á441ñNIACIN OR NIACINAMIDE ASSAY

USP Reference Standards á11ñ— USP Niacin RS.USP Niacinamide RS.[NOTE—The previously dried Reference Standards may be stored in a desiccator over silica gel,protected from light.]

Chemical Method
NOTE—Determine from the labeling if the vitamin in the assay specimen is niacin or niacinamide,and use the corresponding standard preparation (either Standard Niacin Preparationor Standard Niacinamide Preparation)as directed in the Procedure.
Cyanogen Bromide Solution— Dissolve 5g of cyanogen bromide in water to make 50mL.[Caution—Prepare this solution under a hood,as cyanogen bromide volatilizes at room temperature,and the vapor is highly irritating and poisonous. ]
Sulfanilic Acid Solution— To 2.5g of sulfanilic acid add 15mLof water and 3mLof 6Nammonium hydroxide.Mix,add,with stirring,more 6Nammonium hydroxide,if necessary,until the acid dissolves,adjust the solution with 3Nhydrochloric acid to a pHof about 4.5,using bromocresol green TSas an external indicator,and dilute with water to 25mL.
Standard Niacin Stock Solution— Transfer 25.0mg of USP Niacin RSto a 500-mLvolumetric flask,dissolve in alcohol solution (1in 4),dilute with alcohol solution (1in 4)to volume,and mix.Store in a refrigerator.Each mLof this solution contains 50µg of USP Niacin RS.
Standard Niacin Preparation — Transfer 10.0mLof Standard Niacin Stock Solutionto a 100-mLvolumetric flask,dilute with water to volume,and mix.Each mLof this solution contains 5µg of USP Niacin RS.
Standard Niacinamide Stock Solution— Transfer 50.0mg of USP Niacinamide RSto a 500-mLvolumetric flask,dissolve in alcohol solution (1in 4),dilute with alcohol solution (1in 4)to volume,and mix.Store in a refrigerator.Each mLof this solution contains 100µg of USP Niacinamide RS.
Standard Niacinamide Preparation— Transfer 10.0mLof Standard Niacinamide Stock Solutionto a 100-mLvolumetric flask,dilute with water to volume,and mix.Each mLof this solution contains 10µg of USP Niacinamide RS.
Assay Preparation— Prepare as directed in the individual monograph.
Procedure— Pipet into four marked tubes the quantities of the appropriate Standard Preparation,the Assay Preparation,the ammonia dilution,and water indicated in the accompanying table.Then add the other constituents,respectively,as listed in the table,according to the directions given herein.
Reaction Mixtures for Niacin or Niacinamide Assay—Chemical Method
Constituent Tube 1,mL Tube 2,mL Tube 3,mL Tube 4,mL
Standard Preparation 1.0 1.0 — —
Assay Preparation — — 1.0 1.0
Ammonia Dilution (ammonium hydroxide,
diluted to 1in 50)		 0.5 0.5 0.5 0.5
Water 6.5 1.5 6.5 1.5
Cyanogen Bromide Solution — 5.0 — 5.0
Sulfanilic Acid Solution 2.0 2.0 2.0 2.0
Hydrochloric Acid 1drop — 1drop —
To Tube 1add the Sulfanilic Acid Solution,shake well,add the hydrochloric acid,mix,place in a suitable spectrophotometer,and adjust to zero absorbance at 450nm.To Tube 2add the Cyanogen Bromide Solution,mix,and 30seconds,accurately timed,after completion of the addition of the cyanogen bromide,add the Sulfanilic Acid Solution,with swirling.Close the tube,place it in the spectrophotometer,and after 2minutes measure its absorbance at 450nm against Tube 1as a blank,designating the absorbance as AS.Repeat the procedure with Tubes 3(as blank)and 4,designating the absorbance of Tube 4as AU.Calculate the quantity of niacin or niacinamide in the sample as directed in the individual monograph.

Microbiological Method
Test Solution of Material to be Assayed— Place the prescribed amount of the material to be assayed in a flask of suitable size,and proceed by one of the methods given below.The concentrations of the sulfuric acid and sodium hydroxide solutions used are not stated in each instance because these concentrations may be varied depending upon the amount of material taken for assay,volume of test solution,and buffering effect of material.
(a)For Dry or Semidry Materials that Contain No Appreciable Amount of Basic Substances—Add a volume of dilute sulfuric acid (1in 35)equal,in mL,to not less than 10times the dry weight of the material,in g,but the resulting solution shall contain not more than 5.0mg of niacin in each mL.If the material is not readily soluble,comminute it so that it may be evenly dispersed in the liquid,then agitate vigorously,and wash down the sides of the flask with dilute sulfuric acid (1in 35).
Heat the mixture in an autoclave at 121to 123for 30minutes,and cool.If lumping occurs,agitate the mixture until the particles are evenly dispersed.Adjust the mixture with sodium hydroxide solution to a pHof 6.8,dilute with water to make a final measured volume that has a concentration of niacin equivalent to that of Standard Niacin Solution,and filter.
(b)For Dry or Semidry Materials that Contain Appreciable Amounts of Basic Substances—Add sufficient sulfuric acid solution to bring the pHof the mixture to between 5.0and 6.0.Add such an amount of water that the total volume of liquid shall be equal in mLto not less than ten times the dry weight of the assay specimen,in g,but the resulting solution shall contain not more than 5.0mg of niacin in each mL.Then add the equivalent of 10mLof dilute sulfuric acid (2in 7)for each 100mLof liquid,and proceed as directed under (a),beginning with the second paragraph.
(c)For Liquid Materials—Adjust the material with either sulfuric acid solution or sodium hydroxide solution to a pHof 5.0to 6.0.Add such an amount of water that the total volume of liquid shall be equal,in mL,to not less than 10times the volume of the specimen,in mL,but the resulting solution shall contain not more than 5.0mg of niacin in each mL.Then add the equivalent of 10mLof dilute sulfuric acid (2in 7)for each mLof liquid,and proceed as directed under (a),beginning with the second paragraph.
Standard Niacin Stock Solution I— Transfer 50.0mg of USP Niacin RSto a 500-mLvolumetric flask,dissolve in alcohol,dilute with alcohol to volume,and mix.Store in a refrigerator.Each mLof this solution contains 100µg of USP Niacin RS.
Standard Niacin Stock Solution II— To 100.0mLof Standard Niacin Stock Solution Iadd water to make 1000.0mL.Store under toluene in a refrigerator.Each mLof this solution contains 10µg of USP Niacin RS.
Standard Niacin Solution— Dilute a suitable volume of Standard Niacin Stock Solution IIwith water to such a measured volume so that after incubation as described in the Assay Procedurethe transmittance of the 5.0-mLlevel of Standard Niacin Solutionis equivalent to that of a dried cell weight of not less than 1.25mg,when the inoculated blank is set at 100percent transmittance.This concentration is usually between 10ng and 40ng of niacin per mL.Prepare a fresh Standard Niacin Solutionfor each assay.
Basal Medium Stock Solution—
Acid-hydrolyzed Casein Solution 25mL
Cystine–Tryptophan Solution 25mL
Dextrose Anhydrous 10g
Sodium Acetate Anhydrous 5g
Adenine–Guanine–Uracil Solution 5mL
Riboflavin–Thiamine Hydrochloride–Biotin Solution 5mL
Aminobenzoic Acid–Calcium Pantothenate–Pyridoxine Hydrochloride Solution 5mL
Salt Solution A 5mL
Salt Solution B 5mL
Dissolve the anhydrous dextrose and sodium acetate in the solutions previously mixed,and adjust with 1Nsodium hydroxide to a pHof 6.8.Finally,add water to make 250mL.
Acid-Hydrolyzed Casein Solution— Mix 100g of vitamin-free casein with 500mLof constant-boiling hydrochloric acid [approximately 20percent (w/w)HCl],and reflux the mixture for 24hours.Remove the hydrochloric acid from the mixture by distillation under reduced pressure until a thick paste remains.Redissolve the resulting paste in water,adjust the solution with 1Nsodium hydroxide to a pHof 3.5(±0.1),and add water to make 1000mL.Add 20g of activated charcoal,stir for 1hour,and filter.Repeat the treatment with activated charcoal if the filtrate does not appear straw-colored to colorless.Store under toluene in a refrigerator.Filter the solution if a precipitate forms upon storage.
Cystine–Tryptophan Solution— Suspend 4.0g of l-cystine and 1.0g of l-tryptophan (or 2.0g of dl-tryptophan)in 700to 800mLof water,heat to 70to 80,and add the 20percent (w/w)hydrochloric acid,dropwise,with stirring,until the solids are dissolved.Cool,and add water to make 1000mL.Store under toluene in a refrigerator at a temperature not below 10.
Adenine–Guanine–Uracil Solution— Dissolve 100mg each of adenine sulfate,guanine hydrochloride,and uracil,with the aid of heat,in 5.0mLof the 20percent (w/w)hydrochloric acid,cool,and add water to make 100mL.Store under toluene in a refrigerator.
Riboflavin–Thiamine Hydrochloride–Biotin Solution— Prepare a solution containing,in each mL,20µg of riboflavin,10µg of thiamine hydrochloride,and 0.04µg of biotin by dissolving crystalline riboflavin,crystalline thiamine hydrochloride,and crystalline biotin (free acid)in dilute glacial acetic acid (1in 850).Store,protected from light,under toluene in a refrigerator.
Aminobenzoic Acid–Calcium Pantothenate–Pyridoxine Hydrochloride Solution— Prepare a solution of neutral 25percent alcohol having a concentration of 10µg of aminobenzoic acid,20µg of calcium pantothenate,and 40µg of pyridoxine hydrochloride per mL.Store in a refrigerator.
Salt Solution A— Dissolve 25g of monobasic potassium phosphate and 25g of dibasic potassium phosphate in water to make 500mL.Add 5drops of hydrochloric acid,and store under toluene.
Salt Solution B— Dissolve 10g of magnesium sulfate,500mg of sodium chloride,500mg of ferrous sulfate,and 500mg of manganese sulfate in water to make 500mL.Add 5drops of hydrochloric acid,and store under toluene.
Stock Culture of Lactobacillus plantarum— Dissolve 2.0g of water-soluble yeast extract in 100mLof water,add 500mg of anhydrous dextrose,500mg of anhydrous sodium acetate,and 1.5g of agar,and heat the mixture with stirring,on a steam bath,until the agar dissolves.Add approximately 10-mLportions of the hot solution to test tubes,plug the tubes with cotton,sterilize for 15minutes in an autoclave at 121to 123,and allow the tubes to cool in an upright position.Prepare stab cultures in three or more of the tubes,using a pure culture of Lactobacillus plantarum,*incubating for 16to 24hours at any selected temperature between 30and 37,but held constant to within ±0.5,and finally store in a refrigerator.Prepare a fresh stab of the stock culture every week,and do not use for inoculum if the culture is more than 1week old.
Culture Medium— To each of a series of test tubes containing 5.0mLof the Basal Medium Stock Solutionadd 5.0mLof water containing 1.0µg of niacin.Plug the tubes with cotton,sterilize for 15minutes in an autoclave at 121to 123,and cool.
Inoculum— Make a transfer of cells from the stock culture of Lactobacillus plantarumto a sterile tube containing 10mLof culture medium.Incubate this culture for 16to 24hours at any selected temperature between 30and 37,but held constant to within ±0.5.The cell suspension so obtained is the inoculum.
Calibration of Spectrophotometer— Add aseptically 1mLof Inoculumto approximately 300mLof Culture Mediumcontaining 1mLof Standard Niacin Solution.Incubate the inoculated medium for the same period and at the same temperature to be employed in the Assay Procedure.
Following the incubation period,centrifuge and wash the cells three times with approximately 50-mLportions of saline TS,and then resuspend the cells in about 25mLof the saline solution.
Dry to constant weight a 10-mLportion,accurately measured,using a steam bath and completing the drying in vacuum at 100,and calculate the dry weight of the cells,in mg per mL,corrected for the amount of sodium chloride present.
Dilute a second portion,accurately measured,of the saline cell suspension with the saline solution so that each mLcontains a known quantity of cells equivalent to 500µg on a dried basis.To test tubes add,in triplicate,0.5mL,1.0mL,1.5mL,2.0mL,2.5mL,3.0mL,4.0mL,and 5.0mL,respectively,of this diluted cell suspension and 5.0mLof Basal Medium Stock Solution,and make the volume in each tube to 10.0mLwith saline solution.Using as the blanks three similar tubes containing no cell suspension,measure the light transmittance of each tube under the same conditions to be employed in the assay.Plot the observations as the ordinate on cross-section paper against the cell content,expressed as mg of dry weight,as the abscissa.
Repeat this procedure at least twice for the spectrophotometer to be used in the assay.Draw the composite curve best representing the three or more individual curves relating transmittance to cell density for the spectrophotometer under the conditions of the assay.
Assay Procedure— Prepare standard niacin tubes as follows.To test tubes add,in duplicate,0.0mL,0.5mL,1.0mL,1.5mL,2.0mL,2.5mL,3.0mL,3.5mL,4.0mL,4.5mL,and 5.0mL,respectively,of Standard Niacin Solution.To each tube add 5.0mLof Basal Medium Stock Solutionand water to make 10.0mL.
Prepare tubes containing the material to be assayed as follows.To test tubes add,in duplicate,1.0mL,2.0mL,3.0mL,and 4.0mL,respectively,of the test solution of the material to be assayed.To each tube add 5.0mLof Basal Medium Stock Solutionand water to make 10.0mL.After mixing,plug the tubes with cotton or cover with caps,and sterilize in an autoclave at 121to 123.(Overheating the assay tubes may produce unsatisfactory results.)Cool,aseptically inoculate each tube with 1drop of Inoculum,and incubate for 16to 24hours at any selected temperature between 30and 37,but held constant to within ±0.5.Contamination of the assay tubes with any foreign organism invalidates the assay.
Determine the transmittance of the tubes in the following manner.Mix the contents of each tube,to which 1drop of a suitable antifoam agent solution may be added,and transfer to an optical container.After agitating its contents,place the container in a spectrophotometer that has been set at a specific wavelength between 540nm and 660nm,and read the transmittance when a steady state is reached.This steady state is observed a few seconds after agitation when the reading remains constant for 30seconds or more.Allow approximately the same time interval for the reading on each tube.
With the transmittance set at 1.00for the uninoculated blank,read the transmittance of the inoculated blank.If this transmittance reading corresponds to a dried cell weight greater than 600µg per tube,or if there is evidence of contamination with a foreign microorganism,disregard the results of the assay.
Then with the transmittance set at 1.00for the inoculated blank,read the transmittance for each of the remaining tubes.Disregard the results of the assay if the difference between the transmittance observed at the highest level of the standard and that of the inoculated blank is less than the difference corresponding to a dried cell weight of 1.25mg per tube.
Calculation— Prepare a standard curve of the niacin standard transmittances for each level of Standard Niacin Solutionplotted against µg of niacin contained in the respective tubes.From this standard curve,determine by interpolation the niacin content of the test solution in each tube.Disregard transmittance values equivalent to less than 0.5mLor more than 4.5mLof Standard Niacin Solution.The niacin content of the test material is calculated from the average values obtained from not less than six tubes that do not vary by more than ±10percent from the average.If the transmittance values of less than six tubes containing the test solution are within the range of the 0.5-to 4.5-mLlevels of the niacin standard tubes,the data are insufficient to permit calculation of the concentration of niacin in the test material.Transmittance values of inoculated blank exceeding readings corresponding to dried cell weights of more than 600µg per tube indicate the presence of an excessive amount of niacin in the Basal Medium Stock Solutionand invalidate the assay.
Multiply the values obtained by 0.992if the results are to be expressed as niacinamide.
*  Pure cultures of Lactobacillus plantarummay be obtained,as number 8014,from the American Type Culture Collection,12301Parklawn Drive,Rockville,MD20852.