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201 Thin-Layer Chromatographic Identification Test

This document provides procedures for thin-layer chromatographic identification tests to verify identities of drug substances and ingredients in dosage forms. It describes preparing test and standard solutions, developing the chromatogram, and comparing results to identify principal spots.

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20 views2 pages

201 Thin-Layer Chromatographic Identification Test

This document provides procedures for thin-layer chromatographic identification tests to verify identities of drug substances and ingredients in dosage forms. It describes preparing test and standard solutions, developing the chromatogram, and comparing results to identify principal spots.

Uploaded by

GOGETTO
Copyright
© © All Rights Reserved
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Printed on: Fri Jan 05 2024, 06:37:55 AM(EST) Status: Currently Official on 05-Jan-2024 DocId: GUID-DC3E6EDA-7CEE-4429-9788-D504DEEC0A1B_2_en-US

Printed by: USP NF Official Date: Official as of 01-May-2018 Document Type: GENERAL CHAPTER @2024 USPC
Do Not Distribute DOI Ref: n88df DOI: https://doi.org/10.31003/USPNF_M98950_02_01
1

á201ñ THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST


PROCEDURES
• GENERAL PROCEDURE
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 á621ñ) 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 R F value of the principal
spot obtained from the test solution corresponds to that obtained from the Standard solution.
• PROCEDURE FOR BACITRACIN, NEOMYCIN, AND POLYMYXIN B
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 á201BNPñ in a monograph signifies that this procedure is intended.
Prepare a Test solution as follows, unless otherwise directed in the individual monograph.

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Test solution
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
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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,
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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.]
Standard bacitracin solution: Dissolve a portion of USP Bacitracin Zinc RS in 0.1 N hydrochloric acid to obtain a solution
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containing 500 USP Bacitracin Units per mL.


Standard neomycin solution: Dissolve a portion of USP Neomycin Sulfate RS in 0.1 N hydrochloric acid to obtain a solution
containing the equivalent of 3.5 mg of neomycin (base) per mL.
Standard polymyxin B solution: Dissolve a portion of USP Polymyxin B Sulfate RS in 0.1 N hydrochloric acid to obtain a
solution containing 10,000 USP Polymyxin B Units per mL. Where the article under test also contains Bacitracin or
Bacitracin Zinc, dissolve a portion of USP Polymyxin B Sulfate RS in 0.1 N hydrochloric acid to obtain a solution
containing 500J USP Polymyxin B Units per mL, J being the ratio of the labeled amount of USP Polymyxin B Units to the
labeled amount of USP Bacitracin Units in each g of Cream, Lotion, or Ointment.
Developing solvent solution: Prepare a mixture of methanol, isopropyl alcohol, methylene chloride, ammonium hydroxide,
and water (4: 2: 2: 2: 1.5).
Procedure: Apply 10 µL of the Test solution and each of the relevant Standard solutions to a suitable thin-layer
chromatographic plate (see Chromatography á621ñ) coated with a 0.25-mm layer of chromatographic silica gel. Place the
plate in a presaturated chromatographic chamber, and develop the chromatogram with the Developing solvent system
until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the chamber, and
dry at 105° for 10 minutes. Spray the plate with a 0.2% solution of ninhydrin in butyl alcohol, and heat at 105° for 5
minutes. The RF value of each principal spot in the chromatogram of the Test solution corresponds to that of the principal
spot in the chromatogram obtained from each relevant Standard solution as appropriate for the labeled active ingredient
or ingredients specified on the label. If the chromatogram of the Test solution yields excessive streaking, proceed as directed
for Modified procedure.
Modified procedure: Transfer the Test solution to a 15-mL centrifuge tube, add 10 mL of saturated aqueous picric acid
solution (1.2%, w/v), vortex for 1 minute, centrifuge for 10 minutes, and discard the supernatant. Wash the residue with
1-mL portions of water until no yellow color is observed in the washing. Discard the washings, and dry the residue under a
stream of nitrogen at 50°. Dissolve the residue in 1 mL of acetone, add 1 mL of a freshly prepared solution of sulfuric acid
in acetone (1 in 100), shake, centrifuge for 5 minutes, and discard the supernatant. Rinse the residue with 1 mL of acetone,
centrifuge briefly, and discard the washing. Repeat the washing until no yellow color is observed. Dry the residue under a
stream of nitrogen at 50°. Dissolve the residue in 0.5 mL of 0.1 N hydrochloric acid (Modified test solution). Repeat the
Procedure using this Modified test solution instead of the Test solution. The R F value of each principal spot in the
chromatogram of the Modified test solution corresponds to that of the principal spot in the chromatogram obtained from
each relevant Standard solution as appropriate for the active ingredient or ingredients specified on the label.

https://online.uspnf.com/uspnf/document/1_GUID-DC3E6EDA-7CEE-4429-9788-D504DEEC0A1B_2_en-US 1/2
Printed on: Fri Jan 05 2024, 06:37:55 AM(EST) Status: Currently Official on 05-Jan-2024 DocId: GUID-DC3E6EDA-7CEE-4429-9788-D504DEEC0A1B_2_en-US
Printed by: USP NF Official Date: Official as of 01-May-2018 Document Type: GENERAL CHAPTER @2024 USPC
Do Not Distribute DOI Ref: n88df DOI: https://doi.org/10.31003/USPNF_M98950_02_01
2

USP Reference Standards á11ñ


USP Bacitracin Zinc RS
USP Neomycin Sulfate RS
USP Polymyxin B Sulfate RS

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https://online.uspnf.com/uspnf/document/1_GUID-DC3E6EDA-7CEE-4429-9788-D504DEEC0A1B_2_en-US 2/2

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