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Abstract Introduction
Data-independent acquisition (DIA) is advantageous in large library screening methods for drug analysis. Using LC/Q-TOF, information can be gathered on targets and suspects simultaneously and further, older sample data files can easily be re-investigated for the existence of emerging analytes of interest. Screening for a large range of analytes at many different concentration levels can, however, result in the need for manual evaluation and possible sample reinjection. Intelligent reflex workflows with MassHunter Acquisition have been developed to increase instrument up time and reduce the need for manual worklist setup. Outlined herein is a demonstration of two intelligent reflex workflows performed using the new Revident LC/Q-TOF. Evaluation of drug analytes over a range of concentrations in solvent, plasma and urine is demonstrated using workflows to automatically manage carryover and detection of values above the calibration curve with adjusted sample reinjections during real-time analyses.
Objective
To demonstrate the capabilities of new intelligent reflex workflows for drug screening using a new software tool for fast reflexive worklist management for carry over and above calibration curve detection through automated worklist appending and injection volume reduction.
Method
Urine and plasma samples prepared by dilution and EMR-Lipid extraction, respectively, were spiked at 8 calibration levels from 1 to 100 ng/mL with 32 scheduled drugs and 16 heavy labeled analytes at 50 ng/mL. An 11-minute reverse phase LC gradient method was optimized on a 100 mm Poroshell 120 EC-C18 column. Non-targeted acquisition was carried out using All Ions methodology at collision energies 0, 20 and 40 eV. MassHunter Quantitation methods were built using library MS/MS spectra for comparison of molecular ion and fragment ions and curated further with retention times specific to the chromatography gradient. Intelligent reflex was enabled in the worklist during data acquisition to address carryover and samples detected above highest calibration levels in real time.
Results
All Ions acquisition was used for non-targeted analysis of spiked calibration curves between 1 and 100 ppb (n=4), and results will be shown for plasma and urine matrix. Initial analysis showed, for the 32 compounds of interest the screening method designated all compounds as identified at the 5 ng/mL level with most identified < 5 ng/mL. The screening method utilizes mass accuracy, retention time, mass match score, and number of verified ions. Most calibration curves showed good linearity over the entire concentration range, with correlations R2 > 0.99. Precision of 95% of the response RSDs for each calibration level were below 10%, and mass accuracies were within ±2 ppm for most compounds. The above calibration curve reflexive logic was tested by the worklist integration of a sample over-spiked with 6 compounds at 500 ng/mL. Upon detection of signal over the calibration levels in the associated MassHunter Quantitation method, the sample was reinjected, and injection volume was reduced from 4 uL to 0.5 uL, placing the new data within the calibration range. Carryover management was engaged by setting a blank concentration threshold of 1 ng/mL, and upon detection of carryover in the blank by the instrument, an additional blank injection was inserted into the worklist and replicated until a workflow limit or no carryover detection.
Conclusion
Intelligent reflex methodology can be utilized for non-targeted drug screening workflows, increasing instrument up time and reducing the requirement for manual interpretation between initial injection and subsequent reinjections. Shown herein, this can be beneficial for automatically handling samples with results above calibration curve levels and those showing carryover in integrated blanks. In combination with succinct LC/Q-TOF data analysis supported by the LC Screener tool, complex analysis can be simplified yielding clear and accurate results.
RA45296.487974537.
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= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
