HPLC Method Development for Quantification of Doxorubicin in Cell Culture and Placental Perfusion Media

Abstract

Assessment of drug transport across the placenta is important in understanding the effect of drugs on placental and fetal health. These phenomena can be studied in both in vitro cell lines and ex vivo placental perfusions. We have successfully developed a sensitive yet simple high performance liquid chromatography (HPLC) method coupled with fluorescence detection to determine the concentration of doxorubicin (DXR) in cell culture media for transport studies in human trophoblast cells (BeWo, b30 clone) and in fetal media for placental perfusion experiments. The method was developed based on a protein precipitation technique and was validated in both media types for linearity, intra-day, and inter-day precision and accuracy. The relationship of peak area to concentration was linear with R2 values of 0.99 or greater obtained over the concentration range of 1.5 to 15,000 ng/mL. Despite the high concentrations of albumin in fetal perfusion media (30 mg/mL), the lower limits of detection and quantification for DXR were found to be 1.5 and 5 ng/mL, respectively. This analytical method may be used to study the transport of DXR across BeWo cells and human placenta during placental perfusion studies.

Keywords

• doxorubicin
• BeWo cell culture media
• placenta
• high performance liquid chromatography
• fluorescence
• anthracycline
• analytical method
• perfusion