Objective: To determine tamoxifen (TAM) and its metabolites, endoxifen (END) and 4-hydroxytamoxifen (4-HT) in patients' DBS simultaneously for monitoring studies purposes. Methods: The UPLC-MS/MS method was developed and validated with clomiphene as the internal standard. Optimization was done by evaluating several parameters that affect the efficiency of DBS preparation and analysis of TAM and its metabolites. Results: Sample preparation was performed by protein precipitation using methanol. 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/minute. The mass detection was performed on Waters Xevo TQD using ESI+for TAM, END, 4-HT, and clomiphene as internal standard with m/z value: 372.2>72.27; 374.29>58.2; 388.29>72.19; dan 406.28>100.17 respectively. This method was linear within the range of 5-200 ng/ml for TAM; 1-40 ng/ml for END; and 0.5-20 ng/ml for 4-HT with r value of ≥0.9983; ≥0.9964; and ≥0.9981. %diff and %CV of the assay were within 15% and within 20% for LLOQ. The method was applied to 29 breast cancer patients, where the results lied between 30.29 and 188.63 ng/ml for tamoxifen, 1.45 and 28.77 ng/ml for endoxifen, 0.21 and 11.28 ng/ml for 4-hydroxytamoxifen. Conclusion: This method has successfully fulfilled validation requirement referring to 2011 EMA and 2013 FDA guidelines. The method was successfully applied to determine TAM, END, and 4-HT in DBS of breast cancer ER+patients. TAM and its metabolites level in patients were showed high variability with END concentration of 2 patients below the clinical threshold.
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.
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