Liquid chromatography tandem mass spectrometry has been a widely used technique for quantifying oligonucleotides in biological samples. However, lack of simple and efficient sample cleanup approach remains a challenge. Our study aimed to evaluate the major factors during the sample pretreatment process for developing optimal sample preparation workflow for oligonucleotides. In this study, we have employed a model formed with rat plasma containing a 16 mer oligonucleotide standard in order to comprehensively optimize the sample preparation procedures. These included liquid-liquid extraction (LLE), solid-phase extraction (SPE), protein precipitation (PPT) and LLE combined with SPE. LLE with phenol: dichloromethane (2:1, v:v) was found to be the most efficient sample cleanup procedure with low cost and less toxicity. Followed by the extraction, ethanol precipitation (-80 °C, 5 min) was determined to be the optimal drying conditions. Also, mass spectrometric parameters were tuned to optimal conditions. It was found that the central composite design suite was proved to be highly practical for optimizing MS parameters. Finally, the thoroughly optimized sample preparation workflow was fully validated. The developed assay provided a quantitative range of 0.25-1000 nM, with accuracy and precision were < 7.45% and < 12.20%, respectively. Matrix effect and carryover were also evaluated and no significant effect was observed.
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