LC-MS/MS Analysis of Amyloid Beta Peptides in Artificial Cerebrospinal Fluid using the Xevo TQ Absolute for Clinical Research

Importance of the Topic

The reliable quantification of amyloid beta (Aβ) peptides in cerebrospinal fluid is critical for advancing Alzheimer’s disease research. Traditional immunoassays often face cross-reactivity and require separate assays for each analyte, leading to increased variability, time, and cost. A robust LC-MS/MS approach offers enhanced selectivity and multiplexing capability in a single analytical run.

Objectives and Study Overview

This study aimed to develop and validate an LC-MS/MS method using the ACQUITY Premier UPLC I-Class System coupled with the Xevo TQ Absolute Mass Spectrometer to quantify Aβ 1-38, 1-40, and 1-42 peptides in artificial cerebrospinal fluid over a concentration range of 0.1–10 ng/mL.

Methodology

Sample preparation involved spiking artificial CSF containing 4 % bovine serum albumin with calibration and QC levels, addition of an internal standard, denaturation with guanidine-HCl and phosphoric acid, and solid-phase extraction on Oasis MCX µElution plates. Eluates were evaporated under nitrogen, reconstituted in acetonitrile:water:ammonia, and injected onto the LC-MS/MS system. Chromatographic separation employed a BEH Peptide C18 column with a 0.3 % NH₄OH/water/acetonitrile gradient. Detection was performed in positive electrospray ionization mode.

Used Instrumentation

• ACQUITY Premier UPLC I-Class FTN System
• ACQUITY UPLC BEH Peptide C18, 2.1 × 150 mm, 1.7 µm, 300 Å column
• Xevo TQ Absolute Mass Spectrometer (positive ESI)

Main Results and Discussion

• Sensitivity: Signal-to-noise (S/N) >10 at the lowest calibrator (0.1 ng/mL) for all Aβ peptides across five runs.
• Linearity: Calibration curves showed r² >0.999 over 0.1–10 ng/mL in five analytical runs.
• Precision: Total and repeatability CVs for QC levels (0.2, 1.0, 7.5 ng/mL) were below 5 % (n=25).
• Accuracy: QC recoveries ranged from 96.5 % to 100.2 % relative to nominal concentrations.

Benefits and Practical Applications

This LC-MS/MS workflow delivers superior analytical selectivity, simultaneous multi-peptide quantification, and improved reproducibility compared to ELISA, reducing assay time and cost. It is well suited for biomarker studies in clinical research settings.

Future Trends and Possibilities

Broader adoption of LC-MS/MS for neurodegenerative biomarker panels, expansion to human CSF samples, integration with high-throughput automation, and coupling with multiplexed assays for comprehensive profiling of Alzheimer’s disease markers.

Conclusion

The validated method using ACQUITY Premier UPLC I-Class and Xevo TQ Absolute achieves high sensitivity, excellent linearity, precision, and accuracy for Aβ peptide quantification, supporting its application in Alzheimer’s disease clinical research.