abstract

Abstract

This technical note demonstrates a sensitive method for the quantitation of peptides in extracted rat plasma using nanoflow LC- Zeno MRM^HR method. Peptide quantitation as low as 2.5 attomoles on column for lower limit of quantitation (LLOQ) was achieved (Figure 1), with excellent accuracy, linearity, and reproducibility.

As the pharmaceutical industry shif ts toward increasingly potent peptide therapeutics, discovery phase bioanalysis faces the dual challenge of extreme sensitivity requirements and limited sample availability. Nanoflow LC enables enhanced ionization efficiency, providing a powerful approach for achieving improved detection sensitivity, particularly for low abundance peptides. In parallel, high-resolution mass spectrometry (HRMS), with its superior mass accuracy and resolving power, allows more effective discrimination between target analytes and interfering background components. This capability significantly reduces matrix-related interferences, thereby improving quantitative ac curacy and analytical robustness. The combination of nanoflow ionization and HRMS offers a compelling solution for reliable, sensitive, and accurate peptide quantitation in complex biological matrices.

Herein, the ZenoTOF 8600 system was used for the quantitation of a series of synthetic peptides in matrix. Multiple hardware improvements on the ion source, the front-end of the mass analyzer and an advanced optical detector significantly boosted the MS/MS sensitivity and %CV for low-level quantitation. This technical note summarizes the application of the Zeno MRM^HR method to evaluate specificity and sensitivity for quantifying peptides on an HRMS platform.

Figure 1. Representative extracted ion chromatograms (XICs) of matrix blanks and LLOQ samples for peptides VSFELFADK and NLSVEDAAR are shown. No matrix interferences were observed at the retention time of the analyte.

image-top

key-benefits

Key benefits or nanoflow-based peptide quantitation using the ZenoTOF 8600 system

• High sensitivity quantitation at nanoflow rate: Achieve as low as 2.5 amol LLOQ for peptide quantitation using the ZenoTOF 8600 system.
• Advanced hardware upgrades enabling improved ion transmission efficiency: Achieve outstanding sensitivity for peptide quantitation with advanced front- end technology, resulting in superior performance on the ZenoTOF 8600 system.
• Meet critical quantitative performance criteria: Achieve accurate quantitative performance with %CV < 5% at all concentration levels across an LDR of up to 4. 8 orders of magnitude.
• Simplified data management: Employ a single platform for streamlined data acquisition, processing, and management with SCIEX OS software.

introduction

Introduction

Quantitation of peptides in pharmaceutical discovery is strongly driven by the need to detect low-level peptides in low sample volumes and highly complex biological matrices. During peptide analysis, co-eluting matrix constituents can introduce significant interferences, leading to ion suppression, diminished analytical selectivity, and reduced quantitative accuracy, particularly for low-abundance analytes. HRMS offers a robust and effective strategy to address these challenges. Its superior mass accuracy and resolving power enable precise discrimination between target peptides and closely related matrix-derived ions that are indistinguishable at lower resolution. By effectively reducing matrix -related background contributions, high-resolution detection enhances selectivity, improves data robustness, and increases confidence in quantitative results. As a result, HRMS offers a practical and reliable approach to peptide analysis in complex biological matrices.

When coupled with a low flow approach, such as nanoflow LC, it significantly enhances ionization efficiency in mass spectrometry by reducing the solvent flow entering the ion source. At lower flow rates, spray droplets formed during nebulization are smaller and undergo more efficient desolvation, facilitating improved ion formation compared to conventional flow conditions. This highly efficient ion ization process reduces ion suppression and increases the proportion of analyte ions transferred into the gas p hase, thereby enhancing sensitivity. Consequently, nanoflow ionization is particularly advantageous for the detection and quantitation of low abundance analytes in complex biological samples, specifically when sample amounts are limited in the drug discovery phase or in proteomics research.

methods

Methods

Sample preparation: Rat plasma samples were subjected to protein precipitation by adding methanol at a 3-fold excess relative to plasma volume. Following vortex mixing, samples were centrifuged at ambient temperature to remove precipitated proteins. The resulting clear super natant was collected and diluted 4-fold with a 2:2:0.1 (v/v/v) mixture of acetonitrile, acetic acid, and formic acid in water. A set of 10 peptides was subsequently spiked into the diluted rat plasma extracts and serially diluted to establish calibration curves for quantitative evaluation.¹

Nanoflow LC conditions: The M-Class system (Waters) was used for analyte separation in direct injection mode. A volume of 1 μL of the sample was injected for analysis. The mobile phase A consisted of water with 0.1% formic acid in water, while mobile phase B was composed of 0.1% formic acid in acetonitrile. For analyte separation, the operating flow rate was set to 300 nL/min using an IonOpticks Aurora Elite C18 column (15 cm × 75 µm). The column oven temperature was set to 60°C. Chromatographic conditions for analyte separation are shown in Table 1.

image-bottom

Mass spectrometry conditions: Samples were analyzed in triplicate. Method details, such as source and gas parameters and MS conditions, are as described in Table 2. Sample analysis was performed on the ZenoTOF 8600 system (SCIEX) using Zeno MRM^HR, with an ion source configured for a <1 μL/min nanoflow electrode.

image-bottom

Data processing: Analysis was performed using SCIEX OS software, version 4.0.0. Peaks were integrated using the MQ4 algorithm, and a weighting of 1/x² was used to quantify all peptides in the peptide mix. An XIC peak width of 0.05 Da was applied for quantitation.

Quantitative performance or peptide analysis with the ZenoTOF 8600 system using a nanoflow LC

The quantitative performance of nanoflow LC with the ZenoTOF 8600 system was evaluated by analyzing 10 peptides in extracted rat plasma. The peptide mixture was spiked into processed rat plasma at different concentrations. Each concentration level was evaluated in triplicate. Zeno MRM^HR mode was employed for sample analysis, enabling data collection across a range of fragment ions, with select fragment ions automatically evaluated and summed for best assay sensitivity using SCIEX OS software.² For this study, all peptides were quantified using the sum of 3 fragment ions.

As shown in Figure 2, the on-column LLOQ for peptides GGPFSDSYR, VLDALQAIK, SADFTNFDPR, EGHLSPDIVAEQK, ESDTSYVSLK, GYSIFSYATK, FEDENFILK, and FSTVAGESGSADTVR were 7.1 amol, 7.1 amol, 7.1 amol, 4.2 amol, 12.7 amol, 12.7 amol, 7.6 amol, and 7.6 amol, respectively.

Figure 2. XICs of the matrix blank and LLOQ of 10 peptides in extracted rat plasma. A sum of 3 fragment ions was applied for quantitation for all peptides.

image-top

Figure 3. Calibration curves for quantifying 3 representative peptides with a weighing factor 1/x² using nanoflow LC. Peptides GGPFSDSYR, GYSIFSYATK, and NLSVEDAAR showed a wide linear range where asum of 3 fragment ions was applied for quantitation.

image-top

Calibration curves from analysis of 3 representative peptides are shown in Figure 3. Overall, an LDR of up to 4.8 orders of magnitude was achieved for peptide analysis using nanoflow LC, demonstrating measurement of a wide range of concentrations (Table 3).

For the bioanalytical performance assessment, the LLOQ was determined based on the requirement that the %CV be below 20% and that accuracy be between 80% and 120%. For concentrations above the LLOQ, the %CV was required to be below 15%, with accuracy between 85% and 115%.³ Figure 4 shows the accuracy and precision values for 3 example peptides across the measured linear range.

Calculated concentrations for all calibration points were within ±13% of the nominal value, where %CV was <5%, demonstrating high reproducibility. Overall, a highly sensitive method for the quantitation of peptides was demonstrated. For all 10 peptides evaluated in this study, low‑attomole sensitivity was achieved using nanoflow LC.

Table 3. Summary of the quantitative performance on the ZenoTOF 8600 system using nanoflow LC. Samples were analyzed in triplicate.

image-bottom

compliance-ready

Figure 4. Quantitative performance of 3 representative peptides. Accuracy and precision successfully met the bioanalytical criteria across all concentration levels. For the analysis of peptides GGPFSDSYR, GYSIFSYATK, and NLSVEDAAR, a sum of 3 fragment ions was applied.

image-top

Compliance-ready SCIEX OS software

Equivalent SCIEX OS software capabilities for regulated bioanalysis can be executed on the ZenoTOF 8600 system, ensuring high fidelity when performing method transfers while retaining critical compliance features.

SCIEX OS software is a closed system and requires records and signatures to be stored electronically, in compliance with 21 CFR Part 11. SCIEX OS software can open raw data files from any visible storage location within a closed network by using designated processing workstations.

Figure 5 illustrates the features of SCIEX OS software used to monitor the audit trail, acquire and process data, and configure user access. The audit trail feature enables users to audit critical user actions and locks in data integrity. The Central Administrator Console (CAC) feature allows users to centralize acquisition and processing on a single platform, maximizing efficiency for multi-instrument laboratories, regardless of compliance standards. The configuration module allows users to assign roles and access as the administrator, method developer, analyst, and reviewer.

Figure 5. Features of SCIEX OS software for monitoring user access and evaluating the audit trail. The audit trail view allows users to filter for high-risk events easily and enables data integrity features to meet compliance requirements. The software features a Central Administrator Console (CAC) to manage users and groups, role definitions, workstations, and projects across all systems. The CAC feature supports both regulated and non-regulated compliance standards. The configuration module enables users to quickly set up roles and access levels for the administrator, method developer, analyst, and reviewer.

image-top

conclusions

Conclusions

• LLOQs as low as single -digit amol (on column) levels were achieved for peptide quantitation using nanoflow LC with the ZenoTOF 8600 system.
• A simplified Zeno MRM^HR approach was demonstrated with the collection of several high intensity product ions from TOF MS/MS spectra, which were automatically evaluated and summed using SCIEX OS software on the ZenoTOF 8600 system for optimal sensitivity.
• Accurate quantitative performance was achieved with %CV <5% at all concentration levels across an LDR of up to 4.8 orders of magnitude.
• Retain data management and compliance-readiness (21 CFR Part 11) features using SCIEX OS software to support quantitative analysis on the ZenoTOF 8600 system.

references

References

1. Enhanced sensitivity and quantitative performance featuring a novel quadrupole time of flight mass spectrometer. SCIEX technical note. MKT-33800-A.
2. Automated peptide fragment ion selection and summation for streamlined HRMS quantitative workflows. SCIEX technical note. MKT-36303-A.
3. Bioanalytical Method Validation, May 2018.