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Levothyroxine / Official Monographs

IDENTIFICATION
THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST 201
Adsorbent: 0.1-mm layer of cellulose
Diluent: Methanol and ammonium hydroxide (19:1)
Standard solution: 0.03 mg/mL of USP Levothyroxine RS in
Diluent
Sample solution: Shake an amount of powdered Tablets,
equivalent to 60 g of levothyroxine sodium with 2 mL of
Diluent in a centrifuge tube for 10 min, and centrifuge.
Application volume: 10 L
Developing solvent system: Tert-amyl alcohol, ammonium
hydroxide and water (5:1:4)
[NOTEShake and allow to stand. Transfer the upper phase
to a suitable chromatographic chamber arranged for thinlayer chromatography, pour it over the paper lining, cover
the chamber, and allow to stand for 1 h.]
Spray reagent: Add 65 mL of 2 N hydrochloric acid to 50
mL of a 1-in-10 solution of sodium arsenite in 1 N sodium
hydroxide, with vigorous stirring. Mix 1 volume of this
solution with 5 volumes of a 27-in-1000 solution of ferric
chloride in 2 N hydrochloric acid, and 5 volumes of a
freshly-prepared 35 mg/mL potassium ferricyanide solution.
Analysis: Develop the plate in the Developing solvent system
until the solvent front has moved NLT 10 cm beyond the
point of application of the Sample solution, air-dry, and spray
the plate with Spray reagent.
Acceptance criteria: The chromatogram of the Sample
solution shows a blue spot corresponding in RF value to that
of the Standard solution.
ASSAY
PROCEDURE
Mobile phase: Acetonitrile and water (4:6) that contains
0.5 mL/L of phosphoric acid
Solution A: 400 mg of sodium hydroxide in 500 mL of
water. Cool, add 500 mL of methanol.
Levothyroxine stock solution: 0.4 mg/mL of USP
Levothyroxine RS in Solution A
Liothyronine stock solution: 0.4 mg/mL of USP
Liothyronine RS in Solution A. Make a 1:100 dilution of this
solution using Mobile phase.
Standard solution: 10 g/mL of levothyroxine from
Levothyroxine stock solution and 0.2 g/mL of liothyronine
from Liothyronine stock solution, in Mobile phase
Sample solution: Weigh and finely powder NLT 20 Tablets.
Transfer a portion of the powder equivalent to 100 g of
levothyroxine sodium to a centrifuge tube, add two glass
beads, pipet 10 mL of Mobile phase into the tube, and mix
using a vortex mixer for 3 min. Centrifuge to obtain a clear
supernatant, filtering if necessary.
Chromatographic system
(See Chromatography 621, System Suitability.)
Mode: LC
Detector: UV 225 nm
Column: 4.6-mm 25-cm; packing L10
Flow rate: 1.5 mL/min
Injection size: 100 L
System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 5.0 between liothyronine and
levothyroxine
Relative standard deviation: NMT 2.0% of levothyroxine
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of C15H10I4NNaO4 in the portion
Tablets taken:
Result = (rU/rS) (CS/CU) (Mr1/Mr2) 100
rU

= peak response of levothyroxine from the Sample

solution

USP 32
rS

= peak response of levothyroxine from the

Standard solution
= concentration of USP Levothyroxine RS in the
CS
Standard solution (mg/mL)
= nominal concentration of levothyroxine sodium
CU
in the Sample solution (mg/mL)
= molecular weight of levothyroxine sodium,
Mr1
798.85
= molecular weight of levothyroxine, 776.87
Mr2
Acceptance criteria: 90.0%110.0%
PERFORMANCE TESTS
DISSOLUTION 711
[NOTEAll containers that are in contact with solutions
containing levothyroxine sodium are to be made of glass.]
Test 1
Medium: 0.01 N hydrochloric acid containing 0.2%
sodium lauryl sulfate; 500 mL
Apparatus 2: 50 rpm
Time: 45 min
Determine the amount of C15H10I4NNaO4 dissolved by
employing the following method.
Mobile phase: Methanol and 0.1% phosphoric acid (6:4)
Standard stock solution: 0.1 mg/mL of USP
Levothyroxine RS in methanol
Standard solution: Dilute the Standard stock solution with
Medium to obtain a solution having a concentration similar
to that expected in the Sample solution.
Sample solution: Sample per Dissolution 711. Dilute with
Medium to a concentration that is similar to the Standard
solution, and filter.
[NOTEBefore use, check the filters for absorptive loss of
drug.]
Chromatographic system
(See Chromatography 621, System Suitability.)
Mode: LC
Detector: UV 225 nm
Column: 4.6-mm 25-cm; packing L1
Flow rate: 2 mL/min
Injection size: 800 L
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 1.5
Relative standard deviation: NMT 4.0% of
levothyroxine
Analysis
Samples: Standard solution and Sample solution
Calculate the amount of C15H10I4NNaO4 dissolved.
Tolerances: NLT 70% (Q) of the labeled amount of
C15H10I4NNaO4 is dissolved.
Test 2
(If the product complies with this test, the labeling indicates
that it meets USP Dissolution Test 2.)
Medium, Apparatus, Mobile phase, Standard solution,
Sample solution, Chromatographic system, and System
suitability: Proceed as directed for Test 1.
Time: 15 min
Analysis: Calculate the amount of C15H10I4NNaO4
dissolved.
Tolerances: NLT 80% (Q) of the labeled amount of
C15H10I4NNaO4 is dissolved.
Test 3
(If the product complies with this test, the labeling indicates
that it meets USP Dissolution Test 3.)
Medium, Apparatus, Time, Standard solution, and Sample
solution: Proceed as directed in Test 1.
[NOTEFilter the Standard solution in a manner identical to
the Sample solution.]
Determine the amount of C15H10I4NNaO4 dissolved by
employing the following method.

Copyright 2008 The United States Pharmacopeial Convention. All Rights Reserved.
For Discussion Purposes Only Not for Dissemination