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in a photoelectric colorimeter. The suspension may be used for 2 weeks if it is stored under refrigeration.Staphylococcus epidermidis (ATCC 12228), 1 which is maintained on agar as described in $ 440.80a(b)(1)(ii)(a) of this chapter, may also be used as the test organism. From a stock slant, inoculate a Roux bottle containing this medium and incubate for 24 hours at 32° C.-35° C. Wash the resulting growth from the agar surface, using approximately 30 milliliters of sterile sodium chloride solution. Standardize the suspension by determining the dilution that will permit 80 percent light transmission through a filter of 6500 Angstrom units in a photoelectric colorimeter. The suspension may be stored for 2 weeks under refrigeration.

(vi) Preparation of plates. Using the agar described in subdivision (ii) of this subparagraph and approximately a 0.5 percent inoculum of the suspension described in paragraph (b)(1)(v) of this section, prepare the plates as directed in $ 440.80a(b)(1)(v) of this chapter.

(vii) Assay. Dissolve volumetrically in 0.1 M potassium phosphate buffer, PH 7.8 to 8.0, the sample to be tested to make a convenient stock solution. Further dilute volumetrically this solution with 0.1 M potassium phosphate buffer, pH 7.8 to 8.0, to a final concentration of 10.0 micrograms (estimated) per milliliter, if the test organism is Staphylococcusaureus or 1.0 microgram per milliliter (estimated) if the test organism is Staphylococcus epidermidis.

(2) Toxicity. Proceed as directed in $ 440.80a(b)(4) of this chapter, using 0.5 milliliter of a solution prepared by diluting the sample to approximately 200 micrograms per milliliter with physiological salt solution.

(3) Moisture. In an atmosphere of about 10 percent relative humidity, transfer about 100 milligrams of the finely powdered sample to a tared weighing bottle equipped with groundglass top and stopper. Weigh the bottle and place it in a vacuum oven, tilting the stopper on its side so that there is no closure during the drying period. Dry at a temperature of 60° C. and a pressure of 5 millimeters of mercury or less for 3 hours. At the end of the drying period fill the vacuum oven with air dried by passing it through a drying

$ 436.542 Acid resistance/dissolution

test for enteric-coated erythro

mycin pellets. (a) Equipment. Use Apparatus 1 as described in the United States Pharmacopeia XX dissolution test.

(b) Immersion fluids. All immersion fluids may be degassed by heating immediately prior to use.

(1) Acid resistance medium. Use 0.06N hydrochloric acid, pH 1.2.

(2) Dissolution medium. Dissolve 6.8 grams of monobasic potassium phosphate in 250 milliliters of water. Add 109 milliliters of 0.2N sodium hydroxide and 400 milliliters of water and adjust the resulting solution with 0.2N sodium hydroxide to a pH of 6.8+0.1. Dilute to 1 liter.

(c) Procedure. Warm the immersion fluids to a temperature of 37° +5.0° C. Place the contents of one capsule into the basket. Lower the basket into 900 milliliters of acid resistance medium contained in the beaker. Ensure that all air is displaced from the immersed basket and that the pellets remain in the basket. Rotate the basket at the speed of 50 revolutions per minute for an accurately timed period of 1 hour. Remove the basket from the fluid and immediately lower the basket into 900 milliliters of dissolution medium contained in the beaker. Again ensure that all air is displaced from the immersed basket and that the pellets remain in the basket. Rotate the basket at 50 reyolutions per minute for an accurately timed dissolution period of 45 minutes. Withdraw a 25-milliliter sample of the dissolution medium from a point midway between the stirring shaft and the wall of the vessel and approximately midway in depth. Filter the sample through a Whatman 541 filter paper or equivalent, discarding the first 2 milliliters. Assay for erythromycin using

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Milligrams of doxycycline dissolved

As

where:

Av=Absorbance of sample; As=Absorbance of standard; c=Concentration of working standard in

milligrams; and d=Dilution factor of sample withdrawn

from beaker. *If more than 15 milliliters of dissolution

medium is removed, correct for the volume removed.

(e) Evaluation. The pellet-filled capsule passes the test if no more than 50 percent of the drug is dissolved at 20 minutes. If one pellet-filled capsule fails to meet this requirement, repeat the test on six additional pellet-filled capsules. No more than 2 pellet-filled capsules in 12 may exceed 50 percent of the drug dissolved at 20 minutes.

(50 FR 41679, Oct. 15, 1985; 50 FR 45603, Nov. 1, 1985)

8 436.543 Acid resistance test for pel

let-filled doxycycline hyclate cap

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

(b) Acid resistance medium. Use 0.06N hydrochloric acid, pH 1.2. Maybe degassed by heating immediately prior to use.

(c) Procedure. Warm the acid resistance medium to a temperature of 37+2.0 • C. Place the contents of one pelletfilled capsule into the basket. Lower the basket into a beaker containing 900 milliliters of acid resistance medium. Ensure that all air is displaced from the immersed basket and that the contents of the pellet-filled capsule remain in the basket. Rotate the basket at the speed of 50 revolutions per minute for an accurately timed period of 20 minutes. Withdraw a 5-milliliter sample of the acid resistance medium from a point midway between the stirring shaft and the wall of the vessel and approximately midway in depth (this is the sample solution). Assay the sample solution for doxycycline as described in paragraph (d) of this section. Repeat the test on five additional pellet-filled capsules.

(d) Doxycycline content-(1) Preparation of working standard solution. Dissolve an accurately weighed portion of doxycycline hyclate working standard with 0.1M hydrochloric acid to obtain a concentration of 0.01 milligram per milliliter.

(2) Preparation of sample solution. Dilute the 5-milliliter sample portion to 25 milliliters with 0.1M hydrochloric acid.

(3) Procedure. Using a suitable spectrophotometer and 0.1M hydrochloric acid as the blank, determine the absorbance of each standard and sample solution at the absorbance peak at ap

8 436.544 Dissolution test for pellet

filled doxycycline hyclate capsules. (a) Equipment. Use Apparatus 1 as described in the United States Pharmacopeia XXI dissolution test.

(b) Dissolution medium. Prepare the dissolution medium as follows: Dissolve 10.21 grams

of potassium biphthalate and 1.4 grams of sodium hydroxide in approximately 950 milliliters of distilled water and adjust the pH to 5.5 using 1M sodium hydroxide solution. Dilute with distilled water to 1,000 milliliters.

(c) Procedure. Proceed as directed in the United States Pharmacopeia XXI dissolution test. Ensure that all air is displaced from the immersed basket and that the contents of the pelletfilled capsule remain in the basket. Rotate the basket at the speed of 50 revolutions per minute for an accurately timed period of 30 minutes. Withdraw a 5-milliliter sample of the dissolution medium from a point midway between the stirring shaft and the wall of the

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medium. Rotate the paddle at the speed of 50 revolutions per minute for an accurately timed period of 1 hour. Withdraw a 50-milliliter sample of the dissolution medium from a point midway between the stirring shaft and the wall of the vessel and approximately midway in depth. Filter the sample through a Whatman No. 1 filter paper or equivalent, discarding the first 5.0 milliliters. Assay for dissolved erythromycin as directed in paragraph (d) of this section using the filtrate as the sample solution. Repeat the test on five additional tablets.

(d) Arsenomolybdate colorimetric assay for dissolved erythromycin-(1) Apparatus. Automatic analyzer consisting of (i) a liquid sampler, (ii) a proportioning pump, (iii) suitable spectrophotometers equipped with matched flow cells and analysis capability at 660 nanometers, (iv)

means of recording spectrophotometric readings, and (v) a manifold consisting of the components illustrated in the diagram in paragraph (d)(4) of this section.

(2) Reagents4i) Arsenomolybdate solutions-(a) Stock solution. Dissolve 100 grams of ammonium molybdate in approximately 1,700 milliliters of water contained in a 2-liter volumetric flask. Insert an inert plastic coated stirring bar into the flask, and begin mixing. While mixing, slowly add 84 milliliters of sulfuric acid (temperature of solution should not exceed 50 °C). Dissolve 12 grams of sodium arsenate in 100 milliliters of water, and add to the solution in the flask. Remove the stirring bar, dilute with water to volume, and mix. Store in an amber bottle for 24 hours before using. (This solution should not be allowed to come into contact with rubber.)

(b) Working solution. Dilute 1 part of stock solution with 2 parts of water, and mix. This solution is freshly prepared on the day of use.

(ii) Acetate buffer, pH 4.8. Dissolve 133 grams of ACS grade sodium acetate crystals in about 3.5 liters of water. Adjust the pH to 4.8+0.1 with glacial acetic acid. Dilute with water to 4,000 milliliters, and mix.

(iii) 9N Sulfuric acid. Place a 2-liter volumetric flask containing an inert plastic coated magnetic stirring bar and about 1,500 milliliters of water in

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an ice bath, and begin mixing. While mixing, cautiously add 300 milliliters of sulfuric acid. Allow the solution to cool. Remove the stirring bar, dilute with water to volume, and mix.

(3) Preparation of working standard solutions—(i) Working standard stock solution. Accurately weigh approximately 400 milligrams of USP Erythromycin Reference Standard, previously dried at 60 °C for 3 hours under vacuum (pressure of 5 millimeters of mercury or less), and transfer to a 100-milliliter volumetric flask. Dissolve and dilute with acetate buffer, pH 4.8 to volume, and mix.

(ii) Working standard solutions. Pipet 5, 10, 15, and 20 milliliters of the standard stock solution into separate 500milliliter volumetric flasks, add acetate buffer, pH 4.8 to volume, and mix. The approximate concentrations of these solutions (before adjusting for the standard potency) are 40, 80, 120,

and 160 micrograms of erythromycin per milliliter, respectively.

(4) Procedure. Use the working standard solutions prepared as described in paragraph (d)(3) of this section. The arrangement of the apparatus and flow of the samples and reagents are shown in the manifold diagram set forth following this paragraph. The sampler rate is usually 60 per hour, but may be varied. Establish a steady state by pumping reagents until the record trace becomes constant. Place cups containing the four concentrations of working standard solutions in the sampler followed by no more than 12 cups of sample solutions. Then place four more cups containing the four concentrations of working standard solutions in the sampler. Repeat the sequence above for additional samples by bracketing standards around no more than 12 sample solutions at a time.

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