Authorized USP Pending Monograph Version 1

BRIEFING Tenofovir Disoproxil Fumarate. This monograph was posted on the USP Website as a draft USP Pending Monograph on February 25, 2011, and has been available for public comment for more than 90 days. A comment was received to correct the equation in the test for Tenofovir Disoproxil Related Compound B. The comment was incorporated, and the SM1 Expert Committee approved the monograph as an Authorized USP Pending Monograph. It is proposed to use a flexible monograph approach to indicate that manufacturers should perform either Organic Impurities, Procedure 1 or Organic Impurities, Procedure 2 based on labeling instructions and impurity profile. The liquid chromatographic procedures in the Assay and Organic Impurities, Procedure 1 were validated with the YMC-Pack ODS-AQ brand of L1 column, in which tenofovir disoproxil elutes at about 6 min with the isocratic conditions used in the Assay and about 21 min with the gradient conditions used for Organic Impurities, Procedure 1. The HPLC procedure in the test for Organic Impurities, Procedure 2 was validated with the Intersil ODS 3V brand of L1 column, in which tenofovir disoproxil elutes at about 19 min. The gas chromatographic procedure in the test for Chloromethyl Isopropyl Carbonate is based on analyses performed with the J&W Scientific DB1 brand of G1 column. The HPLC procedure in the test for Enantiomeric Purity is based on analyses performed with the Chromtech Chiral AGP brand of L41 column, in which tenofovir disoproxil elutes at about 7 min. The HPLC procedure in the test for Tenofovir Disoproxil Related Compound B is based on analyses performed with the YMC-Pack ODS-AQ brand of L1 column, in which tenofovir disoproxil elutes at 5.2 min. Description and Solubility: White to off-white crystalline powder. Freely soluble in methanol and in dimethylformamide; sparingly soluble in water. (SM1: L. Santos.) Correspondence NumberC63868

Tenofovir / 1
IDENTIFICATION A. INFRARED ABSORPTION 197K B. The retention time of the major peak from the Sample solution corresponds to that of the Standard solution, as obtained in the test for Enantiomeric Purity. ASSAY PROCEDURE Buffer: 1 mL/L of triethylamine in water. Adjust with phosphoric acid to a pH of 6.0. Mobile phase: Acetonitrile and Buffer (9:11) Diluent: Acetonitrile and water (1:19) Standard solution: 0.05 mg/mL of USP Tenofovir Disoproxil Fumarate RS in Diluent Sample solution: 0.05 mg/mL of Tenofovir Disoproxil Fumarate in Diluent Chromatographic system (See Chromatography 621, System Suitability.) Mode: LC Detector: UV 260 nm Column: 4.6-mm 25-cm; 5-m packing L1 Column temperature: 30 Flow rate: 1 mL/min Injection size: 10 L Run time: 2.5 times the retention time of tenofovir disoproxil System suitability Sample: Standard solution Suitability requirements Tailing factor: NMT 2.0 Relative standard deviation: NMT 2.0% Analysis Samples: Standard solution and Sample solution Calculate the percentage of tenofovir disoproxil fumarate (C19H30N5O10P C4H4O4) in the portion of Tenofovir Disoproxil Fumarate taken: Result = (rU/rS) (CS/CU) 100 = peak response from the Sample solution = peak response from the Standard solution = concentration of USP Tenofovir Disoproxil Fumarate RS in the Standard solution (mg/mL) CU = concentration of Tenofovir Disoproxil Fumarate in the Sample solution (mg/mL) Acceptance criteria: 98.0%102.0% on the anhydrous basis IMPURITIES RESIDUE ON IGNITION 281: NMT 0.2% HEAVY METALS, Method II 231: NMT 20 ppm [NOTEUse Organic Impurities, Procedure 1 when the impurity profile includes adenine or tenofovir disoproxil monoester. Use Organic Impurities, Procedure 2 when the impurity profile includes desmethyl tenofovir disoproxil.] ORGANIC IMPURITIES, PROCEDURE 1 Buffer: 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.
Table 1 Time (min) 0 2 30 45 50 60 Solution A (%) 100 100 0 0 100 100 Solution B (%) 0 0 100 100 0 0

rU rS CS

Tenofovir Disoproxil Fumarate

.

v.1 Authorized September 1, 2011

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(1methylethyl) ester, 5-oxide, (E)-2-butenedioate (1:1); Bis(hydroxymethyl) [[(R)-2-(6-amino-9H-purin-9-yl)-1methylethoxy]methyl]phosphonate, bis(isopropyl carbonate) (ester), fumarate (1:1); 9-[(R)-2-[Bis[[(isopropoxycarbonyl)oxy]methoxy]phosphinyl]methoxy]propyl]adenine fumarate (1:1) [202138-50-9]. DEFINITION Tenofovir Disoproxil Fumarate contains NLT 98.0% and NMT 102.0% of C19H30N5O10P C4H4O4, calculated on the anhydrous basis.

This monograph has been developed under USPs Pending Monographs Guideline and is not a USPNF monograph. http://www.usp.org 2011 The United States Pharmacopeia. All Rights Reserved.

2 / Tenofovir
System suitability solution: 0.75 g/mL of USP Tenofovir RS and USP Adenine RS in Solution A Standard solution A: 0.5 g/mL of USP Tenofovir Disoproxil Fumarate RS in Solution A Standard solution B: 0.1 mg/mL of USP Fumaric Acid RS in Solution A Sample solution: 500 g/mL of tenofovir disoproxil fumarate in Solution A. [NOTEPrepare fresh before injection.] Chromatographic system (See Chromatography 621, System Suitability.) Mode: LC Detector: UV 260 nm Column: 4.6 mm 25 cm; 5-m packing L1 Sample temperature: 4 Column temperature: 35 Flow rate: 1 mL/min Injection size: 10 L System suitability Samples: System suitability solution and Standard solution A Suitability requirements Tailing factor: NMT 2.0, Standard solution A Resolution: NLT 1.5 between tenofovir and adenine, System suitability solution Relative standard deviation: NMT 10.0%, Standard solution A Analysis Samples: Standard solution A, Standard solution B, and Sample solution [NOTEIdentify the impurities using the relative retention times shown in Table 2. Disregard the fumaric acid peak. Tenofovir disoproxil ethyl ester may elute as a split peak; consider it a single peak.] Calculate the percentage of each impurity in the portion of Tenofovir Disoproxil Fumarate taken: Result = (rU/rS) (CS/CU) (1/F) 100 = peak response of each impurity from the Sample solution rS = peak response from Standard solution A CS = concentration of USP Tenofovir Disoproxil Fumarate RS in Standard solution A (g/mL) CU = concentration of Tenofovir Disoproxil Fumarate in the Sample solution (g/mL) F = relative response factor (see Table 2) Acceptance criteria: See Table 2.
Table 2 Relative Retention Time 0.12 0.14 0.16 0.24 0.80 Relative Response Factor 2.1 4.4 1.5 0.72 Acceptance Criteria, NMT (%) 0.15 0.15 1.0 0.15

Authorized USP Pending Monograph Version 1

Table 2 (Continued) Relative Retention Time 0.82 1.00 1.40 1.76 Relative Response Factor 0.94 0.73 1.0 1.0 Acceptance Criteria, NMT (%) 0.30 0.15 0.15 0.10 2.0

Name Tenofovir isopropyl isoproxilf Tenofovir disoproxil Tenofovir disoproxil carbamateg Tenofovir disoproxil dimerh Any Individual unspecified impurity Total impurities
a b

Process impurity. Do not quantify. (R)-(1-(6-Amino-9H-purin-9-yl)propan-2-yloxy)methylphosphonic acid. c 9H-Purin-6-amine. d ({[(R)-1-(6-Amino-9H-purin-9-yl)propan-2yloxy]methyl}(hydroxy)phosphoryloxy)methyl isopropyl carbonate. e O-(Ethoxycarbonyloxymethyl)-O-(isopropoxycarbonyloxymethyl)-{(R)-[1-(6amino-9H-purin-9-yl)propan-2-yloxy]}methylphosphonate. f O-(Isopropoxycarbonyloxymethyl)-O-isopropyl-{(R)-[1-(6-amino-9H-purin-9yl)propan-2-yloxy]}methylphosphonate. g O,O-Bis(isopropoxycarbonyloxymethyl) {(R)-[1-[(6isopropoxycarbonylamino)-9H-purin-9-yl]propan-2yloxy]}methylphosphonate. h Tetra(isopropoxycarbonyloxymethyl) (2S)-1,1-[6,6methylenebis(azanediyl)bis(9H-purine-9,6-diyl)]bis(propane-2,1diyl)bis(oxy)bis(methylene)diphosphonate.

ORGANIC IMPURITIES, PROCEDURE 2 Solution A: 0.1 M of ammonium acetate in water. Adjust with glacial acetic acid to a pH of 3.8. Solution B: Methanol and acetonitrile (7:3) Mobile phase: See Table 3.
Table 3 Time (min) 0.0 10.0 25.0 50.0 60.0 70.0 Solution A (%) 95 50 50 20 95 95 Solution B (%) 5 50 50 80 5 5

rU

Name Fumaric acida Tenofovirb Adeninec Tenofovir isoproxil monoesterd Tenofovir disoproxil ethyl estere
a

Process impurity. Do not quantify. b (R)-(1-(6-Amino-9H-purin-9-yl)propan-2-yloxy)methylphosphonic acid. c 9H-Purin-6-amine. d ({[(R)-1-(6-Amino-9H-purin-9-yl)propan-2yloxy]methyl}(hydroxy)phosphoryloxy)methyl isopropyl carbonate. e O-(Ethoxycarbonyloxymethyl)-O-(isopropoxycarbonyloxymethyl)-{(R)-[1-(6amino-9H-purin-9-yl)propan-2-yloxy]}methylphosphonate. f O-(Isopropoxycarbonyloxymethyl)-O-isopropyl-{(R)-[1-(6-amino-9H-purin-9yl)propan-2-yloxy]}methylphosphonate. g O,O-Bis(isopropoxycarbonyloxymethyl) {(R)-[1-[(6isopropoxycarbonylamino)-9H-purin-9-yl]propan-2yloxy]}methylphosphonate. h Tetra(isopropoxycarbonyloxymethyl) (2S)-1,1-[6,6methylenebis(azanediyl)bis(9H-purine-9,6-diyl)]bis(propane-2,1diyl)bis(oxy)bis(methylene)diphosphonate.

Standard solution: 2.0 g/mL of USP Tenofovir Disoproxil Fumarate RS in methanol Sample solution: 2 mg/mL of Tenofovir Disoproxil Fumarate in methanol. [NOTEPrepare fresh before injection.] Chromatographic system (See Chromatography 621, System Suitability.) Mode: LC Detector: UV 260 nm Column: 4.6 mm 25 cm; 5-m packing L1 Column temperature: 30 Flow rate: 1.5 mL/min Injection size: 10 L System suitability Sample: Standard solution Suitability requirements Tailing factor: NMT 2.0 Relative standard deviation: NMT 5.0% Analysis Samples: Standard solution and Sample solution [NOTEIdentify the impurities using the relative retention times shown in Table 4. Disregard the fumaric acid peak.] Calculate the percentage of each impurity in the portion of Tenofovir Disoproxil Fumarate taken: Result = (rU/rS) (CS/CU) (1/F) 100 rU = peak response of each impurity from the Sample solution

This monograph has been developed under USPs Pending Monographs Guideline and is not a USPNF monograph. http://www.usp.org 2011 The United States Pharmacopeia. All Rights Reserved.

Authorized USP Pending Monograph Version 1

= peak response from the Standard solution = concentration of USP Tenofovir Disoproxil Fumarate RS in the Standard solution (g/mL) CU = concentration of Tenofovir Disoproxil Fumarate in the Sample solution (g/mL) F = relative response factor (see Table 4) Acceptance criteria: See Table 4.
Table 4 Relative Retention Time 0.21 0.50 0.79 0.87 1.00 Relative Response Factor 2.0 1.6 1.0 1.0 Acceptance Criteria, NMT (%) 0.15 0.80 0.15 0.15

Tenofovir / 3
Carrier gas: Nitrogen at 0.5 mL/min Split ratio: 20:1 Injection size: 1 mL Vial pressure: 14 psi Pressurization time 0.20 min Injection time: 1.0 min Vial equilibration time: 45 min System suitability Sample: Standard solution Suitability requirements Resolution: NLT 1.0 between tertiary butyl alcohol and methylene chloride Relative standard deviation: NMT 10.0% Analysis Samples: Standard solution and Sample solution Calculate the percentage of chloromethyl isopropyl carbonate in the portion of Tenofovir Disoproxil Fumarate taken: Result = (rU/rS) (CS/CU) 100 = peak response of chloromethyl isopropyl carbonate from the Sample solution rS = peak response of chloromethyl isopropyl carbonate from the Standard solution CS = concentration of USP Chloromethyl Isopropyl Carbonate RS in the Standard solution (mg/mL) CU = concentration of Tenofovir Disoproxil Fumarate in the Sample solution (mg/mL) Acceptance criteria: NMT 0.15% of chloromethyl isopropyl carbonate ENANTIOMERIC PURITY Buffer: 0.1 M ammonium acetate in water. Adjust with 5% ammonia to a pH of 6.8. Mobile phase: Methanol and Buffer (15:85) System suitability solution: 2.5 g/mL of USP Tenofovir Disoproxil Fumarate RS and USP Tenofovir Disoproxil Related Compound A RS in Mobile phase. [NOTEPrepare fresh before injection.] Standard solution: 0.25 mg/mL of USP Tenofovir Disoproxil Fumarate RS in Mobile phase. [NOTEFor Identification test B.] Sample solution: 0.25 mg/mL of Tenofovir Disoproxil Fumarate in Mobile phase Chromatographic system (See Chromatography 621, System Suitability.) Mode: LC Detector: UV 260 nm Column: 4.0 mm 15-cm; 5-m packing L41 Column temperature: 15 Flow rate: 0.8 mL/min Injection size: 10 L System suitability Sample: System suitability solution Suitability requirements Resolution: NLT 1.5 between tenofovir disoproxil and tenofovir disoproxil related compound A Analysis Samples: Standard solution and Sample solution [NOTEThe relative retention times for tenofovir disoproxil and tenofovir disoproxil related compound A are 1.0 and 1.4, respectively.] Calculate the percentage of tenofovir disoproxil related compound A in the portion of Tenofovir Disoproxil Fumarate taken: Result = (rU/rT) 100 = peak response of tenofovir related compound A from the Sample solution rT = sum of the peak responses of tenofovir disoproxil and tenofovir disoproxil related compound A from the Sample solution Acceptance criteria: NMT 1.0% of tenofovir disoproxil related compound A rU rU

rS CS

Name Tenofovira Tenofovir isoproxil monoesterb Tenofovir isopropyl isoproxilc Desmethyl tenofovir disoproxild Tenofovir disoproxil Specified unidentified impurity Any individual unspecified impurity Total impurities
a b

1.05

1.0 1.0

0.15 0.10 1.0

(R)-(1-(6-Amino-9H-purin-9-yl)propan-2-yloxy)methylphosphonic acid. ({[(R)-1-(6-Amino-9H-purin-9-yl)propan-2yloxy]methyl}(hydroxy)phosphoryloxy)methyl isopropyl carbonate. c O-(Isopropoxycarbonyloxymethyl)-O-isopropyl-{(R)-[1-(6-amino-9H-purin-9yl)propan-2-yloxy]}methylphosphonate. d O,O-Bis(isopropoxycarbonyloxymethyl) [(6-amino-9H-purin-9yl)ethyloxy]methylphosphonate.

CHLOROMETHYL ISOPROPYL CARBONATE Standard solution: 0.3 mg/mL of USP Chloromethyl Isopropyl Carbonate RS, 0.05 mg/mL of tertiary butyl alcohol, and 0.05 mg/mL of methylene chloride in Nmethyl-2-pyrrolidone Sample solution: 200 mg/mL of Tenofovir Disoproxil Fumarate in N-methyl-2-pyrrolidone in a suitable headspace vial Chromatographic system (See Chromatography 621, System Suitability.) Mode: GC/Headspace Detector: Flame ionization Column: 0.32 mm 30-m fused silica column coated with 3-m G1 Temperature Headspace oven: 85 Loop: 110 Transfer line: 120 Injector: 200 Detector: 270 Column: See Table 5.
Table 5 Initial Temperature () 45 45 Temperature Ramp (/min) 0 10 Final Temperature () 45 220 Hold Time at Final Temperature (min) 5 10

This monograph has been developed under USPs Pending Monographs Guideline and is not a USPNF monograph. http://www.usp.org 2011 The United States Pharmacopeia. All Rights Reserved.

4 / Tenofovir
TENOFOVIR DISOPROXIL RELATED COMPOUND B Buffer: 0.01 M ammonium acetate in water Mobile phase: Acetonitrile and Buffer (40:60) Standard solution: 0.01 g/mL of USP Tenofovir Disoproxyl Related Compound B RS in methanol Sample solution: 2 mg/mL of Tenofovir Disoproxil Fumarate in methanol Chromatographic system (See Chromatography 621, System Suitability.) Mode: LC Detector: Electrospray mass spectrometer operating in positive ion mode. The protonated species of tenofovir disoproxil related compound B (m/z 176) and tenofovir disoproxil fumarate (m/z 520) are monitored. Column: 4.6 mm 15-cm; 3-m packing L1 Column temperature: 30 Flow rate: 1 mL/min Injection size: 10 L System suitability Sample: Standard solution Suitability requirements Relative standard deviation: NMT 10.0% Analysis Samples: Standard solution and Sample solution [NOTEThe relative retention times for tenofovir disoproxil related compound B and tenofovir disoproxil are 0.4 and 1.0, respectively.] Calculate the quantity, in ppm, of tenofovir related compound B in the portion of Tenofovir Disoproxil Fumarate taken: Result = (rU/rS) (CS/CU) 106 = peak response of tenofovir related compound B from the Sample solution rS = peak responses of tenofovir related compound B from the Standard solution CS = concentration of USP Tenofovir Disoproxil Related Compound B RS in the Standard solution (g/mL) CU = concentration of Tenofovir Disoproxil Fumarate in the Sample solution (g/mL) Acceptance criteria: NMT 5 ppm of tenofovir disoproxil related compound B FUMARIC ACID Sample solution: 5 mg/mL of Tenofovir Disoproxil Fumarate in water rU

Authorized USP Pending Monograph Version 1

Analysis: Titrate 250 mL of the Sample solution with 0.1 N sodium hydroxide, determining the endpoint potentiometrically. Perform a blank determination, and make any necessary correction (see Titrimetry 541). Each mL of 0.1 N sodium hydroxide solution is equivalent to 5.804 mg of fumaric acid (C4H4O4). Acceptance criteria: 17.5%19.0% of fumaric acid on the anhydrous basis SPECIFIC TESTS WATER, Method I 921: NMT 1.0% ADDITIONAL REQUIREMENTS PACKAGING AND STORAGE: Preserve in tight, light-resistant containers. Store at 28. LABELING: If a test for Organic Impurities other than Procedure 1 is used, then the labeling states with which Organic Impurities test the article complies. USP REFERENCE STANDARDS 11 USP Adenine RS 1H-Purin-6-amine. C5H5N5 135.13 USP Chloromethyl Isopropyl Carbonate RS Chloromethyl isopropyl carbonate. C5H9ClO3 152.58 USP Fumaric Acid RS 2-Butenedioic acid. C4H4O4 116.07 USP Tenofovir RS (R)-[[2-(6-Amino-9H-purin-9-yl)-1methylethoxy]methyl]phosphonic acid monohydrate. C9H14N5O4P.H2O 305.23 USP Tenofovir Disoproxil Fumarate RS USP Tenofovir Disoproxil Related Compound A RS (S)-5-[[2-(6-Amino-9H-purin-9-yl)-1methylethoxy]methyl]-2,4,6,8-tetraoxa-5phosphanonanedioic acid, bis(1-methylethyl) ester, 5oxide, (E)-2-butenedioate (1:1)]. C19H30N5O10P.C4H4O4 635.51 USP Tenofovir Disoproxil Related Compound B RS (E)-9-(Prop-1-enyl)-9H-purin-6-amine. C8H9N5 175.19

This monograph has been developed under USPs Pending Monographs Guideline and is not a USPNF monograph. http://www.usp.org 2011 The United States Pharmacopeia. All Rights Reserved.