§ 436.514 21 CFR Ch. I (4–1–98 Edition)
chlortetracycline hydrochloride tro- meyer flask, held in a horizontal posi- ches proceed as directed in tion. Add 500 milliliters of 0.1 N HCl § 446.10a(b)(1) of this chapter, except and shake to dissolve the contents. Im- § 446.10a(b)(1)(x), and in lieu of the di- mediately remove aliquots of this solu- rections in § 446.10a(b)(1)(iv) and tion and, using 0.1 M potassium phos- (viii)(c) of this chapter prepare the phate buffer, pH 4.5, for further dilu- sample as follows: Place 12 troches in a tions, proceed as directed in glass blending jar containing 500 milli- § 446.10a(b)(1) of this chapter if it is liters of 0.01N HCl. Using a high-speed chlortetracycline hydrochloride pow- blender, blend for 3 to 5 minutes and der or § 446.81a(b)(1) of this chapter if it then make the proper estimated dilu- is tetracycline hydrochloride powder. tions in the buffer solution. The aver- Calculate the average total amount of age potency of the troches is satisfac- antibiotic expelled from the contain- tory if they contain not less than 85 ers. The total potency is satisfactory if percent of the number of milligrams it contains not less than 85 percent of they are represented to contain. the number of milligrams of chlor- (b) Moisture. Proceed as directed in tetracycline hydrochloride or tetra- § 440.80a(b)(5)(i) of this chapter. cycline hydrochloride that it is rep- resented to contain. § 436.514 Chlortetracycline hydro- (b) Moisture. Proceed as directed in chloride powder topical; tetra- § 440.80a(b)(5)(i) of this chapter, except cycline hydrochloride powder topi- if it is packaged with inert gases pro- cal. ceed as directed in § 536.513(c) of this (a) Potency—(1) Dry powder. Using a chapter. 3.0-gram sample or the entire contents of the immediate container for each de- [39 FR 18944, May 30, 1974, as amended at 40 FR 13497, Mar. 27, 1975] termination, prepare the sample as fol- lows: Using a high-speed blender, blend § 436.515 Capsules tetracycline and a 3.0-gram sample in a glass blending oleandomycin phosphate; capsules jar containing 500 milliliters of 0.01 N tetracycline and troleandomycin; HCl (use 0.1 N HCl if it is tetracycline), capsules tetracycline hydrochloride or reconstitute in the immediate con- and oleandomycin phosphate; cap- tainer as directed in the labeling of the sules tetracycline hydrochloride drug. Transfer an appropriate aliquot and troleandomycin. of 1.0 milliliter to 5.0 milliliters to a (a) Potency—(1) Tetracycline or tetra- 100-milliliter volumetric flask and cycline hydrochloride content by turbi- make to mark with 0.01 N HCl (use 0.1 dimetric assay—(i) Test culture and N HCl if it is tetracycline). Withdraw media. Maintain the test organism an aliquot from the volumetric flask, Escherichia coli (ATCC 10536) 1 on the and if it is chlortetracycline hydro- agar described in § 440.80a(b)(1) (ii)(a) of chloride dilute to 0.06 µ g. per milli- this chapter. For use in the assay, pre- liter, using 0.1 M potassium phosphate pare a suspension of the organism buffer, pH 4.5, and proceed as directed every 2 weeks, as follows: Transfer the in § 446.10a(b)(1) of this chapter. If it is organism to a fresh agar slant and in- tetracycline hydrochloride, dilute to cubate at 37°C. overnight. Wash the 0.24 µ g. per milliliter, using 0.1 M po- growth from the slant with the aid of 2 tassium phosphate buffer, pH 4.5, and milliliters of sterile distilled water and proceed as directed in § 446.81a(b)(1) of sterile glass beads into a Roux bottle this chapter. The average potency is containing 300 milliliters of the main- satisfactory if it contains not less than tenance medium. Incubate overnight at 85 percent of the number of milligrams 37° C. and then harvest the growth with of chlortetracycline hydrochloride or 50 milliliters of sterile distilled water tetracycline hydrocloride per gram or and sterile glass beads. Standardize per immediate container that it is rep- this suspension by determining the di- resented to contain. lution that will permit 40-percent light (2) Powder packaged with inert gases. Spray, as directed in the labeling, the 1 Available from: American Type Culture entire contents of each container to be Collection, 12301 Parklawn Drive, Rockville, tested into a separate 2-liter Erlen- MD 20852.
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transmission in a photoelectric col- through the points, either by inspec- orimeter using a 650-millimicron filter tion or by means of the following equa- and an 18-millimeter diameter test tions: tube as an absorption cell. Prepare the daily inoculum by adding 10 milliliters L=(3a+2b+c¥e)/5, of that dilution to each liter of nutri- H=(3e+2d+c¥a)/5, ent broth, prepared as directed in where: § 440.80a (b)(1)(ii)(c) of this chapter, L=absorbance value for the lowest con- needed for the test. centration of the standard curve. H=absorbance value for the highest con- (ii) Working standard and solutions. centration of the standard curve. Dissolve an appropriate amount of the a, b, c, d, e=average absorbance values for working standard in sufficient 0.1 N each concentration of the standard HCl to give a concentration of 1,000 curve. micrograms per milliliter. This stock Plot the values obtained for L and H solution may be kept in the refrig- and connect the points with a straight erator for 1 week. Make daily dilutions line. Average the absorbance values for of the stock solution with 0.1 M potas- the sample and read the tetracycline or sium phosphate buffer (pH 4.5) to ob- tetracycline hydrochloride concentra- tain concentrations of 0.146, 0.187, 0.240, tion from the standard curve. Multiply 0.308, and 0.395 micrograms per milli- the concentration by appropriate dilu- liter. Add 1.0 milliliter of each such tion factors to obtain the tetracycline concentration to each of three 16 milli- or tetracycline hydrochloride content meters x 125 millimeters test tubes. of the sample. Its potency is satisfac- (iii) Preparation of sample. Dissolve tory if it contains the equivalent of not the contents of a representative num- less than 85 percent of the number of ber of capsules in sufficient 0.1 N HCl milligrams of tetracycline hydro- to give a stock solution of convenient chloride that it is represented to con- concentration. Further dilute the tain. stock solution with 0.1 M potassium (2) Oleandomycin content. (i) If phosphate buffer (pH 4.5) to obtain a oleandomycin phosphate is used, pro- final concentration of 0.24 microgram ceed as directed in paragraph (c)(1) of per milliliter (estimated). Add 1.0-mil- this section, except prepare the sample liliter of this dilution to each of three as follows: Dissolve the contents of a 16 millimeters x 125 millimeters test representative number of capsules in tubes. sufficient 0.1 M potassium phosphate (iv) Procedure. To each of the 16 milli- buffer (pH 8.0) to give a stock solution meters x 125 millimeters test tubes pre- of convenient concentration. Further pared in paragraph (a)(1)(ii) and (iii) of dilute with 0.1 M potassium phosphate this section, add 9.0 milliliters of the buffer (pH 8.0) to obtain a final con- inoculated nutrient broth described in centration of 5.0 µ g. of oleandomycin paragraph (a)(1)(i) of this section and activity per milliliter (estimated). place immediately in a 37° C. water (ii) If troleandomycin is used, pro- bath for 3 to 4 hours. After incubation, ceed as follows: Dissolve the contents add 0.5 milliliter of a 12-percent form- of a representative number of capsules aldehyde solution to each tube and in chloroform to give a stock solution read the absorbance values in a suit- of 1.0 milligram of oleandomycin activ- able photoelectric colorimeter using a ity per milliliter. Transfer 30 milli- wavelength of 530 millimicrons. Set the liters of the chloroform solution to a instrument at zero absorbance with glass-stoppered test tube (200 millime- clear uninoculated broth prepared as ters x 22 millimeters) and add 20 milli- described in § 440.80a(b)(1)(ii)(c) of this liters of 1 N sodium hydroxide. Shake chapter. for 1 minute and centrifuge briefly to (v) Estimation of potency. Plot the av- aid in the separation of the layers. erage values for each concentration of With the aid of a syringe and needle, the standard on arithmetic graph paper remove and discard the aqueous layer. with absorbance values on the ordinate Repeat the washing procedure with two and tetracycline or tetracycline hydro- more 20-milliter portions of 1 N sodium chloride concentrations on the ab- hydroxide solution. Filter the chloro- scissa. Construct the best straightline form layer through a pledget of cotton.
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Dilute an aliquot of this solution with iological saline solution onto a large chloroform to give a solution contain- agar surface such as that provided by a ing approximately 25 µ g. of Roux bottle containing 300 milliliters oleandomycin per milliliter. Transfer a of the agar described in paragraph 5.0 milliliter aliquot to a 40 milliliter (c)(1)(ii)(a) of this section. Spread the glass-stoppered centrifuge tube, dilute suspension of organisms over the entire to 20 milliliters, with chloroform, and agar surface with the aid of sterile determine the oleandomycin content glass beads. Incubate for 4 hours at 32° as directed in paragraph (d)(1)(i) of this C. and then wash the resulting growth section. from the agar surface with about 30 Its content of oleandomycin is satisfac- milliliters of sterile physiological sa- tory if it contains not less than 85 per- line solution. Standardize the suspen- cent of the number of milligrams that sion by determining the dilution that it is represented to contain. will give 80-percent light transmission, (b) Moisture. Proceed as directed in using a suitable photoelectric colorim- § 440.80a(b)(5)(1) of this chapter. eter with a 650-millimicron filter and (c) Oleandomycin phosphate used in an 18-millimeter-diameter test tube as making the capsules—(1) Potency—(i) an absorption cell. Run test plates to Cylinders (cups). Used cylinders de- determine the quantity of the diluted scribed in § 440.80a(b)(1)(i) of this chap- suspension (usually 1.5 milliliters) that ter. should be added to each 100 milliliters (ii) Culture media. (a) Use the nutri- of agar to give clear, sharp zones of in- ent agar described in § 440.80a hibition of appropriate size. (b)(1)(ii)(a) of this chapter for the seed (vi) Preparation of plates. Add 21 milli- layer and base layer, except that its pH liters of the agar prepared as described after sterilization is 7.8 to 8.0. in paragraph (c)(1)(ii)(a) of this section (b) Use the nutrient agar described in to each Petri dish (20 millimeters × 100 § 440.80a(b)(1)(ii)(a) of this chapter for millimeters). Distribute the agar even- maintaining the test organism. ly in the plates and allow it to harden. (iii) Working standard. Dissolve a Use the plates the same day they are suitable weighed quantity (usually 25 prepared. Melt a sufficient amount of milligrams or less) of the working the agar described in paragraph standard (obtained from the Food and (c)(1)(ii)(a) of this section, cool to 48° Drug Administration) in 2 milliliters of C., add the proper amount of the test ethanol, then add sufficient 0.1 M po- organism as described in paragraph tassium phosphate buffer, pH 8.0, to (c)(1)(v) of this section and mix thor- give a concentration of 1,000 oughly. Add 4 milliliters of this inocu- micrograms of oleandomycin base per lated agar to each Petri dish. Distrib- milliliter. This stock solution may be ute the agar evenly in the plates, cover kept in the refrigerator for 3 days. with porcelain covers glazed on the (iv) Preparation of sample. Dissolve outside, and allow to harden. After the the sample in sufficient 0.1 M potas- agar has hardened, place 6 cylinders on sium phosphate buffer (pH 8.0) to give a the agar surface so that they are at ap- convenient stock solution. Further di- proximately 60° intervals on a 2.8-centi- lute in 0.1 M potassium phosphate buff- meter radius. er (pH 8.0) to give a final concentration (vii) Standard curve. Prepare the daily of 5.0 micrograms per milliliter (esti- standard curve by further diluting the mated). 1,000 micrograms per milliliter stock (v) Preparation of test organism. The solution in 0.1 M potassium phosphate test organism is Staphylococcus buffer (pH 8.0) to obtain concentrations epidermidis (ATCC 12228) 1 which is of 3.2, 4.0, 5.0, 6.25 and 7.80 micrograms maintained on slants or agar described per milliliter. Use 3 plates for the de- under paragraph (c)(1)(ii)(a) of this sec- termination of each point on the curve, tion. Wash the organism from the agar except the 5.0 micrograms per milli- slant with 3 milliliters of sterile phys- liter concentration, a total of 12 plates. On each of 3 plates fill 3 cylinders with 1 Available from: American Type Culture the 5.0 micrograms per milliliter stand- Collection, 12301 Parklawn Drive, Rockville, ard, and the other 3 cylinders with the MD 20852. concentration under test. Thus, there
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will be 36 five-microgram determina- with the standard 5.0 micrograms per tions and 9 determinations for each of milliliter solution and 3 cylinders with the other points on the curve. After in- the 5.0 micrograms per milliliter (esti- cubation, read the diameters of the cir- mated) sample, alternating standard cles of inhibition in the plates. Average and sample. Incubate all plates, includ- the readings of the 5.0 micrograms per ing those containing the standard milliliter concentration and the read- curve, at 32° C.–35° C. overnight, and ings of the point tested for each set of measure the diameter of each circle of 3 plates and average also all 36 readings inhibition. To estimate the potency of of the 5.0 micrograms per milliliter the sample, average the zone readings concentration. The average of the 36 of the standard and the zone readings readings of the 5.0 micrograms per mil- of the sample on the 3 plates used. If liliter concentration is the correction the sample gives a larger zone size than point for the curve. Correct the aver- the average of the standard, add the age value obtained for each point to difference between them to the 5.0 the figure it would be if the 5.0 micrograms per milliliter zone on the micrograms per milliliter reading for standard curve. If the average sample that set of 3 plates were the same as the correction point. Thus, if in cor- value is lower than the standard value, recting the 4.0-microgram concentra- subtract the difference between them tion, the average of the 36 readings of from the 5.0 micrograms per milliliter the 5.0-microgram concentration were value on the curve. From the standard 20.0 millimeters, and the average of the curve, read the potencies corresponding 5.0-microgram concentration of this set to these corrected values of zone sizes. of 3 plates were 19.8 millimeters, the (2) Toxicity. Proceed as directed in correction would be +0.2 millimeter. If § 440.80a(b)(4) of this chapter, except use the average reading of the 4.0- physiological salt solution as the dilu- microgram concentration of these ent, and inject 0.5 milliliter of a solu- same 3 plates were 19.0 millimeters, the tion containing 8 milligrams per milli- corrected value would be 19.2 millime- liter. ters. Plot these corrected values, in- (3) Moisture. Proceed as directed in cluding the average of the 5.0 § 440.80(b)(5)(i) of this chapter. micrograms per milliliter concentra- (4) pH. Proceed as directed in tion, on 2-cycle semilog paper, using § 440.80a(b)(5)(ii) of this chapter, using a the concentration in micrograms per solution containing 100 milligrams per milliliter as the ordinate (the loga- milliliter. rithmic scale) and the diameter of the (5) Crystallinity. Proceed as directed zone of inhibition as the abscissa. Draw in § 440.80a(b)(5)(iii) of this chapter. the standard curve through these (d) Troleandomycin used in making the points, either by inspection or by capsules—(1) Potency—(i) Chemical meth- means of the following equations: od—(a) Reagents and equipment. (1) L=(3a+2b+c¥e)/5, Methyl orange reagent: Shake 0.5 M H=(3e+2d+c¥a)/5, boric acid solution for about 12 hours (to insure saturation) with an excess of where: L=corrected zone diameter for the lowest methyl orange indicators. An alter- concentration of the standard curve, native method is to heat the mixture H=corrected zone diameter for the highest to about 50° C. and shake for about an concentration of the standard curve, hour. Then allow to cool. Filter the c=average zone diameter for 36 readings of saturated dye solution and wash three the 5.0 micrograms per milliliter stand- times with chloroform. Store the dye ard. a, b, d, e=corrected average values for the solution over chloroform. 3.2, 4.0, 6.25, and 7.81 micrograms per (2) Acid-alcohol solution: Add 2 milli- milliliter standard solutions, respec- liters of concentrated sulfuric acid to tively. 98 milliliters of absolute methyl alco- Plot the values obtained for L and H hol. and connect with a straight line. (3) Glycerin: Reagent grade. (viii) Assay. Use 3 plates for each (4) Centrifuge tubes: 40 milliliters, sample. Fill 3 cylinders on each plate glass-stoppered.
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(b) Procedure. Prepare a chloroform milligrams or less) of the solution containing 50.0 milligrams ac- troleandomycin working standard (ob- tivity of standard oleandomycin base tained from the Food and Drug Admin- in 200 milliliters of solution. Transfer istration) in sufficient 80 percent iso- 10.0 milliliters of the solution to a 100- propyl alcohol-water solution to give a milliliter volumetric flask and dilute concentration of 1,000 micrograms per to volume with chloroform. Transfer milliliter (estimated). Use the solution 2.0, 4.0, 6.0, and 8.0 milliliters of this so- the day that it is prepared. lution to glass-stoppered centrifuge (c) In lieu of the directions in para- tubes (40-milliliter size) and dilute to a graph (c)(1)(iv) of this section, dissolve total volume of 20.0 milliliters each the sample in sufficient 80 percent iso- with chloroform. To the 20.0 milliliters propyl alcohol-water solution to give a of the solution present in each (40-mil- convenient stock solution. Further di- liliter size) centrifuge tube add 0.2 mil- lute in 0.2 M potassium phosphate buff- liliter of glacial acetic acid, 0.20 milli- er, pH 10.5 (35 grams of dipotassium liter of glycerin, and 0.40 milliliter of phosphate plus 2 milliliters of 10 methyl orange reagent. Shake for 5 N minutes and centrifuge for 3 minutes. NaOH, q.s. to 1 liter), to give a final Immediately transfer to another tube a concentration of 15 micrograms per 10.0-milliliter aliquot from the chloro- milliliter (estimated). form (lower) layer. Care must be exer- (d) In lieu of the directions in para- cised to see that no portion of the dye- graph (c)(1)(vi) of this section, use the glycerin-phase is included with the agar described in paragraph (d)(1)(ii)(a) chloroform aliquot. Add 1.0 milliliter of this section for both layers. Use the of acid-alcohol solution to this chloro- plates as soon after seeding as is prac- form aliquot, mix well, and read the tical. If they are not to be used shortly absorbancy at 535 mµ, using a 1-centi- after seeding, then they should be re- meter cell and a suitable photometer frigerated until ready for use. and using chloroform, similarly treat- (e) In lieu of the directions for pre- ed, as a blank. Prepare a standard paring the standard curve in paragraph curve, plotting the absorbance values (c)(1)(vii) of this section, prepare the of the standard solutions against the standard curve by diluting the stock concentration expressed in micrograms solution in 0.2 M potassium phosphate per aliquot. Accurately weigh the sam- buffer, pH 10.5, to give concentrations ple to be tested to give 50 milligrams of 9.6, 12.0, 15.0, 18.8, and 23.4 (estimated) of oleandomycin activity, micrograms per milliliter. The 15.0 dissolve in chloroform, and make to 200 micrograms per milliliter is the ref- milliliters with chloroform. Transfer erence concentration. 10.0 milliliters to a 100-milliliter volu- (f) In lieu of the directions in para- metric flask and make to volume with graph (c)(1)(viii) of this section, incu- chloroform. Transfer 5.0 milliliters to a bate the plates at 37° C. overnight. The glass-stoppered centrifuge tube and concentration of the sample and stand- proceed as above. Determine the po- tency of the sample from the standard ard being tested is 15.0 micrograms per curve. milliliter. (ii) Microbiological assay. Proceed as (2) Toxicity. Administer orally, by directed in paragraph (c)(1) of this sec- means of a cannula or other suitable tion, except: device, to each of five mice within the (a) In lieu of the directions in para- weight range of 18 grams to 25 grams, graph (c)(1)(ii)(a) of this section, use 0.5 milliliter of a suspension containing the nutrient agar described in 200 milligrams per milliliter in normal § 440.80a(b)(1)(ii)(a) of this chapter for saline solution. If no animal dies with- the seed and base layers, except add 2.0 in 48 hours, the sample is nontoxic. If milliliters of polysorbate 80 to each 100 one or more animals die within 48 milliliters of agar. Its pH after steri- hours, repeat the test, using for each lization is 7.8 to 8.0. test five or more previously unused (b) In lieu of the directions in para- mice weighing 20 grams (±0.5 gram) graph (c)(1)(iii) of this section, dissolve each; if the total deaths within 48 a suitable weighed quantity (usually 25 hours is no greater than 10 percent of
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the total number of animals tested, in- (6) Acetyl determination—(i) Apparatus cluding the original test, the sample is and reagents. (a) One three-necked nontoxic. Pyrex flask of approximately 45 milli- (3) Moisture. Proceed as directed in liters capacity, pear-shaped with T- § 440.80a(b)(5)(i) of this chapter. joints, agar inlet tube, glass-stoppered (4) pH. Proceed as directed in funnel, glass condenser, and bubble § 440.80a(b)(5)(ii) of this chapter, using a counter. saturated aqueous-ethanol (1:1) solu- (b) 50-milliliter Pyrex Erlenmeyer tion prepared by adding 100 milligrams flask. per milliliter. (5) Paper chromatograph method—(i) (c) 10-milliliter burette, calibrated in Apparatus and reagents—(a) 0.02 milliliter. Chromatographic chamber (cylinder (d) Anhydrous methanol, reagent glass-stoppered museum jar 11.5 inches grade. × 3.5 inches). (e) 2 N sodium hydroxide solution. (b) Chromatographic paper (8 inches × (f) Sulfuric acid solution prepared by 8 inches Whatman No. 1). adding 100 milliliters of concentrated (c) 0.1 N hydrochloric acid. H2SO4 to 200 milliliters of water. (d) Resolving solvent: Butyl acetate, (g) 1 N barium chloride solution. benzene, nitromethane, pyridine (5:5:5:1 (h) Phenolphthalein solution (1 per- by volume). cent in ethanol). (e) Spray reagent: 15 grams antimony trichloride per 100 milliliters of chloro- (i) Water-pumped nitrogen. form. (j) NaOH solution, 0.015 N. (ii) Procedure. Dissolve the sample in (ii) Procedure. Weight accurately (to chloroform to give a solution contain- 0.01 milligram) approximately 30 milli- ing 10 milligrams to 20 milligrams per grams of the sample into the three- milliliter. Prepare a sheet of necked acetyl flask. Add 2.0 milliliters chromatographic paper by drawing a of methanol to dissolve the sample, line of origin parallel to and 1 inch then add slowly with gentle swirling, from the edge of the paper. Wet the 1.0 milliliter of NaOH solution. Connect paper thoroughly with the 0.1 N hydro- the gas inlet tube with bubble counter chloric acid and blot it firmly between attached, and adjust nitrogen flow to sheets of absorbent paper. Starting 2 about two bubbles a second. Put glass- inches in from the edges and at 1-inch stoppered funnel in centerneck of ace- intervals, apply 3 microliters to 5 tyl flask and put about 5 milliliters of microliters of the sample solutions to H O in the funnel. Add a boiling chip to the starting line. Allow a few minutes 2 the solution and attach condenser in for the paper to dry partially. While the paper is still damp, form a cylinder the refluxing position with water cool- by bringing the outer edges together, ing. Adjust burner flame under acetyl allowing about 1-inch overlap, and se- flask to reflux solution gently. Reflux cure with a paper clip. Stand the paper for 30 minutes. Cool assembly slightly in the chromatographic chamber, then rinse down condenser (still in which has been filled to a depth of 1⁄2- reflux position) with a few milliliters inch with the resolving solvent. After of H2O. Reassemble condenser to the the solvent front rises to a height of 4 distillation position and add water inches to 5 inches above the origin, re- through the funnel to make a total of move the paper from the tank and hang approximately 5 milliliters of H2O it up to air dry. Spray the dried paper added to acetyl flask. Adjust burner with the antimony trichloride reagent. flame so that about 5 milliliters of H2O Hang the paper in a 100° C. oven for 3 and methanol is distilled over in ap- minutes. A purple spot becomes visible proximately 10 minutes. Discard this for trioleandomycin at an Rf value of distillate. Cool acetyl flask slightly. about 0.85. The approximate Rf values Acidify solution in flask by adding 1 for diacetyloleandomycin, milliliter of the sulfuric acid solution monoacetyloleandomycin, and through the funnel. Adjust burner oleandomycin are, respectively, 0.72, flame and distill over approximately 20 0.27, and 0.13.
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milliliters of distillate into an Erlen- § 436.517(b)(1)(iii) is used to prepare the meyer flask in about 20 minutes, add- standard curve, by further diluting ing water through the funnel as nec- with pH 8.0 buffer to final concentra- essary. It is important to keep the liq- tions of 0.64, 0.80, 1.0, 1.25, and 1.56 µ g. uid volume in the acetyl flask around 2 per milliliter. The 1.0 µ g per milliliter milliliters to 3 milliliters in order to solution is the reference concentration. obtain a quantitative recovery of the In lieu of the method described in this acetic acid. Collect a second fraction of subparagraph, the neomycin content distillate, about 10 milliliters in vol- may also be determined as follows. ume. As the second fraction is distill- Using the aqueous solution described, ing, process the first fraction. Heat the prepare the sample and proceed as di- first reaction and boil gently about 20 rected in § 436.517(b)(1), except use seconds. Add a few drops of BaCL2 solu- Staphylococcus aureus (American Type tion to check if any sulfate was dis- Culture Collection 12715) 1 as the test tilled over. If the sulfate is present, organism, which is grown and main- discard and repeat the whole deter- tained on agar containing 100 µ g. of mination. If the sulfate is absent im- tetracycline hydrochloride per milli- mediately titrate the solution with the liter of agar. Its neomycin content is 0.015 N NaOH solution to a faint pink satisfactory if it contains not less than endpoint, using one drop of phenol- 85 percent of the number of milligrams phthalein solution as the indicator. Re- that it is represented to contain. peat the above procedure with the sec- (2) Tetracycline hydrochloride-neomy- ond fraction. If the second fraction re- cin sulfate powder—(i) Tetracycline hy- quires less than 0.10 milliliter of the drochloride content. Prepare the sample 0.015 N NaOH solution and all the ace- as directed in § 436.514(a)(2). Use an ap- tic acid has been distilled over, the de- propriate aliquot and proceed as di- termination is completed. If greater rected in § 446.81a(b)(1) of this chapter. than this, collect a third fraction of ap- Its tetracycline hydrochloride content proximately 10 milliliters and titrate is satisfactory if it contains not less this as before. Total volumes of NaOH than 85 percent of the number of milli- used and calculate results as follows: grams that it is represented to contain. (Milliliters of NaOH × N NaOH × 0.043 × 100)/ (ii) Neomycin content. Use an appro- Weight sample in grams=Percent acetyl. priate aliquot of the solution prepared (7) Crystallinity. Proceed as directed in paragraph (a)(2)(i) of this section in § 440.80a(b)(5)(iii) of this chapter. and proceed as directed in paragraph (a)(1)(ii) of this section. Its neomycin § 436.516 Tetracycline-neomycin com- content is satisfactory if it contains plex powder topical; tetracycline not less than 85 percent of the number hydrochloride-neomycin sulfate of milligrams that it is represented to powder topical. contain. (a) Potency—(1) Tetracycline-neomycin (b) Sterility. Thoroughly cleanse with complex powder—(i) Tetracycline content. a suitable disinfectant the value (do Proceed as directed in § 436.514(a)(2), ex- not flame) of each container to be test- cept use water in lieu of 0.1 N HCl for ed. Into each of two empty, sterile Er- dissolving the sample. Its tetracycline lenmeyer flasks stoppered with a cot- content is satisfactory if it contains ton plug, spray quantitites sufficient not less than 85 percent of the equiva- to yield a residue of approximately the lent number of milligrams of tetra- equivalent of 50 milligrams from 10 cycline hydrochloride that it is rep- separate cans by removing the plug resented to contain. temporarily and using aseptic tech- (ii) Neomycin content. Using 0.1 M po- nique while spraying; allow propellant tassium phosphate buffer, pH 8.0, dilute to evaporate, add 250 milliliters to 500 an appropriate aliquot of the aqueous milliliters of diluting fluid B in lieu of solution, prepared as directed in para- diluting fluid A, and swirl the flasks to graph (a)(1) of this section, to a final dissolve the contents. Then proceed as concentration of 1 µ g. per milliliter (estimated), and proceed as directed in 1 Available from: American Type Culture § 436.515(c)(1), except that the neomycin Collection, 12301 Parklawn Drive, Rockville, standard stock solution described MD 20852.
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