Analysis of keto enol tautomers of curcumin by liquid chromatography mass spectrometry

Analysis of keto enol tautomers of curcumin by liquid chromatography mass spectrometry Analysis of keto enol tautomers of curcumin by liquid chromatography mass spectrometry Analysis of keto enol tautomers of curcumin by liquid chromatography mass spectrometry Analysis of keto enol tautomers of curcumin by liquid chromatography mass spectrometry Analysis of keto enol tautomers of curcumin by liquid chromatography mass spectrometry

Original article Analysis of keto-enol tautomers of curcumin by liquid chromatography/mass spectrometry Shin-ichi Kawano a,b , Yusuke Inohana c , Yuki Hashi b , Jin-Ming Lin a, * a Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China b Shimadzu Global COE for Application & Technical Development, Shimadzu (China) Co., Ltd., Shanghai 200052, China c Global Application Development Center, Shimadzu Corporation, Kyoto 604-8511, Japan 1. Introduction Curcumin (C 21 H 20 O 6 ) is a well-known compound found in turmeric. This yellow pigment has been reported to show various pharmacological activities. Because of its anti-inflammatory and anti-cancer effects, curcumin is expected to be a potential drug for cancer or Alzheimer’s disease [1–5]. Quantitative analysis of curcumin and its analogues, such as demethoxycurcumin and bisdemethoxycurcumin, is important for the quality assurance of turmeric products [6–8] or for monitoring their concentration in biological fluids [9–11]. High performance liquid chromatography (HPLC) with UV detection is a useful and popular technique for those purposes. It is known that curcumin shows prototropic (keto-enol) tautomerism [2,4,5]. Interest about curcumin’s form in solution has arisen because there is a strong relationship between tautomeric structure and effects on biological systems [12,13].In our study, keto-enol tautomers of curcumin were analyzed by quadrupole ion trap/time-of-flight mass spectrometry (QIT/ TOFMS). Tautomers of curcumin were separated using an ODS column with water/acetonitrile as the mobile phase. Hydrogen/ deuterium (H/D) exchange LC/MS technique [14,15] with accurate mass measurement was applied for confirmation of both tautomers. 2. Experimental Standard curcumin solutions (5–500 m g/mL in water/acetoni- trile (50/50, v/v)) were prepared. The liquid chromatograph was a Shimadzu (Kyoto, Japan) Prominence UFLC system with an SPD- M20A photodiode array (PDA) detector. A Shimadzu Shim-pack XR-ODS II (30 mm  1.5 mm, 2.2 m m) analytical column was kept at 40 8C. Mobile phase was water/acetonitrile (45/55). The flow rate of the mobile phase was set at 0.2 mL/min. The wavelength of the PDA detector ranged from 200 nm to 600 nm. Mass spectrom- etry was conducted using a Shimadzu LCMS-IT-TOF hybrid QIT/ TOF mass spectrometer equipped with an electrospray ionization (ESI) interface in positive/negative ion mode. The probe voltage for ESI was set at +4.5 kV for positive detection, or À3.5 kV for negative detection. MS/MS spectra of curcumin were obtained with the conditions as follows: precursor ion for positive detection, m/z 369.1333; precursor ion for negative detection, m/z 367.1187. For H/D exchange experiment, D 2 O (Sigma–Aldrich) was introduced to the ESI interface with an auxiliary LC pump. The flow rate was set at 0.8 mL/min. Precursor ion was set at m/z 369.1305 for negative detection. 3. Results and discussion On a mass chromatogram at m/z 367.1187 in negative detection (Fig. 1), a small peak (peak 1) appeared at an earlier retention time (0.40 min) and a large peak of curcumin (peak 2) was observed at 1.45 min. UV spectra of these peaks were different from each other Chinese Chemical Letters 24 (2013) 685–687 ARTICLE INFO Article history: Received 4 March 2013 Received in revised form 16 April 2013 Accepted 23 April 2013 Available online 5 June 2013 Keywords: Keto-enol tautomer Curcumin Liquid chromatography/mass spectrometry ABSTRACT Keto-enol tautomers of curcumin were confirmed by reversed-phase liquid chromatography (RPLC)/ hybrid quadrupole ion trap/time-of-flight mass spectrometry (QIT/TOFMS). Tautomers gave different MS/MS spectra in negative mode. Different mass spectra were also obtained by hydrogen/deuterium exchange LC/MS/MS in positive mode. Our results suggest that enol form is the major form in the solution (water/acetonitrile). ß 2013 Jin-Ming Lin. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. * Corresponding author. E-mail address: jmlin@mail.tsinghua.edu.cn (J M. Lin). Contents lists available at SciVerse ScienceDirect Chinese Chemical Letters journal homepage: www.elsevier.com/locate/cclet 1001-8417/$ – see front matter ß 2013 Jin-Ming Lin. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. http://dx.doi.org/10.1016/j.cclet.2013.05.006 ( l max at 353 nm for peak 1, l max at 429 nm for peak 2). With either wavelength, peak 1 showed lower absorbance than peak 2 in PDA chromatograms. To confirm whether peak 1 represents an impurity of curcumin with a different structure or another form of curcumin, after fractionation of peak 2, a portion of the fraction was subjected to analysis again. The chromatogram was similar to the previous one with a small peak at earlier retention time (0.40 min) than that of main peak (1.45 min). A computational approach was tried to determine the stable structure of curcumin by Kolev et al. [16]. They proposed that the most stable configuration of the enol form had a planar structure and that of the keto form had a folded (non-planar) structure at C-4 position of heptadiene. If peak 1 represents either of the forms, then peak 1 would be the keto form and peak 2 would be the enol form because of the retentive properties according to the size of molecules onto the ODS surface. For further identification of the peaks 1 and 2, MS/ MS experiments were conducted. MS/MS spectra of these peaks by positive detection were very similar each other. As studied by Jiang et al. [17], ions C 11 H 11 O 2 + were detected at m/z 175.0764 (error: 2.9 ppm, Fig. 2a) and at m/z 175.0773 (error: 8.0 ppm, Fig. 2c). Ions C 14 H 13 O 4 + were detected at m/z 245.0827 (error: 5.3 ppm, Fig. 2a) and at m/z 245.0799 (error: À6.1 ppm, Fig. 2c). For positive detection, the ketone moiety would be the position of protonation. At the earlier stage of fragmentation pathways, both the keto form and enol form would produce the same ions. MS/MS spectra by negative detection were different (Fig. 2b and d). Peak 1 gave ions at m/z 175.0421 ([C 10 H 7 O 3 ] À , error: 14.8 ppm), m/z 160.0189 ([C 9 H 4 O 3 ] À , error: 18.1 ppm) and peak 2 gave ions at m/z 173.0623 ([C 11 H 9 O 2 ] À , error: 11.5 ppm), m/z 158.0383 ([C 10 H 6 O 2 ] À , error: 9.5 ppm) in negative detection. For negative detection, these forms would take different fragmentation pathways reflecting the difference in structure as a phenolic hydroxyl group would be deprotonated. Furthermore, H/D exchange technique was applied [(Fig._1)TD$FIG] Fig. 1. Mass chromatogram (m/z 367.1187, negative detection). [(Fig._2)TD$FIG] Fig. 2. MS/MS spectra of peaks 1 and 2 in positive and negative detections. (a) Peak 1, positive, (b) peak 1, negative, (c) peak 2, positive, and (d) peak 2, negative. [(Fig._3)TD$FIG] Fig. 3. Mass spectra of peaks 1 and 2 with H/D exchange MS. (a) Peak 1, positive, MS, (b) peak 1, negative, MS/MS, (c) peak 2, positive, MS, and (d) peak 2, negative, MS/MS. S. Kawano et al. / Chinese Chemical Letters 24 (2013) 685–687 686 because the number of exchangeable hydrogens of the enol form and the keto form are different. Two phenolic hydrogens of keto form, and two phenolic hydrogens and one aliphatic hydroxyl hydrogen of enol form are exchangeable. Mass spectra of curcumin tautomers showed apparent differences (Fig. 3). In positive detection, curcumin gave vast sodium adduct ions while proton- ated molecules were rarely observed because of high content of sodium ion in the D 2 O solvent. Although both peaks gave ions at m/z 393 and 394, the intensity of m/z 394 for peak 2 was doubled compared with that for peak 1. Ions [C 21 H 18 D 2 O 6 + Na] + (m/z 393.1274, error: À2.3 ppm) as the base peak for peak 1 (Fig. 3a) and ions [C 21 H 17 D 3 O 6 + Na] + (m/z 394.1349, error: 0.8 ppm) as the base peak for peak 2 (Fig. 3c) were observed. This result shows difference in H/D exchange at aliphatic hydroxyl group of enol. In negative detection, mass spectra (Fig. 3b and d) were similar to those without use of deuterium (Fig. 2b and d). This indicates that major product ions by negative MS/MS do not contain exchangeable hydrogen. As a consequence, representative fragmentation positions in negative detection were summarized in Fig. 4. The results by LC/UV and LC/MS/MS indicate that the major form of curcumin in water/acetonitrile is enol form. 4. Conclusion Our experiment with a PDA detector and a QIT/TOF mass spectrometer supposed that the enol form of curcumin is the major component in the solution. Two forms were separately confirmed by LC/MS/MS. H/D exchange LC/MS/MS was informative to confirm forms of curcumin. The results obtained here have good agreement with those by spectroscopic [16] and NMR [18] studies. Acknowledgment This work was supported by the Research Fund for the Doctoral Program of Higher Education (No. 20110002110052). References [1] B.B. Aggarwal, K.B. Harikumar, Potential therapeutic effects of curcumin, the anti- inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, met- abolic, autoimmune and neoplastic diseases, Int. J. Biochem. Cell Biol. 41 (2009) 40–59. [2] R.A. Sharma, A.J. Gescher, W.P. Steward, Curcumin: the story so far, Eur. J. Cancer 41 (2005) 1955–1968. [3] S. 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Tautomers of curcumin were separated using an. article Analysis of keto-enol tautomers of curcumin by liquid chromatography/mass spectrometry Shin-ichi Kawano a,b , Yusuke Inohana c , Yuki Hashi b , Jin-Ming Lin a, * a Department of Chemistry,. online 5 June 2013 Keywords: Keto-enol tautomer Curcumin Liquid chromatography/mass spectrometry ABSTRACT Keto-enol tautomers of curcumin were confirmed by reversed-phase liquid chromatography (RPLC)/ hybrid