SVP & Global Head Bioanalytical (Mass Spec) QPS newark, Delaware
The physicochemical properties of oligonucleotide therapeutics (antisense oligonucleotides, microRNA, small interfering RNA, nucleic acid aptamers, and antibody-oligonucleotide conjugates) make quantitation of these compounds in biological matrices very challenging. Different quantitative approaches have been used, such as hybridization ELISA, hybridization HPLC fluorescent, HPLC UV, LC MS/MS, and LC HR(high resolution accurate mass)MS. This presentation will discuss the bioanalytical differences between LC MS/MS and LC HRMS for this modality.
We routinely develop and validate plasma, urine, and tissues assays for oligonucleotides using both LC MS/MS and LC HRMS platform. The LC-(HR)MS/MS methodology can quantitate oligonucleotides, ranging from 5000 to 15000 Da, and meet the assay acceptance criteria per the latest regulatory bioanalytical method validation guidance. These methods are used to support preclinical and clinical studies, having LLOQ ~2 ng/mL with 3 order of linear dynamic range using as little 50 μL sample volume. Furthermore, the antisense strand, the sense strand, and their corresponding metabolites can be quantitated and/or monitored simultaneously in a N-in-1 format.
Our typical sample recovery is 85%+ despite oligonucleotides having intrinsic high protein binding and require complicated extraction steps. The presentation will briefly touch on the various steps taken in sample preparation, column selection, mobile phase selection, and mass isolation in these mass spectrometric assays to address cations adducts in the mass spectrometer which can have negative effects on the assay accuracy and precision.
Fundamentally, there are little differences in running LC MS/MS assays or LC HRMS assays. The perception is the HRMS assay format is complicated, the instrument complex, and relatively rare in most bioanalytical laboratories, while the MS/MS assay format has higher sensitivity and is very common. In our hands, we found MS/MS and QTOF (quadrupole time of flight) HRMS give similar sensitivity. Therefore, we are using HRMS instrument in the full scan mode so we can perform qual quan methodology to detect, monitor, and quantitate oligonucleotide drugs and their full range of potential metabolites in one assay.
Learning Objectives:
Upon completion, participant will be able to select the chromatographic conditions to separate oligonucleotide drugs and their metabolites
Upon completion, participant will be able to understand the differences between MS/MS format or HRMS format for oligonucleotides quantitation.
Upon completion, participant will be able to understand the 'working' mass resolution requires to quantitate oligonucleotides in HRMS.