Development and Validation of Doxorubicin Hydrochloride and Doxorubicinol Quantification Method in Dried Blood Spot by Liquid Chromatography-Tandem Mass Spectrometry

Aldhi Anarta¹, Yahdiana Harahap¹, Herman Suryadi¹

Faculty of Pharmacy, Universitas Indonesia, Depok 16424, West Java, Indonesia

Corresponding author: yahdiana03@yahoo.com

Doxorubicin is an antracycline anticancer drug which is used as the first line therapy of breast cancer. Doxorubicin will be metabolized to the main metabolite named doxorubicinol. According to some studies, doxorubicinol that accumulates in human body could increase the risk of  cardiotoxicity. Therefore, an analysis method is needed to determine doksorubicin and doxorubicinol concentration. Nowadays, there is one biosampling technique known as dried blood spot (DBS) which is being developed because some advantages of this technique such as less invasive, easier procedure, and better stability. This study aims to develop validated analysis method of doxorubicin hydrochloride and coxorubicinol simultaneously in dried blood spot with hexamethylphosphoramide as the internal stanndard. Sample preparation  was performed by protein precipitation using water and methanol. The separation was performed on UPLC Class BEH C18 Acquity UPLC BEH C₁₈ (2.1 × 100 mm; 1.7 μm), with 0.15 mL/menit flow rate and using acetic acid 0.1% and acetonitrile gradient for 9 minutes. Quantification analysis was performed by a triple quadrupole mass spectrometry with electrospray ionization (ESI) in positive ion mode. The multiple reaction monitoring (MRM) was set at m/z 544.22 > 397.06 for doxorubicin hydrochloride; m/z 546.22>361.05 for doxorubicinol; and m/z 180.03>135.16 for  hexamethylphosporamide. This method was linear within the concentration range of 2–500 ng/mL for doxorubicin and 1–250 ng/mL for doxorubicinol.

Keywords: Doxorubicin, doxorubicinol, cardiotoxicity, LC-MSMS, dried blood spot, hexamethylphosphoramide, validation