Macroscopic— Subglobular compound bulbs,3to 5cm in width,consisting of 8to 20cloves,the whole surrounded by 2to 5layers of white scale leaves attached to a flattened,circular base;cloves ovoid and 3-to 4-sided,summit acute,narrowed into a threadlike portion of fiber base,truncate,each clove covered with a white scale leaf and a pinkish white epidermis,easily separated from the solid portion,consisting of two flaky scale leaves and two yellowish green conduplicate foliage leaves.

Microscopic— The protective leaf contains an epidermis enclosing a mesophyll free from chlorophyll.The outer epidermis consists of lignified sclereid cells of thick,pitted walls,elongated,covered with thin cuticle,long fibers up to 500µm in length and 30µm in width.

A: Thin-Layer Chromatographic Identification Test á201ñ—

Test solution— Cut a freeze-dried garlic bulb into small pieces,transfer about 1g of the cut pieces to an extractor,and extract with two 20-mLportions of a mixture of methanol and water (1:1),combining the extracts.Concentrate to a small volume (about 5mL),using a rotary evaporator.

Standard solution A: 0.5mg of USPL-Methionine RSper mL.

Standard solution B: 0.5mg of USP Alliin RSper mL,in a mixture of methanol and water (1:1).

Application volume: 20µL,applied separately as 10-mm bands.

Developing solvent system: a mixture of butyl alcohol,n-propyl alcohol,glacial acetic acid,and water (3:1:1:1).

Procedure— Proceed as directed in the chapter.Spray with a 0.2in 100solution of ninhydrin in a mixture of butyl alcohol and 2Nacetic acid (95:5),heat at 100to 105for about 10minutes,and immediately examine the plate.The chromatogram of the Test solutionshows many orange and pinkish violet zones:a violet zone having an RFvalue of about 0.89;a pink zone having an RFvalue of about 0.5and corresponding in color and RFvalue to that obtained from the chromatogram of Standard solution A;a pinkish zone having an RFvalue of about 0.43;a strong orange zone having an RFvalue of about 0.38;a pinkish violet zone having an RFvalue of about 0.3and corresponding in color and RFvalue to that obtained from the chromatogram of Standard solution B;and additional pinkish orange zones situated very close to each other just below the zone attributed to alliin in the chromatogram of Standard solution B.

B: Transfer about 10g of garlic bulbs that have been cut into small pieces to a suitable flask.Add 10mLof 1Nsodium hydroxide and 10mLof water,heat the flask in boiling water for 10minutes,cool,and filter.Add a few drops of freshly prepared sodium nitroferricyanide TSto 2mLof the filtrate:appearance of a red or orange-red color indicates the presence of sulfur-containing compounds in the test specimen.

C: The retention time of the major peak in the chromatogram of the Test solutioncorresponds to that in the chromatogram of the Standard solution,as obtained in the test for Content of alliin.

D: Thin-Layer Chromatographic Identification Test á201ñ—

Extraction column— Use a 1-cm ×5-cm solid-phase extraction column that contains styrene-divinylbenzene copolymer packing with a 75-to 150-µm diameter and a 400-to 600-Åpore size.Condition column prior to use by washing with 50mLof methanol and with 50mLof a mixture of water and methanol (7:3).[NOTE—Do not allow the column to dry.]

Test solution— Transfer about 10g of freshly peeled garlic clove to a 37-mLhomogenizing cup,and homogenize with 25mLof methanol at the highest speed for 1minute.Centrifuge the mixture,and decant the supernatant to a flask.Add 70mLof water,and mix.Transfer to Extraction column,allow to drain,and discard to eluate.Wash the column with 50mLof a mixture of water and methanol (7:3),allow the solvent mixture to drain,and discard the eluate.Finally,elute the crude saponin fraction on the column with 20mLof methanol,and collect the eluate.Evaporate the solvent to dryness.Dissolve the residue in 4mLof a mixture of 8%sulfuric acid and alcohol (1:1),transfer the solution to a screw-capped test tube,and heat on a boiling water bath for 5hours.Cool the test tube,add 20mLof water,and transfer the solution to a freshly conditioned Extraction column,allow to drain,and discard the eluate.Wash the column with 30mLof a mixture of methanol and water (7:3),and discard the eluate.Finally,elute the column with 50mLof methanol.Collect the eluate,evaporate it to dryness,and dissolve the residue in 0.5mLof methanol.

Standard solution: 0.2mg each of USPb-Chlorogenin RSand USP Agigenin RSper mL,in methanol.

Developing solvent system: a mixture of methylene chloride and methanol (15:2).

Application volume: 20µL,as 7-mm bands.

Spray reagent— Dissolve 0.5mLof 4-methoxybenzaldehyde and 0.5mLof sulfuric acid in alcohol to make 10mL.

Procedure— Proceed as directed in the chapter.Spray the plate with Spray reagent,heat the plate at about 100to 105for about 5minutes,and examine the plate:the chromatogram of the Test solutionexhibits,among several yellowish and grayish green spots,a grayish green spot at an RFvalue of about 0.4,corresponding to the grayish green spot due to b-chlorogenin obtained from the Standard solution.The chromatogram of the Test solutionexhibits no spot at an RFvalue of about 0.2corresponding to agigenin obtained from the Standard solution.

0.045M Phosphate buffer— Dissolve 1.24g of monobasic sodium phosphate in 100mLof water,adjust with 0.2Msodium hydroxide to a pHof 7.1,dilute with water to 200.0mL,and mix.

0.05M Phosphate buffer— Dissolve 1.38g of monobasic sodium phosphate in 100mLof water,adjust with 0.2Msodium hydroxide to a pHof 9.5,dilute with water to 200.0mL,and mix.

0.01M Carboxymethoxylamine hemihydrochloride solution— Dissolve 109mg of carboxymethoxylamine hemihydrochloride in 100.0mLof water.

Derivatization reagent— Dissolve 140mg of o-phthaldialdehyde in 5mLof methanol in a 50-mLvolumetric flask,add 100µLof t-butylthiol,dilute with 0.05M Phosphate bufferto volume,and mix.[NOTE—This reagent may occasionally become opaque during preparation.Store at room temperature,and use within one week.]

Mobile phase— Prepare a mixture of 0.045M Phosphate buffer,acetonitrile,1,4-dioxane,and tetrahydrofuran (69.9:25.0:2.9:2.2).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard solution— Dissolve an accurately weighed quantity of USP Alliin RSin a mixture of methanol and water (1:1),and dilute quantitatively,and stepwise if necessary,with a mixture of methanol and water (1:1)to obtain a solution having a known concentration of 0.05mg per mL.Using a 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.

Test solution— Transfer about 10.0g of freshly peeled garlic cloves,accurately weighed,to a 110-mLhomogenizing cup.Add 70.0mLof 0.01M Carboxymethoxylamine hemihydrochloride solution,and blend at the highest speed for 30seconds.Centrifuge,and decant the supernatant into a 100-mLvolumetric flask.Mix the remaining solids in the cup with 20mLof 0.01M Carboxymethoxylamine hemihydrochloride solution,centrifuge,and add the supernatant to the volumetric flask.Dilute the contents of the flask with 0.01M Carboxymethoxylamine hemihydrochloride solutionto volume,and mix.Transfer 10.0mLof the supernatant homogenate to a 100-mLvolumetric flask,dilute with a mixture of methanol and water (1:1)to volume,and mix.Using a 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.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 337-nm detector and a 4-mm ×10-cm column that contains packing L1.The flow rate is about 1.0mLper minute.[NOTE—Alliin exhibits two major peaks representing its diastereomers.]Chromatograph replicate injections of the Standard solution,and record the peak areas as directed for Procedure:the relative standard deviation for replicate injections is not more than 2.0%for each of the major peaks.

Procedure— Separately inject equal volumes (about 10µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the areas of the responses of the alliin diastereomer peaks.Calculate the percentage of alliin in the portion of Garlic taken by the formula: in which Cis the concentration,in mg per mL,of USP Alliin RSin the Standard solution;Wis the weight,in g,of garlic cloves taken for the Test solution;and rUand rSare the sums of the peak responses for alliin diastereomers obtained from the Test solutionand the Standard solution,respectively:not less than 0.5%,calculated on the dried basis,is found.

0.05M Phosphate buffer— Dissolve 6.80g of monobasic potassium phosphate in 900mLof water,and adjust with phosphoric acid to a pHof 2.6.Dilute with water to 1000.0mL,and mix.

Mobile phase— Prepare a mixture of 0.05M Phosphate bufferand methanol (85:15).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard solution— Dissolve an accurately weighed quantity of USPg-Glutamyl-(S)-Allyl-L-Cysteine RSin a mixture of methanol and water (1:1),and dilute quantitatively,and stepwise if necessary,with a mixture of methanol and water (1:1)to obtain a solution having a known concentration of about 0.08mg per mL.

Test solution— Transfer about 10g of freshly peeled garlic clove,accurately weighed,to a 110-mLhomogenizing cup.Add 80mLof a mixture of methanol and water (1:1),and homogenize at the highest speed for 1minute.Centrifuge the mixture,and decant the supernatant into a 250-mLvolumetric flask.Mix the remaining solids with two 70-mLportions of a mixture of methanol and water (1:1),centrifuge,and gather the supernatants to the volumetric flask.Dilute the contents of the flask with a mixture of methanol and water (1:1)to volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 205-nm detector and a 4.6-mm ×15-cm column that contains packing L1.The flow rate is about 0.8mLper minute.Chromatograph the Standard solution,and record the peak areas as directed for Procedure: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 areas for the major peaks.Calculate the percentage of g-glutamyl-(S)-allyl-L-cysteine in the portion of Garlic taken by the formula: in which Cis the concentration,in mg per mL,of USPg-Glutamyl-(S)-Allyl-L-Cysteine RSin the Standard solution;Wis the weight,in g,of garlic cloves taken for the Test solution;and rUand rSare the g-glutamyl-(S)-allyl-L-cysteine peak responses obtained from the Test solutionand the Standard solution,respectively.