Real-time, on-line monitoring of organic chemical reactions using extractive electrospray ionization tandem mass spectrometry Liang Zhu 1 , Gerardo Gamez 1 , Huan Wen Chen 2 ** , Hao Xi Huang 1 , Konstantin Chingin 1 and Renato Zenobi 1 * 1 Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland 2 Department of Applied Chemistry, East China Institute of Technology, Fuzhou 344000, P. R. China Received 13 June 2008; Revised 31 July 2008; Accepted 31 July 2008 Extractive electrospray ionization mass spectrometry (EESI-MS) for real-time monitoring of organic chemical reactions was demonstrated for a well-established pharmaceutical process reaction and a widely used acetylation reaction in the presence of a nucleophilic catalyst, 4-dimethylaminopyridine (4-DMAP). EESI-MS provides real-time information that allows us to determine the optimum time for terminating the reaction based on the relative intensities of the precursors and products. In addition, tandem mass spectrometric (MS/MS) analysis via EESI-MS permits on-line validation of proposed reaction intermediates. The simplicity and rapid response of EESI-MS make it a valuable technique for on-line characterization and full control of chemical and pharmaceutical reactions, resulting in maximized product yield and minimized environmental costs. Copyright # 2008 John Wiley & Sons, Ltd. Obtaining comprehensive information on chemical reactions is crucial for the characterization of reaction mechanisms as well as the maximization of production efficiency in the chemical and pharmaceutical industries. Usually, detection of process deviations and prompt modification of reaction conditions are key to achieving the best control of chemical reactions. However, this demands techniques that are suited for real-time, on-line monitoring of the chemical reaction processes. Among many other benefits, real-time, on-line characterization allows identification of theoretically pro- posed transients, which are usually short-lived species of low concentration, resulting in a better understanding of the reaction mechanisms. This improved understanding will allow the design of superior reaction schemes with higher efficiency and minimized cost. Suitable techniques for on-line monitoring of chemical reactions require high sensitivity, high specificity and fast response. Mass spec- trometry-based methods are of particular interest for the on- line analysis of reactions, 1 due to their high sensitivity and high specificity. Tandem mass spectrometry (MS n ) is often used to acquire kinetic information on chemical reactions and to characterize the reaction intermediates in solution, providing advances in mechanistic studies in organic chemistry. 2,3 Although direct infusion electrospray ioniz- ation spectrometry (ESI-MS) 4–9 and membrane introduction mass spectrometry (MIMS) 10–12 are gaining popularity in this field, both techniques require a series of steps and specially designed equipment to complete the sample pre-treatments (e.g. extraction, separation, dilution, etc.), and this can cause a delay of several minutes in the analysis. 8–10 Moreover, ESI signal variations can occur due to changes in solution composition. 13 To address the delay problem, rapid mixing has been coupled to direct infusion ESI-MS to acquire pre- steady-state information of fast reactions, decreasing the delay to several tens of ms. 14 Even so, rapid mixing is not suitable for on-line monitoring of process scale reactions. MIMS is more amenable to compounds with appreciable vapor pressure and favorable permeability, which depends on the properties of the membrane used and the compounds being studied. Therefore, MIMS cannot be generally used for monitoring of organic chemical reactions. Recently, direct analysis in real time (DART) has been applied for reaction monitoring in drug discovery. 15 In the DART approach, the end of a tube was dipped into a solution to fetch analytes, and then put in front of a heated DART ion source. After volatilization of the solvent, the analytes on the glass surface were ionized, and then directed to the mass spectrometer for analysis. 15 However, the high temperature (up to 2508C) could cause degradation of sensitive compounds. 15 Alternatively, neutral analytes in gaseous, liquid, aerosol form or liberated from a surface can be rapidly and directly detected by extractive electrospray ionization (EESI)-MS, 16–22 without any sample pre-treatment. In addition, EESI may be applicable to reaction suspensions and heterogeneous reaction mixtures which would otherwise be impossible to RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2008; 22: 2993–2998 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.3700 *Correspondence to: R. Zenobi, Department of Chemistry and Applied Biosciences, ETH Zurich, HCI E 329, CH-8093 Zurich, Switzerland. E-mail: zenobi@org.chem.ethz.ch **Correspondence to: H. W. Chen, Department of Applied Chem- istry, East China Institute of Technology, Fuzhou 344000, P.R. China. E-mail: chw8868@gmail.com Copyright # 2008 John Wiley & Sons, Ltd.