Abstract
Objective: Tamoxifen (TAM) is a hormonal therapy that is clinically proven to reduce breast cancer recurrence by blocking estrogen receptor, mainly through its active metabolites, 4-hydroxytamoxifen (4HT) and endoxifen (END), which have a higher affinity to ER than TAM itself. The objective of the present study was to develop and validate simple and rapid LC-MS/MS method for analysis TAM and its metabolites simultaneously in dried blood spot (DBS) sample for monitoring studies purposes.
 Methods: Optimization was done by evaluating several parameters that affect the efficiency of DBS preparation, such as blood spot volume, drying time and extraction method from the DBS paper. The effectiveness of chromatographic conditions was also optimized by varying flow rate, mobile phase combination and gradient. Clomiphene was used as the internal standard.
 Results: The result showed that preparation of 20 µl blood spot volume with 120 min of drying time and 25 min of extraction time using 1 ml methanol was the most efficient condition and also fulfilled recovery and matrix effect requirement according to FDA and EMA guidelines. The separation was performed on UPLC Class BEH C18 using formic acid 0.1%-formic acid 0.1% in acetonitrile (35:65) as the mobile phase in isocratic mode at 0.25 ml/min with a total analysis time of 4 min.
 Conclusion: This method has successfully fulfilled all validation requirements referring to EMA and FDA guidelines.
Highlights
Tamoxifen (TAM) is a hormonal therapy that is given to premenopausal patients with estrogen receptor-positive type of breast cancer
Optimization was done by evaluating several parameters that affect the efficiency of dried blood spot (DBS) preparation, such as blood spot volume, drying time, and extraction method from the DBS paper
DBS card used in this research was Perkin Elmer 226 paper obtained from PerkinElmer (USA), and whole blood for validation was obtained from the Indonesian Red Cross
Summary
Tamoxifen (TAM) is a hormonal therapy that is given to premenopausal patients with estrogen receptor-positive type of breast cancer. TAM treatment for 5 y after surgery has been proven to reduce the recurrence rate of ER+breast cancer by 50% in treatment five years after surgery and reduce the 30% mortality rate [1, 2]. TAM is a prodrug that go through metabolism in hepatic mediated by cytochrome P450 enzyme via N-demethylation followed by 4hydroxylation. Demethylation product of tamoxifen via CYP3A4, namely N-desmetyltamoxifen (NDT) is the most abundant metabolite but not active to estrogen receptors. There are other metabolic pathways through hydroxylation of tamoxifen to 4-hydroxytamoxifen (4HT) This pathway is mediated by CYP2D6 and by CYP3A4 and CYP2C19. This pathway is classified as minor [3]
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