Sensitive quantification of a tripeptide with a wide linear dynamic range (LDR) using an accurate mass spectrometer


Quantification of the tripeptide, Val-Tyr-Val, using the ZenoTOF 7600 system

Lakshmanan Deenadayalan1 , Sashank Pillai1 , Rahul Baghla2 , Elliott Jones2 and Eshani Nandita2
1SCIEX, India; 2SCIEX, USA

Abstract


This technical note demonstrates the sensitive quantification of a tripeptide (Val-Tyr-Val) using the ZenoTOF 7600 system. A lower limit of quantification (LLOQ) of 2.5 fg/µL was achieved using an accurate and highly reproducible (%CV <10%) method. Linearity was achieved between 2.5 fg/µL and 1000 pg/µL, generating an LDR spanning 5 orders of magnitude with a coefficient of determination (r2 ) >0.99 (Figure 1). The improved MS/MS sampling efficiency and the 5 GHz, 10-bit ADC detector of the ZenoTOF 7600 system provide the enhanced sensitivity and dynamic range that are desired for quantitative assays. A 7-fold improvement in signal-to-noise (S/N) (peak-to-peak) ratio was reached with the Zeno trap turned on.

Introduction


Drug discovery programs aim to develop compounds that are effective at low dosages, therefore necessitating sufficient sensitivity to quantify analytes in challenging matrices during routine analyses. As therapeutic compounds become more complex, accurate mass spectrometers are becoming more popular for routine analysis because they offer improved selectivity. 1-2 Even though modern TOF platforms have quantitative capabilities, ion transmission is lost between pulses, reducing sensitivity.

This technical note presents a reliable and highly sensitive workflow to support routine quantitative analysis using an accurate mass spectrometer with increased MS/MS sampling efficiency. The Zeno trap on the ZenoTOF 7600 system accumulates the fragment ions and pulses them into the TOF, reducing ion loss during the traditional pulse cycle and raising the duty cycle to more than 90% from the 30% duty cycle characteristic of a classic TOF system. The improved efficiency for total MS/MS sampling on the ZenoTOF 7600 system makes it a valuable tool for quantitative workflows that require high sensitivity and selectivity.

Figure 1. Calibration curve of tripeptide (Val-Tyr-Val) using Zeno MRMHR . Linearity was established between 2.5 fg/µL and 1000 pg/µL, generating an LDR spanning 5 orders of magnitude with an r 2 >0.99.

Key features for the identification of metabolites using the ZenoTOF 7600 system
 

  • Achieve low levels of quantification: An LLOQ of 2.5 fg/µL was reached for the tripeptide using the ZenoTOF 7600 system

  • Wide linear dynamic range: An LDR spanning 5 orders of magnitude was achieved for routine quantification assays

  • Quantitative sensitivity: Quantitative limits were improved as much as 7-fold using the Zeno trap, based on the peakto-peak S/N ratio

  • Accurate and reproducible results: Accurate and reproducible (%CV <10%) quantitative performance was achieved at low concentrations 

  • Streamlined data management: Data acquisition and processing are integrated into SCIEX OS software, a 21 CFR Part 11-compliant platform

Methods


Standard preparation: A 1 mg/mL stock solution of tripeptide (Val-Tyr-Val) was prepared by dissolving the standard in water. Calibration curve samples were prepared by serial dilution using water with 5% acetonitrile and 0.5% formic acid by volume. The calibration curve samples were prepared at concentrations ranging from 2.5 fg/µL to 1000 pg/µL.

Chromatography: An ExionLC system with a Phenomenex Aries Peptide XB-C18 analytical column (1.7 µm, 100 x 2.1 mm) was used for chromatographic separation at a flow rate of 0.25 mL/min. The column was operated at 40°C. Mobile phase A was 0.1% (v/v) formic acid in water and mobile phase B was 0.1% (v/v) formic acid in acetonitrile. A 10 µL injection volume was used for analysis. Table 1 summarizes the LC gradient conditions used.

Table 1. LC gradient.

Mass spectrometry: Samples were analyzed using a ZenoTOF 7600 system operating in positive ion mode. The data were acquired using an MRMHR experiment. The optimized MS parameters are listed in Table 2. The summary of the Zeno MRMHR parameters is displayed in Table 3.

Data processing: Data processing was performed using SCIEX OS software, version 3.0. Peaks were automatically integrated using the MQ4 algorithm with a weighting of 1/x2 . The extracted ion chromatogram (XIC) peak width applied for quantification was 0.02 Da.

Table 2. MS and source parameters.

Table 3. Zeno MRMHR parameters for the tripeptide.

Zeno trap provides enhanced sensitivity


In traditional TOF MS/MS acquisition, ions are often lost between TOF pulses due to differences in velocity. As a result, the typical duty cycle ranges from 5% to 25%. Due to loss in ion transmission, there is a subsequent decline in overall sensitivity as fewer ions arrive at the detector. The Zeno trap increases ion transmission by controlling the ion beam from the collision cell into the TOF accelerator. Ions exit the Zeno trap in an order based on their potential energy.3-4

Figure 2. Representative extracted ion chromatograms (XICs) for the tripeptide. The left and right panels show results from the analysis of 50 fg/µL of the Val-Tyr-Val tripeptide with Zeno MRMHR and MRMHR, respectively. A 7-fold S/N improvement (peak-to-peak) was observed using the Zeno trap.

Quantitative performance


A calibration range between 2.5 fg/µL and 1000 pg/µL was evaluated. Each concentration was analyzed in triplicate.

Analytical performance was evaluated for accuracy and precision. The accuracy of the calculated mean was expected to be between 80% and 120% at the LLOQ and between 85% and 115% at higher concentrations. The %CV of the calculated mean for each concentration was expected to be <20% at the LLOQ and <15% for all higher concentrations.

Calculated percent accuracy and %CV values were within the acceptance criteria at each concentration level (Figure 3). Overall, the assay accuracy was within ±9% of the nominal concentration and %CV was <10% for the Val-Tyr-Val peptide at all concentrations, demonstrating an accurate and highly reproducible workflow.

An LLOQ of 2.5 fg/µL was achieved for the quantification of the ValTyr-Val tripeptide (Figure 4). No interferences were observed in the diluent blank at the retention time of the peptide.

Figure 3. Summary of the quantitative performance. Reproducibility and accuracy results were determined from the calibration curve across 3 replicates at each concentration. The concentrations were calculated for each replicate at each concentration (right, yellow box). Statistical results were summarized using the Analytics module in SCIEX OS software.

Figure 4. Representative XICs of the tripeptide. The left panel shows the XIC of the diluent blank. The middle and right panels show the XICs for the Val-Tyr-Val tripeptide spiked at 2.5 fg/µL (LLOQ) and 5 fg/µL, respectively. No interferences were observed in the blank at the retention time of the peptide.

Figure 5. Features of the 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 nonregulated compliance standards. The configuration module enables users to quickly set up roles and levels of access for the administrator, method developer, analyst and reviewer levels.

Compliance-ready SCIEX OS software



SCIEX OS software is a closed system and requires records and signatures to be stored electronically, meeting the regulations outlined by 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 that are used for monitoring the audit trail, acquiring and processing data and configuring 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 using a single platform to maximize efficiency for multi-instrument laboratories, independent of compliance standards. The configuration module allows users to assign roles and access as the administrator, method developer, analyst and reviewer.

Conclusion
 

  • An LLOQ of 2.5 fg/µL was achieved for a tripeptide (Val-TyrVal) using the ZenoTOF 7600 system

  • Linearity was achieved between 2.5 fg/µL and 1000 pg/µL, generating an LDR spanning 5 orders of magnitude with an r 2 >0.99

  • A 7-fold improvement in S/N ratio (peak-to-peak) was reached for the quantification of the tripeptide using the Zeno trap

  • The method demonstrated accurate and highly reproducible (%CV <10%) quantitative performance at all concentrations

  • The assay exhibited the ability to routinely quantify low-fg/µL levels of tripeptide in a 5 min runtime, enabling highthroughput delivery of high-quality data

  • SCIEX OS software is compliant with 21 CFR Part 11 and integrates with an accurate mass spectrometer to support data acquisition, processing and management on an single platform

References
 

  1. Mike-Qingtao Huang, Zhongping (John) Lin, Naidong Weng (2013). Applications of high-resolution MS in bioanalysis. Bioanalysis 5(10):1269-1276.

  2. Qualitative flexibility combined with quantitative power. SCIEX technical note, RUO-MKT-02-13053-A.

  3. Enhanced sensitivity for peptide quantification in a complex matrix using high-resolution LC-MS/MS. SCIEX technical note, RUO-MKT-02-13324-A.

  4. High sensitivity MRM workflow for signature peptide quantification. SCIEX technical note, RUO-MKT-02- 11882-A.
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