Roots,rhizomes,bark,and herbs	500g
Leaves,flowers,seeds,and fruit	250g
Cut vegetable drugs (average weight of the pieces is less than 0.5g)	50g

Accurately weigh a quantity of the Test Sample,representing 2to 4g of the air-dried material,in a tared crucible,and incinerate,gently at first,and gradually increase the temperature to 675±25,until free from carbon,and determine the weight of the ash.If a carbon-free ash cannot be obtained in this way,extract the charred mass with hot water,collect the insoluble residue on an ashless filter paper,incinerate the residue and filter paper until the ash is white or nearly so,then add the filtrate,evaporate it to dryness,and heat the whole to a temperature of 675±25.If a carbon-free ash cannot be obtained in this way,cool the crucible,add 15mLof alcohol,break up the ash with a glass rod,burn off the alcohol,and again heat the whole to a temperature of 675±25.Cool in a desiccator,weigh the ash,and calculate the percentage of total ash from the weight of the drug taken.

Boil the ash obtained as directed under Total Ash,above,with 25mLof 3Nhydrochloric acid for 5minutes,collect the insoluble matter on a tared filtering crucible or ashless filter,wash with hot water,ignite,and weigh.Determine the percentage of acid-insoluble ash calculated from the weight of drug taken.

Boil the ash obtained as directed for Total Ashwith 25mLof water for 5minutes.Collect the insoluble matter in a sintered-glass crucible or on an ashless filter paper.Wash with hot water,and ignite for 15minutes at a temperature not exceeding 450.Subtract the weight of this residue,in mg,obtained under Total Ash,and calculate the percentage of water-soluble ash with reference to the weight of sample as determined under Total Ash.

Exhaust a weighed quantity of the Test Sample,representing about 2g of the drug,with ether.Add 200mLof boiling dilute sulfuric acid (1in 78)to the ether-exhausted marc,in a 500-mLflask,and connect the flask to a reflux condenser.Reflux the mixture for 30minutes,accurately timed,then filter through a linen or hardened-paper filter,and wash the residue on the filter with boiling water until the effluent washing is no longer acid.Rinse the residue back into the flask with 200mLof boiling sodium hydroxide solution,adjusted to 1.25percent by titration and free from sodium carbonate.Again reflux the mixture for 30minutes,accurately timed,then rapidly filter through a tared filter,wash the residue with boiling water until the last washing is neutral,and dry it at 110to constant weight.Incinerate the dried residue,ignite to constant weight,cool in a desiccator,and weigh the ash:the difference between the weight obtained by drying at 110and that of the ash represents the weight of the crude fiber.

Malt Extract— Use clean new barley malt of known efficacy,and grind just before use.Prepare malt extract just prior to use.For every 80mLof malt extract needed,digest 5g of ground malt with 100mLof water at room temperature for 2hours.[NOTE—If an electric mixer is used,stir the mixture for 20minutes.]Filter to obtain a clear extract,filtering again,if necessary,and mix the infusion well.

Test Solution— Extract about 5g of the finely ground test specimen with five 10-mLportions of ether,using a filter that will completely retain the smallest starch granule.Allow the ether to evaporate from the residue,and wash with 250mLof aqueous alcohol solution (10in 100).Carefully wash the residue from the paper into a 500-mLbeaker with about 100mLof water.Heat to about 60(avoiding,if possible,gelatinizing starch),and allow to stand for about 1hour,stirring frequently to effect complete solution of sugars.Transfer to a wide-mouth bottle,rinse the beaker with a little warm water,and cool.Add an equal volume of alcohol,mix,and allow to stand for not less than 1hour.

General Procedure— Transfer 200mLof the Test Solutionto a flask fitted with a reflux condenser,add 20mLof hydrochloric acid,and heat in a boiling water bath for 2½hours.Cool,nearly neutralize with sodium hydroxide TS,complete neutralization with sodium carbonate TS,dilute with water to 500mL,mix,and filter.The volume of aliquot taken depends on the starch content of the specimen under test (see Table 1).The aliquot should contain between 100and 200mg of dextrose.Transfer 50mLof the filtrate to a 400-mLalkali-resistant glass beaker,add 50mLof alkaline cupric tartarate TS,cover the beaker with a water glass,and heat.Adjust the flame in the burner so that the contents of the flask begin to boil in 4minutes and continue boiling for exactly 2minutes.Filter the hot solution at once through a sintered-glass filter.Wash the precipitate of cuprous oxide thoroughly with water at about 60,then with 10mLof alcohol,and finally with 10mLof ether.

METHOD1B—

Glucoamylase Solution— Prepare a solution of glucoamylase in water containing 30International Units (IU)per mL.Use glucoamylase obtained preferably from Rhizopus delemar.The total glucoamylase activity of the test specimen being used should be not less than 150IU.

Acetate Buffer Solution— Dissolve 16.4g of sodium acetate in 100mLof water,add 12.0mLof glacial acetic acid,and mix.The pHof this solution is 4.8.

Phosphate Buffer— Dissolve 3.63g of tris (hydroxymethyl)aminomethane and 5.0g of monobasic sodium phosphate in 50.0mLof water.At 37,adjust with phosphoric acid to a pHof 7.0,dilute with water to 100.0mL,and mix.[NOTE—The pHof the buffer medium is sensitive to temperature and should be adjusted to the desired pHat the temperature to be used during incubation.]

Enzyme Solution— Dissolve 30mg of glucose oxidase (Type IIfrom Aspergillus niger),3mg of peroxidase (Type Ifrom horseradish),and 10mg of potassium ferrocyanide in 100mLof Phosphate Buffer.[NOTE—This mixture can be stored in a refrigerator for up to 10days.]

18N Sulfuric Acid— Add slowly,while stirring,54mLof sulfuric acid to 102mLof water,allow to cool to 25,and mix.

Standard Solutions— Dissolve an accurately weighed quantity of USP Dextrose RSin water to obtain a solution containing 1.0mg of USP Dextrose RSper mL.Quantitatively dilute a known volume of this solution with water to obtain Standard Solutions A,B,C,D,and E,having known concentrations of 10,20,25,40,and 50µg per mLof USP Dextrose RS,respectively.[NOTE—Allow 4hours for complete mutarotation before use.]

Test Solutions— Extract about 5g of finely ground test specimen with five 25-mLportions of 80%alcohol,and filter.Remove all the alcohol from the residue by drying in an air oven at 105for about 8hours.[NOTE1—Any traces of alcohol remaining in the residue will inhibit glucoamylase.]Cool,and transfer the flask containing the dried test specimen to a desiccator.Transfer about 1g,accurately weighed,of the test specimen to a previously tared flask,add 25mLof water,and adjust with phosphoric acid to a pHbetween 5.0and 7.0,if necessary.Boil the suspension for about 3minutes,transfer the flask to an autoclave,and heat to 135for 2hours.Remove the flask from the autoclave,maintain the temperature near 55,and add 2.5mLof Acetate Buffer Solutionand sufficient water to adjust the total weight of the solution to 45±1g.Immerse the flask in a water bath maintained at 55±1,and add 5mLof Glucoamylase Solution.Continuously swirl the flask for 2hours to effect hydrolysis,filter through filter paper into a 250-mLvolumetric flask,wash quantitatively with water,and collect all the washings in the flask.Dilute the contents of the flask with water to volume,and mix.Transfer 1mLof an aliquot containing 20to 60µg of D-glucose to each of five test tubes.[NOTE2—In order to obtain the range of concentration of glucose in the hydrolysate,quantitatively dilute,if necessary,with water to volume.]Add 2mLof Enzyme Solutionto each of the five test tubes,and place the test tubes in the dark at 37±1for exactly 30minutes to develop the color.At the end of 30minutes,add 2mLof 18N Sulfuric Acidto each of the test tubes to stop the reaction,and mix.

Control Solution— Transfer an accurately weighed quantity of about 0.4g of starch to a previously tared flask and proceed as directed under Test Solutionsbeginning with “add 25mLof water and,adjust the pHwith phosphoric acid.”

Procedure— Concomitantly determine the absorbances of the Standard Solutionsand the Test Solutionsat the wavelength of maximum absorbance at about 540nm,with a suitable spectrophotometer,using the Control Solutionas the blank to set the instrument.Plot the absorbance values of the Standard Solutionsversus concentration,in µg per mL,of dextrose,and draw the straight line best fitting the five plotted points.From the graph so obtained,determine the concentration,C,in µg per mL,of dextrose in each of the Test Solutions,calculate the average concentration,in µg per mL,of the solution under test.The percentage of starch content in the weight of the test specimen taken by the equation is calculated by the formula: in which Eis the weight,in g,of the test specimen taken;V0is the volume,in mL,of the aliquot taken from the 250-mLvolumetric flask;Wis the percentage of dry weight of the test specimen;and V1is the volume,in mL,if extra dilution is done (see Note 2under Test Solutions).[NOTE—V0is 1.0when no extra dilution is done.]

Set up a round-bottom,shortneck,1-liter flask in a heating mantle set over a magnetic stirrer.Insert an egg-shaped stirring bar magnet in the flask,and attach a cold-finger condenser and an appropriate volatile oil trap of the type illustrated.

Coarsely comminute a sufficient quantity of the drug to yield from 1to 3mLof volatile oil.Small seeds,fruits,or broken leaves of herbs ordinarily do not need comminution.Very fine powders are to be avoided.If this is not possible,it may be necessary to mix them with purified sawdust or purified sand.Place a suitable quantity of the drug,accurately weighed,in the flask,and fill it one-half with water.Attach the condenser and the proper separator.Boil the contents of the flask,using a suitable amount of heat to maintain gentle boiling for 2hours,or until the volatile oil has been completely separated from the drug and no longer collects in the graduated tube of the separator.

If a proper quantity of the volatile oil has been obtained in the graduated tube of the separator,it can be read to tenths of 1mL,and the volume of volatile oil from each 100g of drug can be calculated from the weight of the drug taken.The graduations on the separator “for oils heavier than water”are so placed that oil remains below the aqueous condensate that automatically flows back into the flask.

For unground or unpowdered drugs,prepare about 10g of the Laboratory Sampleby cutting,granulating,or shredding,so that the parts are about 3mm in thickness.Seeds or fruits smaller than 3mm should be cracked.Avoid the use of high-speed mills in preparing the sample,and exercise care that no appreciable amount of moisture is lost during the preparation and that the portion taken is representative of the Laboratory Sample.Determine the water content as directed for Procedure for Articles of Botanical Originin the Method III(Gravimetric)under Water Determination á921ñ.

This test is provided to detect the possible presence of aflatoxins B1,B2,G1,and G2in any material of plant origin.Unless otherwise specified in the individual monograph,use the following method.