats. It has been reported
that narasin can be used as a growth stimulant, and monensin and lasalocid improve the efficiency of
feed utilization. These antibiotics are a new class of drug known as carboxylic polyethers and have
similar chemical structures.

Derivatization of monensin, salinomycin and naracin employs post-column method with vanilline
followed by detection at VIS 522 nm. This method has been applied for the analysis of bovine tissues,
milk, chicken tissues and feed [176-178]. Sample pretreatment employs extraction with aqueous
methanol, followed by liquid-liquid extraction with dichloromethane, and clean-up with SPE (Sep-pak
silica). The detection limits were 5-25 ppb. The post-column method was also applied for the analysis
of semduramicin, a relatively new polyether antibiotic [179].

On the other hand, a method using pre-column technique was developed for the analysis of polyether
antibiotics, i.e. allylic hydroxyl group of salinomycin was acidified with pyridinium dichromate to give
α,β-unsaturated keton and detected at UV 225 nm [180]. However, this method requires column-
switching as well as off-line clean-up to eliminate interfering peaks from co-existents in the residue
analysis of livestock products. Derivatization with ADAM (λex365 nm. λem418 nm) and 1-
bromoacetylpyrene (BAP) has been widely used [181]. The carboxylic acid group in the structures of
polyether antibiotics made it possible to form the fluorescent derivatives of each antibiotic by reacting
with ADAM or BAP. Application of ADAM was reported for the analysis of monensin, salinomycin,
naracin and lasalocid in beef liver [182,183]. For the use of BAP, analysis of polyether antibiotics in
feed, monensin and salinomycin in chicken was reported [184,185]. These polyether antibiotics have
ionophore action, and carboxylic and hydroxyl group form complex compounds with alkaline metal-
ion, thus direct derivatization by ADAM proceeds with difficulty. Therefore, the extraction with
chloroform under weak acidic condition in order to obtain free monensin (acid form) [186], acetylation
of hydroxyl group with acetic anhydride to release ionophore state [182,183], or addition of crown ether
such as Kryptofix 222 as a catalyst of ADAM has been tried [184,185]. The crown ether reacts with
alkaline metal-ion to form complex compounds and the reaction is supposed to be the ionization of
carboxyl group resulted in catalyzation of the reaction.

1.2.4.3—
Antiparasitic Agents

Avermectins

Avermectins, which are isolated from the myceria of Streptmyces avermitilis, are a family of

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