Methodology Development for Complete Nucleoside and Nucleotide Monitoring within Cell Samples



Tuesday, February 8th, 2022 at 9am EST | 3pm CET | 2pm GMT and 2pm EST | 11am PST | 7pm GMT Approaches and lessons learned on developing an HPLC methodology to cover a complex and broad range of polar analytes: the complete cellular pool of nucleosides and nucleotides plus our drug candidates and associated prodrugs. The utility of PEEK-lined columns and newer HILIC-Z column phases is highlighted in this anecdote of HPLC methodology development.

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Event Overview:

Our drug development campaign produced a need to assay a complex array of metabolites simultaneously: the complete nucleoside and nucleotide pool in cells along with our drug candidates that are intended to perturb them. This particular array of metabolites represents a challenge in that it spans an extremely broad range of physical properties that must be properly retained, separated, and eluted by the intended chromatographic method. This webinar covers our strategy to develop a proper chromatography method to approach this difficult array of metabolites starting from our humble beginning using the entirely wrong phases, all the way to probing final optimization on an effective HPLC methodology. Agilent support, such as through existing white papers, and direct correspondence proved critical for our success. Key lessons are:

  • Value of PEEK-lined columns for dealing with metabolites that interact strongly with stainless steel
  • Some strategies and thoughts on dealing with the chemoclines that can arise when working with HILIC phases and can make the chromatography look far worse than it may actually be

Key Learning Objectives:

  • Considering PEEK-lined columns for dealing with metabolites that interact strongly with stainless steel
  • Phase selection: some considerations on when to move from simple “standard” reverse phases to HILIC phases and our example of how to think about narrowing down the proper HILIC phase for your application
  • Identifying and troubleshooting chemoclines that can occur when working in HILIC phase

Who Should Attend:

  • Technicians that only have a low familiarity with HILIC phases or PEEK-lined columns and that are interested in LC methodology development for targeting complex mixtures of polar analytes especially those that cover a wide range of LogD values.


Nathaniel Sherden, Ph.D.
VP Chemistry
Octagon Therapeutics Inc.

Nathaniel (Nat) Sherden obtained his PhD at California Institute of Technology in organometallic chemistry under the tutelage of Professor Brian Stoltz where his research focused on studying the mechanism and catalyst design around a specific unusual palladium catalyzed allylic alkylation method that proved to function by a non-standard mechanism. He did his Postdoctoral work under Professor Sarah O’Connor which started at the Massachusetts Institute of Technology but continued mostly at the John Innes Centre in the UK. For his postdoctoral work he pursued gene discovery of previously unknown key secondary metabolic enzymes critical to the synthesis of the complex alkaloids in the medicinal plant C. Roseus via a combination of transcriptomics, heterologous gene expression, and untargeted metabolomics. Nat then began work at Octagon Therapeutics Inc. where he is head of chemistry and individually responsible for any analytical chemistry work done at the company so far.

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