An electrospray mass spectrometric method for accurate mass determination of highly acid-sensitive phosphoramidites Zolta ´ n Kupiha ´r 1,2 * , Zolta ´n Tima ´r 1,2 , Zsuzsanna Darula 4 , Douglas J. Dellinger 1,3 and Marvin H. Caruthers 1 1 Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO, USA 2 Department of Medicinal Chemistry, University of Szeged, Szeged, Hungary 3 Agilent Laboratories, 5555 Airport Road, Boulder, CO 80301, USA 4 Proteomics Laboratory, Biological Research Center, Szeged, Hungary Received 24 October 2007; Revised 29 November 2007; Accepted 11 December 2007 An accurate mass determination method utilizing electrospray ionization mass spectrometry is described for analysis of several different types of phosphoramidites that are extremely acid-sensitive compounds. An earlier method, which applied a LiCl/acetonitrile system, was extended for this special application by using polymeric standards including poly(ethylene glycol) (PEG), poly(ethylene glycol) dimethyl ether (PDE) and poly(propylene glycol) (PPG). Concentrations of standards, samples and LiCl were optimized and potential impurities that affect the analyses were also investigated. Copyright # 2008 John Wiley & Sons, Ltd. In the early 1980s, 2 0 -deoxynucleoside phosphoramidites as monomer units revolutionized nucleic acid synthesis. 1,2 These 2 0 -deoxynucleoside derivatives are relatively stable but after mild acidic activation become highly reactive for the condensation reaction that forms the sugar-phosphate backbone of nucleic acids. Following this early research, 3 several phosphoramidite derivatives have been synthesized for different purposes (e.g. fluorescent dye or biotin- containing molecules, 3 0 - and 5 0 -amino or thiol modifiers, sugar or nucleobases with diverse protecting groups). 4–12 However, chemical characterization of these compounds remains problematic. Currently, 1 H, 13 C and 31 P NMR are used to provide important data but a complete characteriz- ation, including elemental analysis, is not possible as phosphoramidites are isolated as foams or oils. Accurate mass determination could replace elemental analysis but the commonly used mild ionization techniques (MALDI, FAB, LSIMS, ESI) apply acidic media or matrices that are incompatible with the acid-labile phosphoramidites. Additionally, many phosphoramidites contain acid labile moieties such as substituted trityl protecting groups which provide strong, undesired signals in the positive detection mode. A method using electrospray ionization mass spec- trometry (ESI-MS) has been used with LiCl in acetonitrile instead of an acidic aqueous methanol solution in order to characterize phosphoramidites in both positive and negative mode. 13 However, for accurate mass determination, which requires an internal standard, there is an additional problem. This is because the internal standard and the analyte molecule mutually perturb one another’s ionization in the ion source. Application of a dual ESI spray ion source can reduce the analyte-reference competition during the formation of ions but this procedure also requires constant optimization of the reference standards and analyte concentrations. 14,15 Addition- ally, these two channel sources are not widely available. Recently, a method has been described by Fujitake et al. for measuring the accurate mass of phosphoramidites using liquid secondary ion mass spectrometry (LSIMS) with a double focusing mass spectrometer and a novel triethano- lamine-NaCl matrix system. 16 These workers analyzed nucleoside phosphoramidites but only in the positive ion detection mode. Although they could measure several phosphoramidites with very high accuracy, the relative intensities of the sample peaks compared to the undesired, substituted trityl peaks were only 0.4–20.7%, which results in lower sensitivity and reproducibility. Our aim was to develop a one-channel ESI-MS procedure in combination with different polymeric standards such as poly(ethylene glycol) dimethyl ether (PDE), poly(ethylene glycol) (PEG), and poly(propylene glycol) (PPG) 15,17,18 as a simple method for accurate mass determination of various phosphoramidites. In our preliminary experiments, as reported in a poster session, 19 5 0 -O-dimethoxytrityl-2 0 - deoxythymidine-3 0 -O-(2-cyanoethyl-N,N-diisopropyl) phos- phoramidite, a PEG standard, and LiCl in acetonitrile were used to optimize the sample/standard/LiCl ratio. Based on these promising results, we investigated many additional phosphoramidites and polymer standards in order to explore the general use of this procedure. These results are summarized in this manuscript. RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2008; 22: 533–540 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.3394 *Correspondence to: Z. Kupiha ´r, Department of Medicinal Chem- istry, University of Szeged, Do ´m te ´r 8, Szeged 6720, Hungary. E-mail: kupi@ovrisc.mdche.u-szeged.hu Contract/grant sponsor: Agilent Technologies and the Univer- sity of Colorado. Copyright # 2008 John Wiley & Sons, Ltd.