DOING MORE WITH LESS: ADDRESSING THE MICROSAMPLING SENSITIVITY CHALLENGE IN DMPK STUDIES USING VACUUM JACKETED COLUMN & UHPLC-CYCLIC ION MOBILITY MS

Significance of the Topic

The increasing demand for high-sensitivity pharmacokinetic and metabolite identification in drug metabolism and pharmacokinetic (DMPK) studies has driven the need for analytical methods capable of delivering robust results from minimal sample volumes. Microsampling strategies reduce animal use, streamline workflows, and address ethical and logistical concerns while requiring enhanced sensitivity and chromatographic performance.

Goals and Overview

This study aims to develop a rapid and sensitive UHPLC-MS/MS approach leveraging vacuum jacketed columns (VJC) and cyclic ion mobility-mass spectrometry (cIM-MS) to characterize the pharmacokinetics and biotransformations of fasiglifam (TAK-875) in rat models using microsamples. Specific objectives include:

• Quantitative profiling of IV and oral dosing regimens
• Identification of novel metabolites, including potentially toxic species
• Optimization of data analysis workflows for rapid turn-around

Methodology and Instrumentation

The workflow combined microsampling with advanced chromatography and mass spectrometry:

• Sample Preparation: 10 µL plasma protein precipitation with acetonitrile (4:1), dilution 1:100 in 25 % water
• Quantitative Analysis: ACQUITY Premier UPLC with vacuum jacketed BEH C18 column (2.1×50 mm, 1.7 µm) at 60 °C; reversed-phase gradient (0.1 % formic acid vs. acetonitrile) over 0.9 min; Xevo TQ-S micro MS, positive ESI, MRM transitions
• Metabolite Identification: ACQUITY Premier UPLC with Select Series Cyclic IMS (HSS T3 C18, 2.1×100 mm) at 40 °C; HDMSe acquisition (50–1200 m/z); nitrogen drift gas; UNIFI software for automated library screening, neutral loss and product ion matching, trend analysis, and reporting

Main Results and Discussion

• Quantitative Performance: IV (5 mg/kg) peak plasma concentrations of ~9 µg/mL; oral (10 and 50 mg/kg) peaks of 12–13 µg/mL and 76–84 µg/mL, bioavailability 85–120 %; half-lives ~11–12 h
• Chromatographic Improvements: Vacuum jacketed columns reduced extra-column dispersion, delivering improved resolution from endogenous interferences and enabling faster run times
• Metabolite Characterization: Identification of 15 biotransformation products, including 3 novel metabolites (α-keto acid, side-chain shortened species, acyl glucuronide), with structural elucidation supported by drift-time aligned spectral data
• Data Workflow: Automated UNIFI library creation and use of trend plots streamlined screening, reduced false positives, and accelerated reporting

Benefits and Practical Applications

• Microsampling: Full PK profiling and metabolite screening achieved with minimal sample volumes and only three animals per dose group, supporting ethical research and resource efficiency
• Enhanced Sensitivity: Improved limit of quantification during elimination phase and better detection of minor metabolites
• Operational Efficiency: Faster data turn-around and simplified data analysis support high-throughput DMPK studies

Future Trends and Opportunities

• Integration with Automated Data Analysis and AI: Further reduce manual data review and enhance metabolite annotation
• Expansion to Other Modalities: Applying VJC and cIM-MS to peptide, lipid, and large molecule profiling
• Miniaturized Workflows: Continued development of sub-microliter sampling techniques to minimize invasiveness
• Advanced Ion Mobility: Leveraging higher-resolution drift separation for complex isomer differentiation

Conclusion

The combination of vacuum jacketed UHPLC columns, cyclic ion mobility-MS, and a streamlined data workflow delivers a powerful solution for microsampling DMPK studies. This approach enables high-sensitivity quantitation and comprehensive metabolite identification, enhancing efficiency while reducing sample and animal use.

Used Instrumentation

• Waters ACQUITY Premier UPLC
• BEH C18 columns in vacuum jacketed format
• Xevo TQ-S micro mass spectrometer
• Select Series Cyclic IMS
• UNIFI, MassLynx, and TargetLynx software

References

1. Kaku K et al. Long-term safety and efficacy of fasiglifam (TAK-875)... Diabetes Obes Metab. 2016;18(9):925–929.
2. Li X et al. Fasiglifam inhibits hepatobiliary transporters... Drug Metab Dispos. 2015;43(11):1751–1759.
3. Molloy BJ et al. Investigation of the Pharmacokinetics and Metabolic Fate of Fasiglifam... Xenobiotica. 2023;16:1–30.
4. Tanna N et al. Enhanced chromatographic efficiency with vacuum jacketed columns... Talanta. 2023;254:124089.