Title:Development of Doxorubicin Quantification by Reductive Amination
VOLUME: 13 ISSUE: 4
Author(s):Yi-Reng Lin, Chia-En Wu, Mei-Fang Huang, You-Ying Wu, Jing-Heng Huang, Meng-Chieh Liu, Ziyu Chen, Yow-Ling Shiue and Shih-Shin Liang*
Affiliation:Department of Biotechnology, Fooyin University, School of Environment and Life Sciences, Kaohsiung, Department of Pharmacy and Master Program, College of Pharmacy and Health Care, Tajen University, Pingtung, Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Department of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University, Kaohsiung, Institute of Biomedical Science, College of Science, National Sun Yat-Sen University, Kaohsiung, Department of Biotechnology, Kaohsiung Medical University, P.O. Box: 80708, Kaohsiung
Keywords:Doxorubicin, doxorubicinol, reductive amination, absolute quantification, high-performance liquid-phase chromatography
(HPLC), tandem mass spectrometry (MS/MS).
Abstract:Background: Doxorubicin (Dox) is a medicine for cancer chemotherapy in various cancers,
such as leukaemia, lympyoma and breast cancer. However, the Dox metabolite induced cardiotoxicity.
Therefore, how to monitor exactly the concentration of Dox and its metabolites in plasma or serum is an
important issue.
Methods: Doxorubicin was dissolved in ethanol to prepare 10000 μg/mL solutions. Doxorubicin solution
(20 μL) was transferred into two tubes and each tube was adjusted the pH value to 5.6 using sodium
acetate buffer (160 μL). The samples were allowed to react with 10 μL 4% formaldehyde-H2 and 10 μL
4% formaldehyde-D2 solutions for 5 min, respectively. Eventually, the modified samples were reduced
for 1 h in the presence of 0.6 M NaBH3CN (10 μL). Individual samples were injected into the mass
spectrometer by syringe to determine m/z values and establish MRM transition. The signal enhancement,
illustration of calibration curve, and method validation were according to the MS detection of
MRM transitions between modified and unmodified compounds.
Results: The signal of modified Dox was enhanced by reductive amination, and the signal enhancement
was improved 190.8% which was compared with unmodified Dox. The linearity of doxorubicin was
illustrated from the range of 5.0-1000.0 ng/mL, and the limit of detection and the limit of quantitation
were 1.37 and 1.46 ng/mL, respectively. The method validation was spiking doxorubicin with low, intermediate,
and high concentrations including 20 ng/mL, 100 ng/mL, and 500 ng/mL in human serum,
and recoveries were between 80% to 98%.
Conclusion: Via reductive amination of formaldehyde, a novel, laborless, inexpensive and convenient
method was developed for the absolute quantification of doxorubicin. Furthermore, doxorubicin would
be reduced to generate doxorubicinol which is a metabolite of doxorubicin. Therefore, this method
could be utilized to quantitate doxorubicin as well as doxorubicinol. The quantification of doxorubicin
and its metabolite, doxorubicinol, could share the same internal standard and standard for calibration
curve illustration. Besides, the novel modification of reductive amination enhanced MS signal intensities,
facilitating the absolute quantification of doxorubicin.
