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6495 LC/TQ Best Practices Guide

New Best Practices Guide

Agilent 6495 LC/TQ

Agilent 6495C LC/TQ

Get the highest performance from your Agilent 6495 triple quadrupole LC/MS

Your samples are precious, and turnaround time is critical to your organization’s success. The rugged 6495 LC/TQ delivers day-in/day-out performance for the highest levels of confidence.

To help you maximize instrument sensitivity and workflow efficiency, we’ve put together this collection of resources—all in one convenient place.

Agilent 6495C LC/TQ

Use these tabs to select the resources you need

By ensuring that your HPLC system is properly plumbed, conditioned, and primed, you can achieve the low detection limits you need for rigorous quantitative analysis. These guides will help you maximize resolution, sensitivity, and workflow efficiency.

A clean baseline and clearly separated endogenous peaks are a must for quantitative accuracy. That’s why it is critical to avoid sample carryover at every point in your LC flow path. You can reduce carryover to a negligible amount using the Multiwash function of the Agilent 1290 Infinity II multisampler. These two guides will show you how.

Agilent Jet Stream (AJS) thermal gradient focusing enhances ESI-MS sensitivity by improving ion desolvation and spatial focusing. Superheated nitrogen sheath gas confines the nebulizer spray to effectively concentrate and desolvate droplets in a thermal confinement zone. The benefits include less noise, reduced peak tailing, and the elimination of sample recirculation. What’s more, improved ion production results in higher MS and MS/MS signal intensities and improved limits of detection.

Agilent iFunnel technology combines the efficient ion generation and focusing of Agilent Jet Stream technology with a hexabore capillary sampling array. Together, they enable a much larger fraction of the ESI spray plume to enter the mass spectrometer ion optics. A dual-stage ion funnel with optimized pressure allows increased ion capture and transmission for robust, precise quantitation at high MRM acquisition speeds requiring short dwell times. This innovative design greatly improves detection limits by collecting more ions while reducing system noise.

iFunnel

Parameter optimization is a lengthy process because you need time between temperature setpoints to achieve full thermal equilibration. Source and iFunnel Optimizer—included with Agilent MassHunter Acquisition software—helps you create and run source parameter ramps.

The software sets the number of replicates per experiment, as well as parameter step size. It also allows you to set the desired time for thermal equilibration. Once all parameters are set, the program creates all methods and runs a worklist with these parameters. You can then analyze each data file to determine the best setpoints.

An appropriate RF amplitude ensures the highest possible transmission efficiency, while minimizing precursor ion fragmentation. Source and iFunnel Optimizer can quickly create high- and low-pressure ramps for easier method development.

Although you can examine the TIC of each voltage setpoint, more information is gained by plotting “Abundance vs RF Amplitude” for the precursor ions of interest. With this plot, you can find the most desirable funnel settings for your method. Keep in mind that an ideal voltage is not always possible. However, we recommend choosing the lowest required high- and low-pressure RF amplitudes that optimize transmission across the m/z range of ions being measured.

When developing targeted methods, or when following prescriptive methods such as EPA 537.1, it is crucial to choose at least two of the most abundant product ions.

Once you have chosen the unique MRM transitions, collision energies (CE) should be ramped systematically to determine the maximum signal produced with good peak shape. Similarly, by plotting abundance versus collision cell voltage, a clearer picture of the effects of MRM collision energy emerges. In this example, the ideal CE values are 16 and 32 V.

To simplify method development, MassHunter software includes an Optimizer tool that helps you develop MRM parameters and manage libraries. To learn more, use these links.

The analysis of peptides poses unique challenges that are better addressed using third-party software. MassHunter easily integrates with open-source Skyline software, helping you develop MRM methods for peptide sequences. Check out these resources to gain insights for peptide-centric workflows.

Application notes

To maximize instrument sensitivity, it is important to consider MRM dwell time, which influences the stochastic sampling of the ion beam. Generally, limits of detection are characterized with considerable time spent sampling the ion beam, which allows a stable, consistent flux of ions to hit the detector.

Due to the need for increased throughput, lab productivity, and regulatory compliance, labs are monitoring analytes using shorter chromatographic runs. As a result, more MRM transitions are being monitored simultaneously to reduce dwell times. Therefore, it is important to understand the impact of dwell time and ion flux on your analytical assay precision.

At lower instrument dwell times (typically <5 ms), ion beam sampling becomes less precise due to the insufficient number of ions involved in the measurement. In the context of chromatographic peak area, lower dwell times can increase the uncertainty (% RSD) of measurements within a set of replicates. The figure compares the results for the same analyte at the same concentration, but with different dwell times—emphasizing its effect on % RSD.

These references explore dwell time in greater detail:

Need help with familiarization and troubleshooting? Use our convenient Triple Quadrupole LC/MS Resources app, which is included with every 6495C LC/TQ installation. You can find the App under the "Agilent" folder within the local PC’s start menu.

The app includes a collection of resources that cover:

  • Software installation
  • MassHunter Data Acquisition software and workflow
  • Mass spectrometer maintenance, including consumable part numbers

These application notes will help you maximize instrument sensitivity and guide your sample analysis.

Food

Environmental

Miscellaneous applications

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