Identification—
Solvent system—
Mix 5 volumes of tert-amyl alcohol, 4 volumes of water, and 1 volume of ammonium hydroxide, shake, and allow to stand. Transfer the upper phase to a suitable chromatographic chamber arranged for thin-layer chromatography, pouring it over the paper lining, cover the chamber, and allow to stand for 1 hour.
Detection 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 freshly prepared potassium ferricyanide solution (35 in 1000).
Standard solution—
Prepare a solution of about 15 mg of
USP Levothyroxine RS, accurately weighed, in 100 mL of a mixture of 19 volumes of methanol and 1 volume of ammonium hydroxide. Dilute 10.0 mL of this solution with the same solvent to 50.0 mL, and mix.
Test solution—
Shake an amount of powdered Tablets, equivalent to about 60 µg of levothyroxine sodium, with 2 mL of a mixture of 19 volumes of methanol and 1 volume of ammonium hydroxide in a centrifuge tube for 10 minutes, and centrifuge.
Procedure—
Apply 10-µL volumes of the Test solution and of the Standard solution, respectively, to a thin-layer chromatographic plate coated with a 0.1-mm layer of cellulose. Develop the plate in the Solvent system until the solvent front has moved not less than 10 cm beyond the point of application of the Test solution, air-dry, and spray the plate with Detection reagent: the chromatogram of the Test solution shows a blue spot corresponding in RF value to the chromatogram from the levothyroxine Standard solution.
Change to read:
Dissolution 
711
—
[NOTE—All 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 minutes.
Determine the amount of C15H10I4NNaO4 dissolved by employing the following method.
Mobile phase—
Prepare a filtered and degassed mixture of methanol and 0.1% phosphoric acid (60:40). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard solution—
Prepare a stock solution of
USP Levothyroxine RS in methanol having a known concentration of about 0.1 mg per mL. Dilute this stock solution with
Medium to obtain a solution having a concentration similar to that expected in the
Test solution.
Test solution—
[NOTE—Prior to use, check the filters for absorptive loss of drug.] Use a filtered portion of the solution under test.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 225-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph the
Standard solution, and record the peak responses as directed for
Procedure: the tailing factor is not more than 1.5; and the relative standard deviation is not more than 4.0%.
Procedure—
Separately inject equal volumes (about 800 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the amount of C15H10I4NNaO4 dissolved.
Tolerances—
Not less than 70% (Q) of the labeled amount of C15H10I4NNaO4 is dissolved in 45 minutes.
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, Test solution, Chromatographic system, and Procedure—
Proceed as directed for Test 1.
Time:
15 minutes.
Tolerances—
Not less than 80% (Q) of the labeled amount of C15H10I4NNaO4 is dissolved in 15 minutes.
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 Test solution—
Proceed as directed for Test 1. [Note—Filter the Standard solution in a manner identical to the Test solution.]
Determine the amount of C15H10I4NNaO4 dissolved by employing the following method.
Mobile phase—
Prepare a filtered and degassed mixture of water and acetonitrile (65:35) with 0.5 mL of phosphoric acid per L. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 225-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L10. The column temperature is maintained at 30
. The flow rate is about 1.5 mL per minute. Chromatograph the
Standard solution, and record the peak responses as directed for
Procedure: the tailing factor is not more than 1.5; and the relative standard deviation is not more than 4.0%.
Procedure—
Separately inject equal volumes (about 100 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the amount of C15H10I4NNaO4.
Tolerances—
Not less than 80% (Q) of the labeled amount of C15H10I4NNaO4 is dissolved in 45 minutes.
USP29
TEST 4—
If the product complies with this test, the labeling indicates that it meets USP
Dissolution Test 4.
NOTE—Do not use paddle stirrers with synthetic coating.
Medium:
0.01 N hydrochloric acid; 500 mL for tablets labeled to contain between 25 µg and 175 µg of levothyroxine sodium; 900 mL for tablets labeled to contain 200 µg or 300 µg of levothyroxine sodium.
Apparatus 2:
75 rpm.
Time:
45 minutes.
Determine the amount of C15H10I4NNaO4 dissolved by employing the following method.
Mobile phase—
Prepare a filtered and degassed mixture of water, acetonitrile, and 85% orthophosphoric acid (700:500:2). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard solution—
Prepare a stock solution by transferring about 100 mg of
USP Levothyroxine RS, accurately weighed, to a 100-mL volumetric flask. Add 80 mL of alcohol and 1 mL of 1 N hydrochloric acid, sonicate for about 2 minutes, dilute with alcohol to volume, and mix. Dilute this stock solution with a mixture of alcohol and water (1:1) to obtain a solution having a concentration of 0.01 mg of levothyroxine per mL. Dilute this intermediate solution with
Medium to obtain a solution having a concentration similar to that expected in the
Test solution.
Test solution—
Use a centrifuged portion of the solution under test.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 225-nm detector and a 4.0-mm × 12.5-cm column that contains packing L7. The flow rate is about 1.5 mL per minute. Chromatograph the
Standard solution, and record the peak responses as directed for
Procedure: the tailing factor is not more than 1.5; and the relative standard deviation is not more than 4.0%.
Procedure—
Separately inject equal volumes (about 500 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the amount of C15H10I4NNaO4.
Tolerances—
Not less than 80% (Q) of the labeled amount of C15H10I4NNaO4 is dissolved in 45 minutes.
Change to read:
Limit of liothyronine sodium—
Mobile phase and Chromatographic system—
Proceed as directed in the
Assay under
Levothyroxine Sodium.
Standard solution—
Use the Standard preparation, prepared as directed in the Assay.
Test solution—
Use the Assay preparation.
Procedure—
Proceed as directed in the
Assay under
Levothyroxine Sodium. Calculate the
percentage
USP29 of liothyronine sodium (C
15H
11I
3NNaO
4) in the portion of Tablets taken by the formula:
(672.96/650.98)(1000C/W)(rU / rS)
USP29in which 672.96 and 650.98 are the molecular weights of liothyronine sodium and liothyronine, respectively;
C is the concentration, in µg per mL, of
USP Liothyronine RS in the
Standard solution; W is the amount, in µg, of levothyroxine sodium in the portion of Tablets, based on the label claim, taken to prepare the
Test solution;USP29 and
rU and
rS are the liothyronine peak responses obtained from the
Test solution and the
Standard solution, respectively: not more than 2.0% of liothyronine is found.
Assay—
Mobile phase, Standard preparation, and Chromatographic system—
Proceed as directed in the
Assay under
Levothyroxine Sodium.
Assay preparation—
Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 100 µg of levothyroxine sodium, to a centrifuge tube, add 2 glass beads, pipet 10 mL of Mobile phase into the tube, and mix on a vortex mixer for 3 minutes. Centrifuge to obtain a clear supernatant, filtering if necessary.
Procedure—
Proceed as directed in the
Assay under
Levothyroxine Sodium. Calculate the quantity, in µg, of levothyroxine sodium (C
15H
10I
4NNaO
4) in the portion of Tablets taken by the formula:
(798.85/776.87)(10C)(rU / rS)
in which 798.85 and 776.87 are the molecular weights of levothyroxine sodium and levothyroxine, respectively;
C is the concentration, in µg per mL, of
USP Levothyroxine RS in the
Standard preparation; and
rU and
rS are the levothyroxine peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
