Development of an ambient temperature post-column oxidation system for high-performance liquid chromatography on-line coupled with cold vapor atomic fluorescence spectrometry for mercury speciation in seafood

Abstract

An efficient ambient temperature post-column oxidation system was developed for high-performance liquid chromatography online coupled with cold vapor atomic fluorescence spectrometry for mercury speciation analysis in seafood. The developed system does not require an external heat source, such as a hot water bath, microwave heating or UV irradiation, to decompose organomercury compounds into inorganic mercury ion (Hg(II)). Methylmercury (MeHg), ethylmercury (EtHg), phenylmercury (PhHg) and Hg(II) were baseline separated on an RP-C₁₈ column with a mixture of methanol, acetonitrile and water (75 ∶ 5 ∶ 20) containing 0.01% m/v ammonium pyrrolidine dithiocarbamate (APDC) (pH 3.5) as the mobile phase. Online ambient temperature post-column oxidation of organomercury species eluted from the HPLC column was achieved by mixing the effluent with a 3% m/v K₂S₂O₈ in 10% v/v HCl solution in a knotted reactor made of a 0.5 mm id × 200 cm long PTFE tube. The developed oxidation approach enhanced sensitivity for organomercury by a factor of 4 for MeHg, 3 for EtHg and 6 for PhHg, compared with that obtained without post-column oxidation. The detection limits for 100 μl injection were 0.19–0.27 μg l⁻¹ (as Hg) in extract, corresponding to 4.75–6.75 μg kg⁻¹ (as Hg) in fish tissue for a 50 ml final extract of 2 g tissue. The precision (RSD, n = 5) ranged from 1.8 to 2.8% for peak area, and from 1.7% to 2.9% for peak height at the 1 μg l⁻¹ (as Hg) level. The method was validated by determination of methylmercury in a certified reference material (DORM-2, dogfish muscle) and applied to the speciation analysis of mercury in local seafood samples.