Remediation of undesirable secondary interactions encountered in hydrophilic interaction chromatography during development of a quantitative LC–MS/MS assay for a dipeptidyl peptidase IV (DPP-IV) inhibitor in monkey serum

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2009-02, Vol.877 (5), p.471-476
• Main Authors: Kadar, Eugene P., Wujcik, Chad E.
• Format: Article
• Language: English
• Subjects: Acetonitriles; Analysis; Analytical, structural and metabolic biochemistry; Animals; Bioanalysis; Biological and medical sciences; Buffers; Chromatography, Liquid - methods; Dipeptidyl Peptidase 4 - blood; Dipeptidyl peptidase IV (DPP-IV) inhibitor; Dipeptidyl-Peptidase IV Inhibitors - blood; Dipeptidyl-Peptidase IV Inhibitors - chemistry; Fundamental and applied biological sciences. Psychology; General pharmacology; Haplorhini - blood; HILIC; Hydrophilic interaction chromatography; Injections; LC–MS/MS; Mass Spectrometry - methods; Medical sciences; Particle Size; Pharmacology. Drug treatments; Reproducibility of Results
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2008.11.048

PF-00734200 (3,3-Difluoropyrrolidin-1-yl)-((2S,4S)-4-(4-(pyrimidin-2-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone) is an inhibitor of dipeptidyl peptidase IV (DPP-IV) for the treatment of diabetic complications and other disorders. A sensitive and selective LC–MS/MS assay capable of quantifying PF-00734200 in monkey serum was required to support regulated safety studies. Due to the polar nature of this compound and for ease of sample processing, hydrophilic interaction chromatography (HILIC) was identified as an ideal assay technique. During the initial phase of method development significant peak tailing was observed. The effects of polar organic modifier percentage, buffer concentration, column particle size, and flow rate were assessed to determine the final optimal conditions. PF-00734200 demonstrated a strong dependence on buffer concentration with respect to height equivalent to a theoretical plate (HETP), capacity factor ( k′), and tailing factor ( T). Improvements in chromatography were observed with increasing buffer concentration due to reduction of electrostatic secondary interactions with ionized silanols. A plot of log k′ versus percentage organic modifier at an elevated buffer concentration, produced a linear fit with a correlation coefficient of 0.996, indicating that the primary chromatographic retention mechanism was partitioning. A LC–MS/MS assay was successfully developed and validated for GLP bioanalysis of PF-00734200 in monkey serum utilizing the optimized HILIC conditions. Additionally, carryover was effectively minimized through fortification of ethylene glycol to the sample extract.