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.
Highlights
The occurrence of breast cancer in pregnant women has increased over the past few years, complicating approximately 1 in 3000 pregnancies
In order to study the transplacental transport of DXR across BeWo cells and placenta during placental perfusion studies, it is necessary to have simple, selective, and validated high performance liquid chromatography (HPLC)
A handful of analytical methods for the determination of doxorubicin in biological fluids are available; to the best of our knowledge, none have been published for quantification in DMEM/F12 cell culture media and/or placental perfusion media
Summary
The occurrence of breast cancer in pregnant women has increased over the past few years, complicating approximately 1 in 3000 pregnancies. In order to study the transplacental transport of DXR across BeWo cells and placenta during placental perfusion studies, it is necessary to have simple, selective, and validated high performance liquid chromatography (HPLC). A handful of analytical methods for the determination of doxorubicin in biological fluids are available; to the best of our knowledge, none have been published for quantification in DMEM/F12 cell culture media and/or placental perfusion media. Even though several methods have been used to detect DXR in biological fluids, there is great benefit to developing a simple method with a low limit of quantification (5 ng/mL) for the assessment of DXR in cell culture and placental perfusion media. We believe that the simplicity of the sample preparation method and the portability between the two different types of media makes this an accessible and valuable method for laboratories studying doxorubicin’s interactions with the placenta
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