Retention Time, Product Ion, and CCS Characterization of the Constituents of a FDA-Approved Small Molecule Pharmaceuticals Library

Significance of Topic

A detailed library combining ultra performance liquid chromatography, ion mobility, mass spectrometry, and collision cross section (CCS) metrics offers enhanced specificity for identifying small molecule pharmaceuticals in complex matrices. This multifactor authentication reduces false positives and increases confidence in non-targeted screening in clinical, forensic, and environmental applications.

Study Objectives and Overview

The study aimed to generate a comprehensive UPLC-IM-MS library of FDA-approved small molecule drugs, including retention times, precursor and product ions, and CCS values, in both positive and negative electrospray modes. It also sought to demonstrate the library’s utility in a non-targeted screening of human urine to identify therapeutic xenobiotics and endogenous compounds.

Methodology and Instrumentation

• Sample preparation: 10-fold diluted human urine collected 6 hours after administration of carbamazepine (2×200 mg) and acetaminophen (2×500 mg).
• Liquid chromatography: ACQUITY UPLC I-Class, HSS T3 C18 column at 40 °C, 10 µL injection, 0.5 mL/min gradient with 0.1% formic acid in water and acetonitrile.
• Ion mobility mass spectrometry: SYNAPT G2-Si in ES+ and ES– modes, m/z 50–1200, HDMSE acquisition, collision energy ramp 15–25 eV, traveling wave IMS with nitrogen buffer gas.
• Data processing: MassLynx v4.2 and UNIFI v1.94 for retention time, accurate mass, product ion, and CCS determination.

Main Results and Discussion

• Library composition: 1343 entries in positive mode ([M+H]+, [M+Na]+) and 950 in negative mode ([M–H]–, [M–H+HCOO]–).
• Separation: Chromatographic co-elution resolved in the ion mobility dimension, enabling extraction of single-component spectra.
• Urine screening: Initial 60 candidate matches (±5 ppm, ±0.1 min) were filtered using ΔCCS (<2%) and expected product ions to yield 5 confirmed identifications.
• Identified compounds: Carbamazepine (ΔCCS 0.51%, mass error 3.9 ppm), carbamazepine-10,11-epoxide, theophylline (coffee metabolite), acetaminophen, DL-carnitine, and hydrocortisone.
• Retention time flexibility: Caffeine and acetaminophen were retrospectively identified using only accurate mass, product ions, and CCS despite different chromatographic conditions.

Benefits and Practical Applications

• Enhanced specificity through multifactor authentication: retention time, accurate mass, product ions, and CCS.
• Reduced false positive rates <5% in non-targeted assays.
• Applicability to clinical monitoring, forensic toxicology, drug metabolism, food safety, and environmental screening.
• Flexible post-acquisition workflows allow incorporation of external libraries for retrospective analyses.

Future Trends and Potential Applications

• Expansion of CCS-searchable libraries to encompass broader chemical spaces and emergent contaminants.
• Integration with suspect screening and data-independent acquisition workflows.
• Improvements in ion mobility resolution and reproducibility for robust long-term operation.
• Application to lipidomics, metabolomics, and complex natural product characterization.

Conclusion

The UPLC-IM-MS CCS library of FDA-approved drugs provides an effective tool for non-targeted screening in complex matrices, significantly enhancing identification confidence and reducing false detections. Validation in human urine demonstrates its robustness and versatility across diverse analytical challenges.

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