Mercury speciation in edible seaweed by liquid chromatography - Inductively coupled plasma mass spectrometry after ionic imprinted polymer-solid phase extraction

Talanta Mercury speciation in edible seaweed by liquid chromatography - inductively coupled plasma mass spectrometry after ionic imprinted polymer-solid phase extraction --Manuscript Draft-- Manuscript Number: TAL-D-20-02796R1 Article Type: Research Paper Section/Category: Atomic Spectroscopy Keywords: Ionic imprinted polymer; solid-phase extraction; mercury speciation; edible seaweed Corresponding Author: Antonio Moreda-Pineiro, Ph.D Universidade de Santiago de Compostela Santiago de Compostela, A Coruña SPAIN First Author: Antonio Moreda-Pineiro, Ph.D Order of Authors: Antonio Moreda-Pineiro, Ph.D Kamal K. Jinadasa Paloma Herbello Hermelo Elena Peña-Vázquez Pilar Bermejo-Barrera Abstract: In contrast to most of essential and heavy metals, mercury levels in seaweed are very low, and pre-concentration methods are required for an adequate total mercury determination and mercury speciation in this foodstuff. An ionic imprinted polymerbased solid phase extraction (on column) pre-concentration procedure has been optimized for mercury species enrichment before liquid chromatography hyphenated with inductively coupled plasma mass spectrometry determination. The polymer has been synthesised by the precipitation polymerization method and using a ternary prepolymerization mixture containing the template (methylmercury), a non-vinylated monomer (phenobarbital), and a vinylated monomer (methacrylic acid). Factors affecting the adsorption/desorption of Hg species (extract pH, loading and elution flow rates, volume of eluent, etc.), and parameters such as breakthrough volume and reusability, were fully studied. Mercury species were first isolated from seaweed by ultrasound assisted extraction using a 0.1% (v/v) HCl, 0.12% (w/v) L-cysteine, 0.1% (v/v) mercaptoethanol solution. Under optimized conditions, the limits of detection were 0.007 and 0.02 µg kg-1 dw for methylmercury and Hg(II), respectively. The preconcentration factor (volume of 10 mL of seaweed extract) was 50. Repeatability and reproducibility of the method were satisfactory with relative standard deviations lower than 16%. The proposed methodology was finally applied for the selective preconcentration and determination of methylmercury and Hg (II) in a BCR-463 certified reference material and in several edible seaweeds. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Highlights: -Selective pre-concentration of mercury species by using an ionic imprinted polymer - Trace levels of inorganic mercury and methylmercury are assessed in edible seaweed - Robust and low-cost pre-concentration procedure for total mercury assessment and for mercury speciation Highlights (for review) IIP-SPE 0 500 1000 1500 2000 2500 3000 0 2 4 6 8 Intensity (cps) Time (min) MeHg Hg (II) UAE Graphical Abstract (for review) 1 Mercury speciation in edible seaweed by liquid chromatography - inductively coupled plasma mass spectrometry after ionic imprinted polymer-solid phase extraction Kamal K. Jinadasa, Paloma Herbello Hermelo, Elena Peña-Vázquez, Pilar Bermejo-Barrera, Antonio Moreda-Piñeiro* Trace Element, Spectroscopy and Speciation Group (GETEE), Strategic Grouping in Materials (AEMAT), Department of Analytical Chemistry, Nutrition and Bromatology. Faculty of Chemistry. Universidade de Santiago de Compostela. Avenida das Ciencias, s/n. 15782, Santiago de Compostela. Spain ∗ Corresponding author e-mail: [email protected] Abstract In contrast to most of essential and heavy metals, mercury levels in seaweed are very low, and pre-concentration methods are required for an adequate total mercury determination and mercury speciation in this foodstuff. An ionic imprinted polymer-based solid phase extraction (on column) pre-concentration procedure has been optimized for mercury species enrichment before liquid chromatography hyphenated with inductively coupled plasma mass spectrometry determination. The polymer has been synthesised by the precipitation polymerization method and using a ternary pre-polymerization mixture containing the template (methylmercury), a non-vinylated monomer (phenobarbital), and a vinylated monomer (methacrylic acid). Factors affecting the adsorption/desorption of Hg species (extract pH, loading and elution flow rates, volume of eluent, etc.), and parameters such as breakthrough volume and reusability, were fully studied. Mercury species were first isolated from seaweed by ultrasound assisted extraction using a 0.1% (v/v) HCl, 0.12% (w/v) L-cysteine, 0.1% (v/v) mercaptoethanol solution. Under optimized conditions, the limits of detection were 0.007 and 0.02 µg kg-1 dw for methylmercury and Hg(II), respectively. The pre-concentration factor (volume of 10 mL of Revised Manuscript [Unmarked Text] 2 seaweed extract) was 50. Repeatability and reproducibility of the method were satisfactory with relative standard deviations lower than 16%. The proposed methodology was finally applied for the selective pre-concentration and determination of methylmercury and Hg (II) in a BCR-463 certified reference material and in several edible seaweeds. Keywords Ionic imprinted polymer, solid-phase extraction, mercury speciation, edible seaweed 1. Introduction Seaweed (benthic marine algae or macroalgae) are a source of food for humans since ancient times, and they have also been used in medicine and as animal fodder [ 1 ]. Seaweed can be classified into three main groups according to their dominant pigmentation: red (Rhodophyta), brown (Phaeophyta), and green (Chlorophyta) seaweed. According to the Food and Agriculture Organization (FAO) statistics, world seaweed mariculture production reached 24.9 million tons (valued about six billion USD) in 2014 [ 2 ]. As a food source, edible seaweed are rich in polysaccharides, proteins, dietary fibre, polyunsaturated fatty acids, vitamins (A, C, B2 and B12), iodine, and minerals (magnesium, sodium, and iron) [ 3 , 4 ]. Additionally, seaweed are used as low-calorie food (body weight control), and their consumption has been reported to prevent cancer, and gastrointestinal and cardiovascular diseases [4, 5 ]. Another important application of seaweed is the production/extraction of derivatives such as agar, carrageenan, alginate, and bio-active compounds [5]. However, some studies are showing the safety hazard of seaweed due to their content of non-essential trace metals (Hg, Cd, Pb, As, etc.), radioactive isotopes, dioxins, and pesticides [ 6 - 8 ]. 3 Mercury (Hg) is a highly toxic element that can be bio-accumulated and biomagnified through the food chain, especially in the marine environment [ 9 ]. The World Health Organization (WHO) has classified Hg as “one of the top ten chemicals or groups of chemicals of major public health concern” [ 10 ], and the Agency for Toxic Substances and Disease Registry (ATSDR) of the United States (US) ranked Hg on the third place of their substance’s priority list [ 11 ]. The sources of Hg in the aquatic environment are several natural processes such as volcanism, weathering of rocks and degassing of the earth's crust; and several anthropogenic activities (e.g. coal combustion, mining industry and by-products, use of agriculture fertilizers, and waste incineration) [ 12 , 13 ]. The toxicity of Hg is related to the chemical form, and the path of entry into the organism. As an example, methylmercury (MeHg) has the greatest impact in the digestive tract, and it has shown the ability to affect the nervous system [ 14 ]. The levels of heavy metals in seaweed depend on several factors i.e. pH, salinity, presence of complex organic-inorganic molecules, temperature, light irradiation, oxygen, and nutrient concentration. According to the published scientific data, Hg concentration in seaweed varies with the type of seaweed and sampling location, and in contrast to other non-essential metals, Hg levels in seaweed are very low [ 15 ]. Therefore, many investigations have reported the Hg content as total Hg concentration (tHg), and the levels were frequently below the limits of detection of most of conventional instrumental techniques [6, 16 - 18 ]. An adequate sample pre-treatment is very important when assessing ultra-trace levels of Hg (and Hg species), and accurate quantification of Hg and/or mercury species in seaweed usually requires a pre-concentration technique before instrumental analysis. Microwave assisted acid digestion has been typically used for seaweed solubilisation when total Hg is assessed [ 19 - 22 ]. Other authors have performed alkaline treatments by using aqueous KOH or KOH/methanol for MeHg isolation [ 23 - 25 ] when applying the US Environmental Protection Agency (USEPA) 1630 method [ 26 ] with cold vapour – atomic fluorescence spectrometry (CV-AFS) quantification (total mercury assessment, MeHg assessment, and inorganic Hg by difference) 4 [24,25]. On other occasions, organic solvents such as toluene [ 27 ] and HCl/toluene mixtures [ 28 ] have been proposed, although several additional stages (back extraction step with sodium thiosulfate and a final oxidation of MeHg with acidified BrCl for total Hg determination by CV-AFS) are required [27]. Few developments of hyphenated techniques can be found in the literature, and the speciation method by Brombach et al. [23] consists of CV-AFS coupling with liquid chromatography (LC) and a sample two-stages pre-treatment (microwave assisted extraction with aqueous KOH, followed of a treatment with concentrated HCl) for guaranteeing a quantitative extraction of mercury species. In order to pre-concentrate and/or cleaning the extracts/digests from seaweed, some procedures, mainly based on solid phase extraction (SPE), have been proposed. Because of the great affinity of mercury ions for thiol groups, sulfydryl-based absorbents such as laboratory made sulfydryl cotton fiber (SCF) for total Hg determination by cold vapour atomic absoroption spectrometry (CV-AAS) [19], and thiol-thiourea on silica by LC-CV-AFS [23] have been used. Molecularly imprinted polymers (MIPs) and ionic imprinted polymers (IIPs) are promising absorbents for selective pre-concentration, matrix removal, and medium exchange [ 29 ], and some few applications have been developed for Hg determination/speciation [ 30 ]. Selective MIP-based SPE has been applied for pre-concentrating mercury species from seawater before LC-inductively coupled plasma – mass spectrometry (ICP-MS) [ 31 ], although most of MIP/IIP applications have been focused on total Hg determination or the assessment of a specific species (MeHg) in fish [20,21,28], wine [ 32 ], human hair [21,22], and human serum [ 33 ]. Similarly, IIPs have been also used for developing selective electrodes for potentiometric/voltammetric determination of total mercury in freshwater [ 34 , 35 ]. However, to the best of authors’ knowledge, there are not developments of MIPs/IIPs for ultra-trace pre-concentration of Hg species from extracts from complex materials such as seaweed. In this work, a selective IIP for mercury species has been synthesized by the precipitation polymerization method, and a on column pre-concentration method combined 5 with LC-ICP-MS has been optimized for assessing low concentrations of Hg(II) and MeHg in edible seaweeds. 2. Materials and methods 2.1. Reagents Methylmercury stock solution (1000 mg L-1) was prepared from methylmercury chloride from Sigma Aldrich (St. Louis, MO, USA). This reagent was also used when preparing the IIP (MeHg as a template). The Hg (II) stock solution (1000 mg L-1) was from Scharlab (Barcelona, Spain). Working standard solutions of MeHg and Hg (II) were prepared daily by appropriate dilution of the stocks. Methacrylic acid (MA), ethylene glycol dimethacrylate (EGDMA), and phenobarbital sodium salt (Sigma Aldrich), 2,2′-azobis(2-methyl propionitrile) (AIBN) from Fluka (Steinheim, Germany), and acetonitrile (Merck, Darmstadt, Germany) were used for IIP synthesis. The ammonium chloride/ammonium hydroxide (NH4Cl/NH4OH) buffer solution was prepared from NH4Cl and NH4OH from Merck. Multi-element standard solutions for the cross-reactivity study were prepared by combining single As, Ca, Co and Mg stock standard solutions (1000 mg L-1) from Merck, single Cr, K, P, Pb and Zn stock standard solutions (1000 mg L-1) from Scharlab, and single Cd, Cu, Fe and Na stock standard solutions (1000 mg L-1) from Perkin Elmer (Shelton, CT, USA). Internal standard solutions (Ge, Sc, and Rh) were prepared from single-element standards (1000 mg L-1) purchased from Perkin Elmer. Other reagents were hydrochloric acid (37%), nitric acid (Hyper pure, 69%), and 33% of hydrogen peroxide from Panreac (Barcelona, Spain), ammonia solution from Merck, thiourea from Sigma Aldrich, and ultrapure water (18.2 MΩ/cm resistivity) from a Milli-QA10 water purification system (Millipore Co., Bedford, MA, USA). Due to the non-availability of Hg certified reference material for Hg species in seaweed, a tuna fish certified reference material 6 (BCR-463) from the European Commission Joint Research Centre, Institute for Reference Materials and Measurements (Geel, Belgium) was used to evaluate the accuracy of the method. To avoid contamination with Hg throughout the study, all glassware and plastic-ware were thoroughly rinsed with ultrapure water, soaked 2 days in 10% (v/v) nitric acid, and finally rinsed several times with ultrapure water. 2.2. Instrumentation A Perkin Elmer Nex-Ion 300X ICP–MS (Waltham, MA, USA) coupled with a Flexar LC (LC pump, column oven, and LC autosampler) from Perkin Elmer, was used for Hg speciation. A Kinetex C-18 100 Å analytical column (100 mm × 2.10 mm, 5 µm particle diameter) connected with a C-18 guard column from Phenomenex (Torrance, USA) was used for reverse-phase chromatographic separation. Polymerization was performed in a Boxcult temperaturecontrolled chamber (Stuart Scientific, Surrey, UK), with the support of a low-profile roller (Stovall, Greensboro, NC, USA). The IIP sorbent was packed into 5 mL syringes (Dispomed Witt OHG, Gelnhausen, Germany) between two Teflon frits (Supelco, Bellefonte, PA, USA). SPE was performed by using an 8 channel Minipuls 3 (Gilson, Middleton, WI, USA) peristaltic pump with 2-stop PVC tubing (1.52 mm i.d.) from SCP Sciences (Baie-D'Urfe, Quebec, Canada). IIPs characterization was performed by Fourier transform infrared spectrometry (FTIR) using a Spectrum-Two FT-IR (Perkin Elmer). Other general instrumentation such as an USC60TH ultrasonic cleaner bath (45 kHz, 120 W) from VWR (Leuven, Belgium), a 2K15 ultracentrifuge (Sigma, Osterode, Germany), a Basic 20 pH meter Crison, Barcelona, Spain), a vibrating zircon ball mill (Retsch, Haan, Germany), an oven model 207 (Selecta, Barcelona, Spain), and a Classic ML analytical balance (Mettler Toledo, Columbus, OH, USA) were used throughout this research. 2.3. Preparation of IIPs IIPs were synthesized following a three step-procedure developed by Rodríguez-Reino et al. [31], with minor changes. To prepare the pre-polymerization mixture, 0.053 g of CH3ClHg, 13 graphs and the slopes of aqueous standard calibration graphs (p>0.05), which means that the matrix effect is negligible. Therefore, determinations can be performed by using aqueous calibration and the equations (mean values and standard deviation for the slope), as well as the calibration range and correlation coefficients, are given in Table 3. The lower concentration in the calibration range is the instrumental limit of quantification (expressed as µg L-1) which is discussed in the further section. The EU has established that a correlation coefficient higher than 0.9980 is required to obtain satisfactory linearity using confirmatory methods [ 39 ]. Acceptable linearity was obtained for the aqueous calibration and standard addition curves for both Hg (II) and MeHg (correlation coefficients higher than 0.999). 3.5. Limit of detection and limit of quantification The limit of detection (LOD) was calculated as 3 times the standard deviation (3 σ) of eleven replicate measurements of the blank sample, while the limit of quantification (LOQ) was calculated as 10 times the standard deviation (10 σ). Therefore, eleven reagent blank samples were prepared and treated as described in section 2.5, and the analytical responses were then expressed as concentrations dividing by the mean slope of the calibration graph. LOD and LOQ values referred to the mass sample (seaweed) were calculated after considering the preconcentration factor of 50 of the IIP-based SPE process. The calculated instrumental LOD and LOQ were 0.007 and 0.02 μg L-1 for MeHg and Hg (II), respectively; whereas, LOQs were 0.02 and 0.07 μg L-1 for MeHg and Hg (II), respectively. Taking into account the extraction procedure for isolating the mercury species from the solid seaweed, the LODs and LOQ of the method (expressed as µg kg-1 dw, dried weight) are listed in Table 3. These LODs (Table 3) are much better than some published LODs for Hg assessment in seaweed such as 0.120 µg kg-1 for MeHg by pre-concentration and LC-CV-AFS [23], 1.3 µg kg-1 for MeHg in cyanobacteria [ 40 ], and 0.01 mg kg-1 dw for MeHg using an automatic Hg analyzer [ 41 ]. The LOD obtained 14 by Morrison et al. [24] was 0.435 µg kg-1 ww using the 1630 USEPA method, while the LOD for biota obtained by Shoham-Frider et al. [27] was 0.07 µg kg-1 dw. On the other hand, the Regulation No 464/2018 of the European Parliament and the Council establishes the maximum residue level (MRL) for Hg in algae, prokaryotic organisms, and food products based on seaweed as 0.01 mg kg-1 [ 42 ], value much higher than the LOD found in the present study which demonstrates the applicability of the proposed method edible seaweed analysis. The sensitivity of the proposed method is similar or even better than those reported by other authors when using SPE methods based on MIPs/IIPs for mercury speciation in foodstuff [21,28, 43 ] and also for potentiometric determination of Hg based on IIP modified carbon electrodes [34, 44 ] (Table 4). 3.6. Repeatability, reproducibility, and accuracy Reproducibility (inter-day assay) and repeatability (intraday assay) were studied using seaweed extracts spiked with MeHg and Hg (II) at different concentration levels. Inter-day assay was performed by spiking seven seaweed extracts at three concentration levels of MeHg (0.02, 0.1, 0.4 μg L-1; i.e. concentrations of 1, 5, and 20 μg L-1after pre-concentration), and three Hg (II) concentration levels (0.04, 0.2, 1 μg L-1; i.e., 2, 10, 50 μg L-1 after pre-concentration) and measuring the seven replicates of each concentration level in the same day. Intraday assay was performed by preparing seven standard addition curves in seven different days by spiking in triplicate several seaweed extract aliquots at five concentration levels of MeHg (1, 2, 5, 10 and 20 μg L-1 after pre-concentration) and Hg (II) (2, 5, 10, 20 and 50 μg L-1 after preconcentration). As it can be observed in Table 5, good analytical recovery and precision is obtained since all analytical recoveries ranged between 89-112% for MeHg and 86-108% for Hg (II), and the relative standard deviations (RSDs) were lower than 20% (13% for MeHg and 16% for Hg (II) for all concentration levels). 15 In addition to the analytical recovery, accuracy of the developed method was also tested by analysing a BCR 463 (tuna fish) CRM (a CRM for total Hg and/or Hg species in seaweed is not commercially available). After BCR 463 UAE and IIP-based SPE (section 2.4 and 2.5) and ICP-MS determination, a total Hg content of 3.01±0.06 mg kg-1 was obtained, which is in good agreement with the certified value (2.85±0.16 mg kg-1). LC-ICP-MS analysis gave a Hg(II) concentration of 0.01±0.001 mg kg-1, and an MeHg concentration of 2.86±0.05 mg kg-1. The found MeHg concentration is in good agreement with certified MeHg content in BCR 463 (3.01±0.06 mg kg-1). In addition, the total Hg concentration as a sum of Hg(II) and MeHg concentrations after LC-ICP-MS (2.87±0.07 mg kg-1) also agrees with the certified total Hg content in BCR 463 (2.85±0.16 mg kg-1). 3.8. Applications Three edible seaweed samples were subjected to the optimised IIP-based SPE after UAE extraction and before LC-ICP-MS (Hg(II) and MeHg assessment) and ICP-MS (total Hg assessment) analysis. Results are given in Table 6 and it can be observed that the results obtained by LC-ICP-MS (sum of the species) are in good agreement with the total Hg concentration levels measured directly in the pre-concentrated eluates by ICP-MS. Hg (II) is the major species in the tested seaweed sample, and the highest Hg (II) content was recorded in sea spaghetti species (0.11±0.02 mg kg-1 dw). Conclusion Ionic imprinted polymer based on the interaction between MeHg (template) and phenobarbital (complexing agent) has found to offer excellent recognition capabilities for MeHg and Hg(II). The IIP-based SPE procedure results robust since large sample volumes (seaweed extracts) can be loaded without impairment of the analytical performances. High pre-concentration factors can be therefore achieved, which implies quite sensitive determinations. The prepared material 16 has demonstrated a large absorption capacity and stability, and each 200 mg portions can be reused at least fifteen times (fifteen absorption/desorption cycles). The optimized IIP-based SPE combined with LC-ICP-MS allows low limits of detection, and the methodology can be successfully applied for quantifying mercury species (MeHg and Hg(II)) at very low levels in complex samples such as seaweeds. Acknowledgment This work was supported by the Dirección Xeral de I + D –Xunta de Galicia: Grupos de Referencia Competitiva, project number ED431C2018/19; and Development of a Strategic Grouping in Materials – AEMAT, grant ED431E2018/08. Figures’ captions Figure 1: FT-IR spectra of IIP (before and after template removal) and NIP Figure 2: LC-ICP-MS chromatograms for a 5.0 µg L-1 Hg(II) and 2.0 µg L-1 MeHg aqueous standard (a), and a pre-concentrated extract from a Sea-spaghetti sample (b) Figure 3: Influence of pH of the seaweed extract (a), loading flow rate (b), elution flow rate (c), and elution volume (d) on the IIP-based SPE of Hg(II) and MeHg Figure 4: Effect of the sample volume (breakthrough volume of the IIP-based SPE procedure) on the analytical recovery of Hg(II) and MeHg (a), and analytical recovery of Hg(II) and MeHg after several loading/elution cycles (reusability of each IIP portion) (b) 17 Table 1: Operating ICP-MS conditions for total Hg determination and cross reactivity studies and operating LC-ICP-MS conditions for Hg speciation Operating ICP-MS conditions Radiofrequency power 1600 W Ar flow rate (L min–1) Nebulization 0.92 Auxiliary 1.2 Plasma 16 O2 flow rate (L min–1) 0.01a Standard mode Ca, Cu, K, Mg, Na, P KED collision mode; He flow rate 4.0 mL min-1 (As, Cd, Co, Cr, Fe, Hg, Pb, Zn) Analytes 75As, 43Ca, 111Cd, 59Co, 53Cr, 63Cu, 57Fe, 39K, 202Hg, 26Mg, 23Na, 31P, 208Pb, 66Zn Internal standards 74Ge (As, Co, Cr, Fe, and Zn) 54Sc (Ca, K, Mg, Na, and P) 103Rh (Cd, Hg, and Pb) Operation LC conditions Column Kinetex C-18 100 A (100×2.10 mm, 5 µm) Mobile phase 0.4% mercaptoethanol, 10% methanol, pH 2.0 Flow rate 0.3 mL min-1, 8.5 min Injection volume 50 µL (a) Auxiliary O2 only when operating as LC-ICP-MS 18 Table 2: Extraction efficiency, distribution ratio and selectivity coefficients for the IIP and the NIP applied to the SPE of seaweed extract Ions Extraction efficiency (E) /%a Distribution ratio (D)b Selectivity coefficient (S)c IIP NIP IIP NIP IIP NIP MeHg 96.8 11.7 30 0.13 _ 226 Hg (II) 95.0 9.6 19 0.11 2 282 Cd(II) 78.3 0.2 3.61 0.00 8 ---d Pb(II) 26.0 0.2 0.35 0.00 85 ---d Al(III) 35.6 10.4 0.55 0.12 54 258 Cr(II) 0.9 0.1 0.01 0.00 3249 ---d Fe(III) 5.8 1.2 0.06 0.01 491 2394 Co(II) 0.7 0.2 0.01 0.00 4301 ---d Ni(II) 3.1 0.2 0.03 0.00 940 ---d Cu(II) 0.4 0.0 0.00 0.00 ---d ---d Zn(II) 0.5 0.0 0.00 0.00 ---d ---d As(III) 1.4 0.6 0.01 0.01 ---d 4654 Na(I) 0.0 0.3 0.00 0.00 ---d ---d K(I) 0.0 1.7 0.00 0.02 ---d 1770 Ca(II) 2.5 0.0 0.03 0.00 1174 ---d Mg(II) 1.0 0.8 0.01 0.01 3014 3627 (a) E(%)=A2 ATx100 (b) D= A2 A1 (c) SMeHg/M=DMeHg DM (d) Not calculated A1 = Analyte concentration at equilibrium A2 = Analyte concentration enriched by IIP/NIP SPE at equilibrium AT = Total analyte concentration M = Hg(II), Cd(II), Pb(II), Al(III), Cr(III), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), As(III), Na(I), K(I), Ca(II), Mg(II) 19 Table 3: Linearity, equation of calibration and LOD/LOQ of the method MeHg Hg (II) Calibration range 0.02-20 µg L-1 0.07-50 µg L-1 Aqueous calibration equation (n=3) peak area = 0 + 4987(±166) [MeHg] peak area = 0 + 3012(±547) [Hg(II)] Correlation coefficient >0.999 >0.999 Limit of detection 0.007 µg kg-1 0.02 µg kg-1 Limit of quantification 0.02 µg kg-1 0.07 µg kg-1 20 Table 4: Comparison of characteristic performances obtained by using the proposed (IIP)-SPE method and other SPE methods and potentiometric assays based on IIPs for mercury determination/speciation Sample Analyte(s) Adsorbent for SPE Detection technique LOD Enrichment factor Reference Fish Hg (II) and MeHg IIP AFS _ [21] Fish MeHg MIP HRCS-AAS 6.6 µg kg-1 1 [28] Wine Hg (II) Silica gel-IIP composite CV-AAS 0.02 µg L-1 _ [32] Fish Hg (II) IIP CV-AAS 0.01 µg L-1 120 [43] Fisha Hg (II) MWCNTIIP composite Potentiometry 6.3×10−8 mol L-1 _ [34] Fish and shrimpa Hg (II) Graphene oxide-IIP composite Potentiometry 1.95 × 10−9 mol L−1 [44] Seaweed Hg (II) and MeHg IIP-SPE LC-ICPMS 0.02 µg kg-1 for Hg (II) and 0.007 µg kg-1 for MeHg 50 This work (a) This procedure does not imply a SPE stage AFS: atomic fluorescence spectrometry, CV-AAS: cold vapour atomic absorption spectrometry, HRCSAAS: high resolution continuum source - atomic absorption spectrometry, LC-ICP-MS: liquid chromatography - inductively coupled plasma - mass spectrometry 21 Table 5: Inter-day and intraday analytical recovery and precision (RSD). MeHg Hg(II) Concent ration / µg L-1 Analytical recovery / % RSD / % Concentra tion / µg L-1 Analytical recovery / % RSD / % Inter-day 1 112±8 8 2 86±10 12 5 97±13 13 10 108±18 16 20 98±10 10 50 102±8 8 1 94±7 6 2 98±8 8 2 92±9 9 5 100±7 7 Intraday 5 89±7 9 10 102±10 10 10 89±8 9 20 102±4 4 20 95±6 6 50 97±5 4 22 Table 6: Mercury species concentration in BCR-463 and in commercial edible seaweed samples Sample Hg (II) / mg kg-1a MeHg / mg kg-1a tHg, mg kg-1b tHg / mg kg-1c Wakame 0.06±0.01 0.01±0.002 0.07±0.01 0.07±0.01 Sea-spaghetti 0.11±0.02 0.06±0.02 0.17±0.03 0.19±0.02 Hijiki 0.06±0.01 0.01±0.002 0.07±0.01 0.06±0.003 (a) Hg(II) and MeHg concentrations after IIP-based SPE and LC-ICP-MS determination (b) Total Hg expressed as the sum of Hg (II) and MeHg concentrations after IIP-based SPE and LC-ICP-MS determination (c) Total Hg after IIP-based SPE and ICP-MS determination Dear Editor, I am sending to you the revised version of our manuscript TAL-D-20-02796 ‘Mercury speciation in edible seaweed by high performance liquid chromatography - inductively coupled plasma mass spectrometry after ionic imprinted polymer-solid phase extraction’. Reviewers’ comments have been taken into account and all queries have been answered. Two copies of the manuscript [clean copy and manuscript with Track Changes (red underline) have been uploaded. Waiting your news Best regards, Dr. Antonio Moreda-Piñeiro (corresponding author) Cover Letter The research summaries results regarding the selective and interference-free speciation of mercury (methylmercury and inorganic mercury) in edible seaweeds. Despite seaweed pre-concentrate essential and toxic elements, the levels of mercury in seaweed are very low, and the presence of this toxic element in this foodstuff is usually ignored. However, the assessment of mercury (and mercury species) must be controlled on the basis of several food safety regulations. The current research proposes the use of an ionic imprinted polymer-based solid phase extraction procedure for selectively preconcentrating mercury species (methylmercury and inorganic mercury) from seaweed extracts. The developed procedure is robust and the high pre-concentration factor allows the determination of total mercury after applying atomic spectrometric techniques, and also mercury speciation when using hyphenated techniques such as HPLC-ICP-MS. Novelty Statement 75 80 85 90 95 100 105 400 900 1400 1900 2400 2900 3400 3900 Transmitance (%) Wavenumber (cm-1) NIP IIP-with template IIP-without template Figure 1 0 500 1000 1500 2000 2500 3000 0 2 4 6 8 Intensity (cps) Time (min) Sample Standard MeHg Hg (II) Figure 2 0 3 6 9 12 15 0 20 40 60 80 100 5678910 11 [Hg (II)] (µg L-1) MeHg analytical recovery (%) pH MeHg Hg (II) (a) 0 5 10 15 20 0 20 40 60 80 100 0.5 1.0 2.0 [Hg (II)] (µg L-1) MeHg analytical recovery (%) Loading flow rate (mL min-1) MeHg Hg (II) (b) 0 10 20 30 0 20 40 60 80 100 120 0.5 1.0 2.0 3.0 4.0 [Hg (II)] (µg L-1) MeHg analytical recovery (%) Volume of the eluting solution (mL) MeHg Hg (II) (d) (c) 0 20 40 0 20 40 60 80 0.5 1.0 2.0 [Hg (II)] (µg L-1) MeHg analytical recovery (%) Elution flow rate (mL min-1) MeHg Hg (II) Figure 3 (a) 0 20 40 60 80 100 120 10.0 25.0 37.5 50.0 100.0 Analytical recovery (%) Sample volume (mL) Hg (II) MeHg (b) 0 20 40 60 80 100 120 140 012345678910 11 12 13 14 15 Analytical recovery (%) Number of sorption/desorption cycles Hg (II) MeHg Figure 4