Trace analysis of fullerenes in biological samples by simplified liquid–liquid extraction and high-performance liquid chromatography

Fullerene (C60) has several potential biomedical and industrial applications. While pure fullerene is not soluble in water, nanoparticles of the fullerene aggregates (nano-C60) can be prepared in water solutions. The concentration of nano-C60 in biological media after systemic exposure could be very low and requires trace analytical methods to be developed for the toxicological and pharmacokinetic studies of the nanomaterial. A serious drop in extraction efficiency was observed when the concentration was under 0.5 μg/mL using traditional liquid–liquid extraction (LLE) protocols. The evaporation of the solvent extract to dryness was found to be the main reason for the efficiency drop and an improved evaporation method was proposed to overcome this problem. Optimal proportion of glacial acetic acid (GAA) was used to solublize the proteins and surfactants in the biological samples, so that the emulsion problem was eliminated during LLE. Magnesium perchlorate was used to destabilize the nano-C60 particles in the water solution and promoted the solvent extraction. A simplified LLE method was developed for high throughput while preserved the advantages of the traditional LLE. The developed method was used for trace analysis of fullerenes in protein containing media and tape-stripped skin samples. Under optimal experimental conditions, the detection limit was 0.34 ng/mL and the recovery was in the range of 94–100% (n = 5) at a concentration of 10 ng/mL nano-C60 in the biological media.