Rapid LC-MS Analysis of Oligonucleotides: Application to Genotyping and Quality Control

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Kenneth J. Fountain, Martin Gilar, Claude R. Mallet, John C. Gebler [Waters]
Fall ACS 2003, 226th ACS National Meeting; New York September 7-11, ISPPP 2003, Delray Beach, Florida November 9-12
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XTerra MS C18 2.5 µm Steel 1.0 mm 50 mm
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Synthetic oligonucleotides are utilized for many diagnostic and therapeutic purposes. Therefore, their quality control (QC) is imperative for monitoring the reliability of synthesis. Traditionally, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) has been used for this purpose; however, oligonucleotides greater than 50mer in length are difficult to analyze, due to decreasing signal intensity and mass accuracy. A capillary liquid chromatography-mass spectrometry (LC-MS) method was recently developed for the sensitive analysis and characterization of long oligonucleotides. Duty cycle times were in the range of 20-40 minutes per sample, depending on the size of the oligomer. The method proved to be sensitive at subpicomole levels, and was shown to have an average mass accuracy of 125 ppm for oligonucleotides up to 110mer in length. The method was successful for the separation and characterization of guanine-rich oligonucleotides and phosphorothioate (PS) drugs, and their failure sequences/metabolites. However, for routine QC of oligonucleotides, faster analysis is desirable. Here, an alternative rapid LC-MS method is presented for the high throughput quality control of synthetic oligonucleotides up to 110mer. The method essentially consists of two alternating isocratic LC runs (one for desalting and the other for elution) controlled by a dual position switching valve. Duty cycle times for the current method are as low as 1.5 minutes per sample. This allows for the analysis of approximately 950 samples per 24 hour time period, which is suitable for medium to high throughput applications. The developed method utilizes LC only as a desalting tool, with separation and synthesis component identification being achieved by mass spectrometry. As little as 100 picomoles of sample was needed for analysis, and average mass accuracy was determined to be ~ 80 ppm.

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