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

Importance of the Topic

The accumulation of amyloid beta peptides in the brain is a hallmark of neurodegenerative disorders such as Alzheimer disease and reliable measurement of these peptides in cerebrospinal fluid is critical for clinical research on disease progression and therapeutic development.

Objectives and Study Overview

This work demonstrates a sensitive and selective liquid chromatography tandem mass spectrometry method for the simultaneous quantitation of amyloid beta isoforms Aβ1-38 Aβ1-40 and Aβ1-42 in artificial cerebrospinal fluid using sample preparation based on solid phase extraction and multiple reaction monitoring detection.

Methodology and Sample Preparation

Calibrators and quality controls were spiked into a bovine serum albumin fortified artificial cerebrospinal fluid surrogate matrix. Samples were denatured with guanidine hydrochloride and phosphoric acid then extracted on mixed mode cation exchange solid phase extraction plates. Eluates were evaporated under nitrogen and reconstituted in a mixture of acetonitrile water and ammonia prior to analysis. Chromatographic separation employed a reversed phase peptide column with a gradient of aqueous ammonium hydroxide and acetonitrile.

Used Instrumentation

Instrumentation included the ACQUITY Premier UPLC I-Class FTN system coupled to the Xevo TQ Absolute Mass Spectrometer and an ACQUITY UPLC BEH Peptide C18 column. Detection was performed using optimized multiple reaction monitoring transitions for each peptide and corresponding internal standards.

Key Results and Discussion

The method achieved a lower limit of quantitation of 0.1 ng/mL with signal to noise ratios exceeding ten to one for all isoforms. Calibration curves were linear from 0.1 to 10 ng/mL with correlation coefficients above 0.999. Intra and interday precision was below five percent coefficient of variation and accuracy ranged from 96.1 to 100.4 percent across quality control levels.

Benefits and Practical Applications

This single LC-MS/MS assay eliminates the cross reactivity and batch variability limitations of immunoassays enables multiplexed measurement of multiple biomarkers in one run and reduces overall analysis time and cost for clinical research studies.

Future Trends and Opportunities

Further work may extend the assay to authentic cerebrospinal fluid clinical samples incorporate additional amyloid beta isoforms and integrate automation for higher throughput in biomarker discovery and pharmacodynamic studies.

Conclusion

The described LC-MS/MS workflow provides a robust sensitive and selective platform for quantification of amyloid beta peptides in artificial cerebrospinal fluid supporting its application in clinical research and therapeutic evaluation.

References

• Jensen M Hartmann T Engvall B Wang R Uljon SN Sennvik K Naslund J Muehlhauser F Nordstedt C Beyreuther K et al Quantification of Alzheimer amyloid beta peptides ending at residues 40 and 42 by novel ELISA systems Mol Med 2000 6 4 291 302
• Lachno DR Evert BA Maloney K Willis BA Talbot JA Vandijck M Dean RA Validation and clinical utility of ELISA methods for quantification of amyloid beta peptides in cerebrospinal fluid specimens from Alzheimer disease studies J Alzheimers Dis 2015 45 2 527 542
• Salcedo J Ph L Davey L Lame M Dunning C Chambers E Amyloid beta peptides quantification by SPE LC MS MS with automated sample preparation for preclinical research and biomarker discovery Waters Application Note 720006517