Quantification of new antiepileptic drugs by liquid chromatography/electrospray ionization tandem mass spectrometry and its application to cellular uptake experiment using human placental choriocarcinoma BeWo cells

摘要

A method for quantification of new antiepileptic drugs, including lamotrigine (LTG), levetiracetam (LEV), gabapentin (GBP), and topiramate (TPM), in cellular samples, using liquid chromatography/electrospray ionization tandem mass spectrometry was developed to better understand the membrane transport mechanisms of these drugs. Cell lysate was deproteinized by methanol containing LEV-d(3) as an internal standard (IS). Chromatographic separation was performed on a C18 column using gradient elution with methanol-water-formic acid (10:90:0.1, v/v/v) and methanol-formic acid (100:0.1, v/v). Analytes were detected in positive ion electrospray mode with selected reaction monitoring (SRM). This method was applicable for a linear range of 5 to 500 pmol for LTG; 5 to 1000 pmol for LEV; 10 to 10,000 pmol for GBP; and 5 to 5000 pmol for TPM. The intra-day precision, inter-day precision, and accuracy data were assessed and found to be acceptable. This developed and validated method was then successfully applied to the investigation of uptake of the new antiepileptic drugs in placental choriocarcinoma BeWo cells. The intracellular concentration of these drugs in BeWo cells, accumulating over 30 min at 37 degrees C was in the order of GBP > LTG > LEV approximate to TPM. Furthermore, the uptake of GBP at 4 degrees C was much lower than that at 37 degrees C. The uptake of GBP was saturated at high concentrations. The kinetic parameters calculated for GBP uptake in BeWo cells were determined as K-m of 105.4 +/- 6.4 mu M and Vmax at 8153 +/- 348 pmol/mg protein/min. The novel method described here should enable investigators to elucidate the transport mechanisms of these antiepileptic drugs in BeWo cells. (C) 2015 Elsevier B.V. All rights reserved.