In the world of scientific research and development, certain analytes pose unique challenges that require specialized expertise to overcome. Oligonucleotides, a class of analytes consisting of short DNA or RNA sequences, are notorious for their complexity and demanding nature. With their increasing importance in the development of therapeutic applications, understanding and effectively analyzing oligonucleotides has become crucial. As a Senior Scientific Advisor at KCAS, John Perkins brings a wealth of knowledge and experience to the table. John recently delivered a presentation highlighting the intricacies and hurdles associated with oligonucleotide analysis at the International Oligonucleotide and Peptides Conference in Milan, Italy. The presentation covered many of the routine issues we see during oligonucleotide analysis and the steps we take to ensure that our assays are robust and successful.
Unraveling the Challenges of Oligonucleotides
Oligonucleotides present a series of obstacles throughout the analytical process, making them a particularly difficult analyte class to work with. One of the major challenges is absorption. Oligos have a tendency to bond to surfaces, whether during extraction, within LC tubing, or on columns. To mitigate this issue, low-binding plasticware and dedicated lab supplies are employed. Traditional protein precipitation techniques, although simple to sue can be less desirable due to nonspecific binding and co-precipitation risks. Liquid-liquid extraction is also a simple approach but we’ve seen that extracts can still be relatively dirty leading to interferences. Our methodology of choice tends rto be a combination of liquid-liquid extraction followed by anion exchange solid-phase extraction where we can take advantage of phases that have been developed specifically with oligonucleotide analysis in mind.. Similarly, recent advancements in chromatography have led to the development of specialized LC column phases specifically tailored for oligonucleotide analysis. However, even with these specialist columns, tailing and non-retention can still pose challenges.
Optimizing Analytical Processes
John emphasized the need to optimize the three-step process involved in LC-MS/MS analysis: extraction, chromatography, and mass spectrometry. By fine-tuning each step, researchers can achieve optimal performance for oligonucleotide analysis. Passivating the LC-MS system overnight through repeat injections of the analytes helps eliminate initial binding and ensures accurate quantification. Optimizing the extraction and LC-MS/MS process is essential for leveraging the selectivity and capabilities each step offers while dealing with the unique challenges posed by oligonucleotides.
KCAS: A Leading Force in Overcoming Challenges
As a Senior Scientific Advisor at KCAS, John Perkins has a wealth of experience with bioanalysis using LC-MS/MS. KCAS, a renowned research organization, has emerged as a frontrunner in providing solutions to bioanalytical challenges. Leveraging their depth of knowledge and extensive experience, the team at KCAS has analyzed a significant number of oligonucleotides, ranging from 20 to 35-mer building blocks. This expertise enables them to offer invaluable insights and solutions to clients seeking assistance in this field. KCAS has positioned itself as a leading voice in the industry, actively addressing the complexities of oligonucleotide analysis.
Leading the Charge
John’s presentation at the International Oligonucleotide and Peptides Conference serves as a testament to KCAS’s commitment to advancing the understanding and successful analysis of oligonucleotides. As a Senior Scientific Advisor, John shared KCAS’ experiences, knowledge, and best practices with the scientific community. By disseminating information and shedding light on the intricacies of oligonucleotide analysis, John and the team at KCAS hope to lead the charge in overcoming the challenges associated with these analytes.
Oligonucleotides increasingly play a vital role in addressing unmet therapeutic needs. However, their complex nature presents unique challenges throughout the analytical process. The bioanalytical LC-MS teams at KCAS, have been at the forefront of tackling these challenges head-on. Through John’s presentation and the collective knowledge of the team at KCAS, they are poised to lead the charge in unraveling the complexities of oligonucleotide analysis.