S0308-8146(14)01281-3
http://dx.doi.org/10.1016/j.foodchem.2014.08.066
FOCH 16289
To appear in:
Food Chemistry
Received Date:
Revised Date:
Accepted Date:
30 November 2013
11 April 2014
13 August 2014
Please cite this article as: Syed, H.K., Liew, K.B., Loh, G.O.K., Peh, K.K., Stability indicating HPLC-UV method
for detection of Curcumin in Curcuma longa Extract and Emulsion Formulation, Food Chemistry (2014), doi: http://
dx.doi.org/10.1016/j.foodchem.2014.08.066
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Haroon Khalid Syed, Kai Bin Liew, Gabriel Onn Kit Loh and Kok Khiang Peh*
First author: Haroon Khalid Syed, B.Pharm, M.Phil. Department of Pharmaceutical Technology,
School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang,
Malaysia. Telephone: 604-6533296. Email: haroonkhalid80@gmail.com.
Co-author: Kai Bin, Liew, B.Pharm, MSc. Department of Pharmaceutical Technology, School of
Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia.
Telephone: 604-6533296. Email: liewkaia@yahoo.com.
Co-author: Gabriel Onn Kit Loh, B.Sc. Department of Pharmaceutical Technology, School of
Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia.
Telephone : 604-6533296. Email: lohonnkitgabriel@yahoo.com.
Abstract
A stability-indicating HPLC-UV method for the determination of curcumin in Curcuma longa
extract and emulsion was developed. The system suitability parameters, theoretical plates (N),
tailing factor (T), capacity factor ( ), height equivalent of a theoretical plate (H) and resolution
(Rs) were calculated. Stress degradation studies (acid, base, oxidation, heat and UV light) of
curcumin were performed in emulsion. It was found that N > 6500, T < 1.1, was 2.68 - 3.75,
HETP about 37 and Rs was 1.8. The method was linear from 2 200 g/mL with a correlation
coefficient of 0.9998. The intra-day precision and accuracy for curcumin were 0.87% and 2.0
%, while the inter-day precision and accuracy values were 2.1% and -1.92. Curcumin
degraded in emulsion under acid, alkali and UV light. In conclusion, the stability-indicating
method could be employed to determine curcumin in bulk and emulsions.
Key words: Curcumin; System suitability; Stress degradation study; Stability indicating HPLCUV method.
1. Introduction
Curcuma longa is a famous and important food spice with a rich source of polyphenolic
compounds, namely curcuminoids (Saleemulla, Makhija, Khamar & Rani, 2010). This spice has
been extensively used in traditional medicine and its demand is increasing in the field of
pharmaceutical, food and cosmetic industries. Apart from its daily use in food as a condiment
and spice, it has been used in the treatment of a number of ailments such as cough, fever,
jaundice, wounds, eczema, inflammatory joints, parasitic skin diseases, cold, liver and urinary
diseases (Antony, Elumalai & Benny, 2011). The rhizome has also been found useful in the
treatment of anemia, bacterial and viral infections (Nadkarni, 1976; Bakhru, 1997).
Curcuma longa extract has three important compounds called as curcumin (Cur),
demethoxycurcumin and bis-demethoxycurcumin, which are present in approximately 70% -77
%, 18% - 20 % and 5% - 10 % (Ahsan, Parveen, Khan & Hadi, 1999). The chemical structures of
the three compounds are shown in Fig. 1.
Methods that have been used to quantify curcumin include thin layer chromatography
(Ravindranath & Chandrasekhara, 1981), thin layer chromatography-UV and thin layer
chromatography- densitometric (Cooray, 1988), high-performance liquid chromatographyultraviolet (HPLC-UV) spectrophotometric and electrochemical detection (Smith & Witowska,
1994), HPLC- UV and LC- MS (Asai & Miyazawa, 2000) and HPLC-UV (Heath, Milagros,
Dean & Cherly, 2002). Most of these methods could only detect one single peak of curcumin
except a method reported by Asai & Miyazawa (2000), which could simultaneously detect the
three
curcumin
peaks
corresponding
to
curcumin,
demethoxycurcumin
and
bis-
demethoxycurcumin. Surash &Yadav (2009) reported a sensitive RP-HPLC- UV method for the
determination of curcumin but system suitability and stability studies were not performed.
Recently, a stability indicating HPLC-UV method was reported by Gugulothu & Patravale
(2012) for determination of curcumin and celecoxib in nanoparticle formulation. The result
showed that curcumin in solution was not stable and degraded after 1hr of stress studies.
Curcumionoids are polyphenols and decomposed when exposed to high temperature (Schieffer,
2002).
The objective of the present study was to develop a simple, rapid and reproducible stability
indicating HPLC-UV method for the determination of curcumin in Curcuma longa extract and
emulsion. The method could simultaneously detect curcumin, demethoxycurcumin and bisdemethoxycurcumin. Stress degradation studies (acid, base, oxidation, heat and UV light) were
carried out on emulsion formulation.
2.2. Instrumentation
The HPLC system was comprised of a Shimadzu (VP series, Kyoto, Japan) pump (LC-20AT vp)
with solvent cabinet, a degasser (DGU-20A3), a column oven (CTO-10S VP), an auto-injector
(SIL-20A HT vp), UV/VIS detector (SPD-20AD vp) and a computer software (LC- solution
VP).
The number of theoretical plates (N) is used to describe the quality of chromatographic column.
HETP or H, the height equivalent of a theoretical plate, measures the column efficiency per unit
length (L) of the column.
volumetric flask containing methanol. The flask was heated in water-bath at 60 C until the
emulsion melted and diluted to the mark with methanol. The flask was shaken for 15min
manually. The flask was then subjected to sonication for 15 minutes. The resulting solution was
centrifuged at 4000 rpm for 10 min. 0.2 mL of the supernatant was diluted with methanol in a
10mL volumetric flask to give a concentration of 20g/ml. The content of curcumin in emulsion
was calculated using the following equation,
%
=
neutralized immediately and adjusted to volume with methanol. These samples served as zero
hour samples. Another set of flasks was left on the bench at room temperature (26C / 65% RH)
and the same neutralization procedure was repeated after 24 hours. The neutralized solutions
were injected in triplicate.
2.17. Linearity
Calibration standards of curcumin at concentrations of 2.0, 4.0, 12.5, 25.0, 50.0, 100.0, 200.0
g/mL in methanol were prepared. The standard calibration curve was constructed using peak
area versus known concentrations of curcumin. The linear regression line was used to determine
the linearity and concentration of the samples. The linearity of curcumin was determined using
six sets of the calibration standards.
temperature (26 C). The stock solution was then diluted to a concentration within the standard
10
calibration linear range (160g/mL). The instrumental responses at 24 hours were compared with
that of fresh samples at zero hour.
2.20. Recovery of curcumin
1.0 mg of curcumin was added into 50 ml volumetric flask containing emulsion base (total
weight of 1gm) with methanol. The flask was heated in water-bath at 60 C until the emulsion
base melted and diluted to the mark with methanol. The flask was shaken for 15 min manually
and subjected to sonication for 15 minutes. The resulting solution was centrifuged at 4000 rpm
for 10 min. 0.2 mL of supernatant was diluted with methanol in a 10mL volumetric flask to give
a concentration of 20g/ml. 25 L of sample was injected into the HPLC system. The analysis
was repeated three times. The recovery of curcumin was calculated from the slope and the
intercept of the calibration curve drawn in the concentration range of 2-200 g/ml.
11
3.5. Specificity
There was no peak found at the retention time of the analyte in the blank solution. The
chromatogram of blank (solvent) is presented in Fig. 2a. The results from the stress testing
studies indicated the method was highly specific for curcumin. The degradation products were
completely distinguishable from the parent compound. The placebo chromatogram is presented
in Fig. 2b.
12
An ideal stability-indicating method is one that can quantify the standard drug and resolve its
degradation products (Kadi, Mohammed, Kaseem & Darwish, 2011). The method was able to
separate both analyte and degradation product peak. The results of acid, alkali, oxidation, heat
and UV degradation are shown in Table 3. Curcumin degraded under acid, alkali and UV light
challenges in emulsion. Under alkaline and UV conditions, excipients such as Tween 80 or Span
80 might undergo reactions to form secondary products. These secondary products might interact
with curcumin reducing the curcumin concentration in the sample (Zhang, 2009).
3.7. Linearity
The standard calibration curve exhibited an excellent linearity and a good correlation coefficient
over the given range of 2 200 g/mL of curcumin. The mean linear regression equation from
six calibration curves for curcumin was y = 38267.3 ( 764.2) x + 1279.6 ( 3538.3), (x =
curcumin concentration, y = average peak area) with a correlation coefficient of 0.9998 (0.0001).
13
The percentage of curcumin remaining after twenty four hours kept at room temperature was
100.41%. The result suggests that the stock solution was stable at least for 24 hours at room
temperature.
4. Conclusion
It can be concluded that a stability indicating HPLC-UV method for determination of curcumin
in Curcuma longa extract and emulsion was successfully developed. The method was rapid,
simple, precise, sensitive and specific for the analysis of curcumin in emulsion. Curcumin was
stable against oxidation and heat, but susceptible to acid and alkali and very susceptible to UV
degradation.
Acknowledgement
The authors thanked Universiti Sains Malaysia for the graduate assistant scheme and PRGS grant
(1001/PFARMASI/844074) support.
References
14
Ahsan, H., Parveen, 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. Chem.-Biol. Interact, 163, 161-175.
Antony, S., Elumalai, S., & Benny, M. (2011). Isolation, purification and identification of
curcuminoids from turmeric (Curcuma longa) by column chromatography. Journal of
Experimental Sciences, 2, 21-25.
Asai, A., & Miyazawa, T. (2000). Occurrence of orally administered curcuminoid as glucuronide
and glucuronide/sulfate conjugates in rat plasma. J. Life Sciences,67, 2785-2793.
Bakhru, H. K. (1997). Herbs that heal: natural remedies for good health. (1sted.). New Delhi,
India: Orient Paperbacks, (Chapter 1).
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Coun, Srilanka,16, 39-51.
Gugulothu, D. B., & Patravale, V. B. (2012). A New Stability-Indicating HPLC Method for
Simultaneous Determination of Curcumin and Celecoxib at Single Wavelength: an Application
to Nanoparticulate Formulation. Pharmaceut Anal Acta, 3, 1-6.
15
Heath, D. D., Milagros, A. P., Dean, E. B., & Cherly, L. R. (2002). Curcumin in plasma and
urine: quantification by HPLC. Chromatography J. B, 783, 287-295.
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Improved Reversed Phase-HPLC Method for Simultaneous Determination of Curcumin,
Demethoxycurcumin and Bis-Demethoxycurcumin. Chromatographia, 65, 483-488.
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16
Smith, R. M., & Witowska, B. A. (1994). Comparison of Detectors for the determination of
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E. Q. Marian (6th Ed.), Handbook of Pharmaceutical Excipients (pp. 549- 553). London,
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17
Table 1
Mobile phase composition and observation.
Composition
pH
(Acetonitrile:Methanol:Water)
Flow rate
Observation
(mL/min)
(v/v/v)
35:10:55
3.0
1.3
35:10:55
3.0
1.5
40:15:45
3.0
1.3
40:15:45
3.0
1.5
40:20:40
3.0
1.3
40:20:40
3.0
1.5
45:20:35
3.0
1.3
45:20:35
3.0
1.5
Poor separation
Table 2
Results of system suitability studies of quality control samples of curcumin. Mean SD, N = 6
20
Conc.
(g/mL)
HETP
Capacity
factor
Resolution
factor
6838.37 318.40
1.01 0.01
36.62 1.62
3.45 0.19
1.79 0.03
80
6755.66 274.49
1.01 0.01
37.05 1.50
3.46 0.20
1.77 0.03
160
6715.00 150.86
1.02 0.01
37.24 0.83
3.47 0.21
1.76 0.03
Table 3
Results of stress degradation studies of curcumin in emulsion. Mean SD, N = 3.
Parameters
24
99.45 0.65
86.33 0.64
99.99 0.85
82.21 0.74
96.66 0.08
98.02 0.03
99.47 0.60
101.06 0.62
UV degradation (%)
98.95 1.31
48.69 0.82
21
Table 4
Results of intra-day and inter-day precision and accuracy. The results are presented as mean, N=
6
Intra-day
Inter-day
Conc.
Precision
Accuracy
Precision
Accuracy
(g/mL)
(% CV)
(% Bias)
(% CV)
(% Bias)
0.86
1.99
2.0
-0.59
0.11
0.50
1.28
-0.20
80
0.31
0.48
1.47
-1.92
160
0.36
-1.09
1.30
-1.16
22
Highlights
Determination of curcumin in Curcuma longa extract and emulsion using HPLC method.
System suitability studies were performed.
Stress degradation studies of curcumin in emulsion formulation.
23