This study aimed to develop a high-throughput HPLC-ICP-MS method for the simultaneous quantification of inorganic mercury (Hg²⁺) and methylmercury (MeHg⁺) in human breast milk. The method was validated using a certified reference material, achieving 99% accuracy.A 4-month analysis of breast milk samples from 10 participants examined the effects of collection method and timing on mercury speciation. The results revealed correlations between mercury species, fat content, and selenium, with MeHg⁺ concentrations being higher in hindmilk. The study highlights the importance of accurate mercury speciation and proper breast milk collection methodologies.<blockquote><h4>The original article</h4><h4><a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965">High-throughput mercury speciation analysis of breast milk using HPLC-ICP-MS</a></h4>Kenta Iwai, Miyuki Iwai-Shimada, Nozomi Tatsuta, Yayoi Kobayashi, Kaname Asato, Mitsuo Nishimoto, Kunihiko Nakai, Shoji F. NakayamaMicrochemical Journal, Volume 211, April 2025, 113142<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1016/j.microc.2025.113142">https://doi.org/10.1016/j.microc.2025.113142</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/">CC-BY 4.0</a></h6></blockquote>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.<h3>1. Introduction</h3>Mercury is a global pollutant released into the oceans and atmosphere as a result of economic activities such as gold mining and natural phenomena such as volcanic eruptions <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0005" name="bb0005" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0005">[1]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0010" name="bb0010" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0010">[2]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0015" name="bb0015" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0015">[3]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0020" name="bb0020" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0020">[4]</a>. This element is present in the environment in various chemical forms, and the sources of exposure, health effects, and toxicokinetics differ significantly among chemical species; therefore, it is necessary to estimate the exposure level of each chemical species <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0025" name="bb0025" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0025">[5]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0030" name="bb0030" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0030">[6]</a>. Methylmercury (MeHg+), the chemical form of mercury of greatest concern in terms of human toxicity, is converted from inorganic mercury (Hg2+) by microorganisms, and individuals are exposed to it through the consumption of seafood. Nervous system disorders as well as cardiovascular and other effects have&nbsp;been reported&nbsp;following exposure to MeHg+ <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0035" name="bb0035" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0035">[7]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0040" name="bb0040" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0040">[8]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0045" name="bb0045" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0045">[9]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0050" name="bb0050" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0050">[10]</a>. The Minamata Convention on Mercury was established&nbsp;to regulate anthropogenic mercury emissions and advocate for the global monitoring of mercury exposure levels <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0055" name="bb0055" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0055">[11]</a>. Nevertheless, given that mercury remains a pervasive environmental pollutant, continuous global monitoring continues to be imperative.Exposure to mercury during the vulnerable period from the fetal stage to infancy is a health concern. Prenatal exposure to MeHg+ may adversely affect child development <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0060" name="bb0060" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0060">[12]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0065" name="bb0065" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0065">[13]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0070" name="bb0070" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0070">[14]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0075" name="bb0075" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0075">[15]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0080" name="bb0080" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0080">[16]</a>. Although measurements of total mercury in breast milk—the&nbsp;main&nbsp;source of exposure in infants—have been reported in many regions <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0085" name="bb0085" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0085">[17]</a>, MeHg+ <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0090" name="bb0090" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0090">[18]</a> and Hg2+ <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0095" name="bb0095" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0095">[19]</a> are released in breast milk; therefore, it is necessary to analyze these chemical species. Blood MeHg+ levels positively correlate with seafood consumption in Japan <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0100" name="bb0100" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0100">[20]</a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0105" name="bb0105" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0105">[21]</a>, and inorganic mercury exposure is heavily influenced by dental amalgams <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0110" name="bb0110" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0110">[22]</a> and gold mining <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0115" name="bb0115" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0115">[23]</a>.&nbsp;In Japan,&nbsp;dental amalgams have rarely been used&nbsp;since the 1990s <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0120" name="bb0120" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0120">[24]</a>.&nbsp;In addition, Hg2+ exposure is also lower now because of the reduced use of mercury in thermometers and other devices.Thus fat, MeHg+ in breast milk has been analyzed mainly using gas chromatography equipped with electron capture detector (GC-ECD) <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0190" name="bb0190" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0190">[38]</a>; however, the&nbsp;pretreatment for such measurements is complicated, and the analytical volume required is large. It is especially important to establish a method that can adequately analyze even small amounts of colostrum because sample volumes are small. Consequently, the following prerequisites must be met for a comprehensive investigation of breast milk, including colostrum: 1) the minimum analytical volume of the sample must be feasible; 2) pretreatment must be straightforward; and 3) validation must be viable, with the methodology being proved highly reliable. In this study, we developed a high-throughput and sensitive analytical method for the separate determination of chemical species of mercury in breast milk that satisfies the above requirements.This study was designed to achieve three objectives. The first was to develop a straightforward approach for analyzing mercury speciation in breast milk. The second was to ascertain the impact of the sampling methodology and timing on the concentrations of Hg2+ and MeHg+ in breast milk. The third was to elucidate the relationships between the Hg2+ and MeHg+ contents and the contents of the other elements and fats.<h3>2. Methods</h3><h4>2.3. HPLC apparatus and conditions</h4>Separation was performed on a metal-free YMC-Triart C18 HPLC column (YMC CO., LTD., Kyoto, Japan), installed in an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=1&amp;search=1260+Bio-inert+LC">Agilent 1260 Bio-inert LC</a> system consisting of a G5611A Bio-inert quaternary pump, G5611A Bio-inert high-performance autosampler, and a G1390B isocratic pump (Agilent Technologies Inc., Santa Clara, CA, USA). The outlet of the HPLC column was connected directly to an ICP-MS nebulizer (<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#t0010" name="bt0010" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="t0010">Table 2</a>).<h4>2.4. ICP-MS device and conditions</h4>An <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library?manufacturerItems=1&amp;search=8900+ICP-QQQ">Agilent 8900 ICP-QQQ </a>(Agilent Technologies Inc.) was used for speciation analysis and elemental concentration measurements. The introduction system consisted of a micro-mist nebulizer, a quartz double-pass spray chamber, and a quartz torch with a diameter of 2.5 mm. The total element concentration analysis was performed using the ICP-MS equipped with an SPS 4 Autosampler (Agilent Technologies, Inc.) [49].<h3>3. Results and discussion</h3><h4>3.2.3. Comparison of total mercury levels measured by HPLC-ICP-MS and ICP-MS</h4>To validate the developed method, we compared the sum of the Hg2+ and MeHg+ concentrations obtained using HPLC-ICP-MS with the total mercury concentration quantified via ICP-MS analysis. We analyzed foremilk and hindmilk samples from 10 participants in four sampling rounds (N = 80). The median concentrations (ng g−1) for the 80 samples were 0.109 (5th–95th percentile: 0.014–0.365) for Hg2+ and 0.220 (5th–95th percentile: 0.046–0.792) for MeHg+. The Hg2+ and MeHg+ concentrations in all the breast milk samples were above the MDL. The ICP-MS determination method for the total mercury and other elemental contents has been previously published <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#b0245" name="bb0245" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="b0245">[49]</a>. NIST SRM 1953 was used as the reference material to validate the determination of total mercury, as well as other elements using ICP-MS (<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#s0120" name="bs0120" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="s0120">Supplemental Table 2</a>). No mercury chemical species other than Hg2+ and MeHg+ were present in breast milk, as determined by HPLC-ICP-MS; therefore, the sum of Hg2+ and MeHg+ was taken to be the total mercury in breast milk. To validate the analytical method, the distributions of total mercury as measured using HPLC-ICP-MS and ICP-MS were plotted. The results from the two methods were found to be strongly correlated (R2&nbsp;=&nbsp;0.895, Pearson’s correlation coefficient&nbsp;=&nbsp;0.946) (<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#f0010" name="bf0010" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="f0010">Fig. 2</a> and <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#s0120" name="bs0120" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="s0120">Supplemental Table 3</a>). The effects of adjusting the fat content were also investigated. The values showed better agreement in samples with low fat contents, whereas there was more variation in samples with high fat contents. For samples with high fat contents, the correlation improved upon fat content adjustment (<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#s0120" name="bs0120" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="s0120">Supplemental Table 4</a>). A Bland–Altman plot was used to assess the agreement between the two methods (<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0026265X25004965#f0015" name="bf0015" data-sd-ui-side-panel-opener="true" data-xocs-content-type="reference" data-xocs-content-id="f0015">Fig. 3</a>). The results of the two measurement methods were generally in agreement. However, discrepancies were observed at lower concentrations. These discrepancies are thought to be due to errors in the ICP-MS measurements, which increase as the measured concentration approaches the limit of quantification.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Microchemical_Journal_Volume_211_April_2025_113142_Fig_2_Regression_correlation_between_the_total_mercury_concentration_as_obtained_via_ICP_MS_analysis_02f63d9d9f.jpg" alt="Microchemical Journal, Volume 211, April 2025, 113142: Fig. 2. Regression correlation between the total mercury concentration as obtained via ICP-MS analysis and the sum of Hg2+ and MeHg+ as obtained via HPLC-ICP-MS analysis (N = 80). THg: total mercury." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Microchemical_Journal_Volume_211_April_2025_113142_Fig_2_Regression_correlation_between_the_total_mercury_concentration_as_obtained_via_ICP_MS_analysis_02f63d9d9f.jpg 168w," sizes="100vw" width="1400" height="1300"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/MICROC_3_c4a9ebef9e.jpg" alt="Microchemical Journal, Volume 211, April 2025, 113142: Fig. 3. A Bland–Altman plot and regression line (dotted line) and 95% limits of agreement (dashed lines) for total mercury in 80 samples as measured by ICP-MS and HPLC-ICP-MS." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_MICROC_3_c4a9ebef9e.jpg 156w," sizes="100vw" width="1400" height="1404"><h3>4. Conclusion</h3>We developed a high-throughput and sensitive method for analyzing mercury speciation in breast milk using HPLC-ICP-MS. The method was validated by analyzing NIST SRM 1953, comparing the values obtained with the total mercury concentrations obtained via ICP-MS analysis.&nbsp;Hg2+ and MeHg+ were detected in all the samples of foremilk and hindmilk provided monthly for 4&nbsp;months by the 10 participants. MeHg+ concentrations differed significantly between foremilk and hindmilk samples but not between samples obtained 2 and 5&nbsp;months postpartum. In contrast, Hg2+ concentrations in the foremilk and hindmilk did not differ and were correlated with selenium concentrations. Breast milk composition differs significantly between colostrum, transitional milk, and mature milk. Hence, a large cross-sectional study using our method will reveal the level of infant exposure to each chemical species of mercury.

High-throughput mercury speciation analysis of breast milk using HPLC-ICP-MS

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