Claims:1. A process for the preparation of Liothyronine Sodium (Formula I) which is easily scalable, comprising the steps of:
a. Iodination of L-Tyrosine (Formula VIII) in presence of Iodine and a suitable organic base to give 3,5-Diiodo-L-Tyrosine hydrate (Formula VII);
b. Reacting 3,5-Diiodo-L-Tyrosine dihydrate (Formula VII) with aqueous copper sulphate solution to give 3, 5-diiodo L-Tyrosine copper complex (Formula VI);
c. Coupling reaction between 3, 5-diiodo L-Tyrosine copper complex (Formula VI) and Bis (p-anisyl) iodonium iodide (Formula V) in the presence of an organic base in alcoholic solvent to obtain 2-Amino-3- (3,5-diiodo-4-(4-methoxy phenoxy)phenyl) propanoic acid (Formula IV);
d. De-methylation of 2-Amino-3-(3,5-diiodo-4-(4- methoxyphenoxy)phenyl) propanoic acid (Formula IV) using a mixture of Acetic acid and hydroiodic acid to obtain 3,5-diiodothyronine (Formula III);
e. Iodinizing 3,5-diiodothyronine (Formula III) with aqueous monomethylamine and Iodine to obtain Liothyronine (Formula II);
f. Reacting Liothyronine (Formula II) with sodium hydroxide or sodium carbonate solution in alcoholic or ether solvents or a mixture thereof and isolating pure Liothyronine Sodium (Formula I).

2. The process as claimed in claim 1 wherein the organic base used in step (a) is selected from triethylamine, Diisopropylamine and aqueous or alcoholic monomethylamine.

3. The process as claimed in step (a) of claim 1 wherein the monomethylamine used is alcoholic monomethylamine.

4. The process as claimed in claim 1 wherein step (a) is carried out optionally in the presence of alkyl halide.

5. The process as claimed in claim 1 step (c) wherein the organic base is Diisopropylamine and the alcoholic solvent is n-butanol.

6. The process as claimed in Step (f) of claim 1 wherein the solvent is an alcoholic solvent.

7. The process as claimed in claim 6 wherein the alcoholic solvent is n-butanol.

8. The process as claimed in claim 1 wherein Liothyronine sodium is obtained with Levothyroxine levels of below 1.0% wt/wt.

9. The process as claimed in claim 1 wherein the coupling reagent Bis (p-anisyl)iodonium Iodide (Formula V) of step (c) is synthesized by coupling of para iodoanisole (Formula Va) in presence of anisole and acetic anhydride and methane sulphonic acid to give Bis (p-anisyl)iodonium Iodide.

10. A pharmaceutical formulation comprising the Liothyronine prepared by the process as claimed in claim 1 along with a pharmaceutically acceptable carrier, and at least one pharmaceutically acceptable excipient.
, Description:FIELD OF THE INVENTION
The present invention relates to a novel and easily scalable process for the preparation of pure Liothyronine Sodium, i.e., Sodium (S)-2-amino-3-[4-(4-hydroxy-3-iodophenoxy)-3,5-diiodophenyl]propanoate or (S)-2-Amino-3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]propanoic acid sodium salt.

BACKGROUND OF THE INVENTION

Liothyronine Sodium is a synthetic form of thyroid hormone Thyroxine, which is secreted from follicular cells of thyroid gland. Liothyronine is ideally used for the treatment of thyroid hormone deficiencies such as hypothyroidism. Due to its ability to lower thyroid- stimulating hormone, Liothyronine is also used in the treatment of goiter and also to prevent the recurrence of thyroid cancer.

Liothyronine Sodium has the following chemical structure.

[Formula I]
For the preparation of Liothyronine Sodium several methods have been known.

US2889363 demonstrates the use of animal natural sources as starting material for the preparation of thyroxine/Diiodotyrosine while US2889364 discloses an enzymatic or bio-mimetic method of synthesis for Levothyroxine /Diiodotyrosine.
The invention disclosed in US2889363 provides a process for the production of esters of N- acylthyroxine with a substantial reduction in the time period and involves digestive coupling reactions in which alkyl esters of N-acyldiidotyrosine yield alkyl esters of N-acylthyroxine in a greatly reduced time in the presence of optimal catalyst concentration, pH range and alcohol concentration. This prior art further involves a process for production of thyroxine by incubating alkyl esters of N-acyldiidotyrosine derived from lower alkanols and lower alkanoic acids in aqueous solution of ethanol (70%) of pH of 9.5-10.5 and in the presence of between 1.5 and 5% by weight of a manganese containing catalyst while passing substantially pure oxygen through the solution and while maintaining the temperature in the range of 25°C to 78°C, and hydrolytically removing acyl and ester substituents from the ester of N-acylthyroxine obtained from the incubation step.
PCT publication No. WO 1996011904 discloses further improvements to a six stage process for production of sodium 1-thyroxine from 1-tyrosine as described in U.S. Pat. No. 2,889,363 and U.S. Pat. No. 2,889,364. These improvements comprise the oxidative coupling of a diiodo-L-tyrosine to form a biphenyl ether derivative, catalysed by a manganese salt in which the amine and acid functionality of the diiodo-L-tyrosine have been protected by suitable protecting groups, characterized in that the reaction is performed at a pressure of about 20 atmospheres in the presence of an organic amine additive using a gaseous oxidant comprising oxygen and optionally an inert diluent. The process optionally further comprises acid hydrolysis of the biphenyl ether derivative with hydrochloric acid to form 1-thyroxine hydrochloride salt and generation of sodium-1-thyroxine from the 1-thyroxine hydrochloride salt.

PCT publication number WO2009136249 provides a process for the preparation of Levothyroxine.

EP3024326 relates to a process for preparation of Levothyroxine sodium.

Liothyronine sodium obtained by this invention is substantially free from d- enantiomer of thyroxine / 3, 5-Diiodothyronine impurity. The process also reports the d- enantiomer of thyroxine / 3, 5-Diiodothyronine levels below the limit of detection and liothyronine impurity below 0.5% wt / wt. The end product derived from this process is also free from coloured impurities. Many patent and non-patent literature describe the use of an intermediate called Bis (p- anisyl)iodonium bromide in the preparation of Liothyronine sodium.

Hillmann G. (Z. Naturforch 1956b; Tiel B 11, 424-425) describes a process for the assembly of the biphenyl-ether system present in Liothyronine, wherein a key coupling reaction is initiated between N- Acetyl 3,5- diiodo-L-tyrosine ethyl ester, derived from the stepwise protective conversion of amine as amide and acid as ester of 3,5 diido-L-tyrosine, and Bis (p-anisyl)iodonium bromide in the presence of copper metal or powder as a catalyst to afford (S)-N-acetyl-3,5-diiodo-4-p- methoxyphenoxyphenylalanine ethyl ester, with 87% yield. All the three protective groups in (S)-N-acetyl-3,5-diiodo-4-p-methoxyphenoxyphenylalanine ethyl ester ,viz. acetamide, methyl ether, ethyl ester were cleaved using a mixture of hydroiodic acid and hydrobromic acid to give 3,5-Diiodothyronine. 3,5-Diiodothyronine on subsequent iodination with iodine gave L-thyroxine of an yield that corresponds to 92%.

The use of Bis (p-anisyl)iodonium bromide in the synthesis of Liothyronine leads to the formation of an impurities called monobromodiiodothyronine and dibromomonoiodothyronine which may be categorized as "Genotoxic impurities" based on structure alerts. The Liothyronine compositions thus prepared are not generally preferred due to their genotoxic impurity content associated with purified Liothyronine.

The aforesaid prior art processes cannot be considered for industrial scale preparation in view of their shortcomings such as less yield of the product, high cost involved, pressure reactions which are less feasible for commercialization of the product, more number of reaction steps, cycle time and concerns about the purity of the end product. Hence there is a long felt need for the development of an improved process that circumvents the above disadvantages and provides an industrially feasible, cost effective process involving the use of commercially available, simple raw materials in the process. At the same time it is also required that the process or technology developed should be easily adaptable for multi kilo manufacturing plants for scaling-up the most demanding API product Liothyronine sodium at a very cheap cost, compared to the current import costs of the material and contemporary technologies in vogue.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide an improved and industrially realizable process for preparing Liothyronine Sodium.
It is another object of the present invention to provide an improved process for preparing Liothyronine Sodium which is simple, convenient and economical with commercial feasibility.
Yet another object of the present invention is to provide an improved process for preparing Liothyronine Sodium by involving process intermediates namely 3,5-Diiodo L- Tyrosine copper complex and Bis (p-anisyl)iodonium Iodide.
A further object of the present invention is to provide an improved process for preparing Liothyronine Sodium by using aqueous monomethylamine during mono iodination in the reaction.
A further object of the present invention is to provide an improved process for preparing Liothyronine Sodium by using unprotected L-Thyrosine in the reaction.
Yet another object of the invention is to provide a process for preparing pure Liothyronine Sodium that is devoid of any genotoxic impurities generated in the process.
Another object of the invention is to provide a process for preparing pure Liothyronine sodium using sodium carbonate and an alcoholic / ether mixed solvent.
Another object of the invention is to provide a process for preparing pure Liothyronine Sodium obtained with other unidentified / uncharacterized impurities <0.1%.
These and other objects of the present invention will be realized by way of practice of the invention described hereinafter.

SUMMARY OF THE INVENTION
According to an aspect of the present invention there is provided a process for the preparation of Liothyronine Sodium (Formula I) which is easily scalable, comprising the steps of:
a. Iodination of L-Tyrosine (Formula VIII) in presence of Iodine and a suitable organic base to give 3,5-Diiodo-L-Tyrosine hydrate (Formula VII);
b. Reacting 3,5-Diiodo-L-Tyrosine dihydrate (Formula VII) with aqueous copper sulphate solution to give 3, 5-diiodo L-Tyrosine copper complex (Formula VI);
c. Coupling reaction between 3, 5-diiodo L-Tyrosine copper complex (Formula VI) and Bis (p-anisyl) iodonium iodide (Formula V) in the presence of an organic base in alcoholic solvent to obtain 2-Amino-3- (3,5-diiodo-4-(4-methoxy phenoxy)phenyl) propanoic acid (Formula IV);
d. De-methylation of 2-Amino-3-(3,5-diiodo-4-(4- methoxyphenoxy)phenyl)propanoic acid (Formula IV) using a mixture of Acetic acid and hydroiodic acid to obtain 3,5-diiodothyronine (Formula III);
e. Iodinizing 3,5-diiodothyronine (Formula III) with aqueous monomethylamine and Iodine to obtain Liothyronine (Formula II);
f. Reacting Liothyronine (Formula II) with sodium hydroxide or sodium carbonate solution in alcoholic or ether solvents or a mixture thereof and isolating pure Liothyronine Sodium (Formula I).

DETAILED DESCRIPTION OF THE INVENTION
It has been surprisingly found that by using two simple intermediates namely 3,5-Diiodo L- Tyrosine copper complex and Bis (p-anisyl)iodonium Iodide the process can be made economically and industrially feasible and scalable. The use of 3,5-Diiodo L-Tyrosine copper complex reduces the number of steps and increases the yield of the product while the use of Bis (p-anisyl)iodonium Iodide reduces the probable genotoxic impurities formation. In this method highly pure Liothyronine Sodium (99.0%) is obtained and the Levothyroxine impurities observed, corresponds to only 1.0%.

According to one aspect of the present invention there is provided a process for the preparation of Liothyronine Sodium (Formula I) which is easily scalable, comprising the steps of:
a. Iodination of L-Tyrosine (Formula VIII) in presence of Iodine and a suitable organic base to give 3,5-Diiodo-L-Tyrosine hydrate (Formula VII);
b. Reacting 3,5-Diiodo-L-Tyrosine dihydrate (Formula VII) with aqueous copper sulphate solution to give 3, 5-diiodo L-Tyrosine copper complex (Formula VI);
c. Coupling reaction between 3, 5-diiodo L-Tyrosine copper complex (Formula VI) and Bis (p-anisyl) iodonium iodide (Formula V) in the presence of an organic base in alcoholic solvent to obtain 2-Amino-3- (3,5-diiodo-4-(4-methoxy phenoxy)phenyl) propanoic acid (Formula IV);
d. De-methylation of 2-Amino-3-(3,5-diiodo-4-(4- methoxyphenoxy)phenyl)propanoic acid (Formula IV) using a mixture of Acetic acid and hydroiodic acid to obtain 3,5-diiodothyronine (Formula III);
e. Iodinizing 3,5-diiodothyronine (Formula III) with aqueous monomethylamine and Iodine to obtain Liothyronine (Formula II);
f. Reacting Liothyronine (Formula II) with sodium hydroxide or sodium carbonate solution in alcoholic or ether solvents or a mixture thereof and isolating pure Liothyronine Sodium (Formula I).

The organic base used in step (a) of the above process is preferably triethylamine or Diisopropylamine or aqueous or alcoholic monomethylamine and more preferably methanolic monomethylamine.
Optionally step (a) can be carried out additionally in the presence of alkyl halide.

The organic base used in step (c) of the above process is preferably Diisopropylamine and the alcoholic solvent is preferably n-butanol.
The solvent used in step (f) of the above process is preferably an alcoholic solvent which is more preferably n-butanol.
The quality of water used in the synthesis process of Liothyronine sodium meets with USP / EP and water for injection grade.
The coupling reagent i.e. Bis (p-anisyl)iodonium Iodide, used in the process of the present invention has been described in EP3024326. Bis (p-anisyl)iodonium Iodide (Formula V) may be synthesized by coupling of para iodoanisole (Formula Va) in presence of anisole and acetic anhydride methane sulphonic acid to give Bis (p-anisyl)iodonium Iodide.

Representation of the Scheme
Scheme 1: Representation of overall chemical route of synthesis of Liothyronine sodium according to the present invention is depicted below:
SCHEME 1:

Storage condition of Liothyronine sodium made by above described process is preferably under standard packing / vacuum packing / nitrogen blanketing / -20 degress Celsius to 20-35 degrees Celsius.
According to another aspect of the present invention, there is provided pharmaceutical compositions comprising a therapeutically effective amount of Linothyronine sodium prepared by the present invention along with a pharmaceutically acceptable carrier, and at least one pharmaceutically acceptable excipient. Various pharmaceutically acceptable carriers and pharmaceutically acceptable excipients are known in the art. Liothyronine sodium may be formulated into tablets, capsules, suspensions, dispersions, injectables or other pharmaceutical forms. In a preferred embodiment Liothyronine sodium may be formulated into injectables.
It will be apparent to the skilled person that certain changes and modifications may be practiced within the scope of the invention. Following examples illustratively demonstrate the process of the present invention, without posing any limitation to it.

EXAMPLES
Example-1
Preparation of 3,5-Diiodo-L-Tyrosine dihydrate (Formula VII):
RBF (Round bottom Flask) was charged with 1000 ml of methanolic monomethyl amine, 100 g of L-Tyrosine (Formula VIII) and 1000 ml of methanol at 20-25°C, cooled the reaction mixture to 0-5°C and charged 308.16 g of Iodine, maintained for 2-2.5 hours at 0-5°C. After reaction completion, 1000 ml of 20 % Aq. Potassium bisulfate, potassium acetate was added to the reaction mixture then the temperature of the reaction mixture was raised to 20-25°C. pH of the reaction mixture was adjusted to 6.3-6.8 with 1000 ml of aqueous hydroiodic acid. Cooled the reaction mixture to 0-5°C, maintained for 2-2.5 hours, filtered, washed the wet cake with 200 ml of chilled Methanol, suck dried. Again charged 1000 ml of Methanol and the above obtained wet cake into another RBF at 20-25°C, heated the reaction mixture to 50-55°C, maintained for 2-2.5 hours at 50-55°C, then cooled to 0-5°C, stirred for 60-75 min, filtered, washed the wet cake with 2x200 ml of Methanol and dried at NMT 55°c to get 230 g of 3,5-Diiodo-L-Tyrosine dihydrate (Formula VII); purity NLT 97 %; yield 90 %.

Experimental yield details:
S.No Experiment No.
[Each experiment followed the above process steps] Input (L-Tyrosine) Output (3,5-Diiodo-L-Tyrosinedihydrate) Yield (%)
01 LVT-A133-004 100 g 230 g 89
02 LVT-A133-005 100 g 236 g 91
03 LVT-A133-006 100 g 232 g 90

Example-2
Preparation of Bis (p-anisyl)iodonium Iodide (Formula V):
RBF (Round bottom Flask) was charged with 930 ml of Acetic acid, 100 g of Para iodo anisole (Formula Va) at 25-30°C, 19.76 g of was added slowly over a period of 30-45 min. 103.9 g of methanesulfonic acid was added slowly over a period of 30-45 min, charged 92.3 g of anisole at 25-30°C. The reaction mixture was heated to 50-55°C over a period of 1-2 hours. 246.68 g of ammonium persulfate was added slowly in 10 lots between 15 min interval, maintained the reaction mixture for 15-16 hrs at 50-55°C. After reaction completion, cooled to 20-25°C, maintained for 1-1.5 hours. potassium iodide solution (85.12 g of potassium iodide dissolved in 220 ml of purified water) was added slowly to reaction mixture over a period of 20-30 mins, stirred for 15-30 min, filtered, washed the wet cake with 3x600 ml of Purified water, suck dried. Charged 500 ml of Acetone and the above obtained wet cake into another RBF at 25-30°C, stirred for 2.5-3 hours, filtered, washed the wet cake with 2x100 ml of Acetone and dried under vacuum at NMT 55°C to get 160 g of Bis (p-anisyl)iodonium Iodide (Formula V); purity NLT 96 %; yield 82 %.

Experimental yield details:
S. No Experiment No.
[Each experiment followed the above process steps] Input (Para iodo anisole) Output (Bis (p-anisyl)iodonium Iodide) Yield (%)
01 LVT-A104-030 100 g 160 g 80
02 LVT-A104-040 100 g 164 g 82
03 LVT-A104-041 100 g 167 g 84

Example-3
Preparation of 3,5-Diiodo L-Tyrosine copper complex (Formula VI):
RBF (Round bottom Flask) was charged with 1200ml of water and 15.01g of sodium hydroxide at 20-25°C and stirred to get a clear solution. 100g of 3,5-Diiodo-L-tyrosine dihydrate (Formula VII) was added and to the above mixture and stirred for an hour to get a clear solution. Copper sulphate solution (35.94gms dissolved in 225ml of water at 40°C) was then added into the reaction mixture over a period of l hour and maintained at 20-25°C for l hour. The resultant reaction mixture is filtered and washed with 1500ml of DM water and dried under vacuum at 55-60°C to get 95g of 3,5-Diiodo L-Tyrosine copper complex (Formula VI). The purity of obtained complex is NLT (not less than) 97% with yield of 95% will be taken directly for next stage.

Example-4:
Preparation of 3,5-Diiodothyronine (Formula III):
RBF (Round bottom Flask) was charged with l00gms of 3,5-Diiodo L-Tyrosine copper complex (Formula VI) and 1200ml of water to which 1460ml of n-butanol was slowly added over a period of 30-45min at 20-25°C. RBF was then charged with 36.6g of diisopropylamine at 20°C and 92g of Bis(p-anisyl)iodonium iodide (Formula V) was added into the reaction mixture at 20-25°C. The reaction mixture was heated to 90°C and maintained for 2hrs at 90°C, then cooled to 20°C, followed by addition of 278ml of toluene. To the above mixture 226ml of 10% aqueous citric acid solution was added at 20-30°C and maintained for 2hrs at 20°C followed by filtration and washing with 41ml of water followed by 181ml of Methylisobutylketone wash to get 230g of wet 2-Amino-3-(3,5-diiodo-4-(4-methoxyphenoxy)phenyl) propanoic acid. The above wet cake was stirred with 400ml of water and 40g of citric acid for an hour at 20- 25°C, filtered and washed with dilute citric acid solutions to get 160g of wet and pure 2- Amino-3-(3,5-diiodo-4-(4-methoxyphenoxy)phenyl)propanoic acid. The above wet cake was charged with 400ml of acetic acid and 300ml of hydroiodic acid and the mixture was heated to 100°C and maintained for 5 hours and then cooled to 25-30°C. The reaction mixture was quenched in 3000ml of 5% potassium bisulfite solution and 5% potassium dihydrogen phosphate mixture solution. The pH of the reaction mixture was adjusted to 4 using 250ml of 50% aqueous lithium hydroxide solution and maintained for 30min at 25-30°C. The reaction mixture was then filtered and washed with 500ml of water followed by 200ml of 1-propanol (2 times), dried under vacuum at 50-55°C for 12-15hrs to get 80g of 3, 5 –Diiodothyronine (Formula III) of purity NLT 98% and yield corresponding to 74%.

Experimental yield details:
S.No Experiment No.
[Each experiment followed the above process steps] Input (3,5-Diiodo-L-Tyrosinedihydrate) Output (3,5-Diiodothyronine) Yield (%)
01 LVT-A105-008 100 g 85 g 76
02 LVT-A105-009 100 g 84 g 75
03 LVT-A105-010 100 g 83 g 74

Example-5
Preparation of Liothyronine (Formula II):
RBF was charged with l00gms of 3,5-Diiodothyronine and 1000ml of Aqueous monomethylamine at 25-30°C and stirred for 15-20min to get clear solution. The reaction mixture was then cooled to -8 to 0°C, and added with Iodine solution(1.0equ) at -8 to 0°C over a period of 2 hours and maintained for 30-45min at -8 to 0°C.The temperature of the reaction mixture was slowly brought to 15-20°C and Charged 600g of Sodium acetate. The temperature of the reaction mixture was adjusted to 25-30°C and maintained for 30 - 45min at 25-30°C. The resultant mixture was filtered and washed with 200ml of water followed by 200ml of acetonitrile, dried under vacuum for 12-15 hours at 55-60°C to get 100g of Liothyronine with purity NLT 98.5% and yield of 50-70%.
Experimental yield details:
S.No. Experiment No.
[Each experiment followed the above process steps] Input (3,5-Diiodothyronine) Output (Liothyronine)
Yield (%)
1 PRD-A128-45 100 g 64 g 52
2 PRD-A128-46 100 g 66 g 53
3 PRD-A128-47 100 g 65 g 52

Example-6
Preparation of Liothyronine Sodium (Formula I):
RBF (Round bottom Flask) was charged with 1500ml of n-butanol and l00g of Liothyronine (Formula II). The mixture was then heated to 60-65°C with sodium carbonate (1equ) and maintained for 5-6 hours at 60-65°C, followed by cooling to 40-45°C, added Norit charcoal(5%) for 30min maintenance further cooled to 25°C and filtered through hyflow at 25°C. The filtrate was then distilled under vacuum at NMT45°C and added 200ml of ethanol/ THF mixture, further the reaction mixture is cooled to 0- 5°C and charged 200ml of ethanol/THF mixture maintained for 1 hour, filtered and the filtered solid washed with 100ml of ethanol/THF mixture followed by vacuum drying at 45-50°C for 12-15 hours to get 65g of pure Liothyronine sodium (Formula I) of 64% yield.
Experimental yield details:
S.No Experiment No Input (Liothyronine) Output (Liothyronine Sodium)
Yield (%)
01 PRD-A128-089 100 67 65
02 PRD-A128-091 100 66 64
03 PRD-A128-092 100 66 64

Example-5
Chemical analysis of Liothyronine Sodium (Formula I):
The product was chemically characterized by purity analysis through USP and EP methods (depicted in Tables 1), 1H NMR (depicted in Structure -1 and Table-2), 13C NMR (Structure-2 and Table-3), Mass (Structure-3, Table-4), Elemental analysis (Table-5).

Analytical summary of the three isolated batches as per EP/USP (European / US Pharmacopoeia) monograph.

Table: 1A
S. No. Test Parameter Specification PRD-A128-089-05 PRD-A128-091-03 PRD-A128-092-03
1. Description Light tan, Odorless, Crystalline powder Off-White Crystalline Powder Off-White Crystalline Powder Off-White Crystalline Powder
2. Solubility Slightly soluble in alcohol, very slightly soluble in water, Practically insoluble in most other organic solvents. Complies Complies Complies
3. Identification by
A) UV Absorptivity on dried basis (%) Do not differ more than 5.0 Complies Complies Complies
B) Test for Iodine Violet vapors should be evolved Complies Complies Complies
C) Test for Sodium White precipitate should be produced Complies Complies Complies
D) By HPLC Retention time of sample should conform to that of standard in assay method. Complies Complies Complies
4. Chloride Content (%) Not more than 1.2 Less than 1.2 Less than 1.2 Less than 1.2
5. Sodium Content (%) Not less than 2.9 and Not more than 4.0 3.2 3.2 3.2
6. Specific optical rotation (°) @ 25°C (On dried basis) Between +18to +22 +21.98 +20.46 +20.94
7. Loss on drying (% w/w) Not more than 4.0 2.2 2.6 2.4
8. Limit of Levothyroxine sodium (%) Not more than 5.0 0.8 0.2 0.2
9. Assay By HPLC (%w/w)
(On dried basis) Not less than 95.0 and Not more than 101.0 99.7 99.57 99.39
Additional information
10. Related substances by HPLC (%w/w) (Ph. Eur, 8.0)
Impurity-A Not more than 1.0 0.30 0.08 0.18
Impurity-B Not more than 0.3 Not detected Not detected Not detected
Impurity-C Not more than 0.3 Not detected Not detected Not detected
Impurity-D Not more than 0.2 0.06 Not detected BRT
Impurity-E Not more than 0.5 0.16 0.08 0.12
Uncharacterized impurities Not more than 0.10 Below Reporting Threshold Below Reporting Threshold Below Reporting Threshold
Total impurity Not more than 2.0 0.59 0.23 0.40

Table: 1B
S.No Code Chemical Name Structure
1 Impurity-A (S)-2-Amino-3-(4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl)propanoic acid

2 Impurity-B (2S)-2-Amino-3-(4-hydroxy-3,5-diiodophenyl)propanoic acid

3 Impurity-C [4-(4-Hydroxy-3-iodophenoxy)- 3,5-diiodophenyl]acetic acid

4 Impurity-D [4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]acetic acid

5 Impurity-E (2S)-2-Amino-3-[4-(4-hydroxyphenoxy)-3,5-diiodophenyl]propanoic acid

Structure-1 – Sodium Salt of Liothyronine:

Table-2:
1H NMR SPECTRUM ASSIGNMENTS
PROTON CHEMICAL SHIFT ?(ppm) MULTIPLICITY #OF PROTONS
5 & 51 7.78 s 2H
3 6.97 - 6.96 d 1H
4 6.84 - 6.82 d 1H
2 6.54 - 6.51 dd 1H
9 4.04 s 2H
8 3.17 m 1H
6 & 61 2.98 – 2.93 & 2.57 - 2.54 dd 2H

s-singlet, d-doublet, dd-doublet of doublet & m-multiplet

The Proton Nuclear Magnetic Resonance experiment was done using DMSO-d6 as solvent.

Structure-2 – Sodium Salt of Liothyronine:

Table-3:
13 C NMR SPECTRUM ASSIGNMENTS
CARBON CHEMICAL SHIFT ? (PPM)
13 177.34
7 154.45
4 151.58
1 147.84
10 141.64
9 & 91 140.66
5 124.25
3 116.30
2 115.48
8 & 81 91.84
6 86.02
12 56.97
11 37.00

The Carbon Nuclear Magnetic Resonance experiment was done using DMSO-d6 as solvent

Structure-3 – Sodium Salt of Liothyronine:

Molecular mass: 672.96 g/mol

Table-4:
Details Expected mass Obtained results
(M+H-Na) + 672.96 651.6

Elemental analysis (C, H, N)
Theoretical and experimental values for the elemental analysis of PRD-A128-089-05 are given in the Table. Data obtained from elemental analysis is matching with the theoretical values of Molecular formula C15H11I3NNaO4.

Table-5:
Element Theoretical (% w/w) Determined (% w/w)
Carbon 26.77 26.82
Hydrogen 1.65 1.77
Nitrogen 2.08 2.07