Saw Palmetto Capsules
DEFINITION
Saw Palmetto Capsules contain Saw Palmetto Extract. Capsules contain NLT 22.0% of lauric acid and NMT 34.0% of the labeled amount of Saw Palmetto Extract. The ratio of the concentrations of lauric acid to caprylic acid is NLT 8.5 and NMT 17.5. The ratio of the concentrations of lauric acid to myristic acid is NLT 2.2 and NMT 2.8.
IDENTIFICATION
• A.
The retention times of the peaks for methyl caprate, methyl caproate, methyl caprylate, methyl laurate, methyl linoleate, methyl linolenate, methyl myristate, methyl oleate, methyl palmitate, methyl palmitoleate, and methyl stearate of the Sample solution correspond to those of the Standard solution, as obtained in the test for Content of Lauric Acid and the Ratios of the Concentrations of Lauric Acid to Caprylic Acid and Lauric Acid to Myristic Acid.
• B. Presence of Sterols
-Cholestanol solution:
-cholestanol in chloroform (1 in 100)
Derivatizing stock solution:
N,O-bis(trimethylsilyl)-acetamide, trimethylsilylimidazole, and trimethylchlorosilane (3:3:2)
Derivatizing solution:
Derivatizing stock solution, bis(trimethylsilyl)trifluoroacetamide, and pyridine (1:1:1)
Internal standard solution:
10 mg/mL of eicosanol and 5 mg/mL of cholesterol in chloroform
Standard stock solution:
0.75 mg/mL of USP Hexacosanol RS and 1.4 mg/mL of USP -Sitosterol RS in chloroform
-Sitosterol RS in chloroform
Standard solution:
Mix 5.0 mL of Standard stock solution with 1.0 mL of the Internal standard solution. Evaporate 0.75 mL of this solution to dryness using a stream of nitrogen. Dissolve the residue in 1.0 mL of Derivatizing solution, and allow to stand for NLT 15 min at room temperature.
System suitability stock solution A:
2 mg/mL each of tetracosanol, octacosanol, USP Hexacosanol RS, and triacontanol in chloroform
System suitability solution A:
Mix 5.0 mL of System suitability stock solution A with 1.0 mL of Internal standard solution. Evaporate 0.75 mL of this solution to dryness using a stream of nitrogen. Dissolve the residue in 1.0 mL of Derivatizing solution, and allow to stand for NLT 15 min at room temperature.
System suitability stock solution B:
2 mg/mL each of campesterol, stigmasterol, and USP -Sitosterol RS, and 0.37 mg/mL of stigmastanol
-Sitosterol RS, and 0.37 mg/mL of stigmastanol
System suitability solution B:
Mix 5.0 mL of System suitability stock solution B with 1.0 mL of Internal standard solution. Evaporate 0.75 mL of this solution to dryness using a stream of nitrogen. Dissolve the residue in 1.0 mL of Derivatizing solution, and allow to stand for NLT 15 min at room temperature.
-Cholestanol solution:
-cholestanol in chloroform (1 in 100)
Sample solution
Sample:
A number of Capsules, equivalent to 10 g of Saw Palmetto Extract
Open the Capsules, and transfer the shells and contents to a suitable container.
Transfer 5 g of the Sample to a 250-mL round-bottom flask, and evaporate in vacuum at a temperature of NMT 50
. Add 50 mL of a solution prepared by dissolving 130 mg/mL of potassium hydroxide in methanol and water (4:1). Attach a condenser, and reflux in a bath at 100 until a clear solution is obtained. Reflux for an additional 10 min, and cool by adding 50 mL of water through the condenser. Transfer to a separation funnel, rinsing the flask with a total of 50 mL of water in small portions. Extract with 80 mL of ether, shaking for 30 s, and repeat twice. [Note—If an emulsion forms, it can be eliminated by adding small quantities of methanol. ] Transfer the combined ether layers to a separation funnel, and wash with successive portions of 50 mL of water until a neutral washing is obtained. [Note—If an emulsion forms, it can be eliminated by adding small quantities of methanol. ] Pass the ether extract through filter paper containing anhydrous sodium sulfate, wash the filter with 30 mL of ether, and evaporate to dryness in vacuum. Dissolve the residue in 2.0 mL of chloroform. Extract the sterols using the following chromatographic system.
. Add 50 mL of a solution prepared by dissolving 130 mg/mL of potassium hydroxide in methanol and water (4:1). Attach a condenser, and reflux in a bath at 100 until a clear solution is obtained. Reflux for an additional 10 min, and cool by adding 50 mL of water through the condenser. Transfer to a separation funnel, rinsing the flask with a total of 50 mL of water in small portions. Extract with 80 mL of ether, shaking for 30 s, and repeat twice. [Note—If an emulsion forms, it can be eliminated by adding small quantities of methanol. ] Transfer the combined ether layers to a separation funnel, and wash with successive portions of 50 mL of water until a neutral washing is obtained. [Note—If an emulsion forms, it can be eliminated by adding small quantities of methanol. ] Pass the ether extract through filter paper containing anhydrous sodium sulfate, wash the filter with 30 mL of ether, and evaporate to dryness in vacuum. Dissolve the residue in 2.0 mL of chloroform. Extract the sterols using the following chromatographic system.
Chromatographic extraction system
Mode:
TLC
Absorbent:
Chromatographic plate coated with 0.25-mm silica gel having an application zone that was previously dipped under 3 cm of a solution prepared by dissolving 13 mg/mL of potassium hydroxide in methanol and water (49:1)
Developing solvent system:
Hexanes and ether (7:3)
Application volumes:
200 µL of chloroform solution containing Capsule residue and 20 µL of -Cholestanol solution
-Cholestanol solution
After the spots have been applied, allow the plate to dry, and heat it to 100 for 1 h before use. The plate can be stored in a desiccator containing calcium chloride until the time of use. Develop the plates until the solvent front has moved 17–19 cm. Keep the chamber temperature between 15 and 20. Dry the plate in a current of warm air, then spray with an alkaline solution of 2,7-dichlorofluorescein in alcohol (0.2 in 100). Observe the plate under 366-nm wavelength light, and identify the bands corresponding to the sterols by referring to the -cholestanol spot. Scrape off these bands and transfer them to a test tube. Add 10 mL of warm chloroform, and shake for 2 min with the aid of several glass beads. Filter the chloroform solution, wash the filter with chloroform, and evaporate the combined filtrate and washings to dryness in vacuum. Dissolve the residue with some drops of anhydrous acetone, and evaporate in vacuum. Dry the residue in an oven at 105 for 15 min. Dissolve the residue in 0.2 mL of Derivatizing solution. Use this resulting solution as the Sample solution for GC analysis.
for 1 h before use. The plate can be stored in a desiccator containing calcium chloride until the time of use. Develop the plates until the solvent front has moved 17–19 cm. Keep the chamber temperature between 15 and 20. Dry the plate in a current of warm air, then spray with an alkaline solution of 2,7-dichlorofluorescein in alcohol (0.2 in 100). Observe the plate under 366-nm wavelength light, and identify the bands corresponding to the sterols by referring to the -cholestanol spot. Scrape off these bands and transfer them to a test tube. Add 10 mL of warm chloroform, and shake for 2 min with the aid of several glass beads. Filter the chloroform solution, wash the filter with chloroform, and evaporate the combined filtrate and washings to dryness in vacuum. Dissolve the residue with some drops of anhydrous acetone, and evaporate in vacuum. Dry the residue in an oven at 105 for 15 min. Dissolve the residue in 0.2 mL of Derivatizing solution. Use this resulting solution as the Sample solution for GC analysis.
Chromatographic system
Mode:
GC
Detector:
Flame ionization
Column:
0.2-mm × 25-m capillary, coated with a 0.33-µm thickness of phase G1
Temperature
Detector:
325
Injector:
325
Column:
See the temperature program table below.
| Initial Temperature ( ) |
Temperature Ramp ( /min) |
Final Temperature ( ) |
Hold Time at Final Temperature (min) |
|---|---|---|---|
| 200 | 0 | 200 | 3 |
| 200 | 10 | 300 | 35 |
Carrier gas:
Helium
Flow rate:
0.5 mL/min
Make up gas flow:
25 mL/min
Split ratio:
1:40
Injection size:
1 µL
Injection type:
Split injection system
System suitability
Samples:
System suitability solution A and System suitability solution B
[Note—The relative retention times for tetracosanol, octacosanol, hexacosanol, and triacontanol are 0.89, 1.00, 1.15, and 1.36, respectively, System suitability solution A; and the relative retention times for cholesterol, campesterol, stigmasterol, -sitosterol, and stigmastanol are 0.85, 0.92, 0.95, 1.00, and 1.01, respectively, System suitability solution B. ]
-sitosterol, and stigmastanol are 0.85, 0.92, 0.95, 1.00, and 1.01, respectively, System suitability solution B. ]
Suitability requirements
Resolution:
NLT 2 between -sistosterol and stigmastanol, System suitability solution B
-sistosterol and stigmastanol, System suitability solution B
Column efficiency:
NLT 200,000 theoretical plates for the eicosanol peak, System suitability solution A; and NLT 150,000 theoretical plates for the cholesterol peak, System suitability solution B
Tailing factor:
NMT 2.0 for each relevant peak, System suitability solution A; and NMT 2.0 for each relevant peak, System suitability solution B
Analysis
Samples:
Standard solution and Sample solution
Identify the signals corresponding to the relevant analytes by comparison with the chromatograms obtained with System suitability solutions A and B.
Acceptance criteria:
The Sample solution exhibits peaks for campesterol, -sitosterol, and stigmasterol, identified by their retention times relative to the -sitosterol peak in the Standard solution.
-sitosterol, and stigmasterol, identified by their retention times relative to the -sitosterol peak in the Standard solution.
STRENGTH
• Content of Lauric Acid and the Ratios of the Concentrations of Lauric Acid to Caprylic Acid and Lauric Acid to Myristic Acid
Internal standard solution:
12 mg/mL of nonadecane in hexanes
Standard stock solution:
Dissolve quantities of USP Methyl Laurate RS, USP Methyl Oleate RS, USP Methyl Myristate RS, USP Methyl Palmitate RS, USP Methyl Linoleate RS, USP Methyl Caproate RS, USP Methyl Caprylate RS, USP Methyl Caprate RS, USP Methyl Palmitoleate RS, USP Methyl Stearate RS, and USP Methyl Linolenate RS in hexanes to obtain concentration of each methyl ester as given in the table below.
| Methyl Ester | Concentration (mg/mL) |
|---|---|
| Methyl laurate | 5 |
| Methyl oleate | 5 |
| Methyl myristate | 2 |
| Methyl palmitate | 2 |
| Methyl linoleate | 1 |
| Methyl caproate | 0.4 |
| Methyl caprylate | 0.4 |
| Methyl caprate | 0.4 |
| Methyl palmitoleate | 0.4 |
| Methyl stearate | 0.4 |
| Methyl linolenate | 0.4 |
Standard solution:
Add 1.0 mL of Internal standard solution to 5.0 mL of the Standard stock solution.
Sample solution:
Take a number of Capsules, equivalent to 10 g of Extract, open the Capsules, and transfer the shells and contents to a suitable container. Transfer 100 mg to a pressure-proof screw-capped vial, and add 3.0 mL of a solution of sulfuric acid in methanol (5 in 100). Heat in an oil bath at 100 for 2 h, shaking from time to time. Allow to cool, and add 1.0 mL of Internal standard solution, 10.0 mL of water, 1 g of sodium chloride, and 5 mL of hexanes. Shake well, and allow the layers to separate completely. Use the hexanes layer. [Note—Store this solution in a refrigerator until use. ]
for 2 h, shaking from time to time. Allow to cool, and add 1.0 mL of Internal standard solution, 10.0 mL of water, 1 g of sodium chloride, and 5 mL of hexanes. Shake well, and allow the layers to separate completely. Use the hexanes layer. [Note—Store this solution in a refrigerator until use. ]
Chromatographic system
Mode:
GC
Detector:
Flame ionization
Column:
0.25-mm × 30-m fused silica capillary, coated with a 0.25-µm film of phase G16
Temperature
Detector:
300
Injector:
250
Column:
See the temperature program table below.
| Initial Temperature ( ) |
Temperature Ramp ( /min) |
Final Temperature ( ) |
Hold Time at Final Temperature (min) |
|---|---|---|---|
| 120 | 0 | 120 | 3 |
| 120 | 50 | 220 | 12 |
Carrier gas:
Helium
Flow rate:
1 mL/min
Injection size:
1 µL
System suitability
Sample:
Standard solution
[Note—The relative retention times for methyl caproate, methyl caprylate, methyl caprate, methyl laurate, nonadecane (internal standard), methyl myristate, methyl palmitate, methyl palmitoleate, methyl stearate, methyl oleate, methyl linoleate, and methyl linolenate are about 0.39, 0.56, 0.76, 0.94, 1.0, 1.1, 1.3, 1.35, 1.65, 1.7, 1.8, and 2.0, respectively. ]
Suitability requirements
Resolution:
NLT 1.5 between the methyl stearate and methyl oleate peaks
Tailing factor:
NMT 2.0 for each of the methyl ester peaks
Relative standard deviation:
NMT 5.0% for each of the methyl ester peaks
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentages of lauric acid, myristic acid, and caprylic acid in the labeled amount of Saw Palmetto Extract in the portion of Capsules taken:
Result = (RU/RS) × (CS × V/W) × (Mr1/Mr2) × AW/LE × 100
| RU | = | = ratio of the response of the relevant methyl ester peak and the internal standard peak from the Sample solution |
| RS | = | = ratio of the response of the relevant methyl ester peak and the internal standard peak from the Standard solution |
| CS | = | = concentration of the respective methyl ester in the Standard stock solution (mg/mL) |
| V | = | = volume of the Standard stock solution taken to prepare the Standard solution (5.0 mL) |
| W | = | = weight of sample used to prepare the Sample solution (mg) |
| Mr1 | = | = molecular weight of the relevant fatty acid |
| Mr2 | = | = molecular weight of the methyl ester of the relevant fatty acid |
| AW | = | = average weight of the Capsule contents (mg/Capsule) |
| LE | = | = labeled amount of Saw Palmetto Extract per Capsule (mg/Capsule) |
Using these percentages, calculate the individual ratios of the concentration of lauric acid to caprylic acid and of lauric acid to myristic acid in the portion of Capsules taken.
Acceptance criteria:
22.0%–34.0% of lauric acid in the labeled amount of Saw Palmetto Extract. The ratio of lauric acid to caprylic acid is 8.5–17.5. The ratio of lauric acid to myristic acid is 2.2–2.8.
PERFORMANCE TESTS
• Disintegration and Dissolution of Dietary Supplements 
2040
:
Meet the requirements for Rupture Test for Soft Shell Capsules
2040:
Meet the requirements for Rupture Test for Soft Shell Capsules
• Weight Variation of Dietary Supplements 2091:
Meet the requirements
2091:
Meet the requirements
CONTAMINANTS
• Microbial Enumeration Tests 2021:
The total bacterial count does not exceed 104 cfu/g, the total combined molds and yeasts count does not exceed 1000 cfu/g, the coliform count does not exceed 100 cfu/g, and the count for enterobacteria does not exceed 100 cfu/g.
2021:
The total bacterial count does not exceed 104 cfu/g, the total combined molds and yeasts count does not exceed 1000 cfu/g, the coliform count does not exceed 100 cfu/g, and the count for enterobacteria does not exceed 100 cfu/g.
• Microbial Procedures for Absence of Specified Microorganisms 2022:
Capsules meet the requirements of the tests for absence of Salmonella species, Escherichia coli, and Staphylococcus aureus.
2022:
Capsules meet the requirements of the tests for absence of Salmonella species, Escherichia coli, and Staphylococcus aureus.
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight, light-resistant containers.
• Labeling:
The label states the Latin binomial and, following the official name, the name of article from which the Capsules were prepared. Label it to indicate the amount of Extract in mg/Capsule.
• USP Reference Standards 11
11
USP Hexacosanol RS 
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USP Methyl Caprate RS
USP Methyl Caproate RS
USP Methyl Caprylate RS
USP Methyl Laurate RS
USP Methyl Linoleate RS
USP Methyl Linolenate RS
USP Methyl Myristate RS
USP Methyl Oleate RS
USP Methyl Palmitate RS
USP Methyl Palmitoleate RS
USP Methyl Stearate RS
USP -Sitosterol RS
-Sitosterol RS ') + '&img=../../images/v35300/c11rs1496.gif',600,500);)
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Maged H. Sharaf, Ph.D.
Principal Scientific Liaison 1-301-816-8318 |
(DS2010) Monographs - Dietary Supplements |
| 2021 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| 2022 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 1437
Pharmacopeial Forum: Volume No. 36(1) Page 163