A recent paper published in Trends in Analytical Chemistry (TrAC) by scientists from the Institute of Pharmaceutical Sciences of Western Switzerland at the University of Geneva (1), presents recent advances, as well as current challenges, relating to hydrophilic interaction chromatography (HILIC) for the analysis of therapeutic oligonucleotides (ONs).
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HILIC is a liquid chromatography (LC) technique that uses a polar stationary phase (for example, silica or a polar bonded phase) in conjunction with a mobile phase containing an appreciable quantity of water (usually at least 2.5% by volume) combined with a higher proportion of a less polar solvent (often acetonitrile). Most commonly, separations are carried out using 5–40% water (or aqueous buffers); the technique also is compatible with gradient elution (2).
ON therapeutics have begun to emerge as a strong alternative to treat a variety of diseases. Drug manufacturers view these therapeutics as a rapidly expanding category of products that may evolve into more personalized treatment. However, the chemical synthesis of ON therapeutics has limited use and development, as well as created concerns with stability, purity, and cost of development (3,4).
The TrAC paper says that there have been significant advances in understanding the retention mechanism of ONs in HILIC mode. These advances have resulted in the expansion of analytical methods which are able to effectively resolve ONs mainly based on chain length and are influenced by accessible groups involved in the HILIC retention mechanism; these groups include phosphorothioate groups, as well as base nature or sugar modifications). Furthermore, the susceptibility of adsorption of ONs onto column hardware has resulted in advances in specially adapted columns which were established to lessen this problem. As a result, the use of bioinert HILIC hardware has begun to occur as a necessity in the avoidance of sensitivity loss, and to diminish problems such as peak tailing and broadening, as well as inadequate general execution.
The paper notes there has been positive developments in improving the performance of HILIC-MS performance. Among these developments are the significant enhancement of the technique’s effectiveness via the systematic optimization of chromatographic conditions to favor negative ionization of ONs, along with fine tuning of MS nebulization parameters. The authors said that this analytical strategy may represent an intriguing substitute for ion-pair reversed-phase mass spectroscopy (IP-RPLC-MS), especially in cases where when ion-pair free LC-MS methods are more favorable, if not mandatory. While the performance of IP-RPLC-MS appears to still prevail over that of HILIC-MS, especially in terms of MS sensitivity, adopting of new technological strategies (such as miniaturization, for example) may help in overcoming these challenges, which would then allow for greater flexibility in the selection of the most suitable method for the characterization of ONs.
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