Liquid chromatographic assay of tylosin in animal feeds

A liquid chromatographic (LC) method is described for the assay of tylosin (tylosin A, tylosin B, and tylosin-urea adduct [TUA]) in animal feeds at approximately 4-200 g/ton. Samples are extracted with an equal mixture of methanol and 0.1M pH 8 phosphate buffer. An acidic alumina column cleanup step is used prior to reversed-phase LC. Determination is accomplished with UV detection at 280 nm. A 15 cm C8 column is used for sample concentration. Elution is performed at 1.5 mL/min with a gradient containing an increasing methanol concentration with 0.5% acetic acid and tetramethylammonium chloride (5 g/L) as the aqueous portion. Baseline separation of tylosin B, TUA, and tylosin A is achieved with retention times of approximately 7.4, 9.3, and 10.7 min, respectively. Conversion factors of 1.0 and 1.2 are used to convert peak areas of tylosin B and TUA, respectively, to tylosin A equivalent as an alternative to standards being used for these components of some feeds. The precision of the procedure ranged from a coefficient of variation (CV) of 1.6% for tylosin A at 94 g/ton to a CV of 11.3% for TUA at 10 g/ton. The average percent recovery of spiked feed extracts was 100.4-102.2% for the 3 tylosin components. A correlation with the microbiological assay is presented for 12 feeds, 1 premix, and 1 injectable tylosin product.