Mass spectrometry approaches for the selection of primary calibrators in the development of reference method

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Abstract Background Neurofilament‐light chain (NF‐L) is a known early marker for neurodegeneration. This marker which is essential for the axonal structure, is quantifiable in biological fluids after neuronal damage but is not specific to a given pathology. Currently, the methods for detecting/quantifying this biomarker are mainly based on immunoassays. While the performance of immunoassay‐based assays is good in term of sensitivity and precision, mass spectrometry (MS) approaches are required to achieve standardisation. Those aiding to improved characterisation/quantification of the calibrator and potential for developing a reference measurement procedure traceable to the International System of Units. Furthermore MS has the potential of multiplexing NF‐L with other specific biomarkers for dementia. The characterization and quantification of a number of primary calibrators and their suitability for harmonization of immunoassay based results and development of a reference measurement procedure is here assessed. Method Microwaver hydrolysis and isotope dilution MS was used for quantification of NF‐L from different manufactures. An Orbitrap mass spectrometer (Q‐Exactive, Thermofischer) was used to provide high resolution intact protein analysis information. A QQQ mass spectrometer (Xevo TQ‐XS, Waters) was utilized to assess the potential of developing a reference measurement procedure for NF‐L. Result The quantitative analysis of the three NF‐L by amino acid analysis (AAA) showed for all samples discrepancy between the nominal concentrations and the experimental results with a general overestimation for the nominal concentration due to water and salt contents. Separation, identification and quantification of impurities present in the preparations was attempted to obtain a total protein content from the AAA and the NF‐L content from the AAA results corrected for the impurities. The sequence of NF‐L was verified by high resolution intact protein MS and tryptic digestion. A QQQ method was developed to monitor the tryptic digest peptides and assess their suitability to be used for the development of a reference measurement procedure. This was based on specificity, limits of detection and limits of quantification. Conclusion These studies allow us to characterize three NF‐L proteins from different suppliers and to perform a first selection of the most relevant ones as primary calibrators for the development of a reference method.

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