Simple continuous and simultaneous determination of tetracycline residues

A new continuous separation method was developed for the determination of five different tetracyclines (oxytetracycline, tetracycline, chlortetracycline, methacycline and doxycycline). A bioassay using minimum medium (MM) seeded with Bacillus subtilis ATCC 6633 was carried out for the detection, with simple extraction from the agar block of the clear inhibition zone on the MM produced by the mixed tetracyclines. The extract was subjected to continuous identification by high performance liquid chromatography using a mu-Bondapack C18 column. The tetracyclines were separated at ambient temperature using a mobile phase of 0.01 M oxalic acid: acetonitrile: N,N-dimethylformamide (74:18:8, v/v/v) at a flow rate of 1.0 ml min-1. A variable-wavelength detector set at 355 nm and recorder set at 4 mm min-1 were used for the detection. The entire mixture was resolved as five peaks with retention times ranging from 2.75 to 9.65 minutes. This continuous, simple and rapid method of detection, extraction and identification may be useful for routine laboratory testing of residual antimicrobial agents in food.