Volumetric Absorptive Microsampling as a New Biosampling Tool for Monitoring of Tamoxifen, Endoxifen, 4-OH Tamoxifen and N-Desmethyltamoxifen in Breast Cancer Patients

Introduction: In this research, we used a volumetric absorptive microsampling (VAMS) technique to collect blood samples from the patients. A rapid and simple sample preparation method and LC-MS.MS assay was then developed and validated for the simultaneous analysis of tamoxifen and its three active metabolites. Methods: VAMS extraction was performed in methanol by sonication-assisted extraction method for 25 min after 2 hof VAMS drying. Separation was carried out using Acquity UPLC BEH C18 column (2.1 x 100 mm; 1.7 µm), with a flow rate of 0.2 mL/min, and the mobile phase gradient of formic acid 0.1% and formic acid 0.1% in acetonitrile for 5 min. The multiple reaction monitoring (MRM) values were set at m/z 358.31>58.27 for N-desmethyltamoxifen, m/z 372.33>72.28 for tamoxifen, m/z 388.22>72.28 for 4-hydroxytamoxifen, m/z 374.25>58.25 for endoxifen, and m/z 260.26>116.12 for propranolol. Results and Discussion: The lower limit of quantification value (LLOQ) was 2.50 ng/mL for tamoxifen, 2.50 ng/mL for endoxifen, 1.50 ng/mL for 4-hydroxitamoxifen, and 2.00 ng/mL for N-desmethyltamoxifen. Accuracy (%bias) and precision (%CV) were within 20% for LLOQ and 15% for other concentrations. There were no interference responses >20% of the LLOQ and 5% of the internal standard. The level of ion suppression in all analytes was less than 7%. The preparation system developed in this study successfully extracted more than 90% of analytes from the matrix with precision below 15%. Carryover was shown to be below 6% in all analytes. Stability of analytes in VAMS was demonstrated for up to 30 days, under room temperature storage in a sealed plastic bag with desiccant. This method was successfully applied to analyze tamoxifen and the metabolites level in 30 ER+ breast cancer patients.