A single laboratory validation study for the determination of curcuminoids in dietary supplements...
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ABSTRACTDemand for botanical products has grown dramatically over the past ten years, and markets remain robust. The World Health Organization estimates that up to 80% of the worlds population rely on traditional, primarily herbal remedies; herbal products are becoming increasingly important in conventional medicine too. More than 120 million Americans try herbs, vitamins, and dietary supplements to treat a variety of illnesses. Turmeric, the powdered rhizome of the herb Curcuma longa, is used extensively in curries and mustards as a coloring and flavoring agent. In the Ayurvedic medicine, turmeric has traditionally been used as a treatment for inflammation, skin wounds and tumors. Curcuminoids - a group of phenolic compounds, namely curcumin, demethoxycurcumin and bisdemethoxycurcumin - represent the yellow pigments of turmeric. These compounds exhibit anti-inflammatory and anti-oxidant properties, and have been recently discussed as potential chemopreventive agents. A number of studies demonstrate that the contents of the marketed dietary supplements vary. One of the reasons of this inconsistency is that the quantity and quality of botanical raw material is affected by many factors: the climate during the growing season, when and how the product was harvested, how and how long the raw product was stored, etc. We purchased turmeric dietary supplements and spices from various stores in Tucson, AZ. We developed a fast, reversed phase, rapid resolution HPLC method for the separation and quantitation of curcuminoids. An optimized extraction method was applied for each turmeric sample, the soluble and insoluble parts were separated, then the amount of curcuminoids were determined from the soluble fraction. The standard curves and assay repeatability for each curcuminoids were measured on five independent days.
EXPERIMENTAL PROCEDURESChromatography: Curcumin, demethoxycurcumin, and bisdemethoxycurcumin standards were obtained from ChromaDex Inc. Turmeric samples were purchased from local health/food syores in Tucson, Arizona. The HPLC system consisted of an Agilent 1200 RR series binary gradient pump, diode-array detector, thermostated
column compartment, autosampler and a Zorbax XDB C18 column (4.6x150 mm, 1.8 m particle size). Separation was performed using a reversed phase, gradient HPLC method with a flow rate of 1.5 mL/min at 40oC.
The mobile phases were water and acetonitrile, containg 0.01% TFA. Ten L of each of the standards and the extracts were injected onto the HPLC column. Signals were observed at 425, 280 and 260 nm. The AUAC values reported on this poster were calculated from the signals detected at 425 nm.
Extraction optimization: In order to optimize the extraction procedure, a ground turmeric sample (SWAD, 0.50.01 g) was mixed with five
different solvent mixtures (methanol, 80:20, 50:50 and 20:80 methanol:water mixtures and acetonitrile) and shaken at 40C for 1 hour.
The effect of extraction time on the extraction efficiency was compared by shaking and ultrasonicating the turmeric sample for 0.5, 1, 4 and 24 hours.
Finally, repetitive dilutions were used to determine the extraction efficiency and determine the number of extractions needed to ensure complete extraction.
Optimized extraction procedure: Whole samples were ground and passed through a 250 m sieve. Each sample was accurately weighed and transferred
into a 100 mL volumetric flask. Approx. 80 mL of 80:20 methanol:water solvent mixture was added to the flask and the content was mixed well. The slurry was sonicated at room temperature for 1 hr, then diluted to volume. The samples were centrifuged and 1.5 mL of the supernatant was transferred into HPLC vials. Two replicate sample preparations were performed that way.
CONCLUSION A fast RP HPLC method was developed and a single-laboratory validation study was
performed for the determination of curcuminoids in dietary supplements and food products The method succesfully separates the curcuminoids; curcumin, demethoxycurcumin
and bis-demethoxycurcumin within the first 4 minutes of the HPLC run. The method is repeatable for the determination of curcuminoids; within day repeatibility
RSD of the analyzed turmeric samples ranged from 1.0 to 13.8% (data not presented).
(mg/g)Relative standard deviation
SWAD whole turmeric
bis-demethoxycurcumin 3.3 0.16 5.0demethoxycurcumin 4.2 0.25 6.0
curcumin 13.4 1.06 7.9
bis-demethoxycurcumin 4.5 0.22 4.8demethoxycurcumin 5.7 0.36 6.5
curcumin 16.9 1.37 8.1
bis-demethoxycurcumin 12.5 0.56 4.5demethoxycurcumin 7.8 0.77 9.9
curcumin 18.6 1.54 8.3
bis-demethoxycurcumin 2.7 0.15 5.7demethoxycurcumin 3.0 0.22 7.2
curcumin 9.7 0.66 6.9
Finest NaturalsTurmeric extract capsules
bis-demethoxycurcumin 16.7 1.05 6.3demethoxycurcumin 102.4 5.96 5.8
curcumin 254.1 26.93 10.6
Nattures BountysTurmeric Curcumin capsules
bis-demethoxycurcumin 5.7 0.35 6.1demethoxycurcumin 16.2 0.93 5.7
curcumin 74.8 5.12 6.9
New Chapters Turmeric force gel capsules
bis-demethoxycurcumin 7.2 1.79 25.0demethoxycurcumin 4.4 1.24 28.1
curcumin 6.8 2.21 32.3
Analyte InjectionRetention time
(min)Tailing factor Correlation coefficient
of the calibration curve
Curcumin1 2.9660.008 0.607 0.9996 2.452 2.9900.008 0.603 0.9992 2.593 2.9950.004 0.598 0.9993 2.56
Demethoxycurcumin1 2.5520.006 0.598 0.99952 2.5710.007 0.599 0.99923 2.5750.004 0.595 0.9993
Bis-demethoxycurcumin1 2.1860.005 0.551 0.9995 2.302 2.2000.006 0.579 0.9994 2.473 2.2040.002 0.582 0.9993 2.46
REPRESENTATIVE CHROMATOGRAM OF THE CURCUMINOIDS STANDARDS
EXTRACTION EFFICIENCYSYSTEM SUITABILITY
THE EFFECT OF SOLVENT COMPOSITION
THE EFFECT OF EXTRACTION TIME AND MODE
THE EFFECT OF REPETITIVE EXTRACTION
CURCUMINOIDS CALIBRATION CURVES @ 425 nm
CHROMATOGRAMS OF TURMERIC SAMPLES VALIDATION RESULTS
Bis-demethoxycurcumin Demethoxycurcumin Curcumin
Linear range 0.62-156.1 g/mL 0.89-224.0 g/mL 0.76-189.1 g/mL
Linearity of standard R2 > 0.999 R2 > 0.999 R2 > 0.999
Limit of quantitation (LOQ) 2.07 g/mL 1.88 g/mL 1.31 g/mL
Limit of detection (LOD) 0.68 g/mL 0.62 g/mL 0.43 g/mL
INTRADAY (5 DAYS) REPEATIBILITY OF 14 SAMPLES
LOD and LOQ was calculated according to LOD = 3.3 SD/S and LOQ = 10 SD/S, where SD is the standard deviation of y-intercepts of the regression line, and S is the mean of the slope of the calibration curve.CHEMICAL STRUCTURE OF CURCUMINOIDS
WHOLE TURMERIC (SWAD)
TURMERIC SPICE (McCORMICK)
TURMERIC EXTRACT DIETARY SUPPLEMENT (Finest Natural: 900 mg turmeric and 5 mg black pepper extract)
A SINGLE LABORATORY VALIDATION STUDY FOR THE DETERMINATION OF CURCUMINOIDS IN DIETARY SUPPLEMENTS AND FOODS BY RAPID RESOLUTION HPLC USING PDA DETECTION
ANIK M. SLYOM, GAAS Corporation, Tucson, AZ 85747
CURCUMIN DEMETHOXYCURCUMIN BIS-DEMETHOXYCURCUMIN
LEVEL 4 CALIBRATION STANDARD MIX (7.56 g/mL curcumin, 8.96 g/mL demethoxycurcumin and 6.24 g/mL bis-demethoxycurcumin)