Real time monitoring of accelerated chemical reactions by ultrasonication-assisted spray ionization mass spectrometry Shu-Hsuan Lin, Ta-Ju Lo, Fang-Yin Kuo and Yu-Chie Chen* Ultrasonication has been used to accelerate chemical reactions. It would be ideal if ultrasonication-assisted chemical reactions could be monitored by suitable detection tools such as mass spectrometry in real time. It would be helpful to clarify reaction intermediates/products and to have a better understanding of reaction mechanism. In this work, we developed a system for ultrasonication-assisted spray ionization mass spectrometry (UASI–MS) with an ~1.7 MHz ultrasonic transducer to monitor chemical reactions in real time. We demonstrated that simply depositing a sample solution on the MHz-based ultrasonic transducer, which was placed in front of the orifice of a mass spectrometer, the analyte signals can be readily detected by the mass spectrometer. Singly and multiply charged ions from small and large molecules, respectively, can be observed in the UASI mass spectra. Furthermore, the ultrasonic transducer used in the UASI setup accelerates the chemical reactions while being monitored via UASI–MS. The feasibility of using this approach for real-time acceleration/monitoring of chemical reactions was demonstrated. The reactions of Girard T reagent and hydroxylamine with steroids were used as the model reactions. Upon the deposition of reactant solutions on the ultrasonic transducer, the intermediate/product ions are readily generated and instantaneously monitored using MS within 1 s. Additionally, we also showed the possibility of using this reactive UASI–MS approach to assist the confirmation of trace steroids from complex urine samples by monitoring the generation of the product ions. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: ultrasonication; real time monitoring; mass spectrometry; electrospray ionization; steroids Introduction Acoustic cavitation induced by ultrasound forms vapor-filled bubbles in liquids, followed by expansion, which leads to the generation of small and energetic bubbles. [1,2] These small bubbles can be considered as micro reactors that facilitate the efficiency of chemical reactions. [3] Consequently, ultrasonication has been widely used to accelerate chemical reactions. [4–8] For example, ultrasonication remarkably accelerates enzymatic diges- tion. [7,8] The reaction time can be shortened from hours to minutes. The main advantage of ultrasonication-assisted reac- tions is the improved efficiency of chemical reactions in terms of reaction time and yield. Ultrasound has been used to assist ionization in mass spectro- metric analysis. [9–18] For example, a low-frequency ultrasonicator (~40 kHz) has been used to facilitate ionization in ultrasonication- assisted spray ionization mass spectrometry (UASI–MS). [12–14] Capillary tubes are used as sampling tubes for delivering samples to facilitate sample delivery and to generate the ultrasonic spray at the outlet of the capillary. The ultrasonic spray containing analyte ions can then be directly detected using UASI–MS although no electric contact is made with the UASI capillary. Furthermore, UASI–MS with a low-frequency ultrasonicator (~40 kHz) has been readily used for online monitoring of chemical reactions. [14] How- ever, the acceleration of the chemical reactions was not observed by the low-frequency ultrasonic transducer-based UASI setup. Reactive MS was lately popularized by desorption/ionization spray ionization (DESI) [19] that can facilitate chemical reactions in situ during desorption/ionization process. In DESI, a stream of charge droplets generated from electrospray is directed hit onto the sample deposited on a solid substrate at ambient condi- tions. [19–21] As a result, the secondary ions are generated from the interaction between the charged droplets with analyte on the DESI substrate for MS analysis. When the charge droplets are composed of reactive reagents, the droplets can selectively react with their target analytes from the sample deposited on the sample substrate through ion/molecule reactions during desorption/ionization process. The reaction products derived from target analytes are simultaneously detected by MS. Thus, reactive MS can be defined as a technique in which target analytes are selectively reacted with reactive additives during ionization process and allow the generated product ions to be detected by MS instantaneously. High-frequency ultrasonicators are known to prominently accelerate chemical reactions. [22–26] We propose herein using MHz-based UASI–MS as the reactive ion source. Namely, when liquid solution containing reactants and samples is deposited directly on a MHz based-ultrasonic transducer, it is expected that chemical reactions and ionization occur simultaneously. The generated product ions can be * Correspondence to: Yu-Chie Chen, Department of Applied Chemistry, National Chiao Tung University, Taiwan. E-mail: yuchie@mail.nctu.edu.tw Department of Applied Chemistry, National Chiao Tung University, Hsinchu 300, Taiwan J. Mass Spectrom. 2014, 49, 50–56 Copyright © 2014 John Wiley & Sons, Ltd. Research article Received: 9 September 2013 Revised: 10 November 2013 Accepted: 26 November 2013 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jms.3319 50