- Email: [email protected]
Rapid quantitation of curcumin in turmeric via NMR and LC–tandem mass spectrometry
Food Chemistry 113 (2009) 1239–1242
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Rapid quantitation of curcumin in turmeric via NMR and LC–tandem mass spectrometry Ahmet C. Gören a,*, Simay Çıkrıkçı a, Muhiddin Çergel b, Gökhan Bilsel a a b
_ TÜBITAK, UME, Group of Chemistry, P.O. Box 54, 41470 Gebze-Kocaeli, Turkey _ TÜBITAK, Marmara Research Center, EMSI, P.O. Box 21, 41470 Gebze-Kocaeli, Turkey
a r t i c l e
i n f o
Article history: Received 2 January 2008 Received in revised form 11 June 2008 Accepted 3 August 2008
Keywords: Curcumin Turmeric Curcuma longa Quantitative nuclear magnetic resonance LC–tandem mass spectrometry Validation Uncertainty
a b s t r a c t A rapid, sensitive and accurate 1H NMR method has been developed for the quantitation of curcumin isolated from Curcuma longa rhizome (turmeric) extract, the results of which were compared with a validated LC–MS/MS method. The relative standard deviations of the methods were found to be 2.49% and 3.48% for the 1H NMR and LC–MS/MS methods, respectively. The correlation coefficients were 0.998 for 1H NMR and 0.995 for LC–MS/MS assay in the calibration range. The recoveries at 5 mg mL1 and 50 lg mL1 concentrations averaged to 99–101% for both techniques, respectively. The uncertainty of the measurement of curcumin via 1H NMR spectroscopy was determined to be 5.80% while in LC–ESIMS/MS method was 7.38%. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Turmeric, the ‘‘golden spice”, has been unique for its medicinal uses and socio-religious practices since ancient times, for at least 6000 years. Many species of Curcuma are known to occur in the wild, where turmeric is probably a native, originated from Southeast Asia (Ravindran, 2007). Turmeric, which is a rich source of phenolic compounds, curcuminoids, is widely used as a dietary spice and colouring agent in food and herbal medicine. It is valuable for its pharmacological activities, such as anti-inflammatory, anti-microbial, anti-oxidant, anti-parasitic, anti-mutagenic and anti-cancer affects (Ahsan, Parween, Khan, & Hadi, 1999; Çıkrıkçı, Moziog˘lu, & Yılmaz, 2008; Péret-Almeida, Cherubino, Alves, Dufossé, & Glória, 2005). Because of these specific features, turmeric is cultivated mostly in India, followed by Bangladesh, China, Cambodia, Malaysia, Thailand, Philippines and Indonesia. Limited production of turmeric is also observed in tropical regions of Africa, America and Pacific Ocean Islands (Ravindran, 2007). The powdered rhizome (turmeric) of the perrenial herb Curcuma longa L. extract generally contains three different diaryl heptanoids, which are curcumin (Fig. 1), demethoxycurcumin and bisdemethoxycurcumin (Jayaprakasha, Rao, & Sakariah, 2002; Péret-Almeida et al., 2005). The main constituent of Curcuma spe* Corresponding author. Tel.: +90 262 679 50 00x6102; fax: +90 262 679 50 01. E-mail addresses: [email protected], [email protected] (A.C. Gören). 0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2008.08.014
cies is curcumin, which is responsible for the biological activity of turmeric. These properties have been attracting the interest of various scientists that, only in 2007, 72 curcumin related papers have been reported. Cultivar diversity of turmeric and their quality parameters are well documented, which indicate that the curcumin contents of crude cultivated species change from 0.02% to 10.9% (Ravindran, Nirmal Babu, & Shiva, 2007). Curcumin exists in equilibrium, possessing keto-enol and diketo forms and is known for its ability to form coloured complexes as a result of the carbonyl and olefinic groups (Shen & Ji, 2007; Sundaryono, Nourmamode, Gardrat, Fritsch, & Castellan, 2003). Several methods including HPLC-UV, GC, LC/MS and spectrophotometric methods, expressing the total colour content of the sample have been described for the determination of curcumin content in turmeric (ASTA Method, 1985; Holzgrabe, Deubner, Schollmayer, & Waibel, 2005; Jayaprakasha et al., 2002; Michaleas & Antoniadou-Vyza, 2006; Péret-Almeida et al., 2005). These techniques are time consuming and their uncertainty is not as good as the NMR method, which provides simultaneous detection of both identity and quantity. It had been observed that particularly quantitative NMR spectroscopy is less time consuming, easy to perform, produces more accurate and precise results and leads to a higher reproducibility compare with standard HPLC methods (Holzgrabe et al., 2005; Michaleas & Antoniadou-Vyza, 2006). In this study, curcumin content in commercial turmeric samples was determined by applying NMR technique and the results were
1240
A.C. Gören et al. / Food Chemistry 113 (2009) 1239–1242
9
HO
9'
8 10 3
7 5 H3CO
6
3'
1
7'
2'
2
5' 4'
4 O
OH
8'
10'
6'
OCH3
O H
Curcumin Fig. 1. Structure of curcumin.
compared with LC–MS/MS method. It has been demonstrated that the crude plant powders could be analysed in a short time, such as within three minutes via 1H NMR method, which is much shorter than the conventional methods.
jor fragment ion at m/z 245. A Phenomenex Luna silica column (50 mm 4.6 mm 5 lm) with a flow rate of 1 mL min1 was used. 2.4. Optimisation of NMR procedure
2. Experimental 2.1. Chemicals 2.1.1. Standards and solutions Curcumin (97%) and HPLC grade methanol were obtained from Merck (Darmstadt, Germany). 1,3,5-Trimethoxybenzene (99%) and DMSO-d6 (99.8%) were purchased from Sigma–Aldrich (Darmstad, Germany) and Acros Organics (New Jersey, USA) respectively. The powdered curry species were obtained from local market (Istanbul, Turkey). The turmeric samples were obtained from ROHA Caleb UK Ltd. (Monmouthshire, UK) and PROLAB (Istanbul, Turkey). 2.1.2. Preparation of standard and sample solutions 2.1.2.1. 1H NMR experiment. A stock solution was prepared by dissolving 50 mg of curcumin in DMSO-d6 in 10 mL volumetric flask, from which 5 mg mL1, 2.5 mg mL1, 1 mg mL1, 0.5 mg mL1 and 0.2 mg mL1 solutions were prepared for the calibration curve. Dilutions were performed using automatic pipettes and in 5 mL volumetric amber flasks (A class). About 25 mg of 1,3,5-trimethoxy benzene, which was used as an internal standard, was dissolved in 10 mL DMSO-d6, 200 lL of which was added to each sample before NMR experiment. The samples were prepared freshly and carefully protected from light during the NMR experiments. 2.1.2.2. LC–MS/MS experiment. A stock solution was prepared by dissolving 5 mg of curcumin in HPLC grade methanol in a 1 L volumetric flask, from which 10 lg L1, 50 lg L1, 100 lg L1 and 200 lg L1 solutions were prepared for the calibration curve. The samples were prepared freshly with the same method.
After the trial of couple of solvents, DMSO-d6 was found to be the best solvent as the separation and resolution of the peaks, which was used as described in the literature (Anderson, Mitchell, & Mohan, 2000). As a second important parameter, 1,3,5-trimethoxybenzene was determined to be the correct internal standard as it gave simple and sharp peaks at 6.06 (3H, s) and 3.68 (9H, s) in DMSO-d6. These signals are very close to the curcumin peaks, 6.03 (1H, s) and 3.81 (6H, s) (Fig. 2). 2.5. Optimisation of LC–MS/MS procedure The ion source, ionisation technique and analysis voltage are very important in LC–MS/MS technique to obtain the best result. Initially Thermo LCQ Deca ion trap and Waters Premier XE quadrupole–quadrupole tandem mass spectrometries were applied. Experiments in the ion trap mass spectrometry, in Thermo LCQ Deca, assays indicated that the relative abundance of fragmented peaks is not stable like quadrupole–quadrupole system. Then, the quadrupole–quadrupole tandem mass spectrometry system was decided to be used for the further experiments. ESI ion source was determined to be the best. In APCI experiment, m/z 217, which was formed as a result of the fragmentation of m/z 369, was observed as a main peak. However, it was found not to be stable like the fragment m/z 245 obtained from ESI source. Considering all these experiences, quadrupole–quadrupole LC–MS/MS system coupled to ESI source with a 25 collision energy level was decided to be used. 3. Results and discussion 3.1. Validation
2.2. Instruments 2.2.1. 1H NMR apparatus and parameters All the spectra were recorded in DMSO-d6 using Varian 600 MHz spectrometer with an ID-6508 indirect probe (S/ N = 1084). The following parameters were used during the NMR experiments: number of scans, 64; relaxation delay, 1.0 s; and pulse degree, 45°. About 800 lL of solution was added to each 5 mm NMR tubes. Peak height was calculated for the quantitative analysis. 2.2.2. LC–MS/MS apparatus and parameters LC–ESI-MS/MS experiments were performed in Waters Premier XE quadrupole–quadrupole LC–tandem mass spectrometry. (+) ESI spectrum gave a molecular ion peak [M + 1]+ at m/z 369 and a ma-
All validation procedures in 1H NMR were performed using standard curcumin and turmeric. 1,3,5-Trimethoxybenzene was used as an internal standard. In all LC–MS/MS experiments, external calibration was applied. 3.1.1. Linearity Calibration curves were obtained from calibration solutions, which were prepared from the reference curcumin obtained from Merck. The lowest concentration level in the calibration curve was established as a practical determination limit. Linearity of 1H NMR method was assayed by analysing the calculation of a fivepoint linear plot in the range of 0.2–5 mg mL1 with 10 replicates, based on linear regression and squared correlation coefficient, r2, which should be 0.998. Linear regression equation was
A.C. Gören et al. / Food Chemistry 113 (2009) 1239–1242
1241
Fig. 2. 1H NMR spectrum of turmeric sample (5 mg mL1) along with IS (1,3,5-trimethoxy benzene) and LC/MS/MS chromatogram of turmeric sample (200 lg L1).
y = 11.033x 0.003. Linearity of LC–MS/MS method was assayed by analysing the calculation of a four-point linear plot in the range of 10–200 lg L1 with 10 replicates, based on linear regression and squared correlation coefficient, r2, which should be 0.995. Linear regression equation was y = 4.1189x. 3.1.2. LOD and LOQ The limit of detection (LOD) of the 1H NMR method was found to be 0.002 mg mL1 with 64 number of scans. The limit of quantification value of the measurement was 10 times of the LOD values.
Then, LOQ value was determined as 0.02 mg mL1. The lower detection limits could be obtained by increasing the number of scans to such as 2048. In LC–MS/MS experiments, LOD and LOQ levels were determined as 1 lg L1 and 10 lg L1, respectively. 3.1.3. Recovery and repeatability Levels of recovery concentrations were determined at three fortification levels (1 mg mL1, 2.5 mg mL1 and 5 mg mL1) of the turmeric extract. The unspiked turmeric was also analysed to determine the curcumin concentrations in blank sample. The
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A.C. Gören et al. / Food Chemistry 113 (2009) 1239–1242
recoveries of each curcumin at each fortification levels were evaluated. Recoveries were calculated according to the following formula (Eq. 1), which changed in between 98 and 101. Recovery ð100%Þ ¼
Measured concentration Endogenous concentration 100 Spiked concentration
ð1Þ 1
Mean relative standard deviations (RSD) of H NMR and LC–MS/ MS methods were determined as 2.49% and 3.48%, respectively. 3.2. Estimation of uncertainty Sources and quantification of the uncertainty for the applied method were evaluated using EURACHEM/CITAC guide and reported as follows: impurity of reference standard, the sample weighing, the IS addition (200 lL/5 mL) and the calibration curve were identified as the uncertainty sources for the NMR assay. In this reported method, the source of the main uncertainties is the calibration curve and the purity of the standard. The percent relative uncertainty of curcumin in 1H NMR measurement was found to be 5.8 % at 95 % confidence level (k = 2) for 2500 mg L1 turmeric sample solution. Source of uncertainty of LC–MS/MS method was also determined as impurity of reference standard, the sample weighing, calibration curve and dilution of the solutions. According to these parameters, uncertainty of the method was determined as 7.38 % at 95 % confidence level (k = 2) for 200 lg L1 in the LC–MS/ MS method. 4. Conclusion To solve the time consuming problem of the quantitation of curcumin in turmeric, isolated from the C. longa, an 1H NMR method has been developed. Three candidate reference peaks were observed at 7.29 (d, 2H, J = 1.8 Hz, H6 and H06 ), 6.03 (s, 1H, H-1) and 3.81 (OCH3, 6H) in the 1H NMR spectrum of curcumin in DMSOd6. All the proton peaks were well separated at 600 MHz NMR instrument. A sharp singlet peak at 9.62 indicated the hydroxyl groups of curcumin. Since its intensity and chemical shift change depending on the concentration of the sample, it was not taken into account for the quantitation via 1H NMR. Considering the NMR signals of curcumin, 1,3,5-trimethoxybenzene (IS) was suggested to be used as an internal standard, as it gives two well resolved singlet peaks in the same area curcumin gives, i.e., 6.06 (3H, s) and 3.68 (9H, s). These peaks were selected for the validation of the measurement. In the next step, detection limit of curcumin was determined as 64 scans sample of turmeric, and LOD and LOQ values were obtained as 0.002 mg mL1 and 0.02 mg mL1, respectively. Validation and uncertainty information were given in the previous section. The QQ-LC–ESI-MS/MS technique is known to be useful for determination of curcumin in turmeric extract, and lower detection levels could be determined with this technique (Jiang, Timmermann, & Gang, 2006; Tamvakopoulos, Sofianos, Garbis, & Pantazis, 2007). When the same turmeric sample was analysed with the both techniques, 1H NMR and LC–ESI-MS/MS, purity of samples were found to be 93.1 ± 5.0% and 89.7 ± 6.61%, respectively. The results showed that both techniques are reliable for the purity analysis of turmeric samples.
In conclusion, Curcumin content isolated from C. longa rhizome (turmeric) extract was determined by 1H NMR method. The curcumin signal at 6.03 was well separated and no interference was observed for all the samples. However, the main problem in curcumin analysis was observed to be its instability in solutions. This problem was overcome by preparing fresh calibration solutions, considering that curcumin is a light sensitive substance. This study indicated that curcumin content of commercial turmeric samples and the crude plant powders could be analysed in short time, only 3 min, by applying simple NMR technique. The proposed method has the additional advantages to the conventional HPLC-UV, GC and LC/MS methods such as it is fast, selective, accurate and reliable. For the trace analysis of curcumin, LC–MS/MS method will be more suitable, however, in tracing small impurities and/or analogues of curcumin, 1H NMR will be the best spectroscopic technique. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.foodchem.2008.08.014. References Ahsan, H., Parween, N., Khan, N. U., & Hadi, S. M. (1999). Pro-oxidant, anti-oxidant, and cleavage activities on DNA of curcumin and its derivatives demethoxycurcumin and bisdemethoxycurcumin. Chemico-Biological Interaction, 121, 161–175. Anderson, A. M., Mitchell, M. S., & Mohan, R. S. (2000). Isolation of curcumin from turmeric. Journal of Chemical Education, 77, 359–360. ASTA Method (1985). Official analytical methods of the American spice trade association (3rd ed.). Englewood Cliffs, NJ: American Spice Trade Association. Çıkrıkçı, S., Moziog˘lu, E., & Yılmaz, H. (2008). Biological activity of curcuminoids isolated from Curcuma longa. Records of Natural Products, 2(1), 19–24. Holzgrabe, U., Deubner, R., Schollmayer, C., & Waibel, B. (2005). Quantitative NMR spectroscopy-applications in drug analysis. Journal of Pharmaceutical and Biomedical Analysis, 38, 806–812. Jayaprakasha, G. K., Rao, L. J. M., & Sakariah, K. K. (2002). Improved HPLC method for the determination of Curcumin, Demethoxycurcumin, and bisdemethoxycurcumin. Journal of Agricultural and Food Chemistry, 50, 3668–3672. Jiang, H., Timmermann, B. N., & Gang, D. R. (2006). Use of liquid chromatographyelectrospray ionization tandem mass spectrometry to identify diarylheptanoids in turmeric (Curcuma longa L.) rhizome. Journal of Chromatography A, 1111, 21–31. Michaleas, S., & Antoniadou-Vyza, E. (2006). A new approach to quantitative NMR: Fluoroquinolones analysis by evaluating the chemical shift displacements. Journal of Pharmaceutical and Biomedical Analysis, 42, 405–410. Péret-Almeida, L., Cherubino, A. P. F., Alves, R. J., Dufossé, L., & Glória, B. A. M. (2005). Separation and determination of the physico-chemical characteristics of curcumin, demethoxycurcumin and bisdemethoxycurcumin. Food Research International, 38, 1039–1044. Ravindran, P. N. (2007). Turmeric the genus Curcuma: The golden spice of life. In P. N. Ravindran, K. Nirmal Babu, & K. Sivaraman (Eds.), Medicinal and aromatic plants-industrial profiles (pp. 1–13). New York: Taylor and Francis Group, CRC Press. Ravindran, P. N., Nirmal Babu, K., Shiva, K. N. (2007). Turmeric the genus Curcuma: Botany and crop improvement of turmeric. In P. N. Ravindran, K. Nirmal Babu, & K. Sivaraman (Eds.), Medicinal and aromatic plants-industrial profiles (pp. 2–70). New York: Taylor and Francis Group, CRC Press. Shen, L., & Ji, H. (2007). Theoretical study on physicochemical properties of curcumin. Spectrochimica Acta Part A, 67, 619–623. Sundaryono, A., Nourmamode, A., Gardrat, C., Fritsch, A., & Castellan, A. (2003). Synthesis and complexation properties of two new curcuminoid bearing a diphenylmethane linkage. Journal of Molecular Structure, 649, 177–190. Tamvakopoulos, C., Sofianos, Z. D., Garbis, S. D., & Pantazis, P. (2007). Analysis of the in vitro matabolites of diferuloylmethane (curcumin) by liquid chromatography–tandem mass spectrometry on a hybrid quadrupole linear ion trap system: Newly identified metabolites. European Journal of Drug Metabolism and Pharmacokinetics, 32(1), 51–57.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Rapid quantitation of curcumin in turmeric via NMR and LC–tandem mass spectrometry Ahmet C. Gören a,*, Simay Çıkrıkçı a, Muhiddin Çergel b, Gökhan Bilsel a a b
_ TÜBITAK, UME, Group of Chemistry, P.O. Box 54, 41470 Gebze-Kocaeli, Turkey _ TÜBITAK, Marmara Research Center, EMSI, P.O. Box 21, 41470 Gebze-Kocaeli, Turkey
a r t i c l e
i n f o
Article history: Received 2 January 2008 Received in revised form 11 June 2008 Accepted 3 August 2008
Keywords: Curcumin Turmeric Curcuma longa Quantitative nuclear magnetic resonance LC–tandem mass spectrometry Validation Uncertainty
a b s t r a c t A rapid, sensitive and accurate 1H NMR method has been developed for the quantitation of curcumin isolated from Curcuma longa rhizome (turmeric) extract, the results of which were compared with a validated LC–MS/MS method. The relative standard deviations of the methods were found to be 2.49% and 3.48% for the 1H NMR and LC–MS/MS methods, respectively. The correlation coefficients were 0.998 for 1H NMR and 0.995 for LC–MS/MS assay in the calibration range. The recoveries at 5 mg mL1 and 50 lg mL1 concentrations averaged to 99–101% for both techniques, respectively. The uncertainty of the measurement of curcumin via 1H NMR spectroscopy was determined to be 5.80% while in LC–ESIMS/MS method was 7.38%. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Turmeric, the ‘‘golden spice”, has been unique for its medicinal uses and socio-religious practices since ancient times, for at least 6000 years. Many species of Curcuma are known to occur in the wild, where turmeric is probably a native, originated from Southeast Asia (Ravindran, 2007). Turmeric, which is a rich source of phenolic compounds, curcuminoids, is widely used as a dietary spice and colouring agent in food and herbal medicine. It is valuable for its pharmacological activities, such as anti-inflammatory, anti-microbial, anti-oxidant, anti-parasitic, anti-mutagenic and anti-cancer affects (Ahsan, Parween, Khan, & Hadi, 1999; Çıkrıkçı, Moziog˘lu, & Yılmaz, 2008; Péret-Almeida, Cherubino, Alves, Dufossé, & Glória, 2005). Because of these specific features, turmeric is cultivated mostly in India, followed by Bangladesh, China, Cambodia, Malaysia, Thailand, Philippines and Indonesia. Limited production of turmeric is also observed in tropical regions of Africa, America and Pacific Ocean Islands (Ravindran, 2007). The powdered rhizome (turmeric) of the perrenial herb Curcuma longa L. extract generally contains three different diaryl heptanoids, which are curcumin (Fig. 1), demethoxycurcumin and bisdemethoxycurcumin (Jayaprakasha, Rao, & Sakariah, 2002; Péret-Almeida et al., 2005). The main constituent of Curcuma spe* Corresponding author. Tel.: +90 262 679 50 00x6102; fax: +90 262 679 50 01. E-mail addresses: [email protected], [email protected] (A.C. Gören). 0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2008.08.014
cies is curcumin, which is responsible for the biological activity of turmeric. These properties have been attracting the interest of various scientists that, only in 2007, 72 curcumin related papers have been reported. Cultivar diversity of turmeric and their quality parameters are well documented, which indicate that the curcumin contents of crude cultivated species change from 0.02% to 10.9% (Ravindran, Nirmal Babu, & Shiva, 2007). Curcumin exists in equilibrium, possessing keto-enol and diketo forms and is known for its ability to form coloured complexes as a result of the carbonyl and olefinic groups (Shen & Ji, 2007; Sundaryono, Nourmamode, Gardrat, Fritsch, & Castellan, 2003). Several methods including HPLC-UV, GC, LC/MS and spectrophotometric methods, expressing the total colour content of the sample have been described for the determination of curcumin content in turmeric (ASTA Method, 1985; Holzgrabe, Deubner, Schollmayer, & Waibel, 2005; Jayaprakasha et al., 2002; Michaleas & Antoniadou-Vyza, 2006; Péret-Almeida et al., 2005). These techniques are time consuming and their uncertainty is not as good as the NMR method, which provides simultaneous detection of both identity and quantity. It had been observed that particularly quantitative NMR spectroscopy is less time consuming, easy to perform, produces more accurate and precise results and leads to a higher reproducibility compare with standard HPLC methods (Holzgrabe et al., 2005; Michaleas & Antoniadou-Vyza, 2006). In this study, curcumin content in commercial turmeric samples was determined by applying NMR technique and the results were
1240
A.C. Gören et al. / Food Chemistry 113 (2009) 1239–1242
9
HO
9'
8 10 3
7 5 H3CO
6
3'
1
7'
2'
2
5' 4'
4 O
OH
8'
10'
6'
OCH3
O H
Curcumin Fig. 1. Structure of curcumin.
compared with LC–MS/MS method. It has been demonstrated that the crude plant powders could be analysed in a short time, such as within three minutes via 1H NMR method, which is much shorter than the conventional methods.
jor fragment ion at m/z 245. A Phenomenex Luna silica column (50 mm 4.6 mm 5 lm) with a flow rate of 1 mL min1 was used. 2.4. Optimisation of NMR procedure
2. Experimental 2.1. Chemicals 2.1.1. Standards and solutions Curcumin (97%) and HPLC grade methanol were obtained from Merck (Darmstadt, Germany). 1,3,5-Trimethoxybenzene (99%) and DMSO-d6 (99.8%) were purchased from Sigma–Aldrich (Darmstad, Germany) and Acros Organics (New Jersey, USA) respectively. The powdered curry species were obtained from local market (Istanbul, Turkey). The turmeric samples were obtained from ROHA Caleb UK Ltd. (Monmouthshire, UK) and PROLAB (Istanbul, Turkey). 2.1.2. Preparation of standard and sample solutions 2.1.2.1. 1H NMR experiment. A stock solution was prepared by dissolving 50 mg of curcumin in DMSO-d6 in 10 mL volumetric flask, from which 5 mg mL1, 2.5 mg mL1, 1 mg mL1, 0.5 mg mL1 and 0.2 mg mL1 solutions were prepared for the calibration curve. Dilutions were performed using automatic pipettes and in 5 mL volumetric amber flasks (A class). About 25 mg of 1,3,5-trimethoxy benzene, which was used as an internal standard, was dissolved in 10 mL DMSO-d6, 200 lL of which was added to each sample before NMR experiment. The samples were prepared freshly and carefully protected from light during the NMR experiments. 2.1.2.2. LC–MS/MS experiment. A stock solution was prepared by dissolving 5 mg of curcumin in HPLC grade methanol in a 1 L volumetric flask, from which 10 lg L1, 50 lg L1, 100 lg L1 and 200 lg L1 solutions were prepared for the calibration curve. The samples were prepared freshly with the same method.
After the trial of couple of solvents, DMSO-d6 was found to be the best solvent as the separation and resolution of the peaks, which was used as described in the literature (Anderson, Mitchell, & Mohan, 2000). As a second important parameter, 1,3,5-trimethoxybenzene was determined to be the correct internal standard as it gave simple and sharp peaks at 6.06 (3H, s) and 3.68 (9H, s) in DMSO-d6. These signals are very close to the curcumin peaks, 6.03 (1H, s) and 3.81 (6H, s) (Fig. 2). 2.5. Optimisation of LC–MS/MS procedure The ion source, ionisation technique and analysis voltage are very important in LC–MS/MS technique to obtain the best result. Initially Thermo LCQ Deca ion trap and Waters Premier XE quadrupole–quadrupole tandem mass spectrometries were applied. Experiments in the ion trap mass spectrometry, in Thermo LCQ Deca, assays indicated that the relative abundance of fragmented peaks is not stable like quadrupole–quadrupole system. Then, the quadrupole–quadrupole tandem mass spectrometry system was decided to be used for the further experiments. ESI ion source was determined to be the best. In APCI experiment, m/z 217, which was formed as a result of the fragmentation of m/z 369, was observed as a main peak. However, it was found not to be stable like the fragment m/z 245 obtained from ESI source. Considering all these experiences, quadrupole–quadrupole LC–MS/MS system coupled to ESI source with a 25 collision energy level was decided to be used. 3. Results and discussion 3.1. Validation
2.2. Instruments 2.2.1. 1H NMR apparatus and parameters All the spectra were recorded in DMSO-d6 using Varian 600 MHz spectrometer with an ID-6508 indirect probe (S/ N = 1084). The following parameters were used during the NMR experiments: number of scans, 64; relaxation delay, 1.0 s; and pulse degree, 45°. About 800 lL of solution was added to each 5 mm NMR tubes. Peak height was calculated for the quantitative analysis. 2.2.2. LC–MS/MS apparatus and parameters LC–ESI-MS/MS experiments were performed in Waters Premier XE quadrupole–quadrupole LC–tandem mass spectrometry. (+) ESI spectrum gave a molecular ion peak [M + 1]+ at m/z 369 and a ma-
All validation procedures in 1H NMR were performed using standard curcumin and turmeric. 1,3,5-Trimethoxybenzene was used as an internal standard. In all LC–MS/MS experiments, external calibration was applied. 3.1.1. Linearity Calibration curves were obtained from calibration solutions, which were prepared from the reference curcumin obtained from Merck. The lowest concentration level in the calibration curve was established as a practical determination limit. Linearity of 1H NMR method was assayed by analysing the calculation of a fivepoint linear plot in the range of 0.2–5 mg mL1 with 10 replicates, based on linear regression and squared correlation coefficient, r2, which should be 0.998. Linear regression equation was
A.C. Gören et al. / Food Chemistry 113 (2009) 1239–1242
1241
Fig. 2. 1H NMR spectrum of turmeric sample (5 mg mL1) along with IS (1,3,5-trimethoxy benzene) and LC/MS/MS chromatogram of turmeric sample (200 lg L1).
y = 11.033x 0.003. Linearity of LC–MS/MS method was assayed by analysing the calculation of a four-point linear plot in the range of 10–200 lg L1 with 10 replicates, based on linear regression and squared correlation coefficient, r2, which should be 0.995. Linear regression equation was y = 4.1189x. 3.1.2. LOD and LOQ The limit of detection (LOD) of the 1H NMR method was found to be 0.002 mg mL1 with 64 number of scans. The limit of quantification value of the measurement was 10 times of the LOD values.
Then, LOQ value was determined as 0.02 mg mL1. The lower detection limits could be obtained by increasing the number of scans to such as 2048. In LC–MS/MS experiments, LOD and LOQ levels were determined as 1 lg L1 and 10 lg L1, respectively. 3.1.3. Recovery and repeatability Levels of recovery concentrations were determined at three fortification levels (1 mg mL1, 2.5 mg mL1 and 5 mg mL1) of the turmeric extract. The unspiked turmeric was also analysed to determine the curcumin concentrations in blank sample. The
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A.C. Gören et al. / Food Chemistry 113 (2009) 1239–1242
recoveries of each curcumin at each fortification levels were evaluated. Recoveries were calculated according to the following formula (Eq. 1), which changed in between 98 and 101. Recovery ð100%Þ ¼
Measured concentration Endogenous concentration 100 Spiked concentration
ð1Þ 1
Mean relative standard deviations (RSD) of H NMR and LC–MS/ MS methods were determined as 2.49% and 3.48%, respectively. 3.2. Estimation of uncertainty Sources and quantification of the uncertainty for the applied method were evaluated using EURACHEM/CITAC guide and reported as follows: impurity of reference standard, the sample weighing, the IS addition (200 lL/5 mL) and the calibration curve were identified as the uncertainty sources for the NMR assay. In this reported method, the source of the main uncertainties is the calibration curve and the purity of the standard. The percent relative uncertainty of curcumin in 1H NMR measurement was found to be 5.8 % at 95 % confidence level (k = 2) for 2500 mg L1 turmeric sample solution. Source of uncertainty of LC–MS/MS method was also determined as impurity of reference standard, the sample weighing, calibration curve and dilution of the solutions. According to these parameters, uncertainty of the method was determined as 7.38 % at 95 % confidence level (k = 2) for 200 lg L1 in the LC–MS/ MS method. 4. Conclusion To solve the time consuming problem of the quantitation of curcumin in turmeric, isolated from the C. longa, an 1H NMR method has been developed. Three candidate reference peaks were observed at 7.29 (d, 2H, J = 1.8 Hz, H6 and H06 ), 6.03 (s, 1H, H-1) and 3.81 (OCH3, 6H) in the 1H NMR spectrum of curcumin in DMSOd6. All the proton peaks were well separated at 600 MHz NMR instrument. A sharp singlet peak at 9.62 indicated the hydroxyl groups of curcumin. Since its intensity and chemical shift change depending on the concentration of the sample, it was not taken into account for the quantitation via 1H NMR. Considering the NMR signals of curcumin, 1,3,5-trimethoxybenzene (IS) was suggested to be used as an internal standard, as it gives two well resolved singlet peaks in the same area curcumin gives, i.e., 6.06 (3H, s) and 3.68 (9H, s). These peaks were selected for the validation of the measurement. In the next step, detection limit of curcumin was determined as 64 scans sample of turmeric, and LOD and LOQ values were obtained as 0.002 mg mL1 and 0.02 mg mL1, respectively. Validation and uncertainty information were given in the previous section. The QQ-LC–ESI-MS/MS technique is known to be useful for determination of curcumin in turmeric extract, and lower detection levels could be determined with this technique (Jiang, Timmermann, & Gang, 2006; Tamvakopoulos, Sofianos, Garbis, & Pantazis, 2007). When the same turmeric sample was analysed with the both techniques, 1H NMR and LC–ESI-MS/MS, purity of samples were found to be 93.1 ± 5.0% and 89.7 ± 6.61%, respectively. The results showed that both techniques are reliable for the purity analysis of turmeric samples.
In conclusion, Curcumin content isolated from C. longa rhizome (turmeric) extract was determined by 1H NMR method. The curcumin signal at 6.03 was well separated and no interference was observed for all the samples. However, the main problem in curcumin analysis was observed to be its instability in solutions. This problem was overcome by preparing fresh calibration solutions, considering that curcumin is a light sensitive substance. This study indicated that curcumin content of commercial turmeric samples and the crude plant powders could be analysed in short time, only 3 min, by applying simple NMR technique. The proposed method has the additional advantages to the conventional HPLC-UV, GC and LC/MS methods such as it is fast, selective, accurate and reliable. For the trace analysis of curcumin, LC–MS/MS method will be more suitable, however, in tracing small impurities and/or analogues of curcumin, 1H NMR will be the best spectroscopic technique. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.foodchem.2008.08.014. References Ahsan, H., Parween, N., Khan, N. U., & Hadi, S. M. (1999). Pro-oxidant, anti-oxidant, and cleavage activities on DNA of curcumin and its derivatives demethoxycurcumin and bisdemethoxycurcumin. Chemico-Biological Interaction, 121, 161–175. Anderson, A. M., Mitchell, M. S., & Mohan, R. S. (2000). Isolation of curcumin from turmeric. Journal of Chemical Education, 77, 359–360. ASTA Method (1985). Official analytical methods of the American spice trade association (3rd ed.). Englewood Cliffs, NJ: American Spice Trade Association. Çıkrıkçı, S., Moziog˘lu, E., & Yılmaz, H. (2008). Biological activity of curcuminoids isolated from Curcuma longa. Records of Natural Products, 2(1), 19–24. Holzgrabe, U., Deubner, R., Schollmayer, C., & Waibel, B. (2005). Quantitative NMR spectroscopy-applications in drug analysis. Journal of Pharmaceutical and Biomedical Analysis, 38, 806–812. Jayaprakasha, G. K., Rao, L. J. M., & Sakariah, K. K. (2002). Improved HPLC method for the determination of Curcumin, Demethoxycurcumin, and bisdemethoxycurcumin. Journal of Agricultural and Food Chemistry, 50, 3668–3672. Jiang, H., Timmermann, B. N., & Gang, D. R. (2006). Use of liquid chromatographyelectrospray ionization tandem mass spectrometry to identify diarylheptanoids in turmeric (Curcuma longa L.) rhizome. Journal of Chromatography A, 1111, 21–31. Michaleas, S., & Antoniadou-Vyza, E. (2006). A new approach to quantitative NMR: Fluoroquinolones analysis by evaluating the chemical shift displacements. Journal of Pharmaceutical and Biomedical Analysis, 42, 405–410. Péret-Almeida, L., Cherubino, A. P. F., Alves, R. J., Dufossé, L., & Glória, B. A. M. (2005). Separation and determination of the physico-chemical characteristics of curcumin, demethoxycurcumin and bisdemethoxycurcumin. Food Research International, 38, 1039–1044. Ravindran, P. N. (2007). Turmeric the genus Curcuma: The golden spice of life. In P. N. Ravindran, K. Nirmal Babu, & K. Sivaraman (Eds.), Medicinal and aromatic plants-industrial profiles (pp. 1–13). New York: Taylor and Francis Group, CRC Press. Ravindran, P. N., Nirmal Babu, K., Shiva, K. N. (2007). Turmeric the genus Curcuma: Botany and crop improvement of turmeric. In P. N. Ravindran, K. Nirmal Babu, & K. Sivaraman (Eds.), Medicinal and aromatic plants-industrial profiles (pp. 2–70). New York: Taylor and Francis Group, CRC Press. Shen, L., & Ji, H. (2007). Theoretical study on physicochemical properties of curcumin. Spectrochimica Acta Part A, 67, 619–623. Sundaryono, A., Nourmamode, A., Gardrat, C., Fritsch, A., & Castellan, A. (2003). Synthesis and complexation properties of two new curcuminoid bearing a diphenylmethane linkage. Journal of Molecular Structure, 649, 177–190. Tamvakopoulos, C., Sofianos, Z. D., Garbis, S. D., & Pantazis, P. (2007). Analysis of the in vitro matabolites of diferuloylmethane (curcumin) by liquid chromatography–tandem mass spectrometry on a hybrid quadrupole linear ion trap system: Newly identified metabolites. European Journal of Drug Metabolism and Pharmacokinetics, 32(1), 51–57.