Premium
Comparison of direct infusion and on‐line liquid chromatography/electrospray ionization mass spectrometry for the analysis of nucleic acids
Author(s) -
Huber Christian G.,
Krajete Alexander
Publication year - 2000
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/1096-9888(200007)35:7<870::aid-jms11>3.0.co;2-d
Subject(s) - chemistry , electrospray ionization , acetonitrile , chromatography , triethylamine , high performance liquid chromatography , electrospray , mass spectrometry , aqueous solution , solvent , organic chemistry
The applicability of ion‐pair reversed‐phase high‐performance liquid chromatography/electrospray ionization mass spectrometry (IP‐RP‐HPLC/ESI‐MS) and direct infusion/ESI‐MS to the characterization of nucleic acid mixtures was evaluated by the analysis of the reaction products obtained from solid‐phase synthesis of a 39‐mer oligonucleotide. IP‐RP‐HPLC/ESI‐MS was performed using 200 µm i.d. capillary columns packed with octadecylated, micropellicular poly(styrene–divinylbenzene) particles and applying gradients of acetonitrile in 50 m M triethylammonium bicarbonate (TEAB). Three different solvent systems were utilized for direct infusion/ESI‐MS with removal of metal cations by on‐line cation exchange: (1) 10 m M triethylamine (TEA) in 50% aqueous acetonitrile, (2) 2.2 m M TEA, 400 m M hexafluoro‐2‐propanol (HFIP) in 20% aqueous methanol and (3) 50 m M TEAB in 10% aqueous acetonitrile. Owing to its separation capability, the highest selectivity and specificity were achieved with IP‐RP‐HPLC/ESI‐MS, which, apart form the 39‐mer target sequence, allowed the identification of two isobutyryl‐protected target sequences and a 10‐mer and 20‐mer failure sequence. Direct infusion/ESI‐MS with TEA–acetonitrile or TEA–HFIP–methanol as solvent revealed signals for the 39‐mer in the m/z range 700–1600. The presence of derivatives containing one, two, three and four isobutyryl groups indicated that the hydrolysis of the protecting groups after solid‐phase synthesis was not complete. Failure sequences could not be identified by direct infusion/ESI‐MS under conditions favoring multiple charging of the analytes owing to the high chemical background and coincidental overlapping of m/z signals. However, efficient charge state reduction upon addition of carbonic acid to the electrosprayed solvent shifted the signals of the 39‐mer and derivatives to m/z values >2400 and allowed the detection of seven different failure sequences, ranging from the 8‐mer to the 23‐mer, in the mixture. Copyright © 2000 John Wiley & Sons, Ltd.