Hybridization liquid chromatography-mass spectrometry for quantitative bioanalysis of oligonucleotides: The upcoming paradigm shift

• Hybridization LC-MS is rapidly growing as a new bioanalytical technique for oligonucleotides • This paper provides a comprehensive review of hybridization LC-MS methodologies • Strategies and challenges for both single-stranded and double-stranded oligonucleotides discussed • Key considerations, novel applications and future opportunities were also discussed • Hybridization LC-MS has great potential to become the primary method for oligonucleotide bioanalysis Therapeutic oligonucleotides, which are short, synthetic, single- or double-stranded oligonucleotides, have been rapidly growing in drug discovery and development as a new class of modality. Major types of approved oligonucleotide therapeutics include antisense oligonucleotide (ASO), small interfering RNA (siRNA), and aptamers. Various bioanalytical techniques, including hybridization immunoassay, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography (LC)-fluorescence, and quantitative polymerase chain reaction (qPCR), have been used for the quantitative bioanalysis of oligonucleotides, but all have their own drawbacks on either specificity or sensitivity. Recently, hybridization LC-MS has been rapidly growing as a new bioanalytical technique for the quantification of oligonucleotides. This technique offers a unique advantage of achieving both high specificity and good sensitivity by integrating hybridization extraction with LC-MS. With the continued growing demands for sensitive, specific, accurate, and reliable bioanalytical methods, hybridization LC-MS has great potential to be more widely applied and to become the primary method for oligonucleotide bioanalysis. In this review, this technique will be reviewed and compared with the traditional techniques. The strategies and challenges of hybridization LC-MS for the quantification of oligonucleotides, including both single-stranded oligonucleotides (e.g., ASO) and double-stranded oligonucleotides (e.g., siRNA), will be discussed. Considerations, novel applications (e.g., microsampling, microflow LC), and future opportunities of this new technique will also be discussed.