441 NIACIN OR NIACINAMIDE ASSAY
USP Reference Standards 11 USP Niacin RS. USP Niacinamide RS. [NoteThe previously dried Reference Standards may be stored in a desiccator over silica gel, protected from light. ]
NoteDetermine from the labeling if the vitamin in the assay specimen is niacin or niacinamide, and use the corresponding standard preparation (either Standard Niacin Preparation or Standard Niacinamide Preparation) as directed in the Procedure.
Cyanogen Bromide Solution Dissolve 5 g of cyanogen bromide in water to make 50 mL. [CautionPrepare this solution under a hood, as cyanogen bromide volatilizes at room temperature, and the vapor is highly irritating and poisonous. ]
Sulfanilic Acid Solution To 2.5 g of sulfanilic acid add 15 mL of water and 3 mL of 6 N ammonium hydroxide. Mix, add, with stirring, more 6 N ammonium hydroxide, if necessary, until the acid dissolves, adjust the solution with 3 N hydrochloric acid to a pH of about 4.5, using bromocresol green TS as an external indicator, and dilute with water to 25 mL.
Standard Niacin Stock Solution Transfer 25.0 mg of USP Niacin RS to a 500-mL volumetric flask, dissolve in alcohol solution (1 in 4), dilute with alcohol solution (1 in 4) to volume, and mix. Store in a refrigerator. Each mL of this solution contains 50 µg of USP Niacin RS.
Standard Niacin Preparation Transfer 10.0 mL of Standard Niacin Stock Solution to a 100-mL volumetric flask, dilute with water to volume, and mix. Each mL of this solution contains 5 µg of USP Niacin RS.
Standard Niacinamide Stock Solution Transfer 50.0 mg of USP Niacinamide RS to a 500-mL volumetric flask, dissolve in alcohol solution (1 in 4), dilute with alcohol solution (1 in 4) to volume, and mix. Store in a refrigerator. Each mL of this solution contains 100 µg of USP Niacinamide RS.
Standard Niacinamide Preparation Transfer 10.0 mL of Standard Niacinamide Stock Solution to a 100-mL volumetric flask, dilute with water to volume, and mix. Each mL of this solution contains 10 µg 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 AssayChemical Method
To Tube 1 add the Sulfanilic Acid Solution, shake well, add the hydrochloric acid, mix, place in a suitable spectrophotometer, and adjust to zero absorbance at 450 nm. To Tube 2 add the Cyanogen Bromide Solution, mix, and 30 seconds, 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 2 minutes measure its absorbance at 450 nm against Tube 1 as a blank, designating the absorbance as AS. Repeat the procedure with Tubes 3 (as blank) and 4, designating the absorbance of Tube 4 as AU. Calculate the quantity of niacin or niacinamide in the sample as directed in the individual monograph.
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 SubstancesAdd a volume of dilute sulfuric acid (1 in 35) equal, in mL, to not less than 10 times the dry weight of the material, in g, but the resulting solution shall contain not more than 5.0 mg 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 (1 in 35).
Heat the mixture in an autoclave at 121 to 123 for 30 minutes, and cool. If lumping occurs, agitate the mixture until the particles are evenly dispersed. Adjust the mixture with sodium hydroxide solution to a pH of 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 SubstancesAdd sufficient sulfuric acid solution to bring the pH of the mixture to between 5.0 and 6.0. Add such an amount of water that the total volume of liquid shall be equal in mL to not less than ten times the dry weight of the assay specimen, in g, but the resulting solution shall contain not more than 5.0 mg of niacin in each mL. Then add the equivalent of 10 mL of dilute sulfuric acid (2 in 7) for each 100 mL of liquid, and proceed as directed under (a), beginning with the second paragraph.
(c) For Liquid MaterialsAdjust the material with either sulfuric acid solution or sodium hydroxide solution to a pH of 5.0 to 6.0. Add such an amount of water that the total volume of liquid shall be equal, in mL, to not less than 10 times the volume of the specimen, in mL, but the resulting solution shall contain not more than 5.0 mg of niacin in each mL. Then add the equivalent of 10 mL of dilute sulfuric acid (2 in 7) for each mL of liquid, and proceed as directed under (a), beginning with the second paragraph.
Standard Niacin Stock Solution I Transfer 50.0 mg of USP Niacin RS to a 500-mL volumetric flask, dissolve in alcohol, dilute with alcohol to volume, and mix. Store in a refrigerator. Each mL of this solution contains 100 µg of USP Niacin RS.
Standard Niacin Stock Solution II To 100.0 mL of Standard Niacin Stock Solution I add water to make 1000.0 mL. Store under toluene in a refrigerator. Each mL of this solution contains 10 µg of USP Niacin RS.
Standard Niacin Solution Dilute a suitable volume of Standard Niacin Stock Solution II with water to such a measured volume so that after incubation as described in the Assay Procedure the transmittance of the 5.0-mL level of Standard Niacin Solution is equivalent to that of a dried cell weight of not less than 1.25 mg, when the inoculated blank is set at 100 percent transmittance. This concentration is usually between 10 ng and 40 ng of niacin per mL. Prepare a fresh Standard Niacin Solution for each assay.
Basal Medium Stock Solution
Dissolve the anhydrous dextrose and sodium acetate in the solutions previously mixed, and adjust with 1 N sodium hydroxide to a pH of 6.8. Finally, add water to make 250 mL.
Acid-Hydrolyzed Casein Solution Mix 100 g of vitamin-free casein with 500 mL of constant-boiling hydrochloric acid [approximately 20 percent (w/w) HCl], and reflux the mixture for 24 hours. 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 1 N sodium hydroxide to a pH of 3.5 (±0.1), and add water to make 1000 mL. Add 20 g of activated charcoal, stir for 1 hour, 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.
CystineTryptophan Solution Suspend 4.0 g of l-cystine and 1.0 g of l-tryptophan (or 2.0 g of dl-tryptophan) in 700 to 800 mL of water, heat to 70 to 80, and add the 20 percent (w/w) hydrochloric acid, dropwise, with stirring, until the solids are dissolved. Cool, and add water to make 1000 mL. Store under toluene in a refrigerator at a temperature not below 10.
AdenineGuanineUracil Solution Dissolve 100 mg each of adenine sulfate, guanine hydrochloride, and uracil, with the aid of heat, in 5.0 mL of the 20 percent (w/w) hydrochloric acid, cool, and add water to make 100 mL. Store under toluene in a refrigerator.
RiboflavinThiamine HydrochlorideBiotin Solution Prepare a solution containing, in each mL, 20 µg of riboflavin, 10 µg of thiamine hydrochloride, and 0.04 µg of biotin by dissolving crystalline riboflavin, crystalline thiamine hydrochloride, and crystalline biotin (free acid) in dilute glacial acetic acid (1 in 850). Store, protected from light, under toluene in a refrigerator.
Aminobenzoic AcidCalcium PantothenatePyridoxine Hydrochloride Solution Prepare a solution of neutral 25 percent alcohol having a concentration of 10 µg of aminobenzoic acid, 20 µg of calcium pantothenate, and 40 µg of pyridoxine hydrochloride per mL. Store in a refrigerator.
Salt Solution A Dissolve 25 g of monobasic potassium phosphate and 25 g of dibasic potassium phosphate in water to make 500 mL. Add 5 drops of hydrochloric acid, and store under toluene.
Salt Solution B Dissolve 10 g of magnesium sulfate, 500 mg of sodium chloride, 500 mg of ferrous sulfate, and 500 mg of manganese sulfate in water to make 500 mL. Add 5 drops of hydrochloric acid, and store under toluene.
Stock Culture of Lactobacillus plantarum Dissolve 2.0 g of water-soluble yeast extract in 100 mL of water, add 500 mg of anhydrous dextrose, 500 mg of anhydrous sodium acetate, and 1.5 g of agar, and heat the mixture with stirring, on a steam bath, until the agar dissolves. Add approximately 10-mL portions of the hot solution to test tubes, plug the tubes with cotton, sterilize for 15 minutes in an autoclave at 121 to 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 16 to 24 hours at any selected temperature between 30 and 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 1 week old.
Culture Medium To each of a series of test tubes containing 5.0 mL of the Basal Medium Stock Solution add 5.0 mL of water containing 1.0 µg of niacin. Plug the tubes with cotton, sterilize for 15 minutes in an autoclave at 121 to 123, and cool.
Inoculum Make a transfer of cells from the stock culture of Lactobacillus plantarum to a sterile tube containing 10 mL of culture medium. Incubate this culture for 16 to 24 hours at any selected temperature between 30 and 37, but held constant to within ± 0.5. The cell suspension so obtained is the inoculum.
Calibration of Spectrophotometer Add aseptically 1 mL of Inoculum to approximately 300 mL of Culture Medium containing 1 mL of 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-mL portions of saline TS, and then resuspend the cells in about 25 mL of the saline solution.
Dry to constant weight a 10-mL portion, 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 mL contains a known quantity of cells equivalent to 500 µg on a dried basis. To test tubes add, in triplicate, 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL, 3.0 mL, 4.0 mL, and 5.0 mL, respectively, of this diluted cell suspension and 5.0 mL of Basal Medium Stock Solution, and make the volume in each tube to 10.0 mL with 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, 0.0 mL, 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL, 3.0 mL, 3.5 mL, 4.0 mL, 4.5 mL, and 5.0 mL, respectively, of Standard Niacin Solution. To each tube add 5.0 mL of Basal Medium Stock Solution and water to make 10.0 mL.
Prepare tubes containing the material to be assayed as follows. To test tubes add, in duplicate, 1.0 mL, 2.0 mL, 3.0 mL, and 4.0 mL, respectively, of the test solution of the material to be assayed. To each tube add 5.0 mL of Basal Medium Stock Solution and water to make 10.0 mL. After mixing, plug the tubes with cotton or cover with caps, and sterilize in an autoclave at 121 to 123. (Overheating the assay tubes may produce unsatisfactory results.) Cool, aseptically inoculate each tube with 1 drop of Inoculum, and incubate for 16 to 24 hours at any selected temperature between 30 and 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 1 drop 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 540 nm and 660 nm, 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 30 seconds or more. Allow approximately the same time interval for the reading on each tube.
With the transmittance set at 1.00 for the uninoculated blank, read the transmittance of the inoculated blank. If this transmittance reading corresponds to a dried cell weight greater than 600 µg 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 1.00 for 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 1.25 mg per tube.
Calculation Prepare a standard curve of the niacin standard transmittances for each level of Standard Niacin Solution plotted 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 0.5 mL or more than 4.5 mL of 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 ±10 percent 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-mL levels 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 600 µg per tube indicate the presence of an excessive amount of niacin in the Basal Medium Stock Solution and invalidate the assay.
Multiply the values obtained by 0.992 if the results are to be expressed as niacinamide.
* Pure cultures of Lactobacillus plantarum may be obtained, as number 8014, from the American Type Culture Collection, P.O. Box 1549, Manassas, VA 20108.
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