Customizable Intelligent Reflex Workflows: Embedding User-Defined Reaction Logic in Automatic High Throughput LC/MS Analysis

Importance of the Topic

Automated, data-dependent decision making during LC/MS acquisition addresses a central challenge in high-throughput targeted analysis: maintaining analytical quality while minimizing manual intervention and sample rework. Intelligent reflex workflows reduce the risk of carryover, out-of-range quantitation, and missed confirmations by enabling the acquisition system to react in real time to chromatographic or mass spectrometric outliers. This capability is particularly important for regulated and high-volume applications such as pharmaceutical QA/QC, food pesticide and veterinary-drug surveillance, and large-cohort bioanalytical studies where throughput, reproducibility and sample integrity are critical.

Objectives and Study Overview

This work presents enhancements in Agilent MassHunter Acquisition for LC/MS version 13 that extend automated reinjection and decision-making (intelligent reflex) logic. The primary aims were to: define flexible, user-configurable reflex logic; expand dilution and reinjection strategies for over-range analytes; allow carryover handling across blanks and reinjected samples; and provide a fully custom workflow option enabling users to tailor reflex actions to their laboratory needs. The approach was demonstrated using stress-test worklists containing blanks, samples, QCs and calibration standards on Agilent triple quadrupole and Q-TOF platforms.

Methodology and Used Instrumentation

Workflows were implemented and evaluated using Agilent LC/MS triple quadrupole (TQ) and quadrupole time-of-flight (Q-TOF) systems controlled by MassHunter Acquisition for LC/MS version 13 paired with an Agilent Infinity III 1290 HPLC system. Quantitative methods with pre-established calibration curves and outlier thresholds (based on analyte concentration and retention time windows or other chromatographic/mass spectral criteria) were created for each workflow. The acquisition software evaluates the outlier logic in real time during a running worklist and triggers reflex actions such as pausing/stopping the queue, inserting or appending reinjections, and reinjecting blanks or QCs.

Key Results and Discussion

The updated intelligent reflex functionality demonstrated several practical capabilities and improvements:

• Custom workflow logic: Users can define outlier criteria for blanks, QCs and samples and choose specific reflex actions (pause, stop, insert or append injections). Options include automatically adding blanks before/after reinjections and inserting QCs after a user-specified number of appended injections.
• Above-calibration handling: When a sample exceeds the upper limit of quantitation, the system can automatically (a) reduce injection volume to provide an estimated concentration, (b) apply a defined dilution factor using a diluent vial and reinject the sample, or (c) trigger a user-defined sample-preparation routine. The worklist tags reinjected samples with dilution details and inserts blanks to mitigate carryover.
• Carryover management: Carryover detected in blanks results in automatic insertion of new blank injections. The carryover workflow can operate on all blanks present in the worklist, including those produced by other reflex actions, ensuring contamination is identified and isolated in real time.
• Concurrent workflows: Multiple reflex workflows (e.g., Carryover, Above Calibration Range, Fast Screening, Custom, plus Q-TOF specific targeted MS/MS or iterative MS/MS) can execute concurrently within a single worklist, reducing manual oversight and avoiding unnecessary re-runs.

The system was stress-tested with mixed worklists to confirm the logic performs reliably in realistic high-throughput scenarios. Results emphasize how live decision-making during acquisition prevents propagation of outliers through a batch and preserves valuable sample material.

Benefits and Practical Applications

The enhancements deliver several tangible benefits for routine and regulated laboratories:

• Increased throughput and reduced operator time by automating reinjection, dilution and pause/stop decisions directly during acquisition.
• Improved data quality and consistency by defining objective, method-linked outlier criteria rather than relying on manual judgment calls.
• Protection of precious or limited samples through automated dilution and controlled reinjection strategies.
• Minimized risk of carryover contaminating subsequent injections via automated blank insertion and carryover checks.
• Greater method flexibility for diverse applications—pharmaceutical QC, residue testing in food, clinical population studies—through a customizable reflex framework.

Future Trends and Possible Uses

Several developments could extend the value and scope of intelligent reflex workflows:

• Integration with laboratory information management systems (LIMS) and instrument fleet management to propagate reflex decisions to sample tracking and reporting systems.
• Adaptive, machine-learning–driven thresholds that evolve with incoming data to reduce false positives/negatives for outlier detection.
• Closer coupling with automated sample preparation systems to execute complex dilution or cleanup protocols without manual intervention.
• Expansion of reflex capabilities to high-resolution and hybrid instruments with built-in automated decision trees for targeted and non-targeted workflows.
• Standardized validation and audit trails for reflex actions to support regulatory compliance in GLP/GMP environments.

Conclusion

MassHunter Acquisition v13’s enhanced intelligent reflex workflows provide flexible, real-time decision-making during LC/MS acquisition that improves throughput, reduces rework and safeguards sample integrity. The new custom workflow and expanded dilution/carryover options deliver practical tools for laboratories handling large sample volumes or critical analyses. By enabling multiple concurrent reflex strategies and allowing user-defined logic, the platform supports robust, automated batch processing while preserving the traceability required for regulated testing.

Reference

Rennie EE, Shah D, Wavre A, Bertram L, Zhu Y. Customizable Intelligent Reflex Workflows: Embedding User-Defined Reaction Logic in Automatic High Throughput LC/MS Analysis. ASMS 2026 Poster MP389. Agilent Technologies Inc., Santa Clara, CA, 2026.

Agilent Technologies, Inc. MassHunter Acquisition for LC/MS version 13 software and Infinity III 1290 HPLC system. Agilent Technologies, 2026.