Determination of pirlimycin residue in milk by liquid chromatographic analysis of the 9-fluorenylmethyl chloroformate derivative

A procedure to determine pirlimycin residues in bovine milk was developed, Pirlimycin was extracted from milk after protein precipitation and 2 stages of liquid-liquid partitioning, The extract was evaporated to dryness, redissolved in dilute base, and derivatized with 9-fluorenylmethyl chloroformate (Fmoc) to impart a chromophore for ultraviolet (UV) detection, The derivatized extract was analyzed by reversed-phase liquid chromatography (LC), Pirlimycin concentration was calculated from the peak height of the extract by using a linear regression standard curve prepared from a series of derivatized standards, The procedure's performance in the range of the regulatory tolerance (0.4 ppm) was validated by replicate analysis of control, fortified control, and residue-incurred bovine milk, Average recoveries at 0.2, 0.4, and 0.8 ppm were 91, 88, and 87%, respectively, and coefficients of Variation were 5, 4, and 1%, respectively, Overall recovery for all samples was 89%, with 4% coefficient of variation, Linear regression analysis of LC/UV results versus results from a previously published LC/mass spectrometric assay gave a slope of 0.95, with a correlation coefficient of 0.98.