The use of capillary high performance liquid chromatography with electrospray mass spectrometry for the analysis of small volume blood samples from serially bled mice to determine the pharmacokinetics of early discovery compounds

The methodology described demonstrates, for the first time, the feasibility of performing pharmacokinetic studies in the serially bled mouse model to support the early development of discovery compounds. Sample analysis, usin g capillary high performance liquid chromatography combined with tandem mas s spectrometry, has facilitated the achievement of this milestone and has s uccessfully been applied to determine pharmacokinetic information following both intravenous and oral administration of a single discovery compound. T he methodologies described demonstrate potential for a reduction in the amo unt of new chemical entity required to undertake pharmacokinetic studies. T ypically, such studies are performed in larger rodents with a significantly increased body mass (ten times in the case of the rat) and therefore it fo llows that to undertake the same experiment in the mouse would require ten times less compound to effect an equivalent dose. Conventionally, pharmacok inetic studies to obtain both intravenous and oral information, e.g. cleara nce and half-life, and the resultant bioavailability have been performed us ing two parallel groups of rodents, collecting blood by exsanguination, sep arating off the plasma and analysing this using conventional liquid chromat ography/tandem mass spectrometry. The use of capillary high performance liq uid chromatography (HPLC) has facilitated the analysis of small volume bloo d samples by increasing the effective sensitivity of the analytical method. Consequently, we have established a protocol for serially bleeding mice th us reducing the number of animals and so further reducing the amount of com pound required for such experiments. This paper reports data obtained from collected and processed blood volumes of <20 mu L with the subsequent injec tion of only 1 mu L of precipitated extract onto a capillary column. Copyri ght (C) 1999 John Whey & Sons, Ltd.