SIMPLE AND RAPID METHOD FOR THE SIMULTANEOUS ANALYSIS OF TAMOXIFEN, ENDOXIFEN, AND 4-HYDROXYTAMOXIFEN IN DRIED BLOOD SPOT USING LIQUID CHROMATOGRAPHY–TANDEM MASS SPECTROMETRY
Keywords:Tamoxifen, Endoxifen, 4-hydroxytamoxifen, Chlomiphene, Dried blood spot, Optimization
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.
Early Breast Cancer Trialists’ Collaborative Group (EBCTCG). Relevance of breast cancer hormone receptors and other factors to the efficacy of adjuvant tamoxifen: Patient-level meta-analysis of randomized trials; Lancet 2011. p. 771–84.
NGL Jager, SC Linn, JHM Schellens, JH Beijnen. Tailored tamoxifen treatment for breast cancer patients: a perspective. Clin Breast Cancer 2015;15:241–4.
MJ Higgins, V Stearns. CYP2D6 polymorphisms and tamoxifen metabolism: clinical relevance. Curr Oncol Rep 2010;12:7–15.
AHM de Vries Schultink, W Zwart, SC Linn, JH Beijnen, ADR Huitema. Effects of pharmacogenetics on the pharmacokinetics and pharmacodynamics of tamoxifen. Clin Pharmacokinet 2015;54:797–810.
L Madlensky, L Natarajan, S Tchu, M Pu, J Mortimer, SW Flatt, et al. Pierce, tamoxifen metabolite concentration, CYP2D6 genotype and breast cancer outcomes. Clin Pharmacol Ther 2007;86:573–9.
P Saladores, T Murdter, D Eccles, B Chowbay, NK Zgheib, S Winter, et al. Brauch, Tamoxifen metabolism predicts drug concentrations and outcome in premenopausal patients with early breast cancer. Pharmacogenomics J 2015;15:84–94.
MP Goetz, K Sangkuhl, HJ Guchelaar, M Schwab, M Province, M Whirl Carrillo, et al. Klein, clinical pharmacogenetics implementation consortium (CPIC) guideline for CYP2D6 and tamoxifen therapy. Clin Pharmacol Ther 2018;103:770–7.
HI Woo, SK Lee, J Kim, SW Kim, J Yu, SY Bae, et al. Lee, Variations in plasma concentrations of tamoxifen metabolites and the effects of genetic polymorphisms on tamoxifen metabolism in Korean patients with breast cancer. Oncotarget 2017;8:100296–311.
SF Teunissen, NGL Jager, H Rosing, AH Schinkel, JHM Schellens, JH Beijnen. Development and validation of a quantitative assay for the determination of tamoxifen and its five main phase I metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 2011;879:1677–85.
J Peris Vicente, E Ochoa Aranda, D Bose, J Esteve Romero. Determination of tamoxifen and its main metabolites in plasma samples from breast cancer patients by micellar liquid chromatography. Talanta 2015;131:535–40.
MV Antunes, DD Rosa, T dos, S Viana, H Andreolla, TO Fontanive R. Linden, sensitive HPLC-PDA determination of tamoxifen and its metabolites N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen in human plasma. J Pharm Biomed Anal 2013;76:13–20.
SF Teunissen, NGL Jager, H Rosing, AH Schinkel, JHM Schellens, JH Beijnen. Development and validation of a quantitative assay for the determination of tamoxifen and its five main phase I metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry. J Chromatogr B: Anal Technol Biomed Life Sci 2011;879:1677–85.
AJ Wilhelm, JCG den Burger, EL Swart. Therapeutic drug monitoring by dried blood spot: progress to date and future directions. Clin Pharmacokinet 2014;53:961–73.
Y Harahap, BP Manggadani, T Ribka, J Sitorus, CA Mulyadi, DJ Purwanto. Clinical application of dried blood spot for monitoring studies of tamoxifen, endoxifen, and 4-hydroxytamoxifen in breast cancer patient using liquid chromatography–tandem mass spectrometry. Int J Appl Pharm 2019;11:59-63.
MV Antunes, S Raymundo, V de Oliveira, DE Staudt, G Gossling, GP Peteffi, et al. Linden, Ultra-high performance liquid chromatography tandem mass spectrometric method for the determination of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen in dried blood spots-development, validation and clinical application during breast c. Talanta 2015;132:775–84.
EM Agency. Guideline on bioanalytical method validation. Guideline Bioanalytical Method Validation Table Contents 2012;44:1–23.
Food and Drug Administration, Guidance for Industry Bioanalytical Method Validation Guidance for Industry Bioanalytical Method Validation; 2018.
M Tre-Hardy, A Capron, MV Antunes, R Linden, P Wallemacq. Fast method for simultaneous quantification of tamoxifen and metabolites in dried blood spots using an entry level LC–MS/MS system. Clin Biochem 2016;49:1295–8.
RA Koster, B Greijdanus, DJ Touw, JC Alffenaar. The performance of five different dried blood spot cards for the analysis of six immunosuppressants. Bioanalysis 2015;7:1225–35.
NG Jager, H Rosing, JH Schellens, JH Beijnen. Determination of tamoxifen and endoxifen in dried blood spots using LC–MS/MS and the effect of coated DBS cards on recovery and matrix effects. Bioanalysis 2014;6:2999–3009.
BS Mohammed, GA Cameron, L Cameron, GH Hawksworth, PJ Helms, JS McLay. Can finger-prick sampling replace venous sampling to determine the pharmacokinetic profile of oral paracetamol. Br J Clin Pharmacol 2010;70:52-6.