m3429_authorized.pdf

Authorized USP Pending MonographVersion 1 Tenofovir / 1

BRIEFING IDENTIFICATION• A. INFRARED ABSORPTION ⟨197K⟩• B. The retention time of the major peak from the Sample

solution corresponds to that of the Standard solution, asTenofovir Disoproxil Fumarate. This monograph wasobtained in the test for Enantiomeric Purity.posted on the USP Website as a draft USP Pending

Monograph on February 25, 2011, and has been available ASSAYfor public comment for more than 90 days. A comment was • PROCEDUREreceived to correct the equation in the test for Tenofovir Buffer: 1 mL/L of triethylamine in water. Adjust with phos-Disoproxil Related Compound B. The comment was phoric acid to a pH of 6.0.incorporated, and the SM1 Expert Committee approved the Mobile phase: Acetonitrile and Buffer (9:11)monograph as an Authorized USP Pending Monograph. Diluent: Acetonitrile and water (1:19)It is proposed to use a flexible monograph approach to Standard solution: 0.05 mg/mL of USP Tenofovir Dis-indicate that manufacturers should perform either Organic oproxil Fumarate RS in DiluentImpurities, Procedure 1 or Organic Impurities, Procedure 2 Sample solution: 0.05 mg/mL of Tenofovir Disoproxilbased on labeling instructions and impurity profile. The Fumarate in Diluentliquid chromatographic procedures in the Assay and Chromatographic systemOrganic Impurities, Procedure 1 were validated with the (See Chromatography ⟨621⟩, System Suitability.)YMC-Pack ODS-AQ brand of L1 column, in which tenofovir Mode: LCdisoproxil elutes at about 6 min with the isocratic Detector: UV 260 nmconditions used in the Assay and about 21 min with the Column: 4.6-mm × 25-cm; 5-µm packing L1gradient conditions used for Organic Impurities, Procedure 1. Column temperature: 30°The HPLC procedure in the test for Organic Impurities, Flow rate: 1 mL/minProcedure 2 was validated with the Intersil ODS 3V brand of Injection size: 10 µLL1 column, in which tenofovir disoproxil elutes at about 19 Run time: 2.5 times the retention time of tenofovirmin. The gas chromatographic procedure in the test for disoproxilChloromethyl Isopropyl Carbonate is based on analyses System suitabilityperformed with the J&W Scientific DB1 brand of G1 Sample: Standard solutioncolumn. The HPLC procedure in the test for Enantiomeric Suitability requirementsPurity is based on analyses performed with the Chromtech Tailing factor: NMT 2.0Chiral AGP brand of L41 column, in which tenofovir Relative standard deviation: NMT 2.0%disoproxil elutes at about 7 min. The HPLC procedure in Analysisthe test for Tenofovir Disoproxil Related Compound B is based Samples: Standard solution and Sample solutionon analyses performed with the YMC-Pack ODS-AQ brand Calculate the percentage of tenofovir disoproxil fumarateof L1 column, in which tenofovir disoproxil elutes at 5.2 (C19H30N5O10P · C4H4O4) in the portion of Tenofovir Dis-min. oproxil Fumarate taken:Description and Solubility: White to off-white crystallinepowder. Freely soluble in methanol and in Result = (rU/rS) × (CS/CU) × 100dimethylformamide; sparingly soluble in water.

rU = peak response from the Sample solutionrS = peak response from the Standard solutionCS = concentration of USP Tenofovir Disoproxil(SM1: L. Santos.)

Fumarate RS in the Standard solutionCorrespondence Number—C63868(mg/mL)

CU = concentration of Tenofovir DisoproxilFumarate in the Sample solution (mg/mL)

. Acceptance criteria: 98.0%–102.0% on the anhydrousbasisTenofovir Disoproxil Fumarate

v.1 Authorized September 1, 2011 IMPURITIES• RESIDUE ON IGNITION ⟨281⟩: NMT 0.2%• HEAVY METALS, Method II ⟨231⟩: NMT 20 ppm

[NOTE—Use Organic Impurities, Procedure 1 when theimpurity profile includes adenine or tenofovir disoproxilmonoester. Use Organic Impurities, Procedure 2 when theimpurity profile includes desmethyl tenofovir disoproxil.]

• ORGANIC IMPURITIES, PROCEDURE 1Buffer: 0.01 M of dibasic sodium phosphate in water.Adjust with phosphoric acid to a pH of 5.5.

Solution A: Methanol, tertiary butyl alcohol, and Buffer(11:1:28)

Solution B: Methanol, tertiary butyl alcohol, and Buffer (27:1:12)

Mobile phase: See Table 1.C19H30N5O10P · C4H4O4 635.51(R)-5-[[2-(6-Amino-9H-purin-9-yl)-1-methylethoxy]methyl]-

2,4,6,8-tetraoxa-5-phosphanonanedioic acid, bis(1- Table 1methylethyl) ester, 5-oxide, (E)-2-butenedioate (1:1);

Time Solution A Solution BBis(hydroxymethyl) [[(R)-2-(6-amino-9H-purin-9-yl)-1-(min) (%) (%)methylethoxy]methyl]phosphonate, bis(isopropyl carbonate)

0 100 0(ester), fumarate (1:1);9-[(R)-2-[Bis[[(isopropoxycarbonyl)oxy]methox- 2 100 0

y]phosphinyl]methoxy]propyl]adenine fumarate (1:1) 30 0 100[202138-50-9]. 45 0 100

50 100 0DEFINITION60 100 0Tenofovir Disoproxil Fumarate contains NLT 98.0% and NMT

102.0% of C19H30N5O10P · C4H4O4, calculated on the anhy-drous basis.

This monograph has been developed under USP‘s Pending Monographs Guideline and is not a USP–NF monograph.http://www.usp.org

2011 The United States Pharmacopeia. All Rights Reserved.

Authorized USP Pending Monograph2 / Tenofovir Version 1

System suitability solution: 0.75 µg/mL of USP Tenofovir Table 2 (Continued)RS and USP Adenine RS in Solution A Relative Relative AcceptanceStandard solution A: 0.5 µg/mL of USP Tenofovir Retention Response Criteria,Disoproxil Fumarate RS in Solution A Name Time Factor NMT (%)Standard solution B: 0.1 mg/mL of USP Fumaric Acid RS

Tenofovir isopropylin Solution Aisoproxilf 0.82 0.94 0.30Sample solution: 500 µg/mL of tenofovir disoproxil

Tenofovir disoproxil 1.00 — —fumarate in Solution A. [NOTE—Prepare fresh beforeTenofovir disoproxilinjection.]carbamateg 1.40 0.73 0.15Chromatographic system

(See Chromatography ⟨621⟩, System Suitability.) Tenofovir disoproxilMode: LC dimerh 1.76 1.0 0.15Detector: UV 260 nm Any Individual

—Column: 4.6 mm × 25 cm; 5-µm packing L1 unspecified impurity 1.0 0.10Sample temperature: 4° Total impurities — — 2.0Column temperature: 35°

a Process impurity. Do not quantify.Flow rate: 1 mL/minb (R)-(1-(6-Amino-9H-purin-9-yl)propan-2-yloxy)methylphosphonic acid.Injection size: 10 µLc 9H-Purin-6-amine.System suitabilityd ({[(R)-1-(6-Amino-9H-purin-9-yl)propan-2-Samples: System suitability solution and Standard solution yloxy]methyl}(hydroxy)phosphoryloxy)methyl isopropyl carbonate.

A e O-(Ethoxycarbonyloxymethyl)-O-(isopropoxycarbonyloxymethyl)-{(R)-[1-(6-Suitability requirements amino-9H-purin-9-yl)propan-2-yloxy]}methylphosphonate.

Tailing factor: NMT 2.0, Standard solution A f O-(Isopropoxycarbonyloxymethyl)-O-isopropyl-{(R)-[1-(6-amino-9H-purin-9-yl)propan-2-yloxy]}methylphosphonate.Resolution: NLT 1.5 between tenofovir and adenine,g O,O-Bis(isopropoxycarbonyloxymethyl) {(R)-[1-[(6-System suitability solutionisopropoxycarbonylamino)-9H-purin-9-yl]propan-2-Relative standard deviation: NMT 10.0%, Standardyloxy]}methylphosphonate.solution Ah Tetra(isopropoxycarbonyloxymethyl) (2S)-1,1’-[6,6’-Analysis methylenebis(azanediyl)bis(9H-purine-9,6-diyl)]bis(propane-2,1-

Samples: Standard solution A, Standard solution B, and diyl)bis(oxy)bis(methylene)diphosphonate.Sample solution

• ORGANIC IMPURITIES, PROCEDURE 2[NOTE—Identify the impurities using the relative retentionSolution A: 0.1 M of ammonium acetate in water. Adjusttimes shown in Table 2. Disregard the fumaric acid peak.with glacial acetic acid to a pH of 3.8.Tenofovir disoproxil ethyl ester may elute as a split peak;

Solution B: Methanol and acetonitrile (7:3)consider it a single peak.]Mobile phase: See Table 3.Calculate the percentage of each impurity in the portion

of Tenofovir Disoproxil Fumarate taken:Table 3

Result = (rU/rS) × (CS/CU) × (1/F) × 100Time Solution A Solution B(min) (%) (%)rU = peak response of each impurity from the

0.0 95 5Sample solutionrS = peak response from Standard solution A 10.0 50 50CS = concentration of USP Tenofovir Disoproxil 25.0 50 50

Fumarate RS in Standard solution A (µg/mL) 50.0 20 80CU = concentration of Tenofovir Disoproxil

60.0 95 5Fumarate in the Sample solution (µg/mL)70.0 95 5F = relative response factor (see Table 2)

Acceptance criteria: See Table 2. Standard solution: 2.0 µg/mL of USP Tenofovir DisoproxilFumarate RS in methanol

Table 2 Sample solution: 2 mg/mL of Tenofovir DisoproxilFumarate in methanol. [NOTE—Prepare fresh beforeRelative Relative Acceptanceinjection.]Retention Response Criteria,

Chromatographic systemName Time Factor NMT (%)(See Chromatography ⟨621⟩, System Suitability.)Fumaric acida 0.12 — — Mode: LC

Tenofovirb 0.14 2.1 0.15 Detector: UV 260 nmAdeninec 0.16 4.4 0.15 Column: 4.6 mm × 25 cm; 5-µm packing L1Tenofovir isoproxil Column temperature: 30°monoesterd 0.24 1.5 1.0 Flow rate: 1.5 mL/min

Injection size: 10 µLTenofovir disoproxilSystem suitabilityethyl estere 0.80 0.72 0.15Sample: Standard solutiona Process impurity. Do not quantify. Suitability requirementsb (R)-(1-(6-Amino-9H-purin-9-yl)propan-2-yloxy)methylphosphonic acid. Tailing factor: NMT 2.0c 9H-Purin-6-amine. Relative standard deviation: NMT 5.0%d ({[(R)-1-(6-Amino-9H-purin-9-yl)propan-2- Analysisyloxy]methyl}(hydroxy)phosphoryloxy)methyl isopropyl carbonate.Samples: Standard solution and Sample solutione O-(Ethoxycarbonyloxymethyl)-O-(isopropoxycarbonyloxymethyl)-{(R)-[1-(6-

amino-9H-purin-9-yl)propan-2-yloxy]}methylphosphonate. [NOTE—Identify the impurities using the relative retentionf O-(Isopropoxycarbonyloxymethyl)-O-isopropyl-{(R)-[1-(6-amino-9H-purin-9- times shown in Table 4. Disregard the fumaric acidyl)propan-2-yloxy]}methylphosphonate. peak.]g O,O-Bis(isopropoxycarbonyloxymethyl) {(R)-[1-[(6- Calculate the percentage of each impurity in the portionisopropoxycarbonylamino)-9H-purin-9-yl]propan-2- of Tenofovir Disoproxil Fumarate taken:yloxy]}methylphosphonate.h Tetra(isopropoxycarbonyloxymethyl) (2S)-1,1’-[6,6’- Result = (rU/rS) × (CS/CU) × (1/F) × 100methylenebis(azanediyl)bis(9H-purine-9,6-diyl)]bis(propane-2,1-diyl)bis(oxy)bis(methylene)diphosphonate.

rU = peak response of each impurity from theSample solution



Authorized USP Pending MonographVersion 1 Tenofovir / 3

rS = peak response from the Standard solution Carrier gas: Nitrogen at 0.5 mL/minCS = concentration of USP Tenofovir Disoproxil Split ratio: 20:1

Fumarate RS in the Standard solution (µg/mL) Injection size: 1 mLCU = concentration of Tenofovir Disoproxil Vial pressure: 14 psi

Fumarate in the Sample solution (µg/mL) Pressurization time 0.20 minF = relative response factor (see Table 4) Injection time: 1.0 min

Acceptance criteria: See Table 4. Vial equilibration time: 45 minSystem suitabilitySample: Standard solutionTable 4Suitability requirements

Relative Relative Acceptance Resolution: NLT 1.0 between tertiary butyl alcohol andRetention Response Criteria, methylene chloride

Name Time Factor NMT (%) Relative standard deviation: NMT 10.0%Tenofovira 0.21 2.0 0.15 Analysis

Samples: Standard solution and Sample solutionTenofovir isoproxilCalculate the percentage of chloromethyl isopropylmonoesterb 0.50 1.6 0.80carbonate in the portion of Tenofovir Disoproxil FumarateTenofovir isopropyltaken:isoproxilc 0.79 1.0 0.15

Desmethyl tenofovir Result = (rU/rS) × (CS/CU) × 100disoproxild 0.87 1.0 0.15

Tenofovir disoproxil 1.00 — — rU = peak response of chloromethyl isopropylSpecified carbonate from the Sample solutionunidentified rS = peak response of chloromethyl isopropylimpurity 1.05 1.0 0.15 carbonate from the Standard solution

CS = concentration of USP Chloromethyl Isopropyl Any individual— Carbonate RS in the Standard solutionunspecified impurity 1.0 0.10

(mg/mL)Total impurities — — 1.0CU = concentration of Tenofovir Disoproxil

a (R)-(1-(6-Amino-9H-purin-9-yl)propan-2-yloxy)methylphosphonic acid. Fumarate in the Sample solution (mg/mL)b ({[(R)-1-(6-Amino-9H-purin-9-yl)propan-2- Acceptance criteria: NMT 0.15% of chloromethylyloxy]methyl}(hydroxy)phosphoryloxy)methyl isopropyl carbonate.

isopropyl carbonatec O-(Isopropoxycarbonyloxymethyl)-O-isopropyl-{(R)-[1-(6-amino-9H-purin-9-• ENANTIOMERIC PURITYyl)propan-2-yloxy]}methylphosphonate.

Buffer: 0.1 M ammonium acetate in water. Adjust withd O,O-Bis(isopropoxycarbonyloxymethyl) [(6-amino-9H-purin-9-5% ammonia to a pH of 6.8.yl)ethyloxy]methylphosphonate.

Mobile phase: Methanol and Buffer (15:85)• CHLOROMETHYL ISOPROPYL CARBONATE System suitability solution: 2.5 µg/mL of USP Tenofovir

Standard solution: 0.3 mg/mL of USP Chloromethyl Disoproxil Fumarate RS and USP Tenofovir DisoproxilIsopropyl Carbonate RS, 0.05 mg/mL of tertiary butyl Related Compound A RS in Mobile phase. [NOTE—Preparealcohol, and 0.05 mg/mL of methylene chloride in N- fresh before injection.]methyl-2-pyrrolidone Standard solution: 0.25 mg/mL of USP Tenofovir

Sample solution: 200 mg/mL of Tenofovir Disoproxil Disoproxil Fumarate RS in Mobile phase. [NOTE—ForFumarate in N-methyl-2-pyrrolidone in a suitable Identification test B.]headspace vial Sample solution: 0.25 mg/mL of Tenofovir Disoproxil

Chromatographic system Fumarate in Mobile phase(See Chromatography ⟨621⟩, System Suitability.) Chromatographic systemMode: GC/Headspace (See Chromatography ⟨621⟩, System Suitability.)Detector: Flame ionization Mode: LCColumn: 0.32 mm × 30-m fused silica column coated Detector: UV 260 nmwith 3-µm G1 Column: 4.0 mm × 15-cm; 5-µm packing L41

Temperature Column temperature: 15°Headspace oven: 85° Flow rate: 0.8 mL/minLoop: 110° Injection size: 10 µLTransfer line: 120° System suitabilityInjector: 200° Sample: System suitability solutionDetector: 270° Suitability requirementsColumn: See Table 5. Resolution: NLT 1.5 between tenofovir disoproxil and

tenofovir disoproxil related compound ATable 5 Analysis

Samples: Standard solution and Sample solutionHold Time [NOTE—The relative retention times for tenofovirInitial Temperature Final at Final disoproxil and tenofovir disoproxil related compound ATemperature Ramp Temperature Temperature are 1.0 and 1.4, respectively.](°) (°/min) (°) (min) Calculate the percentage of tenofovir disoproxil related45 0 45 5 compound A in the portion of Tenofovir Disoproxil45 10 220 10 Fumarate taken:

Result = (rU/rT) × 100

rU = peak response of tenofovir related compoundA from the Sample solution

rT = sum of the peak responses of tenofovirdisoproxil and tenofovir disoproxil relatedcompound A from the Sample solution

Acceptance criteria: NMT 1.0% of tenofovir disoproxilrelated compound A



Authorized USP Pending Monograph4 / Tenofovir Version 1

• TENOFOVIR DISOPROXIL RELATED COMPOUND B Analysis: Titrate 250 mL of the Sample solution with 0.1 NBuffer: 0.01 M ammonium acetate in water sodium hydroxide, determining the endpointMobile phase: Acetonitrile and Buffer (40:60) potentiometrically. Perform a blank determination, andStandard solution: 0.01 µg/mL of USP Tenofovir make any necessary correction (see Titrimetry ⟨541⟩). EachDisoproxyl Related Compound B RS in methanol mL of 0.1 N sodium hydroxide solution is equivalent to

Sample solution: 2 mg/mL of Tenofovir Disoproxil 5.804 mg of fumaric acid (C4H4O4).Fumarate in methanol Acceptance criteria: 17.5%–19.0% of fumaric acid on the

Chromatographic system anhydrous basis(See Chromatography ⟨621⟩, System Suitability.)

SPECIFIC TESTSMode: LC• WATER, Method I ⟨921⟩: NMT 1.0%Detector: Electrospray mass spectrometer operating in

positive ion mode. The protonated species of tenofovir ADDITIONAL REQUIREMENTSdisoproxil related compound B (m/z 176) and tenofovir • PACKAGING AND STORAGE: Preserve in tight, light-resistantdisoproxil fumarate (m/z 520) are monitored. containers. Store at 2°–8°.Column: 4.6 mm × 15-cm; 3-µm packing L1 • LABELING: If a test for Organic Impurities other thanColumn temperature: 30° Procedure 1 is used, then the labeling states with whichFlow rate: 1 mL/min Organic Impurities test the article complies.Injection size: 10 µL • USP REFERENCE STANDARDS ⟨11⟩System suitability USP Adenine RSSample: Standard solution 1H-Purin-6-amine.Suitability requirements C5H5N5 135.13Relative standard deviation: NMT 10.0% USP Chloromethyl Isopropyl Carbonate RSAnalysis Chloromethyl isopropyl carbonate.Samples: Standard solution and Sample solution C5H9ClO3 152.58[NOTE—The relative retention times for tenofovir USP Fumaric Acid RSdisoproxil related compound B and tenofovir disoproxil 2-Butenedioic acid.are 0.4 and 1.0, respectively.] C4H4O4 116.07 Calculate the quantity, in ppm, of tenofovir related USP Tenofovir RScompound B in the portion of Tenofovir Disoproxil (R)-[[2-(6-Amino-9H-purin-9-yl)-1-Fumarate taken: methylethoxy]methyl]phosphonic acid monohydrate.C9H14N5O4P.H2O 305.23Result = (rU/rS) × (CS/CU) × 106

USP Tenofovir Disoproxil Fumarate RSUSP Tenofovir Disoproxil Related Compound A RSrU = peak response of tenofovir related compound(S)-5-[[2-(6-Amino-9H-purin-9-yl)-1-B from the Sample solutionmethylethoxy]methyl]-2,4,6,8-tetraoxa-5-rS = peak responses of tenofovir related compoundphosphanonanedioic acid, bis(1-methylethyl) ester, 5-B from the Standard solutionoxide, (E)-2-butenedioate (1:1)].CS = concentration of USP Tenofovir Disoproxil

C19H30N5O10P.C4H4O4 635.51Related Compound B RS in the StandardUSP Tenofovir Disoproxil Related Compound B RSsolution (µg/mL)(E)-9-(Prop-1-enyl)-9H-purin-6-amine.CU = concentration of Tenofovir DisoproxilC8H9N5 175.19Fumarate in the Sample solution (µg/mL)

Acceptance criteria: NMT 5 ppm of tenofovir disoproxilrelated compound B

• FUMARIC ACIDSample solution: 5 mg/mL of Tenofovir DisoproxilFumarate in water



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