Development of a Solid Phase Extraction-Based Method for the Quantitative Analysis of Methylmercury in Soil and Sediment

A simple, universal method to quantify soil methylmercury (MeHg) is not available. Here, we developed a solid-phase extraction-based method using gas chromatography mass spectrometry. MeHg was purified from the soil matrix using an optimized solid-phase extraction method, which reduced the use of organic solvents and eliminated the requirement for harmful reagents. The sample limit of quantification was 7.5 ng/g. MeHg recovery in the reference samples was 96.2%–102.6%; the intra- and inter-assay coefficients of variation were 3.4%–7.1% and 4.3%–7.1%, respectively, indicating high validation performance. This analysis method is simple as it can be performed using general-purpose reagents and instruments; has a high degree of trueness and accuracy; could be useful for soil MeHg quantification with improved sensitivity; and can provide reliable data to prevent MeHg contamination and improve food safety.

The amount of Hg in the top layer of the oceans worldwide, to a depth of 100 m, is now approximately twice as high as that 100 years ago and is still increasing (United Nations Environment Programme, UNEP 2013). Hg in the atmosphere is emitted primarily from Asia, accounting for approximately 50% of global emissions (UNEP 2013). Hg is transported from the emission area to the environment through the atmosphere and ocean currents.
As some inorganic Hg is converted to methylmercury (MeHg) by sulfate-and iron-reducing bacteria under anaerobic conditions, such as paddy soils (Sunderland et al. 2009), the soils polluted by inorganic Hg can cause problems in MeHg-polluted rice in China (Lin et al. 2021). A previous study indicated that MeHg generated in paddy soil is transferred to rice grains during rice plant growth, with the highest Hg concentration in the embryo (Kodamatani et al. 2020). Furthermore, MeHg bioaccumulates in edible organisms, such as loaches and eels, living in Hg-polluted paddy fields (Du et al. 2021). Although MeHg exposure through rice and fish consumption is a concern, particularly in Southeast Asia, detailed information in this regard is lacking (Rothenberg et al. 2014). If paddy soil continues to be polluted by inorganic Hg in Japan, more MeHg-polluted rice will be produced posing increased health risks through longterm and chronic MeHg exposure from staple rice.
Therefore, a universal method to measure the concentration of MeHg in soil is required to prevent pollution. The Japanese official Hg analytical method for soils in the "Mercury Analysis Manual" (The Ministry of the Environment 2004) can be used for a wide variety of samples, including biological samples such as blood, and environmental samples such as seafood and soil; the accuracy of the analysis values for MeHg is recognized worldwide. However, this method involves the use of harmful reagents that are difficult to handle, such as dithizone toluene solution for extraction, alkaline solution for decomposition, and toluene in pre-cleaning reagents. The method also requires a device with restricted use, such as a gas chromatography system connected to a radioactive 63 Ni electron capture detector (GC-ECD). In addition, the official analytical method used in the United States for analyzing extractable Hg species in soil or sediment (EPA 3200) (US Environmental Protection Agency 2014) combines an acid treatment using microwaves and a solid-phase extraction step comprising of sulfhydryl cotton fiber. Hence, this method requires devices with low versatility. Alternatively, a solid-phase cartridge for sample * Sachiko Kakimoto kakimoto@iph.osaka.jp 1 Division of Hygienic Chemistry, Osaka Institute of Public Health, 1-3-69 Nakamichi, Higashinari-ku, Osaka 537-0025, Japan preparation is a more user-friendly tool for MeHg quantification than alkaline solutions and toluene-based techniques. Furthermore, there are highly sensitive analytical methods that use solid-phase microextraction (SPME) (Mishra et al. 2005), a type of solid phase extraction, and highly sensitive measurement instruments such as cold vapor atomic fluorescence spectrometry (CVAFS). However, these analytical methods require SPME or special CVAFS instruments. Thus, MeHg in soil could be more efficiently quantified with high specificity and validation performance using gas chromatography-mass spectrometry (GC-MS), which is a universal technique. In this study, we aimed to develop a simple and universal method to quantify MeHg in soil. Using this newly developed method, we also assessed the MeHg levels in reference samples.

Materials and Methods
Methylmercury chloride (98% purity) was purchased from GL Science Inc. (Tokyo, Japan). Potassium bromide, sodium dihydrogen phosphate dihydrate, and disodium hydrogen phosphate dodecahydrate (guaranteed reagent grade); anhydrous sodium sulfate, acetonitrile, acetone, and n-hexane (for pesticide residue-PCB analysis); polyethylene glycol 200 (PEG200) and formic acid (Wako first grade); and hydrochloric acid (for analysis of poisonous metals) were purchased from FUJIFILM Wako Pure Chemical Corporation, Ltd. (Osaka, Japan). Tetraphenyl sodium borate (Kalibor, analytical grade) was purchased from Dojindo Laboratories Co., Ltd. (Kumamoto, Japan). The Oasis PRiME HLB cartridge (200 mg/6 cc) and Sep-Pak C18 Vac 3 cc (200 mg) were purchased from Waters (Milford, MA, USA). Glass beads (cat. no. BZ-1, soda glass) were purchased from As One Co., Ltd. (Tokyo, Japan) and washed with acetone and n-hexane before use. Potassium bromide solution (1 mol/L) was prepared by dissolving 59.5 g of potassium bromide in 500 mL of water. Hydrochloric acid (5 mol/L) solution was prepared by adding 150 mL of concentrated hydrochloric acid to 210 mL of water. Sodium phosphate buffer (0.2 mol/L; pH 7.0) was prepared by mixing 0.2 mol/L sodium dihydrogen phosphate solution with 0.2 mol/L disodium hydrogen phosphate solution and adjusting the pH. Sodium tetraphenylborate solution (1%) was prepared using sodium phosphate buffer. Ultrapure water was used in all experiments. Glassware was washed with acetone and n-hexane before use.
Certified estuarine sediment samples of ERM CC-580 containing 132 ± 3 mg/kg Hg and 0.075 ± 0.004 mg/ kg MeHg were obtained from the Institute for Reference Materials and Measurements (Geel, Belgium). The following samples were obtained from the National Institute of Advanced Industrial Science and Technology (Ibaraki, Japan): Geological Survey of Japan (GSJ) geochemical reference samples of sediments in Japan (JLK-1), which were collected from Biwa Lake; samples of JSD-1, -2, -3, and -4, which were collected from the Kanto area; and andosol samples (JSO-1), which were collected from Tokyo. Environmental certified reference material of landfill cover soil-No. 33-collected from north of Tokyo in Japan was obtained from the National Institute for Environmental Studies (Ibaraki, Japan), which was irradiated at a high dose of 21 kGy with a cobalt source. As instructed in Material Technical Information, the material was stored as follows: JSD-1, -2, -3, -4, JSO-1, No. 33, and JLK-1 were stored at room temperature (approximately 5-35°C), away from sunlight and high temperature/humidity; and ERM-CC580 was stored in a refrigerator.
MeHg was extracted from 1.0 g of each sample using 10 mL of 5 mol/L hydrochloric acid solution, 5 mL of 1 mol/L bromide potassium solution, and 4 g of glass beads for 5 min with shaking at 2,500 rpm. After centrifuging the samples at 20°C and 3589×g for 5 min, the residue was extracted again as described above. The resulting supernatants were collected as extracts.
The extracts were loaded into an Oasis PRiME HLB solid-phase cartridge conditioned with 6 mL of acetonitrile and 6 mL of water before use. The cartridge was washed with 10 mL of water and eluted with 5 mL of acetonitrile containing 10% formic acid. Conditioning, loading, washing, and elution of the sample solution were performed without vacuum or positive pressure.
The experiment was performed in a laboratory fume hood. The elute was adjusted with the same solution until a total volume of 5 mL, and 4 mL of elute was used for phenyl-derivatization by mixing with 5 mL of phosphate buffer, 1 mL of sodium tetraphenylborate solution, and 2 mL of n-hexane, followed by shaking at 2,500 rpm for 2 min. After centrifugation at 20°C for 3 min 3589×g, the n-hexane phase was dehydrated by adding anhydrous sodium sulfate, and then, 0.2 mL of n-hexane phase was added to 0.1 mL of 1.5 mg/mL PEG 200. The resulting solution was used as the test sample for the subsequent GC-MS analysis.
A spiking agent consisting of a MeHg solution (3 µg/ mL) was prepared by dissolving 11.64 mg of methylmercury chloride in water to 100 mL with ultrasound treatment and diluting 3 mL with water to 100 mL. A 1000 mg/L MeHg stock standard solution was prepared by dissolving 11.68 mg of methylmercury chloride in 10 mL of toluene. The stock standard solution was diluted with n-hexane to cover a 1-50 ng/mL concentration range, providing a full calibration curve.
The test sample solution was assayed with a 5973 inert gas chromatograph equipped with a mass spectrometry detector (GC-MSD; Agilent Technologies, Inc., Santa Clara, CA, USA). Separation was carried out in an InertCap 5MS/ 1 3 NP (30 m × 0.25 mm i.d., 0.25 μm film thickness) column (GL Sciences Inc.) using helium as the carrier gas (flow rate: 1.0 mL/min). The injector temperature was 250°C, and the injection volume was 1 µL (splitless). The oven temperature was programmed from 70 (1 min) to 280°C (5 min) at a rate of 20°C/min, and the transfer line temperature was set to 280°C. The mass spectrometer was operated in the electron ionization mode with selected ion monitoring (SIM). Selected ions were monitored at m/z 294 (quantitative ion) and 292 (confirmative ion), and the ion source temperature was set to 230°C. The concentrations obtained from the GC-MSD analysis were converted to units of nanograms per gram of sample weight by multiplying the corresponding dilution factor to obtain the MeHg levels.
One gram of certified estuarine sediment sample (ERM CC-580) was extracted without spiking; 1 g of GSJ geochemical reference samples (both JLK-1 and JSO-1) was spiked before extraction with MeHg at a concentration of 20 ng/g. The MeHg level of spiked and non-spiked test sample solutions were measured twice per assay and once per assay, respectively, for six independent experiments in a single laboratory, performed according to the official Japanese guideline of analytical methods for metals in foods (The Ministry of Health 2012). The average recovery and intraand inter-assay reproducibility were calculated using a oneway analysis of variance (one-way ANOVA). As a result, a six-point calibration curve was generated. The concentration was calculated in the range with signal-to-noise (S/N) ratios of more than 3 in the qualitative analysis and more than 10 in quantitative ion detection.

Results and Discussion
Solid-phase cartridges, which are simple and easy-to-use, can be extracted and purified without hazardous reagents; hence, they have been widely used in pesticide analyses (Amendola et al. 2010;Schenck et al. 2008). Liem-Nguyen et al. (2020) reported a MeHg analytical method using a solid-phase cartridge to concentrate water samples and improve the test sensitivity. However, the solid-phase cartridge is also potentially suitable for removing the soil matrix, thus facilitating the use of GC-MSD, which is susceptible to the negative effects of the soil matrix. After loading the Oasis PRiME HLB solid-phase cartridge with the extracts, the hydrophobic methyl group of MeHg bound to the HLB cartridge, and the aqueous matrix was removed by washing with water, followed by elution with acetonitrile containing 10% formic acid. This analytical method is effective in developing a GC-MS-based method combining a solid-phase cartridge with high selectivity and validation performance.
To optimize sample preparation, we first tested the extraction volume with 1 mol/L potassium bromide solution and 5 mol/L hydrochloric acid solution. The former was determined to be 10 mL in total. The latter was determined to be 20 mL in total, as the recovery rates of MeHg in ERMCC-580 were the highest at 92.1%, 99.7%, and 105.2%, corresponding to 10, 20, and 30 mL of the total volume of 5 mol/L hydrochloric acid solution, respectively (data not shown). Therefore, to recover as much MeHg as possible and to reduce the amount of reagent used, the total volume of 5 mol/L HCl was set to 20 mL. When the potassium bromide solution was changed to 10 and 20 mL with 5 mol/L hydrochloric acid set to 20 mL, the MeHg recovery rates of ERMCC-580 were 99.7% and 103.5%, respectively, and there was almost no difference in the recovery rates; therefore, the amount of potassium bromide used was set to 10 mL.
Next, to determine the optimal type of solid-phase cartridge, we tested the recovery rates between the Sep-Pak C18 Vac (200 mg, 3 cc) and Oasis PRiME HLB cartridges (200 mg, 6 cc) by adding 100 ng of MeHg spiking agent to the extracts from the GSJ geochemical reference sample of JSO-1, with the MeHg level lower than the detection limit. Both Sep-Pak C18 Vac and Oasis PRiME HLB cartridges could retain MeHg with hydrophobic bonds. However, with the use of 10 mL of acetonitrile, MeHg was not recovered from the former, whereas approximately 46% of MeHg was recovered from the latter. We speculated that this was because the Sep-Pak C18 Vac cartridge had a higher log P of 9.18 and silanol groups that absorbed basic compounds of MeHg with ion-exchanges, whereas the polymer-based Oasis PRiME HLB cartridge had a lower log P of 3.59 and did not have silanol groups. The solid-phase extraction cartridge was determined to be HLB, and the elution solvent was investigated. As the recovery rate with the Oasis PRiME HLB cartridge was similar with loading volumes of 60, 200, and 500 mg, 200 mg was selected to minimize the flow time and elution volume. To improve the MeHg elution efficiency with the Oasis PRiME HLB cartridge, acetonitrile containing formic acid, which enhanced the dissociation of the ionic bond between MeHg and bromide and chloride ions, was tested as an elution solvent. As a result, the recovery rates of JSO-1 extracts spiked with 100 ng of MeHg were 102% and 106%, eluted with 10 mL of 10% and 15% formic acid/ acetonitrile, respectively. We used 10% formic acid solution for elution to minimize the amount of formic acid.
According to a previous report (The Ministry of the Environment 2014), the average MeHg concentration of soil and sediment (n = 288) is 21.39 ng/g with a maximum value of 444.35 ng/g. To verify the applicability of the elution method to the MeHg concentration level of the real sample and the MeHg retention capacity of the solid-phase cartridge, JSO-1 extracts spiked with 20 and 600 ng of MeHg were tested. MeHg was eluted with 2.5 mL of 10% formic acid/acetonitrile and fractioned four times. The recovery rates of MeHg are summarized in Table 1. Although approximately 100% of MeHg was recovered in the first fraction during testing of both 20 and 600 ng of MeHg spiked samples, small amounts (approximately 3 ng/mL, namely 8 ng) of MeHg were confirmed in the second fraction during the testing of 600 ng of MeHg spiked samples. Therefore, the elution volume was set to 5 mL for full recovery. Thus, the newly developed elution method had sufficient performance to cover MeHg levels in real sample analysis.
In this method, the extracted and purified MeHg was phenyl derivatized and analyzed using GC-MS in the SIM mode, monitoring m/z 294 ion (quantitative ion) and m/z 292 ion (confirmative ion), respectively. Typical chromatograms of the phenyl-derivatized MeHg in the standard solution and test sample solutions are shown in Fig. 1. The approximate retention time of the MeHg was 5.5 min. Quantitative ion calibration curves (y = 195.1 (± 6.4)x − 2.0 (± 163.9), confidence limit 95%) of phenyl-derivatized MeHg were generated with excellent linearity (r 2 greater than 0.99) in the range of 1-50 ng/mL (Fig. 2).
The limit of detection (LOD) and quantification (LOQ) were determined to be 1 ng/mL and 3 ng/mL (2.5 ng/g and 7.5 ng/g each converting to per sample weight), calculated at S/N ratios greater than 3 and 10, respectively.
The validation parameters were determined in six independent experiments, including trueness and reproducibility, and are summarized in Table 2. For the ERMCC-580, JLK-1, and JSO-1 extracts, the trueness values were 98.8%, 102.6%, and 96.2%, respectively. Furthermore, the repeatability (3.4%, 7.1%, and 7.0%, respectively) and reproducibility (4.3%, 7.1%, and 7.0%, respectively) were high as determined using the intra-assay coefficient of variations (CVs) and the inter-assay CVs. The trueness and intra-and inter-assay CVs were considered acceptable, according to the Japanese official guidelines for the validation of test methods for metals (The Ministry of Health, Labour and Welfare of Japan 2012).
As a high level of inorganic Hg is distributed biasedly in soil and sediment samples, there is a risk of overestimating MeHg concentration values unless a reliable separation of MeHg from inorganic Hg is achieved (Morita et al. 1982).
As mentioned above, we demonstrated the high validation performance of the GC-MSD method in samples despite certain difficulties. Specifically, the difficulties were that ERMCC-580 had a significantly biased methyl and inorganic Hg content ratio (approximately 1760-fold difference), and JSO-1 of andosol soil had a complex matrix causing several analytical problems (Łozowicka et al. 2017). Therefore, this newly developed solid-phase extraction-based method is a clean and simple method that can be used with a universal GC-MSD device, which reduces the use of harmful reagents, overcoming challenges encountered in previous studies.
The developed method was used to assess MeHg concentrations in GSJ geochemical reference samples JSD-1-4 and the environmental certified reference material No. 33, and Table 1 Optimizing the elution volume of the Oasis PRiME HLB cartridge: recovery rates of MeHg  Table 3. The MeHg concentration in JSD-4 was determined to be 64.1 ng/g, whereas MeHg was not detected in other samples of JSD-1-3 and No. 33 (less than LOQ). However, these MeHg-negative samples contained certified concentrations of total Hg as follows: 16, 106, 254, and 89 ng/g, respectively. For JSD-4, no information was available on the certified concentration of total Hg. Overall, the resulting concentrations of MeHg in Table 3 were acceptable because they were well below 15 mg/kg, the maximum limit set by the Japanese law for the regulation of Hg and its compounds to prevent soil pollution. As some inorganic Hg is converted to MeHg by anaerobic bacteria remaining in soil and sediment samples during storage (Kodamatani et al. 2017), the MeHg levels can vary. Therefore, the collected samples were stored frozen (Kodamatani et al. 2017). As shown in Table 3, except for No. 33, which was irradiated at a high dose for sterilization, the MeHg concentrations in JSD-1-4 may be variable in other assays because JSD-1-4 was not verified as completely sterilized by the manufacturer. Nonetheless, the concentration of JLK-1 and JSO-1, which were not verified for complete sterilization and obtained from the same manufacturer as JSD-1-4, was considered constant in method validation; therefore, the JSD-1-4 concentration could also be constant, and the range of variation was considered small. Overall, this method can be applied to the surveillance of MeHg pollution in soil, but future studies will require optimal soil sterilization treatment and verification of concentration variance to obtain more accurate concentrations.
In conclusion, a solid-phase extraction method was developed for the quantitative analysis of MeHg in soil and sediment samples. This method required 9 h of pretreatment, and the lower limit of quantification in the samples was 7.5 ng/ mL, which was slightly higher than that of the conventional method. However, the advantages of this method are that it does not require equipment such as SPME; it uses HLB, which a general-purpose solid-phase extraction cartridge, in the purification process; it is a clean analysis method that does not use reagents harmful to the human body such as benzene and toluene; and it uses GC/MS equipment, which is a general-purpose instrument.
In addition, the method can demonstrate good trueness, reproducibility, and repeatability. Measurements of MeHg concentrations in reference soil and sediment samples showed values in compliance with Japanese regulatory standards. If the sensitivity of this method is further   improved, it will be useful for MeHg analyses of soil samples in the field, and can provide environmentalists and agricultural professionals with reliable data to prevent MeHg contamination and improve the safety of food harvested from paddy field soil.