The determination of tamoxifen and its main metabolites (4-hydroxytamoxifen, N-desmethyl-tamoxifen, tamoxifen-N-oxyde and endoxifen) in human blood is needed to understand the mechanism of tamoxifen metabolization and to predict the effect of tamoxifen treatment for breast cancer patient.  A method was developed for the quantification of tamoxifen and its main derivatives in plasma using micellar liquid chromatography coupled to fluorescence detection. The analytes were activated by off-line UV-irradiation at 254 nm during 20 to form the photocycled fluorescent derivatives. No extraction step was needed, then the experimental procedure was strongly expedited.  Tamoxifen and its metabolites were separated in < 40 min using a mobile phase containing 0.08 M SDS–4.5% n-butanol at pH 3 running at 1.5 mL/min through a C18 column at 40 °C, without interferences from the matrix. Excitation and emission wavelengths were 260 and 380 nm, respectively. The methodology was validated following the International Conference on Harmonization of (ICH) guidelines in terms of: selectivity, linear range (0.3–15 μg/mL), linearity (r²>0.999), sensitivity (LOD, 65-80 ppb; LOQ, 165-200 ppb), intra- and interday accuracy (12.2 to 11.5 %)  and prevision (< 9.2 %) and robustness (< 6.3 %). The method was used to quantify tamoxifen and tamoxifen derivatives in several breast cancer patients from a local Hospital, to relate the genotype of the patient and its way to metabolize tamoxifen.

Acknowledgments: This work was supported by Projects P1.1B2012-36 of Pla de Promoció de la Investigació de la Universitat Jaume I and Res. Nº 162/12 of Secretaria de Ciencia y Técnica – UNC.