The following procedure is applicable as an aid in verifying the identities of many compendial drug substances as such and in their respective dosage forms.

Prepare a test solution as directed in the individual monograph. On a line parallel to and about 2 cm from the edge of a suitable thin-layer chromatographic plate, coated with a 0.25-mm layer of chromatographic silica gel mixture (see Chromatography 621) apply 10 µL of this solution and 10 µL of a Standard solution prepared from the USP Reference Standard for the drug substance being identified, in the same solvent and at the same concentration as the test solution, unless otherwise directed in the individual monograph. Allow the spots to dry, and develop the chromatogram in a solvent system consisting of a mixture of chloroform, methanol, and water (180:15:1), unless otherwise directed in the individual monograph, until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow the solvent to evaporate. Unless otherwise directed in the individual monograph, locate the spots on the plate by examination under short-wavelength UV light. The RF value of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.

The following thin-layer chromatographic procedure is applicable as an aid in verifying the identities of bacitracin, neomycin, and polymyxin B active ingredients and in dosage forms when present singly and in two- and three-component mixtures. The reference 201BNP in a monograph signifies that this procedure is intended.

Prepare a Test Solution as follows, unless otherwise directed in the individual monograph.

FOR DRUG SUBSTANCES— Dissolve a portion of Bacitracin, Bacitracin Zinc, Neomycin Sulfate, or Polymyxin B Sulfate in 0.1 N hydrochloric acid to obtain a solution containing about 500 USP Bacitracin Units per mL, 3.5 mg of neomycin (base) per mL, or 10,000 USP Polymyxin B Units per mL.

FOR SOLUTIONS— Where the Solution contains neomycin and polymyxin B, dilute a portion of it with 0.1 N hydrochloric acid to obtain a solution containing the equivalent of about 3.5 mg of neomycin (base) per mL. Where the Solution contains polymyxin B but not neomycin, dilute a portion of it with 0.1 N hydrochloric acid to obtain a solution containing about 10,000 USP Polymyxin B Units per mL.

FOR CREAMS, LOTIONS, AND OINTMENTS— Where the Cream, Lotion, or Ointment contains Bacitracin or Bacitracin Zinc, transfer a portion of it equivalent to about 500 USP Bacitracin Units, to a 15-mL centrifuge tube. Where the Cream, Lotion, or Ointment contains neomycin, but not Bacitracin or Bacitracin Zinc, transfer a portion of it equivalent to about 3.5 mg of neomycin (base) per mL to a 15-mL centrifuge tube. Add 4 mL of chloroform to the centrifuge tube, and shake well to disperse the Cream, Lotion, or Ointment. Add 1 mL of 0.1 N hydrochloric acid, vortex for 4 minutes, centrifuge, and use the clear supernatant.

NOTE—The Modified Test Solution as described below in the Modified Procedure may be used in lieu of the Test Solution.