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Printed by: Le Tran Official Date: Official as of 01-May-2020 Document Type: USP @2021 USPC
1

ASSAY
Warfarin Sodium • PROCEDURE
Buffer: Transfer 1.36 g of monobasic potassium phosphate
to a 200-mL volumetric flask, and dissolve in 50 mL of
water. Add 39.1 mL of 0.2 N sodium hydroxide, and dilute
with water to volume. Adjust with sodium hydroxide or
phosphoric acid to a pH of 7.4 ± 0.1.
Mobile phase: Methanol, glacial acetic acid, and water
(64:1:36)
Standard stock solution: 0.376 mg/mL of USP Warfarin RS
prepared as follows. Transfer USP Warfarin RS to a suitable
volumetric flask, and dissolve in 0.1 N sodium hydroxide
equivalent to 39% of the final volume. Add 0.2 M
monobasic potassium phosphate, equivalent to 25% of the
final volume, and dilute with water to volume.
C19H15NaO4 330.31 Standard solution: Transfer 5 mL of Standard stock solution
2H-1-Benzopyran-2-one, 4-hydroxy-3-(3-oxo-1-phenylbutyl)- and 15 mL of Buffer into a conical flask, and mix.
, sodium salt; Sample stock solution: 0.4 mg/mL of Warfarin Sodium
3-(α-Acetonylbenzyl)-4-hydroxycoumarin sodium salt; prepared as follows. Transfer 100 mg of Warfarin Sodium,
Sodium 2-oxo-3-(3-oxo-1-phenylbutyl)-2H-chromen-4-olate accurately weighed, to a 250-mL volumetric flask, and
[129-06-6]. dissolve in 97.8 mL of 0.1 N sodium hydroxide. Add
62.5 mL of 0.2 M monobasic potassium phosphate, and
DEFINITION dilute with water to volume.

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Warfarin Sodium is an amorphous solid or a crystalline Sample solution: Transfer 5 mL of Sample stock solution and
clathrate. The crystalline form consists principally of warfarin 15 mL of Buffer into a conical flask, and mix.
sodium and isopropyl alcohol in a 2:1 molecular ratio. It Chromatographic system
contains NLT 8.0% and NMT 8.5% of isopropyl alcohol. (See Chromatography á621ñ, System Suitability.)
Warfarin Sodium contains NLT 97.0% and NMT 102.0% of Mode: LC
warfarin sodium (C19H15NaO4), calculated on the anhydrous
basis for the amorphous form or on the anhydrous and
isopropyl alcohol-free basis for the crystalline form.
ci Detector: UV 280 nm
Column: 4.6-mm × 25-cm; packing L7
Flow rate: 1.4 mL/min
IDENTIFICATION Injection volume: 20 µL
System suitability
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Change to read: Sample: Standard solution
Suitability requirements
• A. ▲SPECTROSCOPIC IDENTIFICATION TESTS á197ñ, Infrared Relative standard deviation: NMT 1.0%
Spectroscopy: 197K▲ (CN 1-May-2020) Analysis
Standard: Use USP Warfarin RS. Samples: Standard solution and Sample solution
Sample: Dissolve 100 mg in 25 mL of water, and adjust with Calculate the percentage of warfarin sodium (C19H15NaO4)
O

hydrochloric acid to a pH of less than 3, using short-range in the portion of Warfarin Sodium taken:
pH indicator paper. Stir the mixture, and allow the
precipitate to coagulate. Filter the mixture, wash the Result = (r U/r S) × (C S/C U) × (M r1/M r2) × 100
precipitate with four, 5-mL portions of water, and dry under
vacuum over phosphorus pentoxide for 4 h. Use the rU = peak response of warfarin from the Sample
warfarin obtained. solution
• B. The retention time of the major peak of the Sample rS = peak response of warfarin from the Standard
solution corresponds to that of the Standard solution, as solution
obtained in the Assay. CS = concentration of USP Warfarin RS in the Standard
• C. solution (mg/mL)
Methoxyphenylacetic reagent: Dissolve 2.7 g of CU = concentration of Warfarin Sodium in the Sample
methoxyphenylacetic acid in 6 mL of 10% solution (mg/mL)
tetramethylammonium hydroxide aqueous solution, and M r1 = molecular weight of warfarin sodium, 330.31
add 20 mL of absolute alcohol. M r2 = molecular weight of warfarin, 308.33
Diluted ammonia: Dilute 41 g of ammonium hydroxide
with water to 100 mL. Acceptance criteria: 97.0%–102.0% on the anhydrous
Ammonium carbonate solution: 158 mg/mL of basis for the amorphous form or on the anhydrous and
ammonium carbonate in water isopropyl alcohol-free basis for the crystalline form
Sample solution: Dissolve 30 mg of Warfarin Sodium in
0.5 mL of water. IMPURITIES
Analysis: Add 1.5 mL of Methoxyphenylacetic reagent to the • ORGANIC IMPURITIES
Sample solution, and cool in ice water for 30 min. Diluent: Methanol and water (25:75)
Acceptance criteria: A voluminous, white, crystalline Mobile phase: Acetonitrile, glacial acetic acid, and water
precipitate is formed. Place the precipitate in a water bath (32:1:68)
at 20° and stir for 5 min. The precipitate does not disappear. Standard stock solution: 0.12 mg/mL each of USP
Add 1 mL of Diluted ammonia. The precipitate dissolves Warfarin RS and USP Warfarin Related Compound A RS
completely. Add 1 mL of Ammonium carbonate solution. No prepared as follows. Transfer USP Warfarin RS and USP
precipitate is formed. Warfarin Related Compound A RS to a suitable volumetric
flask, and add 0.1 N sodium hydroxide and methanol
equivalent to 2% and 25% of the final volume, respectively.
Dilute with water to volume.

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Standard solution: 2.4 µg/mL each of USP Warfarin RS and Table 1

USP Warfarin Related Compound A RS in Diluent, from Relative Relative Acceptance
Standard stock solution Retention Response Criteria,
Sample solution: 0.8 mg/mL of Warfarin Sodium in Diluent Name Time Factor NMT (%)
System suitability solution: 2.4 µg/mL of USP Warfarin 4-Hydroxycoumarina 0.4 2.0 0.3
Related Compound A RS and 0.8 mg/mL of Warfarin
Sodium in Diluent prepared as follows. Transfer 0.5 mL of Benzalacetoneb 0.6 2.0 0.3
Standard stock solution to a 25-mL volumetric flask and Warfarin 1.0 — —
dilute with Sample solution to volume.
Chromatographic system Alice’s ketonec 1.2 1.0 0.3
(See Chromatography á621ñ, System Suitability.) Any individual
—
Mode: LC unspecified impurity 1.0 0.10
Detector: UV 260 nm
Total impurities — — 1.0
Column: 4.6-mm × 25-cm; packing L10
Column temperature: 35° a 4-Hydroxy-2H-chromen-2-one.
Flow rate: 1.5 mL/min b (E)-4-Phenylbut-3-en-2-one.
Injection volume: 50 µL c Sodium salt of warfarin related compound A; 3-(o-Hydroxyphenyl)-5-phenyl-
Run time: NLT 2 times the retention time of the 2-cyclohexen-1-one sodium salt.
warfarin peak
System suitability SPECIFIC TESTS
Samples: Standard solution and System suitability solution • ISOPROPYL ALCOHOL CONTENT (CRYSTALLINE CLATHRATE
Suitability requirements FORM)
Internal standard solution: 4.25 mg/mL of n-propyl

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Resolution: NLT 3 between warfarin and warfarin related
compound A peaks, System suitability solution alcohol in water
Relative standard deviation: NMT 5.0% for the warfarin Standard stock solution: 4.25 mg/mL of isopropyl alcohol
and warfarin related compound A peaks, Standard in water
solution Standard solution: Transfer 2.0 mL of the Standard stock
Analysis solution and 2.0 mL of the Internal standard solution to a
Samples: Standard solution and Sample solution
Calculate the percentage of Alice’s ketone (sodium salt of
warfarin related compound A) in the portion of Warfarin
ci headspace vial, seal, and mix.
Sample solution: Transfer 100 mg of Warfarin Sodium,
2.0 mL of water, and 2.0 mL of the Internal standard
Sodium taken: solution to a headspace vial, seal, and mix.
Blank solution: Transfer 2.0 mL of water and 2.0 mL of the
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Result = (r U/r S) × (C S/C U) × (M r1/M r2) × 100 Internal standard solution to a headspace vial, seal, and mix.
Chromatographic system
rU = peak response of warfarin related compound A (See Chromatography á621ñ, System Suitability.)
from the Sample solution Mode: GC
rS = peak response of warfarin related compound A Detector: Flame ionization
from the Standard solution Column: 0.32-mm × 30-m; 1.8-µm coating of phase G43
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CS = concentration of USP Warfarin Related Temperatures

Compound A RS in the Standard solution Injector: 140°
(mg/mL) Detector: 240°
CU = concentration of Warfarin Sodium in the Sample Column: See Table 2.
solution (mg/mL)
M r1 = molecular weight of Alice’s ketone, 286.30 Table 2
M r2 = molecular weight of warfarin related compound Hold Time at Fi-
A, 264.32 Initial Temperature Final nal
Temperature Ramp Temperature Temperature
Calculate the percentage of any other individual impurity (°) (°/min) (°) (min)
in the portion of Warfarin Sodium taken: 40 0 40 20

Result = (r U/r S) × (C S/C U) × (M r1/M r2) × (1/F) × 100 40 10 240 10

rU = peak response of any other individual impurity Carrier gas: Nitrogen

from the Sample solution Flow rate: 1 mL/min
rS = peak response of warfarin from the Standard Injector type: Headspace; split ratio, 80:1
solution Temperatures
CS = concentration of USP Warfarin RS in the Standard Equilibration: 65°
solution (mg/mL) Needle: 75°
CU = concentration of Warfarin Sodium in the Sample Transfer line: 85°
solution (mg/mL) Headspace carrier pressure: 15 psi
M r1 = molecular weight of warfarin sodium, 330.31 Times
M r2 = molecular weight of warfarin, 308.33 Equilibration: 20 min
F = relative response factor for each individual Pressurization: 3.0 min
impurity (see Table 1) Loop fill: 0.2 min
Injection: 0.1 min
Acceptance criteria: See Table 1. Disregard any impurity System suitability
peak less than 0.06%. Sample: Standard solution

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[NOTE—The relative retention times for isopropyl Acceptance criteria: 8.0%–8.5%

alcohol and n-propyl alcohol are about 0.66 and 1.0, • PH á791ñ
respectively.] Sample solution: 10 mg/mL
Suitability requirements Acceptance criteria: 7.2–8.3
Resolution: NLT 5.0 between isopropyl alcohol and • WATER DETERMINATION á921ñ, Method I: NMT 4.5% for the
n-propyl alcohol amorphous form; NMT 0.3% for the crystalline
Tailing factor: NMT 1.3 for the isopropyl alcohol peak clathrate form
Relative standard deviation: NMT 2.0%, peak response • ABSORBANCE IN ALKALINE SOLUTION
ratio of isopropyl alcohol to n-propyl alcohol Sample solution: 125 mg/mL in sodium hydroxide solution
Analysis (1 in 20). Pass through a membrane filter.
Samples: Standard solution, Sample solution, and Blank Blank: Sodium hydroxide solution (1 in 20)
solution Analysis: Determine the absorbance of the solution in a
Calculate the percentage of isopropyl alcohol in the portion 1-cm cell at 385 nm, with a suitable spectrometer, within
of Warfarin Sodium taken: 15 min.
Acceptance criteria: NMT 0.1
Result = (R U/R S) × (C S/C U) × 100
ADDITIONAL REQUIREMENTS
RU = peak response ratio of isopropyl alcohol to • PACKAGING AND STORAGE: Preserve in well-closed,
n-propyl alcohol from the Sample solution light-resistant containers.
RS = peak response ratio of isopropyl alcohol to • LABELING: Label it to indicate whether it is the amorphous
n-propyl alcohol from the Standard solution or the crystalline form.
CS = concentration of isopropyl alcohol in the Standard • USP REFERENCE STANDARDS á11ñ
solution (mg/mL) USP Warfarin RS

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CU = concentration of Warfarin Sodium in the Sample USP Warfarin Related Compound A RS
solution (mg/mL) 3-(o-Hydroxyphenyl)-5-phenyl-2-cyclohexen-1-one.
C18H16O2 264.32
ci
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