Electrospray ionization mass spectrometric analysis of nucleic acids using high-throughput on-line desalting

A rapid on-line desalting method utilizing ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) was employed in tandem with negative electrospray ionization mass spectrometry (ESI-MS) for the routine analysis of nucleic acids. Desalting was performed on a short 10 x 2.1 mm guard column packed with 3.5 microm C(18) sorbent. The HPLC system was connected in-line to an orthogonal ESI-TOF mass spectrometer via a six-port, two-position switching valve, allowing desalting followed by mass analysis of nucleic acids. Duty cycle times for the method were as low as 1.5 min per sample. This allowed for the analysis of approximately 950 samples per 24-h time period, which is suitable for medium- to high-throughput applications. Average mass accuracy was determined to be 80 ppm for oligonucleotides up to 110 mer in length with external calibration. The method was utilized for synthetic oligonucleotide quality control and analysis of DNA genotyping fragments.