Determination of tamoxifen and four metabolites in serum by low-dispersion liquid chromatography.

In this assay of tamoxifen and four metabolites in human serum, the serum samples are deproteinized with an equal volume of acetonitrile, then injected into a small (0.21 X 2 cm) precolumn packed with 5-micron-diameter octadecylsilane (ODS) particles. The samples are concentrated on-column by equilibrating the column with an equivolume solution of water and acetonitrile containing 3 mmol of acetic acid and 2 mmol of diethylamine per liter. The drugs are then directed into an analytical ODS column (0.21 X 10 cm) by changing the mobile phase followed by column switching. The primary alcohol of tamoxifen ("metabolite Y"), 4-hydroxytamoxifen ("metabolite B"), tamoxifen, N-desdimethyltamoxifen ("metabolite Z"), N-desmethyltamoxifen ("metabolite X"), and 4-methoxytamoxifen (internal standard) are eluted in this order at a flow rate of 0.3 mL/min with a mobile phase of acetonitrile/water (91/9 by vol) at low ionic strength (1 mmol of acetic acid and 0.67 mmol of diethylamine per liter) and detected by post-column fluorescence activation by passage through a capillary quartz tube exposed to ultraviolet light. Analytical recovery was close to 100%. Within-day precision corresponded to a CV of 1-5% at serum concentrations of tamoxifen or metabolites greater than 10 micrograms/L; the detection limit of the assay for these compounds was about 1 microgram/L. This fully automated assay has the advantage of simple sample processing, high sample output, low solvent consumption, high analytical recovery of tamoxifen and four metabolites in serum, and determination of all these compounds plus an internal standard in a single run.