High performance liquid chromatography in biopharmaceutical analysis using column switching techniques

High performance liquid chromatographic methods are described for the determination of various drugs in biological fluids, using direct injection and a column switching valve. The methods are based on the enrichment of the drug on a reversed-phase concentration column followed by chromatographic analysis using various mobile phases. Members of three major drug groups were examined, Tricyclic Antidepressants, Antihistamines and Benzodiazepines. One of the fundamental requirements of a bioassay is the capability to isolate and detect mixtures of polar and non-polar substances simultaneously, as is often the case with a drug and its metabolites. In the determination of amitriptyline and its metabolites, nortriptyline, 10-hydroxynortriptyline and 10-hydroxyamitriptyline, a direct injection/column switching procedure is described which determines all four analytes simultaneously with excellent recovery. A conventional liquid-liquid extraction procedure is also described which failed to isolate the non-polar metabolites. Both methods are fully validated, compared and applied to samples from patients undergoing treatment with amitriptyline. For the determination of tripelennamine (an antihistamine), in bovine plasma and milk an on-line solid phase extraction technique is described. Bovine plasma proved to be chromatographically cleaner than human plasma but operationally more difficult to handle due to its viscous nature. The extension of the method for clean-up of milk samples was also investigated. With centrifugation prior to injection this proved possible. The method was fully validated. Setistine, a novel antihistamine was also determined using a solid-phase extraction technique. In the final section a direct injection/column switching procedure for protein bound drugs is described. It is applied to the determination of diazepam and its metabolites desmethyl diazepam, temazepam and oxazepam, in plasma. The method is fully validated and compared to a classical liquid-liquid extraction scheme.