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 world’s 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 µm 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 µL 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, 0.5±0.01 g) was mixed with five

different solvent mixtures (methanol, 80:20, 50:50 and 20:80 methanol:water mixtures and acetonitrile) and shaken at 40°C 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 µm 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).

Sample Analyte Concentration(mg/g)

Standard deviation(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

Sproutsturmeric powder

bis-demethoxycurcumin 4.5 0.22 4.8demethoxycurcumin 5.7 0.36 6.5

curcumin 16.9 1.37 8.1

McCormickturmeric spice

bis-demethoxycurcumin 12.5 0.56 4.5demethoxycurcumin 7.8 0.77 9.9

curcumin 18.6 1.54 8.3

Laxmiturmeric powder

bis-demethoxycurcumin 2.7 0.15 5.7demethoxycurcumin 3.0 0.22 7.2

curcumin 9.7 0.66 6.9

Finest Natural’s“Turmeric extract” capsules

bis-demethoxycurcumin 16.7 1.05 6.3demethoxycurcumin 102.4 5.96 5.8

curcumin 254.1 26.93 10.6

Natture’s Bounty’s“Turmeric Curcumin” capsules

bis-demethoxycurcumin 5.7 0.35 6.1demethoxycurcumin 16.2 0.93 5.7

curcumin 74.8 5.12 6.9

New Chapter’s “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 Injection Retention time(min)

Tailing factor Correlation coefficient of the calibration curve

Resolution

C-DMC DMC-BDMC

Curcumin1 2.966±0.008 0.607 0.9996 2.452 2.990±0.008 0.603 0.9992 2.593 2.995±0.004 0.598 0.9993 2.56

Demethoxycurcumin1 2.552±0.006 0.598 0.99952 2.571±0.007 0.599 0.99923 2.575±0.004 0.595 0.9993

Bis-demethoxycurcumin1 2.186±0.005 0.551 0.9995 2.302 2.200±0.006 0.579 0.9994 2.473 2.204±0.002 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 μg/mL 0.89-224.0 μg/mL 0.76-189.1 μg/mL

Linearity of standard R2 > 0.999 R2 > 0.999 R2 > 0.999

Limit of quantitation (LOQ) 2.07 μg/mL 1.88 μg/mL 1.31 μg/mL

Limit of detection (LOD) 0.68 μg/mL 0.62 μg/mL 0.43 μg/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

ANIKÓ M. SÓLYOM, GAAS Corporation, Tucson, AZ 85747

• CURCUMIN • DEMETHOXYCURCUMIN • BIS-DEMETHOXYCURCUMIN

LEVEL 4 CALIBRATION STANDARD MIX (7.56 µg/mL curcumin, 8.96 µg/mL demethoxycurcumin and 6.24 µg/mL bis-demethoxycurcumin)

Curcumin

DemethoxycurcuminBis-demethoxycurcumin