(d) Procedure. Using a volume of from 1 to 2 milliliters of standard or sample solution, add an equal volume of water and mix. Add the following reagents in the specified volumetric proportions with respect to the sample or standard solutions: Add 1.25 volumes of neutral hydroxylamine reagent and allow to react for 5 minutes. Add 1.25 volumes of ferric ammonium sulfate reagent, mix, and after 3 minutes determine the absorbance of the resulting solution at the wavelength of 480 millimicrons, using a suitable spectrophotometer and a reagent blank prepared by treating a volume of water in the same manner as the standard or sample solution. The time elapsed after the addition of the ferric ammonium sulfate reagent and the reading of the absorbance must be precisely the same (within 10 seconds) for each solution. Calculate the potency of the sample in units or micrograms per milligram as follows:

Units or micrograms per milligram of sample

A1=Absorbance of sample solution. A2-Absorbance of standard solution.

[39 FR 18944, May 30, 1974, as amended at 39 FR 34032, Sept. 23, 1974; 39 FR 44012, Dec. 20, 1974; 42 FR 59856, Nov. 22, 1977; 44 FR 10378, Feb. 20, 1979; 45 FR 16474, Mar. 14, 1980; 46 FR 2981, Jan. 13, 1981; 46 FR 25602, May 8, 1981; 46 FR 61072, Dec. 15, 1981; 49 FR 34350, Aug. 30, 1984]

§ 436.206 Test for metal particles in ophthalmic ointments.

(a) Procedure. Extrude the contents of each of 10 tubes as completely as practicable into separate, clear, glass Petri dishes (60 millimeters in diameter), cover the dishes, and heat to 80° C. to 85° C. for at least 2 hours or until the ointment has melted completely and evenly in the dishes. A higher temperature of 100° C.±2° C. may be used if necessary to allow adequate settling of metal particles. Allow the ointment to

cool to room temperature without agitation. Invert each Petri dish on the stage of a suitable microscope adjusted to furnish 30 times magnification and equipped with an eye-piece micrometer disc which has been calibrated at the magnification being used. In addition to the usual source of light, direct an illuminator from above the ointment at a 45° angle. Examine the entire bottom of the Petri dish for metal particles. By varying the intensity of the illuminator from above, such metal particles are recognized by their characteristic reflection of light. Count the total number of metal particles exceeding 50 microns in any single dimension.

(b) Evaluation. The batch is acceptable if (1) a total of not more than 50 such particles is found in 10 tubes; and (2) not more than one tube is found to contain more than eight such particles. If the batch fails the above test, repeat

the test on 20 additional tubes of ointment. The total number of metal particles exceeding 50 microns in any single dimension from the 30 tubes tested shall not exceed 150, with not more than three tubes containing more than eight such particles.

[39 FR 18944, May 30, 1974; 40 FR 11869, Mar. 14, 1975]

§ 436.207 Residue on ignition.

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

(a) Method 1. Place approximately 1 gram of the sample, accurately weighed, in a tared porcelain crucible and carefully ignite at a low temperature until thoroughly charred. The crucible may be loosely covered with a porcelain lid during the charring. Add 2 milliliters of nitric acid and 5 drops of sulfuric acid to the contents of the crucible and cautiously heat until white fumes are evolved, then ignite, preferably in a muffle furnace, at 500° C. to 600° C. until the carbon is all burned off. Cool the crucible in a desiccator and weigh. From the weight of residue obtained, calculate the sulfated ash content.

(b) Method 2. Proceed as directed in paragraph (a) of this section, except use 2 milliliters of sulfuric acid and do not use the nitric acid.

§ 436.208 Heavy metals determination. (a) Reagents—(1) Ammonia solution. Prepare an aqueous solution containing not less than 9 grams and not more than 10 grams of ammonia (NH3) per 100 milliliters.

(2) 6 percent acetic acid. Dilute 60 milliliters of glacial acetic acid with sufficient water to give a solution of 1,000 milliliters.

(3) Hydrogen sulfide solution. Prepare a saturated solution of hydrogen sulfide by passing hydrogen sulfide into cold water for a sufficient time. It is suitable if it produces an immediate copious precipitate when added to an equal volume of 1N ferric chloride. Prepare a fresh hydrogen sulfide solution each time a heavy metals test is to be performed.

(4) Lead nitrate stock solution. Dissolve 159.8 milligrams of lead nitrate with 100 milliliters of 0.15N nitric acid, and dilute with water to a volume of 1,000

milliliters. Prepare and store this solution in glass containers free from soluble lead salts.

(5) Standard lead solution. Dilute a 10milliliter aliquot of the lead nitrate stock solution to 100 milliliters with water. This solution must be freshly prepared each time a heavy metals test is performed. One milliliter of this standard lead solution represents a lead level of 10 parts per million in a 1.0-gram sample or 20 parts per million in a 0.5-gram sample.

(b) Preparation of the sample. Use the sulfated ash obtained as described in § 436.207(a). If the heavy metal limit is greater than 30 parts per million, the sulfated ash may be obtained from a 0.5-gram sample. Add 2 milliliters of hydrochloric acid to the sulfated ash and slowly evaporate to dryness on a steam bath. Moisten the residue with 1 drop of hydrochloric acid, add 10 milliliters of hot water, and digest by heating on the steam bath for 2 minutes. After cooling to room temperature, add ammonia solution dropwise until a pH of 7.2 is reached, then add 2 milliliters of 6 percent acetic acid. Filter the solution, if necessary, and wash the crucible and the filter with about 10 milliliters of water. Combine the washings with the filtrate and dilute to exactly 25 milliliters with water.

(c) Procedure. Prepare a series of five standard lead solutions, in increments of 10 parts per million, in which the solution of lowest concentration contains 20 parts of lead per million less than the maximum limit of heavy metals permitted for the sample. Transfer the necessary quantities of standard lead solution described in paragraph (a)(5) of this section directly into metal-free 50-milliliter Nessler tubes of uniform diameter, add 2 milliliters of 6 percent acetic acid to each, and adjust each to a final volume of 25 milliliters with water. Transfer the 25-milliliter solution of the sample described in paragraph (b) of this section to another Nessler tube. Add 10 milliliters of hydrogen sulfide solution to each standard and sample solution, mix well, and allow to stand for 10 minutes. View downward over a white surface; the color of the solution of the sample should be no darker than the standard

that contains the lead equivalent of the heavy metals limit of the test.

§ 436.209 Melting range or temperature.

(a) Apparatus. Melting range apparatus consists of a glass container for a bath of colorless fluid, a suitable stirring device, an accurate thermometer, and a controlled source of heat. Any apparatus or method of equal accuracy may be used. The accuracy should be checked periodically by use of melting point standards, preferably those that melt near the expected melting range of the product to be tested. The bath fluid is selected with a view to the temperature required, but light paraffin is used generally and certain liquid silicones are well adapted to the higher temperature ranges. The fluid is deep enough to permit immersion of the thermometer to its specified immersion depth so that the bulb is still 2 centimeters above the bottom of the bath.

(b) Sample preparation. If necessary, reduce the sample to a fine powder and store it in a desiccator over sulfuric acid for 24 hours. If a method for loss on drying is included in the section for the antibiotic to be tested, a sample dried by that method may be used.

(c) Test procedure. Use a capillary glass tube about 10 centimeters long and 0.8 to 1.2 millimeters internal diameter with the wall 0.2 to 0.3 millimeter in thickness. Charge the tube with a sufficient amount of the dry power to form a column 2.5 to 3.5 millimeters high from the sealed end when packed down as closely as possible by moderate tapping on a solid surface. Heat the bath until a temperature 10°+1° C. below the expected melting range is reached, then introduce the charged tube, and heat at a rate of rise of 3o±0.5° C. per minute until melting is completed. The temperature at which the column of the sample is observed to collapse definitely against the side of the tube at any point is defined as the beginning of melting, and the temperature at which the sample becomes liquid throughout is defined as the end of melting or the melting point.

[39 FR 18944, May 30, 1974, as amended at 41 FR 24883, June 21, 1976]

§ 436.210 Specific rotation.

(a) Test procedure. The appropriate solvent, test concentration, and polarimeter tube length are specified in the section for each antibiotic to be tested. Accurately weigh the sample to be tested in a glass-stoppered volumetric flask, dissolve in the appropriate solvent, and dilute to the specified test concentration at 25° C. Maintain the solution at 25° C. and transfer to the appropriate polarimeter tube. Determine the angular rotation of both solvent and sample solution in a suitable polarimeter, using a sodium light source or a white light source with a 589.3-millimicron filter. The zero correction is the average of the blank readings and is subtracted from the average observed rotation of the sample solution if the two figures are of the same sign, or is added if they are opposite in sign, to give the corrected angular rotation of the sample solution. The determination must be completed within one-half hour from the time the solution is prepared.

(b) Calculations. Determine the specific rotation. [a], by the following formula:

where:

100a [a]t, = lc

a=The corrected angular rotation of the sample solution in degrees at temperature t using a light source of a wavelength of x millimicrons;

1=The length of the polarimeter tube in decimeters;

c-The concentration of the solution expressed as number of grams of substance in 100 milliliters of solution.

§ 436.211 Identity test by infrared spectrophotometry.

(a) Apparatus-(1) Spectrophotometer. A suitable spectrophotometer capable of recording the infrared absorption spectrum in the 2 to 15 micron range.

(2) Hydraulic press. A 30-ton hydraulic press with 12-inch square platens.

(b) Sample preparation methods. Use the sample preparation method specified in the individual section for each antibiotic.

(1) Potassium bromide discs. Quantities of materials specified are for a 13-millimeter die. Appropriate adjustments

should be made in the quantities of materials when dies of other sizes are used. To prepare a 1.0 percent mixture weigh approximately 2 milligrams of the sample and mix thoroughly with 200 milligrams of dried potassium bromide (infrared spectrophotometric quality). For a 0.5 percent potassium bromide mixture, use 1 milligram of sample. For a 0.25 percent potassium bromide mixture, use 0.5 milligram of sample. A mortar and pestle, a ball mill, or other suitable mixing device may be used. Transfer the uniformly milled mixture to the die, evacuate gradually while raising the pressure to 3,000 pounds per square inch until evacuation is complete, then raise the pressure to 16,000 pounds per square inch, and hold that pressure for 2 to 3 minutes. Release the pressure, dismantle the die, and recover the potassium bromide disc. Mount the disc in a suitable holder and proceed as directed in paragraph (c) of this section.

(2) Mineral oil mull. Weigh approximately 20 milligrams of the sample into an agate mortar and add 2 drops of mineral oil. Triturate thoroughly with a pestle until a uniform consistency is obtained. Use two rock salt plates as an absorption cell. Place a small drop of the mull in the center of one of the plates. Gently put the other plate on the mull and slowly squeeze the plates together to spread the mull uniformly. Clamp the two plates firmly together in a metal holder. Examine the assembled cell by holding it up to the light. It should appear smooth and free of any air bubbles. Proceed as directed in paragraph (c) of this section.

(3) 1 percent solution. Prepare a 1 percent solution of the sample in chloroform and use 1.0 millimeter matched absorption cells. Proceed as directed in paragraph (c) of this section.

(c) Procedure. Place the sample, prepared as directed in paragraph (b) of this section, in the spectrophotometer. Determine the infrared absorbance spectrum between the wavelengths of 2 to 15 microns. To be suitable the spectrum should have a transmittance of between 20 and 70 percent at most of the wavelengths showing significant absorption. Compare the spectrum to that of an authentic sample of the same antibiotic prepared in an iden

tical manner. To pass the infrared identity test, the absorption spectrum of the sample should compare qualitatively with that of the authentic sample.

§ 436.212 Disintegration test.

(a) Apparatus-(1) Basket-rack assembly. The basket-rack assembly consists of 6 open-ended glass tubes, each 7.75±0.25 centimeters long and having an inside diameter of approximately 21.5 millimeters and a wall approximately 2 millimeters thick; the tubes are held in a vertical position by two plastic plates, each about 9 centimeters in diameter and 6 millimeters in thickness, with six holes, each about 24 millimeters in diameter, equidistant from the center of the plate and equally spaced from one another. Attached by screws to the undersurface of the lower plate is 10-mesh No. 23 (0.025 inch) W. and M. gauge woven stainless steel wire cloth. The glass tubes and the upper plastic plate are secured in position at the top by means of a stainless steel plate, about 9 centimeters in diameter and 1 millimeter in thickness, having six perforations each about 20 millimeters in diameter, which coincide with those of the upper plastic plate and the upper open ends of the glass tubes. A central shaft about 8 centimeters in length, the upper end of which terminates in an eye through which a string or wire may be inserted, is attached to the stainless steel plate. The parts of the apparatus are assembled and rigidly held by means of three bolts passing through the two plastic plates and the steel plate. The design of the basket-rack assembly may be varied somewhat provided the specifications for the glass tubes and the screen mesh size are maintained.

(2) Disks. Each tube is provided with a slotted and perforated cylindrical disk 9.5±0.15 millimeters thick and 20.7±0.15 millimeters in diameter. The disk is made of a suitable, transparent plastic material having a specific gravity of between 1.18 and 1.20. Five 2-millimeter holes extend between the ends of the cylinder, one of the holes being through the cylinder axis and the others parallel with it and equally spaced on a 6-millimeter radius from it.

Equally spaced on the sides of the cylinder are four notches that form Vshaped planes perpendicular to the ends of the cylinder. The dimensions of each notch are such that the openings on the bottom of the cylinder are 1.60 millimeters square and those on the top are 9.5 millimeters wide and 2.55 millimeters deep. All surfaces of the disk are smooth.

(3) Raising and lowering device. Use a device for raising and lowering the basket in the immersion fluid at a constant rate between 28 and 32 cycles per minute through a distance of not less than 5 centimeters and not more than 6 centimeters.

(b) Immersion fluids. During the performance of the tests all immersion fluids are maintained at a temperature of 37o±2° C. by using a thermostatically controlled water bath.

(1) Distilled water.

(2) Simulated gastric fluid: Dissolve 2.0 grams of sodium chloride and 7.0 milliliters of hydrochloric acid in about 500 milliliters of water. Dissolve 3.2 grams of pepsin in this solution and add sufficient water to make 1,000 milliliters. This solution has a pH of about 1.2.

(3) Simulated intestinal fluid: Dissolve 6.8 grams of monobasic potassium phosphate in 250 milliliters of water, mix and add 190 milliliters of 0.2N sodium hydroxide and 400 milliliters of water. Add 10.0 grams of pancreatin, mix, and adjust the resulting solution with 0.2N sodium hydroxide to a pH of 7.5±0.1. Dilute to 1,000 milliliters.

(c) Immersion vessel. Use a suitable vessel, such as a 1-liter beaker.

(d) Operation. Add enough immersion fluid to the immersion vessel so that when the basket-rack assembly is placed on the raising and lowering device at the highest point of the upward stroke, the wire mesh remains at least 2.5 centimeters below the surface of the fluid and descends to not less than 2.5 centimeters from the bottom of the immersion vessel.

(e) Procedure―(1) Uncoated or filmcoated tablets. Place one tablet into each of the six tubes of the basket, add a disk to each tube, and operate the apparatus, using simulated gastric fluid as the immersion fluid. At the end of the time limit specified in the individ

ual section for the particular antibiotic tablet being tested, lift the basket from the fluid and observe the tablets.

(2) Plain-coated tablets. Place one tablet in each of the six tubes of the basket, add a disk to each tube, and operate the apparatus, using simulated gastric fluids as the immersion fluid. After 30 minutes, lift the basket from the fluid and observe the tablets. If the tablets have not disintegrated completely, substitute simulated intestinal fluid as the immersion fluid and continue the test for a total period of time (including previous immersion in simulated gastric fluid) equal to the time limit specified in the individual section for the particular antibiotic tablet being tested. Lift the basket and observe the tablets.

(3) Enteric-coated tablets. Place one tablet in each of the six tubes of the basket and operate the apparatus, using simulated gastric fluid as the immersion fluid. One hour later, lift the basket from the fluid and observe the tablets. If the tablets show no distinct evidence of dissolution or disintegration, add a disk to each tube and operate the apparatus, using simulated intestinal fluid as the immersion fluid, for a total period of time (including the previous immersion in simulated gastric fluid) equal to the time limit specified in the individual section for the particular antibiotic tablet being tested. Lift the basket and observe the tablets.

(4) Pastilles. Place one pastille into each of the six tubes of the basket, add a disk to each tube, and operate the apparatus, using distilled water as the immersion fluid. At the end of the time limit specified in the individual section for the particular antibiotic pastille being tested, lift the basket from the fluid and observe the pastilles.

(5) Capsules. Place one capsule into each of the six tubes of the basket, add a disk to each tube, and operate the apparatus, using distilled water as the immersion fluid. At the end of the time limit specified in the individual section for the capsules being tested, lift the basket from the fluid and observe the capsules.

(f) Evaluation. Complete disintegration is defined as the state in which any residue of the tablet, pastille, or