Experimental design optimisation and validation by accuracy profile of a novel method for Hg speciation analysis by...

This study addresses the development and validation of an analytical method for speciation analysis of mercury (inorganic/Hg²⁺ and methylmercury/CH₃Hg⁺) in fishery products. The Hg species are separated by reversed-phase (RP) high-performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS). The effective separation of Hg²⁺ and CH₃Hg⁺ was achieved in <8 min using a peptide mapping RP column and a mobile phase containing 2-mercaptoethanol at 0.25% (v/v) and methanol at 1% (v/v). The optimization was carried out using an experimental design through response surface methodology (RSM) with central composite design (CCD), addressing both the HPLC separation and the sample extraction. The method validation was carried out based on the accuracy profile approach. For this purpose, six series of measurements were carried out in duplicate over a time span of 2 months. The limits of quantification (LOQ) were 2.5 µg/kg (wet weight, ww) for CH₃Hg⁺ and 1.2 µg/kg (ww) for Hg²⁺. The intermediate reproducibility in terms of coefficient of variation (CV_R) was <6%. The bias (%) obtained for the analysis of four certified reference materials (CRMs), namely TORT-3 (lobster hepatopancreas), SRM 1566-b (oyster tissue), SQID-1 (cuttlefish) and NMIJ CRM 7402-a (cod fish tissue) was <7%. This demonstrates the method robustness and suitability for routine speciation analysis of CH₃Hg⁺ and Hg²⁺ in fishery products. The method is intended to be applied for the analysis of the panel of fishery products and fish-based foods in the framework of the (ongoing) third French Total Diet Study.