Chemical assay of avermectins by high performance liquid chromatography with fluorescence detection.

Chemical methods for determining the concentrations of the avermectins in feeds, dosage forms, and biological samples are reviewed. The earliest methods for measuring the avermectins made use of the intense ultraviolet absorption due to the conjugated diene (olefinic bonds between carbons 8 and 9 and carbons 9 and 10) that has an ultraviolet absorption maximum at 245 nm and a molar absorptivity of over 30,000 l mol-cm-1. High performance liquid chromatographic (HPLC) separation of the avermectins and photometric measurement of their absorption at 245 nm has permitted the determination of plasma or serum concentrations to approximately 10 ng ml-1. Increased sensitivity of detection has been achieved by dehydration of the dihydroxycyclohexene ring of the avermectins in the presence of various catalysts that produces an intensely fluorescent derivative with an absorption maximum at 365 nm and emission maximum at 475 nm. These derivatives have been separated by HPLC and detected by fluorescence detectors at concentrations less than 1.0 ng ml-1 of plasma or serum. Recent improvements in the use of more efficient catalysts to effect the dehydration have reduced analysis time and decreased the formation of by-products, thereby improving the performance of these methods. A method based on HPLC with fluorescence detection of the dehydrated derivative was developed to determine ivermectin in horse dung after oral ivermectin doses of 200 micrograms kg-1 of body weight. Ivermectin concentrations greater than 0.05 microgram g-1 of wet feces were detectable for 1-2 days after dosing.