High-sensitivity quantitative analysis of therapeutic peptides in plasma using UPLC and Xevo TQ-S

High-sensitivity quantitative analysis of therapeutic peptides in plasma using UPLC and Xevo TQ-S

Robert S. Plumb, Gareth Booth and Paul D. Rainville

Waters Corporation, Milford, MA, USA

Application advantage

The Xevo TQ-S is a new tandem quadrupole mass spectrometer system that provides the highest sensitivity for peptide bioanalysis. Its fast data capture rate and dual-scan multi-reaction monitoring capability allow accurate quantification of narrow peaks generated by UPLC ® and accurate identification of peak characteristics.

Waters Solutions

XevoTM TQ-S
ACQUITY UPLC ®
ACQUITY UPLC BEH Column Chemistry
MassLynxTM in conjunction with TargetLynxTM

Key words

StepWaveTM, T-Wave, TM Peptide Bioanalytical, Desmopressin

Introduction

Polypeptides are increasingly being used as therapeutic drugs due to their high tolerance, target receptor selectivity and high potency properties. To accurately quantify these therapeutic peptides in biological fluids, selective separation techniques, high-resolution chromatography systems, and high-sensitivity detectors are required. A high-resolution chromatography system separates analytes of interest from endogenous polypeptides in plasma and blood, some of which may be ions of the same mass or fragment ions of the same mass. Since these therapeutic polypeptides mimic or mimic the activity of endogenous polypeptides, these endogenous and exogenous polypeptides must be distinguished during the assay.

StepWave ion transport technology

Traditional ion transport technology

Figure-1 UPLC/MS/MS quantitative analysis of desmopressin in rat plasma extracts using MRM mode. The chromatogram above shows the peak response of the new StepWave ion transport technology using the Xevo TQ-S; the lower chromatogram shows the peak response with conventional ion transport techniques.

Chemical structure of desmopressin

Although we observed multi-charged compounds and the molecular mass of the peptides was 1000-4000 amu, the detection of these peptides required the mass detection limit of 2000 m/z to be successfully analyzed. In this application note, for high-sensitivity peptide bioanalysis, we introduced the use of the ACQUITY UPLC system and the new high-sensitivity tandem quadrupole mass spectrometer Xevo TQ-S.

Experimental condition

Chromatography

Liquid Chromatography (LC) System: ACQUITY UPLC (Dual Solvent Management System, Sample Manager, HT Column Incubator)
Column conditions: ACQUITY UPLC BEH C 18 column, 1.7 micron, 2.1 × 50 mm column temperature: 40 °C
Gradient: Acetonitrile (containing 0.1% formic acid), eluted from 5% to 45% for more than 1.5 minutes Flow rate: 450 μl/min Injection volume: 10 μl

Mass spectrometry condition

Mass Spectrometry System: Xevo TQ-S and Xevo TQ operate in electrospray positive ion mode
MRM data acquisition 535 => 328
Voltage: Optimal optimization of the cone flow and capillary voltage, cone voltage and collision voltage of the mass spectrometer Source temperature: 140 °C
Solvent removal temperature: 625 °C
Spray gas flow: 1200 liters / hour

Data management

MassLynx 4.1
TargetLynx Application Management System

Results and discussion

Peptide therapeutics have excellent efficacy, but they are also rapidly excreted by metabolism or by prototype drugs. In order to accurately determine the pharmacokinetic parameters (PK) of these drugs, it is necessary to have a highly sensitive LC/MS/MS system to determine the late time points to determine the elimination phase of the PK curve. The Xevo TQ-S is a new tandem quadrupole mass spectrometer with an innovative source design. This new design allows more analyte ions to migrate to the analyzer, greatly increasing the efficiency of the ion sampling process. It uses a larger sampling hole and differential pumping zone as well as off-axis multilayer ring ion transport technology to prevent neutral compounds from entering the analytical region of the instrument.

To assess the increase in sensitivity when performing peptide analysis in biological fluids, desmopressin was injected into the plasma and extracted by acetonitrile precipitation of protein (2:1). The data obtained using conventional optics and the new StepWave optics is shown in Figure-1. Here we can see that the peak of desmopressin is almost invisible in instruments using traditional ion transport technology, while the obvious peaks are seen in the Xevo TQ-S mass spectrometer. Extending the baseline of the traditional source and comparing the data in Figure-2, we can clearly see that there is a peak of desmopressin at 0.73 minutes in the traditional instrumental chromatogram. In this way, the sensitivity is increased by 25 times.

StepWave ion transport technology

Traditional ion transport technology

Figure-2 UPLC/MS/MS quantitative analysis of desmopressin in rat plasma extracts using MRM mode. The chromatogram above shows the peak response of the new StepWave ion transport technology using the Xevo TQ-S; the chromatogram below shows the peak response with conventional ion transport techniques (the desensitization of the baseline is extended to show 0.74 minutes of desmopressin chromatography) peak).

in conclusion

Polypeptide therapeutics can be used to treat new diseases with minimal side effects, drug-drug interactions, and toxicity. The combination of Xevo TQ-S and ACQUITY UPLC provides an ideal platform for the analysis of peptides in biological fluids. The combination of the two can provide:

* Highest sensitivity
* Fast analysis
* Endogenous peptide resolution
* Sufficient mass range in the analyzer for quantitative analysis of macromolecular peptides

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