Ion-pairing reversed-phase chromatography coupled to inductively coupled plasma mass spectrometry as a tool to determine mercurial species in freshwater fish.

Most of analytical community is focused on reversed phase high performance liquid chromatography (RP-HPLC) for mercury speciation by employing mobile phases comprising of high salts and moderate amounts of organic solvents. This study aims at rapid mercury speciation analysis by ion-pairing RP-HPLC with inductively coupled plasma mass spectrometry (ICP-MS) detection only using low salts for the sake of green analytical chemistry. Two ion-pairing HPLC methods were developed on individual usage of positively and negatively charged ion-pairing reagents (tetrabutylammonium hydroxide -TBAH and sodium dodecylbenzene sulfonate -SDBS), where sodium 3-mercapto-1-propysulfonate (MPS) and l-cysteine (Cys) were individually added in mobile phases to transform mercury species into negative and positive Hg-complexes for good resolution. Addition of phenylalanine was also utilized for rapid baseline separation in combination of short C18 guard columns. Optimum mobile phases of 2.0mM SDBS+2.0mM Cys+1.0mM Phe (pH 3.0) and 4.0mM TBAH+2.0mM MPS+2.0mM Phe (pH 6.0) both achieved baseline separation of inorganic mercury (Hg2+ ), methylmercury (MeHg), ethylmercury (EtHg) and phenylmercury (PhHg) on two consecutive 12.5-mm C18 columns. The former mobile phase was selected for mercury speciation in freshwater fish because of short separation time (3.0min). Detection limits of 0.015 for Hg2+ , 0.014 for MeHg, 0.028 for EtHg and 0.042μgL-1 for PhHg were obtained along with satisfactory precisions of peak height and area (1.0-2.8% for 5.0μgL-1 Hg-mixture standard). Good accordance of determined values of MeHg and total mercury in certified reference materials of fish tissue (GBW 10029) and tuna fish (BCR-463) with certified values as well as good recoveries (91-106%) proved good accuracy of the proposed method. An example application to freshwater fish indicated its potential in routine analysis, where MeHg was presented at 3.7-20.3μgkg-1 as the dominate species.