21
Food and Drugs
5
1998-04-01
1998-04-01
false

Iodometric assay.
436.204

Section 436.204
DRUGS FOR HUMAN USE
TESTS AND METHODS OF ASSAY OF ANTIBIOTIC AND ANTIBIOTIC-CONTAINING DRUGS
General Chemical Tests for Antibiotics
§ 436.204
Iodometric assay.
(a) Reagents. (1) 0.01N Sodium thiosulfate (2.482 grams Na2S2O35H2O and 125 milligrams Na2CO3 per liter).
(2) 1.0N Sodium hydroxide.
(3) 1.2N Hydrochloric acid.
(4) 0.01N Iodine solution (prepared from 0.1N iodine U.S.P.).
(5) Starch iodide paste, T.S. (U.S.P.).
(b) Preparation of sample and working standard solutions—(1) Workingstandard solutions. From the following table, select the initial solvent, diluent, and final concentration as listed for each antibiotic working standard. Dissolve and dilute an accurately weighed portion to the specified final concentration and proceed as directed in paragraphs (c) and (d) of this section.
Antibiotic | Initial solvent | Diluent (solution number as listed in § 436.101(a)) | Final concentration in units or milligrams of activity per milliliter of standard solution
Amoxicillin | None | Distilled water | 1.0 mg.
Ampicillin | ......do | ......do | 1.25 mg.
Cephalexin | ......do | ......do | 2 mg.
Cephaloridine | ......do | ......do | 2 mg.
Cephalothin | ......do | ......do | 2 mg.
Cephapirin | ......do | ......do | 2 mg.
Cloxacillin | ......do | ......do | 1.25 mg.
Cyclacillin | ......do | ......do | 1.0 mg.
Dicloxacillin | ......do | 1 | 1.25 mg.
Methicillin | ......do | 1 | 1.25 mg.
Nafcillin | ......do | 1 | 1.25 mg.
Oxacillin | ......do | 1 | 1.25 mg.
Penicillin G | ......do | 1 | 2,000 units.
Penicillin V potassium | ......do | 1 | 2,000 units.
(2) Bulk antibiotic solutions. From the following table, select the initial solvent, diluent, and final concentration as listed for each antibiotic. Dissolve an accurately weighed aliquot (approximately 30 to 60 milligrams) of the sample, dilute to the appropriate final concentration, and proceed as directed in paragraphs (c) and (d) of this section.
Antibiotic | Initial solvent | Diluent (solution number as listed in § 436.101(a)) | Final concentration in units or milligrams of activity per milliliter of sample
Amoxicillin trihydrate | None | Distilled water | 1.0 mg.
Ampicillin | ......do | ......do | 1.25 mg.
Ampicillin sodium | ......do | 1 | 1.25 mg.
Ampicillin trihydrate | ......do | Distilled water | 125 mg.
| Bacampicillin hydrochloride | None | ......do | 1.25 mg.^2
Cephalexin | ......do | ......do | 2 mg.
Cephaloridine | ......do | ......do | 2 mg.
Cephalothin sodium | ......do | ......do | 2 mg.
Cephapirin sodium | ......do | ......do | 2 mg.
Cloxacillin sodium monohydrate | ......do | ......do | 1.25 mg.
Cloxacillin sodium monohydrate | ......do | ......do | 1.25 mg.
Dicloxacillin sodium monohydrate | ......do | 1 | 1.25 mg.
Cyclacillin | ......do | Distilled water | 1.0 mg.
Methicillin sodium monohydrate | ......do | 1 | 1.25 mg.
Mezlocillin | ......do | Distilled water | 2.0 mg.
Nafcillin sodium monohydrate | ......do | 1 | 1.25 mg.
Oxacillin sodium monohydrate | ......do | 1 | 1.25 mg.
Penicillin G benzathine blank solution | ......do | Distilled water | 2,000 units.
Penicillin G benzathine inactivated solution | ......do | 1N NaOH | 2,000 units.^1
Penicillin G potassium | ......do | 1 | 2,000 units.
Penicillin G procaine | 2 ml methyl alcohol | 1 | 2,000 units.
Penicillin G sodium | None | 1 | 2,000 units.
Penicillin V | 2 ml methyl alcohol | 1 | 2,000 units.
Penicillin V potassium | ......do | 1 | 2,000 units.
^1 Allow to stand in 1N NaOH for 15 minutes before assaying.
^2 The final concentration of bacampicillin hydrochloride is calculated in milligrams of ampicillin activity per milliliter of sample. The ampicillin working standard is used for the assay of bacampicillin hydrochloride.
(3) Finished product solutions. Prepare the sample for assay as directed in the individual section for each antibiotic product to be tested by diluting to the concentration prescribed in the table in paragraph (b)(2) of this section and proceed as described in paragraphs (c) and (d) of this section.
(c) Inactivated sample and standard solutions. (1) Transfer 2.0 milliliters each of the sample and the appropriate working standard solutions to glass-stoppered Erlenmeyer flasks.
(2) Add 2.0 milliliters of 1N sodium hydroxide, except if the sample has been diluted in 1N sodium hydroxide, and allow to stand at room temperature for 15 minutes.
(3) Add 2.0 milliliters of 1.2N hydrochloric acid.
(4) Add 10.0 milliliters of 0.01N iodine solution, stopper, allow to stand at room temperature for 15 minutes, and proceed as directed in paragraph (e) of this section.
(d) Blank determination. Transfer 2.0 milliliters each of the sample and the appropriate working standard solutions to glass-stoppered Erlenmeyer flasks. Add 10.0 milliliters of 0.01N iodine solution and immediately proceed as directed in paragraph (e) of this section.
(e) Titration procedure. Titrate the excess iodine using 0.01N sodium thiosulfate. Toward the end of the titration, add 1 drop of the starch iodide paste. Continue the titration by the addition of 0.01- to 0.02-milliliter portions of 0.01N sodium thiosulfate, shaking vigorously after each addition. The endpoint is reached when the blue color of the starch-iodine complex is discharged. Calculate the antibiotic content as described in paragraph (f) of this section.
(f) Calculations—(1) F factor determination. Using the appropriate working standard for the particular antibiotic to be tested, assay the standard as directed in this section. Calculate the F factor (the units of micrograms of activity equivalent of each milliliter of 0.01N sodium thiosulfate consumed) by means of the following formula:
where:
W s=Actual weight in milligrams of standard in the 2.0 milliliters titrated;
P=Potency of the working standard in units or micrograms per milligram;
V s=Milliliters of sodium thiosulfate used in the working standard blank determination minus the milliliters of sodium thiosulfate used in the titration of the inactivated working standard solution (the difference is the equivalent of the number of milliliters of 0.01N iodine absorbed by the inactivated standard).
(2) Bulk antibiotic. Calculate the potency of the sample in units or micrograms per milligram by means of the following formula:
where:
V u=Milliliters of sodium thiosulfate used in the sample blank determination minus the milliliters of sodium thiosulfate used in the titration of the inactivated sample solution (the difference is the equivalent of the number of milliliters of 0.01 N iodine absorbed by the inactivated sample);
W u=Actual weight in miligrams of sample in the 2.0 milliliters titrated.
(3) Finished products. Calculate the potency of the sample in units or milligrams by means of the appropriate one of the following formulas:
where:
d=Dilution factor for the sample;
n=Number of doses or items in the sample assayed.
[39 FR 18944, May 30, 1974, as amended at 39 FR 34032, Sept. 23, 1974; 42 FR 59856, Nov. 22, 1977; 44 FR 10378, Feb. 20, 1979; 46 FR 2980, Jan. 13, 1981; 46 FR 25602, May 8, 1981; 46 FR 46312, Sept. 18, 1981; 46 FR 58298, Dec. 1, 1981; 46 FR 61072, Dec. 15, 1981; 49 FR 6091, Feb. 17, 1984]