PENICILLIN ASSAY- Chart for Determining Potency as Percent of Standard from Two-Dose Plate Method

the 0.8 unit concentration, the average of the 81 readings of the 1.0 unit concentration is 20.0 mm., and the average of the 1.0 unit concentration of this set of 3 plates is 19.8 mm., the correction is 0.2 mm. If the average reading of the 0.8 unit concentration of these same 3 plates is 19.0 mm. the corrected value is then 19.2 mm. Plot these corrected values including the average of the 1.0 unit/ml. concentration on 2-cycle semilog paper using the concentration in units per ml. as the ordinate (the logarithmic scale) and the diameter of the zone of inhibition as the abscissa. Draw the standard curve through these points. The 10 points selected to determine the curve are arbitrary and should be so chosen that the limits of the curve will fill the needs of the laboratory. However, the potency of the sample under test should fall in the interval of from 60 percent to 150 percent of the correction point of the standard curve.

To estimate the potency of the sample average the zone readings of the standard and the zone readings of the sample on the three plates used. If the sample gives a larger average zone size than the average of the standard, add the difference between them to the 1.0 unit zone on the standard curve. If the average sample value is lower than the standard value, subtract the difference between them from the 1.0 unit value on the curve. From the curve read the potencies corresponding to these corrected values of zone sizes.

(i) Potency. The potency of sodium penicillin, calcium penicillin, and potassium penicillin is satisfactory when assayed by the methods described in this section if the immediate containers are represented to contain:

(1) 200,000 units or less and contain 85 percent or more of the number of units so represented;

(2) More than 200,000 units and contain 90 percent or more of the units so represented.

§ 141a.2 Sodium penicillin, calcium penicillin, potassium penicillin; sterility-(a) Culture medium. In the test for bacteria, use U. S. P. fluid thioglycolate medium I or a dehydrated mixture which, when reconstituted with distilled water, has the same composition as such medium and has growth-promoting, buffering, and oxygen-tension-controlling properties equal to or better than those of such medium. In the preparation of

In

the medium from either the individual ingredients or any dehydrated mixture avoid contamination with calcium. the test for molds and yeasts use U. S. P. Sabouraud Liquid Medium (Modified).

(b) Conduct of test for bacteria. Add not more than 10 milliliters of sterile distilled water, or sterile physiological salt solution, to each immediate container in the sample to be tested. From each of not less than seven immediate containers transfer aseptically the equivalent of approximately 300 milligrams, or the entire contents if the container is packaged to contain less than 300 milligrams, to individual tubes (38 x 200 millimeter size) each containing 75-100 milliliters of thioglycolate medium and sufficient penicillinase to completely inactivate the penicillin used in the test. (Prior to use, the tubes containing the medium with added penicillinase are incubated at 32° C.-35° C. for not less than 24 hours and examined for sterility.) After adding the penicillin to the tubes let them stand at room temperature for 2 hours, with frequent shaking. To one of such tubes add 1.0 milliliter of a 1:1,000 dilution of an 18-24 hour broth culture of M. pyogenes var. aureus (P. C. I.-209P and American Type Culture Collection 6538P). Incubate all tubes at 32° C.-35° C. for 5 days. The batch meets the requirements of the test for bacteria if on the first or second test the control tube and no other tube shows growth, or if the number of tubes (excluding the control tubes) that show growth in three or more consecutive tests is not more than 10 percent (to compensate for contamination that may have been induced during the test) of the total number of samples tested.

(c) Conduct of test for molds and yeasts. Add not more than 10 milliliters of sterile distilled water or sterile physiological salt solution to each immediate container in the sample to be tested. From each of not less than four immediate containers transfer aseptically the equivalent of approximately 300 milligrams, or the entire contents if the container is packaged to contain less than 300 milligrams, to individual tubes each containing 75–100 milliliters of U. S. P. Sabouraud Liquid Medium. Incubate all tubes at approximately 25° C. for 5 days. The batch meets the requirements of the test for molds and yeasts if on the first or second test no tube shows growth. or if the number of tubes that show

growth in three or more consecutive tests is not more than 10 percent (to compensate for contamination that may have been induced during the test) of the total number of samples tested.

§ 141a.3 Sodium penicillin, calcium penicillin, potassium penicillin; pyrogens-(a) Test animal. Use healthy rabbits, weighing 1,500 grams or more, which have been maintained for at least 1 week on a uniform, unrestricted diet and have not lost weight during this period. For subsequent tests, animals utilized for previous tests may be used after a rest period of not less than 2 days. Use a clinical rectal thermometer after it has been tested in a rabbit to determine the time required to reach maximum temperature. (Other recording devices of equal sensitivity are acceptable.) Insert the thermometer or other recording device beyond the internal sphincter and allow it to remain a sufficient time to reach maximum temperature as determined above. Make four rectal temperature readings on each of the animals to be used in the test at 2-hour intervals, 1 to 3 days before such use (this may be omitted for any animal that has been used in such tests during a preceding period of 2 weeks). House the test animals in individual cages and protect them from disturbances likely to cause excitement. Exercise particular care to avoid exciting the animals on the day of taking the control temperatures and on the test day. Maintain the animals in an environment of uniform temperature (±5° F.) at all times.

(b) Conduct of test. Heat all syringes and needles to be used in a muffle furnace at 250° C. for not less than 30 minutes to render them pyrogen-free and sterile. Perform the test in a room held at the same temperature as that in which the animals are housed. During the test restrain the animals in individual stocks. On the day of the test do not feed the animals used, until after completion of the test. Take a control temperature reading not more than 15 minutes after the animal is removed from the cage. Use three animals for each test, but do not use those with control temperatures of 38.8° C. or under and 39.9° C. or over. Dilute the sample with pyrogen-free, sterile, distilled water to a concentration of 2,000 units per milliliter and warm to approximately 37° C. Inject 2,000 units (estimated) per kilogram of rabbit intra

venously through an ear vein within 15 minutes subsequent to the control temperature reading. Read temperatures 1 hour after injection and each hour thereafter until three readings, have been made. The sample is non-pyrogenic if when so tested no animal shows a rise in any of the temperature readings, after injection, of 0.6° C. or more above the control temperature of such animal. If one or more of the animals shows such a rise in temperature, or if the sum of the temperature rises of the three animals exceeds 1.4° C., repeat the test on five additional animals. The sample is nonpyrogenic if not more than one of these five animals shows a rise in temperature of 0.6° C. or more above the control temperature of such animal.

§ 141a.4 Sodium penicillin, calcium penicillin, potassium penicillin; toxicity. Inject intravenously each of five mice, within the weight range of 18 to 25 grams, with 0.5 milliliter of a solution of the sample prepared by diluting with sterile distilled water to approximately 4,000 units per milliliter. The injection should be made over a period of not more than 5 seconds. If no animal dies within 48 hours, the sample is nontoxic. If one or more animals die within 48 hours, repeat the test with five unused mice weighing 20 grams (±0.5 gram) each; if all animals survive the repeat test, the sample is nontoxic.

§ 141a.5 Sodium penicillin, calcium penicillin, potassium penicillin--(a) Moisture. In an atmosphere of about 10 percent relative humidity, transfer about 100 milligrams of the finely powdered sample to a tared weighing bottle or weighing tube equipped with a capillary-tube stopper, the capillary having an inside diameter of 0.20 millimeter0.25 millimeter. Weigh the bottle or tube and place it in a vacuum oven without removing the stopper and 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 agent such as sulfuric acid or silica gel. Place weighing bottle or tube in a desiccator over a desiccating agent such as phosphorous pentoxide or silica gel, allow to cool to room temperature, and reweigh.

(b) pH. Dilute the sample to be tested with carbon-dioxide-free distilled water so that the resulting solution contains 5,000 to 10,000 units per milliliter.

(iii) N-ethyl piperidine solution. Nethyl piperidine (boiling range 129.5°131.0° C.) should be stored in brown bottles in a refrigerator. Dilute 1.0 milliliter of this reagent with 4.0 milliliters of amyl acetate. Saturate this solution with the N-ethyl piperidine salt of penicillin G, using about 3 milligrams of the salt for each 1.0 milliliter of solution. Cool this solution to 0°-8° C. and filter it through a sintered-glass filter immediately before use.

(iv) Phosphoric acid solution. Prepare by dissolving 1.0 milliliter of reagent grade phosphoric acid (85 percent) in 4.0 milliliters of water. Cool to 0° to 8° C. and shake before using.

(v) Silica gel. Use dry silica gel (mesh size 6-16, Tyler standard). Place about 0.5 gram of the silica gel in a micro filter funnel (approximately 10millimeter diameter) having a frittedglass disc of medium porosity.

(2) Procedure. Accurately weigh from 60 to 70 milligrams of the sample to be tested in a glass test tube or glass vial of approximately 10 milliliters capacity. Add 2.0 milliliters of water to dissolve the penicillin and cool the solution to 0° to 5° C. Add 2 milliliters of the amyl acetate solution and 0.5 milliliter of the phosphoric acid solution, stopper and shake vigorously for approximately 15 seconds. Centrifuge to obtain a clear separation of the two layers

Percent of sodium penicillin G

Percent of potassium penicillin G

(approximately 20 seconds). After centrifuging, remove as much of the amyl acetate layer as possible (usually about 1.7 milliliters-1.8 milliliters) with a 2milliliter hypodermic syringe equipped with a suitable needle, and add it to the filter funnel containing the silica gel. Allow the amyl acetate to remain in contact with the silica gel for exactly 20 seconds, then apply suction and collect the filtrate in a small test tube placed in a suction flask surrounded by cracked ice. Pipette a 1.0-milliliter aliquot of the amyl acetate filtrate into a tared flat bottom glass tube (approximately 15 x 50 millimeters) containing 1.0 milliliter of the acetone solution and 0.5 milliliter of the N-ethyl piperidine solution. The time elapsing between acidification and the addition of the filtrate to the above reagents should not be more than 3 minutes. Place the glass tube containing this mixture in a large weighing bottle, stopper the bottle and allow to stand for not less than 2 hours in a refrigerator at 0° to 8° C. Remove the liquid from the precipitate by means of a tared micro filter stick and wash with a total of 1 milliliter of the acetone solution adding the latter by means of a hypodermic syringe equipped with a fine needle. Place the filter stick inside the glass tube. dry under vacuum at room temperature for not less than 1 hour, and weigh. (Save all N-ethyl piperidine penicillin G residues for saturating reagents.)

(f) Penicillin K content. Determine the content of penicillin K by the following method:

Dilute a weighed sample or the contents of a vial with 0.3 M phosphate (Na, HPO, and KH,PO.) buffer pH 6.0 to give a solution containing approximately 1,000 units/ml. In the case of calcium penicillin where a precipitate of calcium phosphate occurs, remove the precipitate by filtration and use the clear filtrate. Place a 15.0-milliliter aliquot of this solution in a 125-milliliter separatory funnel, add 30.0 milliliters of chloroform U. S. P. and shake for 1 minute. (Carry out all operations at room temperature.) Allow the mixture to stand with occasional swirling to settle

the droplets of chloroform until the top layer is clear (usually about 10 minutes). Draw off all but about 2 milliliters of the lower chloroform layer through a small pledget of cotton into a glass-stoppered flask. Take a 4.0-milliliter aliquot of the original solution, a 4.0-milliliter aliquot of the buffer solution remaining in the separatory funnel and a 10.0-milliliter aliquot of the chloroform solution and determine the mg./ml. of penicillin in each by the iodometric assay procedure described in paragraph (d) of this section using 4.0 milliliters of the 1 N NaOH and 4.0 milliliters of the 1.2 N HCl for each of the above aliquots. Make blank determinations on the same size aliquots. Calculate the percent