A Reproducible Method for the Quantification of Pioglitazone and Metabolites in Human Plasma Using the ACQUITY UPLC H-Class System and Xevo TQD MS with UNIFI

Importance of the Topic

Pioglitazone is a key thiazolidinedione used to manage type 2 diabetes by activating PPAR-γ and regulating glucose and lipid metabolism. Accurate measurement of pioglitazone and its active metabolites in plasma is essential for pharmacokinetic studies, dose optimization and safety monitoring.

Objectives and Study Overview

This study aimed to develop a highly sensitive and reproducible UPLC–MS/MS method for quantifying pioglitazone, keto pioglitazone and hydroxy pioglitazone in human plasma with a lower limit of quantification of 10 pg/mL. The approach focused on improving extraction efficiency, reducing carryover and ensuring robust quantification over a wide dynamic range.

Methodology and Instrumentation

The sample preparation employed Oasis HLB μElution solid-phase extraction of 300 μL plasma spiked with deuterated internal standards. After conditioning, loading and washing, analytes were eluted, diluted and injected. Chromatographic separation used an ACQUITY UPLC BEH C18 column (2.1×50 mm, 1.7 μm) under a 2-minute gradient at 600 μL/min with 0.1% ammonium hydroxide and methanol mobile phases. Detection was performed on a Xevo TQD in positive MRM mode monitoring transitions 357>134, 371>148 and 373>150 with corresponding deuterated analogues. Data processing utilized the UNIFI Scientific Information System.
Instrumentation:

• Xevo TQD Mass Spectrometer
• ACQUITY UPLC H-Class System
• UNIFI Scientific Information System
• Oasis HLB μElution Plate

Main Results and Discussion

All three analytes eluted within 1.34–1.59 minutes, producing narrow symmetric peaks (<3 s at baseline). Carryover was negligible, enabling full sensitivity exploitation. The method achieved an LLOQ of 10 pg/mL with signal-to-noise ratios of 44:1, 21:1 and 58:1. Calibration curves were linear (r2 0.997–0.999) using 1/x weighting. Intra-day precision (%CV) ranged from 1.1% to 13.4% and accuracy bias from −7.0% to +1.8% across QC levels.

Benefits and Practical Applications

• High sensitivity with LLOQ of 10 pg/mL for all analytes
• Minimal carryover supporting high-throughput analysis
• Robust accuracy and precision suitable for clinical and preclinical studies
• Applicability to pharmacokinetic profiling and therapeutic monitoring

Future Trends and Applications

Advances in UPLC-MS/MS will continue to enhance sensitivity and throughput. Automation of sample preparation, integration with high-resolution instruments and advanced data analytics will expand metabolite profiling capabilities. Personalized medicine approaches may benefit from rapid, low-volume quantification of drug exposures and metabolites.

Conclusion

The described UPLC–MS/MS assay delivers a reproducible, high-sensitivity platform for quantifying pioglitazone and its active metabolites in human plasma. Its performance in terms of low LLOQ, minimal carryover and robust validation metrics makes it valuable for pharmacokinetic and clinical research applications.

References

1. Baughman TM, Graham RA, Wells-Knecht K, Silver IS, Tyler LO, Wells-Knecht M, Zhao Z. Metabolic activation of pioglitazone identified from rat and human liver microsomes and freshly isolated hepatocytes. Drug Metab Dispos. 2005;33:733-738.