Improved GLP-1 Receptor Agonist Peptide Recovery Using a QuanRecovery™ With MaxPeak™ High Performance Surfaces (HPS) Collection Plate
This is an Application Brief and does not contain a detailed Experimental section.
Abstract
GLP-1 (glucagon-like peptide-1) receptor agonist therapeutics are used to treat metabolic diseases like type 2 diabetes and obesity. However, analyzing them with liquid chromatography-mass spectrometry (LC-MS) can be challenging due to their large size and the hydrophobicity of their fatty acid conjugate moieties. A major issue with these peptides is non-specific binding (NSB), where the peptides interact with and adhere to surfaces or materials used during sample preparation, resulting in peptide loss and unreliable results. In this study, we demonstrate that the use of a Waters QuanRecovery with MaxPeak HPS 700 µL Collection Plate effectively reduces non-specific binding and improves recovery of GLP-1 peptides such as semaglutide, exenatide, liraglutide, and tirzepatide. Ultimately, this is an improvement in analyte sample handling that can help improve assay sensitivity and reliability.
Experimental
A stock solution mix of the GLP-1 (10 µg/mL) receptor agonist therapeutic peptides (Table 1) was prepared from individual peptide solutions (1 mg/mL) of semaglutide, liraglutide, exenatide, and tirzepatide. The organic composition of this stock solution mix was 80/20/1 water/acetonitrile/formic acid. LC-MS analysis samples of the combined peptides were prepared directly in the QuanRecovery with MaxPeak HPS 700 µL Collection Plates or standard polypropylene plates at 1 and 10 ng/mL concentrations in an organic composition of 80/20/1 water/acetonitrile/formic acid. A 4 minute reversed-phase LC analysis was performed using an ACQUITY™ UPLC Peptide CSH C18 Column and ACQUITY UPLC I-Class PLUS System with MS detection on a Xevo™ TQ-XS Tandem Quadrupole Mass Spectrometer operating with multiple reaction monitoring (MRM) scans. Peptide transitions are listed in Table 2. A 10 µL volume of the 1 and 10 ng/mL GLP-1 therapeutic peptide mixes were injected for analysis.
Table 1. Physiochemical properties of the GLP-1 receptor agonist therapeutic peptides, illustrating their large, hydrophobic, and acid nature. *higher number=more hydrophobic; †Contains a fatty acid conjugate.
LC Conditions
|
UPLC: |
ACQUITY UPLC I-Class PLUS System , FL with Column Manager (CMA) |
|
Mobile phase A: |
0.1% Formic acid in water |
|
Mobile phase B: |
0.1% Formic acid in acetonitrile |
|
Column: |
ACQUITY UPLC Peptide CSH C18 Column, 130 Å, 1.7 µm 2.1 mm x 50 mm (p/n: 186006933) |
|
Column temperature: |
65 °C |
|
Sample temperature: |
10 °C |
|
Injection volume: |
5–10 µL |
|
Analysis time: |
4 min |
|
WNW: |
90:10 Water:Acetonitrile+0.1% Formic Acid |
Gradient Table
MS Conditions
|
MS: |
Xevo TQ-XS Mass Spectrometer |
|
Capillary (kV): |
2 |
|
Cone voltage: |
32 V |
|
Desolvation temperature: |
500 °C |
|
Desolvation flow: |
1100 L/Hr |
Table 2. MRM transitions and collision energy for the GLP-1 receptor agonist therapeutic peptides.
Results and Discussion
Figure 1. Representative LC-MRM-MS chromatograms demonstrating mitigation of non-specific binding to collection plates for the GLP-1 receptor agonist therapeutic peptides, semaglutide, exenatide, liraglutide, and tirzepatide (1 ng/mL prepared in a solution of 80/20/1 water, acetonitrile, and formic acid) prepared in a QuanRecovery with MaxPeak HPS, 700 µL Collection Plate vs standard polypropylene sample collection plates.
Figure 2. Illustration of improved GLP-1 receptor agonist therapeutic peptide recovery using QuanRecovery with MaxPeak HPS Collection Plates vs standard polypropylene collection plates for 1 and 10 ng/mL mixes (Diluent Composition: 80/20/1 Water/Methanol/Formic Acid. MS response of standard plate was normalized to QuanRecovery with MaxPeak HPS Collection Plates MS response.
Conclusion
Use of QuanRecovery with MaxPeak HPS Collection Plates effectively mitigate NSB of the GLP-1 receptor agonist therapeutic peptides, significantly improving LC-MS detection and robustness of the assay.
Ordering Information
720008717, March 2025
