Capillary gas chromatography of hexadecylphosphocholine in Caco-2T cells and cell culture media.

W F Steelant, E A Bruyneel, M M Mareel, E G Van den Eeckhout

Index: Anal. Biochem. 227(1) , 246-50, (1995)

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Abstract

The gas-chromatographic determination of hexadecylphosphocholine (HePC), an experimental antitumor agent of the alkyllysophospholipid group, in Caco-2T cell culture and cell culture media is described. The Caco-2T cells were treated with HePC at a concentration of 40 micrograms/ml (9.8 microM) and the uptake of the drug into the cells (calculated per milligram protein) was measured after 48 h culture (37 degrees C, 10% CO2). Also, a reversibility test for another 48 h was carried out in which the retention of the drug was measured. The toxicity of HePC on Caco-2T cells in viability assays was determined. Before the capillary gas-chromatographic determination, sample cleanup was performed by solid-phase extraction (SPE) on a weak cation-exchange column of the CBA (carboxylic acid) type. For quantitation, racemic 1-O-octadecyl-2-O-methylglycero-3-phosphorylcholine (ET-18-OMe) was added as internal standard, followed by derivatization with trimethylsilylbromide. The results showed that HePC taken up by the cells during 48 h of treatment was still detectable 48 h after removal of the drug from the medium.