New Method Overcomes Challenges of High Polarity, Small Molecular Size
Results to be Published in Biomedical Chromatography
Though metformin is reportedly the most prescribed medication for Type II Diabetes1, it has traditionally presented distinct challenges to researchers who want to use the compound in further research.
Analysis is especially difficult because metformin has high polarity and small molecular size, which lead to poor retention on reversed-phase liquid chromatographic columns.
Working with clients from Janssen Pharmaceuticals, the Frontage Bioanalytical group responded to this challenge by developing a new method using hydrophilic interaction chromatography tandem mass spectrometry (HILIC-MS/MS).
Method developed for preclinical studies of metformin and an experimental drug substance
Combined with Tecan automated sample preparation, this high-throughput method was developed for several preclinical studies to determine metformin levels in rat plasma when dosed together with an experimental drug substance.
Scheduled for publication in an upcoming issue of Biomedical Chromatography2, the method used direct injection of the extracted samples. It achieved acceptable intra-run and inter-run assay precision (coefficient of variation = 3.9%) and accuracy (99.0%-101.8%) over a linear range of 50-5,000 ng/mL. Following the FDA guidance, the HILIC-MS/MS method was validated for accuracy, precision, sensitivity, stability, matrix effect, recovery, and calibration range. Finally, the incurred sample reproducibility test demonstrated the ruggedness of the validated method.
This achievement is significant because previous published applications failed to support direct injection of extracted sample solution3. As a result, they required additional time consuming evaporation and reconstitution procedures that could lead to analyte loss.
Method may add to interest in hydrophilic interaction chromatography
The new Frontage-Janssen method may add to the growing interest in hydrophilic interaction chromatography (HILIC) as a means to overcome some of the limitations of liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.
The main reasons for the increased interest are that HLIC can:
•Provide better chromatographic retention of highly polar or ionizable analytes
•Separate analytes of widely different polarity in a reasonable time
•Enhance sensitivity with electrospray LC-MS, and
•Resolve an analyte and its counter-ion in the same analysis4.
Unlike reversed-phase, organic solvent has weaker elution strength on HILIC. Therefore, HILIC also has the advantage of direct injection of extracted samples with high organic concentration generated by protein precipitation, liquid–lquid extraction and solid phase extraction5.
This direct injection of samples leads to shortened sample preparation and eliminates the potential analyte loss during sample drying down and reconstitution, which in turn increases speed and throughput. Owing to the high organic concentration in the mobile phase, HILIC usually enhances ionization in mass spectrometry detection, and thus the sensitivity.
For further information or to obtain a copy of the publication, please contact Zhongping (John) Lin, VP Bioanalytical Services, Frontage Laboratories, johnlin@frontagelab.com.
REFERENCES
1.Bailey CJ and Day C. Metformin: its botanical background. Practical Diabetes International 2004; 21(3): 115-117.
2.Zhang W, Han F, Zhao H, Lin Z, Huang Q, Weng N. Determination of metformin in rat plasma by HILIC-MS/MS combined with Tecan automation and direct injection. Biomedial Chromatography 2011 (wileyonlinelibraray.com) DOI 10.1002/bmc.2673.
3.Sengupta et al., 2009; Zhao et al., 2007; Mistri et al., 2007; Chen et al., 2004; Marques et al., 2007; Wang et al., 2004.
4.Jian et al., 2010; Grumbach et al., 2004.
5.Lin et al., 2004.
About Frontage
Frontage Laboratories, Inc. is a global contract research, development and manufacturing organization, offering a full range of pharmaceutical R&D services. Frontage operates in the US and China, using one seamless GXP platform. Frontage runs three Phase 1 Clinical units, a 72-bed Phase 1 Unit in Hackensack, NJ, a 120-bed Phase 1 Unit in Zhengzhou, Henan Province China, and a-80 bed Phase 1 Unit in Changchun, Jiling Province China. Frontage has an AALAC certified preclinical animal facility in Pennsylvania, where it also maintains bioanalytical and CMC facilities. In Shanghai and Beijing, China, the company also operates bioanalytical and CMC facilities. As a rapidly expanding CRO with ten years of success providing high-quality GXP services, Frontage has established an international standard in pharmaceutical product research, development, quality and management systems.