Determination of urapidil and its metabolites in human serum and urine: comparison of liquid-liquid and fully automated liquid-solid extraction.

Direct injection of biological fluids into an automated pre-column system for high-performance liquid chromatographic quantitation of urapidil and its metabolites was compared with sample preparation by liquid-liquid extraction. On-line sample purification and enrichment proved to be a superior method for quantitation of the parent compound and its metabolites in serum, plasma, and urine. The automatic procedure circumvents the disadvantages of conventional sample work-up by extraction, such as time-consuming and complicated sample preparation, the need for large samples, poor recovery, and formation of artefacts. Injection of 100 microliters of serum into the automated pre-column system, followed by high-performance liquid chromatography with electrochemical detection, gave a detection limit for urapidil and metabolites in serum of 5 ng/ml. For urine samples with injection of 50 microliters the detection limit for the same compounds was 100 ng/ml using UV detection, which is adequate for evaluating renal excretion. Accuracy as well as precision of the analyses were better than 10% for concentrations above 100 ng/ml. In contrast to the direct injection method, liquid-liquid extraction requires 1-2 ml of serum, plasma, or urine for clean-up. Moreover, the extraction yield for the main human metabolite is significantly lower, and its stability after extraction is inadequate for using an autosampler. The direct injection method was applied in studying the pharmacokinetics and metabolism of urapidil in healthy volunteers and patients. So far, ca. 15 000 urapidil samples (both serum or plasma and urine) have been analysed by this technique.