Autumn Breaud (Presenter)
The Johns Hopkins University
Bio: Autumn Breaud is the technical manager of the clinical reference and contract labs of Dr. William Clarke at the Johns Hopkins University.
Authorship: Autumn Breaud and William Clarke
The Johns Hopkins University
| Short Abstract We describe the development and validation of a method for quantification of doxorubicin and doxorubicinol in human serum and oral fluid. During development of the method, we performed stability studies for this compound in various primary solutions and matrices, based upon previous studies that indicate degradation under varying conditions. |
Long Abstract
Introduction
We describe the development and validation of a UPLC-MS/MS method for quantification of anthracycline chemotherapeutic agent doxorubicin and its cardiotoxic metabolite doxorubicinol in human serum and oral fluid.
Methods
We performed chromatographic separation on the Thermo Scientific Prelude UPLC system using a Hypersil Gold PFP column with dimensions 50 x 2.1 mm; 1.9 um particle size. Mobile phases were water (A) or methanol (B) with 0.1% formic acid and elution was performed by a ramp to 100% B over 90 seconds. Detection of doxorubicin and doxorubicinol were achieved with a Thermo Scientific Vantage triple quadrupole mass spectrometer equipped with a heated electrospray probe operating in positive ionization mode.
We performed validation studies: simple and complex precision, limit of quantification, linearity, carryover, recovery, and matrix effects. We investigated the correlation between serum and oral fluid concentrations of spiked samples.
During development of the method, we performed stability studies for this compound in various primary solutions and matrices, based upon previous studies that indicate degradation under varying conditions.
Results
We found that inter-day precision varied by less than 5% at three concentrations tested for either analyte. Intra-day precision varied by less than 10% at these concentrations. The method was found to be linear in the range of 10-1000 ng/mL. The limit of quantification was determined to be 10 ng/mL. No carryover was determined in blank injections following injections analyzed at 5 times the ULMI. Recovery was found to be better than 90% at all concentrations tested. Matrix influence on ionization was found to occur in both serum and oral fluid, but was corrected for by the common influence on internal standards, in both matrices. Ionization differed to a great extent between oral fluid and serum.
Conclusions
We found that stability studies for doxorubicin and doxorubicinol were important to conduct and propose similar studies be performed in future method development schemas. Over the course of time of typical method development in our laboratory, we assume stability of primary solutions of analytes, based upon standard manufacturer recommendations, but care should be taken to characterize such stability empirically.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | yes | Thermo Scientific |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |