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Optimization and Validation of High-Performance Liquid Chromatography Method for Individual Curcuminoids in Turmeric by Heat-Refluxed Extraction Young-Jun Kim, Hyong Joo Lee, and Youngjae Shin J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 28 Oct 2013 Downloaded from http://pubs.acs.org on November 3, 2013

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Journal of Agricultural and Food Chemistry

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Optimization and Validation of High-Performance Liquid Chromatography

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Method for Individual Curcuminoids in Turmeric by Heat-Refluxed Extraction

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Young-Jun Kim1, Hyong Joo Lee1,2, and Youngjae Shin 3*

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1. Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea

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2. WCU Biomodulation Major, Department of Agricultural Biotechnology, College of

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Agriculture and Life Sciences, Seoul National University, Seoul 151-742, Republic of

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Korea

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3. Department of Environmental Horticulture, Dankook University, Cheonan 330-714, Republic of Korea

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*Corresponding author E-mail: [email protected] Tel: +82-41-550-3648.

Fax: +82-41-559-7881

Short title: HPLC Optimization of Curcuminoids analysis in Turmeric Section selection: Analytical Methods

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ACS Paragon Plus Environment


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ABSTRACT

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Turmeric is a plant in the Zingiberaceae family which contains curcuminoids as anti-cancer

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agents and has been widely used as a main ingredient in curry powder. However, there is a

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lack of suitable high performance liquid chromatography-diode array detection (HPLC-DAD)

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methods for reducing sample preparation time and peak resolution improvement of

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curcuminoids (bisdemethoxycurcumin, demethoxycurcumin and curcumin). No significant

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differences in yield concentrations were observed after 60 minutes of heat-refluxed extraction

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(p < 0.05). Simultaneous chromatographic separation of all three curcuminoids achieved

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satisfactory results with a separation factor of 1.08 and a resolution factor of 3.39 with

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validation results in compliance with FDA guidelines. The expanded relative measurement

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uncertainty results 5.71-6.60 complied with CODEX draft. The method was successfully

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applied to the turmeric samples (n = 107, range 2.70–4.41 g/100g, total curcuminoids 3.58

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g/100g). These results show that heat-refluxed extraction can be carried out easily with

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excellent precision and accuracy of total curcuminoids in turmeric samples.

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KEYWORDS: turmeric; curcumin; demethoxycurcumin; bisdemethoxycurcumin; method

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validation; high-performance liquid chromatography (HPLC)

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INTRODUCTION

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Turmeric (from the rhizome of Curcuma longa L) is a plant in the Zingiberaceae family and

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the main ingredient of curry, which has been widely used as a coloring and flavoring agent 1-2.

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Turmeric was originally consumed as a food additive in curries and used to enhance storage,

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preservation and palatability of food3. Turmeric is cultivated mostly in India and surrounding

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regions such as Bangladesh, China, Cambodia, Malaysia, Thailand, Philippines, and

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Indonesia4. It is particularly a staple in India, where 94% of the turmeric in the world

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originates5. Furthermore, it has long been used traditionally as an herbal medicine for treating

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a variety of inflammatory conditions such as coryza, cough, diabetic wounds, hepatitis, and

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other diseases6.

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The major bioactive compounds in turmeric are three different curcuminoids consisting of

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curcumin as the dominant constituent, demethoxycurcumin and bisdemethoxycurcumin.

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Pharmacological studies conducted so far on turmeric have shown that curcuminoids have

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several biological activities, such as an antioxidant, anti-protozoal, anti-microbial, anti-

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venom, anti-HIV, anti-tumor, anti-inflammatory, anti-cancer, and anti-carcinogenic3,7-12. Thus,

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turmeric has a wide range of health benefits and antioxidant activity in food and biological

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systems.

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Curcuminoids make up 3–5% of turmeric depending on the variety13. However, there is a

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lack of information about individual curcuminoids according to the diverse varieties and

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origins. Various methods have been established to determine individual curcuminoids in

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turmeric powder and extracts, such as spectrophotometric measurements, capillary

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electrophoresis14,

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chromatography-electrospray ionization tandem mass spectrometry (LC-ES-MS/MS)1,19,

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near-infrared spectroscopy20 and nuclear magnetic resonance21. Refluxing for 1–2.5 h

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Soxhlet extraction for 2–5 h

high

performance

liquid

15,16

chromatography

, shaking for 20 min

19,20

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(HPLC)2,15-18,

liquid

1,22,23

,

sonication14 for 2 min, and


24

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magnetic stirring for 20 h

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However, none of these studies compared different refluxing times during sample extraction

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or conducted measurement uncertainty of individual curcuminoids analysis using HPLC. The

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use of a conventional HPLC method is preferred for analysis of curcuminoid concentrations

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in a large number of turmeric samples because of its low-cost and ease of application.

have been reported as methods used to extract curcuminoids.

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The objectives of this study were (1) to improve a routine analytical method to determine

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curcuminoids in turmeric samples, including optimization of sample preparation (heat-

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refluxed extraction) and chromatographic conditions to enhance peak resolution; (2) to

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validate linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy,

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recovery, stability and measurement uncertainty for reliability of analysis; and (3) to further

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elucidate differences in the curcuminoid content in turmeric consumed in Korea.

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MATERIALS AND METHODS

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Reagents and Chemicals. The chemicals used were all analytical grade. Pure reference

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standards of curcumin (96.4% purity, CAS no. 458-37-7), demethoxycurcumin (95.1% purity,

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CAS no. 22608-11-3), and bisdemethoxycurcumin (91.8% purity, CAS no. 22608-12-4) were

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supplied by Chromadex Co. (Irvine, CA, USA). The chemical structures of these compounds

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are shown in Figure 1. Methanol and acetonitrile were obtained from J.T. Baker Chemical Co.

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(Deventer, The Netherlands) and Fisher Scientific (Pittsburgh, PA, USA), respectively. High

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purity water (18.2 MΩ) as the HPLC eluent was purified using a Milli-Q Water system

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(Millipore, Bedford, MA, USA). Liquid chromatography solvents were filtered with a 47 mm,

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0.45 µm HVLP (Millipore, Billerica, MA, USA).

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Preparation of Standard Solutions. Individual standard solutions were prepared by

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dissolving 5 mg of each compound in 10 mL of methanol to obtain a final concentration of

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500 mg/L. The working standard solutions were prepared at concentrations of 0.2, 1.0, 5, 10,

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25, 50, and 100 mg/L by serially diluting the curcuminoid solutions in methanol to fit the

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calibration curves and determine the linearity of the responses.

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Turmeric Sample Preparation. Dried turmeric samples were powdered using a Knifetec

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1095 sample mill (Foss Tecator, Höganäs, Sweden). They were then passed through a 250 µm

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stainless steel sieve before extraction. One hundred mg of turmeric was weighed and

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dissolved in about 30 mL of methanol in a 100 mL round-bottomed flask and refluxed in a

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cooled condenser, according to the modified procedures described in the ASTA method22. We

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observed refluxing time at 30 min intervals for up to 180 min to determine optimum

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extraction efficiency. After cooling, the extract was transferred to a 100 mL volumetric flask

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with a methanol rinse and penetrated through glass wool. Extracted sample solutions were

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filtered through 15 mm, 0.45 µm RC membrane filters (Sartorius, Goettingen, Germany) for

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HPLC analysis. The extract was placed in a light resistant amber colored glass vial, and 5 µL

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of the sample solution was injected into the HPLC system.

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Apparatus. All chromatographic analyses were carried out using an Agilent Technologies

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HPLC (1200 series, Palo Alto, CA, USA) with an automatic degasser, quaternary pump,

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autosampler, column thermostat, and DAD for analysis. A Capcell Pak C18 UG120 column

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(4.6 mm i.d.× 250 mm; 5 µm; Shiseido, Inc., Tokyo, Japan) was employed for analytical

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separation and quantification with the column temperature set to 25 °C. HPLC analysis was

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performed using mobile phase A (1% acetic acid in distilled water) and B (acetonitrile) in a

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gradient program at a flow rate of 1.0 mL/min. The mobile phase started with an initial

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gradient of 45% A and then increased from 45% solvent A to 50% in 10 min. The system was 5



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then kept at the initial mobile phase condition for another 5 min for the next run. The DAD

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was positioned at a wavelength of 424 nm (at 4 nm bandwidth) with a 550 nm reference

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wavelength (at 50 nm bandwidth). Full spectral scanning was also performed from 200 nm to

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600 nm, with a range step of 2 nm. Agilent Chemstation software was used to control all

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analytical conditions and the chromatographic data processing. Sample quantification was

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calculated by comparing peak area with the external calibration curve from neat standard

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solution. The content of each curcuminoid was estimated as follows: Cspl = (Aj – Bo)/B1 × F,

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where Cspl is the curcuminoid concentration; Aj, is the jth measurement of the area of the ith

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calibration standard; B1 is the slope of the calibration curve; Bo is the intercept of the

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calibration curve, and F is a dilution factor.

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Column performance test. Peak asymmetry (As) was estimated at 10% of peak height from

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the ratio of the widths of the rear and front sides of the peak25. Retention factors (k’) were

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calculated as k’ = (tr–t0)/t0, where t0 is retention time of un-retained solvent and tr is retention

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time of the analyte. The k’ values were within the optimum range (1 ≤ k’ ≤ 10) for

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satisfactory chromatographic elution of curcuminoids26.

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which indicated acceptable separation26. The resolution factor (Rs), which is a measurement

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of how well two peaks are separated, was calculated as Rs = 2 × (t1– t2)/(w1+w2), where t1 and

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t2 are the retention time for two adjacent peaks; and w1, w2 are peak widths at the bases. In

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addition, the number of theoretical plates (N), which is a measurement of peak dispersion,

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was calculated from peak width at half height (w0.5) using the formula N = 5.54(tr/w0.5)2 25.

Separation factors (α) were > 1,

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HPLC Method Validation. The HPLC-DAD method was fully validated for selectivity,

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LOD, LOQ, linearity, precision, accuracy, recovery, and stability according to the FDA

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guideline27. 6


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a. Selectivity.

Selectivity is the ability of an analytical method to differentiate and

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quantify the individual curcuminoids that are expected to be present. Blank matrix samples

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were tested for interference, and selectivity was evaluated by comparing the chromatograms

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of blank and turmeric sample solutions by confirming DAD. Retention times were

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determined to verify peaks which were compared to a ratio of sample peaks from DAD to a

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ratio of reference standard peaks.

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b. LOD, LOQ, and Linearity.

The LOD and LOQ were defined according to Knoll’s

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research28 as the lowest concentration of analyte in the standard solution that triggers a

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significantly different instrumental signal from the blank or background noise, which is equal

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to signal-to-noise ratios of 3 and 10, respectively. Linearity of the detector response was

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verified with bisdemethoxycurcumin, demethoxycurcumin, and curcumin standard solutions

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over the range of 0.2–100 mg/L. Calibration curves were prepared each experiment day, and

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the concentration of the analytes in the samples was calculated.

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c. Precision and Accuracy.

The intraday precision of the HPLC–DAD method was

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tested six times/day with a quality control sample (fortified turmeric solution), which was a

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standard solution of bisdemethoxycurcumin, demethoxycurcumin, and curcumin. For

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interday precision, three measurements/day on three different days were conducted. The

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precision of the method was expressed as the relative standard deviation (RSD) for the

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repeated measurements. The accuracy of the method was calculated as the relative difference

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between the determined and nominal concentrations of the analyzed samples.

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d. Recovery. Turmeric samples were spiked with curcuminoid standards. Turmeric

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samples (bisdemethoxycurcumin for Indian turmeric, demethoxycurcumin for Chinese

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turmeric, and curcumin for Korean turmeric sample 100 mg used) were analyzed in triplicate

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to quantify each curcuminoid concentration as a blank. Standard-added samples were

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prepared containing individual curcuminoids (0.05 mL, 0.5 mL, and 2.5 mL of 1,000 mg/L

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stock solution) to give final results at three concentrations (for 0.05 g/100g, 0.5 g/100g, and

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2.5 g/100g, dilution factor = final volume;100 mL volumetric flask / sample weight; 100 mg,

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that is 1,000). The recovery (R) was calculated by the method of Rodriguez et al.29 as R =

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(Cfound – Csample)/Cadded, where Cfound is the concentration in the standard added sample, Csample

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is the concentration as a blank sample, and Cadded is the added concentration.

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e. Stability. The stability of curcuminoids in turmeric extracted solution was

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conducted under various conditions; freeze-thaw stability (at -20°C and room

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temperature for three cycle), short-term stability (at room temperature for 12 h), long-

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term stability (-20°C for 6 weeks), which were analyzed through triplicate

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determinations in low, mid and high concentration (1 mg/L, 5 mg/L and 25 mg/L in

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matrix solution), respectively. Furthermore, post-preparative stability was evaluated in

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the sample solution by tests in HPLC autosampler (sample solution, at room

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temperature, for 12h).

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f. Measurement Uncertainty Assessment.

Uncertainty of a measurement is defined as

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“a parameter, which is associated with the result of a measurement and characterizes the

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dispersion of the values that could reasonably be attributed to the measurand”

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measurement uncertainty budget for the methods was evaluated based on the modified of

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EURACHEM/CITAC Guide and Guide to the Expression of Uncertainty in Measurement

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(GUM) method31. The sources of measurement uncertainty (i.e. standard stock solution (uSSS),

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sample preparation (uSP), calibration curve (uCal) and repeatability for the determination of

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curcuminoids in turmeric sample (uRP)) in associated with the analysis of curcuminoids were

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evaluated. There error components were estimated and calculated as an expanded uncertainty

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(U c) using a coverage factor (k) of 2 at the confidential level of 95%.

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. The

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Statistical Analysis. Samples were prepared and analyzed in triplicate. The results are

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reported as mean values and standard deviations. Differences were detected by one-way

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analysis of variance (ANOVA) using the SPSS 12.0.1 version 4 software package (SPSS, Inc.,

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Chicago, IL, USA) with a significance level of 0.05.

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RESULTS AND DISCUSSION

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Analytical Characteristics

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a. Optimizing Sample Preparation Efficiency. The sample preparation procedure for

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HPLC is one of the most important and time-consuming steps, which may also affect errors.

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Various extraction methods for turmeric have been described in the literature. Turmeric

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powder samples have been extracted with hexane for 30 min using a Soxhlet extractor and re-

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extracted with methanol for 2 h

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pentane and methanol for 2 and 3 h using a Soxhlet extractor, respectively, and the methanol

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solution was concentrated using a rotary evaporator. These studies may not be suitable for

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routine analysis of a large number of turmeric samples because sample extraction takes a long

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time. The ASTA method, which is a common method for measuring turmeric color, uses a

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reflux time of 2.5 h

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determining curcuminoids by HPLC is scarce. To determine the reflux time (0, 30, 60, 90,

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120, 150, and 180 min, at 65 °C) for optimum extraction efficiency, turmeric powder was

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extracted at each reflux time with three replicate extractions. Each compound concentration

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was analyzed by HPLC to determine the effectiveness of sample preparation. As shown in

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Figure 2, significant differences were observed between un-refluxed (0 min) and refluxed

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samples (30-180 min). In case of demethoxycurcumin and curcumin, no significant

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. Bos et al.

16

extracted turmeric powder samples with

22

. However, information related to turmeric reflux extraction time for

9



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differences in yield concentrations with good precision and no thermal decomposition were

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observed by comparing 30–180 min heat-refluxed samples. However, bisdemethoxycurcumin

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showed significant differences between 30 min and 60-180 min refluxing time period (p
0.999 on each of the 3 days in which calibration curves

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were run. The calibration parameters, LOD, and LOQ of the three curcuminoids are shown in

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Table 2.

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c. Precision and Accuracy.

Table 3 shows the intraday and interday precision and

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accuracy values of the entire procedure for each curcuminoid. Excellent intraday precision

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data (n = 6) and interday precision data (n = 9) were obtained for the turmeric powder

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samples containing all analytes. The RSDs ranged from 1.02% to 1.11% for the intraday

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precision tests, and 1.41% to 1.56% for the 3-day interday precision tests. These values were

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considered in high compliance with FDA criteria (

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97.08% with the RSD for each analyte < 0.53%. Table 4 shows the recoveries (average

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100.32%) that were close to 100%. This observation was similar to previous studies16,17,20.

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Further, recoveries of the method were in compliance with the FDA in the range of 80–120%

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.

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e. Stability. Lechtenberg et al.14 and Gören et al.21 reported that the main problem in

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curcumin analysis was that curcuminoids are light sensitive compounds. All experiments

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including stability test for curcuminoids analysis used light resistant amber colored glass vials

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to protect the curcuminoids from light. For the stability evaluation, the average and standard

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deviations between initial concentration and the found concentration of the solution were

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compared. The fortified matrix sample solution had an acceptable stability at room

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temperature for 12 h (S.D.

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