0117-0119 [115] Dexpanthenol Assay

8/15/2019 0117-0119 [115] Dexpanthenol Assay

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USP 35 Biological Tests / 〈115〉 Dexpanthenol Assay 117

Glossary of Symbols (Continued) Glossary of Symbols (Continued)

P probability of observing a given result, or of the xS mean log-dose for Standard [Equation 9].tabular value of a statistic, usually P = 0.05 or xU mean log-dose for Unknown [Equation 9].0.95 for confidence intervals [Tables 1, 2, 9]. X log-potency from a unit response, as interpolated

P* potency, P* = antilog M or computed directly. from a standard curve [Equations 7a, 7b, 19].R ratio of a given dose of the Standard to the cor- XM confidence limits for an estimated log-potency M

responding dose of the Unknown, or assumed [Equations 25, 30].

potency of the Unknown [Equations 2, 30, 33]. XP* confidence limits for a directly estimated potencyR* ratio of largest of k ranges in a series to their P* (see Digitalis assay) [Equation 33].

sum [Table 2]. y an observed individual response to a dose of s = √s2 standard deviation of a response unit, also of a drug in the units used in computing potency

single estimated log-potency in a direct assay and the error variance [Equations 13 to 16]; a[Equation 24]. unit difference between paired responses in 2-

s2 error variance of a response unit. dose assays [Equations 17, 18].

Si a log-dose of Standard [Tables 6, 7 ]. y1...yN observed responses listed in order of magnitude, for computing G1, G2, or G3 in Table 1.Σ “the sum of.”

y′ replacement for a missing value [Equation 1].t Student’s t for n degrees of freedom andprobability P = 0.05 [Table 9]. y mean response in a set or assay [Equation 5].

T total of the responses y in an assay [Equation 16]. yt mean response to a given treatment [Equations 3, 6].T′ incomplete total for an assay in randomized sets

with one missing observation [Equation 1]. Y a response predicted from a dosage-response re-lationship,often with qualifying subscripts [Equa-T1 Σ(y) for the animals injected with the Standardtions 3 to 5].

on the first day [Equations 18, 36]. z threshold dose determined directly by titrationT2 Σ(y) for the animals injected with the Standard(see Digitalis assay) [Equation 11].on the second day [Equations 18, 36].

z mean threshold dose in a set (see Digitalis assay)Ta Ti for the difference in the responses to the Stan-[Equations 31, 32, 33].dard and to the Unknown [Tables 6 to 8].

Tab Ti for testing the difference in slope betweenStandard and Unknown [Tables 6 to 8].

Taq Ti for testing opposed curvature in the curves for Standard and Unknown [Tables 6 to 8].

Tb Ti for the combined slope of the dosage-responsecurves for Standard and Unknown [Tables 6 to8]. 〈115〉 DEXPANTHENOL ASSAY

Tb′ Σ(x1Tt) or Σ(x1 y) for computing the slope of thelog-dose response curve [Equations 10, 23, 28].

Ti sum of products of Tt multiplied by the corre-The following procedure is provided for the determinationsponding factorial coefficients in each row of

of dexpanthenol as an ingredient of multiple-vitamin prepa-Tables 6 to 8. rations. It is applicable also to the determination of the dex-

Tq Ti for testing similar curvature in the curves for trorotatory component of racemic panthenol and of other Standard and Unknown [Tables 6 to 8]. mixtures containing dextrorotatory panthenol.

Tr row or set total in an assay in randomized sets Media may be prepared as described hereinafter, or dehy-[Equation 16]. drated mixtures yielding similar formulations may be used

Tr ′ incomplete total for the randomized set with a provided that, when reconstituted as directed by the manu-missing observation in Equation 1. facturer or distributor, they have growth-promoting proper-

ties equal to or superior to those obtained from the formu-Tt total of f responses y for a given dose of a prepa-las given herein.ration [Tables 6 to 8; Equations 6, 13, 14, 16].

Tt′ incomplete total for the treatment with a missing USP Reference Standards 〈11〉—USP Dexpanthenol RS .observation in Equation 1. Standard Stock Solution of Dexpanthenol—Dissolve an

Ui a log-dose of Unknown [Tables 6 to 8]. accurately weighed quantity of USP Dexpanthenol RS inwater, dilute with water to obtain a solution having av variance for heterogeneity between assays [Equa-known concentration of about 800 µg per mL, and mix.tion 45].Store in a refrigerator, protected from light, and use within V = 1/w variance of an individual M [Equations 44 to 47 ].30 days.

w weight assigned to the M for an individual assay

Standard Preparation—On the day of the assay, prepare[Equation 38], or to a probit for computing ana water dilution of the Standard Stock Solution of Dexpanthe-LD50 [Equations 2a, 2b].nol to contain 1.2 µg of dexpanthenol per mL.w′ semi-weight of each M in a series of assays

Assay Preparation—Proceed as directed in the individual[Equations 47, 48].monograph for preparing a solution expected to contain ap-x a log-dose of drug in a bioassay [Equation 5];proximately the equivalent of the dexpanthenol concentra-also the difference between two log-thresholdtion in the Standard Preparation.doses in the same animal [Equation 12].

Modified Pantothenate Medium—x* coefficients for computing the lowest and high-est expected responses yL and yH in a log-doseresponse curve [Table 4; Equation 3]. Acid-Hydrolyzed Casein Solution 25 mL

x1 a factorial coefficient that is a multiple of (x – x) Cystine–Tryptophane Solution 25 mL for computing the slope of a straight line [Table Polysorbate 80 Solution 0.25 mL5; Equation 6].

Dextrose, Anhydrous 10 gx mean log-dose [Equation 5].

Sodium Acetate, Anhydrous 5 g

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118 〈115〉 Dexpanthenol Assay / Biological Tests USP 35

hol to make 500 mL, and mix. Store in a refrigerator, and Adenine–Guanine–Uracil Solution 5 mLuse within 30 days.Riboflavin–Thiamine Hydrochloride–Biotin Solution 5 mL

Stock Culture of Pediococcus acidilactici —Dissolve inPara-aminobenzoic Acid–Niacin–Pyridoxine Hydro- 5 mLabout 800 mL of water, with the aid of heat, 6.0 g of pep-chloride Solutiontone, 4.0 g of pancreatic digest of casein, 3.0 g of yeast

Salt Solution A 5 mLextract, 1.5 g of beef extract, 1.0 g of dextrose, and 15.0 g

Salt Solution B 5 mL of agar. Adjust with 0.1 N sodium hydroxide or 0.1 N hy-Pyridoxal-Calcium Pantothenate Solution 5 mL drochloric acid to a pH between 6.5 and 6.6, adjust thePolysorbate 40–Oleic Acid Solution 5 mL volume with water to 1000 mL, and mix. Add approxi-

mately 10-mL portions of the solution to culture tubes,place caps on the tubes, and sterilize at 121° for 15 min-Dissolve the anhydrous dextrose and sodium acetate inutes. Cool on a slant, and store in a refrigerator. Prepare athe solutions previously mixed, and adjust with 1 N sodiumstock culture of Pediococcus acidilactici * on a slant of thishydroxide to a pH of 6.8. Finally, dilute with water to 250medium. Incubate at 35° for 20 to 24 hours, and store in amL, and mix.refrigerator. Maintain the stock culture by monthly transfer Double-Strength Modified Pantothenate Medium—onto fresh slants.Prepare as directed under Modified Pantothenate Medium,

Inoculum—Inoculate three 250-mL portions of Modified but make the final dilution to 125 mL instead of 250 mL.Pantothenate Medium from a stock culture slant, and incu-Prepare fresh.bate at 35° for 20 to 24 hours. Centrifuge the suspensionAcid-Hydrolyzed Casein Solution—Mix 100 g of vita- from the combined portions, and wash the cells with Modi-min-free casein with 500 mL of 6 N hydrochloric acid, andfied Pantothenate Medium. Resuspend the cells in sufficientreflux the mixture for 8 to 12 hours. Remove the hydrochlo-Modified Pantothenate Medium so that a 1:50 dilution, whenric acid from the mixture by distillation under reduced pres-tested in a 13-mm diameter test tube, gives 80% lightsure until a thick paste remains. Redissolve the resultingtransmission at 530 nm. Transfer 1.2-mL portions of thispaste in about 500 mL of water, adjust the solution with

stock suspension to glass ampuls, seal, freeze in liquid nitro-1 N sodium hydroxide to a pH of 3.5 ± 0.1, and add water gen, and store in a freezer. On the day of the assay, allowto make 1000 mL. Add 20 g of activated charcoal, stir for 1the ampuls to reach room temperature, mix the contents,hour, and filter. Repeat the treatment with activated char-and dilute 1 mL of thawed culture with sterile saline TS tocoal. Store under toluene in a refrigerator at a temperature150 mL. [NOTE—This dilution may be altered, when neces-not below 10°. Filter the solution if a precipitate forms dur-sary, to obtain the desired test response.]ing storage.

Procedure—Prepare in triplicate a series of eight cultureCystine–Tryptophane Solution—Suspend 4.0 g of L-cys-tubes by adding the following quantities of water to thetine and 1.0 g of L-tryptophane (or 2.0 g of D,L-trypto-tubes within a set: 5.0 mL, 4.5 mL, 4.0 mL, 3.5 mL, 3.0 mL,phane) in 700 mL to 800 mL of water, heat to 75 ± 5°, and2.0 mL, 1.0 mL, and 0.0 mL. To these same tubes, and inadd dilute hydrochloric acid (1 in 2) dropwise, with stirring,the same order, add 0.0 mL, 0.5 mL, 1.0 mL, 1.5 mL, 2.0until the solids are dissolved. Cool, add water to make 1000mL, 3.0 mL, 4.0 mL, and 5.0 mL of the Standard mL, and mix. Store under toluene in a refrigerator at a tem-Preparation.perature not below 10°.

Prepare in duplicate a series of five culture tubes by add-Adenine–Guanine–Uracil Solution—Dissolve 200 mg ing the following quantities of water to the tubes within a

each of adenine sulfate, guanine hydrochloride, and uracil, set: 4.0 mL, 3.5 mL, 3.0 mL, 2.0 mL, and 1.0 mL. To thesewith the aid of heat, in 10 mL of 4 N hydrochloric acid, same tubes, and in the same order, add 1.0 mL, 1.5 mL,cool, add water to make 200 mL, and mix. Store under

2.0 mL, 3.0 mL, and 4.0 mL of the Assay Preparation.toluene in a refrigerator. Add 5.0 mL of Double-Strength Modified Pantothenate Me-Polysorbate 80 Solution—Dissolve 25 g of polysorbate dium to each tube, and mix. Cover the tubes with metal

80 in alcohol to make 250 mL, and mix. caps, and sterilize in an autoclave at 121° for 5 minutes.Cool to room temperature in a chilled water bath, and inoc-Riboflavin–Thiamine Hydrochloride–Biotin Solution—ulate each tube with 0.5 mL of the Inoculum. Allow to incu-Prepare a solution containing, in each mL, 20 µg of ribofla-bate at 37° for 16 hours. Terminate growth by heating to avin, 10 µg of thiamine hydrochloride, and 0.04 µg of biotin,temperature not below 80°, such as by steaming at atmos-by dissolving riboflavin, thiamine hydrochloride, and biotinpheric pressure in a suitable sterilizer, for 5 to 10 minutes.in 0.02 N acetic acid. Store, protected from light, under Cool, and concomitantly determine the percentage trans-toluene in a refrigerator.mittance of the suspensions, in cells of equal pathlength, onPara-aminobenzoic Acid–Niacin–Pyridoxinea suitable spectrophotometer, at 530 nm.Hydrochloride Solution—Prepare a solution in neutral 25

Calculation—Draw a dose-response curve on arithmeticpercent alcohol to contain 10 µg of para-aminobenzoicgraph paper by plotting the average response, in percentacid, 50 µg of niacin, and 40 µg of pyridoxine hydrochlo-transmittance, for each set of tubes of the standard curveride in each mL. Store in a refrigerator.against the standard level concentrations. The curve isSalt Solution A—Dissolve 25 g of monobasic potassiumdrawn by connecting each adjacent pair of points with aphosphate and 25 g of dibasic potassium phosphate instraight line. From this standard curve, determine by inter-water to make 500 mL. Add 5 drops of hydrochloric acid,polation the potency, in terms of dexpanthenol, of eachmix, and store under toluene.tube containing portions of the Assay Preparation. Divide the

Salt Solution B—Dissolve 10 g of magnesium sulfate, 0.5 potency of each tube by the amount of Assay Preparationg of sodium chloride, 0.5 g of ferrous sulfate, and 0.5 g of added to it, to obtain the individual responses. Calculate themanganese sulfate in water to make 500 mL. Add 5 drops mean response by averaging the individual responses thatof hydrochloric acid, mix, and store under toluene. vary from their mean by not more than 15%, using not less

Pyridoxal–Calcium Pantothenate Solution—Dissolve 40 than half the total number of tubes. Calculate the potencymg of pyridoxal hydrochloride and 375 µg of calcium pan- of the portion of the material taken for assay, in terms of tothenate in 10 percent alcohol to make 2000 mL, and mix.

* American Type Culture Collection No. 8042 is suitable.Store in a refrigerator, and use within 30 days.Polysorbate 40–Oleic Acid Solution—Dissolve 25 g of

polysorbate 40 and 0.25 g of oleic acid in 20 percent alco-

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USP 35 Biological Tests / 〈121〉 Insulin Assays 119

dexpanthenol, by multiplying the mean response by the ap- Preparation of Animal—Select suitable, healthy rabbitspropriate dilution factor. each weighing not less than 1.8 kg. Keep the rabbits in the

laboratory for not less than 1 week before use in the assay,maintaining them on an adequate uniform diet, with water available at all times.

Procedure—Divide the rabbits into four equal groups of preferably not less than six rabbits each. On the precedingday, approximately 20 hours before the assay, provide eachrabbit with an amount of food that will be consumed within〈121〉 INSULIN ASSAYS 6 hours. Follow the same feeding schedule before each testday. During the assay, withhold all food until after the finalblood specimen is taken. Handle the rabbits with care in

The most prominent manifestation of insulin activity, an order to avoid undue excitement, and inject subcutaneouslyabrupt decrease in blood glucose, was the basis for biologic the doses indicated in the following design (see Table 1),assay from the time of its first clinical use. The procedure, the second injection being made on the day after the firstalthough relatively cumbersome, has the great merit of ac- injection, or not more than 1 week later. The time betweencurately reflecting the effect on the diabetic patient. The the first and second injection is the same for all rabbits.advent of practical yet sophisticated physicochemical meth-ods (e.g., liquid chromatography) to measure insulin po-

Table 1tency quantitatively has resulted in a more accurate andprecise compendial test for insulin and insulin products. Group First Injection Second InjectionHowever, the bioidentity of insulin and insulin products can- 1 Standard Solution 2 Assay Solution 1not be assessed by these methods. Thus, a qualitative test in

2 Standard Solution 1 Assay Solution 2rabbits is included in this chapter, and its use is called for in3 Assay Solution 2 Standard Solution 1the appropriate monographs.

4 Assay Solution 1 Standard Solution 2The Rabbit Blood Sugar Method—Quantitative is used todetermine the potency of Insulin Reference Standards, for the validation of the stability of new insulin preparations, Blood Samples— At 1 hour ± 5 minutes and 21/2 hours ±and to determine the specific activities of insulin analogs. 5 minutes after the time of injection, obtain from each

rabbit a suitable blood specimen from a marginal ear vein.Blood can also be collected effectively from the central au-RABBIT BLOOD SUGAR METHOD—ricular artery.

QUANTITATIVEDextrose Determination—Determine the dextrose con-

tent of the blood specimens by a suitable procedure that isUSP Reference Standards 〈11〉—USP Dextrose RS. USP adapted to automated analysis. The following procedure

Insulin RS. USP Insulin (Beef) RS. USP Insulin Human RS. USP may be used.Insulin (Pork) RS.

Anticoagulant Solution— Dissolve 1 g of edetate sodiumDiluent—Prepare an aqueous solution containing 0.1% and 200 mg of sodium fluoride in 1 L of water, and mix.

to 0.25% (w/v) of either cresol or phenol, 1.4% to 1.8%Dextrose Standard Preparations— Transfer known concen-(w/v) of glycerin, and sufficient hydrochloric acid to pro-

trations of USP Dextrose RS to suitable vessels, and diluteduce a pH between 2.5 and 3.5, unless otherwise directedquantitatively and stepwise with Anticoagulant Solution (1:9)in the individual monograph.to obtain a range of Dextrose Standard Preparations contain-Standard Stock Solution—Dissolve either a suitable ing between 20 and 100 mg per 100 mL, having known

quantity of accurately weighed USP Insulin RS or a vial of concentrations similar to the concentrations in the rabbitlyophilized USP Insulin RS of the appropriate species in Dilu- blood samples.ent to make a Standard Stock Solution containing 40 USP

Test Preparations— Pipet into separate, suitable vessels 0.1Insulin Units per mL and having a pH between 2.5 and 3.5,mL of each Blood Sample and 0.9 mL of Anticoagulant unless otherwise directed in the individual monograph.Solution.Store in a cold place, protected from freezing, and use

Procedure— Subject the Test Preparations to dialysis acrosswithin 6 months.a semipermeable membrane for a sufficient time so that theStandard Solutions—Dilute portions of the Standard dextrose passes through the membrane into a saline TS so-Stock Solution with Diluent to make two solutions, one tolution containing glucose oxidase, horseradish peroxidase,contain 1.0 USP Insulin Unit per mL (Standard Solution 1),3-methyl-2-benzothiazolinone hydrazone hydrochloride TS,and the other to contain 2.0 USP Insulin Units per mL (Stan-and N,N -dimethylaniline. The absorbances of the Test Prepa-dard Solution 2).rations are determined at 600 nm in a recording colorime-

Assay Stock Solution—Proceed as directed under Stan- ter. The absorbances of the Dextrose Standard Preparations dard Stock Solution, except to use a suitable quantity of the are similarly determined at the start and the end of eachpreparation under test in place of USP Insulin RS. The Assay run.Stock Solution contains about 40 USP Insulin Units per mL. Calculation—Calculate the response of each rabbit to

Assay Solutions—Dilute portions of the Assay Stock Solu- each injection from the sum of the two blood-sugar values,tion with Diluent to make two dilutions of the preparation and subtract its response, disregarding the chronological or-under test, one of which may be expected, on the basis of der in which the responses were observed, to obtain thethe assumed potency, to contain 1.0 USP Insulin Unit per individual differences, y, as shown in Table 2.mL (Assay Solution 1), and the other to contain 2.0 USP When the data for one or more rabbits are missing in anInsulin Units per mL (Assay Solution 2). In the case of neutral assay, do not use the confidence interval formulas giveninsulin injection, adjust to a pH of 2.5 to 3.5 prior to mak- here, but seek statistical help. The data can still be analyzeding the dilutions. with proper analysis of variance.

Doses of the Solutions To Be Injected—Select on the When the number of rabbits, f, carried through the assaybasis of trial or experience the dose of the dilutions to be is the same in each group, total the y’s in each group andinjected, the volume of which usually will be between 0.30 compute Ta = –T1 + T2 + T3 – T4 and Tb = T1 + T2 + T3 + T4.mL and 0.50 mL. For each animal the volume of the Stan- The logarithm of the relative potency of the test dilutions isdard Solution is the same as that of the Assay Solution. M′ = 0.301Ta/Tb. The potency of the injection in USP Units

per mg equals the antilog (log R + M ′), where R = vS/vU, in

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0117-0119 [115] Dexpanthenol Assay