<p><span><strong>This study evaluates headspace gas chromatography–ion mobility spectrometry (HS-GC-IMS) for characterizing the volatile profiles of five </strong></span><i><span><strong>Origanum</strong></span></i><span><strong> species and detecting blends with nonoregano leaves. MS-supported compound identification revealed species-specific differences, particularly in carvacrol/thymol and sesquiterpene profiles, while the method showed good repeatability and stable profiles over six weeks.</strong></span></p><p><span>Authenticity experiments using oregano blended with marjoram, myrtle, thyme, olive, and hoary rock-rose showed that potential marker compounds could be identified for marjoram and myrtle. The results demonstrate that HS-GC-IMS offers a sensitive and cost-effective approach for </span><i><span>Origanum</span></i><span> characterization and selected oregano adulteration screening.</span></p><blockquote><h4><strong>The original article</strong></h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/afsthl/article/doi/10.1021/acsfoodscitech.6c00607/5428045/Gas-Chromatography-Ion-Mobility-Spectrometry-for"><strong>Gas Chromatography-Ion Mobility Spectrometry for Characterizing Different Species of Origanum and Detecting Blends of Its Products</strong></a></h4><p>Modestus Wigger* ; Phil Carbow; Daria Garder; Silke Hillebrand; Ursula Telgheder; Stefanie Sielemann*</p><p>ACS Food Sci. Technol. (2026)&nbsp;</p><div><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acsfoodscitech.6c00607">https://doi.org/10.1021/acsfoodscitech.6c00607</a></div><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6></blockquote><p><strong>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.</strong></p><div><div><div><div><div><div><div><p>Gas chromatography-ion mobility spectrometry (GC-IMS) has emerged as a complementary technique to GC-MS for food-aroma analysis and offers several advantages for aroma profile characterization. Applications include the authentication of olive oils, (<a href="javascript:;">14</a>) monofloral honeys, (<a href="javascript:;">15</a>) and many more. (<a href="javascript:;">16</a>)</p></div><div><p>Many GC-IMS approaches in food analysis use HS sampling. (<a href="javascript:;">16</a>) It eliminates the need for sample preparation and is optimal for transferring the compounds of interest into the GC system. After GC separation, IMS provides an additional dimension via drift time. Furthermore, it is a very sensitive, less costly method compared to a conventional quadrupole MS. Unfortunately, the capabilities for identifying compounds in IMS are limited, as it lacks the powerful databases that MS have. Therefore, in the analytical setup used, a quadrupole MS is installed on a separate flow line, as described by Budzyńska et al. (<a href="javascript:;">17</a>) and Augustini et al. (<a href="javascript:;">18</a>) Coupled with the NIST MS database and the peak-assignment approach of Augustini et al., (<a href="javascript:;">18</a>) this method enables reliable identification of most GC-IMS signals. Using this HS-GC-MS/-IMS configuration, it is demonstrated how GC-IMS can characterize <i>Origanum</i> volatilomes by analyzing five (sub)species, including one species represented by material from two geographic origins. To minimize environmental effects on volatile expression, reference seeds were grown under identical conditions in an indoor greenhouse prior to analysis. This approach helps to compare chemotypic differences among taxa and origins under controlled growth conditions.</p></div><div><p>Beyond chemotaxonomy and quality control, a further motivation for robust analytical fingerprinting of <i>Origanum</i> is the widespread issue of oregano adulteration. According to the European Spice Association, only the leaf product derived from <i>Origanum vulgare</i> ssp. <i>hirtum</i> and <i>Origanum onites</i> may be called oregano, while the International Organization for Standardization defines oregano in ISO 7925:1999–10 as leaves from the genus <i>Origanum</i> except <i>Origanum majorana</i>. ISO also specifies that the proportion of stalks and other plant parts shall not exceed 3% and that extraneous matter shall not exceed 1%, a requirement often violated in commercial oregano for economic reasons. Maquet et al. (<a href="javascript:;">19</a>) reported that 48% of oregano samples collected across the supply chain in the European Union were adulterated. Common adulterants include leaves from <i>Cistus</i> spp. for a dark green color, from olive (<i>Olea europaea</i>), and myrtle (<i>Myrtus communis</i>) for financial reasons. Another possibility could be an accidental admixture with other herbs, such as marjoram (<i>O. majorana</i>) or thyme (<i>Thymus spp</i>.). (<a href="javascript:;">20</a>) These types of food fraud are challenging for consumers and regulatory authorities to identify. One possibility is to analyze leaf material macroscopically or microscopically for foreign matter. (<a href="javascript:;">21</a>) However, a variety of analytical approaches have been developed to detect oregano adulteration. Fourier-transform infrared (FT-IR) spectroscopy has been used to screen for olive, myrtle, sumac, hoary rock-rose, and hazelnut leaf adulterants. (<a href="javascript:;">22</a>) Flügge et al. (<a href="javascript:;">23</a>) combined next-generation sequencing (NGS) with nuclear magnetic resonance (NMR) spectroscopy to detect olive, myrtle, hazelnut, and pink rock-rose (<i>Cistus</i>) leaves. Liquid chromatography–mass spectrometry (LC-MS) targeting oleuropein has been developed specifically to identify olive leaf adulteration (<a href="javascript:;">24</a>) and is also used by Maquet et al. (<a href="javascript:;">19</a>) as an analytical method for an EU-wide control of adulteration. Rocomora-Rivera et al. (<a href="javascript:;">25</a>) were the first to apply GC-IMS for the detection of oregano adulteration. Using orthogonal partial least-squares discriminant analysis (OPLS-DA), they identified admixtures containing olive leaves, but they did not test other potential adulterants. They also applied the method to commercially available oregano samples. However, their results were not verified by other analytical techniques. In the presented study, the feasibility of GC-IMS for detecting common adulterants in oregano, namely the leaves of hoary rock-rose, olive, myrtle, thyme, and marjoram, is tested by identifying marker compounds. Because only one authentic oregano type and no commercially adulterated samples were available, blends were prepared by combining oregano with candidate adulterant leaf material to simulate adulteration scenarios.</p></div><div><p>The objectives of this study are to evaluate HS-GC-IMS for volatilome profiling of five <i>Origanum</i> (sub)species grown under controlled conditions and assess the technique’s ability to detect common leaf adulterants in oregano blends.</p></div><h4><strong>2. Material and Methods</strong></h4><h5><strong>2.3. Analysis</strong></h5><div><p>An <a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/en-us/gcms/library?mainauthorItems=1&amp;search=AOC5000"><strong>AOC5000</strong></a> (Shimadzu Corporation, Kyoto, Japan) HS sampler incubating for 30 min at 120 <span>°</span>C was used for the static HS analysis of the samples. 500 μL of the HS was injected into a <a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/en-us/gcms/library?mainauthorItems=1&amp;search=2010"><strong>GC 2010-Plus</strong></a> (Shimadzu Corporation), equipped with two split injectors with a split of 1:20 and two identical analytical columns (HP-5 MS UI 30 m × 0.25 mm × 0.5 μm; Agilent, Santa Clara, CA, USA), each leading to one spectrometer (IMS or MS). A constant flow rate (35 cm/s) of helium 5.0 (Messer Industriegase, Siegen, Germany) was used. The GC temperature program started at 40 <span>°</span>C, held for 4 min, then heated with 3 <span>°</span>C/min to 150 <span>°</span>C, then heated 10 <span>°</span>C/min to 220 <span>°</span>C, held for 12.33 min, for a total program time of 60 min.</p></div><div><p>One analytical column was connected to the <strong>quadrupole MS QP-2010 Ultra</strong> (Shimadzu Corporation) in scan mode (<i>m</i>/<i>z</i> 32–300 at 3.3 Hz) with electron ionization (70 eV) and the other to the ion mobility spectrometer (G.A.S. Gesellschaft für analytische Sensorsysteme mbH, Dortmund, Germany). The drift tube (dimensions: 15.2 mm × 53 mm; temperature: 80 <span>°</span>C) was flushed with 75 mL/min drift gas (nitrogen 5.0, produced by an in-house generator), and ionization was performed by atmospheric-pressure chemical ionization with a <sup>3</sup>H source. The field strength was 500 V/cm.</p><h4><strong>3. Results and Discussion</strong></h4><h5><strong>3.1. Identification of Volatiles in Origanum</strong></h5><div><p>For the characterization of the volatilome of different <i>Origanum</i> species, it is necessary not only to detect compounds sensitively using HS-GC-IMS but also to identify them. For identification, HS-GC-MS was used in addition to HS-GC-IMS. The HS-GC-MS enables identification of unknowns through database comparison of fragmentation patterns.</p></div><div><p><a href="javascript:;">Figure 1</a> shows the total ion current (TIC) chromatograms for both HS-GC-IMS and HS-GC-MS of a fresh leaf from <i>O. vulgare</i> ssp. <i>hirtum</i>, with retention time on the <i>x</i>-axis, the integrated ion current for the IMS, and the intensity for MS on the <i>y</i>-axis. The <i>y</i>-axis of the HS-GC-MS chromatogram is logarithmically scaled, because it shows significant intensity differences (3 orders of magnitude between baseline and the highest peak), and linear scaling would discriminate small peaks in visualization. Most analytes are properly separated within the 60 min analysis time. When comparing the signals in both chromatograms, it is noticeable that they are shifted by several minutes. For example, an analyte (marked with an arrow), eluting at 35.8 min in the IMS flow line, elutes at 27.6 min in the MS flow line. However, the shift in retention times does not allow direct peak assignment. Using the 2-alkanone standard mixture as a reference, the retention times can be converted to retention indices using <a href="javascript:;">eqs 1</a> and <a href="javascript:;">2</a>, which can then be used to derive retention times for the GC-MS. An example calculation is provided in the <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/afsthl/article-supplement/5428045/pdf/fs6c00607_si_001/">Supporting Information</a>. To demonstrate the effectiveness of this method, the TIC chromatograms shown in <a href="javascript:;">Figure 1</a> are replotted in <a href="javascript:;">Figure 2</a>, with the calculated retention index on the <i>x</i>-axis instead of the retention time. The marked substance in <a href="javascript:;">Figure 1</a> is visible at a retention index of 992 in the HS-GC-IMS and 986 in the HS-GC-MS, corresponding to a deviation of only 6 RI units (≈0.6%), indicating very good assignment between the two methods. It is noticeable that, due to the logarithmic scaling of the intensity in the HS-GC-MS chromatogram, the peak pattern resembles that of the HS-GC-IMS. The IMS exhibits a nonlinear response over the measured intensity range. Therefore, logarithmic scaling of the MS signal intensity provides a representation that more closely matches the response characteristics of the IMS. Although the retention index still shows a slight shift, peak assignments are possible by comparing peak patterns. Additionally, within the retention index range of 600 to 700 (highlighted in gray), it is noticeable that the HS-GC-IMS exhibits more intense peaks than the HS-GC-MS. Peaks with a retention index above 700 become broader in the HS-GC-IMS, which can be explained by a less heated transfer and detector setup. Regarding sensitivity and separation power, the HS-GC-IMS is therefore more suitable for higher volatiles, while the HS-GC-MS is more suitable for lower volatiles, as it is also shown in Schanzmann et al. (<a href="javascript:;">33</a>) The results show that the IMS has comparable analytical power to the MS, but is more cost-effective, as it requires no vacuum and less maintenance.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Food_Sci_Technol_2026_Figure_1_Total_ion_current_TIC_chromatogram_of_a_leaf_from_O_vulgare_ssp_hirtum_cover_fc6d9adaee.jpg" alt="ACS Food Sci. Technol. (2026): Figure 1. Total ion current (TIC) chromatogram of a leaf from O. vulgare ssp. hirtum measured by headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) (upper part) and HS-GC mass spectrometry (HS-GC-MS) (lower part). The intensity of the HS-GC-MS-TIC is given on a log10 scale for better visualization, since the intensity range is 3 orders of magnitude and small peaks would not be visible in linear scaling." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Food_Sci_Technol_2026_Figure_1_Total_ion_current_TIC_chromatogram_of_a_leaf_from_O_vulgare_ssp_hirtum_cover_fc6d9adaee.jpg 201w," sizes="100vw" width="1600" height="1247"></p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Food_Sci_Technol_2026_Figure_2_Total_ion_current_TIC_chromatogram_of_a_leaf_from_O_vulgare_ssp_hirtum_bb33b1d86a.png" alt="ACS Food Sci. Technol. (2026): Figure 2. Total ion current (TIC) chromatogram of a leaf from O. vulgare ssp. hirtum measured by headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) (upper part) and HS-GC mass spectrometry (HS-GC-MS) (lower part) with the calculated retention index based on 2-alkanones on the x-axis. The intensity of the HS-GC-MS-TIC is given on a log10 scale for better visualization, since the intensity range is 3 orders of magnitude and small peaks would not be visible in linear scaling." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Food_Sci_Technol_2026_Figure_2_Total_ion_current_TIC_chromatogram_of_a_leaf_from_O_vulgare_ssp_hirtum_bb33b1d86a.png 201w," sizes="100vw" width="2508" height="1946"></p></div><h5><strong>3.4. Comparison of the Species</strong></h5><p>The first main topic of this study is to investigate species-specific variations in the volatilome. The HS-GC-IMS chromatograms for each species are illustrated in <a href="javascript:;">Figure 6</a>, where all <i>Origanum</i> species can be compared visually. Distinct differences in peak pattern and intensity between the samples are evident.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Food_Sci_Technol_2026_Figure_6_Headspace_gas_chromatography_ion_mobility_spectrometry_HS_GC_IMS_chromatograms_962a045868.png" alt="ACS Food Sci. Technol. (2026): Figure 6. Headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) chromatograms of five different species, one from two origins, Germany (DE) and Greece (GR), with the drift time on a reactant ion peak (RIP) relative scale (RIP position: ∼4.9 ms), the retention time in seconds on the y-axis, and the intensity in V on a color scale." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Food_Sci_Technol_2026_Figure_6_Headspace_gas_chromatography_ion_mobility_spectrometry_HS_GC_IMS_chromatograms_962a045868.png 152w," sizes="100vw" width="1254" height="1289"></p><p>It can be seen that the species <i>O. vulgare</i> ssp. <i>hirtum</i> contains more monoterpenes/monoterpenoids, especially carvacrol, than <i>O. vulgare</i> ssp. <i>vulgare</i>, which shows higher amounts of sesquiterpenes. This comparison provides insight into the profile of each species, as even two different plants of the same species can exhibit distinct profiles.</p><h5><strong>3.5. Detection of Blends in Oregano</strong></h5><p>After investigating the volatilomes of different <i>Origanum</i> species, the potential of this technique to detect food fraud can be demonstrated by comparing the volatilomes of oregano and its adulterants. The capabilities and limitations of GC-IMS for identifying oregano adulteration were assessed within this feasibility study by using self-made blends, as no intentionally fraudulent samples were available. For this purpose, common adulterants for oregano, namely leaves from olive, thyme, marjoram, myrtle, and hoary rock-rose, were added in different mass ratios (10%, 30%, and 50%) to ground oregano known to be pure and from <i>O. vulgare</i> ssp. <i>hirtum</i>. The sample material is shown in <a href="javascript:;">Figure 9</a> as a photograph.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Food_Sci_Technol_2026_Figure_9_Photograph_of_the_sample_material_80493b7a68.png" alt="ACS Food Sci. Technol. (2026): Figure 9. Photograph of the sample material: Oregano (O. vulgare ssp. hirtum), olive (O. europaea L.), thyme (Thymus vulgaris L.), marjoram (O. majorana L.), myrtle (M. communis L.), hoary rock-rose (C. incanus L.)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Food_Sci_Technol_2026_Figure_9_Photograph_of_the_sample_material_80493b7a68.png 245w," sizes="100vw" width="1254" height="291"></p><div><p>These pure samples were analyzed by HS-GC-IMS, and the chromatograms in the <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/afsthl/article-supplement/5428045/pdf/fs6c00607_si_001/">Supporting Information</a> generally show lower numbers and lower intensities of volatiles in olive leaves and hoary rock-rose than in oregano. In contrast to oregano, the volatilome of thyme and marjoram is comparably complex, with thyme exhibiting a very similar pattern with high proportions of thymol/carvacrol and a variety of monoterpenes and sesquiterpenes. Marjoram also contains many monoterpenes and some sesquiterpenes, but no carvacrol/thymol. The chromatogram of myrtle shows some monoterpenes, but not in the same numbers and intensity as oregano.</p></div><div><p>Potential marker substances were identified by analyzing all individual signals in the blending material and excluding those present in dried oregano or the fresh leaves of the different <i>Origanum</i> plants. Additional GC-MS measurements, the previously described peak assignment, and the NIST database enabled tentative identifications.</p></div><div><p>In marjoram piperityl acetate (RI = 1305, DT = 6.15 ms) is found, which has been tentatively identified by the described procedure. The peak intensity has a coefficient of variation of 29.9% for the lowest blend (10% marjoram in oregano) in technical triplicate. The high variation can be explained by coelution of the peak with the dominant carvacrol. The limit of detection (LOD) for marjoram in oregano using piperityl acetate as the marker was estimated at 4.1% based on the signal-to-noise (S/N) approach of Schanzmann et al. (<a href="javascript:;">33</a>) The S/N was determined for the 10% blend, and the blend ratio corresponding to S/N = 3 was calculated assuming proportionality between signal intensity and blend level. Creydt et al. identified blumeatin as a marker for marjoram adulterations using liquid chromatography–mass spectrometry and established a LOD of approximately 5%. (<a href="javascript:;">41</a>) Consequently, both volatile and nonvolatile analytes yielded comparable detection limits.</p></div></div></div></div></div></div></div></div>

Gas Chromatography-Ion Mobility Spectrometry for Characterizing Different Species of Origanum and Detecting Blends of Its Products

<p><span><strong>This study examines how freely dissolved concentrations influence the comparative hazard ranking of bisphenol A (BPA) and 24 alternative chemicals in </strong></span><i><span><strong>in vitro</strong></span></i><span><strong> assays. Chemical stability and bioavailable concentrations were assessed experimentally and modeled, with predicted freely dissolved concentrations generally agreeing with measurements within one order of magnitude.</strong></span></p><p><span>Exposure correction had little effect for hydrophilic compounds but substantially increased the apparent potency of hydrophobic, highly protein-bound alternatives. For example, BPAF and TCBPA ranked considerably higher when freely dissolved rather than nominal concentrations were used. The findings show that accounting for actual </span><i><span>in vitro</span></i><span> exposure is essential for reliable hazard assessment of BPA alternatives.</span></p><blockquote><h4><strong>The original article</strong></h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/crtoec/article/doi/10.1021/acs.chemrestox.6c00300/5442587/In-Vitro-Exposure-Assessment-Improves-Hazard"><strong>In Vitro Exposure Assessment Improves Hazard Ranking of Bisphenol A Alternatives</strong></a></h4><p>Luise Henneberger*; Sophia Bardehly; Arina Kremser; Vanessa Srebny; Beate I. Escher</p><p>Chem. Res. Toxicol. (2026)&nbsp;</p><div><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acs.chemrestox.6c00300">https://doi.org/10.1021/acs.chemrestox.6c00300</a></div><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6></blockquote><p><strong>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.</strong></p><div><div><div><div><div><div><div><p>Bisphenol A (BPA) was used for decades as a plastic chemical, but due to its endocrine disrupting properties it is now banned for food-contact materials (<a href="javascript:;">1</a>) and thermal paper (<a href="javascript:;">2</a>) in the European Union. Numerous BPA replacement chemicals have therefore been developed, but for most of them effects to human and environmental health have not been thoroughly assessed. (<a href="javascript:;">3, 4</a>) Cell-based <i>in vitro</i> bioassays allow a fast screening for adverse effects and can help to identify which alternatives show similar toxicity compared to BPA itself and which ones can be considered more benign. Numerous <i>in vitro</i> toxicity studies have been published, but the majority has either focused on only a few of the BPA alternatives or few specific end points like estrogenicity and androgenicity (<a href="javascript:;">5, 6</a>) or adipogenesis. (<a href="javascript:;">7, 8</a>) The most comprehensive <i>in vitro</i> toxicity datasets for BPA and its alternatives can be found in the recent publication by Srebny et al. (<a href="javascript:;">9</a>) that reports data from six assays for BPA and 26 alternatives and in the Tox21 database, which contains data from various assays for BPA and 15 alternatives as reviewed by Adamovsky et al. (<a href="javascript:;">4</a>)</p></div><div><p>To date, comparative hazard assessment using <i>in vitro</i> toxicity data for BPA and its alternatives was exclusively done based on nominal effect concentrations. (<a href="javascript:;">9−12</a>) However, nominal concentrations do not necessarily correlate with the bioactive concentration, because the test concentration of the chemicals added at the beginning of the bioassay can be reduced by plastic sorption, volatilization, cellular metabolism, abiotic degradation and binding to cell culture medium constituents (<a href="javascript:;">Figure 1</a>). (<a href="javascript:;">13</a>) Some of these binding and loss processes are only relevant for certain compound classes or assay types. For example, plastic sorption was shown to be negligible for assays that use a high serum content, i.e., 10% fetal bovine serum (FBS) in 384-well plates. (<a href="javascript:;">14</a>) Furthermore, many cell lines applied in high-throughput screening assays (e.g., HEK293 cells) have limited metabolic capacity, which makes this loss process also less relevant. (<a href="javascript:;">15</a>) Volatility can be evaluated using the medium-air partition constant (<i>K</i><sub>medium/air</sub>) of the test chemicals. Previous studies have shown that chemicals with log <i>K</i><sub>medium/air</sub> &lt;4 are lost from the assay plates or even cross-contaminate neighboring wells and cannot be tested in open well plate formats. (<a href="javascript:;">16, 17</a>)</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Chem_Res_Toxicol_2026_Figure_1_Binding_and_loss_processes_in_cell_based_in_vitro_bioassays_9255cda1ae.png" alt="Chem. Res. Toxicol. (2026): Figure 1. Binding and loss processes in cell-based in vitro bioassays and their relevance for BPA and its alternatives. Created in BioRender. Henneberger, L. (2026) " srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Chem_Res_Toxicol_2026_Figure_1_Binding_and_loss_processes_in_cell_based_in_vitro_bioassays_9255cda1ae.png 192w," sizes="100vw" width="750" height="608"></p><div><p>Following these considerations, previous studies identified abiotic stability as a key parameter for <i>in vitro</i> exposure assessment that should always be measured in the first place. (<a href="javascript:;">18−20</a>) Unfortunately, stability measurements in bioassay medium are rarely published. For BPA and its two alternatives Bisphenol S (BPS) and F (BPF), medium concentrations were recently measured over time by Brenner et al. (<a href="javascript:;">21</a>) and were found to be stable for 24 h. The diglycidyl ether of BPA (BADGE) was reported to hydrolyze quickly in aqueous solution, (<a href="javascript:;">9, 22, 23</a>) which will occur in medium as well. However, for most BPA replacement chemicals the stability in assay medium is not known.</p></div><div><p>For stable compounds the binding to medium proteins and lipids will determine the bioavailable concentration. In previous studies, free fractions (<i>f</i><sub>free</sub>) in cell culture medium were measured for BPA, BPS and BPF, (<a href="javascript:;">21, 24</a>) but experimental data for other BPA alternatives are lacking. Prediction of <i>f</i><sub>free</sub> is also possible with a variety of different <i>in vitro</i> distribution models, (<a href="javascript:;">13</a>) but the required chemical input parameters for protein and lipid binding are also not available experimentally for the majority of the BPA alternatives, which may lead to high prediction errors.</p></div><div><p>To address the aforementioned data gaps, BPA and 24 of its alternatives with diverse physicochemical properties were tested for their chemical stability in cell culture medium in the present study. For the subset of stable chemicals, binding to bovine serum albumin (BSA) and phospholipid membranes was quantified. BSA is the most abundant protein in FBS and can be considered to be the predominant sorptive phase for BPA and its alternatives. (<a href="javascript:;">25−27</a>) The measured distribution ratios to proteins and lipids were used to validate the quantitative structure-activity relationships (QSARs) reported in previous studies (<a href="javascript:;">28, 29</a>) that allow a prediction of protein and lipid binding from the octanol-water partition constant (<i>K</i><sub>ow</sub>) and speciation of the BPA alternatives. Additionally, <i>f</i><sub>free</sub> in cell culture medium was measured and compared to <i>f</i><sub>free</sub> predicted using a simple mass balance model. (<a href="javascript:;">30</a>) The experimental <i>f</i><sub>free</sub> were used to calculate relative effect potencies (REPs) of the BPA alternatives based on freely dissolved concentrations and the derived REPs were compared to published REPs based on nominal concentrations.</p></div></div><h4><strong>Materials and Methods</strong></h4><h5><strong>Instrumental Analysis</strong></h5><p>Chemical concentrations in the PBS samples after SPME extraction, in the corresponding sample extracts, as well as in the samples from the stability and membrane partitioning experiments were determined using a liquid chromatography instrument (<a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/en-us/lcms/library?mainauthorItems=10&amp;search=1260+Infinity+II"><strong>1260 Infinity II, Agilent</strong></a>) coupled to either a fluorescence detector (<strong>1100/1200 FLD</strong>), a diode array detector (DAD) or a triple quadrupole mass spectrometer (<a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/en-us/lcms/library?mainauthorItems=10&amp;search=6420"><strong>Triple Quad 6420, Agilent</strong></a>). Further details on the instrumentation including column types, instrumental conditions and limits of quantification (LOQs) are provided in the <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/crtoec/article-supplement/5442587/pdf/tx6c00300_si_001/">SI Tables S2-4</a>. Calibration standards with a concentration range of 1–10.000 ng/mL in desorption solvent or PBS (for the determination of isotherms, pin-water distribution and membrane-water partitioning) and acetonitrile blanks were measured together with the samples. The PBS samples were diluted 1:1 or 1:10 with acetonitrile according to the composition of the calibration standards.</p><h4><strong>Results</strong></h4><h5><strong>Stability Measurements in Bioassay Medium</strong></h5><p>BPA and 22 of its alternatives were found to be stable in bioassay medium (OptiMEM base medium supplemented with 2% cs-FBS). The total medium concentration for these chemicals determined after 48 h of incubation at 37<span>°</span>C differed less than 20% from the total medium concentration determined in the freshly prepared medium (<a href="javascript:;">Fig. 2</a>). For the most hydrophobic test chemical Bisphenol PH (BPPH) losses of 37% and 10% were observed in assay medium with 2% and 10% FBS, respectively. BADGE was completely degraded within 48 h in both types of media (<a href="javascript:;">Fig. 2</a>).</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Chem_Res_Toxicol_2026_Figure_2_Stability_of_BPA_and_24_alternatives_f4ada9114e.png" alt="Chem. Res. Toxicol. (2026): Figure 2. Stability of BPA and 24 alternatives at 37°C in assay medium (OptiMEM supplemented with 2% cs-FBS) expressed as %loss after 48 h, different symbols indicate independent experimental replicates. Stability of BADGE and BPPH was also tested in assay medium consisting of 90% DMEM with 10% untreated FBS." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Chem_Res_Toxicol_2026_Figure_2_Stability_of_BPA_and_24_alternatives_f4ada9114e.png 107w," sizes="100vw" width="750" height="1090"></p><h5><strong>Bovine Serum Albumin Binding and Membrane-water Partitioning</strong></h5><div><p>The SPME method was successfully optimized for BPA and 15 of the alternatives. Binding to the pin coating as well as equilibration times were found to increase with increasing hydrophobicity (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/crtoec/article-supplement/5442587/pdf/tx6c00300_si_001/">Figure S4</a>). Chemical recoveries (sum of amount in PBS and pin coating) for the optimization experiments in PBS were high (76-111%). Several chemicals showed nonlinear sorption to the pin coating, which had to be considered in the <i>C</i><sub>free</sub> calculation (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/crtoec/article-supplement/5442587/pdf/tx6c00300_si_001/">Figure S3</a>). Despite the good performance of the SPME method, the testable concentration range was limited for some BPA alternatives due to high LOQs (e.g., 1 μg/mL for BPP and 0.5 μg/mL for BPZ).</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Chem_Res_Toxicol_2026_Figure_3_Comparison_of_measured_EXP_and_modelled_MBM_A_bovine_serum_albumin_water_distribution_ratios_d5813c1071.jpg" alt="Chem. Res. Toxicol. (2026): Figure 3. Comparison of measured (EXP) and modelled (MBM) (A) bovine serum albumin-water distribution ratios (DBSA/w) and (B) membrane or liposome-water distribution ratios (Dm/w or Dlip/w), (C) dependence of measured (EXP) and modelled (MBM) free fractions (ffree) in assay medium (OptiMEM base medium +2% cs-FBS) of the test chemicals on the membrane-water distribution ratio (Dm/w), (D) comparison of average measured (EXP) and modelled (MBM) ffree, (E) comparison of measured freely dissolved concentrations (Cfree) with dosed nominal concentration (Cnom), (F) comparison of measured (EXP) and modelled (MBM) Cfree. Solid lines in A, B, D, E and F indicate the 1:1 line, the dotted lines indicate the 1:10 and 10:1 lines. AAFE (absolute average fold error)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Chem_Res_Toxicol_2026_Figure_3_Comparison_of_measured_EXP_and_modelled_MBM_A_bovine_serum_albumin_water_distribution_ratios_d5813c1071.jpg 216w," sizes="100vw" width="1600" height="1158"></p></div><div><p>Binding to BSA was measured for BPA and 15 of the alternatives (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/crtoec/article-supplement/5442587/pdf/tx6c00300_si_001/">Figure S5</a>, abbreviations in <a href="javascript:;">Table 1</a>). For BPF, BPAF, BP-MIBK, BPA, BPS-MAE and D8 binding isotherms were linear, while for BPT, BPZ, BzBP, Bz, BPAP, BPB, BPE and BPS-MPE binding to BSA was concentration-dependent (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/crtoec/article-supplement/5442587/pdf/tx6c00300_si_001/">Table S6</a>). <a href="javascript:;">Table 2</a> reports the mean <i>D</i><sub>BSA/w</sub>. BPS and 24BPS showed weak affinities for BSA. For BPS <i>D</i><sub>BSA/w</sub> could only be determined at one concentration level and no isotherm was fitted. For 24BPS no binding could be measured at any of the tested concentrations indicating <i>f</i><sub>bound</sub> &lt; 20% at the tested BSA concentration of 1.56 g/L. <a href="javascript:;">Figure 3</a>A shows a comparison of the measured <i>D</i><sub>BSA/w</sub> with the values predicted from log <i>K</i><sub>ow</sub> by Srebny et al. (<a href="javascript:;">9</a>) Because regression models based on log <i>K</i><sub>ow</sub> do not consider concentration dependence, the average <i>D</i><sub>BSA/w</sub> was calculated and used for the comparison in <a href="javascript:;">Fig. 3</a>A even for the chemicals with nonlinear binding isotherms, resulting in generally larger error bars (see also <a href="javascript:;">Table 2</a>). On average, the measured <i>D</i><sub>BSA/w</sub> agreed with the modelled values within a factor of 4.7 (AAFE). For most chemicals the measured and predicted <i>D</i><sub>BSA/w</sub> agreed within a factor of 7. For benzylparabene (BzBP), BPS-MAE and D8 the measured <i>D</i><sub>BSA/w</sub> were a factor of 17, 24, and 35 higher compared to the value predicted from log <i>K</i><sub>ow</sub>. For BPA the measured log <i>D</i><sub>BSA/w</sub> of 3.57 agrees well with literature data (<a href="javascript:;">29</a>) (<i>D</i><sub>BSA/w</sub> = 3.65). This literature value was also used for the comparison in <a href="javascript:;">Figure 3</a>A.</p></div><h4><strong>Conclusion</strong></h4><div><p>The same concept as applied in the present study can also be used for other classes of chemicals. Abiotic stability should always be measured first. For unstable chemicals hazard assessment will need to include the formed transformation products as well and a concentration metric that considers temporal changes (e.g., area under the curve). The same applies to stable chemicals if cell lines with high metabolic capacity are used for <i>in vitro</i> testing (e.g., HepG2 or HepaRG). For stable and not quickly metabolized chemicals EC<sub>free</sub> should be used for hazard ranking, if the chemicals show significant binding to medium proteins and lipids or high chemical diversity like the BPA alternatives investigated in the present study. Simple prediction models can provide a fast alternative to experimental determination of <i>f</i><sub>free</sub>, but they rely on the quality of the used chemical input parameters and are less accurate for organic acids that show nonlinear protein binding.</p></div><div><p>Finally, for IVIVE, metabolism of the BPA alternatives needs to be considered as well. Metabolization does not only differ between BPA and its alternatives, but also between different cell lines and between <i>in vitro</i> and <i>in vivo</i>. For mechanistic understanding of metabolism and its influence on toxicity it might be advisible to use a defined external metabolic system as used by Srebny et al. (<a href="javascript:;">9</a>) with non-metabolically competent cell lines before testing directly in metabolically competent cells. Metabolization generally decreases estrogenicity and cytotoxicity of BPA and its alternatives, (<a href="javascript:;">9</a>) but data for other end points are largely missing.</p></div></div></div></div></div></div></div>

In Vitro Exposure Assessment Improves Hazard Ranking of Bisphenol A Alternatives

<p><span><strong>This study establishes an integrated workflow for quantitative whole-animal elemental imaging of </strong></span><i><span><strong>Caenorhabditis elegans</strong></span></i><span><strong> by LA-ICP-TOF-MS. Optimized on-slide preparation using petrolatum improved specimen preservation, while oversampling during laser ablation enabled a spatial resolution of 2 μm.</strong></span></p><p><span>Quantitative elemental maps were generated using matrix-matched gelatin standards designed to mimic tissue ablation depth. The workflow addresses key challenges in sample handling, preservation, spatial resolution, and quantification, expanding the use of LA-ICP-TOF-MS for microscale metal biology and other small model organisms.</span></p><blockquote><h4><strong>The original article</strong></h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/ancham/article/98/38/28440/5431951/On-Slide-Preparation-of-Caenorhabditis-elegans"><strong>On-Slide Preparation of Caenorhabditis elegans toward Quantitative, High-Resolution LA-ICP-TOF Mass Spectrometry Imaging</strong></a></h4><p>Aidan J. Reynolds# ; Aaron Sue# ; Keith MacRenaris; Thomas V. O’Halloran* ; Tian Autumn Qiu*</p><p>Anal. Chem. (2026) 98 (38): 28440–28453</p><div><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acs.analchem.6c04271">https://doi.org/10.1021/acs.analchem.6c04271</a></div><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6></blockquote><p><strong>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.</strong></p><div><div><div><div><div><div><p>In this study, we established an integrated workflow consisting of sample preparation, imaging, and quantitative data analysis for whole-animal elemental imaging of <i>C. elegans</i> by LA-ICP-TOF-MS using petrolatum as a preferred embedding medium and achieved 2-μm spatial resolution with laser ablation oversampling. Compared to water-based embedding media, hydrocarbon-based petrolatum showed excellent preservation of <i>C. elegans</i> while retaining sufficient optical transparency for microscopy visualization, possessed the necessary nonvolatility for long-term storage, and was elementally pure for high-quality LA-ICP-MS imaging. With the assistance of a home-designed 3D-printed tool, uniform layer media application tool (ULMAT), embedding medium was applied as a uniform layer on top of charged microscope slides for on-slide embedding, freezing, and subsequent elemental analysis of <i>C. elegans</i>. An optional and additional step was also used to extract embedded worms from the embedding media layer to prepare petrolatum-coated worms. Comparisons between petrolatum-embedded worms and petrolatum-coated worms were also made to evaluate sample integrity and imaging data quality. Through several time-series experiments at ambient conditions, we evaluated sample integrity under different sample preparation approaches to determine optimal imaging time frames such that tissue decay and metal delocalization did not impede the accuracy of elemental imaging. We then characterized LA-ICP-TOF-MS imaging artifacts indicative of the stages of sample desiccation. We applied an oversampling technique in LA-ICP-TOF-MS imaging and achieved a 2-μm spatial resolution, providing high resolution imaging of <i>C. elegans</i> elemental content. Finally, using metal-spiked gelatin standards and ImageJ-based data analysis pipeline, we developed quantitative LA-ICP-TOF-MS imaging workflow and successfully produced quantitative elemental profiling of key biological elements in <i>C. elegans</i>.</p><h4><strong>Experimental Section</strong></h4><h5><strong>Microscopy and LA-ICP-TOF-MS Imaging</strong></h5><p>After sample processing including embedding, freezing and optional desiccation, brightfield, GFP and tdTomato fluorescence imaging of embedded and coated worms were taken using a Zeiss Axio Imager.Z2 upright fluorescence microscope. Embedded and coated worms were then taken for elemental analysis by LA-ICP-TOF-MS imaging using an ESL Bioimage 266 nm laser ablation system coupled to a TofWerk icpTOF S2. Samples were ablated with a laser power of 90% using a 5-μm spot size and a 3-μm overlap. Imaging cup and imaging chamber flow rates were set to 200 mL/min and 250 mL/min, respectively, using helium gas. Laser repetition rate was set to 125 Hz with a washout time of 10 ms. Plasma, auxiliary, and nebulizer gas flow were set to 14.0 L/min, 8.0 L/min, and 0.9–1.0 L/min, respectively. Measurement mode was kinetic energy discrimination (KED) using a helium gas flow of 5 mL/min. Time-of-flight elemental analysis was done using a <i>m</i>/<i>z</i> range of 14–256 at a mass resolving power of 1000. All essential microscopy and LA-ICP-TOF-MS parameters for <i>C. elegans</i> imaging are shown in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/ancham/article-supplement/5431951/pdf/ac6c04271_si_001/">Table S2</a> and <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/ancham/article-supplement/5431951/pdf/ac6c04271_si_001/">Table S3</a>.</p><h4><strong>Results and Discussion</strong></h4><h5><strong>Selection of Embedding Media To Preserve C. elegans Structural Integrity</strong></h5><p>Preparation of tissue samples for LA-ICP-MS imaging requires preserving tissue morphology and native distribution of elements over possibly hours of instrumental run time. Traits for an ideal embedding medium should be elementally pure to introduce none-to-little background noise, be optically transparent for microscopy, be capable of preserving <i>C. elegans</i> over hours, and not be difficult for the laser to ablate through. We first evaluate the capability of embedding media and application approach to preserve <i>C. elegans</i> structural integrity postprocessing (i.e., after embedding, freezing, and optional desiccation) (<a href="javascript:;">Figure 2</a>A). Two types of embedding media were tested, including aqueous-based hydrophilic media such as OCT and wax-like hydrophobic media such as petrolatum. An isogenic strain of N2, SJ4144, with GFP fluorescence reporters in intestinal cells, was used to better visualize tissues, and brightfield and fluorescence microscopy were used to evaluate tissue morphology.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Anal_Chem_2026_98_38_28440_28453_Figure_2_Summary_of_three_C_elegans_sample_preparation_and_short_term_visualization_of_processed_C_elegans_74a4294b6d.png" alt="Anal. Chem. (2026) 98 (38): 28440–28453: Figure 2. Summary of three C. elegans sample preparation and short-term visualization of processed C. elegans from different preparation. (A) A combined C. elegans embedding workflow for OCT-embedding, petrolatum-embedding, and petrolatum-coating. A synchronized population of day 1 gravid adult C. elegans was rinsed with M9 and transferred to an unseeded NGM plate to let the extra M9 buffer absorb into nematode growth media prior to the embedding workflows. (B) Appearance of day 1 gravid adult SJ4144 C. elegans embedded in OCT, embedded in petrolatum, and coated in petrolatum observed before and after a 1.5-hour time period, visualizing differences in brightfield, GFP fluorescence, and preservation effectiveness. Brightfield black contrast was set to 0 for all microscopy images. OCT-embedded and petrolatum-coated brightfield white contrast was set to 16 000 and 9000, respectively. Petrolatum-coated brightfield white contrast was set to 6000. Green fluorescence black contrast was set to 150, and white contrast was set to 1500 for all microscopy images. White scale bars in microscopy images represent 100 μm." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Anal_Chem_2026_98_38_28440_28453_Figure_2_Summary_of_three_C_elegans_sample_preparation_and_short_term_visualization_of_processed_C_elegans_74a4294b6d.png 245w," sizes="100vw" width="1289" height="789"></p><p>We found that the tested water-based embedding media were ineffective in preventing embedded <i>C. elegans</i> from desiccation and subsequent structural deformation (<a href="javascript:;">Figure 2</a>B, <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/ancham/article-supplement/5431951/pdf/ac6c04271_si_001/">Figure S2</a>). In OCT-embedded worms, the morphology and fluorescence reporter were clearly preserved immediately after the slide was taken out of the freezer (hour 0); however, signs of desiccation appeared after 1.5 h (<a href="javascript:;">Figure 2</a>B). Hygroscopic dopants such as glycerol can be added to improve water retention capabilities of these media; however, these molecules typically function as osmolytes (<a href="javascript:;">40</a>) or penetrating cryoprotectants. These additives likely displaced water from tissues, resulting in more rapid tissue desiccation. Compared to OCT, fish gelatin and glycerol-doped CMC showed better preservation of reporter fluorescence and worm body, while the wrinkled appearance of the worm cuticle indicated deformation (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/ancham/article-supplement/5431951/pdf/ac6c04271_si_001/">Figure S2</a>).</p><h5><strong>Simultaneous Mapping of Multiple Elements by High-Resolution LA-ICP-TOF-MS</strong></h5><div><p>The LA-ICP-TOF-MS technology provides the capability to image most elements on the periodic table simultaneously with the TOF analyzer. Using this technology, we successfully demonstrated simultaneous mapping of biologically relevant elements such as <sup>31</sup>P, <sup>55</sup>Mn, <sup>56</sup>Fe, <sup>63</sup>Cu, and <sup>66</sup>Zn in <i>C. elegans</i>, exceeding previous quadrupole-based LA-ICP-MS studies on <i>C. elegans</i> with one or two elements and their respective isotopes. (<a href="javascript:;">31−33</a>) The metallome of <i>C. elegans</i> consists of Na, Mg, K, Ca, Mn, Fe, Cu, and Zn representing the most abundant metals, and lower-abundance metals such as Co, Mo, and Se. (<a href="javascript:;">53−56</a>) While LA-ICP-TOF-MS as a technology provided simultaneous visualization of all relevant elements in analyzed <i>C. elegans</i>, in this work, we present the five most abundant biologically relevant elements, particularly for later quantitative analysis, for their biological importance.</p></div><div><p>We visualize <sup>31</sup>P, <sup>55</sup>Mn, <sup>56</sup>Fe, <sup>63</sup>Cu, and <sup>66</sup>Zn simultaneously in day 1 gravid adult <i>C. elegans</i> prepared by both petrolatum coating (<a href="javascript:;">Figure 4</a>A) and petrolatum embedding (<a href="javascript:;">Figure 4</a>B). By comparing elemental images with brightfield microscope images, we were able to observe discrete visualization of <i>C. elegans</i> anatomy for the assignment of elemental localization. In petrolatum-coated worms, <sup>31</sup>P was found throughout the worm’s body and most strongly accumulated in the intestinal cells and posterior region of the worm. <sup>31</sup>P is an essential element in biomolecules such as DNA, phospholipids, energy-storage molecules such as ATP, and as such, its abundance can reflect the cell size and activity across regions of tissue. (<a href="javascript:;">57</a>)<sup>55</sup>Mn exhibited the most discrete localization of imaged elements throughout the intestinal tract of <i>C. elegans</i> with a punctate pattern. This defined distribution of <sup>55</sup>Mn has previously only been visualized using synchrotron-XFM technology and was first achieved in this work with LA-ICP-MS imaging. (<a href="javascript:;">52</a>)<sup>56</sup>Fe showed some increased background noise due to the presence of the isobaric interference ArO<sup>+</sup>; however, we used kinetic energy discrimination within the collision cell and improved <sup>56</sup>Fe signals. <sup>56</sup>Fe and <sup>55</sup>Mn showed higher relative abundances in the anterior intestine in likely the grinder, with significant localization to the intestinal cells throughout the worm body. Meanwhile, <sup>56</sup>Fe did not exhibit a punctate distribution like <sup>55</sup>Mn throughout intestinal cells. While an essential element, <sup>63</sup>Cu was found in low abundance throughout <i>C. elegans</i>, potentially due to the organism’s reduced use of the element in biological processes. (<a href="javascript:;">58</a>) Improved imaging of <sup>63</sup>Cu can be achieved by using LA-ICP-QQQ-MS imaging if copper biology is of interest. While <sup>55</sup>Mn and <sup>56</sup>Fe showed enrichment in <i>C. elegans</i>’ anterior intestine, <sup>66</sup>Zn showed little or no enrichment in the anterior region of <i>C. elegans</i> and instead showed increased abundance in the posterior region of imaged worms. Synchrotron-XFM imaging has revealed similar zinc distributions with posterior intestinal cells on day 5 <i>C. elegans</i> possessing high zinc content; (<a href="javascript:;">52</a>) however, our 2-μm spatial resolution is not sufficient to resolve the zinc-rich gonad arms from the intestine. Additionally, the orientation of <i>C. elegans</i> during imaging may confound resolving the gonad arms from the intestine. We also noticed that images from petrolatum-coated worms showed a noisier background compared to others (<a href="javascript:;">Figures 2</a>B and <a href="javascript:;">4</a>A). One likely reason is that the laser was directly ablating the bare glass slides without embedding media on top of it and therefore introduced more interfering signals from glass slides contaminants such as <sup>56</sup>Fe from iron oxide (Fe<sub>2</sub>O<sub>3</sub>) and <sup>66</sup>Zn from zinc oxide (ZnO). Additionally, metal contamination could be introduced during the transfer step in creating petrolatum-coated worms and increased background noises. Metal impurities may be found in adhesives such as glue, (<a href="javascript:;">59, 60</a>) which was used to construct the eyelash tool for transferring worms. Repeated cleaning of the eyelash pick between transfers using 70% ethanol solutions may result in a slow dissolution of the glue, which may have subsequently resulted in leaching of metal impurities onto the glass slide when dragged across the surface during transfer. Further investigations should be made to examine possible sources of metal contamination during sample preparation.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Anal_Chem_2026_98_38_28440_28453_Figure_4_Brightfield_microscopy_and_elemental_imaging_of_day_1_gravid_adult_wildtype_N2_C_elegans_9bdf0f4129.jpg" alt="Anal. Chem. (2026) 98 (38): 28440–28453: Figure 4. Brightfield microscopy and elemental imaging of day 1 gravid adult wildtype N2 C. elegans prepared by petrolatum-based methods. (A) Brightfield microscopy and LA-ICP-TOF-MS imaging of a petrolatum-coated C. elegans. (B) Brightfield microscopy and LA-ICP-TOF-MS imaging of a petrolatum-embedded C. elegans. LA-ICP-TOF-MS imaging was able to perform simultaneous mapping of 31P and essential trace elements including 55Mn, 56Fe, 63Cu, and 66Zn. Lower limits for all elements were set to 0 ppm, and upper limits were set to 99th percentile for all elements except 55Mn which was set to 99.9th percentile. Brightfield black contrast and white contrast were set to 0 and 9000, respectively. White scale bars represent 100 μm." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Anal_Chem_2026_98_38_28440_28453_Figure_4_Brightfield_microscopy_and_elemental_imaging_of_day_1_gravid_adult_wildtype_N2_C_elegans_9bdf0f4129.jpg 221w," sizes="100vw" width="1600" height="1128"></p><p>When comparing petrolatum-coated elemental images (<a href="javascript:;">Figure 4</a>A) to petrolatum-embedded elemental images (<a href="javascript:;">Figure 4</a>B), several notable differences were observed. The background signal was drastically reduced in all ion images of petrolatum-embedded <i>C. elegans</i>. Elemental contaminants are present in glass slides, and with petrolatum serving as a barrier between the laser and the microscope slide, these contaminant signals are not observed through the direct ablation of glass. Additionally, minor differences in elemental localization were observed between petrolatum-embedded and -coated <i>C. elegans</i>. Across petrolatum-embedded replicates, elements that were found to be primarily intestinally localized in petrolatum-coated images showed somewhat broadened localization such as <sup>31</sup>P and <sup>56</sup>Fe. We also observed differences in concentration scaling for <sup>31</sup>P, <sup>55</sup>Mn, <sup>56</sup>Fe, <sup>63</sup>Cu, and <sup>66</sup>Zn across imaged replicates. Such variation could be attributed to embedding media matrix effects altering plume properties and impacting spatial clarity in imaging or could be due to biological variations. Future studies should focus on characterizing the impact of worm orientation, embedding media thickness, and developmental variations on the slight differences in metal distribution.</p></div><h4><strong>Conclusion</strong></h4><p>LA-ICP-MS imaging is an important imaging modality for metal biology research in model animals. Previous studies have performed LA-ICP-MS imaging on <i>C. elegans</i>, but the sample preparation and technological limitations made it challenging to achieve high-resolution, multi-element, and quantitative imaging. (<a href="javascript:;">31−33</a>) We developed an integrated workflow that includes sample preparation, instrumental analysis and data processing to enable high-resolution LA-ICP-TOF-MS imaging with quantification on <i>C. elegans</i>, providing a powerful tool for future metal biology research in this classical biology model. We first developed a 3D-printed tool to create a uniform and a thin layer of viscous embedding media on glass slide for on-slide embedding of <i>C. elegans</i>. Three different sample preparations, OCT-embedded, petrolatum-embedded and petrolatum-coated, were evaluated using microscopy and subsequent LA-ICP-MS imaging. We demonstrated that petrolatum preserved reporter fluorescence, tissue morphology, and elemental distribution for a longer period when compared to conventional embedding media such as OCT. Through oversampling laser shots, we were able to achieve an effective 2-μm spatial resolution in LA-ICP-MS imaging, allowing for resolving cellular elemental compartmentalization that has been previously only observed in XFM studies. (<a href="javascript:;">52, 72</a>) We further established a data analysis pipeline toward quantitative LA-ICP-TOF-MS imaging. With gelatin standards, we demonstrated quantitative imaging of five endogenous elements simultaneously in one sample, allowing colocalization of worm fluorescence reporters, brightfield images and different elements.</p></div></div></div></div></div></div>

On-Slide Preparation of Caenorhabditis elegans toward Quantitative, High-Resolution LA-ICP-TOF Mass Spectrometry Imaging

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