1. OVERVIEW Investigation of Reserpine Oxidation Using On-Line Electrochemistry/Electrospray Mass Spectrometry Vilmos Kertesz and Gary J. Van Berkel Organic and Biological Mass Spectrometry Group, Chemical Sciences Division Oak Ridge National Laboratory, Oak Ridge, TN 37831-6131  Reserpine is widely used in tuning/ calibrating mass spectrometers and undergoes different paths of chemical degradation  Oxidation  Hydrolysis  Oxidation of reserpine results in several major products:  1-hydroxy-reserpine, observed as the protonated molecule at m/z 625  Ions at m/z 623 and m/z 607, identified as 1-hydroxy-3,4-dehydroreserpine and 3,4-dehydroreserpine, respectively  V. K. acknowledges support through an appointment to the Oak Ridge National Laboratory (ORNL) Postdoctoral Research Associates Program administered jointly by the Oak Ridge Institute for Science and Education and ORNL.  ES-MS instrumentation was provided through a Cooperative Research and Development Agreement with MDS SCIEX (CRADA No. ORNL02-0662).  The work carried out at ORNL was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences, United States Department of Energy under Contract DE-AC05- 00OR22725 with ORNL, managed and operated by UT- Battelle, LLC. ACKNOWLEDGEMENTS  Electrochemical reactions of reserpine:  Oxidation intermediate with m/z 625 seems to be a protonated form of 1-hydroxy-reserpine, (hydroxy group on the pyrrole nitrogen, N 1 position).  Oxidation product at m/z 623 is product of oxidation of m/z 625 intermediate. Results suggest that m/z 623 corresponds to 1-hydroxy-3,4-dihydro- reserpine.  Reduction of m/z 623 yields an ion at m/z 607, that corresponds to 3,4-dihydro-reserpine. Also, 3,4- dihydro-reserpine is the only product of reserpine autoxidation in air.  Study of oxidation on compounds that has similar structure to reserpine to provide further proof on the location of oxidation sites  Preparation, collection, separation and NMR study of oxidation intermediates and final products (m/z 625, 623 and 607)  Potential-induced preconcentration/release of 3,4-dihydro- reserpine was detected during the experiments on the PGC working electrode at -1.0V. Detailed investigation on this process is in progress. [1] Allen, M.J. et al., J. Electrochem. Soc. 1958, 105, 541-544. [2] Ebel, S., et al., J. Pharm. Biomed Anal. 1989, 7, 709-713. [3] Awang, D. V. C., et al., J. Org. Chem. 1990, 55, 4443-4448. [4] Sanchez, M., et al., Analyst 1996, 121, 1581-1582. [5] Carmona-Guzman, M. C., et al., J. Chem. Soc. Perkin Trans. 2. 1986, 409-412. [6] Munoz, M. A., et al., J. Chem. Soc. Perkin Trans. 2. 1991, 453-456. [7] Van Berkel, G. J., et al., Anal. Chem. 2004, 76, 1493-1499. [8] Deng, H., et al., Anal. Chem. 1999, 71, 4284-4293. [9] Kertesz, V., et al., Electroanalysis 2001, 13, 1425-1430. NEAR FUTURE REFERENCES EXPERIMENTAL SECTION  This research is focused on understanding the oxidation/reduction reactions and the chemical follow-up reactions of reserpine  To-date investigations of electrochemical [1- 2] and chemical [3-6] oxidation of reserpine concluded different oxidation products: 3,4- dehydroreserpine [2-6], 3,4,5,6- tetradehydroreserpine (lumireserpine) [2-5], 10-hydroxy-reserpine [1], reserpine N 4 -oxide [1].  Mass spectrometric experiments indicated reserpine oxidation products with m/z 607 and m/z 625 [7]  No thorough mass spectrometry study on the oxidation of reserpine has been accomplished until now.  On-line electrochemistry/electrospray mass spectrometry (EC/ES-MS) is a powerful method in identifying products of electrochemical reactions  e.g. initial polymerization products of aniline [8] and methylene blue [9]  In this presentation we show step-by-step identification of major oxidation products of reserpine oxidation using EC/ES-MS method  Oxidation intermediate 1-hydroxy-reserpine, (hydroxy group on the pyrrole nitrogen, N 1 position) that presents in the MS spectrum as protonated molecule at m/z 625  Oxidation product 1-hydroxy-3,4-dihydro- reserpine that shows ionic properties and presents in the MS spectrum at m/z 623  Reduction product of m/z 623 yields an ionic species that corresponds to 3,4- dihydroreserpine exhibiting a peak at m/z 607 in the MS spectrum  3,4-dihydroreserpine is the only product of reserpine autoxidation in air INTRODUCTION RESULTS AND DISCUSSION  Samples and Reagents. Reserpine (Aldrich, Milwaukee, WI) solutions were prepared as a 50/50 (v/v) mixture of water (Milli-Q, Bedford, MA) and acetonitrile or methanol (Burdick and Jackson, Muskegon, MI) containing 5.0 mM ammonium acetate (99.999%, Aldrich) and 0.75% (v/v) acetic acid (PPB/Teflon grade, Aldrich).  ES-MS. Experiments were performed on PE Sciex API 165 single quadrupole (MDS Sciex, Concord, Ontario, Canada) or 4000 Q TRAP LC/MS/MS System (Applied Biosystems, Foster City, CA and MDS Sciex, Ontario, Canada) mass spectrometers. A HP 1099 HPLC system or syringe pump was used to deliver solvent and analyte solutions to the ion source. On the single quadrupole mass spectrometer the spectra were acquired with the TurboIonSpray TM source [7] using a fused silica spray capillary emitter (100 μm-i.d., 330 μm-o.d., 3.5 cm long), while on the 4000 Q TRAP instrument the normal stainless steel spray system was used.  Electrochemistry. The electrochemical experiments were controlled using a CH Instruments model 660 Electrochemical Workstation and the potentiostat unit of CH Instruments model 900 Scanning Electrochemical Microscope (Austin, TX). The off-line cyclic voltammograms were recorded using a freshly polished 3.0 mm diameter glassy carbon disk electrode, platinum wire auxiliary electrode and a Ag/AgCl reference electrode (model RE-5B, BAS) in a 10 mL batch cell. During the on- line flow through experiments two ESA model 5030, DB- 1018 electrochemical cells (ESA Inc., Chelmsford, MA) were inserted between the solvent delivery unit and the mass spectrometer. The fluid flow through the cells was along the radial axis and passed directly through the working electrode located at half cell width. The working electrode was composed of porous graphitic carbon (PGC) (40% total porosity, 99% open porosity with a mean pore size of 0.8 μm) 1.6 mm dia. x 0.38 mm thick. The calculated surface area of the working electrode was 17cm 2. The Pd auxiliary and the Pd quasi-reference electrodes (99.95%) were located upstream of the working electrode. Flow rate was 20  L/min. Figure 1 Exploded view of the EC cell PGC working electrode Reference and auxiliary electrodes (inside ) Figure 2 EC cells coupled to MS Cell 1 (ESA1) Inlet from pump Outlet to MS Cell 2 (ESA2)  Cyclic voltammogram of reserpine (Figure 3) showed that reserpine was relatively easy to oxidize in the solvent system in which the ES-MS experiments were performed. The voltammogram also showed that the oxidation of reserpine was not reversible (missing cathodic peak on the reverse scan).  As cyclic voltammetry alone did not provide detailed information regarding the identity of the oxidation product(s), on-line EC/ES-MS technique was applied. Figure 3 [M+H] + =609 Figure 4 c =20  M 1 mM reserpine  Oxidation of reserpine in the first flow cell at +0.7 V and +1.3 V resulted new peaks in the MS spectrum at m/z 625 and m/z 623.  Reduction of compound with m/z 623 in the second cell resulted in a species with m/z 607  Adding Li-salt to the solution showed that compounds with m/z 609 (reserpine) and m/z 625 are protonated molecules, while the products at m/z 623 and m/z 607 are non-protonated ions  In 50/50 v/v% methanol/water a methoxylated product appeared at m/z 639 (Figure 7a). Experiments in D 2 O indicated that only one exchangeable proton exists in compound with m/z 639 (Figure 7c), while reserpine (m/z 609) exhibited two exchangeable protons (two N-H in the protonated reserpine, Figure 7b). Adding Li-salt to the deuterated solution, results showed (Figure 7d) that the Li-adduct at m/z 646 of compound with m/z 639 do not have exchangeable protons.  These results indicated that methoxylation took place on the pyrrole nitrogen yielding 1-methoxy- reserpine. c =20  M Figure 5 autoxidation b) c) Figure 6 20  M reserpine a) b) c) d) 2  M reserpine, 2mM Li-acetate autoxidation a) 20  M reserpine  The place of the methoxylation suggested that hydroxylation of reserpine (yielding compound with m/z 625) resulted in 1-hydroxy-reserpine.  MS/MS data support that hydroxylation takes place on the pyrrole nitrogen.  Based on the experimental results the following structures and redox processes of reserpine is suggested: Figure 7 a) b) c) d) 20  M reserpine, 20 mM Li-acetate a) MS/MS of m/z 607 Figure 8 c) MS/MS of m/z 623 b) MS/MS of m/z 609 d) MS/MS of m/z 625 m/z 174 m/z 190 Figure 9 Oxidation of reserpine Lack of reserpine reduction wave Oxidation of water Reduction of water CONCLUSIONS