Element Speciation

Element Speciation

The group “Elements and Element Speciation” develops methods along our project demands and for future analytical challenges:

  • Elemental mapping: We use LA-ICP-MS for elemental mapping in different kinds of sample types, such as tissue samples, e.g. brain slices, or environmental samples like sediments from Yellowstone gush springs. Special challenges are analytical protocols for quantitative elemental mapping.
  • Element speciation:For speciation analysis several different coupling methods are developed and optimized based on LC-ICP-MS and LC-ICP-OES. The methods are widely applied in our research on neurodegenerative pathologies. A special focus lays on CE-ICP-MS methods in 2D-identification procedures, speciation of nanoparticles and especially for Iron Redox Speciation of Ferritin, Fe2+ and Fe3+, paralleled by GSH/GSSG differentiation, both helping to characterize oxidative stress and helping to explain steps of ferroptosis mechanism. See the video explanation of the method here.

Our new CE-ICP-DRC-MS based method for quantification of ferrous (Fe2+) and ferric (Fe3+-) iron in tissue and body fluids within ca. 300 seconds:

Background: Oxidative stress (OS) plays a crucial role in a number of serious diseases, especially neurodegenerative diseases of the brain, like Alzheimer's or Parkinson´s disease, as well as in cancer and diabetes (1-6). Oxidative stress is very often related to iron, but not simply to “total iron”, but to the intracellular ratio and balance of the redox couple Fe2+ and Fe3+. While Fe3+ is redox-inactive, reactive oxygen species (ROS) are a direct reaction product of the free Fe2+ ions. Bivalent iron ions (Fe2+) catalyze H2O2 cleavage, being accompanied by the production of hydroxyl radicals, which in turn leads to membrane lipid peroxidation (7,8).

While on the one hand there is a "stable" iron pool in the cells in which iron - mostly Fe3+ - is bound in thermodynamically stable complexes (for example metal chaperones), on the other hand there is a "labile" iron pool as loosely bound, cytoplasmic Fe2+, which easily can be released and is then involved in numerous redox reactions.

At the molecular level, Fe2+-generated ROS and peroxidized phospholipids have a high damaging potential to proteins, lipids and DNA (9,10), resulting even in a programmed necrotic cell death, known as "ferroptosis” (FPT) (11 , 12). Therefore, the quantitative determination of Fe2+ and Fe3+  - i.e. the quantitative iron redox speciation - is of outstanding importance in a broad spectrum of redox-related diseases.

The new CE-ICP-DRC-MS method: Already in 2014 (13) and 2017 (10) the working group “Elements and Element Speciation” under Bernhard Michalke initially developed an LC-ICP-MS based method for iron redox speciation. Although the method works very reliably, it was associated with a higher expenditure of time and material costs. Since capillary electrophoresis (CE) - also coupled to ICP-MS - is often used in the working group, it was logical to develop a fast and reliable, but cheaper method based on CE-ICP-MS coupling (14, 15). The method was first tested on cerebrospinal fluid samples from our research projects on Parkinsonism.

To expand the sample spectrum to other sample types such as cell lysates, our cooperation partner Dr. Vivek Venkataramani from the University of Göttingen (https://pathologie.umg.eu/forschung/redoxmetabolismus-in-tumoren-und-neuronen/) developed a specifically adapted sample preparation technique, where, among other things, the redox equilibrium is not disturbed during sample processing. In this joint cooperation already a series of various cell lysates from different research approaches were successfully operated together with Dr. Vivek Venkataramani. Now, further studies are planned that will show whether quantitative Fe2+ and Fe3+ determinations in biological samples (cell lysates, liquor, tumor samples) can provide information about the extent of Fe2+-mediated tissue damage (biomarkers for "tissue at risk “).

Figure: Electropherogram of Fe2+ and Fe3+ - analysis from an SH-SY5Y cell lysate sample (reproduced from 14).


1 Hare, D. J., M. Arora, N. L. Jenkins, D. I. Finkelstein, P. A. Doble, A. I. Bush. Is early-life iron exposure critical in neurodegeneration? Nat Rev Neurol. 11(9), 536-544 (2015).

2 Ashraf, A., M. Clark, P. W. So. The Aging of Iron Man. Frontiers in Aging Neuroscience. 10. doi.org/10.3389/fnagi.2018.00065 (2018).

3 Hare, D. J., Cardoso, B. R., Szymlek-Gay, E. A., Biggs B. A. Neurological effects of iron supplementation in infancy: finding the balance between health and harm in iron-replete infants. Lancet Child Adolesc Health. 2(2), 144-156 (2018).

4 Torti, S. V., Torti, F.M. Iron and cancer: more ore to be mined. Nat Rev Cancer. 13(5), 342–355 (2013).      

5. Asmat, U., Abad, K., Ismail, K. Diabetes mellitus and oxidative stress—A concise review. Saudi Pharm J. 24(5): 547–553 (2016); doi: 10.1016/j.jsps.2015.03.013

6. Giacco, F., Brownlee, M. Oxidative stress and diabetic complications. Circ Res. 107(9): 1058–1070 (2010). doi: 10.1161/CIRCRESAHA.110.223545

7. Kehrer, J. P. The Haber-Weiss reaction and mechanisms of toxicity. Toxicology. 149(1), 43-50 (2000).

8. Gaschler, M. M., Stockwell B. R. Lipid peroxidation in cell death. Biochemical and Biophysical Research Communications. 482(3), 419-425 (2017).

9. Michalke, B., Halbach S., Nischwitz V. JEM Spotlight: Metal speciation related to neurotoxicity in humans. Journal of Environmental Monitoring. 11(5), 939-954 (2009).

10.Solovyev, N., Vinceti, M., Grill, P., Mandrioli J., Michalke B. Redox speciation of iron, manganese, and copper in cerebrospinal fluid by strong cation exchange chromatography - sector field inductively coupled plasma mass spectrometry. Anal Chim Acta. 973, 25-33 (2017).

11. Dixon, S. J., Lemberg, K. M., Lamprecht, M. R., Skouta, R., Zaitsev, E. M., Gleason, C. E.,. Patel, D. N, Bauer, A. J., Cantley, A. M., Yang, W. S., Morrison, B., Stockwell, B. R. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 149(5), 1060-1072 (2012).

12. Stockwell, B. R., Friedmann, Angeli J. P., Bayir, H., Bush, A. I., Conrad, M., Dixon, S. J., Fulda, S., Gascón, S., Hatzios, S. K., Kagan, V. E., Noel, K., Jiang, X., Linkermann, A., Murphy, M. E., Overholtzer, M., Oyagi, A., Pagnussat, G. C., Park, J., Ran, Q., Rosenfeld, C. S., Salnikow, K., Tang, D., Torti, F. M., Torti, S. V., Toyokuni, S., Woerpel, K. A., Zhang, D. D. Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell. 171(2), 273-285 (2017).

13. Fernsebner, K.., Zorn, J., Kanawati, B., Walker A., Michalke, B. Manganese leads to an increase in markers of oxidative stress as well as to a shift in the ratio of Fe(II)/(III) in rat brain tissue. Metallomics 6(4): 921-931 (2014).

14. Michalke, B., Willkommen, D., Venkataramani V. Iron Redox Speciation Analysis Using Capillary Electrophoresis coupled to Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS) Frontiers in Chemistry, Vol. 7, article 136 (2019). doi: 10.3389/fchem.2019.00136

15. Michalke, B., Willkommen, D., Venkataramani, V. Quantification of iron redox species (Fe(II), Fe(III)) by capillary electrophoresis - inductively coupled plasma mass spectrometry (CE-ICP-MS), Jove-Journal of Visualized Experiments, 159 (2020)

  • Element determination in low-mass solid samples: We operate and develop element determination methods for solid samples with very low sample mass, unsuitable for digestion. For such samples, we use a modern ETV system hyphenated to ICP-OES with transient signal monitoring.
  • Quality control and reference materials: Aside, methods are developed and validated in the frame of our cooperation with the Institute for Reference Materials and Measurements of the European Joint Research Center“, for certification of a broad range of standard reference materials (CRM), for reference value determination within the German EQUAS system or within the workgroup “Analysis in biological materials” of the Deutsche Forschungsgemeinschaft.
Inductively Coupled Plasma Mass Spectrometer
Source: Helmholtz Zentrum München


Selected Publications:

Lucio, M., Barbir, R., Lovrenčić, M. V., Varžić, S. C., Ljubić, S., Duvnjak, L. S., ... & Vrček, I. V. (2020). Association between arsenic exposure and biomarkers of type 2 diabetes mellitus in a Croatian population: A comparative observational pilot study. Science of The Total Environment, 137575.

Violi, F., Solovyev, N., Vinceti, M., Mandrioli, J., Lucio, M., & Michalke, B. (2020). The study of levels from redox-active elements in cerebrospinal fluid of amyotrophic lateral sclerosis patients carrying disease-related gene mutations shows potential copper dyshomeostasis. Metallomics.

Michalke, B., Willkommen, D., & Venkataramani, V. (2019). Iron Redox Speciation Analysis Using Capillary Electrophoresis Coupled to Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS). Frontiers in Chemistry, 7, 136.

Lucio, M., Willkommen, D., Schroeter, M., Sigaroudi, A., Schmitt-Kopplin, P., & Michalke, B. (2019). Integrative Metabolomic and metallomic analysis in a case control cohort with Parkinson´ s disease. Frontiers in aging neuroscience, 11, 331.

Michalke, B., Kramer, M. F., & Brehler, R. (2018). Aluminium (Al) speciation in serum and urine after subcutaneous venom immunotherapy with Al as adjuvant. Journal of Trace Elements in Medicine and Biology, 49, 178-183.