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 Kawanoa,b, Yusuke Inohanac, Yuki Hashib, Jin-Ming Lina,*

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 (C21H20O6) 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 (5–500mg/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, 2.2mm) 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 3.5 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, D2O (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

A R T I C L E I N F O

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

A B S T R A C T

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)

ß2013 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).

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j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / c c l e t

1001-8417/$ – see front matter ß 2013 Jin-Ming Lin Published by Elsevier B.V on behalf of Chinese Chemical Society All rights reserved.

(lmaxat 353 nm for peak 1,lmaxat 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 C11H11O2 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

C14H13O4 were detected at m/z 245.0827 (error: 5.3 ppm,Fig 2a) and at m/z 245.0799 (error: 6.1 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 ([C10H7O3] , error: 14.8 ppm), m/z 160.0189 ([C9H4O3] , error: 18.1 ppm) and peak 2 gave ions at m/z 173.0623 ([C11H9O2] , error: 11.5 ppm), m/z 158.0383 ([C10H6O2] , 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 Mass chromatogram (m/z 367.1187, negative detection).

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.

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 D2O 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 [C21H18D2O6+ Na]+

(m/z 393.1274, error: 2.3 ppm) as the base peak for peak 1

(Fig 3a) and ions [C21H17D3O6+ 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)

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Fig 4 Fragmentation of curcumin in keto form and enol form by negative MS/MS.