Trace analysis of dimethoxytrityl alcohol (DMT-OH) in oligonucleotide matrices using liquid chromatography coupled with tandem mass spectrometry

Abstract

A liquid chromatography (LC) method with ultraviolet (UV) and tandem mass spectrometry (MS/MS) detection was developed to quantify dimethoxytrityl alcohol (DMT-OH), a small molecule byproduct generated during the detritylation reaction in oligonucleotide synthesis. The pros and cons of quantification via multiple analytical methods including LC coupled with UV, selected ion monitoring (SIM), and multiple reaction monitoring (MRM) were evaluated. The MRM method was ultimately selected for further qualification and exhibited good linearity (R² = 0.997 from 0.5 to 64 ng mL⁻¹), accuracy (recoveries ranging 75–90% with ≤ 2% RSD), repeatability (<5% RSD), and sensitivity (LOQ of 1.6 ng mL⁻¹). The MRM method was further applied to analyze DMT-OH in various oligonucleotide intermediates and drug substances. Similar MRM methods for six other small molecule impurities (aniline, benzamide, isobutyramide, 2-phenylacetamide, succinamide, and uny-CTP) as well as their application are also presented.