Evaluation of extraction methods for quantification of aqueous fullerenes in urine

Abstract

There is a growing concern about the human and environmental health effects of fullerenes (e.g., C(60)) 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 C(60) in human and synthetic urine as compared with ultrapure water. Glacial acetic acid, which is necessary to prevent emulsions during LLE, inhibited C(60) detection by LC-MS, but this could be mitigated with evaporation. Aqueous C(60) aggregates (nC(60)) 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, C(60) 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 C(60) 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.

Fig. 1

Interference of the extraction solution on the detection of C₆₀ using LC–MS. Blank samples of water, synthetic urine, and human urine were subjected to SPE and LLE. C₆₀ was spiked into the extraction solutions at 240 µg/L. Detection of C₆₀ spiked into LLE matrices was inhibited by approximately 45%. Error bars represent three LC–MS analyses of one sample

Fig. 2

Impact of LLE solutions on the detection of C₆₀ using LC– MS. a The four columns on the left represent the detection of a C₆₀-toluene standard (240 µg/L final conc.) spiked into the extracted toluene phase of the various components of the LLE protocol. Mg (ClO₄)₂ had a marginal impact on detection of C₆₀, whereas the presence of acetic acid lowered the signal by approximately 50%. Error bars represent three LC–MS analyses of one sample. b nC₆₀ was spiked into water and synthetic urine prior to LLE. The recoveries of nC₆₀ without (white) and with (blue) evaporation of the toluene extract indicate the importance of removing acetic acid from the toluene sample to mitigate signal suppression

Fig. 3

Recovery of C₆₀ from synthetic and human urine matrices using LLE and SPE. C₆₀ was spiked to a final concentration of 180 µg/L and allowed to equilibrate in the media overnight. Error bars indicate the variability in quantification between three extractions of each sample matrix