Performance and reliability of splitless microliter gradient pumps in a metabolic stability study using cytochrome P450/3A4 and capillary liquid chromatography–mass spectrometry

The performance of two commercially available capillary LC pumps (MicroPro (Eldex, USA), Evolution 200 (ProLab, Switzerland)) generating really splitless gradients in the microliter per minute range was tested in detail concerning their applicability for routine drug discovery. A standard method to study metabolic stability against CYP450 isoform 3A4 was selected. This method was transformed into a fast splitless capillary LC–MS method. Both pumps generated reproducible gradients at flows of 5–10 μl/min within 10–15 min. Although gradient formation of the MicroPro system was very reproducible, its equilibration time was too long for fast gradients around 5 μl/min. The Evolution 200 pump offered a good performance with 180 μm i.d. columns at a flow rate of 6 μl/min. The precision of the retention time of the internal standard (ISTD) varied between 1.4 and 3.4% (n=131–152, three different columns tested). Up to 800 injections of sufficient performance on one column and a stable enough response of the ISTD for 16 h sequence duration were obtained. Accuracy between 95 and 105% and precision ≤8.4% for 1′-hydroxylated midozolam were reached. The IC50 values of the miniaturized assay (drug candidate BAL4815 1.7±0.5, itraconazole 0.46±0.06, and ketoconazole 0.12±0.01 μM) agreed well with those of the conventional approach. Details concerning method optimization and limitations in operation are discussed in detail. Still, the overall performance of the capillary LC pumps cannot cope completely with that of conventional HPLC pumps in terms of user-friendliness.