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

Solvent: benzyl alcohol.

Internal standard solution,Standard solution,and Chromatographic system— Prepare as directed forContent of fatty acidsunderSaw Palmetto.

Test solution— Transfer about 100mg of Extract,accurately weighed,to a pressure-proof,screw-capped vial,and add 3.0mLof a solution of sulfuric acid in methanol (5in 100).Heat at 100in an oil bath for 2hours,shaking from time to time.Allow to cool,and add 1.0mLofInternal standard solution,10.0mLof water,1g of sodium chloride,and 5mLof 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 forContent of fatty acidsunderSaw Palmetto.Calculate the percentage of each fatty acid in the portion of Extract taken by the formula: in which Wis 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 ofDerivatizing solution 1,bis(trimethylsilyl)-trifluoroacetamide,and pyridine (1:1:1).

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

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

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

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

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

Chromatographic system(see Chromatography á621ñ)— 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 200for 3minutes,then to increase the temperature from 200to 300at a rate of 10per minute,and to hold the final temperature for 35minutes.The injection port and detector are both maintained at 325.The carrier gas is helium,flowing at a rate of about of 0.5mLper minute,and the split ratio is 1:40.The make up gas flow is 25mLper minute.Chromatograph theSystem suitability solution 1as directed forProcedure:the relative retention times are about 0.89,1.00,1.15,and 1.36for tetracosanol,hexacosanol,octacosanol,and triacontanol,respectively;the column efficiency is not less than 200,000theoretical plates for the eicosanol peak;and the tailing factor for each relevant peak is not more than 2.0.Chromatograph theSystem suitability solution 2as directed forProcedure:the relative retention times are about 0.85,0.92,0.95,1.00,and 1.01for cholesterol,campesterol,stigmasterol,b-sitosterol,and stigmastanol,respectively;the resolution,R,between b-sistosterol and stigmastanol is not less than 2;the column efficiency is not less than 150,000theoretical 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 1µL)of theStandard solution and theTest 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 1and 2.Separately calculate the percentages of tetracosanol,hexacosanol,octacosanol,and triacontanol,respectively,in the portion of Extract taken by the formula: in whichCis the concentration of hexacosanol,in mg per mL,in theStandard solution;Wis the weight,in mg,of the Extract taken to prepare theTest solution;RUis the ratio of the appropriate long-chain alcohol peak to the eicosanol internal standard in the chromatogram of theTest solution;andRSis the ratio of the hexacosanol peak to the eicosanol internal standard in the chromatogram of theStandard solution.Calculate the total content of long-chain alcohols as a percentage by adding the individual percentages.