A COMPARISON OF CONVENTIONAL AND RAPID GRADIENT MICROBORE LC CYCLIC™ ION MOBILITY MASS SPECTROMETRY FOR NON-TARGETED SCREENING OF BIOLOGICAL MATRICES

Significance of the Topic

The integration of ion mobility separation and collision cross section (CCS) metrics into non-targeted LC-MS workflows enhances the specificity of compound identification in complex biological matrices. This approach addresses challenges of false positives in clinical, forensic and environmental screening by providing an additional separation dimension and a complementary physicochemical descriptor.

Objectives and Study Overview

• Compare conventional 12-minute LC-cIM-MS with a rapid 2.5-minute microbore gradient LC-cIM-MS (RGM-LC-cIM-MS) for non-targeted screening of human urine.
• Evaluate the impact of CCS values, accurate mass, retention time and product ion filters on detection specificity and throughput.

Methodology and Instrumentation

• Sample preparation: Human urine collected 5 hours after administration of carbamazepine (2×200 mg), acetaminophen (2×500 mg) and naproxen (1×500 mg), diluted 1:10 in water.
• Conventional LC method: 12 min gradient, C18 column (100 mm×2.1 mm, 1.8 µm), 0.5 mL/min, 0.1% formic acid in water/acetonitrile.
• Rapid gradient microbore LC: 2.5 min gradient, C18 microbore column (50 mm×1.0 mm, 1.8 µm), 0.4 mL/min, same mobile phases.
• Mass spectrometry: Positive electrospray, quadrupole-cIM-ToF MS, IMS resolution ~65.

Used Instrumentation

• Waters cyclic ion mobility separator coupled to quadrupole and time-of-flight mass analyzer.
• Reversed-phase C18 columns: 100 mm×2.1 mm for conventional, 50 mm×1.0 mm for rapid gradients.
• Electrospray ionization source operating in positive mode.

Main Results and Discussion

• Conventional LC-cIM-MS detected 86 candidate features in urine; applying product ion count ≥1 and ΔCCS <2% filters reduced identifications to 6 true positives (carbamazepine, naproxen, acetaminophen and metabolites).
• RGM-LC-cIM-MS identified 140 features before CCS filtering and 8 after applying ΔCCS <2%, demonstrating equivalent specificity with five-fold higher throughput.
• Mass accuracy below 1 ppm, retention time error under 0.1 min and ΔCCS under 1% were achieved for key analytes and their biotransformation products.
• Only one false positive (ketoprofen) was observed in the rapid method, reflecting high reliability of the screening strategy.

Benefits and Practical Applications

• CCS-enabled libraries reduce false detection rates in complex biological samples.
• Rapid gradient microbore LC workflows maintain confidence in identification while significantly increasing sample throughput.
• Simultaneous acquisition of precursor/product ions and CCS values streamlines non-targeted screening.
• Applicable to clinical toxicology, pharmaceutical monitoring, metabolomics and food safety analysis.

Future Trends and Possibilities

• Expansion of CCS-searchable libraries to include broader compound classes and metabolites.
• Automation of CCS calibration and integration with data-independent acquisition methods.
• Deployment of ultrahigh-throughput microbore LC-IM-MS platforms in clinical diagnostics and environmental monitoring.
• Development of advanced informatics for real-time CCS matching and identification confidence scoring.

Conclusion

The incorporation of cyclic ion mobility separation and CCS metrics into LC-MS workflows significantly enhances specificity and throughput for non-targeted screening in biological matrices. Rapid gradient microbore LC-cIM-MS offers comparable analytical performance to conventional methods in a fraction of the time, supporting robust high-throughput bioanalysis.

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