á91ñCALCIUM PANTOTHENATE ASSAY
USP Reference Standards á11ñ— USP Calcium Pantothenate RS
Standard Stock Solution of Calcium Pantothenate— Dissolve 50mg of USP Calcium Pantothenate RS,previously dried and stored in the dark over phosphorus pentoxide and accurately weighed while protected from absorption of moisture during the weighing,in about 500mLof water in a 1000-mLvolumetric flask.Add 10mLof 0.2Nacetic acid and 100mLof sodium acetate solution (1in 60),then dilute with water to volume.Each mLrepresents 50µg of USP Calcium Pantothenate RS.Store under toluene in a refrigerator.
Standard Preparation— On the day of the assay,dilute a measured volume of Standard Stock Solution of Calcium Pantothenatewith sufficient water so that it contains,in each mL,between 0.01µg and 0.04µg of calcium pantothenate,the exact concentration being such that the responses obtained as directed for Procedure,2.0and 4.0mLof the Standard Preparationbeing used,are within the linear portion of the log-concentration response curve.
Assay Preparation— Proceed as directed in the individual monograph for preparing a solution expected to contain approximately the equivalent of the calcium pantothenate concentration in the Standard Preparation.
Basal Medium Stock Solution—
| Acid-hydrolyzed Casein Solution | 25mL |
| Cystine–Tryptophane Solution | 25mL |
| Polysorbate 80Solution | 0.25mL |
| Dextrose,Anhydrous | 10g |
| Sodium Acetate,Anhydrous | 5g |
| Adenine–Guanine–Uracil Solution | 5mL |
| Riboflavin-Thiamine Hydrochloride-Biotin Solution | 5mL |
| Para-aminobenzoic Acid–Niacin–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,dilute with water to 250mL,and mix.
Acid-Hydrolyzed Casein Solution— Mix 100g of vitamin-free casein with 500mLof 6Nhydrochloric acid,and reflux the mixture for 8to 12hours.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.Store under toluene in a refrigerator at a temperature not below 10
.Filter the solution if a precipitate forms during storage.
Cystine–Tryptophane Solution— Suspend 4.0g of L-cystine and 1.0g of L-tryptophane (or 2.0g of D,L-tryptophane)in 700to 800mLof water,heat to 70
to 80,and add dilute hydrochloric acid (1in 2)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 200mg each of adenine sulfate,guanine hydrochloride,and uracil,with the aid of heat,in 10mLof 4Nhydrochloric acid,cool,and add water to make 200mL.Store under toluene in a refrigerator.
Polysorbate 80Solution— Dissolve 25g of polysorbate 80in alcohol to make 250mL.
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 riboflavin,thiamine hydrochloride,and biotin in 0.02Nacetic acid.Store,protected from light,under toluene in a refrigerator.
Para-aminobenzoic Acid–Niacin–Pyridoxine Hydrochloride Solution— Prepare a solution in neutral 25percent alcohol to contain 10µg of para-aminobenzoic acid,50µg of niacin,and 40µg of pyridoxine hydrochloride in each 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,0.5g of sodium chloride,0.5g of ferrous sulfate,and 0.5g 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,suitably close or cover the tubes,sterilize at 121
,and allow the tubes to cool in an upright position.Prepare stab cultures in 3or more of the tubes,using a pure culture of Lactobacillus plantarum,*incubating for 16to 24hours at any selected temperature between 30and 37but 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 Basal Medium Stock Solutionadd 5.0mLof water containing 0.2µg of calcium pantothenate.Plug the tubes with cotton,sterilize in an autoclave at 121
,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 30
and 37but held constant to within ±0.5.The cell suspension so obtained is the inoculum.
Procedure— To similar test tubes add,in duplicate,1.0and/or 1.5,2.0,3.0,4.0,and 5.0mL,respectively,of the Standard Preparation.To each tube and to 4similar tubes containing no Standard Preparationadd 5.0mLof Basal Medium Stock Solutionand sufficient water to make 10mL.
To similar test tubes add,in duplicate,volumes of the Assay Preparationcorresponding to 3or more of the levels listed above for the Standard Preparation,including the levels of 2.0,3.0,and 4.0mL.To each tube add 5.0mLof the Basal Medium Stock Solutionand sufficient water to make 10mL.Place one complete set of Standard and Assay tubes together in one tube rack and the duplicate set in a second rack or section of a rack,preferably in random order.
Cover the tubes of both series suitably to prevent contamination,and heat in an autoclave at 121for 5minutes.Cool,add 1drop of inoculum to each tube,except 2of the 4tubes containing no Standard Preparation(to serve as the uninoculated blanks),and mix.Incubate the tubes at a temperature between 30and 37,held constant to within ±0.5until,following 16to 24hours of incubation,there has been no substantial increase in turbidity in the tubes containing the highest level of standard during a 2-hour period.
for 5minutes.Cool,add 1drop of inoculum to each tube,except 2of the 4tubes containing no Standard Preparation(to serve as the uninoculated blanks),and mix.Incubate the tubes at a temperature between 30and 37,held constant to within ±0.5until,following 16to 24hours of incubation,there has been no substantial increase in turbidity in the tubes containing the highest level of standard during a 2-hour period.Determine the transmittance of the tubes in the following manner:Mix the contents of each tube,and transfer to an optical container if necessary.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 galvanometer 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.With the transmittance set at 1.00for the inoculated blank,read the transmittance for each of the remaining tubes.If there is evidence of contamination with a foreign microorganism,disregard the result of the assay.
Calculation— Prepare a standard concentration-response curve as follows.For each level of the standard,calculate the response from the sum of the duplicate values of the transmittance as the difference,y=2.00-S(of transmittance).Plot this response on the ordinate of cross-section paper against the logarithm of the mLof Standard Preparationper tube on the abscissa,using for the ordinate either an arithmetic or a logarithmic scale,whichever gives the better approximation to a straight line.Draw the straight line or smooth curve that best fits the plotted points.
Calculate the response,y,adding together the two transmittances for each level of the Assay Preparation.Read from the standard curve the logarithm of the volume of the Standard Preparationcorresponding to each of those values of ythat fall within the range of the lowest and highest points plotted for the standard.Subtract from each logarithm so obtained the logarithm of the volume,in mL,of the Assay Preparationto obtain the difference,x,for each dosage level.Average the values of xfor each of three or more dosage levels to obtain bar(x)=M¢,the log-relative potency of the Assay Preparation.Determine the quantity,in mg,of USP Calcium Pantothenate RScorresponding to the calcium pantothenate in the portion of material taken for assay as antilog:
M=antilog (M¢+log R),
in which Ris the number of mg of calcium pantothenate that was assumed to be present in each mg (or capsule or tablet)of the material taken for assay.
Replication— Repeat the entire determination at least once,using separately prepared Assay Preparations.If the difference between the two log-potenciesMis not greater than 0.08,their mean,bar(M),is the assayed log-potency of the test material (see The Confidence Interval and Limits of Potency á111ñ).If the two determinations differ by more than 0.08,conduct one or more additional determinations.From the mean of two or more values of Mthat do not differ by more than 0.15,compute the mean potency of the preparation under assay.
*
American Type Culture Collection No.8014is suitable.This strain formerly was known as Lactobacillus arabinosus17-5.