High-Throughput Mass Spectrometry Assay for Quantifying β-Amyloid 40 and 42 in Cerebrospinal Fluid

Clin Chem. 2019 Dec;65(12):1572-1580. doi: 10.1373/clinchem.2018.300947. Epub 2019 Oct 18.


Background: The ratio of β-amyloid 1-42 (Aβ42) to Aβ40 in cerebrospinal fluid (CSF) may be useful for evaluating Alzheimer disease (AD), but quantification is limited by factors including preanalytical analyte loss. We developed an LC-MS/MS assay that limits analyte loss. Here we describe the analytical characteristics of the assay and its performance in differentiating patients with AD from non-AD dementia and healthy controls.

Methods: To measure Aβ42/Aβ40, we used unique proteolytically derived C-terminal peptides as surrogate markers of Aβ40 and Aβ42, which were analyzed and quantified by LC-MS/MS. The assay was analytically validated and applied to specimens from individuals with clinically diagnosed AD (n = 102), mild cognitive impairment (n = 37), and non-AD dementias (n = 22), as well as from healthy controls (n = 130). Aβ42/Aβ40 values were compared with APOE genotype inferred from phenotype, also measured by LC-MS/MS.

Results: The assay had a reportable range of 100 to 25000 pg/mL, a limit of quantification of 100 pg/mL, recoveries between 93% and 111%, and intraassay and interassay CV <15% for both peptides. An Aβ42/Aβ40 ratio cutoff of <0.16 had a clinical sensitivity of 78% for distinguishing patients with AD from non-AD dementia (clinical specificity, 91%) and from healthy controls (clinical specificity, 81%). The Aβ42/Aβ40 ratio decreased significantly (P < 0.001) with increasing dose of APOE4 alleles.

Conclusions: This assay can be used to determine Aβ42/Aβ40 ratios, which correlate with the presence of AD.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Aged
  • Alzheimer Disease / diagnosis
  • Alzheimer Disease / metabolism
  • Amyloid beta-Peptides / analysis*
  • Amyloid beta-Peptides / cerebrospinal fluid
  • Biomarkers / cerebrospinal fluid
  • Chromatography, Liquid / methods
  • Cognitive Dysfunction / diagnosis
  • Cognitive Dysfunction / metabolism
  • Dementia / diagnosis
  • Dementia / metabolism
  • Female
  • High-Throughput Screening Assays / methods
  • Humans
  • Male
  • Middle Aged
  • Peptide Fragments / analysis*
  • Peptide Fragments / cerebrospinal fluid
  • Sensitivity and Specificity
  • Tandem Mass Spectrometry / methods*
  • tau Proteins / cerebrospinal fluid


  • Amyloid beta-Peptides
  • Biomarkers
  • Peptide Fragments
  • amyloid beta-protein (1-40)
  • tau Proteins