Feed Injection at Elevated Pressure Using the Agilent 1290 Infinity III Hybrid Multisampler

Importance of the Topic

A high-throughput, sensitive, and robust analytical workflow for multipesticide residue screening is essential in food safety, environmental monitoring, and regulatory compliance. Modern UHPLC-MS/MS methods frequently require sub-2 µm columns operated at extreme pressures to achieve rapid separations of hundreds of analytes. However, high injection volumes in strong-eluting solvents can degrade peak shape, complicate integration, and reduce sensitivity. The Agilent 1290 Infinity III Hybrid Multisampler’s Feed Injection mode addresses these challenges by minimizing peak broadening at elevated pressures and enabling reliable quantification even at low limits of quantification (LOQs).

Study Objectives and Overview

The primary goal was to compare the performance of Feed Injection mode versus classical flow-through injection for a multipesticide UHPLC-MS/MS method. Key aims included:

• Assessing peak shape preservation across injection volumes (2, 5, 10 µL).
• Determining LOQs down to 0.02 ppb for circa 120 pesticide compounds.
• Evaluating practical benefits in automated data integration and sensitivity.

Methodology and Instrumentation

A reverse-phase UHPLC separation was conducted on a 2.1 × 150 mm Poroshell 120 Phenyl-Hexyl column (1.9 µm) at 55 °C, with a 9 min gradient from 5 % to 100 % methanol containing 5 mM ammonium formate/0.1 % formic acid at 0.6 mL/min. Injection volumes of 2, 5, and 10 µL were tested in both injection modes. MS detection used dynamic multiple reaction monitoring (dMRM) on an Agilent Ultivo triple quadrupole with JetStream ionization, alternating positive and negative polarity. Calibration curves spanned 100 ppb–0.02 ppb in acetonitrile.

Instrumentation

• Agilent 1290 Infinity III High-Speed Pump and MCT
• Agilent 1290 Infinity III Hybrid Multisampler (Feed Injection capable up to 1300 bar)
• Agilent Ultivo Triple Quadrupole LC/MS with JetStream source
• Agilent MassHunter Acquisition, Qualitative, and Quantitative software suites

Main Results and Discussion

When injecting 2 µL, both modes yielded comparable peak shapes and LOQs. At 5 µL, flow-through injections began to exhibit peak splitting/shouldering for early eluters, whereas Feed Injection preserved sharp, symmetrical peaks and improved signal-to-noise. At 10 µL, classical injection suffered severe fronting, split peaks, and loss of quantifiable signal, while Feed Injection showed only minor broadening on the earliest compounds. Quantitatively, Feed Injection reduced LOQs by up to 5× at 5 µL and up to 25× for late eluters at 10 µL. The maintained peak integrity in Feed Injection mode facilitated automated integration without manual correction.

Benefits and Practical Applications

The Feed Injection mode at elevated pressure offers:

• Enhanced sensitivity and lower LOQs for complex multipesticide assays.
• Improved peak shapes that support reliable, automated data processing.
• Capability to use larger injection volumes in strong solvents without chromatographic distortion.
• Flexibility to employ longer, sub-2 µm columns without gradient delay or peak broadening.

Future Trends and Potential Applications

Advances may include further integration of high-pressure injection modes with ultrahigh-throughput screening for broader analyte classes, such as polar metabolites. Software improvements in real-time peak correction and AI-assisted integration will augment data quality. The approach could extend to other high-complexity matrices (e.g., textiles, biological fluids) and support regulatory demands for ever-lower detection limits.

Conclusion

The Agilent 1290 Infinity III Hybrid Multisampler’s Feed Injection mode delivers superior chromatographic performance for multipesticide residue analysis at elevated pressures. By preserving peak shape at high injection volumes, it enables lower LOQs, streamlined data integration, and robust quantification, meeting the rigorous demands of modern analytical chemistry laboratories.

References

1. Roy J-F, Deckers C, Honnold R, et al. A Sensitive and Robust Workflow to Measure Residual Pesticides and Mycotoxins in Dry Cannabis Flower. Agilent Technologies Application Note 5994-0429EN, 2020.
2. Naegele E. Improved Peak Shape and Lower LOQs in Pesticide Analysis. Agilent Technologies Application Note 5994-6125EN, 2024.