USP 40Official Monographs / Levofloxacin 4831Acceptance criteria: See Table 1.Sample solution: 1mg/mL of Levofloxacin in Mobile phaseChromatographic systemTable 1(See Chromatography 〈621〉, System Suitability .)Relative Relative Acceptance Mode: LCRetention Response Criteria,Detector: UV 360 nmNameTimeFactor NMT (%)Column: 4.6-mm × 25-cm; 5-µm packing L1Levodopa related Column temperature: 45°compound A 0.90.830.1Flow rate: 0.8mL/min Injection size: 25µL Levodopa1.0——System suitabilityLevodopa related Sample: Standard solution compound B 2.80.830.5Suitability requirements 5,6-Dihydroxy-in-Tailing factor: 0.5–1.5dole-2-carboxylic Relative standard deviation: NMT 1.0%acid6.0 2.50.1Analysis0.1Samples: Standard solution and Sample solutionindividual Calculate the percentage of levofloxacin (C 18H 20FN 3O 4)—0.3 total in the portion of Levofloxacin taken:Unknown impurities 1.0unknown Total impurities——1.1Result = (r U /r S ) × (C S /C U ) × 100r U= peak response of levofloxacin from the SampleADDITIONAL REQUIREMENTSsolution•P ACKAGING AND S TORAGE : Preserve in tight, light-resistant r S = peak response of levofloxacin from thecontainers, in a dry place, and prevent exposure to ex-Standard solutioncessive heat.C S = concentration of USP Levofloxacin RS in the•USP R EFERENCE S TANDARDS 〈11〉Standard solution (mg/mL)USP Levodopa RSC U = concentration of Levofloxacin in the SampleUSP Levodopa Related Compound A RS solution (mg/mL)3-(3,4,6-Trihydroxyphenyl)alanine.Acceptance criteria: 98.0%–102.0% on the anhydrous C 9H 11NO 5213.19basisUSP Levodopa Related Compound B RS 3-Methoxytyrosine.IMPURITIESC 10H 13NO 4211.22•R ESIDUE ON I GNITION 〈281〉: NMT 0.2%. Use a platinum crucible.Delete the following:Levofloxacin••H EAVY M ETALS , Method II 〈231〉: NMT 10ppm •(Official 1-Jan-2018)•O RGANIC I MPURITIES , P ROCEDURE 1[N OTE —Procedure 1 is recommended if levofloxacin N -ox-ide is a potential organic impurity. Procedure 2 and Pro-cedure 3 are recommended if levofloxacin related com-pound B is a potential organic impurity.]Solution A, Mobile phase, Sample solution, and Chro-matographic system: Proceed as directed in the C 18H 20FN 3O 4·1/2H 2O 370.38Assay .7H -Pyrido[1,2,3-de ]-1,4-benzoxazine-6-carboxylic acid,System suitability solution: 1mg/mL of USP Levoflox-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-acin RS in Mobile phase7-oxo-hydrate (2:1), (S )-;Sensitivity solution: 0.3µg/mL of USP Levofloxacin RS (−)-(S )-9-Fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piper-in Mobile phase azinyl)-7-oxo-7H -pyrido[1,2,3-de ]-1,4-benzoxazine-6-car-System suitabilityboxylic acid, hemihydrate [138199-71-0].Samples: System suitability solution and Sensitivity Anhydrous [100986-85-41].solutionSuitability requirementsDEFINITIONRelative standard deviation: NMT 1.0%, System suit-Levofloxacin contains NLT 98.0% and NMT 102.0% of ability solutionC 18H 20FN 3O 4, calculated on the anhydrous basis.Signal-to-noise ratio: NLT 10, Sensitivity solution IDENTIFICATIONAnalysis•A . I NFRARED A BSORPTION 〈197K 〉Sample: Sample solution•B . The retention time of the major peak of the Sample Calculate the percentage of each individual impurity in solution corresponds to that of the Standard solution , as the portion of Levofloxacin taken:obtained in the Assay.Result = (r U /r S ) × (1/F ) × 100ASSAY•P ROCEDUREr U = peak response of each impurity Buffer: 8.5g/L of ammonium acetate, 1.25g/L of cu-r S = peak response of levofloxacin pric sulfate, pentahydrate, and 1.3g/L of L -isoleucine in F = relative response factor (see Table 1)waterAcceptance criteria: See Table 1.Mobile phase: Methanol and Buffer (3:7)Standard solution: 1mg/mL of USP Levofloxacin RS in Mobile phase4832Levofloxacin / Official MonographsUSP 40Table 1in methanol from Levofloxacin related compound B stock solutionRelative Relative Acceptance Standard solution: 0.4µg/mL of levofloxacin andRetention Response Criteria,0.8µg/mL of levofloxacin related compound B in aceto-NameTimeFactorNMT (%)nitrile and water (1:10) from Levofloxacin standard solu-N -Desmethyl tion and Levofloxacin related compound B standard levofloxacin a0.47 1.00.3solutionDiamine derivative b 0.520.90.3Sample solution: 0.4mg/mL by dissolving the sample Levofloxacin in acetonitrile at about 8% of final volume and diluting N -oxide c0.63 1.10.3with water to volume. [N OTE —Sonicate if necessary.]Chromatographic system9-Desfluoro levoflox-(See Chromatography 〈621〉, System Suitability .)acin d0.73 1.00.3Mode: LCLevofloxacin 1.0——Detector: 280 nmD -Isomer e1.23 1.00.8Column: 4.0-mm × 15-cm; 3.0-µm packing L1Any unknown Column temperature: 38°—impurity1.00.1Flow rate: 1.0mL/min Total Impurities——0.5*Injection size: 10µL a(S )-9-Fluoro-2,3-dihydro-3-methyl-10-(piperazin-1-yl)-7-oxo-7H -pyrido[1,System suitability2,3-de ][1,4]benzoxazine-6-carboxylic acid.Sample: System suitability solution b (S )-9-Fluoro-2,3-dihydro-3-methyl-10-[2-(methylamino)ethylamino]-7-Suitability requirementsoxo-7H -pyrido[1,2,3-de ][1,4]benzoxazine-6-carboxylic acid.Relative standard deviation: NMT 2.0% for c (S )-4-(6-Carboxy-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H -pyrido-[1,2,3-levofloxacin de ][1,4]benzoxazine-10-yl)-1-methyl-piperazine-1-oxide.Analysisd (S )-2,3-Dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H -pyrido[1,Samples: Standard solution and Sample solution2,3-de ][1,4]benzoxazine-6-carboxylic acid.Calculate the percentage of levofloxacin related com-e (R )-9-Fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H -pyrido[1,2,3-de ][1,4]benzoxazine-6-carboxylic acid.pound B in the portion of Levofloxacin taken:*Do not include the D -isomer in the calculation for total impurities.Result = (r U /r S ) × (C S /C U ) × 100•O RGANIC I MPURITIES , P ROCEDURE 2[N OTE —Solutions of levofloxacin are not stable in light;r U= peak response for levofloxacin relateduse amber bottles.]compound B from the Sample solutionBuffer: Dissolve 3.08g/L of ammonium acetate and r S = peak response for levofloxacin related8.43g/L of sodium perchlorate monohydrate in pound B from the Standard solutionAdjust with phosphoric acid to a pH of 2.2.C S = concentration of USP Levofloxacin RelatedSolution A: Acetonitrile and Buffer (16:84)Compound B RS in the Standard solution Solution B: Acetonitrile, methanol, and Buffer (mg/mL)(30:20:50)C U = concentration of Levofloxacin in the SampleSolution C: 0.4mg/mL of USP Levofloxacin RS by dis-solution (mg/mL)solving in acetonitrile at about 8% of volume and dilut-Calculate the percentage of other impurities in the ing with water to volumeportion of Levofloxacin taken:Solution D: 0.05mg/mL of USP Levofloxacin Related Compound A RS in 0.2% ammonium hydroxide in Result = (r U /r S ) × (C S /C U ) × 100methanolMobile phase: See Table 2.r U= peak response of any other impurity from theSample solutionr S = peak response of levofloxacin from theTable 2Standard solutionTime Solution ASolution BC S = concentration of USP Levofloxacin RS in the(min)(%)(%)standard solution (mg/mL)01000C U = concentration of Levofloxacin in the Samplesolution (mg/mL)51000Acceptance criteria: See Table 3.108218154060Table 330406030.11000Relative Acceptance Retention Criteria,38100NameTimeNMT (%)System suitability solution: 0.1mg/mL of USPLevofloxacin related compound A Levofloxacin RS and 5µg/mL of USP Levofloxacin Re-(N -Desmethyl levofloxacin)a 0.90.20lated Compound A RS in water from Solution C and Levofloxacin1.0—Solution DLevofloxacin related compound B b 2.90.13Levofloxacin stock solution: 0.4mg/mL of USPAny other impurity —0.10Levofloxacin RS. Dissolve USP Levofloxacin RS in aceto-Total impurities—0.50nitrile at about 8% of final volume, sonicate, and dilute with water to volume.a(S )-9-Fluoro-3-methyl-10-(piperazin-1-yl)-7-oxo-2,3-dihydro-7H -pyrido[1,2,3-de ][1,4-benzoxazine-6-carbocylic acid.Levofloxacin standard solution: 0.02mg/mL of USP b (S )-9,10-Difluoro-3-methyl-7-oxo-2,3-dihydro-7H -pyrido[1,2,3-de ][1,4-Levofloxacin RS in acetonitrile and water (1:10) from benzoxazine-6-carbocylic acid.Levofloxacin stock solutionLevofloxacin related compound B stock solution:•O RGANIC I MPURITIES (E NANTIOMERIC P URITY), P ROCEDURE 30.2mg/mL USP Levofloxacin Related Compound B RS Buffer: 1.32g/L of D -phenylalanine and 0.75g/L of in methanol. [N OTE —Sonicate if necessary.]copper(II)sulfate pentahydrate in waterLevofloxacin related compound B standard solution:0.04mg/mL USP Levofloxacin Related Compound B RSUSP 40Official Monographs / Levofloxacin4833Mobile phase: Methanol and Buffer (15:85)ASSAYSystem suitability solution: 0.01mg/mL of USP Oflox-•P ROCEDUREacin RS and 0.01mg/mL of USP Levofloxacin RS in[N OTE—Protect the solutions of levofloxacin from light.] water Diluent: Acetonitrile and water (18:82)Sample solution: 0.08mg/mL in water Mobile phase:Diluent that contains 1mL of trifluoroa-Chromatographic system cetic acid in each 1000mL of solution(See Chromatography 〈621〉, System Suitability.)Standard solution: 102.5µg/mL of USP Levofloxacin Mode: LC RS in DiluentDetector: 294 nm System suitability solution: 102.5µg/mL each of USP Column: 4.6-mm × 15-cm; 3.5-µm packing L1Levofloxacin RS and USP Levofloxacin Related Com-Column temperature: 40°pound A RS in DiluentFlow rate: 0.7mL/min Sample solution: 102.5µg/mL of levofloxacin in Dilu-Injection size: 10µL ent based on the label claim. [N OTE—Mix the solution System suitability well after equilibrating the solution for 4 h at room Sample:System suitability solution temperature while protected from light.][N OTE—The relative retention times for D-ofloxacin and Chromatographic systemlevofloxacin are 0.91 and 1.0, respectively.](See Chromatography 〈621〉, System Suitability.)Suitability requirements Mode: LCResolution: NLT 2.0 between D-ofloxacin (D-isomer)Detector: UV 294 nmand levofloxacin Column: 4.6-mm × 15-cm; 3.5-µm packing L11 Analysis Column temperature: 30°Sample: Sample solution Flow rate: 0.7mL/minCalculate the percentage of D-ofloxacin in the portion Run time: 2.5 times the retention time of the levoflox-of Levofloxacin taken:acin peakInjection size: 20µLResult = (r U/r T) × 100System suitabilitySamples:Standard solution and System suitability r U= peak response for D-ofloxacin solutionr T= sum of responses of all peaks Suitability requirementsAcceptance criteria: NMT 1.0%Resolution: NLT 1.9 between levofloxacin relatedcompound A and levofloxacin, System suitability SPECIFIC TESTS solution•O PTICAL R OTATION, Specific Rotation 〈781S〉Relative standard deviation: NMT 2.0%, Standard Solvent: Methanol solutionSample solution: 5mg/mL in Solvent AnalysisAcceptance criteria:−92° to −106°, at 20°Samples:Standard solution and Sample solution•W ATER D ETERMINATION, Method Ia〈921〉: 2.0%–3.0%Calculate the percentage of the labeled amount oflevofloxacin (C18H20FN3O4) in the portion of Oral Solu-ADDITIONAL REQUIREMENTStion taken:•P ACKAGING AND S TORAGE: Preserve in tight, light-resistantcontainers. Store at room temperature.Result = (rU/r S) × (C S/C U) × 100•L ABELING: If a procedure for Organic Impurities other thanProcedure 1 is used, then the labeling states with which rU= peak response from the Sample solution Organic Impurities procedure the article complies.rS= peak response from the Standard solution •USP R EFERENCE S TANDARDS〈11〉CS= concentration of USP Levofloxacin RS in the USP Levofloxacin RS Standard solution (mg/mL)USP Levofloxacin Related Compound A RS CU= nominal concentration of levofloxacin in the (S)-9-Fluoro-3-methyl-10-(piperazin-1-yl)-7-oxo-2,3-Sample solution (mg/mL)dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carbox-Acceptance criteria: 90.0%–110.0%ylic acid.C17H18FN3O4 347.34IMPURITIESUSP Levofloxacin Related Compound B RS Organic Impurities(S)-9,10-Difluoro-3-methyl-7-oxo-2,3-dihydro-7H-pyrido•P ROCEDURE[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.[N OTE—Protect the solutions of levofloxacin from light.] C13H9F2NO4 281.21Diluent, Mobile phase, Standard solution, System USP Ofloxacin RS suitability solution, Sample solution, Chromato-graphic system, and System suitability: Proceed asdirected in the Assay.AnalysisSamples:Standard solution and Sample solution Levofloxacin Oral Solution Calculate the percentage of each individual impurity inthe portion of Oral Solution taken:DEFINITIONResult = (r U/r S) × (C S/C U) × (1/F) × 100 Levofloxacin Oral Solution contains NLT 90.0% and NMT110.0% of the labeled amount of levofloxacinr U= peak response of each individual impurity(C18H20FN3O4).from the Sample solutionr S= peak response of levofloxacin from the IDENTIFICATIONStandard solution•A. The retention time of the major peak of the SampleC S= concentration of USP Levofloxacin RS in thesolution corresponds to that of the Standard solution, asStandard solution (mg/mL) obtained in the Assay.C U= nominal concentration of levofloxacin in theSample solution (mg/mL)F= relative response factor for each impurity (SeeImpurity Table 1)。