Ivermectin derivatization

Since the avermectins exhibit unprecedented potency, they are used at unusually low doses of 6 —300 )-lg/kg, which makes the detection and isolation of residues and metaboUtes from animal tissue a new challenge. For this reason a sensitive analytical assay requires a derivative suitable for detection at concentrations down to 1/10 or 1/100 of one ppm. Ivermectin and avermectin B are therefore converted into an aromatic derivative which allows detection by fluorescence absorbance. To achieve this derivatization, avermectin B, ivermectin, or their derivatives are heated with acetic anhydride in pyridine at 100°C for 24 h (30). The reaction time can be reduced to 1 h by using /V-methylimidazole as a catalyst (31). The resultant... 

For reliable identification of a residue, detailed information about the molecular structure of the analyte is essential. The total information about the molecular structure of the analyte is the sum of the information derived from each individual analytical step of tire method. Frequently used selective analytical steps based on chromatography or immunoaffinity, provide more or less general indirect information. For example, solid-phase extraction (SPE) cleanup followed by liquid chromatography/ultraviolet detection (LC/UV) has been suggested for screening and quantification of ivermectin residues in liver, but presumptive positive samples can be confirmed by derivatizing an aliquot of the SPE eluate and reanalyzing the fluorescent derivative of ivermectin in an LC-fluorescence system (17). 

Detection in liquid chromatography is mostly performed by fluorescence and/or ultraviolet absorption. In a few instances, electrochemical detection has also been employed (357, 368). For compounds that exhibit inherent intense fluorescence such as albendazole and metabolites (319, 320, 338, 355), closantel (344), and thiabendazole and metabolites (378), fluorometric detection is the preferred detection mode since it allows higher sensitivity. Compounds that do not fluoresce such as eprinomectin, moxidectin, and ivermectin, are usually converted to fluorescent derivatives prior to their injection into the liquid chromatographic analytical column. The derivatization procedure commonly applied for this group of compounds includes reaction with trifluoroacetic anhydride in presence of A-methylimidazole as a base catalyst in acetonitrile (346, 347, 351, 352, 366, 369, 372-374). The formation of the fluorophore is achieved in 30 s at 25 C and results in a very stable derivative of ivermectin and moxidectin (353) but a relatively unstable derivative of eprinomectin (365). However, the derivatized extracts are not pure enough, so that their injection dramatically shortens the life of the liquid chromatographic column unless a silica solid-phase extraction cleanup is finally applied. 

Figure 1. Structure of ivermectin, showing the major analogs and the sites that were derivatized to form the hapten-protein conjugates used as immunizing and screening antigens. The 4" hemisuccinate ( Site 1 ) or the hemisuccinate formed through the oxygen on carbon 5 ( Site 2 ) were conjugated to carrier proteins as described in Methods.

Sher AH et al. [537] extracted abamectin, eprinomectin, moxidectin, ivermectin, and doramectin from liver and derivatized them with 1-methylimidazole/ trifluoFoacetic anhydride prior to separation and analysis on a C g colunm (A = 365 nm, ex 465 nm, em). Baseline resolution and excelloit peak shapes were obtained with a 97/3 methanol/water mobile phase. Elution was complete in <10 min. A standard curve from 25 to lOOppb was used. 

Abamectin, doramectin, ivermectin, moxidectin, and two homologs were extracted from milk and derivatized with trifluoroacetic anhydride. Separation was... 

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