capsule (except fragments of the insoluble coating) remaining on the screen is a soft mass having no palpably firm core. The tablets, pastilles, or capsules pass the disintegration test if all of the units tested disintegrate completely under the conditions and time specified in the individual section for the antibiotic tablet, pastille, or capsule being tested. If one or two tablets, pastilles, or capsules fail to disintegrate completely, repeat the test on 12 additional tablets, pastilles, or capsules. The tablets, pastilles, or capsules pass the disintegration test if not less than 16 of the total 18 tested disintegrate completely. Enteric coated tablets fail the disintegration test if they show any distinct evidence of dissolution or disintegration after 1 hour immersion in simulated gastric fluid.

[39 FR 18944, May 30, 1974, as amended at 52 FR 4617, Feb. 13, 1987; 55 FR 19873, May 14, 1990]

§ 436.213 Nonaqueous titrations.

(a) Equipment—(1) Apparatus. Use a closed system consisting of a suitable titrimeter equipped with a potentiometer, an automatic burette, a chart recorder, and a glass calomel combinatin electrode (with saturated methanolic potassium chloride as the electrolyte).

(2) Titration vessel. Use a 100-milliliter tall form beaker without a spout.

(b) Reagents-(1) Methyl alcohol, reagent grade, anhydrous.

(2) Dimethylsulfoxide, A.C.S., reagent grade.

(3) Glacial acetic acid, A.C.S., reagent grade.

(4) Lithium methoxide reagent: 0.02N lithium methoxide in methyl alcohol, standardized against primary grade benzoic acid.

(5) Perchloric acid reagent: 0.02N perchloric acid in glacial acetic acid, standardized against primary grade potassium acid phthalate.

(c) Preparation of sample solutions. Select the weight of the sample and the solvent listed for for each antibiotic. Transfer the accurately weighed sample to a titration vessel. Add the appropriate solvent, cover, and stir magnetically until the sample is dissolved. Proceed as directed in paragraph (e) of

this section, using the procedure or procedures specified in the individual section for each antibiotic.

Cyclacillin-base titration Tobramycin-base titration. *The methyl alcohol is added after the sample has dissolved in dimethylsulfoxide.

(d) Blank determination. Place the same volume of solvent used to prepare the sample solution into a titration vessel and proceed as directed in paragraph (e) of this section, using the procedure or procedures specified in the individual section for each antibiotic.

(e) Titration procedures—(1) Acid titration. Equilibrate the electrode by soaking it overnight in the solvent used for preparing the sample solution. Start the magnetic stirrer and titrate the sample solution with the lithium methoxide reagent. Record the change in potential of the solution with the addition of the titrant. Determine the number of milliliters of reagent consumed at neutralization (the inflection point of the titration curve). Calculate the antibiotic content as directed in the individual section.

(2) Base titration. Proceed as directed in paragraph (e)(1) of this section, except use the perchloric acid reagent as the titrant and calculate the antibiotic

content as directed in the individual section.

[39 FR 18944, May 30, 1974, as amended at 40 FR 22251, Apr. 22, 1975; 40 FR 23725, June 2, 1975; 40 FR 57797, Dec. 12, 1975; 46 FR 2981, Jan. 13, 1981]

§ 436.214 Heat stability.

Store an accurately weighed portion of the sample of approximately 30 milligrams in an unstoppered 50-milliliter Erlenmeyer flask for 4 days in an electric oven at 100° C±1° C. At the end of this period, remove the flask from the oven and allow to cool in a desiccator. Accurately weigh an unheated portion of the original sample of approximately 30 milligrams. Assay both the heated and unheated samples for potency as directed in §436.204 or § 436.205 of this chapter. Determine the percent loss from the difference in potency between the unheated original sample and the heat-treated sample.

[42 FR 59856, Nov. 22, 1977]

§ 436.215 Dissolution test.

(a) Equipment. Use either Apparatus 1 or 2 as described in the United States Pharmacopeia XXI dissolution test.

(b) Procedure. For each dosage form listed in the table in this paragraph select the appropriate dissolution medium, rotation rate, sampling time, and apparatus, and proceed as set forth in either Apparatus 1 or 2 methodology of the United States Pharmacopeia XXI dissolution test. Determine the amount of drug substance dissolved by performing the assay described in paragraph (c) of this section. The amount of dissolution medium removed for sampling purposes may be disregarded if the amount removed is not more than 15 milliliters. If more than 15 milliliters is removed, then correct for the volume removed.

(c) Antibiotic drug content—(1) Tetracycline hydrochloride—(i) Preparation of working standard solution. Accurately weigh 20 to 30 milligrams of tetracycline hydrochloride working standard into a suitable-sized volumetric flask. Dissolve and dilute to volume with water. Further dilute an accurately measured portion with distilled water to obtain a known concentration of 0.01 to 0.02 milligram of tetracycline hydrochloride per milliliter.

(ii) Preparation of sample solutions. Dilute an accurately measured portion of the sample with sufficient distilled water to obtain a concentration of 0.01 to 0.02 milligram of tetracycline hydrochloride per milliliter (estimated).

(iii) Procedure. Using a suitable spectrophotometer and water as the blank, determine the absorbance of each standard and sample solution at the absorbance peak at approximately 276 nanometers. Determine the exact position of the absorption peak for the particular instrument used.

(iv) Calculation. Determine the total amount of tetracycline hydrochloride dissolved as follows:

T-Total milligrams of drug dissolved;
Au-Absorbance of sample;

c-Concentration of standard in milligrams;
d-Dilution factor of sample filtrate;
A-Absorbance of standard.

*If more than 15 mL of dissolution medium is removed, correct for the volume removed.

(2) Oxytetracycline hydrochloride; preparation of working standard-solution. (1) Accurately weigh 30 milligrams of oxytetracycline-base working standard into a suitable-sized volumetric flask. Add 5 milliliters of 0.1N hydrochloric acid and swirl the flask to dissolve oxytetracycline base. Dilute an accurately measured portion with distilled water to obtain a known concentration of 0.01 to 0.02 milligram of oxytetracycline per milliliter.

(ii) Proceed as directed in paragraphs (c)(1) (ii), (iii), and (iv) of this section except measure the absorbance at the absorption peak at approximately 273 nanometers.

(3) Doxycycline hyclate. Proceed as directed in paragraph (c)(1) of this section, except use the doxycycline working standard.

(4) Bacampicillin hydrochloride. Use the ampicillin working standard as the standard of comparison and assay for ampicillin content by either of the following methods.

(1) Iodometric assay. Proceed as directed in § 436.204 of this chapter, except dilute the working standard to a final concentration of 0.3 milligram of ampicillin per milliliter and use the sample solution as it is removed from the dissolution vessel without further dilution.

(ii) Hydroxylamine colorimetric assay. Proceed as directed in § 442.40(b)(1)(ii) of this chapter, except:

(a) Buffer. In lieu of the buffer described in § 442.40(b)(1)(ii)(b)(2) of this chapter, use the buffer prepared as follows: Dissolve 200 grams of primary standard tris (hydroxymethyl) aminomethane in sufficient distilled water to make 1 liter. Filter before use.

(b) Preparation of the working standard solution. Dissolve and dilute an accurately weighed portion of the ampicillin working standard with sufficient distilled water to obtain a final concentration of 0.3 milligram of ampicillin per milliliter;

(c) Sample solution. Use the sample solution as it is removed from the dissolution vessel without further dilution; and

(d) Calculations. Determine the total amount of ampicillin dissolved as follows:

into a suitable-sized volumetric flask. Dissolve and dilute to volume with 0.12N hydrochloric acid. Further dilute with a buffer solution (prepared by dissolving 27.2 grams of sodium acetate trihydrate in a mixture of 12 milliliters of glacial acetic acid and sufficient distilled water to make 2 liters) to obtain a known concentration of 0.01 to 0.03 milligram of cephradine per milliliter.

(ii) Preparation of sample solution. Filter the sample and dilute an accurately measured portion of the filtrate with sufficient buffer solution, described in paragraph (c)(5)(i) of this section, to obtain a concentration of 0.01 to 0.03 milligram of cephradine per milliliter (estimated).

(iii) Proceed as directed in paragraphs (c)(1) (iii) and (iv) of this section, except measure the absorbance at the absorption peak at approximately 262 nanometers.

(6) Amoxicillin trihydrate. Assay for the amoxicillin content as described in § 440.103d of this chapter, except use the sample as it is removed from the dissolution vessel.

(7) Vancomycin hydrochloride. Assay for the vancomycin content as described in § 436.105 of this chapter, except use the sample as it is removed from the dissolution test.

(8) Erythromycin-(i) Preparation of working standard solution. Accurately weigh approximately 140 milligrams of erythromycin working standard into a 250-milliliter volumetric flask and dissolve in 10 milliliters of methyl alcohol. Add water nearly to volume, mix, and allow the solution to cool. Dilute to volume with water and mix. On the day of use, dilute an accurately measured aliquot with water to obtain a known concentration of 0.28 milligram of erythromycin per milliliter (before adjusting for standard potency).

(ii) Preparation of sample solution. Dilute an accurately measured portion of the filtered sample with sufficient 0.05M potassium phosphate buffer, pH 6.8, to obtain a concentration of about 0.28 milligram of erythromycin per milliliter (estimated).

(iii) Procedure. Transfer 5.0-milliliter aliquots of the working standard solution and sample solution to 25-milliliter volumetric flasks and treat as follows: Add 2.0 milliliters of water, allow

to stand for 5 minutes with intermittent swirling. Add 15.0 milliliters of 0.25N sodium hydroxide, dilute to volume with sufficient 0.05M potassium phosphate buffer, pH 6.8, and mix. Heat to 60 °C for 5 minutes and allow to cool. Using a suitable spectrophotometer and a blank (prepared as per the procedure above except that 2.0 milliliters of 0.5N sulfuric acid is substituted for the 2.0 milliliters of water) for each solution, determine the absorbance of each working standard and sample solution at the absorbance peak at approximately 236 nanometers. Determine the exact position of the absorption peak for the particular instrument used.

(iv) Calculation. Proceed as directed in paragraph (c)(1)(iv) of this section. (9) Cefuroxime axetil tablets and powder for oral suspension—(i) Preparation of working standard solution—(a) Cefuroxime axetil tablets. Accurately weigh approximately 60 milligrams of cefuroxime axetil working standard into a suitable-sized volumetric flask. Dissolve in 5 milliliters of methanol and dilute to volume with 0.07N hydrochloric acid to obtain a known concentration equivalent to 0.01 to 0.02 milligram of cefuroxime activity per milliliter.

(b) Cefuroxime axetil for oral suspension. Accurately weigh approximately 15 milligrams of cefuroxime axetil working standard into a 100-milliliter volumetric flask. Dissolve in 5 milliliters of methanol and dilute to volume with Sorenson's Modified Phosphate Buffer, pH 7.0 (4.2 grams of sodium dihydrogen orthophosphate dihydrate and 14.3 grams of hydrogen disodium orthophosphate dodecahydrate per liter of water).

(ii) Preparation of sample solution-(a) Cefuroxime axetil tablets. Filter through a 0.45-micron filter and dilute an accurately measured portion of the filtrate with sufficient 0.07N hydrochloric acid to obtain a concentration equivalent to 0.01 to 0.02 milligram of cefuroxime activity per milliliter (estimated).

(b) Cefuroxime axetil for oral suspension. Filter the sample through an 8micron filter. A coarse prefilter may be used to prevent clogging. Use the filtrate solution without further dilution.

(iii) Procedure—(a) Cefuroxime axetil tablets. Using a suitable spectrophotometer and 0.07N hydrochloric acid as the blank, determine the absorbance of each standard and sample solution at the absorbance peak at approximately 280 nanometers. Determine the exact position of the absorption peak for the particular instrument used.

(b) Cefuroxime axetil for oral suspension. Using a suitable spectrophotometer and Sorenson's Modified Phosphate Buffer, pH 7.0 (4.2 grams of sodium dihydrogen orthophosphate dihydrate and 14.3 grams of hydrogen disodium orthophosphate dodecahydrate per liter of water) as the blank, determine the absorbance of each standard and sample solution at the absorbance peak at approximately 280 nanometers. Determine the exact position of the absorption peak for the particular instrument used.

(iv) Calculations. Determine the total amount of cefuroxime activity dissolved as follows:

T = Total milligrams of cefuroxime activity dissolved;

Αν = Absorbance of sample;

c = Cefuroxime activity of working standard solution in milligrams per milliliter; d = Dilution factor of sample filtrate; and A1 = Absorbance of standard.

(10) Cefixime—(i) Preparation of working standard solution. Accurately weigh approximately 25 milligrams of cefixime working standard into a 500milliliter volumetic flask. Wet the powder with 0.5 milliliters of methanol, and dilute to volume with 0.05 M potassium phosphate buffer, pH 7.2 (prepared by dissolving 6.8 grams of monobasic potassium phosphate in distilled water to a volume of one liter. The pH is adjusted to 7.2 with 1.0N NaOH). Sonicate to assure dissolution and mix.

(ii) Preparation of sample solution. Forty-five minutes after the beginning of the rotation, withdraw and filter a portion of the solution. For the 400milligram tablets, pipet 10.0 milliliters of the filtered sample solution into a 100-milliliter volumetric flask. For the 200-milligram tablets, pipet 10.0 milli

liters of the filtered sample into a 50milliliter volumetric flask. Dilute to volume with 0.05 M postassium phosphate buffer, pH 7.2.

(iii) Procedure. Proceed as directed in paragraphs (c)(1) (iii) and (iv) of this section, except measure the absorbance of the peak at peak at approximately 320 nanometers using 0.05 M potassium phosphate buffer, pH 7.2 as the blank. (11) Cephalexin hydrochloride monohydrate. Assay for cephalexin activity of the cephalexin hydrochloride monohydrate as directed in §442.28 of this chapter, and use U.S.P. dissolution apparatus 1 (10 mesh basket). Use the sample as it is removed from the dissolution vessel.

(12) Doxycycline monohydrate. Proceed as directed in paragraph (c)(1) of this section, except use the doxycycline standard.

(13) Clarithromycin. Proceed as directed in § 452.50(b)(1) of this chapter except:

phase

(i) Dissolution medium. Instead of the mobile described in § 452.50(b)(1)(i) of this chapter, use 0.10 M sodium acetate buffer prepared as follows: Weigh 13.6 grams of sodium acetate trihyrate into a container sufficient to hold 1 liter of solution. Dissolve the salt in 750 milliliters of distilled water. Adjust the pH of the solution to 5.0±0.05 with glacial acetic acid. Dilute to 1,000 milliliters with distilled water.

(ii) Preparation of the standard and sample solutions—(a) Standard solution. Dissolve (with shaking or sonication) an accurately weighed portion of the clarithromycin working standard, in sufficient methanol to obtain a solution having a known concentration of approximately 625 micrograms per milliliter of clarithromycin. Quantitatively transfer and dilute an aliquot of this solution with mobile phase (described in §452.50(b)(1)(i) of this chapter) and mix to obtain a solution of known concentration of approximately 125 micrograms per milliliter of clarithromycin.

(b) Sample solution. Use the sample solution as it is removed from the dissolution vessel after diluting and mixing with mobile phase (described in § 452.50(b)(1)(i) of this chapter) 1:2 for