Isocratic high-performance liquid chromatographic assay of taxol in biological fluids and tissues using automated column switching.

Abstract

This report describes the analysis of taxol in human plasma, cell culture medium, and dog bladder tissue by isocratic high-performance liquid chromatography (HPLC) with automated column switching. Cephalomannine was used as the internal standard. Biological samples were extracted with ethyl acetate, with a recovery of > 80%. Sample extracts reconstituted in 37.5% acetonitrile were stable in polypropylene tubes at room temperature for 22 h. The HPLC stationary phase consisted of a clean-up column (Nova-Pak C8, 75 x 3.9 mm I.D., 4 microns particle size) and an analytical column (Bakerbond octadecyl, 250 x 4.6 mm I.D., 5 microns particle size). Taxol and cephalomannine were monitored at 229 nm. Samples were injected onto the clean-up column and eluted with the clean-up mobile phase (37.5% acetonitrile in distilled water) at 1 ml/min. Concurrently, the analytical mobile phase (49% acetonitrile in distilled water) was directed through the analytical column at a flow-rate of 1.2 ml/min. A heart-cut fraction from 8 to 15 min was transferred from the clean-up column onto the analytical column. Loading of a second sample onto the clean up column while the first sample was eluting from the analytical column reduced the HPLC analysis time to about 15 min per sample. This method has a lower detection limit of 5 ng/ml and intra- and inter-day variations of < 5%.