Accurate standardization and normalization of hepatic metal contents in Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS)

Background: LA-ICP-MS is a powerful methodology for the precise determination and quantification of metals in nearly any kind of sample [1]. We recently used this method to quantify metals in experimental models and clinical samples of Wilson's disease [2 – 3]. However, measured ion intensities of selected mass-to-charge (m/z) ratios, may vary considerably from measurement to measurement and underlie non-linear drift during time. Beside proper calibration, normalization strategies for measured traces of a specific ion to a well-characterized reference material are required. Other strategies use an endogenous reference element that ideally should have a homogenous distribution within the measured sample. However, normalization methods depend on the chosen experimental setup, the sample material analyzed, and are most often based on one or few isotopes or the total ion current.

Methods: We here compared different normalization methods that either used a separate reference value for each data point – constituting a pixel in the isotope image – or used a constant normalization factor per measurement run. For image generation and visualization of element concentrations, we used Microsoft Excel 2010 with the novel software tool “Excel Laser Ablation Imaging” that is based on Microsoft Excel Visual Basic for Applications [4]. Matrix-matched standards were produced from homogenized tissue that is homologous to the sample and spiked with different concentrations of standard solutions served for calibration.

Results: Best results were obtained using individual isotopes or isotope groups as reference. The total ion current was not suitable for inorganic mass spectrometry as Na and K which dominate the spectrum. In comparison to normalization with an overall factor, pixel per pixel normalization may increase the image noise, but it can be used to attenuate measurement errors and signal drift of long-lasting measurements. Isotopes with low signal-to-noise ratios may show an increased background noise after normalization, which can be significantly reduced by defining an area of interest. With these standardization and normalization we now are able to precisely quantify hepatic metal alterations that are observed in metal overload disease (e.g., Wilson's disease, hemochromatosis, zinc overdose).

Conclusions: The normalization in LA-ICP-MS measurements is essential to minimize deviations of element concentrations that might occur as the consequence of measurement-related fluctuations. The data sets we present show that proper normalization and the definition of an area of interest that is taken for quantification are powerful tools to obtain high-contrast isotope images with precise metal concentrations. These methods are helpful to quantify metal overload in hepatic diseases.

References:

[1] Susnea I, Weiskirchen R. Mass Spectrom Rev. 2015 Feb 11. doi: 10.1002/mas.21454 [Epub ahead of print].

[2] Boaru SG, Merle U, Uerlings R, Zimmermann A, Weiskirchen S, Matusch A, Stremmel W, Weiskirchen R. BMC Neurosci. 2014;15:98.

[3] Boaru SG, Merle U, Uerlings R, Zimmermann A, Flechtenmacher C, Willheim C, Eder E, Ferenci P, Stremmel W, Weiskirchen R. J Cell Mol Med. 2015;19:806 – 14.

[4] Uerlings R, Matusch A, Weiskirchen R. Int J Mass Spectrom. 2016;395:27 – 35.