Retention behavior of Hg, MeHg, thimerosal and phenylmercuric acetate on a C RP-HPLC column.

Humans are exposed to potentially toxic mercuric mercury (Hg) and methylmercury (MeHg) by the ingestion of food, to the bactericidal vaccine additive thimerosal (THI), and/or to the antifungal compound phenylmercuric acetate (PMA) which is used in some lens cleaning ophthalmic fluids. While numerous HPLC methods have been developed to separate Hg and MeHg in environmental samples (e.g. food, surface waters), comparatively few have been reported for THI and PMA, in part owing to their increased hydrophobicity. We investigated the retention behavior of Hg, MeHg, THI and PMA on a reversed-phase (RP) HPLC column using a flame atomic absorption spectrometer (FAAS) as a Hg-specific detector. Mobile phases comprised of 50 mM phosphate buffer (pH 7.4) with acetonitrile (ACN) concentrations of 30-50 % (v:v) produced single Hg-peaks, which eluted in the order THI, Hg, MeHg and PMA. With the 50 % ACN mobile phase, all mercurials eluted within 5 min. While the utilization of a FAAS precludes the analysis of environmental waters with the developed RP-HPLC-FAAS method, the latter is useful to probe the stability of THI and PMA in the presence of physiologically relevant concentrations of salt (100 mM in blood plasma) and l-cysteine (0.5 mM in hepatocyte cytosol), which is important as both mercurials have been recently shown to effectively inhibit the main protease of SARS-CoV-2, though the actual inhibitory Hg-species is unknown.