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f=Milliliters of Karl Fischer reagent equivalent to each milliliter of methanol solution;

VT-Milliliters of Karl Fischer reagent used;

Vm-Milliliters of methanol solution used.

(3) Karl Fischer reagent equivalence of solvents. (i) Solvent A: Use 20 milliliters of solvent A as the sample. Start the stirrer and titrate to the endpoint by adding measured quantities of Karl Fischer reagent.

(ii) Solvent B: Use 10 milliliters of solvent B as the sample. Add an excess of Karl Fischer reagent to the sample and start the stirrer. Titrate to the endpoint with methanol solution.

(iii) Solvent C. Use 20 milliliters of solvent C as the sample. Start the stirrer and titrate to the endpoint by adding measured quantities of Karl Fischer reagent.

(iv) Calculate the Karl Fischer reagent equivalence of the solvents as follows:

VA=Vc=VT,

VB=(VT- Vm) X f

where:

VA, VB, and Vc-Milliliters of Karl Fischer reagent equivalent to the aliquots used of solvents A, B, and C, respectively; V=Milliliters of Karl Fischer reagent used; VM-Milliliters of methanol solution used; f-Milliliters of Karl Fischer reagent equivalent to each milliliter of methanol solution determined as directed in paragraph (c)(2) of this section.

(d) Sample preparation—(1) Powders. In the case of tablets, grind 4 tablets to a fine powder. In the case of capsules containing enteric-coated pellets, grind the pellets to a fine power. If the maximum moisture limit is greater than 1 percent, accurately weigh about 300 milligrams of the sample into a dry titrating vessel. If the maximum moisture limit is less than 1 percent, accurately weigh 1 to 2 grams of the sample. Proceed as directed in paragraph (e)(1) or (2) of this section.

(2) Ointments and oils. (i) Transfer about 1 to 2 grams, accurately weighed, into a dry titrating vessel. Proceed as directed in paragraph (e)(1) of this section; or

(ii) Transfer about 1 to 2 grams, accurately weighed, into a dry titrating vessel. Add 10 milliliters of solvent B and proceed as directed in paragraph (e)(2) of this section.

(3) Aerosols with propellant. Place the immediate container to be tested in a suitable freezing unit having a temperature of not higher than 0° C. for at least 2 hours. Remove the container from the freezing unit, puncture it, mix the entire contents by swirling. Proceed as directed in paragraph (e)(3) of this section, using an accurately measured 10-milliliter aliquot from the container as the sample and allowing the solution to warm to at least 10° C. before determining the endpoint.

(4) Hygroscopic powders. Weigh the immediate container. Using a suitable dry hypodermic needle and syringe, inject 3 milliliters of anhydrous methanol into the container and shake to dissolve the contents. Using the same syringe, remove the withdrawable contents and transfer into the titration vessel. Rinse the syringe and needle by drawing in an additional 3 milliliters of anhydrous methanol. Add the rinsings to the titration vessel. Titrate the solution immediately, proceeding as directed in paragraph (e)(3) of this section. Determine the Karl Fischer equivalent (in milliliters), if any, of the anhydrous methanol by titrating a blank of the same total volume used in preparing the sample and rinsing the syringe and needle. Dry the immediate container and its closure for three hours at 100° C., cool to room temperature in a desiccator, and weigh. Determine the weight of sample tested by subtracting the weight obtained from the original weight of the immediate container.

(5) Solutions. Proceed as directed in paragraph (e)(3) of this section, using about 1 to 2 grams of the sample, accurately weighted.

(e) Titration procedures and calculations (1) Procedure 1. Add 20 milliliters of solvent A to the sample. Start the stirrer and titrate to the endpoint by adding measured quantities of Karl Fischer reagent. Determine the percent moisture in the sample as follows:

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e-Water equivalence of the Karl Fischer reagent determined as directed in paragraph (c)(1) of this section; V=Milliliters of Karl Fischer reagent used;

VA=Milliliters of Karl Fischer reagent equivalent to the 20 milliliters of solvent A, determined as directed in paragarph (c)(3) of this section;

W.-Weight of the sample in milligrams.

(2) Procedure 2. Add an excess of Karl Fischer reagent to the sample, start the stirrer, and titrate to the endpoint with methanol solution. Calculate the percent moisture in the sample as follows:

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VT-Milliliters of Karl Fischer reagent used;

Vm-Milliliters of methanol solution used; f-Milliliters of Karl Fischer reagent equiv

alent to each milliliter of methanol solution determined as directed in paragraph (c)(2) of this section. VB-Milliliters of Karl Fischer reagent equivalent to the 10 milliliters of solvent B determined as directed in paragraph (c)(3) of this section; e-Water equivalence of the Karl Fischer reagent determined as directed in paragraph (c)(1) of this section; W.-Weight of the sample in milligrams. (3) Procedure 3. Add about 20 milliliters of solvent A to a dry titrating vessel and proceed as directed in titration procedure 1 or 2. Disregard the volume of reagents used to determine the endpoint. Promptly introduce an accurately weighed or measured quantity of sample into the titrating vessel and titrate to the endpoint using either titration procedure 1 or 2 without additional solvents. Calculate the percent moisture in the sample as follows:

(1) If titration procedure 1 is used:

Percent moisture in aerosols

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Milliliters of sample x 10

Percent moisture in hygroscopic powders=

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VT-Milliliters of Karl Fischer reagent used;

Vm-Milliliters of methanol solution used; f=Milliliters of Karl Fischer reagent equivalent to each milliliter of methanol solution determined as directed in paragraph (c)(2) of this section;

V=Karl Fischer equivalent (in milliliters) of the methanol used as a sample solvent;

e-Water equivalence of the Karl Fischer reagent determined as directed in paragraph (c)(1) of this section.

[39 FR 18944, May 30, 1974, as amended at 48 FR 51292, Nov. 8, 1983; 50 FR 41679, Oct. 15, 1985; 51 FR 22071, June 18, 1986; 51 FR 27532, Aug. 1, 1986]

§ 436.202 pH.

(a) Apparatus. A suitable potentiometer fitted with two electrodes, one being constructed of glass and sensitive to hydrogen ion activity and the other being a calomel or a silver/silver chloride reference electrode. A combination electrode with glass electrode and reference electrode contained in the same system may be used.

(b) Standardization. Select two standard buffer solutions such that the expected pH value of the sample is within their pH range and is also within 2 pH

unit of one of the standard buffer solutions. Standardize the pH meter with the two buffer solutions. Make any necessary adjustment of the meter if the observed pH value of either standard solution differs by more than 0.05 pH units of its known value.

(c) Sample preparation. If necessary, dilute the sample with carbon dioxidefree distilled water to the concentration specified in the individual section for each antibiotic.

(d) Test procedure. Determine the pH of the sample at 25°±2° C. Rinse the electrode(s) between determinations first with distilled water and then with a portion of the next sample to be tested. Store electrode(s) with tips immersed in water.

[39 FR 18944, May 30, 1974, as amended at 42 FR 29857, June 10, 1977; 42 FR 31449, June 21, 1977]

$436.203 Crystallinity.

Use the method specified in the individual section for each antibiotic.

(a) Method 1. To prepare the sample for examination, mount a few particles in mineral oil on a clean glass slide. Examine the sample by means of a po

larizing microscope. The particles reveal the phenomena of birefringence and extinction positions on revolving the microscope stage.

(b) Method 2. Proceed as directed in paragraph (a) of this section, except to prepare the sample for examination, mount a few particles in mineral oil, add 1 drop of ethyl alcohol, and allow to react for about 30 seconds.

§ 436.204 Iodometric assay.

Sodium

(a) Reagents. (1) 0.01N thiosulfate (2.482 grams Na2S2O35H2O and 125 milligrams Na2CO3 per liter). (2) 1.0N Sodium hydroxide.

(3) 1.2N Hydrochloric acid.

(4) 0.01N Iodine solution (prepared from 0.1N iodine U.S.P.).

(5) Starch iodide paste, T.S. (U.S.P.). (b) Preparation of sample and working standard solutions-(1) Workingstandard solutions. From the following table, select the initial solvent, diluent, and final concentration as listed for each antibiotic working standard. Dissolve and dilute an accurately weighed portion to the specified final concentration and proceed as directed in paragraphs (c) and (d) of this section.

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(2) Bulk antibiotic solutions. From the following table, select the initial solvent, diluent, and final concentration as listed for each antibiotic. Dissolve an accurately weighed aliquot (approximately 30 to 60 milligrams) of the sample, dilute to the appropriate final concentration, and proceed as directed in paragraphs (c) and (d) of this section.

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1 Allow to stand in 1N NaOH for 15 minutes before assaying.

2 The final concentration of bacampicillin hydrochloride is calculated in milligrams of ampicillin activity per milliliter of sample. The ampicillin working standard is used for the assay of bacampicillin hydrochloride.

(3) Finished product solutions. Prepare the sample for assay as directed in the individual section for each antibiotic product to be tested by diluting to the concentration prescribed in the table in paragraph (b)(2) of this section and proceed as described in paragraphs (c) and (d) of this section.

(c) Inactivated sample and standard solutions. (1) Transfer 2.0 milliliters each of the sample and the appropriate working standard solutions to glassstoppered Erlenmeyer flasks.

(2) Add 2.0 milliliters of 1N sodium hydroxide, except if the sample has been diluted in 1N sodium hydroxide, and allow to stand at room temperature for 15 minutes.

(3) Add 2.0 milliliters of 1.2N hydrochloric acid.

(4) Add 10.0 milliliters of 0.01N iodine solution, stopper, allow to stand at room temperature for 15 minutes, and proceed as directed in paragraph (e) of this section.

(d) Blank determination. Transfer 2.0 milliliters each of the sample and the appropriate working standard solutions to glass-stoppered Erlenmeyer flasks. Add 10.0 milliliters of 0.01N iodine solution and immediately proceed as directed in paragraph (e) of this section.

(e) Titration procedure. Titrate the excess iodine using 0.01N sodium thiosulfate. Toward the end of the ti

tration, add 1 drop of the starch iodide paste. Continue the titration by the addition of 0.01- to 0.02-milliliter portions of 0.01N sodium thiosulfate, shaking vigorously after each addition. The endpoint is reached when the blue color of the starch-iodine complex is discharged. Calculate the antibiotic content as described in paragraph (f) of this section.

(f) Calculations—(1) F factor determination. Using the appropriate working standard for the particular antibiotic to be tested, assay the standard as directed in this section. Calculate the F factor (the units of micrograms of activity equivalent of each milliliter of 0.01N sodium thiosulfate consumed) by means of the following formula:

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nents. To 1 volume of this neutralized solution add 8 volumes of distilled water and 2 volumes of 95 percent ethanol. This solution should be used for 1 day only.

(4) Ferric ammonium sulfate. Dissolve 272 grams of ferric ammonium sulfate in a mixture of 26 milliliters of concentrated sulfuric acid and sufficient distilled water to make 1 liter. This reagent may be used for 1 week when stored in a brown bottle at room temperature.

(b) Preparation of working standard solutions. From the following table, select the diluent and final concentration as listed for each antibiotic working standard. Dissolve and dilute an accurately weighed portion to the specified final concentration and proceed as directed in paragraph (d) of this section.

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d=Dilution factor for the sample;

n=Number of doses or items in the sample assayed.

[39 FR 18944, May 30, 1974, as amended at 39 FR 34032, Sept. 23, 1974; 42 FR 59856, Nov. 22, 1977; 44 FR 10378, Feb. 20, 1979; 46 FR 2980, Jan. 13, 1981; 46 FR 25602, May 8, 1981; 46 FR 46312, Sept. 18, 1981; 46 FR 58298, Dec. 1, 1981; 46 FR 61072, Dec. 15, 1981; 49 FR 6091, Feb. 17, 1984]

§ 436.205 Hydroxylamine colorimetric assay.

(a) Reagents-(1) Hydroxylamine hydrochloride solution. Dissolve 350 grams of hydroxylamine hydrochloride in sufficient distilled water to make 1 liter.

(2) Buffer. Dissolve 173 grams of sodium hydroxide and 20.6 grams of sodium acetate in sufficient distilled water to make 1 liter.

(3) Neutral hydroxylamine. Mix 1 volume each of hydroxylamine hydrochloride solution described in paragraph (a)(1) of this section and the buffer described in paragraph (a)(2) of this section. Check the pH and if necessary adjust to pH 7.0±0.1 by adding an additional amount of one of the compo

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'To prepare the working standard solution, proceed as directed in the individual section of the antibiotic drug regulation in this chapter for the antibiotic to be tested.

(c) Preparation of sample solutions. From the following table, select the diluent and final concentration as listed for each antibiotic. Dissolve an accurately weighed portion of the sample, dilute to the appropriate final concentration, and proceed as directed in paragraph (d) of this section; if the product is packaged for dispensing, dilute an aliquot of the stock solution (prepared as described in the individual monograph) to the appropriate concentration and then proceed as directed in paragraph (d) of this section.

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