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Kavalactones Assay by HPLC
Published:2017-12-21 Views:45

Kavalactones Assay by HPLC

Standard Preparation:

Prepare a mixed stock standard by accurately weighing (±0.01 mg) about 10 mg of each kavalactone reference standard into a 10-mL volumetric flask. Add 7.0 mL of methanol and sonicate until dissolved. Dilute to volume with methanol and mix.

Notice: Alternatively, prepare a kavain stock standard in a like manner and use the kavalactone response factors to quantify against.

Dilute the stock standard to create a minimum of a four-point standard curve. Suggested standard dilutions of the stock are 1:10, 1:25, and 1:100 using methanol.

Notice: Store standards at refrigerated temperatures (0 - 5°C). Kavalactones degrade rapidly after 48 hours. Preparation of fresh reference standards before each analysis is recommended.

Apparatus

· Calibrated analytical balance accurate to ±0.01 mg

· Flask, volumetric, Class A, assorted sizes

· Vials, chromatography with cap

· High-Performance Liquid Chromatography System as described in USP chapter <621>. Verify and document that apparatus, software, and subsystems meet performance requirements for Installation Qualification/Operation Qualification (IQ/OQ).

· Filter, 0.45 µm, Gelman Acrodisc® nylon (P/N 4426), Whatman Puradisc™ Polypropylene (Cat. No. 6788-2504), Whatman GD/X Glass (Cat. No. 6894-2504) or equivalent Sonicator

· Column, YMCbasic, 5 µm, 4.6 x 250 mm (Cat. No. BA99S05-2546WT). Equivalent column, YMC J'Sphere H80, 4µ, 4.6 x 250 (Cat. No. JH08S04-2546WT).

Reagents

· Water, HPLC grade or Nanopure

· Methanol, HPLC grade

· Acetonitrile, HPLC grade

· Phosphoric Acid, ACS Reagent grade

Sample Preparation:

Plant Material

Accurately weigh (± 0.1mg) about 750 mg of finely ground stem, peeling, or root material.

Place the material into a 50-mL volumetric flask along with 40 mL of methanol/water (70/30). Sonicate for 60 minutes at room temperature.

Allow flask to cool to room temperature and dilute to volume with methanol/water (70/30) and mix.

Filter and place sample into a HPLC vial and cap.

Powder and Soft Extract (Paste)

Accurately weigh (± 0.1 mg) about 100 mg of extract into a 50-mL volumetric flask. (The 100 mg weight is based upon a 50-60% kavalactone content in the extract.) Notice: Carefully homogenize extract before sampling. This is accomplished by placing the extract in a container immersed in a 60° - 80° C hot water bath until free flowing.

Add 40 mL of methanol and sonicate for 10 minutes or until all of the solids have dissolved.

Allow flask to cool to room temperature and dilute to volume with methanol and mix.

Filter and place sample into a HPLC vial and cap.

Powder Samples

Kava extract is often blended or spray dried onto various carriers.To successfully assay powder samples, it is necessary to develop a sample preparation step that recovers the kavalactones from the carrier. This can be accomplished by first extracting with pure methanol or water.

Chromatographic Conditions:

Column: YMCbasic, 5 µm, 4.6 x 250 mm
Column Temperature: 40°C
Flow Rate: 0.6 mL/min for YMCbasic ; 1.0 mL/min for J'Sphere
Mobile Phase: isocratic 0.1% phosphoric acid:isopropyl alcohol:acetonitrile (64:16:20 v/v)
Detection: 220 nm (246 nm alternatively to improve selectivity)
Injection Volume: 5 µL
Run Time: 30 minutes

Procedure:

Prepare reference standard solutions and sample preparations as directed.
Make a single injection of an extraction solvent blank.
Make single injection of the standard preparations.
Create a linearity plot of standard peak areas versus standard concentrations.
Make single injections of sample preparations.
Calculate the kavalactone concentration in the sample.

System Suitability:

The correlation coefficient of the linear regression must be ≥0.999. The resolution between desmethoxyyangonin and yangonin peaks must be ≥1.8.

Retention Times (approximate):

Methysticin

14.1 minutes

Dihydromethysticin (DHM)

15.0 minutes

Kavalactone 1

16.0 minutes

Kavalactone 2

16.6 minutes

Kavain

18.6 minutes

Dihydrokavain (DHK)

20.7 minutes

Yangonin

24.3 minutes

Desmethoxyyangonin (DMY)

27.1 minutes

Calculations:

 

   % Individual Kavalactone = (A)(SR)(FV)(D)(F)(100%)

                                       (W)

Where:

A = Peak area response of the kavalactone in the sample.

SR = The response factor of the appropriate kavalactone reference standard determined by the least squares fit of the calibration data.

FV = The final volume of the sample preparation.

D = The dilution factor of the sample preparation (if needed).

F = The correction factor for quantitation against kavain (if needed).

Correction Factors (F):

Notice: Response factors are dependent upon column chemistry, detector, separation, and detector wavelength. Always confirm F using individual reference standards on new chromatographic systems.

Compound

Methysticin

0.663

Dihydromethysticin

1.65

Kavain

1.00

Dihydrokavain

1.70

Desmethoxyyangonin

0.904

Yangonin

0.878

W = The sample weight (mg).

Notice: There is no data (LC-MS or spectral) confirming that the peaks labeled Kavalactone 1 & 2 are indeed kavalactones. Until this information is collected, it is recommended that these peaks not be included in the total kavalactones calculation.