2006
`
`USP 29
`THE UNITED STATES PHARMACOPEIA
`
`NF 24
`THE NATIONAL FORMULARY
`
`By authority of The United States Pharmacopeial
`Convention, meeting at Washington, D. C, March 9—13,
`2005. Prepared by the Council ofExperts and published
`by the Board of Trustees
`
`Ofiicial from January I, 2006
`
`The designation on the cover of this publication, “USP NF
`2006,” is for ease of identification only. The publication
`contains two separate compendia: The United States
`Pharmacopeia, Twenty-Ninth Revision, and the National
`Formulary, Twenty—Fourth Edition.
`
`THE UNITED STATES PHARMACOPEIAL CONVENTION
`
`12601 Twinbrook Parkway, Rockville, MD 20852
`
`Argentum EX1044
`Argentum EX1044
`Page 1
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`Page 1
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`NOTICE AND WARNING
`
`Concerning US. Patent or Trademark Rights
`
`The inclusion in The United States Pharmacopeia or in the National Formulary ofa monograph on any drug
`in respect to which patent or trademark rights may exist shall not be deemed, and is not intended as, a grant of,
`or authority to exercise, any right 'or privilege protected by such patent or trademark. All Such rights and
`privileges are vested in the patent or trademark owner, and no other person may exercise the same without
`express permission, authority, or license secured from such patent or trademark owner.
`
`Concerning Use of USP or NF Text
`
`Attention is called to the fact that USP and NF text is fully copyrighted. Authors and others wishing to use
`portions of the text should request permission to do so from the Secretary of the USPC Board of Trustees.
`
`Copyright © 2005 The United States Pharrnacopeial Convention
`12601 Twinbrook Parkway, Rockville, MD 20852
`All rights reserved.
`.
`ISSN 0195-7996
`-
`"
`‘
`ISBN 1-889788—39-2
`Printed in Canada by Webcom Limited, Toronto, Ohtario
`
`-
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`Page 2
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`

`

`2552
`
`(197) Spectrophotometric Identification Tests / Chemical Tests
`
`respective Pharmacopeial dosage forms. Two procedures are
`provided, one based on paper chromatography (Method 1) and the
`other on thin-layer chromatography (Method II). Method 1 is to be
`used unless otherwise directed in the individual monograph.
`Standard Solution—Unless otherwise directed in the individual
`monograph, dissolve the USP Reference Standard for the drug
`substanCe being identified in the same solvent and at the same
`concentration as for the Test Solution.
`
`Test Solution—Prepare as directed1n the individual monograph.
`
`METHOD I
`
`pH 3.5 Buffer—Dissolve 13.4 g of anhydrous citric acid and
`16.3 g of dibasic sodium phosphate in 1000 mL of water, and mix.
`Developing Solvent—On the day of use, mix 10 volumes of
`chloroform, 20 volumes of nitromethane, and 3 volumes of pyridine.
`. Mixed Test Solution—Mix equal volumes of the Standard
`Solution and the Test Solution.
`
`Chromatographic Sheet—Draw a spotting line 2.5 cm from one
`edge of a 20—cm X ‘20-cm sheet of filter paper (Whatman No. l, or
`equivalent). Impregnate the sheet with pH 3.5 Buffer by passing it
`through a trough filled with pH 3.5 Bufler, and remove the excess
`solvent by firmly pressing the sheet between nonfluorescent blotting
`papers.
`Procedure—«To a- suitable chromatographic chamber, prepared for
`ascending chromatography (see Chromatography (621)) add Devel-
`oping Solvent to a depth of 0.6 cm. Apply at 1.5-cm intervals 2 uL
`each of the Standard Solution, the Test Solution, and the Mixed Test
`Solution to the spotting line of the Chromatographic Sheet. Allow the
`sheet to dry partially, and while still damp place .it
`in the
`chromatographic chamber with the bottom edge touching the
`Developing Solvent. When the solvent front has risen about 10 cm,
`remove the sheet from the chamber, and expose the sheet to. ammonia
`vapor. Examine the chromatogram under long—wavelength UV light.
`Record thepositions of the major yellow fluorescent spots: the RF
`value, of the principal spot obtained from the Test Solution and from
`the Mixed Test Solution corresponds to that obtained from. the
`Standard Solution.
`
`METHOD II
`
`Resolution Solution—Unless otherwise directed1n the individual
`monograph, prepare a solutionIn methanol containing 0.5 mg each of
`USP Chlortetracycline Hydrochloride. RS, USP Doxycycline Hyclate
`RS, USP Oxytetracycline RS, and USP Tetracycline Hydrochloride
`IRS per mL.
`. Developing Solvent—Prepare a mixture of 05M oxalic acid,
`previously adjusted with ammonium hydroxide to a pH of 2.0
`acetonitrile, and methanol (80: 20: 20).
`Chromatographic Plate-Use a suitable thin~layer chromato-
`graphic plate (see Thin—layer Chromatography under Chromatogra-
`phy (621))- coated with a 0. 25--mm layer of octylsilanized
`chromatographic silica gel mixture Activate the plate by heating it
`at 1300 for 20 minutes, allow to cool, and use while still warm.
`Procedure—Separately apply 1 11L each of the Standard Solution,
`the Test Solution, and the Resolution Solution to the Chromato-
`graphic Plate. Allow the spots to dry, and develop the chromatogram
`in the Developing Solvent 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 to air-dry.
`Expose the plate to ammonia vapors for 5 minutes, and promptly
`locate the spots on the plate by viewing under long—wavelength UV
`light: the chromatogram of the Resolution Solution shows clearly
`separated spots, and the principal spot obtained from the Test Solution
`corresponds in RF value, intensity, and appearance to that obtained
`from the Standard Solution
`
`1'
`
`
`(197) SPECTROPHOTOMETRIQ}:
`.
`IDENTIFICATION TESTS '1
`
`Spectrophotometric tests contribute meaningfully toward;
`
`identification of many compendial chemical substances. The
`procedures that follow are applicable to substances that abso '6
`
`and/(gr UV radiation (see Spectrophotometry and Light--Sc II
`851 ).
`.-
`The IR absorption spectrum of a substance, compared wi
`I
`
`obtained concomitantly for the corresponding USP Ref
`
`Standard, provides perhaps the most conclusive evidence
`identity ofthe substance that can be realized from any single test.
`
`
`UV absorption spectrum, 0n the other hand, does not exhib'
`
`degree of specificity. Conformance with both IR absorption an
`
`absorption test specifications, as called forIn a large proportr 4'
`compendial monographs leaves little doubt, if any, regardin ‘
`
`identity of the specirnen under examination.
`
`
`. INFRARED ABSORPTION
`Six methods are indicated for the preparation of previously :I
`test specimens 'and Reference Standards for analysis. The ref '7:
`
`the substance 14
`I.
`(197K)
`in a monograph ‘ signifies that
`examination is mixed intimately. with potassium bromide' 1
`
`reference (197M)In a monograph signifies that the substance 11
`examination is finely ground and dispersed in mineral oil; 1
`
`reference (197F) in a monograph signifies that the substance u
`examination is suspended neat between suitable (for'example, s 1
`
`chloride or potassium bromide), platesxThe reference (1978) si
`that a solution of designated concentration .is-prepared in the:
`
`specifiedIn the individual monograph, and the solution is ex:
`in 0.1—mm cells unless a different cell path length15 specified
`
`I
`individual monograph. The reference (197A)
`signifies
`substance under examination is intimately1n contact with an '
`
`reflection element for attenuated total reflectance (ATR) anal
`
`reference (197E)srgnifies that the substance under examin
`pressed as a thin sample against a suitable plate for. IR mic
`analysis The ATR (197A) and the (197E) techniques can b
`alternative methods for (197K), (197M), (197F),'qand (1978)
`
`
`testingrs perfdrmed qualitatively and the Reference Standard s‘I"_-I'I:
`are similarly obtained
`Record the spectra of the test specimen and the corresponding
`Reference Standard over the range from about 2. 6 pm to 15 um
`cm‘l
`to 650 cm“) unless otherwise specified in the in
`
`monograph. The IR absorption spectrum of the preparation 0
`specimen, previously dried under conditions specified to ,‘
`
`corresponding Reference Standard unless otherwise spec‘- '
`‘
`unless the Reference Standard is to be used without drying,
`
`maxima only atthe same wavelengths as that ofa similar pr
`,
`of the corresponding USP Reference Standard
`
`Differences that may be observed in the spectra- so ob
`sometimes are attributed to the presence of polymorphs, wh ~
`
`not always acceptable (see Procedure under Spectrophotome ;,
`
`Light-Scattering (851)) Unless otherWise directedin the indi
`monograph, therefore, continue as fellows. If a difference ap Ff
`
`the IR spectra of the analyte and the standard, dissolve equal 1 1
`ofthe test specimen and the Reference StandardIn equal voIlu
`
`suitable solvent, evaporate the solution to
`containers under identical conditions, and repeat the test,’
`
`I
`residues.
`
`ULTRAVIOLET ABSORPTION
`
`
`The reference (197U)In a monOgraph signifies that a test s6
`
`and a Standard solution are examined spectrbphotometrically;
`cm cells, over the spectral range from 200 to 400 nm unless o .
`specifiedIn the individual monograph
`
`Dissolve a portion of the substance under examinatio
`designated Medium to obtain a test solution having the cone
`
`specifiedIn the monograph for Solution. Similarly prepare a
`solution containing the corresponding USP Reference Stan
`
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`

`

`P29
`
`Chemical Tests / (201) Thin-Layer Chromatographic Identification Test
`
`2553
`
`Record and compare the spectra concomitantly obtained for the test
`lugtion and the Standard solution. Calculate absorptiViti'es and/or
`orbance ratios where these criteria are included111 an individual
`nograph Unless otherwise specified, absorbances indiCated for
`e calculationsare ‘thoSe measured at the maximum absorbanceat
`iwut the wavelength specifiedin the individual monograph. Where
`abSOrbance1s to be measured at about the specified wavelength
`«er than that of maximum absorbance, the abbreviations (min) and
`*1) are used to indicate a minimum and shoulder, respectively,1n an
`orption spectrum. The requirements are met if the UV absorption
`ctra of the test solution and the Standard solution exhibit maxima
`I minirna at
`the same Wavelengths and absorptivities and/0r
`sorbance ratios are within specified limits
`
`(201) THIN-LAYER
`CHROMATOGRAPHIC
`IDENTIFICATION TEST
`
`GENERAL PROCEDURE
`
`1.
`
`;
`'4
`
`1
`
`-;3,The following procedure18 applicable as an aid1n verifying the
`‘ntities of many compendial drug substances as such andIn their
`5Lpective dosage forms.
`Prepare a test solution as directed1n the individual monograph. On
`ine parallel to and about 2 cm from the edge of a suitable thin-layer
`., cmatographic plate, coated with a 0. 25--mm layer of chromato—
`mphic silica gel mixture (see Chromatography (621)) apply 10 11L
`1 this solution and 1011L of a Standard solution prepared from the
`gs? Reference Standard for the drug substance being identified, in
`same solvent and at the same concentration as the testsolutiOn,
`igess otherwise directed in the individual monograph. A110w the
`fits to dry, and develop the chromatogram in a solvent system
`insisting of a mixture of chloroform, methanol, and water
`1» ':0 15. 1), unless otherwise directed1n tlie individual monograph,
`' ii the solvent front has moved about three—fourths of the length of
`plate. Remove the plate from the developing chamber, mark the
`Vent front, and allow the solvent to evaporate. Unless otherwise
`ted1n the individual monograph, locate the spots on the plate by
`ination under short-wavelength UV light. The RF VaII‘Ie of the
`n'cipal spot obtained from the test solution corresponds to that
`'itained from the Standard solution.
`
`ROCEDURE FOR BACITRACIN, NEOMYCIN,
`;
`AND POLYMYXIN B
`3
`
`The following thin-layer chromatographic procedure1s applicable
`, an aid in verifying the identities of bacitracin, neomycin, and
`lymyxin B active ingredients and in dosage forms when present
`,glyv and in two- and three-component mixtures. The reference
`01BNP) in a monograph signifies that this procedureIS intended
`,Prepare a Test Solution as follows, unless otherwise directed1n the
`'1.1 Vidual monograph.
`Test Solution—
`FOR DRUG SUBSTANCES—Dissolve a portion of Bacitracin,
`,:_ itracin Zinc, Neomycin Sulfate, or Polymyxin B Sulfate in
`N hydrochloric acid to obtain a solution contaimng about 500
`isP Bacitracin Units per mL, 3.5 mg of neomycin (base) per mL, or
`1,1,000 USP Polymyxin B Units per mL.
`FOR SOLUTIONS—Where the Solution contains neomycin and
`‘ ymyxin B, dilute a portion of it with 0.1N hydrochloric acid to
`'tain a solution containing the equivalent of about 3.5 mg of
`‘ umycin (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 orNTMENTs—Where the Cream, Letion,
`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—LThe Modfied Test Solution as described belowin the Modified
`Procedure may be usedin 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
`containing 500 USP Bacitracin Units permL.
`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 0.1N 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 500] USP
`Polymyxin B Units per mL, J being the ratio of the labeled amount
`of USP Polymyxin BUnits 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)
`ProcedurFApp1y_10 11L of the Test Solution and each of the
`relevant Standard Solutions to a suitable thin——layer chromatographic
`plate (see Chromatography (621)) coated witha 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° fer 5 minutes.
`The RF value of each principal spot in the chromatogram of theTest
`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 -m_L portions of
`water until no yellow color1s observed1n 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 gwith 1 mL of acetone, centrifuge briefly, and discard the
`washing. Repeat the washing until no yellow color1s observed. Dry
`the residue under a stream of nitrogen at 50°. Dissolve the residueIn
`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 RF 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.
`
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