Establishing pre-analytical requirements and maximizing peptide recovery in the analytical phase for mass spectrometric quantification of amyloid-β peptides 1-42 and 1-40 in CSF

Objectives: Amyloid-beta (A beta) peptides in cerebrospinal fluid (CSF), including A beta 42 (residues 1-42) and A beta 40 (residues 1-40), are utilized as biomarkers in the diagnostic workup of Alzheimer's disease. Careful consideration has been given to the pre-analytical and analytical factors associated with measurement of these peptides via immunoassays; however, far less information is available for mass spectrometric methods. As such, we performed a comprehensive evaluation of pre-analytical and analytical factors specific to A beta quantification using mass spectrometry. Methods: Using our quantitative mass spectrometry assay for A beta 42 and A beta 40 in CSF, we investigated the potential for interference from hemolysate, bilirubin, lipids, and anti-A beta-antibodies. We also optimized the composition of the calibrator surrogate matrix and A beta recovery during and after solid phase extraction (SPE). Results: There was no interreference observed with total protein up to 12 g/L, hemolysate up to 10% (v/v), bilirubin up to 0.5% (v/v), intralipid up to 1% (v/v), or anti-A beta-antibodies at expected therapeutic concentrations. For hemolysate, bilirubin and lipids, visual CSF contamination thresholds were established. In the analytical phase, A beta recovery was increased by similar to 50% via SPE solvent modifications and by over 150% via modification of the SPE collection plate, which also extended analyte stability in the autosampler. Conclusions: Attention to mass spectrometric-specific preanalytical and analytical considerations improved analytical sensitivity and reproducibility, as well as, established CSF specimen acceptance and rejection criteria for use by the clinical laboratory.

Automated summary

This study comprehensively evaluated pre-analytical and analytical factors for quantifying Aβ peptides using mass spectrometry. The results demonstrated minimal interference and improved analytical sensitivity and stability through method optimization, establishing acceptance and rejection criteria for CSF specimens in clinical laboratories.