Evaluation of extraction methods for quantification of aqueous fullerenes in urine

There is a growing concern about the human and environmental health effects of fullerenes (e.g., C₆₀) due to their increasing application in research, medicine, and industry. Toxicological and pharmacokinetic research requires standard methods for extraction and detection of fullerenes from biological matrices such as urine. The present study validates the use of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) methods in conjunction with liquid chromatography-mass spectrometry (LC-MS) for the quantitative determination of C₆₀ in human and synthetic urine as compared with ultrapure water. Glacial acetic acid, which is necessary to prevent emulsions during LLE, inhibited C₆₀ detection by LC-MS, but this could be mitigated with evaporation. Aqueous C₆₀ aggregates (nC₆₀) were spiked at 180 μg/L into the components of a synthetic urine recipe to determine their individual impacts on extraction and detection. Urea, creatinine, and a complex protein (i.e., gelatin) were found to impair SPE, leading to a low recovery rate of 43∈±∈4% for C ₆₀ spiked into human urine. In contrast, C₆₀ was consistently recovered from synthetic matrices using LLE, and recovery in human urine was 80∈±∈6%. These results suggest that LLE combined with LC-MS is suitable for studying the clearance of fullerenes from the body. LLE is a robust technique that holds promise for extracting C₆₀ from other complex biological matrices (e.g., blood, sweat, amniotic fluid) in toxicological studies, enabling a better understanding of the behavior of fullerenes in human and animal systems and facilitating a more comprehensive risk evaluation of fullerenes.