Application Note: Quantification of Amyloid Beta Peptides in Human Cerebrospinal Fluid using Micro HPLC-HRMS

Amyloid peptides (Abeta; Aβ) result from cleavage of amyloid precursor protein (APP) by beta and gamma secretase.The 42-amino acid variant, amyloid β peptide 1–42 is a widely accepted key biomarker for Alzheimer’s disease (AD) together with the total tau (T-tau) and phosphorylated tau (P-tau) protein. Diagnostic accuracy of the Aβ 1–42 /Aβ 1–40 ratio in cerebrospinal fluid (CSF)
compared to the concentration of Aβ 1–42 alone was found to be even more significant.

Currently these peptides are analyzed routinely in CSF by immunoassays, but in recent years, liquid chromatography tandem mass spectrometry (LC-MS/MS) has been investigated for the quantification of Aβ1-38, Aβ1-40, and Aβ1–42.

[Blennow 2010, Lewczuk 2015, Lame 2011]

Figure 1: Solution structure of the Alzheimer’s disease amyloid beta-peptide (1-42)

By Antje Meyer (Probiodrug, Halle, Germany), Christoph
Siethoff, Filip Sucharski, and Matthias Orth ( Swiss BioQuant, Reinach, Switzerland).


HPLC system:

Analytical Pump:
Zirconium Ultra 410F (Prolab, Reinach, Switzerland)

PAL HTS autosampler (CTC Analytics, Zwingen, Switzerland)

Dilution and loading pumps:
Binary pumps 1200 (Agilent Technologies, Santa Clara, USA)

Trapping column: Gemini C18, 5 µm, Zircotrap, 1 mm ID x 8 mm (Phenomenex, Prolab)
Flow rate: 250 µL/min (50 µL/min Sample flow plus 200 µL/min dilution flow)

Analytical column: BEH C18, 300 A, 1.7 µm, 0.3 mm ID x 150 mm (Waters), 50°C
Phase A: water containing 0.2 % ammonia
Phase B: acetonitrile containing 0.2 % ammonia
Flow rate: 4 to 3 µL/min

Figure 2: LC gradient/flow profile

MS system:

LTQ Orbitrap XL (Thermo, Bremen, Germany)
Prolab µSource and Column oven

Scan mode:
Full scan from m/z 900 to 1200
Resolution at 15000
Positive ion mode


Figure 3: Instrumentation Setup with Load and Dilution Pumps, Autosampler, Analytical
Pump, and Mass Spectrometer

The CSF sample was prepared by immuno-precipitation enrichment, after which it contained 50% organic (ACN). 50 µL of the prepared sample were injected with the autosampler (figure 4). After switching the injector valve, the sample was transferred to the trapping column during 3 minutes using 50 µL/min flow through the injection loop, and an additional 200 µL/min H2O added after the loop to dilute the sample for trapping (figure 5).

Figure 4: Flow schematic – loading sample into sample loop
Figure 5: Flow schematic – transfer sample to trapping column
Figure 6: Flow schematic – switch trapping column into analytical flow path

The trapping column was then switched into the analytical LC flow path (figure 6) and extracted at an initial 4 µL/min flow rate, which was reduced to 3 µL/min after 8 minutes in order to broaden the peaks around the retention time of interest (figure 2).

Results and Conclusions

The linearity at the dynamic range of 100 is R² = 0.9917 (figure 7). The sensitivity at the chosen LLOQ of 200 pg/mL suggests a feasible detection limit below 20 pg/mL (figure 8). Reproducibility of both the area and the retention times (figure 12) is excellent.

Figure 7: Calibration Curve. Concentration range: 200 pg/mL to 20.0 ng/mL
Figure 8: LLOQ – Chromatogram (detail)
Figure 9: LLOQ – Chromatogram CSF sample – IP enrichment
Figure 10: LLOQ – Spectrum at 9.71 min.
Figure 11: LLOQ – Spectrum zoomed in at Abeta 1-40
Figure 12: Reproducibility – 8 sequential runs at LLOQ

The use of the combination of microflow HPLC and high resolution mass spectrometry for quantitation was demonstrated by the analysis of beta amyloid peptides 1-40 and 1-42 in human cerebrospinal fluid. Sensitivity in the low pg/mL range could be achieved by reducing the flow rate and the inner diameter of the HPLC column, but still without reducing the injection volume to get the real advantage of microflow HPLC-MS with respect to sensitivity gain. Quantitation by high resolution mass spectrometry was precise and robust, which allows us to use this method for a biomarker clinical study.


Blennow K, Hampel H, Weiner M, Zetterberg H. Cerebrospinal fluid and plasma biomarkers in Alzheimer disease. Nat Rev Neurol. 2010 Mar;6(3):131-44.

Lewczuk P, Lelental N, Spitzer P, Maler JM, Kornhuber J. Amyloid-β 42/40 cerebrospinal fluid concentration ratio in the diagnostics of Alzheimer’s disease: validation of two novel assays. J Alzheimers Dis. 2015;43(1):183-91.

Lame ME, Chambers EE, Blatnik M. Quantitation of amyloid beta peptides Aβ(1-38), Aβ(1-40), and Aβ(1-42) in human cerebrospinal fluid by ultra-performance liquid chromatography-tandem mass spectrometry. Anal Biochem. 2011


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