German Commission E found Kava Kava

"to be beneficial for relieving nervous anxiety, stress, and restlessness. It has been widely used in Europe for its relaxant effects or as an anti-anxiety remedy."

  BENEFITS: To combat mild-to-moderate anxiety, enhance relaxation, can help ease menstrual cramps because of antispasmodic effects and contains two pain-relieving chemicals as effective as aspirin.

Assay of Kavalactones

Assay Title: Determination of kavalactones (a-pyrones and substituted 5,6-dihydro-a-pyrones) in Piper methysticum by High-Performance Liquid Chromatography.

Scope: This assay can be used to determine methysticin, dihydromethysticin (DHM), kavain, dihydrokavain (DHK), desmethoxyyangonin, and yangonin content in kava plant material and kava soft extract (paste).

Reference: Kava - An Overview, Singh, Y.D., Special Review Herbalgram No. 39, pp. 34 - 56
Yu Shao, Kan He, Bolin Zheng, Qun Yi Zheng, J. Chrom. A, 825 (1998) 1 - 8

Principle: Plant material or extract is extracted/dissolved using methanol and analyzed by HPLC against external standards of each kavalactone (alternately, kavain may be used as a standard for all analytes, using correction factors). The HPLC column is YMCbasic J'Sphere H80, 4.6 x 250 mm, the mobile phase is isocratic acetonitrile/water/methanol at 1.0 mL/minute and detection is UV at 220 nm.

Standards: Desmethoxyyangonin (Addipharma, Batch No. RDY 00197, Sales No. RDY 001 004)
Dihydromethysticin (Addipharma, Batch No. RDM 00298, Sales No. VDM 002 002)
Dihydrokavain (Addipharma, Batch No. RDK 00197, Sales No. VDK 001 005)
Methysticin (Addipharma, Batch No. RME 00197, Sales No. VME 001 003)
Yangonin (Addipharma, Batch No. RYA 00197, Sales No. VYA 001 004)
Kavain (Addipharma, Batch No. LHKA 00197, Sales No. VKA 001 007)


Note: This is not an all-inclusive listing of sources for reference standards. World wide there are many reputable suppliers of botanical reference standards whose product may be used. However quality control protocol and/or government regulation may mandate further qualification of the materials for proper identity and purity.

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^TM 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'Shpere 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.

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. This method is not validated for these matrixes. To successfully assay powder samples, it is necessary to develop a sample preparation step that recovers the kavalactones from the carrier. This sometimes can be accomplished by first extracting with pure methanol or water. However, no general guidelines can be given due to the wide range of carriers used and their individual chemical and physical characteristics.

Scope: This assay can be used to determine hypericin and pseudohypericin in Hypericum perforatum flowering tops and powdered extracts.

Reference: Balogh, M.P. and Li, J.B., HPLC Analysis of Hypericin with PDA and MS Detection, LC-GC, Volume 17, No. 6, pp. 556 -562

Häberlein, H., Tschiersch, K.P., Stock, S., Hölzl, J., Johanniskraut (Hypericum perforatum L.), Pharm. Ztg. Wiss., 137, 1992, pp. 169 -174

Krämer, W. and Wiartall, R., Bestimmung von Naphthodianthronen (Gesamthypericin) in Johanniskraut (Hypericum perforatum L.), Pharm. Ztg. Wiss., 137, 1992, pp. 202 -207

von Frauke Gaedcke, Johanniskraut und dessen Zubereitungen, Deutsche Apotheker Zeitung, Nr. 42, 1997, pp. 117-121

Principle: Samples are extracted with methanol and sonication. Conversion of protohypercin and protopseudohypericin is performed by exposure to light for 30 minutes before analysis by HPLC. Exposure of samples to light ensures good inter-laboratory agreement. The HPLC column is Kromasil® RP18, 4.0 x 125 mm, 5µm; the mobile phase is isocratic methanol:pH 2.1 phosphate buffer:ethyl acetate at 1.0 mL/min and detection is at 590 nm. Quantitation is external against hypericin reference standard.

Standards: Hypericin (Addipharma, Hamburg, Germany, Product No. RHY 00496 or Indofine Chemical Co., Somerville, NJ, Product No. 02-0468S)

Apparatus

• Calibrated analytical balance accurate to ±0.1 mg or ±0.01 mg
• Sonicator, VWR Scientific Aquasonic Model 250D or equivalent
• Centrifuge tubes
• HPLC Column, MZ Analytical Kromasil 4.0 x 125 mm, 5µm, RP18 (Cat. No. 51516184)
  • Equivalent Column
    The following columns have been shown to give good chromatography. However, decreasing the percentage of buffer in the mobile phase composition and/or increasing the flow rate may be needed to optimize analyte retention times:
    Waters Symmetry® C18, 3.9 x 150 mm, 5µm, Part No. WAT046980
    Phenomenex Luna® C18, 5µm, PN 00F-4252-EO, SN 314914
• 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).
• Vials, 2 mL HPLC auto-sampler compatible, with crimp top
• Lamp: Lumilux L 18W/11 (Osram Sylvania)
  or Octron® "700" Series F017/735 (Osram Sylvania)

Note: Lamp has a color rendering index (CRI) of 75 – 80. No filter or diffuser is attached
to the lamp. Contact Osram Sylvania National Customer Service Center at (800) 544-4828.

Reagents

• Methanol, HPLC grade
• Ethyl Acetate, HPLC grade
• Sodium dihydrogen phosphate dihydrate, ACS grade
• Phosphoric acids, ACS grade
• Water, HPLC grade or Nanopure

Mobile Phase Buffer Preparation: Dissolve 15.6 g of dihydrogen phosphate dihydrate into water and make up to 1000 mL. Adjust the pH to 2.1 ±0.1 using phosphoric acid.

Standard Preparation:

Accurately weigh (±0.01 mg) 3-4 mg of hypericin reference standard into a 50-mL volumetric flask and add 30 mL of methanol. Sonicate for 5 minutes or until all solids have dissolved. Dilute to volume with methanol and mix. Dilute stock solution 1:10 and 1:25 using methanol for a three-point calibration curve.

NOTICE: If reference standard does not readily dissolve, add a few drops of pyridine and sonicate until dissolved.

Note: Further dilutions may be made for additional datum points.

NOTICE: Store standard solutions at room temperature in darkness when not in use. Standard response should be checked before each use. The hypericin standard is stable up to 3 months.

Sample Preparation:

Powdered Extract

Accurately weigh (±0.1 mg) 750 mg of powdered extract and place into a 50-mL volumetric flask. Add 40 mL of methanol and sonicate for 15 minutes. Cool to room temperature and dilute to volume with methanol and mix. Remove a 10-mL aliquot and centrifuge for 5 minutes at 4,500 rpm. Transfer the centrifuged aliquot (supernatant) into a HPLC vial and crimp seal it. Expose the vial to specified light source for 30 minutes at a distance of about 10 cm.

Plant Material (Herb)

Accurately weigh (±0.1mg) 900-1100 mg of finely ground plant material (25 mesh) and place into a 100-mL round bottom flask fitted with a reflux condenser. Reflux for 20 minutes using 80 mL of methanol. Filter the sample through cotton wool, saving the filtrate. Extract the herb material and cotton wool two additional times with 60 mL of methanol. Filter and combine collected filtrates and washes into a 250-mL round bottom flask. Concentrate to a final volume of about 3 mL using a rotary evaporator or similar apparatus. Quantitatively transfer the concentrated solution to a 25-mL volumetric flask. Dilute to volume using methanol and mix. Remove an aliquot and centrifuge at 4,500 rpm for 5 minutes. Transfer the centrifuged aliquot (supernatant) into a HPLC vial and crimp seal it. Expose the vial to specified light source for 30 minutes at a distance of about 10 cm.

Notice: Do not replace centrifugation with filtering unless non-retention of analytes on the filtering media is first proven. Teflon and cellulose acetate filters have been shown to bind hypericin.

Ginkgoterpenoid Assay

Assay Title: Determination of Ginkgo diterpenes and bilobalide (often collectively referred to as ginkgolides) in Ginkgo biloba by High-Performance Liquid Chromatography

Scope: This assay can be used to determine the diterpenoids ginkgolides A, B, C, and J and the sesquiterpenoid bilobalide in plant materials and powdered extracts.

Reference: Evaporative Light Scattering and Thermospray Mass Spectrometry: Two Alternative Methods for Detection and Quantitative Liquid Chromatography Determination of Ginkgolides and Bilobalide in Ginkgo biloba Leaf Extracts and Phytopharmaceuticals. Kurt Hostettmann, et al, (1995) Phytochem. Anal. 6, 141-148

Safety Precautions: Consult the Material Safety Data Sheet (MSDS) for any chemical used that is unfamiliar. All chemicals should be considered hazardous avoid direct physical contact.

Principle: Plant material and extract are extracted/dissolved using methanol/water and analyzed by HPLC against the external standards bilobalide, ginkgolide A, and ginkgolide B. The HPLC column is a Phenomenex Prodigy^TM C18, 4.6 x 250 mm, the mobile phase is a water:methanol gradient at 1.0 mL/minute and detection is by evaporative light scattering detector (ELSD).

Standards: Ginkgolide A (Sigma, Cat. #G 4028) or (Indofine Chemical, Cat. #02-8821)

Ginkgolide B (Sigma, Cat. #G 6910) or (Indofine Chemical, Cat. #02-8822)

Bilobalide (Sigma, Cat. #B 9031)

Note: This is not an all-inclusive listing of sources for reference standards. World wide there are many reputable suppliers of botanical reference standards whose product may be used. However, quality control protocol and/or government regulation may mandate further qualification of the materials for proper identity and purity.

Standard Preparation: Prepare a mixed stock standard by accurately weighing 5 mg (±0.01mg) of each ginkgolide 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.

Note: If the ginkgolides do not readily dissolve, heat the solution under hot tap water.

Dilute the stock standard to create a minimum of a three-point standard curve. Suggested standard dilutions of the stock are 1:5 and 1:10 using methanol.

Notice: Because of the variability in ELSD designs, dilutions of stock standard solution should be optimized according to detector sensitivity. However, these standard preparation instructions will provide a guideline to establish a linear dynamic range for the user's detector.

Notice: Store standards at refrigerated temperatures.

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 Acrodisk nylon, or equivalent
• Sonicator
• Centrifuge (optional)
• Column, Phenomenex Prodigy^TM C18, 4.6 x 250 mm, Cat #00G-4097-EO, or equivalent

Reagents

• Water, HPLC grade or Nanopure
• Methanol, HPLC grade
• Acetonitrile, HPLC grade
• Nitrogen, compressed gas

Sample Preparation:

Plant Material

Accurately weigh 600Š 800 mg (±0.1mg) of finely ground leaf material and place into a 50-mL round bottom flask fitted with a reflux condenser.
Reflux for 15 minutes in 5 mL of methanol:water (90:10).
Filter the sample through a Büchner funnel, saving the filtrate.
Note: An excellent alternative approach is to centrifuge the samples and decant rather than filter through a Büchner funnel.
Extract the leaf material a second time with 5 mL of methanol:water (90:10).
Filter and combine collected filtrates and washes.
Evaporate to dryness under a stream of N₂.
Reconstitute the residue with 1.0 mL of methanol.
Place the filtered sample into a HPLC vial and cap.

Extract

Accurately weigh approximately 400 mg (±0.1 mg) of extract and place into a 50-mL volumetric flask along with 40 mL of methanol:water (90:10).
Sonicate the sample for 20 minutes at 60°C or until completely dissolved.
Filter the solution and place into a HPLC vial.