Fatty Acid	Minimum Ratio	Maximum Ratio
Capric	9.0	16
Caproic	8.5	24
Caprylic	8.5	17.5
Linoleic	5.0	16
Linolenic	31.5	55
Myristic	2.2	2.8
Oleic	0.60	1.15
Palmitic	2.8	3.9
Stearic	14	26

Internal standard solution, Standard solution, and Chromatographic system— Prepare as directed for Content of fatty acids under Saw Palmetto.

Test solution— Transfer about 100 mg of Extract, accurately weighed, to a pressure-proof, screw-capped vial, and add 3.0 mL of a solution of sulfuric acid in methanol (5 in 100). Heat at 100 in an oil bath for 2 hours, 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, allow the layers to separate completely, and use the hexanes layer. [note—Store in a refrigerator until ready to use.]

Procedure— Proceed as directed for Content of fatty acids under Saw Palmetto. Calculate the percentage of each fatty acid in the portion of Extract taken by the formula: in which W is the weight, in mg, of Extract taken to prepare the Test solution; and the other terms are as defined therein.

Derivatizing solution 1: a mixture of N,O-bis(trimethylsilyl)-acetamide, trimethylchlorosilane, and trimethylsilylimidazole (3:2:3).

Derivatizing solution 2: a mixture of Derivatizing solution 1, bis(trimethylsilyl)-trifluoroacetamide, and pyridine (1:1:1).

Internal standard solution— Prepare a solution containing 10 mg per mL of eicosanol and 5 mg per mL of cholesterol in chloroform.

Standard solution— Prepare a solution of USP Hexacosanol RS and USP -Sitosterol RS in chloroform having a known concentration of about 0.75 mg and 1.4 mg per mL, respectively. Mix 5.0 mL of this solution with 1.0 mL of the Internal standard solution. Evaporate about 0.75 mL of this solution to dryness using a stream of nitrogen. Dissolve the residue in 1.0 mL of Derivatizing solution 2, and allow to stand for not less than 15 minutes at room temperature.

Test solution— Transfer an accurately weighed quantity of about 3.35 g of extract into a 50-mL, round-bottomed flask. Add 1.0 mL of Internal standard solution, and evaporate under vacuum at a temperature not exceeding 50. Add 20 mL of a solution prepared by dissolving 130 g of potassium hydroxide in 200 mL of water and diluting to 1000 mL with methanol. Attach a condenser, and reflux in a bath at 100 for 2 hours. Quantitatively transfer this solution to a 25-mL volumetric flask, and dilute with water to volume. Transfer a 3-mL portion to a cartridge containing diatomaceous earth capable of holding 3 mL of aqueous phase. [note—A suitable cartridge is Extrelut NT3, or an equivalent cartridge.] Absorb the solution into the column under vacuum for 20 minutes until the column is not cold. Elute the analytes from the column with 90 mL of methylene chloride, and evaporate the eluate to dryness. Dissolve the residue in 1.0 mL of Derivatizing solution 2, and allow to stand for not less than 15 minutes at room temperature.

System suitability solution 1— Prepare a solution containing about 2 mg per mL each of tetracosanol, octacosanol, USP Hexacosanol RS, and triacontanol in chloroform. Mix 5.0 mL of this solution with 1.0 mL of Internal standard solution. Evaporate about 0.75 mL of this solution to dryness using a stream of nitrogen. Dissolve the residue in 1.0 mL of Derivatizing solution 2, and allow to stand for not less than 15 minutes at room temperature.

System suitability solution 2— Prepare a solution containing about 2 mg per mL each of campesterol, stigmasterol, and USP -Sitosterol RS, and 0.37 mg per mL of stigmastanol. Mix 5.0 mL of this solution with 1.0 mL of the Internal standard solution. Evaporate about 0.75 mL of this solution to dryness using a stream of nitrogen. Dissolve the residue in 1.0 mL of Derivatizing solution 2, and allow to stand for not less than 15 minutes at room temperature.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector, a split injection system, and a 0.2-mm × 25-m capillary column coated with a 0.33-µm thickness of phase G1. The chromatograph is programmed to maintain the column temperature at 200 for 3 minutes, then to increase the temperature from 200 to 300 at a rate of 10 per minute, and to hold the final temperature for 35 minutes. The injection port and detector are both maintained at 325. The carrier gas is helium, flowing at a rate of about of 0.5 mL per minute, and the split ratio is 1:40. The make up gas flow is 25 mL per minute. Chromatograph the System suitability solution 1 as directed for Procedure: the relative retention times are about 0.89, 1.00, 1.15, and 1.36 for tetracosanol, hexacosanol, octacosanol, and triacontanol, respectively; the column efficiency is not less than 200,000 theoretical plates for the eicosanol peak; and the tailing factor for each relevant peak is not more than 2.0. Chromatograph the System suitability solution 2 as directed for Procedure: the relative retention times are about 0.85, 0.92, 0.95, 1.00, and 1.01 for cholesterol, campesterol, stigmasterol, -sitosterol, and stigmastanol, respectively; the resolution, R, between -sistosterol and stigmastanol is not less than 2; the column efficiency is not less than 150,000 theoretical plates for the cholesterol peak; and the tailing factor for each relevant peak is not more than 2.0.

Procedure— Separately inject equal volumes (about 1 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Identify the signals corresponding to the relevant analytes by comparison with the chromatograms obtained with System suitability solutions 1 and 2. Separately calculate the percentages of tetracosanol, hexacosanol, octacosanol, and triacontanol, respectively, in the portion of Extract taken by the formula: in which C is the concentration of hexacosanol, in mg per mL, in the Standard solution; W is the weight, in mg, of the Extract taken to prepare the Test solution; RU is the ratio of the appropriate long-chain alcohol peak to the eicosanol internal standard in the chromatogram of the Test solution; and RS is the ratio of the hexacosanol peak to the eicosanol internal standard in the chromatogram of the Standard solution. Calculate the total content of long-chain alcohols as a percentage by adding the individual percentages.