Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1288, Issue -, Pages 73-81Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2013.03.003
Keywords
Mobile phase composition; Electrospray efficiency; Retention; Oligonucleotide; Bioanalysis; Sample preparation
Ask authors/readers for more resources
LC-MS based assays are a promising approach for the bioanalysis of oligonucleotide therapeutics due to their selectivity and structure identification capabilities. However, the lack of sensitivity and complicated sample preparation procedures remain a barrier for application of LC-MS based assays to preclinical and clinical studies. Numerous studies have shown that the mobile phase composition, especially organic solvent type, has a significant impact on the MS sensitivity of oligonucleotides. In this study, we systematically investigated the type of organic solvents and concentration of organic modifiers for their effect on electrospray desorption efficiency, chromatographic separation and LC-MS signal intensity and provide mechanisms for these effects. 25 mM HFIP, 15 mM DIEA and the use of ethanol as an organic solvent were observed to achieve a two order of magnitude increase in LC-MS signal intensity when compared to the most commonly used LC-MS mobile phase composition. Phenol chloroform LLE in combination with ethanol precipitation was demonstrated to be effective for quantitative bioanalysis of therapeutic oligonucleotides. Various conditions for ethanol precipitation were evaluated and >75% absolute recovery was achieved using an optimized extraction procedure. No increase in column pressure or deterioration of separation was observed for >500 injections of biological samples. The method run time was 5 min and the LOQ was 2.5 ng/ml. The accuracy (% error) and precision (%RSD) are <5.09% and <10.56%, respectively, over a dynamic range of 2.5-1000 ng/ml. The assay was applied to a proof of concept animal study and similar PK parameters to previous studies were obtained. (C) 2013 Elsevier B.V. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available