á115ñDEXPANTHENOL ASSAY
The following procedure is provided for the determination of dexpanthenol as an ingredient of multiple-vitamin preparations.It is applicable also to the determination of the dextrorotatory component of racemic panthenol and of other mixtures containing dextrorotatory panthenol.
Media may be prepared as described hereinafter,or dehydrated mixtures yielding similar formulations may be used provided that,when reconstituted as directed by the manufacturer or distributor,they have growth-promoting properties equal to or superior to those obtained from the formulas given herein.
USP Reference Standards á11ñ— USP Dexpanthenol RS.
Standard Stock Solution of Dexpanthenol— Dissolve an accurately weighed quantity of USP Dexpanthenol RSin water,dilute with water to obtain a solution having a known concentration of about 800µg per mL,and mix.Store in a refrigerator,protected from light,and use within 30days.
Standard Preparation— On the day of the assay,prepare a water dilution of the Standard Stock Solution of Dexpanthenolto contain 1.2µg of dexpanthenol per mL.
Assay Preparation— Proceed as directed in the individual monograph for preparing a solution expected to contain approximately the equivalent of the dexpanthenol concentration in the Standard Preparation.
Modified Pantothenate Medium—
| 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 |
| Pyridoxal-Calcium Pantothenate Solution | 5mL |
| Polysorbate 40–Oleic Acid Solution | 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.
Double-Strength Modified Pantothenate Medium— Prepare as directed under Modified Pantothenate Medium,but make the final dilution to 125mLinstead of 250mL.Prepare fresh.
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 about 500mLof 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 700mLto 800mLof water,heat to 75±5
,and add dilute hydrochloric acid (1in 2)dropwise,with stirring,until the solids are dissolved.Cool,add water to make 1000mL,and mix.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,add water to make 200mL,and mix.Store under toluene in a refrigerator.
Polysorbate 80Solution— Dissolve 25g of polysorbate 80in alcohol to make 250mL,and mix.
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,mix,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,mix,and store under toluene.
Pyridoxal–Calcium Pantothenate Solution— Dissolve 40mg of pyridoxal hydrochloride and 375µg of calcium pantothenate in 10percent alcohol to make 2000mL,and mix.Store in a refrigerator,and use within 30days.
Polysorbate 40–Oleic Acid Solution— Dissolve 25g of polysorbate 40and 0.25g of oleic acid in 20percent alcohol to make 500mL,and mix.Store in a refrigerator,and use within 30days.
Stock Culture of Pediococcus acidilactici— Dissolve in about 800mLof water,with the aid of heat,6.0g of peptone,4.0g of pancreatic digest of casein,3.0g of yeast extract,1.5g of beef extract,1.0g of dextrose,and 15.0g of agar.Adjust with 0.1Nsodium hydroxide or 0.1Nhydrochloric acid to a pHbetween 6.5and 6.6,adjust the volume with water to 1000mL,and mix.Add approximately 10-mLportions of the solution to culture tubes,place caps on the tubes,and sterilize at 121
for 15minutes.Cool on a slant,and store in a refrigerator.Prepare a stock culture of Pediococcus acidilactici*on a slant of this medium.Incubate at 35for 20to 24hours,and store in a refrigerator.Maintain the stock culture by monthly transfer onto fresh slants.
Inoculum— Inoculate three 250-mLportions of Modified Pantothenate Mediumfrom a stock culture slant,and incubate at 35
for 20to 24hours.Centrifuge the suspension from the combined portions,and wash the cells with Modified Pantothenate Medium.Resuspend the cells in sufficient Modified Pantothenate Mediumso that a 1:50dilution,when tested in a 13-mm diameter test tube,gives 80%light transmission at 530nm.Transfer 1.2-mLportions of this stock suspension to glass ampuls,seal,freeze in liquid nitrogen,and store in a freezer.On the day of the assay,allow the ampuls to reach room temperature,mix the contents,and dilute 1mLof thawed culture with sterile saline TSto 150mL.[NOTE—This dilution may be altered,when necessary,to obtain the desired test response.]
Procedure— Prepare in triplicate a series of eight culture tubes by adding the following quantities of water to the tubes within a set:5.0mL,4.5mL,4.0mL,3.5mL,3.0mL,2.0mL,1.0mL,and 0.0mL.To these same tubes,and in the same order,add 0.0mL,0.5mL,1.0mL,1.5mL,2.0mL,3.0mL,4.0mL,and 5.0mLof the Standard Preparation.
Prepare in duplicate a series of five culture tubes by adding the following quantities of water to the tubes within a set:4.0mL,3.5mL,3.0mL,2.0mL,and 1.0mL.To these same tubes,and in the same order,add 1.0mL,1.5mL,2.0mL,3.0mL,and 4.0mLof the Assay Preparation.
Add 5.0mLof Double-Strength Modified Pantothenate Mediumto each tube,and mix.Cover the tubes with metal caps,and sterilize in an autoclave at 121for 5minutes.Cool to room temperature in a chilled water bath,and inoculate each tube with 0.5mLof the Inoculum.Allow to incubate at 37for 16hours.Terminate growth by heating to a temperature not below 80,such as by steaming at atmospheric pressure in a suitable sterilizer,for 5to 10minutes.Cool,and concomitantly determine the percentage transmittance of the suspensions,in cells of equal pathlength,on a suitable spectrophotometer,at 530nm.
for 5minutes.Cool to room temperature in a chilled water bath,and inoculate each tube with 0.5mLof the Inoculum.Allow to incubate at 37for 16hours.Terminate growth by heating to a temperature not below 80,such as by steaming at atmospheric pressure in a suitable sterilizer,for 5to 10minutes.Cool,and concomitantly determine the percentage transmittance of the suspensions,in cells of equal pathlength,on a suitable spectrophotometer,at 530nm.Calculation— Draw a dose-response curve on arithmetic graph paper by plotting the average response,in percent transmittance,for each set of tubes of the standard curve against the standard level concentrations.The curve is drawn by connecting each adjacent pair of points with a straight line.From this standard curve,determine by interpolation the potency,in terms of dexpanthenol,of each tube containing portions of the Assay Preparation.Divide the potency of each tube by the amount of Assay Preparationadded to it,to obtain the individual responses.Calculate the mean response by averaging the individual responses that vary from their mean by not more than 15%,using not less than half the total number of tubes.Calculate the potency of the portion of the material taken for assay,in terms of dexpanthenol,by multiplying the mean response by the appropriate dilution factor.
*
American Type Culture Collection No.8042is suitable.