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Assay Title: Determination of sterols in Serenoa repens (saw palmetto or sabel) by gas chromatography.
Scope: This assay can be used to determine stigmasterol, campesterol, brassicasterol, and ß-sitosterol in saw palmetto fruit, oil extract, and blended powders. Determination is performed using gas chromatography after hydrolysis, saponification, and derivatization.
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. For more safety information, see http://hazard.com/msds/.
Background: Saw palmetto contains several principles thought to have physiological activity, including phyto-sterols such as ß-sitosterol, ß-sitosterol 3-O-ß-D-glucoside, campesterol, stigmasterol. Furthermore, saw palmetto contains fatty acids and 1 to 2% essential oils. Purified ethanolic or CO2 extracts of saw palmetto usually contain 70 - 80% free fatty acids and sterols. This oil is commonly blended with excipient to form a dry powder at 30% free fatty acids concentration.
Standards: 5-a-cholestane, Matreya, Inc., Pleasant Gap, PA, Cat. No. 1115 (or Sigma, St. Louis, MO, Cat. No. C8003)
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.
Apparatus
Reagents:
Reagent Solution Preparation:
Hydrochloric Acid, 6N
Prepare by carefully pouring one part by volume concentrated HCl into one part by volume deionized water. Perform operation in a fume hood.
Hydrochloric Acid, 1N
To about 400 mL DI water, carefully add 83 mL of 6N HCl. Bring up to a final volume of 500 mL using deionized water. Perform operation in a fume hood.
Phenolphthalein Indicator Solution
Weigh approximately 0.05 g phenolphthalein into a 100-mL beaker. Add 50 mL of ethanol and mix to dissolve. Pour solution into a 125-mL screw cap bottle and add 50 mL of deionized water. Cap and mix. Store at room temperature.
Dichlorodimethylsilane solution
Prepare a 5% dichlorodimethylsilane solution in a 500-mL volumetric flask. To 50 mL dichlorodimethylsilane, add about 300 mL toluene. Mix and bring up to 500 mL with toluene. Store at room temperature in an oven dried glass bottle containing 3Å molecular sieves.
Reference Standards:
5-a-Cholestane Stock Standard Solution
Accurately weigh out 500 mg of 5-a-cholestane reference standard into a 500-mL volumetric flask. Record the weight to the nearest 0.1 mg.
Add approximately 250 mL heptane and invert the flask several times until all of the cholestane is dissolved. Bring to volume.
Dilute 1:10, 1:50 and 1:100 in heptane to create a linearity curve.
Glassware Preparation (Silanization)
Dry glassware overnight in an oven at 110°C. In a fume hood, soak cooled glassware in dichlorodimethylsilane solution. After 1-2 hours, wash the glassware with methanol. Allow glassware to air dry in the hood.
Note: Addition of 3Å molecular sieves to the methanol used for washing glassware is recommended as a drying agent.
Sample Preparation
Sample Hydrolysis
Weigh into a 500-mL Erlenmeyer flask the following quantity of accurately weighed sample.
Saw palmetto oil 0.1 g
Saw palmetto powder 0.5 g
Saw palmetto fruit 2.0 g
Add enough ethanol to wet the sample and break up any lumps with a glass rod. Rinse the glass rod off into the flask with ethanol.
Add 15 mL 6N HCl and heat in a controlled temperature bath at 100°C (BP for water) for 30 minutes, swirling occasionally to break up any lumps.
Remove from bath and cool to room temperature. While swirling the flask under a stream of cold tap water, slowly add 15 g of KOH pellets such that the solution does not spatter or boil. Add 20 mL ethanol, a boiling stone, and enough deionized water (generally 75 - 150 mL) to dissolve the potassium salt. Gently boil the solution for 45 minutes in a fume hood. Remove flask from heat and immerse the flask into cool tap water until cool to the touch. Alternatively, the solution may be gently refluxed on a steam bath using a round-bottom flask and condenser.
Remove flask from heat and immerse the flask into cool tap water until cool to the touch.
Sample Extraction
Transfer the flask contents to a 500-mL separatory funnel. Filtering through cotton placed in a glass funnel will improve solution clarity. Rinse the flask 3x using 10 mL of ethanol each time.
Rinse the flask with about 50 mL of petroleum ether and add the rinse to the separatory funnel. Place the flask under the separatory funnel. Shake the funnel for 60 seconds, venting as needed.
Allow the layers to separate. Drain the lower aqueous layer into the flask and drain the top layer into a second 500-mL separatory funnel containing about 50 mL deionized water.
Rinse the first separatory funnel with about 5 mL of petroleum ether and pour the ether into the second funnel.
Repeat the extraction with petroleum ether 2x and combine extracts into final separatory funnel.
Add 200 mL deionized water to the separatory funnel containing the ether extracts. Slowly rock the funnel for 15 seconds to gently mix the water and the layers. Do not mix vigorously because an emulsion will form.
Allow the layers to separate. Drain the lower layer into the Erlenmeyer flask. Do not drain any emulsion into the flask. Dispose of the water layer in the flask.
Note: An emulsion will usually break with the addition of a small volume of ethanol and a few swirls.
Repeat the water washing of the ether extract 4x . Add 200 mL deionized water to the funnel and shake moderately for 60 seconds. Allow the layers to separate. Drain the lower layer into the Erlenmeyer flask, testing a portion of it for alkalinity using phenolphthalein.
Repeat 200 mL water wash until the lower layer no longer turns the phenolphthalein solution pink.
Drain the ether layer into a 400-mL beaker through anhydrous sodium sulfate supported by a glass wool plug in a funnel. Rinse the separatory funnel with approximately 25 mL of petroleum ether and drain through the sodium sulfate into the 400-mL beaker. Repeat the ether rinse.
Place the 400-mL beaker on a steam table, set on its highest temperature setting (temp may be adjusted to avoid boiling over), to evaporate all the ether. The residue in the beaker is unsaponified material and contains any sterols present in the sample.
Sample Derivitization:
Caution! Perform these steps in a fume hood.
Add 5 mL of cool heptane to the residue. If necessary, sonicate to aid in mixing.
Transfer 2 mL of the solution into a silanized screw cap test tube. Add 1.0 mL of DMF to the tube.
Add 0.2 mL of HMDS to the tube using a 2.5 mL gastight syringe.
Add 0.1 mL of TMCS to the tube using a gastight syringe.
Cap the tube with a Teflon-lined cap and vortex for 30 seconds.
Let the tube stand for 15 minutes, vortexing every few minutes.
Add 6 mL of deionized water to each test tube.
Recap and vortex the tube for 30 seconds.
Allow the layers to separate. The top heptane layer should not be opaque.
If operating the GC under splitless conditions, dilute the sample by mixing 100 µL of the heptane layer with 900 µL of heptane in an autosampler vial to achieve a 1:10 dilution.
Chromatographic Conditions:
| Injector Temperature: | 345°C |
| Detector Temperature: | 355°C |
| Oven Temperature: | Initial temperature at 200°C for 1 minute. Go to 340°C at 15°C/minute. Hold for 10 minutes. |
| Column: | Restek Rtx®-5, 5% diphenyl&Mac220; 95% dimethyl polysiloxane, 60m x 0.25mm, 0.25 µm film thickness, or equivalent |
| Gas Flows Measured at 200°C: | |
| Hydrogen carrier | 2.2 mL/min |
| Hydrogen carrier + Nitrogen makeup | 30 mL/min |
| Zero air | 420 mL/min |
| Hydrogen carrier + H2 fuel | 32 mL/min |
| Septum purge (at septum purge vent) | 4.0 mL/min |
| Splitless injection | |
| Injection volume | 1mL |
Note: Injection may be split at the split/splitless inlet vent. Split flow of 112 mL/min is suggested. Split injection may result in higher variability of results.
Retention Times (approximate)
| 5-a cholestane | 6.2 minutes |
| brassicasterol | 11.2 minutes |
| campesterol | 11.5 minutes |
| stigmasterol | 11.7 minutes |
| ß-sitosterol | 12.0 minutes |
Calculations:
Because of structural similarities between sterols, FID response is approximately the same for all. Quantify brassicasterol, campesterol, stigmasterol, and ß-sitosterol against the external standard 5-a-cholestane.
% w/w total sterols = C x FV x D x 100%
W(1000µg/mg)
Where:
C = The concentration of total sterols determined by linear regression analysis of 5-a-cholestane, µg/mL
FV = The final volume of the sample preparation Procedure, mL
d = Dilution of the sample, if needed
w = The weight of the sample in µg
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109.001
Filtering of sample preparation solution through cotton fiber added to help clarify solution and prevent emulsions. Alternative approach to sample heating, by reflux procedure, added.