On Spontaneous Oscillatory Condensation of S-(+)-Ketoprofen in Acetonitrile M. Sajewicz1, M. Leda2, M. Gontarska1, D. Kronenbach1, E. Berry1, I.R. Epstein2, T. Kowalska1 1Institute of Chemistry, Silesian University, 9 Szkolna Street, 40-006 Katowice, Poland 2Chemistry Department, MS 015, Brandeis University, Waltham, MA, 02454, USA INTRODUCTION This study is a continuation of our earlier investigations of the chemical stability of ketoprofen dissolved in low-molecular-weight solvents and stored for long periods of time in solution (1-5). In those earlier experiments, we discovered the ability of this profen to undergo spontaneous oscillatory in vitro chiral inversion and also to undergo condensation. A kinetic-diffusive model was proposed to illustrate formation of the oscillatory spatiotemporal waves of the locally changing concentration of the respective antimer pairs. AIM In this study, experimental evidence is provided that condensation of ketoprofen carried out at ambient temperature can be facile, if ketoprofen is dissolved in acetonitrile. We present the results of investigations carried out with non-chiral high-performance liquid chromatography with diode array detector (HPLC-DAD) and mass spectrometry (LC-MS) on the dynamics of condensation of S-(+)-ketoprofen dissolved in acetonitrile and stored for various periods of time. HPLC-DAD Sample preparation: A 3.93×10-3 mol L-1 solution of S-(+)-ketoprofen was prepared in acetonitrile. High performance liquid chromatographic analysis was carried out using two different liquid chromatographs equipped with diode array detectors (HPLC-DAD) and two different chromatographic columns, in order to obtain a replicate set of experimental data. Gyncotek HPLC-DAD conditions: The Gyncotek liquid chromatograph (Gyncotek, Macclesfield, UK) was equipped with a Gyncotek Gina 50 model autosampler, Gyncotek P 580A LPG model pump, Gyncotek DAD UVD 340U model diode array detector, and Chromeleon Dionex v. 6.4 software for data acquisition and processing. The analyses were carried out in the isocratic mode, using a Hypersil GOLD (5 m particle size) column (250 mm  4.6 mm i.d.; Thermo Scientific, Waltham, MA, USA), and methanol – water (5:5, v/v) mobile phase at a flow rate of 0.6 mL min-1. The chromatographic column was thermostated at 35oC with a Varian Pro Star 510 model column oven. The analyses with this system were carried out at 25-min intervals for 30 h. Apart from the predominant peak of S-(+)-ketoprofen, two more peaks of low intensity are also observed on the chromatogram (Fig. 1). These additional peaks probably arise from condensation products. The difference among the UV spectra consists in the intensity of absorbance, and the least intense spectrum is attributed to peak 3. In the course of the 30-h sample storage period, the number of peaks on the chromatograms recorded at 25-min intervals periodically changed (Fig. 2). Throughout the entire experiment, peak 1 (attributed to the starting material, i.e., to S-(+)-ketoprofen) is present in the chromatogram. The observed changes in the consecutive chromatograms are irregular, yet oscillatory in nature. It is most probable that the periodic changes in sample composition are due to formation and decomposition of ketoprofen condensates, represented by peaks 2 and 3. The shapes of the two plots shown in Fig. 3 are similar in the sense that they are both non-monotonic but oscillatory. Fig. 1. Chromatogram of a freshly prepared S-(+)-ketoprofen solution in ACN registered with the Gyncotec liquid chromatograph at 259 nm. Retention times: peak 1, 7.66 min; peak 2, 9.68 min; peak 3, 11.80 min. Insets show UV spectra of the separated species recorded at the maxima of the respective peaks. Fig. 3. Time changes of the chromatographic peak heights for the S-(+)-ketoprofen solution in ACN stored at 22ºC for 30 hours. Peak numbers as in Fig. 1. Fig. 2. Sequence of nine chromatographic concentration profiles for an S-(+)-ketoprofen solution in ACN after (a) 0 h; (b) 5.5 h; (c) 9.5 h; (d) 18 h; (e) 19 h; (f) 20 h; (g) 24.5 h; (h) 28 h; and (i) 30 h storage time at 22ºC. Varian HPLC-DAD conditions: The Varian model 920 liquid chromatograph (Varian, Harbor City, CA, USA) was equipped with Galaxie software for data acquisition and processing. The analyses were carried out in the isocratic mode, using a Pursuit 5 C18 (5 m particle size) column (250 mm  4.6 mm i.d.; Varian, Harbor City, CA, USA), and methanol – water (5:5, v/v) mobile phase at a flow rate of 0.6 mL min-1. The chromatogram shown here was recorded for a S-(+)-ketoprofen sample after 8 days of storage time. The chromatogram (Fig. 4a) and the spectrogram-chromatogram (Fig. 4b) both show the presence of more than three peaks in the aged sample (eight days old). The anti-Langmuir shape of peak (II), characterized by a desorption front that is much steeper than the tailing adsorption front, may signal complex intermolecular interactions, e.g., effective lateral interactions through hydrogen bonds. This result suggests the presence of carboxyl OH groups in the condensed ketoprofen present in the sample (peak (II)), not esterified by methanol from mobile phase. The UV spectrum (Fig. 5) is identical for peaks (I) and (II), furhter evidence that peak (II) represents the condensed ketoprofen. (a) (b) Fig. 4. (a) Chromatogram of an S-(+)-ketoprofen solution in ACN after eight days storage registered with a Varian liquid chromatograph at 257 nm. Retention times: peak (I), 8.28 min, peak (II), 28.45 min. (b) 2D spectrogram-chromatogram of the same sample. Fig. 5. UV spectra of the peaks at 8.28 min (S-(+)-ketoprofen) and at 28.45 min (ketoprofen condensate) retention time. LC-MS Sample preparation: A 3.93×10-3 mol L-1 solution of S-(+)-ketoprofen was prepared in acetonitrile. LC-MS conditions: Analysis was carried out using an LC-MS System Varian (Varian, Palo Alto, CA, USA) equipped with a Varian ProStar model pump, Mass Spectrometer Varian 100-MS, and Varian LC-MS software for data acquisition and processing. The analyses were carried out in the isocratic mode, using a Pursuit X RS 3-C18 column (50 mm×2.0 mm i.d.; Merck KGaA, Darmstadt, Germany), and methanol–water (5:5, v/v) mobile phase at a flow rate of 0.20 mL min-1. MS conditions: Samples were analyzed in the ESI mode (full ESI-MS scan; positive ionization: spray chamber temperature 45oC, drying gas temperature 150oC, drying gas pressure 25 psi, capillary voltage 70 V, needle voltage 5 kV). Varian MS Workstation v. 6.9.1 software was used for data acquisition and processing. (a) The changing pattern of concentration profiles seen in the chromatograms presented in Fig. 6 can be divided into the two groups, and we see that one type of chromatogram evolves to the other and vice versa in a cyclic manner. Cyclic changes of peak positions and areas for the peaks appearing at tR  5 and 10 min can be due to the oscillatory formation and decay of ketoprofen condensates in the aging solution. In this view, gowth of the condensate concentration corresponds to the chromatograms in Figs 6a, b, and d. The predominant peak at tR  10 min has an anti-Langmuir shape, which suggests multi-layer adsorption of the condensates on the stationary phase, with the respective condensate molecules held together by H-bonds. The high intensity of the predominant peak at tR  10 min may result from accumulation of a considerable number of phenyl groups per condensate molecule. With chromatograms like those in Figs 6c, e, and f, the peak at tR  1 min tends to be more intense than that at tR  5 min. The location of the second peak at tR  5 min suggests that the molecular weight of the respective condensates is lower than of those appearing at tR  10 min. Fig. 6 shows two chromatograms with insets of mass spectra for each separated peak. The chromatogram shown in Fig. 7a is analogous to those shown in Figs 6a, b, and d, while the chromatogram shown in Fig. 7b represents those shown in Figs 6c, e, and f. In the mass spectra of the peaks with higher retention times, signals with m/z  500 are abundant, and they probably correspond to ketoprofen condensates formed by coupling of several monomer units. (b) Fig. 6. Sequence of the six chromatographic concentration profiles for the S-(+)-ketoprofen solution in ACN after (a) 0 h; (b) 24 h; (c) 43.5 h; (d) 44.5 h; (e) 45.5 h; and (f) 47.5 h storage time at 22ºC. Fig. 7. Chromatogram of an S-(+)-ketoprofen solution in ACN after (a) 44.5 h and (b) 43.5 h storage time. Insets show mass spectra of the separated species recorded at the maxima of the respective peaks. Theoretical Approach We have examined an abstract kinetic model in which oscillations appear as result of coupling between enantiomers [6]. This model is especially appropriate for systems in which the condensate SR has different chemical properties than RS. Oscillations appear in this scheme only if the rate constant for formation of heterodimers in steps (4) and (5) is greater than that for homodimers in step (2). The system is closed with respect to mass exchange: c = [R] + [S] + [R*] + [S*] + 2([R2] + [S2] + [RS] + [SR]) = const. In the general case, RS and SR different chemically, so we have: k4≠k5, k6≠k7≠k12≠k13, k8≠k9≠k14≠k15 and k10≠k11. Oscillations occur for a broad range of parameters. Results of some simulations are shown in Fig. 8. Concentrations of activated forms of monomers S* and R* (not shown) are about 40 times smaller than concentrations of other species. Generally, activated forms can be detected only in situ because they exist only in the reacting mixture. Fig. 8. (a) Simulated concentration profiles of S (solid line), R (dashed line), (b) S2 (solid line) R2 (dashed line), and (c) RS (solid line) and SR (dashed line). (d) Enantiomeric excess ee=([R]-[S]+[R*]-[S*]+2[R2]-2[S2])/c for c=2, k1=k2=k3=k6=k7=1, k-1=k8=k9=k10= k11=0.1, k12=k13=k14=k15=0.02 and k4=k5=100. Initial conditions: [S]=1.9, [R]=0.1 and concentrations of other compounds equal to zero. CONCLUSION Energetically facile condensation of ketoprofen is linked with its ability to undergo spontaneous oscillatory chiral conversion. Our experimental results suggest that condensation of ketoprofen is an oscillatory process. The work of two of the authors (M.G. and D.K.) was partially supported by PhD scholarships granted to them in 2009 within the framework of the ‘University as a Partner of the Economy Based on Science’ (UPGOW) project, subsidized by the European Social Fund (EFS) of the European Union. REFERENCES [1] M. Sajewicz et. al., J. Liq. Chromatogr. Relat. Technol.., 30, 2185-2192 (2007) [2] M. Sajewicz et. al., J. Liq. Chromatogr. Relat. Technol.., 30, 2193-2208 (2007) [3] M. Sajewicz et al., J. Planar Chromatogr. – Modern TLC, 21, 349-353 (2008) [4] M. Sajewicz et. al., J. Liq. Chromatogr. Relat. Technol.., 32, 1359-1372 (2009) [5] M. Matlengiewicz et al., Acta Chromatogr., 22, 81-90 (2010) [6] R. Plasson, H. Bersini, A. Commeyras, Proc. Nat. Acad. Sci. USA 101, 16733-16738 (2004)