ArticleAnalytical and bioanalytical chemistry2026
Evaluation of extraction capabilities of protease, proteinase K, and TMAH for nanoparticle characterization in biological CRMs by spICP-MS.
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Abstract
Reliable method development and validation rely on certified reference materials (CRMs) that ideally provide accurate analyte values independent of sample preparation and analytical methodology. Although inorganic nanoparticles (NPs) are increasingly being investigated in biological systems, biological matrix CRMs certified for NP characterization are not yet available. In this study, five biological CRMs (DOLT-4, DOLT-5, DORM-4, DORM-5, and TORT-3) were extracted using three commonly employed protocols based on proteinase K, protease, and tetramethylammonium hydroxide (TMAH). The resulting extracts were analyzed by single-particle ICP-MS (spICP-MS) to determine particle number concentrations (PNCs) and particle size distributions (PSDs) of Ag-, Al-, Cu-, Cr-, Fe-, Hg-, Se-, Si-, Ti-, and Zn-containing nanoparticles. Both PNCs and PSDs varied substantially among the extraction protocols, demonstrating that sample preparation is a major source of variability in NP characterization. In addition to differences in extraction performance, elevated NP backgrounds were observed for several elements in the TMAH reagent, highlighting the importance of evaluating reagent-derived contamination when interpreting spICP-MS results. Investigation of Hg- and Se-containing NPs in DOLT-4, DORM-5, and TORT-3 further demonstrated that incomplete tissue digestion and residual matrix fragments complicate particle identification and interpretation. Overall, this work provides a systematic comparison of commonly used extraction strategies for the analysis of naturally occurring NPs in biological CRMs, identifies current limitations associated with sample preparation, and provides guidance for future method development and the design of biological CRMs for NP characterization.
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