Retention behavior of Hg²⁺, MeHg⁺, thimerosal and phenylmercuric acetate on a C₁₈ RP-HPLC column

Humans are exposed to potentially toxic mercuric mercury (Hg²⁺) 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 Hg²⁺ 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 Hg²⁺, 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, Hg²⁺, 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.