Development and validation of a rapid HPLC method for the determination of cefdinir in beagle dog plasma integrated with an automatic on-line solid-phase extraction following protein precipitation in the 96-well plate format

The high-performance liquid chromatography (HPLC) coupled with on-line solid phase extraction (SPE) and ultraviolet (UV) detection was developed for determining cefdinir in beagle dog plasma. After simple pretreatment for plasma with 6% perchloric acid, a volume of 100 μL upper layer of the plasma sample was injected into the self-made on-line SPE column. The analytes were retained on the trap column (Lichrospher C18, 4.6 mm × 37 mm, 25 μm), and the biological matrix was washed out with the solvent (20 mM KH2PO4 adjusted pH 3.0) at flow rate of 2 mL/min. By rotation of the switching valve, the target analytes could be eluted from trap column to analytical column in the back-flush mode by the mobile phase (methanol–acetonitrile–20 mM KH2PO4 adjusted pH 3.0, 11.25:6.75:82, v/v/v) at flow rate of 1.5 mL/min, and then separated on the analytical column (Ultimate™ XB-C18, 4.6 mm × 50 mm, 5 μm). The complete cycle of the on-line SPE preconcentration, purification and HPLC separation of the analytes was 4 min. The UV detection was performed at 286 nm. The calibration curves showed excellent linear relationship (R2 = 0.9995) over the concentration range of 0.05–50 μg/mL. The optimized method showed good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This method was successfully applied to quantify cefdinir in beagle dog plasma to support the pre-clinical pharmacokinetic trial.