Development of Doxorubicin Quantification by Reductive Amination

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 mu g/mL solutions. Doxorubicin solution (20 mu L) was transferred into two tubes and each tube was adjusted the pH value to 5.6 using sodium acetate buffer (160 mu L). The samples were allowed to react with 10 mu L 4% formaldehyde-H-2 and 10 mu L 4% formaldehyde-D-2 solutions for 5 min, respectively. Eventually, the modified samples were reduced for 1 h in the presence of 0.6 M NaBH3CN (10 mu L). 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.