Herbst, M. Birgitte Koch

Rasmussen, Rie Romme

Sloth, Jens Jørgen

Svendsen, Maja Erecius

Mercury (Hg) is a naturally occurring element, which is found in the earth’s crust and can be released into the environment through both natural and anthropogenic processes. Mercury exists as elemental mercury (metallic), inorganic mercury and organic mercury (primarily methylmercury). Methylmercury is highly toxic, particularly to the nervous system, and the developing brain is thought to be the most sensitive target organ for methylmercury toxicity. Methylmercury bioaccumulates and biomagnifies along the food chain and it is the most common mercury species in fish and seafood. Human exposure to methylmercury is mainly from fish and other seafood consumption. A simple method for the determination of methylmercury in marine based foods and feeds has been developed and in-house validated. The applied HPLC-ICPMS method was inspired by Vallant et al (2007). Samples were extracted with 5 M hydrochloric acid by sonication. Hereby the protein-bound mercury species are released. The extracts were then centrifuged (10 min at 3170 x g) and the supernatant decanted (extraction step was repeated twice). The combined extracts were added 10 M sodium hydroxide to increase pH, following further dilution in the mobile phase and filtering prior to analysis. Analysis of mercury species were performed using HPLC-ICPMS equipped with a MicroMist nebuliser. Typical plasma conditions were 1500 W RF power, 15 l/min, 0.97 l/min and 0.17 l/min for plasma, carrier and makeup gas, respectively, Analysis was performed in the time resolved analysis mode monitoring the 202Hg, 198Hg, 35Cl (m/z) with 1 s (Hg) and 0.01 s (Cl) integration time per data point. Separation of inorganic mercury and methylmercury was obtained on a polymer-based cation-exchange column (150×2.1 mm id, 10 μm) using isocatic elution (0.2 ml/min at 40 °C). The mobile phase (pH~3) consisted of L-cysteine (0.5% w/w), pyridine (50 mmol/L), methanol (5% v/w) and formic acid (0.8% v/w). Total run time 10 min. External calibration standards (0–10 μg/L) were run before and after the samples in order to quantify the methylmercury species by peak height (m/z 202). The methylmercury method was validated by triplicate analysis of certified reference materials (DORM-2, TORT-2 and DORM-3) and 4 other fish and feed samples of marine origin, repeated on 3 different days. The limit of detection and quantification were 0.027 and 0.054 mg/kg, respectively. The limits were calculated as three and ten times the standard deviation at intra-laboratory reproducibility conditions of a natural fortified sample with low content (0.06 mg/kg) divided by average recoveries for certified reference materials. Mean recoveries of the reference materials were 94–102%. The in-house reproducibility standard deviations were less than ≤12% for samples containing 0.15 to 4.47 mg/kg and less than ≤20% for samples with 0.06 mg/kg. Vallant B, Kadnar R and Goessler W (2007) J Anal Atom Spectrom 22, 322–325. Acknowledgement: Funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 211326

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Downloaded from orbit.dtu.dk on: Dec 18, 2017Mercury speciation analysis in marine samples by HPLC-ICPMSRasmussen, Rie Romme; Svendsen, Maja Erecius; Herbst, Birgitte Koch; Sloth, Jens JørgenPublication date:2011Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Rasmussen, R. R., Svendsen, M. E., Herbst, M. B. K., & Sloth, J. J. (2011). Mercury speciation analysis inmarine samples by HPLC-ICPMS. Poster session presented at 5th International Symposium on RecentAdvances in Food Analysis, Prague, Czech Republic.CONffIDENCE: Contaminants in food and feed: Inexpensive detection for control of exposureMercury speciation analysis inmarine samples by HPLC-ICPMSRie R. Rasmussen, Maja E. Svendsen, Birgitte K. Herbst and Jens J. Sloth*National Food Institute, Technical University of DenmarkDivision of Food Chemistry, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark*corresponding author, email: jjsl@food.dtu.dkA simple method for determination of mercury species in marine origin has been developed and in-house validated. It is based on acid extraction by sonification,HPLC separation of inorganic mercury and methylmercury by cation-exchange column using isocratic elution and detection by ICPMS.IntroductionMercury (Hg) exists as elemental mercury (metallic), inorganic mercury and organic mercury (primarily methylmercury). Methylmercury is probably by far the mosttoxic form of mercury in food. It is highly toxic, particularly to the nervous system, and the developing brain is thought to be the most sensitive target organ formethylmercury toxicity. It bioaccumulates and biomagnifies along the food chain and is the most common mercury species in fish and seafood. Human exposure tomethylmercury is mainly from fish and other seafood consumption. Inorganic mercury is probably the predominant form of mercury in foods other than fish andseafood. The methylmercury species in feed and food is currently not regulated by the European Union. Only for total mercury maximum levels have been laiddown. There is a current need for fully standardized methods for determination of methylmercury.Extraction of methylmercuryThe applied method was inspired by Vallent et al (2007). Samples wereextracted twice with 5 M hydrochloric acid by sonication. Hereby the protein-bound mercury species are released. Before re-extraction the samples werecentrifuged (10 min at 3170 x g) and the supernatant decanted. The combinedsample extracts were added 10 M sodium hydroxide to increase pH, diluted inthe mobile phase and filtered.Method performanceThe method has been in-house validated using three different certifiedreference materials (DORM-2, TORT-2 and DORM-3) and four other fish andfeed samples of marine origin (Table 1). All samples were analysed in triplicateand repeated on three different days. Limit of detection (LOD) andquantification (LOQ) were calculated as three and six times the standarddeviation at intra laboratory reproducibility conditions (SDIR) divided byaverage recovery for certified reference material (Rrec), respectively. LOD was0 027 mg/kg and LOQ 0 054 mg/kgDetection of methylmercury . . .The in-house reproducibility standard deviations (RSDIR) were less than ≤12% for samples containing 0.15 to 0.47 mg/kg methylmercury and less than ≤ 20% for samples with 0.06 mg/kg. Table 1. Performance of the HPLC-ICPMS method for determination of methylmercury.Separation of mercury speciesSince the ICP-MS detector is not selective towards specific mercury species,the HPLC separation is crucial for selective determination Separation ofAnalysis of methylmercury were performed using HPLC-ICPMS equipped witha MicroMist nebuliser. Typical plasma conditions were 1500 W RF power, 15l/min plasma, 0.97 l/min carrier and 0.17 l/min makeup gas. Analysis wasperformed in the time resolved analysis mode monitoring the 202Hg, 198Hg, 35Cl(m/z) with 1 s (Hg) and 0.01 s (Cl) integration time per data point.DORM-2 TORT-2 DORM-3 Fish feed spiked Codfish Salmon Fish feedRef. level (mg/kg) 4.47 0.15 0.36 0.21 0.17 0.06 0.06 Observations (N) 9 15 9 9 9 9 9 .inorganic mercury and methylmercury was obtained on a polymer-basedcation-exchange column (Hamilton PRP-X200,150 x 2.1 mm id, 10 µm) usingisocratic elution (0.2 ml/min at 40º C). The mobile fase (pH3) consisted of L-cysteine (0.5% w/w), pyridine (50 mmol/L), methanol (5% v/w) and formic acid(0.8% v/w) dissolved in water. Separation of the inorganic mercury and methyl-mercury can be seen in Figure 1.Methylmercury was quantified using peak height (m/z 202) and externalcalibration standards (0-10 µg/l) which were run before and after the samples.Fish feedThe method was applied to fish feed in use in Denmark. In total 23 samples(4 complete fish feed products, 5 fish silage samples and 14 fish mealsamples) were collected by the Danish Plant Directorate. All samples werekept at 18C Samples (except fish silage) were homogenised before analysisMean recovery (%) 94 102 96 - - - -Repeatability RSDr (%) 3 4 3 11 5 13 13 Reproducibility RSDIR (%) 8 12 8 11 12 20 15 1000150020002500Response (m/z 202)Methylmercury (CH3‐Hg+)Inorganic mercury (Hg2+)- . .Certified reference material was used for quality control. The results can beseen in Table 2. For all samples the concentrations were below the EUmaximum levels for total mercury in fish feed at 0.2 mg/kg and marine feedmaterials at 0.5 mg/kg (EU dir 2010/6/EC).Feed sample type CH3-Hg+ (µg/kg) Feed sample type CH3-Hg+ (µg/kg)Fish Silage<0.027 <0.027<0.027 <0.027<0.027 <0.027Table 2. Analysis of feed samples.05000 1 2 3 4 5 6 7 8 9 10Retention time (min)Figure 1. HPLC-ICP-MS chromatogram from extract of a TORT-2 sample (0.15 mg/kg methylmercury). The two mercury species inorganic mercury and methylmercury have different retention times. Fish meal<0.027 <0.027<0.027 <0.027Complete feed<0.027 <0.027<0.027 0.030<0.027 0.0320.032 0.0450.0530.0530.0550.0790.125LOD = 0.027 mg/kg, LOQ = 0.054 mg/kgThe research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 211326.Future use and impactThe method present a simple approach for quantification of methylmercury in food and feed of marine origin.Reference. Vallant B, Kadnar R and Goessler W (2007). Journal of Analytical Atomic Spectrometry 22, 322–325.

Mercury speciation analysis in marine samples by HPLC-ICPMS

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Mercury speciation analysis in marine samples by HPLC-ICPMS

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