Determination of virginiamycin in swine, cattle and chicken muscles by HPLC with fluorescence detection

Abstract

A simple and rapid method for the determination of virginiamycin M₁ (M₁) and S₁ (S₁) in swine, cattle and chicken muscles has been developed by means of HPLC with fluorescence detection. M₁ and S₁ were extracted from 10 g of muscle with 50 ml of acetonitrile and 5 g of Hyflo Super-Cel. The volume of filtered extract was made up to 100 ml with acetonitrile. After evaporation of 50 ml of acetonitrile solution at 40 °C, the residue was dissolved in 2 ml- and 1 ml-portions of methanol. This methanol solution, 1.5 ml of water and 3.5 ml of chloroform were placed in a low actinic test tube. M₁ and S₁ contained in methanol solution were extracted with chloroform by using a test tube mixer. After centrifugation for 5 min at 2800 rpm, the upper layer was removed and the lower layer was washed with 1.5 ml of water. After centrifugation for 5 min at 2800 rpm, the upper layer was removed and the lower layer was transferred into another low actinic test tube. The chloroform solution was dried up with nitrogen. After M₁ and S₁ were dissolved in 1 ml of HPLC mobile phase, the solution was passed through a membrane filter of 0.5 μm pore size and was centrifuged for 2 min at 2800 rpm. The upper layer was injected onto the HPLC system and peak height was measured. The condition of HPLC was as follows: Column, TSKgel ODS-120T, 5 μm, 250 × 4.6 mm i. d. (Toyo Soda); mobile phase, methanol-acetonitrile-0.015M NaH₂PO₄- tetrahydrofuran (43:22:34:1); flow rate, 0.5 ml/ min; column temp. 40°C; detection, fluorescence Ex. 311 nm, Em. 427 nm: The calibration curves were linear from 10 ng to 360 ng for M₁ and from 1 ng to 32 ng for S₁. Recoveries of M₁ added to swine, cattle and chicken muscles at the level of 0.59 μg/g were 98.9, 95.7 and 93.8%, respectively. Recoveries of S₁ added at the level of 0.17 μg/g were 99.5, 98.4 and 101%, respectively. The detection limits of M₁ and S₁ were 0.1, 0.01 μ/g, respectively.