Strategies for Bioanalysis of Oligonucleotides Using LC-MS/MS- Assay development for oligonucleotide compounds in urine and skin

Abstract

Quantitation of in vivo oligonucleotide levels is necessary to evaluate pharmacokinetics /pharmacodynamics (PK/PD) and a drug’s exposure-toxicity relationship. Therefore, a fast and reliable bioanalytical method is desired for both preclinical and clinical development. Intact oligonucleotide macromolecule analysis using LC-MS/MS presents challenges due to multiple charging, cation adduction, high protein binding, and nonspecific binding of the analyte during extraction. This analytical method development aims to provide a simpler, faster LC-MS/MS assay for an oligonucleotide in a clinical PK/PD study. Sample preparation consisted of a liquid-liquid extraction (LLE) using a mixed solvent of phenol/chloroform, followed by a wash with dichloromethane under basic conditions. The oligonucleotide analyte was separated on a Waters XBridge OST C18 column at 60°C, using a step-gradient elution. The mobile phases consisted of water and methanol containing 15 mM triethylamine (TEA) and 400 mM hexafluoroisopropanol (HFIP). Analyte-1 was detected using an AB Sciex 4000 mass spectrometer in negative electrospray ionization (ESI) mode, operating in multiple reaction monitoring (MRM) mode with a transition of m/z 778.1 ? 94.9. A linear dynamic range of Analyte-1 from 10–10,000 ng/mL in both urine and skin homogenate was successfully qualified with good accuracy and precision. A two-step LLE LC-MS/MS bioanalytical method was thus successfully developed and qualified for the quantification of intact Analyte-1 in human urine and skin tissue to support a clinical PK study.

Keywords: Oligonucleotide bioanalysis, LC-MS/MS assay development, Liquid-liquid extraction, Urine and skin matrix, Negative electrospray ionization.