Increasing Speed of UHPLC-MS Analysis Using Single-stage Orbitrap Mass Spectrometer

Significance of the Topic

The need for ultrafast and sensitive analysis in modern laboratories has driven the development of UHPLC coupled to high-resolution accurate-mass (HR/AM) systems. Achieving sufficient data points across chromatographic peaks below 5 s width while maintaining high resolving power and mass accuracy is crucial for reliable quantitation and identification in complex matrices such as food feeds.

Objectives and Study Overview

This work evaluates performance enhancements in the Thermo Scientific Exactive Plus Orbitrap mass spectrometer when paired with an UHPLC system. The primary aim is to increase scan speed and resolve coeluting compounds in a 2-minute gradient method, enabling rapid quantitation and broad screening of pesticide residues in horse feed extracts.

Methodology and Instrumentation

Sample Preparation employed a QuEChERS protocol for efficient extraction of multiple pesticides from feed. Chromatographic separation used an Accela 1250 UHPLC with a Hypersil GOLD PFP column (50 x 2.1 mm, 1.9 µm) at 800 µL/min, yielding a total cycle time of 5 min including a 2 min gradient of water and methanol with 0.1% formic acid.
Mass spectrometric analysis was conducted on an Exactive Plus mass analyzer at 70,000 FWHM (m/z 200) using Full-Scan with data-dependent all-ion fragmentation (FS/ddAIF) triggered by an inclusion list. Data processing and both targeted and unknown screening were performed with ExactFinder 2.0 software.

Used Instrumentation

• Thermo Scientific Accela 1250 UHPLC system with open autosampler
• Hypersil GOLD PFP column, 50 x 2.1 mm, 1.9 µm
• Thermo Scientific Exactive Plus Orbitrap mass spectrometer
• ExactFinder 2.0 processing software

Main Findings and Discussion

Instrumental upgrades including an S-Lens ion guide and improved Orbitrap electronics elevated scan rates to 3.7 Hz at 70,000 resolution. This delivered 13 data points across a 3.2 s peak while maintaining high sensitivity. Targeted analysis of 85 pesticides showed clean extracted ion chromatograms with a 5 ppm mass window and linear calibration over 5–150 µg/kg. The balance of full-scan acquisition and selective fragmentation scans minimized data load without sacrificing coverage. General unknown screening of over 15,000 features enabled rapid identification of additional contaminants using isotopic pattern matching and online databases.

Benefits and Practical Applications

• High-throughput workflows with sub-5 min sample cycles
• Accurate quantitation in complex matrices with minimal interference
• Comprehensive target and non-target screening in a single run
• Reduced need for MS/MS experiments through high resolving power

Future Trends and Opportunities

Further developments may focus on real-time data processing, deeper integration of HR/AM instruments into routine QA/QC, and expansion of spectral libraries for broader non-target screening. Advances in column chemistry and faster electronics will drive sub-minute analyses with maintained selectivity.

Conclusion

The second-generation Exactive Plus Orbitrap system demonstrates that optimized hardware and software can meet the demands of ultra-fast UHPLC-MS applications. Enhanced scan speed and resolution support reliable quantitation and versatile screening in high-throughput environments without compromising data quality.

References

1. Kaufmann A Butcher P Maden K Walker S Widmer M Comprehensive comparison of liquid chromatography selectivity as provided by two types of liquid chromatography detectors high resolution mass spectrometry and tandem mass spectrometry Where is the crossover point Anal Chim Acta 673 60-72
2. Improved analysis of biopharmaceutical samples using an MS-only Orbitrap mass spectrometer Poster ThP28 #623