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., 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 C60 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.

Liquid chromatography–mass spectrometry for C60 fullerene analysis: optimisation and comparison of three ionisation techniques

“…A lower IDL is probably achievable under our study conditions when a larger injection volume is used, as reported in some earlier studies [16,20]. This is also consistent with the results reported by Benn et al [17].…”

“…LC-MS has already been used to some extent to detect fullerenes in a variety of matrices. Before mass detection, the fullerenes have been ionised with atmospheric-pressure chemical ionisation (APCI) [14][15][16][17][18], atmospheric-pressure photoionisation (APPI) [15,19], and electrospray ionisation (ESI) [18,20,21].…”

Beyond nC60: strategies for identification of transformation products of fullerene oxidation in aquatic and biological samples

“…The typical method for C 60 extraction exploits the high solubility of C 60 in toluene, with addition of counter-ions to destabilize the n C 60 particles for quantitative extraction of pristine fullerene and its epoxide. In addition, solid-phase extraction of fullerenes from water has been conducted for samples of environmental and biological origin [106, 107]. HPLC–MS and HPLC–UV–visible strategies have been successfully used to measure the fullerenes content of a variety of matrices [18, 64, 96].…”

“…For detection and quantification of C 60 fullerenes, MS-based detection is more specific than UV-based detection, because it measures the mass-to-charge ratio and, in the case of tandem MS, also yields structural information; it is, generally, also more sensitive. However, strong absorbance of UV by fullerenes results in detection limits comparable with those of atmospheric pressure chemical ionization (APCI)-MS and electrospray ionization (ESI)-MS; tandem MS is even more sensitive [45, 96, 106]. Sensitive detection of fullerenes by UV–visible spectroscopy was achieved for spiked water samples (method detection limit, MDL = 0.02 μg mL −1 ), but this is very likely to be insufficient to reveal exposure at the currently expected levels of contamination.…”

“…Solid-phase extraction techniques also have been successfully used for fullerenes [19, 106, 125]. Here, different sorbents could be combined with use of differential elution with solvents of staggered polarity to obtain fractions enriched in chemical targets to serve as starting points for further functionalization and/or defunctionalization strategies.…”