A: Transfer an amount of pulverized Tablets,equivalent to about 30mg of alliin,to a 100-mLvolumetric flask.Add 70mLof a mixture of methanol and water (1:1),shake,and centrifuge.Concentrate to a small volume (about 5mL),using a rotary evaporator.Continue as directed in Identificationtest Aunder Garlic.

B: The retention times of the alliin diastereomer peaks in the chromatogram of the Test solutioncorrespond to those in the chromatogram of the Standard solution,as obtained in the test for Content of alliin.

Apparatus 2: 100rpm.

Time: 60minutes for the Buffer stage.

Mobile phase,Crude alliinase solution,Blank solution,and Chromatographic system— Proceed as directed in the test for Content of potential allicin.

Standard solution— Dissolve an accurately weighed quantity of USP Alliin RSin water,and dilute quantitatively,and stepwise if necessary,with water to obtain a solution having a known concentration of about 50µg per mL.Transfer 1.0mLof this solution to a 5-mLvolumetric flask containing 100µLof Crude alliinase solution,mix,and allow to stand for 5minutes at room temperature.Dilute with water to volume,and pass through a membrane filter having a 0.45-µm or finer porosity.

Test solution— Transfer 1.0mLof the solution under test to a test tube containing 50µLof 0.21Mcarboxymethoxylamine hemihydrochloride.[NOTE—The solution must be transferred immediately upon removal from the dissolution vessel in order to inhibit the alliinase enzyme.]

Procedure— [NOTE—Do not perform the allicin determination in the Acid stage.]Determine the amount of allicin released by injecting equal volumes (about 100µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the allicin peaks.Calculate the amount,in µg,of allicin released in the Buffer stageby the formula: in which Cis the concentration,in µg per mL,of USP Alliin RSin the Standard solution;162.26is the molecular weight of allicin;354.42is twice the molecular weight of alliin;and rUand rSare the peak responses for allicin obtained from the Test solutionand the Standard solution,respectively.[NOTE—Qis the percentage of the labeled amount of potential allicin released only in the Buffer stage.]

0.045M Phosphate buffer,0.05M Phosphate buffer,0.01M Carboxymethoxylamine hemihydrochloride solution,Derivatization reagent,Mobile phase,Standard solution,and Chromatographic system— Proceed as directed in the test for Content of alliinunder Garlic.

Test solution— Pulverize an accurately counted number of Tablets,equivalent to about 50mg of alliin,with a mortar and pestle.Transfer an accurately weighed amount of the powder,equivalent to 5mg of alliin,to a 100-mLvolumetric flask,add about 70mLof 0.01M Carboxymethoxylamine hemihydrochloride solution,and shake for 1minute.Dilute with 0.01M Carboxymethoxylamine hemihydrochloride solutionto volume,and mix.Using a volumetric syringe,transfer 0.1mLof this solution to a septum-capped vial,add 0.5mLof the Derivatization reagent,and mix.Allow a reaction time of not less than 2minutes before injection into the chromatograph.

Procedure— Proceed as directed in the test for Content of alliinunder Garlic.Calculate the quantity,in µg,of alliin in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Alliin RSin the Standard solution;and rUand rsare the sums of the peak responses for the alliin diastereomers obtained from the Test solutionand the Standard solution,respectively.

0.01M Carboxymethoxylamine hemihydrochloride solution— Prepare as directed in the test for Content of alliinunder Garlic.

Mobile phase— Prepare a filtered and degassed mixture of methanol and water (60:40).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Crude alliinase solution— Homogenize about 5g of raw garlic cloves with 25mLof water.Filter,and extract three times with 50mLof tert-butyl methyl ether.Discard the organic phase,and remove the residual solvent from the aqueous phase by rotary evaporation in vacuum for 5minutes.Filter,and store frozen in small vials.[NOTE—This solution is stable for 6months when stored as directed.]Thaw at room temperature just before use.

Blank solution— Dilute 100µLof Crude alliinase solutionwith water to 1mL.

Standard solution— Dissolve an accurately weighed quantity of USP Alliin RSin water,and dilute quantitatively,and stepwise if necessary,with water to obtain a solution having a known concentration of about 50µg per mL.Transfer 1.0mLof this solution to a 5-mLvolumetric flask containing 100µLof Crude alliinase solution,mix,and allow to stand for 5minutes at room temperature.Dilute to volume with water,and pass through a filter having a 0.45-µm or finer porosity.

Test solution— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed quantity of the powder,equivalent to about 5mg of potential allicin,to a 200-mLvolumetric flask,add 25mLof water,and mix.Incubate at room temperature for exactly 30minutes.Stop the enzymatic reaction by diluting with 0.01M Carboxymethoxylamine hemihydrochloride solutionto volume,and mix.Centrifuge a portion of this solution,transfer 1.0mLof the supernatant to a 5-mLvolumetric flask,dilute with water to volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 240-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 1.0mLper minute.Chromatograph the Standard solutionand the Blank solution,and record the peak responses as directed for Procedure:the allicin peak is identified by comparing the chromatograms of the Blank solutionand the Standard solution;and the relative standard deviation for replicate injections is not more than 2.0%.Chromatograph the Test solution,and record the peak responses as directed for Procedure:the resolution,R,between the allicin peak and the preceding peak at a relative retention time of 0.80(allyl methyl thiosulfinates)is not less than 2.0.

Procedure— Inject equal volumes (about 100µL)of the Standard solution,the Blank solution,and the Test solutioninto the chromatograph,record the chromatograms,and measure the peak responses for allicin.Calculate the amount of potential allicin,in µg,in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Alliin RSin the Standard solution;162.26is the molecular weight of allicin;354.42is twice the molecular weight of alliin;and rUand rSare the responses for the allicin peaks,corrected by the response of the blank,obtained from the Test solutionand the Standard solution,respectively.

0.045M Phosphate buffer,0.05M Phosphate buffer,0.01M Carboxymethoxylamine hemihydrochloride solution,Derivatization reagent,Mobile phase,Standard solution,and Chromatographic system— Proceed as directed in the test for Content of alliinunder Garlic.

Test solution— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed quantity of the powder,equivalent to about 5mg of alliin,to a 100-mLvolumetric flask,add 25mLof water,and mix.Incubate at room temperature for exactly 5minutes.Stop the enzymatic reaction by diluting with 0.01M Carboxymethoxylamine hemihydrochloride solutionto volume,and mix.Centrifuge a portion of this solution,and,using a volumetric syringe,transfer 0.1mLof the supernatant to a septum-capped vial.Add 0.5mLof the Derivatization reagent,and mix.Allow a reaction time of not less than 2minutes before injection into the chromatograph.

Procedure— Proceed as directed in the test for Content of alliinunder Garlic.The area of the alliin peak obtained from the Test solutionis not more than 1%of the area of the alliin peak obtained from the Standard solution.