Given the severe health risks posed by mercury (Hg), especially the neurotoxic effects of methylmercury (MeHg) and recognizing the urgent demand for improved mercury analysis across food samples, especially seafood. We herein used the method described by Hight & Cheng (2006), with some modifications. The method combines ultrasonication-assisted extraction with robust reversed phase chromatography-inductively coupled plasma mass spectrometry (RP-LC-ICP-MS). Through this technique, we achieved the separation of MeHg from inorganic Hg (iHg) in less than 3 minutes using a C-18 Kinetex 2.6 µm C18 100 Å column (100 x 4.6 mm), following ultrasonic extraction using L-cysteine·HCl·H2O as the extractant agent. Total Hg content was calculated as the sum of MeHg and iHg.

Validation of our method included analysis of three certified reference materials (CRMs) and three marine-derived samples. Accuracy of MeHg values was consistent with the certified values at 95% confidence level and the recoveries ranged from 94- 100.8% for all samples. We ensured method reliability through triplicate measurements by two operators, yielding relative standard deviations under repeatability conditions (RSD_r) between 1.1- 9.4% and in-house reproducibility conditions (RSD_IR) ranging from 1.7- 12.3%. The MeHg detection limits (LODs) were 0.09 μg/kg and 0.34 μg/kg, while the quantification limits (LOQs) were 0.29 μg/kg and 1.14 μg/kg in edible seafood (fresh, dilution factor 50) and in lyophilized reference materials (dried, dilution factor 200), respectively.

The developed method was proved to be highly sensitive and suitable for routine analysis, aligns with green analytical chemistry principles, featuring reduced toxic waste, safer HPLC mobile phases, and shorter separation times, thus cutting operational costs. Finally, the developed method was applied to 26 seafood samples of different species collected by the Danish Veterinary and Food Administration as part of their annual control program. The concentrations for total Hg and MeHg ranged from 0.002-0.186 and 0.001-0.175 mg/kg, respectively. The percentage of MeHg in relation to total Hg varied from 35 to 97 %, with the lowest proportion of MeHg found in mussel samples. This demonstrated that total Hg is not in all cases a good proxy for MeHg. This enhances our understanding of Hg distribution in various aquatic species, hence contributing to more reliable health risk evaluations.

Acknowledgement: This research project has received funding from the European Union’s HORIZON-CL4-2022 research and innovation program under grant agreement ID 101093042, PHOTONGATE project.

References:

Hight, S. C., & Cheng, J. (2006). Determination of methylmercury and estimation of total mercury in seafood using high performance liquid chromatography (HPLC) and inductively coupled plasma-mass spectrometry (ICP-MS): Method development and validation. Analytica Chimica Acta, 567(2), 160–172. https://doi.org/10.1016/j.aca.2006.03.048