Amyloid Beta Peptides Quantification by SPE-LC-MS/MS with Automated Sample Preparation for Preclinical Research and Biomarker Discovery

Importance of the topic

Amyloid beta peptides are central to Alzheimer disease research and serve as critical biomarkers for early detection and therapeutic development. Traditional immunoassays suffer from cross-reactivity and variability, creating a demand for more selective, robust, and high throughput quantification methods. The combined use of solid phase extraction, ultra performance liquid chromatography, and tandem mass spectrometry delivers specificity and sensitivity suitable for endogenous concentration measurement in human cerebrospinal fluid.

Study objectives and overview

This study aims to refine and validate an SPE LC-MS/MS method for simultaneous quantitation of aβ peptides (1–38, 1–40, 1–42) in human CSF with reduced sample volume, faster throughput, and automated preparation. Performance was assessed against FDA bioanalytical guidelines and existing methods, evaluating sensitivity, accuracy, precision, and compatibility with automation platforms to support preclinical research and biomarker discovery.

Methodology and instrumentation

Sample preparation:

• 100 µL CSF or artificial CSF with 4 g/L BSA spiked with 15N internal standards
• Standard addition for calibration and quality controls
• Denaturation with guanidine-HCl, acidification with phosphoric acid, incubation steps at room temperature and 37 °C
• SPE using Oasis PRiME MCX µElution 96-well plates on Tecan Freedom EVO 100/4
• Load, wash, and elute with acidic and basic solvents, direct injection without evaporation

Chromatography and mass spectrometry:

• ACQUITY UPLC I-Class with fixed loop sample manager
• ACQUITY UPLC Peptide BEH C18, 300Å, 1.7 µm, 2.1 × 150 mm column at 55 °C
• Gradient 10–45% acetonitrile over 5.5 minutes, total run time 9 minutes, flow rate 0.2 mL/min
• Xevo TQ-S micro tandem quadrupole MS in positive ESI, MRM transitions for each peptide and 15N internal standard
• Capillary voltage 2.5 kV, desolvation 650 °C, cone gas 150 L/hr, desolvation gas 1000 L/hr

Used instrumentation

• Waters ACQUITY UPLC I-Class Fixed Loop System
• ACQUITY UPLC Peptide BEH C18 column, 300Å, 1.7 µm, 2.1 × 150 mm
• Waters Xevo TQ-S micro tandem quadrupole mass spectrometer
• Oasis PRiME MCX µElution Plates and collection plates
• Tecan Freedom EVO 100/4 automated pipetting platform
• MassLynx and TargetLynx software

Key results and discussion

The optimized method achieved baseline separation of three aβ isoforms within a 9 minute LC gradient and sustained column durability through over 800 injections. Carryover was eliminated by adjusting needle wash solvents. Samples remained stable in the autosampler for five days. SPE recoveries ranged from 86 to 101% with accuracy between 96 and 100% and precision ≤ 10% CV. The method LLOQ was 0.1 ng/mL using 100 µL of CSF, covering a linear range of 0.1 to 10 ng/mL. Measured endogenous levels in pooled CSF and certified reference material matched theoretical concentrations. Substitution of BSA for rat plasma as carrier protein yielded equivalent performance. Compared to earlier platforms, the Xevo TQ-S micro provided robust sensitivity at lower cost. Automated sample preparation on Tecan produced results statistically equivalent to manual processing, enabling high throughput and reproducibility.

Benefits and practical applications

• High specificity and sensitivity for low level peptide quantification
• Reduced sample volume and simplified workflow
• Fast throughput: 96 samples processed in under 90 minutes
• Full compliance with FDA bioanalytical validation guidelines
• Seamless integration with automation platforms for routine analysis
• Applicability to preclinical research, biomarker discovery, and quality control in pharmaceutical development

Future trends and opportunities

Further improvements may include expanded multiplexing of additional biomarkers, integration with ion mobility separation for isobaric discrimination, miniaturization of sample preparation, and coupling with high resolution mass spectrometry for deeper proteomic profiling. Standardization across laboratories and integration into clinical trial workflows will enhance the utility of LC-MS/MS for Alzheimer disease diagnostics.

Conclusion

An automated SPE-LC-MS/MS method using Oasis PRiME MCX µElution and Xevo TQ-S micro MS has been developed, offering robust, precise, and accurate quantification of aβ 1–38, 1–40, and 1–42 peptides in human CSF. The protocol minimizes sample volume, accelerates processing, and meets regulatory requirements, supporting high throughput biomarker analysis and advancing Alzheimer disease research.

References

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