Extraction column— Use a solid-phase extraction column that contains benzenesulfonylpropyl bonded to silica gel in the hydrogen form having a sorbent mass to column volume ratio of 1g per 6mL,or equivalent.Condition the column prior to use by washing with 10mLof methanol and with 10mLof water.[NOTE—Do not allow the column to dry.]

Test solution— Mix 1mLof Fluidextract with 5mLof water,and transfer to the Extraction column.Allow to drain,and discard the eluate.Wash the column with 10mLof water and 10mLof methanol,discarding the eluates.Elute the amino acid fraction with 3mLof ammonium hydroxide solution in methanol (7in 100),and collect the eluate.

Standard solution: 0.5mg of USPS-Allyl-L-Cysteine RSper mL.

Application volume: 5µL.

Developing solvent system: a mixture of ethyl acetate,methanol,water,acetone,and glacial acetic acid (10:4:3:3:1).

Procedure— Proceed as directed in the chapter.Spray with iodoplatinate TS,and examine the plate:the chromatogram of the Test solutionexhibits,among several yellow spots on the purple plate,a yellow spot at an RFvalue of about 0.4corresponding to that of the yellow spot obtained in the chromatogram of the Standard solution(presence of S-allyl-L-cysteine).

Mobile phase— Transfer 15.8g of sodium citrate dihydrate to a 1000-mLvolumetric flask containing 250mLof water,carefully add 10.5mLof hydrochloric acid,and mix.Using a pHmeter,adjust with 6Nsodium hydroxide to a pHof 4.0.Dilute with water to volume,and mix.

Derivatizing reagent— Dissolve 0.8g of o-phthalaldehyde in 2mLof 2-mercaptoethanol,add to a solution containing 24.70g of boric acid and 22.35g of potassium hydroxide in 1000mL,and mix.

Reactivating solution— Prepare 0.2Nsodium hydroxide by dissolving 0.8g of sodium hydroxide in 100mLof water.

Standard solution— Dissolve an accurately weighed quantity of USPS-Allyl-L-Cysteine RSin water,and dilute quantitatively,and stepwise if necessary,with water to obtain a solution having a known concentration of about 0.01mg per mL.

Test solution— Transfer about 2.0g of Fluidextract,accurately weighed,to a 100-mLvolumetric flask,dilute with trichloroacetic acid solution (5in 100)to volume,and mix.Centrifuge for 5minutes,and filter the supernatant.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 4.6-mm ×12-cm column that contains packing L17.The column temperature is maintained at 40.The Mobile phaseand the Reactivating solutionare pumped separately,each at the rate of about 0.4mLper minute,by pumps connected to the opposing arms of a tee.The outlet of the tee is connected to the injector and the chromatographic column.The outlet of the column is attached to a tee,the opposing arm of which is attached to a tube from which the Derivatizing reagentis constantly pumped through the system at a rate of about 0.6mLper minute.The outlet of the tee is connected to a 0.5-mm ×2.0-m postcolumn polytef reaction coil maintained at 40.The outlet of the reaction coil is connected to a fluorometric detector set at an excitation wavelength of 340nm and an emission wavelength of 455nm.The system is programmed to deliver the Mobile phasefor 10minutes,the Reactivating solutionfor the next 6minutes,and the Mobile phasefor the 24minutes remaining before the next injection.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the capacity factor,k¢,is between 2.5and 4.5;the tailing factor is not more than 2.0;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the heights of the major peaks.Calculate the quantity,in mg,of S-allyl-L-cysteine (C6H11SN)in the portion of Fluidextract taken by the formula: in which Cis the concentration,in mg per mL,of USPS-Allyl-L-Cysteine RSin the Standard solution;Wis the weight,in g,of Fluidextract taken to prepare the Test solution;and rUand rSare the peak heights for S-allyl-L-cysteine obtained from the Test solutionand the Standard solution,respectively:not less than 0.05%is found,calculated on the dried basis.