Assay— Dissolve 80mg,accurately weighed,in 10mLof diluted hydrochloric acid,dilute with water to 50mL,and add 25mLof a 1in 100solution of dimethylglyoxime in alcohol.Allow to stand for 1hour,and filter.Check for complete precipitation with the dimethylglyoxime solution.Ignite the precipitate in a tared platinum crucible at 850for 2hours,cool,and weigh the palladium.The weight of the residue is not less than 59.0%of the weight of the test specimen.

Residue on ignition	not more than 2.5%
Loss on drying	not more than 8%
Free acid (as lactic acid)	not more than 0.25%
Fat	not more than 0.5%
Reducing sugars	not more than a trace
Fineness	all passes through a 20-mesh sieve

Degree of digestion— Dissolve 1g in 10mLof water.

Nitrogen content (Reagent test)— Determine by the Kjeldahl method,using a test specimen previously dried at 105to constant weight:not less than 10.0%is found.

Loss on drying á731ñ— Dry it at 100to constant weight:it loses not more than 7.0%of its weight.

Residue on ignition á281ñ— Ignite 500mg with 1mLof sulfuric acid:the residue weighs not more than 75mg (15%).

Nitrite— To 5mLof a solution of the digest (1in 50)add 0.5mLof sulfanilic-a-naphthylamine TS,mix,and allow to stand for 15minutes:no pink or red color develops.

Microbial content— Dissolve 1g in 10mLof water.Spread 0.01mLon one square centimeter of a glass slide.Stain by the Gram method,and examine with an oil-immersion lens:not more than a total of 50microorganisms,or clumps,are visible in 10consecutive fields.

Bacteriological test— The digest meets the following tests for bacteria-nutrient properties.Prepare media of the following compositions:

Freedom from fermentable carbohydrate— To medium (a)add sufficient phenolsulfonphthalein TSto give a readable color,place in Durham fermentation tubes,and autoclave.Inoculate with a loop of 24-hour culture of Escherichia coli:no acid,or only a trace in the inner tube,and no gas are produced during incubation for 48hours.

Production of indole— Inoculate 5mLof medium (b)with Escherichia coli,incubate for 24hours,and test by addition of about 0.5mLof p-dimethylaminobenzaldehyde TS:it shows a distinct pink or red color which is soluble in chloroform.

Production of acetylmethylcarbinol— Inoculate 5mLof medium (c)with Aerobacter aerogenes,and incubate for 24hours.Test by adding to the culture an equal volume of sodium hydroxide solution (1in 10),shake,and allow to stand at room temperature for several hours:appearance of a pink color indicates the presence of acetylmethylcarbinol.

Production of hydrogen sulfide— Inoculate 5mLof medium (d)with Salmonella typhosa.Hold a strip or loop of lead acetate test paper between the cotton plug and the mouth of the test tube so that it hangs about 5cm above the medium.After incubation for 24hours,the lower tip of the lead acetate test paper shows little if any darkening.After 48hours,it shows an appreciable amount of brownish blackening (lead sulfide).

Growth-supporting properties— In the foregoing tests the media support good growth of Escherichia coli,Aerobacter aerogenes,and Salmonella typhosa.Medium (e)stab-inoculated with a stock culture of Brucella abortusshows good growth in the line of the stab after incubation for 48hours.Slants of medium (e),inoculated with Escherichia coli,Aerobacter aerogenes,Salmonella typhosa,Pseudomonas aeruginosa,Staphylococcus aureus,and Staphylococcus albus,show characteristic growth after incubation for 24hours.Medium (e),to which about 5%of sheep blood or of rabbit blood has been added and which has been inoculated and poured into Petri dishes,shows characteristic alpha or beta zones about colonies of pneumococciand beta hemolytic streptococci(serological groups Aand B),recognizable within 24hours and fully developed after 48hours'incubation.Medium (e),to which about 10%of blood has been added and which then has been heated to 80to 90until the blood has turned chocolate-brown,permits the growth of gonococcuscolonies within 48hours when incubated in an atmosphere containing about 10%of carbon dioxide.

Nitrogen content (Reagent test)— Determine by the Kjeldahl method,using a test specimen previously dried at 105to constant weight:not less than 8.5%is found.

Assay— Accurately weigh about 300mg,previously dried at 105for 2hours,and transfer to a beaker or casserole.Add 5mLof hydrochloric acid and 50mLof water,and stir until dissolved.Cool to about 15,add about 25g of crushed ice,and slowly titrate with 0.1Msodium nitrite VSuntil a glass rod dipped into the titrated solution produces an immediate blue ring when touched to starch iodide paper.When the titration is complete,the endpoint is reproducible after the mixture has been allowed to stand for 1minute.Each mLof 0.1Msodium nitrite is equivalent to 13.71mg of C7H7NO2.Not less than 98.5%is found.

Melting range á741ñ: between 186and 189.

Loss on drying á731ñ— Dry it at 105for 2hours:it loses not more than 0.2%of its weight.

Residue on ignition (Reagent test): not more than 0.1%.

Assay— Transfer about 1g,accurately weighed,to a 250-mLconical flask containing 50.0mLof 1Nsodium hydroxide VS,and mix by swirling.Immediately,and slowly,add 50mLof hydrogen peroxide TS,previously neutralized to bromothymol blue,through a small funnel placed in the neck of the flask.After the reaction moderates,rinse the funnel and inner wall of the flask with water,allow the solution to stand for 30minutes,add bromothymol blue TS,and titrate the excess alkali with 1Nsulfuric acid VS.Each mLof 1Nsodium hydroxide is equivalent to 30.03mg of HCHO:not less than 95%is found.

Residue on ignition: not more than 0.1%.

Solubility in ammonia— Dissolve 5g in 50mLof ammonia TS:a practically clear,colorless solution results.

Reaction— Shake 1g with 20mLof water for about 1minute,and filter:the filtrate is neutral to litmus.

Assay— Inject an appropriate specimen into a gas chromatograph (see Chromatography á621ñ)equipped with a flame-ionization detector,helium being used as the carrier gas.The following conditions have been found suitable:a 0.25-mm ×30-m capillary column coated with a 1-µm layer of phase G2;the injection port temperature is maintained at 280;the detector temperature is maintained at 300;and the column temperature is maintained at 180and programmed to rise 10per minute to 280.The area of the C15H32peak is not less than 99%of the total peak area.

Refractive index á831ñ: between 1.430and 1.434at 20.

Boiling range (Reagent test)— Not less than 95%distils between 34and 36.

Activity—

Degree of digestion— Dissolve 1g in 10mLof water,and use this solution for the following tests:

Nitrogen content,Loss on drying,Residue on ignition,andNitrite— Proceed as directed under Pancreatic Digest of Casein.

Microbial content— Dissolve 1g in 10mLof water.Spread 0.01mLon one square centimeter of a glass slide.Stain by the Gram method,and examine with an oil-immersion lens:not more than a total of 50microorganisms,or clumps,are visible in 10consecutive fields.

Bacteriologic test— It meets the following tests for bacteria-nutrient properties.Prepare media of the following compositions:

Presence of fermentable carbohydrate— Inoculate medium (a)with Escherichia coliand with Streptococcus liquefaciens:acid is produced by E.colibut not by S.liquefaciensduring incubation for 24hours.

Production of indole— Inoculate medium (b)with Escherichia coliand with Aerobacter aerogenes,and incubate for 24hours.Test by adding about 0.5mLof p-dimethylaminobenzaldehyde TS:the appearance of a pink or red color (soluble in chloroform)indicates the production of indole by E.coli.The A.aerogenesculture gives a negative test.

Production of acetylmethylcarbinol— Inoculate medium (c)with Escherichia coliand with Aerobacter aerogenes,and incubate for 24hours.Test by adding to the culture an equal volume of sodium hydroxide solution (1in 10),shaking well,and allowing to stand at room temperature for several hours:the appearance of a pink color indicates the production of acetylmethylcarbinol by A.aerogenes.The E.coliculture gives a negative test.

Production of hydrogen sulfide— Inoculate medium (d)with Salmonella typhosa.Hold a strip or loop of lead acetate test paper between the cotton plug and the mouth of the test tube so that it hangs about 5cm above the medium.Then incubate for 24hours:the lower part of the lead acetate test paper shows an appreciable amount of brownish blackening (lead sulfide).

Nitrogen content(Reagent test)— Determine by the Kjeldahl method,using a test specimen previously dried at 105to constant weight:between 14.2%and 15.5%of nitrogen is found,corresponding to not less than 89%of protein.

Residue on ignition (Reagent test)— Ignite 500mg with 1mLof sulfuric acid:the residue weighs not more than 25mg (5.0%).

Loss on drying á731ñ— Dry it at 105to constant weight:it loses not more than 7.0%of its weight.

Coagulable protein— Heat a filtered solution (1in 20)to boiling:no precipitate forms.

Proteoses— Mix 5mLof a filtered solution (1in 10)with 20mLof a filtered solution of zinc sulfate (made by dissolving 50g of the salt in 35mLof water):not more than a slight,flocculent precipitate is formed.

Melting range á741ñ: between 137and 140.

Absorptivity— Its absorptivity,1%,1cm,in the range of 272nm to 290nm,in chloroform solution is about 178.

Melting range á741ñ: between 149and 150.

Assay— To 2g,accurately weighed,add 10mLof a freshly prepared solution made by dissolving 25g of acetic anhydride in 100g of anhydrous pyridine.Swirl to mix the liquids,heat on a steam bath for 45minutes,add 10mLof water,heat for 2additional minutes,and cool.Add 10mLof normal butyl alcohol,shake vigorously,add phenolphthalein TS,and titrate with 1Nsodium hydroxide VS.Perform a blank test using the same quantities of the same reagents,and in the same manner,and make any necessary correction.Each mLof 1Nsodium hydroxide is equivalent to 138.2mg of C8H10O2.Not less than 99%is found.

Phenol— Add 0.2mLof it to 20mLof water,mix,and to 5mLof the mixture add 0.2mLof Millon's reagent.Warm the solution at 60for 90seconds,and allow to stand:no pink or red color is produced within 1minute.

Assay— Transfer 250mg,accurately weighed,to a glass-stoppered,250-mLflask.Exercise care to avoid loss by volatilization,and avoid breathing the vapor.Add 20mLof butylamine solution (25g of butylamine diluted to 1000mLwith dioxane previously dried over potassium hydroxide pellets),insert the stopper in the flask,and allow to stand for 15minutes.Add a few drops of methyl red TSand 25mLof water,and titrate the excess amine with 0.1Nsulfuric acid VS.Perform a blank titration on 20mLof the butylamine solution (see Residual Titrations á541ñ).Subtract the volume of 0.1Nsulfuric acid consumed in the test specimen titration from that consumed in the blank titration.Each mLof 0.1Nsulfuric acid,representing this difference,is equivalent to 11.91mg of C6H5NCO:not less than 97.0%of C6H5NCOis found.

Melting range á741ñ: between 156and 158.

Assay— When tested by thin-layer chromatography,with the use of plates coated with chromatographic silica gel mixture and a developing system consisting of a mixture of butyl alcohol,water,and acetic acid (12:5:3),and examined under short-wavelength UVlight,a single spot is exhibited,with trace impurities.

Insoluble matter— Dissolve 1g in 10mLof water:the solution is clear and complete.

Molar absorptivity(see Spectrophotometry and Light-scattering á851ñ)— Dissolve 60mg in 100.0mLof water,and mix.Pipet 2mLof this solution into a 50-mLvolumetric flask,dilute with pH7buffer solution to volume,and mix.The molar absorptivity of this solution,at 239nm,is not less than 9000.

Congealing temperature á651ñ: not below 16.

Insoluble matter— Shake 1mLwith 20mLof diluted acetic acid:the resulting solution is clear or practically so.

Residue on ignition (Reagent test)— Ignite 1mLwith 0.5mLof sulfuric acid:the residue weighs not more than 1mg (0.1%).

Melting range á741ñ: between 242and 246,with slight darkening.

Solubility— Separate 500-mg portions dissolve in 10mLof water and in 10mLof alcohol,respectively,to yield solutions that are clear and complete or practically so.

Residue on ignition (Reagent test)— Ignite 1g with 0.5mLof sulfuric acid:the residue weighs not more than 1mg (0.1%).

Add the following:

Assay— Inject an appropriate specimen into a suitable gas chromatograph (see Chromatography á621ñ)equipped with a flame-ionization detector,helium being used as the carrier gas.The following conditions have been found suitable:a 0.25-mm ×30-m capillary column coated with G1;the injection port temperature is maintained at 250;the column temperature is maintained at 150and programmed to rise 15per minute to 250;and the detector temperature is maintained at 310.The area of the 3-phenylphenol peak is not less than 98%of the total peak area.

Melting range á741ñ: between 76and 79.

Insoluble in alcohol— Dissolve 1g in 20mLof alcohol:a clear and complete solution results.

Melting range,Class Ia á741ñ: between 215and 219.

Residue on ignition(Reagent test)— Ignite 1g with 0.5mLof sulfuric acid:the residue weighs not more than 1mg (0.1%).

Diresorcinol— Heat to boiling a solution of 100mg in 10mLof acetic anhydride,cool the solution,and superimpose it upon 10mLof sulfuric acid:no violet color appears at the zone of contact of the liquids.

Amylase activity— Place in a test tube 5mLof a 1in 50solution of soluble starch in 0.2M,pH5sodium acetate buffer (containing 1.6g of anhydrous sodium acetate in each liter and sufficient glacial acetic acid to adjust to a pHof 5),and add 4mLof water.Mix,and place in a water bath at 40.Add 1mLof a solution containing 0.3mg of the phosphatic enzyme,mix,and note the exact time.After 30minutes remove 1.0mLof the mixture,and add it to 5.0mLof 0.0005Niodine in a 20-×150-mm test tube:a clear,red color results.

Yellow phosphorus— Shake 20g with 75mLof carbon disulfide in a glass-stoppered vessel,and allow to stand in the dark overnight.Filter,and wash the residue with carbon disulfide until the filtrate,collected in a graduated cylinder,measures 100mL.Evaporate the solvent to 10mLby immersing the cylinder in hot water.Dip a strip of cupric sulfate test paper in the remaining solvent:no more color is produced than in a similar strip dipped into 10mLof solution in carbon disulfide containing 3mg of yellow phosphorus (0.015%as P).

Soluble substances— Digest 2g with 30mLof acetic acid on a steam bath for 15minutes.Cool,dilute with water to 40mL,and filter.Evaporate 20mLof the filtrate on a steam bath,and dry at 105for 2hours:the residue weighs not more than 6mg (0.6%).

Insoluble matter (Reagent test): not more than 1mg,from 5g (0.02%).

Chloride(Reagent test)— One g shows not more than 0.3mg of Cl (0.03%).

Nitrate— Dissolve 500mg in 10mLof water,and add about 10mg of sodium chloride,0.1mLof indigo carmine TS,and 10mLof sulfuric acid:the blue color does not disappear within 1minute (about 0.01%).

Sulfate(Reagent test,Method I)— A500-mg portion shows not more than 0.1mg of SO4(0.02%).

Melting range á741ñ: between 89and 92.

Assay—

Melting range á741ñ: between 233and 235,with decomposition.

Assay— Inject an appropriate specimen into a suitable gas chromatograph (see Chromatography á621ñ)equipped with a flame-ionization detector,helium being used as the carrier gas.The following conditions have been found suitable:a 2-mm ×2-m glass column packed with 20%liquid phase G16on 80-to 100-mesh support S1C;the injection port temperature is maintained at 140;the detector temperature is maintained at 300;the column temperature is maintained at 90and programmed to rise 3per minute to 140.The area of the C6H7Npeak is not less than 98%of the total peak area.

Refractive index á831ñ: 1.500±0.002at 20.

Melting range á741ñ: between 115and 117.

Residue on ignition(Reagent test): negligible,from 200mg.

Sensitiveness— Dissolve 25mg in 10mLof warm water containing 0.1mLof glacial acetic acid,and filter the solution,if necessary.Dissolve 100mg of calcium chloride in 250mLof water,and mix.Heat 1mLof the calcium chloride solution in a test tube to about 60,then add to it 1mLof the picrolonic acid solution:a bulky precipitate forms in 5minutes or less.

Congealing range á651ñ: between 12and 15.

Boiling range (Reagent test)— Not less than 95%distils between 104and 106.

Refractive index: about 1.454.

Refractive index á831ñ: about 1.3770.

Specific gravity á841ñ: about 0.760.

Refractive index á831ñ: 1.4590at 20.

Density: 1.127at 25.

Viscosity: 4.3centistokes at 98.9.

Viscosity of 25%solution á911ñ— Add 50.0g of test specimen to a 250-mLwide-mouth,screw-cap jar containing 150.0g of water.Attach the cap securely to the jar,and roll on a mechanical roller until the test specimen is completely dissolved,in 2to 4hours.Allow the solution to stand until all air bubbles have disappeared.Another 2to 4hours may be required.Adjust the temperature of the solution to 37.8±0.1,and determine the kinematic viscosity on a suitable viscosimeter of the Ubbelohde type.The viscosity is not less than 100centistokes.

pHá791ñ: between 6.5and 8.0in a solution (1in 20).[NOTE—Afive-fold dilution of the test solution prepared for the Viscosity of 25%solutiontest may be used.]

Residue on ignition á281ñ: not more than 0.7%,the use of sulfuric acid being omitted.

Change to read:

pHá791ñ: between 5.0and 8.0,in a solution (1in 25).

Loss on drying— Dry it at 110to constant weight:it loses not more than 5%of its weight.

Residue on ignition: not more than 0.75%.

Acidity— Dissolve 4g,accurately weighed,in 50mLof water,add phenolphthalein TS,and titrate with 1Nalkali:it contains between 34%and 36%,calculated as H2SO4.

Insoluble matterand ammonium hydroxide precipitate— Dissolve 10g in 100mLof water,add methyl red TS,render slightly alkaline with ammonia TS,boil for 1minute,and digest on a steam bath for 1hour.Filter through a tared filtering crucible,wash thoroughly,and dry at 105for 2hours:the precipitate weighs not more than 1mg (0.01%).

Heavy metals(Reagent test)— To 30mLof Test solutionadd phenolphthalein TS,and neutralize with ammonia TS.Add 0.5mLof glacial acetic acid,dilute with water to 40mL,and add 10mLof hydrogen sulfide TS:any brown color produced is not darker than that of a control containing 10mLof Test solutionand 0.02mg of added Pb (0.001%).

Iron á241ñ— To 5mLof Test solutionadd 2mLof hydrochloric acid,and dilute with water to 47mL:the solution shows not more than 0.01mg of Fe (0.002%).

Assay— Accurately weigh about 300mg,transfer to a 600-mLbeaker,add 20mLof hydrochloric acid,and heat gently if necessary to achieve complete solution.Add zinc granules,slowly,until no more dissolves,then add 2mLof hydrochloric acid,and digest for 1hour on a steam bath to coagulate the reduced platinum.Add more acid,if necessary,to ensure that all of the zinc has dissolved.Filter through paper,rinsing the beaker with diluted hydrochloric acid until all of the precipitate is transferred to the filter,then wash with several small portions of water.Ignite the filter in a tared crucible at 800±25to constant weight.Each mg of residue is equivalent to 1.0mg of platinum.Not less than 40%is found.

Inhibitor content— Prepare as directed in the Assayunder Hyaluronidase for Injection(USPmonograph)a quantity of Standard solutioncontaining 1USP Hyaluronidase Unit in each mL,and a similar quantity of acetate-buffered Standard solutionusing as the solvent 0.1M,pH6sodium acetate buffer (prepared by diluting the 0.2Mbuffer prepared as directed below with an equal volume of water).Prepare from the potassium hyaluronate under test 10mLof Potassium hyaluronate stock solution,and dilute 2mLof it with the specified Phosphate buffer solutionto make a Hyaluronate solution.In the same way,and concurrently,dilute a second 2-mLportion of the stock solution with 0.2M,pH6sodium acetate buffer (containing 16.4g of anhydrous sodium acetate and 0.45mLof glacial acetic acid in each 1000mL).

Turbidity production— The average absorbance of the solutions in the two tubes containing Hyaluronate solution and Diluent for hyaluronidase solutionsprepared in the test for Inhibitor contentis not less than 0.26at a wavelength of 640nm in a suitable spectrophotometer using a 1-cm cell.

Melting point á741ñ: about 197.

Assay— Weigh accurately about 120mg,transfer to a beaker,and dissolve in a mixture of 10mLof nitric acid,10mLof sulfuric acid,and 25mLof water.Heat to boiling,and boil until copious fumes of sulfur trioxide are evolved.Cool,cautiously add 100mLof water,heat to boiling,add 6g of sodium fluoride,and titrate the hot solution with 0.1Npotassium permanganate VS.Each mLof 0.1Npotassium permanganate is equivalent to 12.69mg of K2TeO3.Not less than 98%is found.

Chloride(Reagent test)— One g shows not more than 0.1mg of Cl (0.01%).

Add the following:

Assay— Accurately weigh about 350mg into a tared,glass-stoppered flask containing 50mLof dimethylformamide previously neutralized to the thymol blue endpoint with 0.1Nsodium methoxide in methanol VS.Titrate with 0.1Nsodium methoxide in methanol VSto the thymol blue endpoint.Perform a blank determination,and make any necessary correction.Each mLof 0.1Nsodium methoxide is equivalent to 13.014mg of C6H10O3.Not less than 97.0%is found.

Refractive index á831ñ: between 1.4035and 1.4045at 20.

Acid-and water-soluble substances— Boil 2.0g of powdered pumice with 50mLof diluted hydrochloric acid under a reflux condenser for 30minutes.Cool,and filter.To half of the filtrate add 5drops of sulfuric acid,evaporate to dryness,ignite,and weigh:the residue weighs not more than 60mg (6.0%).

Melting range á741ñ: between 214and 217.

Melting range á741ñ: between 67and 71.

Assay— Transfer about 9mg,accurately weighed,to a 100-mLvolumetric flask,dissolve in methanol,dilute with methanol to volume,and mix.Transfer 2.0mLof this solution to a 100-mLvolumetric flask,dilute with methanol to volume,and mix.Using a suitable spectrophotometer,1-cm cells,and methanol as the blank,record the absorbance of the solution at the wavelength of maximum absorbance at about 238nm.From the observed absorbance,calculate the absorptivity (see Spectrophotometry and Light-scattering á851ñ):the absorptivity is not less than 432.9,corresponding to not less than 98%of C16H10.

Melting range á741ñ: between 149and 153over a 2range.

Melting range á741ñ: between 171and 175with some decomposition.

Residue on ignition (Reagent test): not more than 0.1%.

Loss on drying á731ñ— Dry it at 105for 2hours:it loses not more than 0.5%of its weight.

Nitrogen content (Reagent test)— Determine by the Kjeldahl method,using a test specimen previously dried at 105for 2hours:between 6.7%and 7.1%of Nis found.

Chloride content— Accurately weigh about 500mg,previously dried at 105for 2hours,and dissolve in 50mLof water.Add 3mLof nitric acid and 50.0mLof 0.1Nsilver nitrate VS,then add 5mLof nitrobenzene,shake for about 2minutes,add ferric ammonium sulfate TS,and titrate the excess silver nitrate with 0.1Nammonium thiocyanate VS:each mLof 0.1Nsilver nitrate is equivalent to 3.545mg of Cl.Between 17.2%and 17.7%is found.

Melting range á741ñ: between 225and 230,with some decomposition.

Residue on ignition(Reagent test): not more than 0.15%.

Loss on drying á731ñ— Dry it at 105for 2hours:it loses not more than 0.5%of its weight.

Nitrogen content (Reagent test)— Determine by the Kjeldahl method,using a test specimen previously dried at 105for 2hours:between 11.3%and 11.8%of Nis found.

Chloride content— Determine as directed in the test for Chloride contentunder Pyridoxal Hydrochloride:between 29.1%and 29.6%of Cl is found.

Melting range á741ñ: between 140and 142.

Sensitiveness— Add 0.1mLof a 1in 1000solution of it in alcohol to a mixture of 10mLof water and 1mLof a buffer solution prepared by mixing 80mLof 0.2Macetic acid and 20mLof sodium acetate solution (8.2in 100),and mix.To this solution add 1mLof a mixture of 1mLof cupric sulfate TSand 2mLof water,and mix:the color changes from yellow to red.

Boiling range(Reagent test)— Not less than 90%distills between 128and 132.

Refractive index á831ñ: about 1.43at 20.

Assay— Accurately weigh about 1g,transfer to a suitable container,and add 100mLof water.Mix,add phenolphthalein TS,and titrate with 0.5Nsodium hydroxide VS.Each mLof 0.5Nsodium hydroxide is equivalent to 44.03mg of CH3COCOOH:not less than 98%of CH3COCOOHis found.