OMEGA FATTY ACIDS-RICH OIL DISPERSION FORMULATIONS

Abstract

The invention comprises an oral Omega-3-fatty acid-rich oil emulsion composition comprising DHA 50-100 mg/ml, made from all natural and biocompatible ingredients for use in management of treatment of a disorder to overcome the same. The composition may be a thixotropic emulsion in nanometric size range having better absorption, better stability at room temperature and refrigerated one (2-8° C.), either alone or in combination with vitamins, minerals, Generally Regarded As safe (GRAS) natural ingredients. in therapeutically effective amount. The disorders comprise pre-term birth disorder in pregnant women, cognitive disorders in children and cardiovascular disorders. The said emulsion is made by adding an oil phase comprising a natural emulsifier to an aqueous phase comprising a gum. Vitamin E TPGS, a preservative and a high intensity sweetener; under mechanical stirring. This invention also embodies a High Performance Liquid Chromatography (HPLC) method for assay of omega-3 fatty acids.

Claims

1. An oral Omega-3-fatty acid-rich oil emulsion composition for use in management of treatment of a disorder to overcome the same.

2. The oral Omega-3-fatty acid-rich oil emulsion composition according to claim 1, wherein the Omega-3-fatty acid-rich oil is microalge oil containing 40% DHA and the emulsion comprising DHA 50-100 mg/ml.

3. The oral Omega-3-fatty acid-rich oil emulsion composition according to claim 1, wherein all ingredients are natural and biocompatible ingredients, compatible for administration in Children including neonates.

4. The oral Omega-3-fatty acid-rich oil emulsion composition according to claim 1, wherein size of the dispersed phase is in nanometric range.

5. The oral Omega-3-fatty acid-rich oil emulsion composition according to claim 1, wherein the same has stability at room temperature (about 30° C.) and refrigerated one (2-8° C.).

6. The oral Omega-3-fatty acid-rich oil emulsion composition according to claim 1, wherein the emulsion comprises Vitamins and Minerals in therapeutically effective amounts.

7. The oral Omega-3 fatty acid-rich oil emulsion composition according to claim 1, wherein the disorder is selected from a group consisting of pre-term birth disorder in pregnant women, cognitive disorders in children and cardiovascular disorders.

8. The oral Omega-3 fatty acid-rich oil emulsion composition according to claim 7, wherein: a. pre-term birth disorder in pregnant women is overcome by achieving normal delivery, b. cognitive disorders in children is overcome by improvement in their cognitive ability, and c. cardiovascular disorder is overcome by return to healthy condition.

9. An oral Omega-3 fatty-acid-rich oil thixotropic emulsion in nanomeric size range and having better absorption the oral Omega-3-fatty-acid-rich oil thixotropic emulsion according to claim 4 comprising emulsion in nanometric size range having higher surface area and absorption.

10. The oral Omega-3-fatty-acid-rich oil thixotropic emulsion according to claim 9 comprising natural emulsifier and their derivatives and bio surfactants alone or in combination with vitamins, minerals, Generally Regarded As safe (GRAS) natural ingredients.

11. The oral Omega-3-fatty-acid-rich oil thixotropic emulsion according to claim 10, wherein the natural emulsifier comprises, one or more selected form the group consisting of natural gums, clays and polymers.

12. The oral Omega-3-fatty-acid-rich oil thixotropic emulsion according to claim 11, wherein the composition comprises additives further comprising one or more selected from the group consisting of rheology modifiers, anti-oxidants, preservatives, stabilizers, sweetening and flavoring agents.

13. An oral Omega-3 fatty-acid-rich oil emulsion composition having dispersed phase having nanometric size comprising natural emulsifier and their derivative surfactants alone or in combination with vitamins, minerals, Generally Regarded As safe (GRAS) natural ingredients.

14. The oral Omega-3-fatty-acid-rich oil emulsion according to claim 13, wherein the natural emulsifier comprises natural gums.

15. The oral Omega-3-fatty-acid-rich oil according to claim 13, wherein the composition comprises additives further comprising one or more selected form the group consisting of rheology modifiers, anti-oxidants, preservatives, stabilizers, sweetening and flavoring agents.

16. A High Performance Liquid Chromatography (HPLC) method for assay of omega-3 fatty acids.

17. The High Performance Liquid Chromatography (HPLC) method for assay of omega-3 fatty acids according to claim 16 comprising steps of: a. separately injecting blank, standard solutions-1, standard solutions-2 and sample solution into the chromatograph, b. recording the chromatograms, and c. measuring the peak responses for Docosahexaenoic acid (DHA).

18. The High Performance Liquid Chromatography (HPLC) method for assay of omega-3 fatty acids according to claim 17, wherein: (a) the blank injected is in a single replicate, (b) standard solutions-1 injected are in five replicates, (c) standard solutions-2 injected are in two replicates, and (d) sample solution injected is in a single replicate, (e) column used is Thermo Syncronis C18 (250×4.6 mm), 5 μm or Equivalent, (f) pump mode is isocratic, (g) flow rate is 1.0 ml/min, (h) detection is at UV, 210 nm, (i) injection volume is 20 μl, (i) column oven temperature is 45° C., (k) run time is 20 minutes.

19. The High Performance Liquid Chromatography (HPLC) method for assay of omega-3 fatty acids according to claim 18, wherein: a. the solution-1 comprises DHA working standard, b. the solution-2 comprises DHA test solution, c. sample solution comprises known quantity of DHA-rich algal oil sonicated for a period of time with n-Heptane in around bottom flask, adding Methanolic Sodium hydroxide solution and refluxed for 10 minutes with a stirrer, cooled in ice bath without removing the round bottom flask, slowly and cautiously adding Boron Triflouride Methanol Complex Solution, refluxing further with magnetic with stirrer, cooled in ice bath without removing the round bottom flask, slowly with cautiously adding n-heptane and refluxing, cooling the mixture and removing the round bottom flask, adding saturated Sodium chloride solution, shaking well and transferring the contents to a centrifuge tube. Centrifuging with low speed, diluting upper Heptane layer with Isopropyl Alcohol and mixing the same. Further diluting this solution with Methanol and mixing the same.

20. The oral Omega-3-fatty acid-rich oil emulsion according to claim 9 wherein: a. the natural emulsifiers comprise one or more selected from the group consisting of i. Vitamin E TPGS (d-α-Tocopheryl polyethylene glycol 1000 succinate); ii. Phospholipids comprise one or more selected from the group consisting of soya- and egg phosphatidylcholine, distearyl phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine; iii. gums comprise one or more selected from the group consisting of gum acacia, guar gum, xanthan gum, and gum tragacanth; and iv. polymers comprise, one or more selected from the group consisting of pectin, gelatin and alginate, b. emulsion stabilizers comprise one or more selected from the group consisting of Xanthan gum, guar gum, gum acacia, Bentonite, glycerol and mixture thereof, c. The oral Omega-3-fatty acid-rich oil comprise of one or more selected from the group consisting of microalgae oil, fish oil or flaxseeds oil. d. antioxidants comprise one or more selected from the group consisting of butylated hydroxytoluene, rosemary oil, sodium ascorbate, Vitablend (consisting of Vitamin E and ascorbyl palmitate), sodium metabisulphite, ascorbyl palmitate and Vitamin E, e. vitamins comprise one or more selected from the group consisting of oil soluble vitamin A, vitamin D, vitamin E, vitamin K; water soluble Vitamin B1, Vitamin B2, Vitamin B3, Vitamin B5, Vitamin B6, Vitamin B12, folic acid and vitamin C. Minerals include zinc, copper, magnesium, potassium, calcium as calcium phosphate or calcium carbonate, iron and β-carotene etc. f. buffers comprise one or more selected from the group consisting of sodium citrate sodium carbonate and phosphate buffer flavoring agent comprise one or more flavors selected from the group consisting of orange, strawberry, raspberry, mango, peach, vanilla, lime flavors, g. sweetening agents comprise one or more selected from the group consisting Sorbitol, xylitol, mannitol, Sucralose, Stevia, Aspartame, Neotame, Acesulfame K and mixtures thereof, h. preservative comprises rosemary extract, sodium benzoate, sodium azide, Methyl and propyl Paraben.

21. A process of making oral Omega-3-fatty acid-rich oil emulsion comprising steps of: a. making an oil phase of omega-3-fatty acid-rich microalgae oil by mixing a natural emulsifier, a blend of vitamins comprising an anti-oxidant with DHA-rich microalgae oil in a manufacturing tank with stirrer at room temperature b. making an aqueous phase in a tank with stirrer comprising steps of: i. soaking a gum in purified water for a period of time required for dissolution, ii. dissolving Vitamin E TPGS (d-α-Tocopheryl polyethylene glycol 1000 succinate) in another vessel under mechanical stirring, and other water soluble ingredients comprising a preservative and a high intensity sweetener were mixed with this solution, iii. thereafter, both gum and Vitamin E and Ascorbyl Palmitate solutions were mixed under mechanical stirring for a period of time required to form uniform mixture, iv. thereafter, oil phase was added to the aqueous phase under mechanical stirring maintaining both phases at room temperature, v. flavoring agent was added, and vi. stirring was continued for a further period of time.

22. The oral Omega-3-fatty acid-rich oil emulsion according to claim 9 wherein: a. the natural emulsifiers comprise one or more selected from the group consisting of i. Vitamin E TPGS (d-α-Tocopheryl polyethylene glycol 1000 succinate); ii. phospholipidstone or more selected from the group consisting of soya- and egg phosphatidylcholine, distearyl phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine; iii. gums consisting of gum acacia, guar gum, xanthan gum, and gum tragacanth; and iv. polymers consisting of pectin, gelatin and alginate, b. emulsion stabilizers comprise one or more selected from the group consisting of Xanthan gum, guar gum, gum acacia, Bentonite, glycerol and mixture thereof, c. antioxidants comprise one or more selected from the group consisting of butylated hydroxytoluene, rosemary oil, sodium ascorbate, Vitablend (consisting of Vitamin E and ascorbyl palmitate), sodium metabisulphite, ascorbyl palmitate and Vitamin E, d. vitamins comprise one or more selected from the group consisting of oil soluble vitamin A, vitamin D, vitamin E, vitamin K; water soluble Vitamin B1, Vitamin B2, Vitamin B3, Vitamin B5, Vitamin B6, Vitamin B12, folic acid and vitamin C. Minerals include zinc, copper, magnesium, potassium, calcium as calcium phosphate or calcium carbonate, iron and β-carotene etc. e. buffers comprise one or more selected from the group consisting of sodium citrate and sodium carbonate, f. flavoring agent comprise one or more flavors selected from the group consisting of orange, strawberry, raspberry, mango, peach, vanilla, lime flavors, g. sweetening agents comprise one or more selected from the group consisting Sorbitol, xylitol, mannitol, Sucralose, Stevia, Aspartame, Neotame, Acesul fame K and mixtures thereof, h. preservative comprises one or more selected from the group consisting of rosemary extract, sodium benzoate, sodium azide, Methyl and propyl Paraben,

23. The process of making oral Omega-3-fatty acid-rich oil emulsion according to claim 17, wherein, a. the natural emulsifier comprises Soya phosphatidyl choline, b. the oil phase comprises a mix of: i. omega-3-fatty acid-rich microalgae oil 12.5-25% w/v, ii. Anti-oxidant mix (Vitablend™) 0.05 to 0.5%, iii. butylated hydroxytoluene 0.1%, iv. DHA-rich microalgae oil 12.5-25% w/v, v. the Manufacturing tank is stainless steel Jacketed, vi. stirring is done at 40-50° C. with stirring speed of 100-300 RPM, vii. the gum is Xanthan gum at 0.3-1.5% w/v), viii. soaking was done in purified water at 40-50° C. for 1-5 h period, ix. Vitamin E TPGS (d-α-Tocopheryl polyethylene glycol 1000 succinate) is added at 1-5% w/v), mechanical stirring is done at 1000-1500 RPM at 1000-1500 RPM, preservative is sodium benzoate 0.02-0.1% w/v, x. high intensity sweetener is Sucralose 0.1-0.5% w/v), xi. uniform mixture is done by stirring and mixing of both gum and Vitamin E TPGS solutions is done under mechanical stirring at 1000-1500 RPM at 40-50° C. for 30-60 minutes. xii. finally, adding the oil phase was to the aqueous phase under mechanical stirring is done at 1000-1500 rpm, maintaining both phases at 40-50° C., xiii. flavor being added is orange oil at 0.5% w/v to 1.0% w/v and stirring continued at 1-2 hrs.

24. The oral Omega-3-fatty acid-rich oil emulsion according to claim 21, wherein: a. the natural emulsifiers comprise one or more selected from the group consisting of i. Vitamin E TPGS (d-α-Tocopheryl polyethylene glycol 1QGG succinate); ii. Phospholipids comprise one or more selected from the group consisting of soya- and egg phosphatidylcholine, distearyl phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine; iii. gums comprise one or more selected from the group consisting of gum acacia, guar gum, xanthan gum, and gum targacanth, and iv. polymers comprise, one or more selected from the group consisting of pectin, gelatin and alginate, b. emulsion stabilizers comprise one or more selected from the group consisting of Xanthan gum, guar gum, gum acacia, Bentonite, glycerol and mixture thereof, c. The oral Omega-3-fatty acid-rich oil comprise of one or more selected from the group consisting of microalgae oil, fish oil or flaxseeds oil. d. antioxidants comprise one or more selected from the group consisting of butylated hydroxytoluene, rosemary oil, sodium ascorbate, Vitablend (consisting of Vitamin E and ascorbyl palmitate), sodium metabisulphite, ascorbyl palmitate and Vitamin E, e. vitamins comprise one or more selected from the group consisting of oil soluble vitamin A, vitamin D, vitamin E, vitamin K; water soluble Vitamin B1, Vitamin B2, Vitamin B3, Vitamin B5, Vitamin B6, Vitamin B12, folic acid and vitamin C. Minerals include zinc, copper, magnesium, potassium, calcium as calcium phosphate or calcium carbonate, iron and b-carotene etc. f. buffers comprise one or more selected from the group consisting of sodium citrate sodium carbonate and phosphate buffer flavoring agent comprise one or more flavors selected from the group consisting of orange, strawberry, raspberry, mango, peach, vanilla, lime flavors, g. sweetening agents comprise one or more selected from the group consisting Sorbitol, xylitol, mannitol, Sucralose, Stevia, Aspartame, Neotame, Acesulfame K and mixtures thereof h. preservative comprises rosemary extract, sodium benzoate, sodium azide, Methyl and propyl Paraben.

25. The oral Omega-3-fatty acid-rich oil emulsion according to claim 13, wherein: a. the natural emulsifiers comprise one or more selected from the group consisting of i. Vitamin E TPGS (d-α-Tocopheryi polyethylene glycol 1000 succinate); ii. phospholipidstone or more selected from the group consisting of soya- and egg phosphatidylcholine, distearyl phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine; iii. gums consisting of gum acacia, guar gum, xanthan gum, and gum targacanth, and iv. polymers consisting of pectin, gelatin and alginate, b. emulsion stabilizers comprise one or more selected from the group consisting of Xanthan gum, guar gum, gum acacia, Bentonite, glycerol and mixture thereof, c. antioxidants comprise one or more selected from the group consisting of butylated hydroxytoluene, rosemary oil, sodium ascorbate, Vitablend (consisting of Vitamin E and ascorbyl palmitate), sodium metabisulphite, ascorbyl palmitate and Vitamin E, d. vitamins comprise one or more selected from the group consisting of oil soluble vitamin A, vitamin D, vitamin E, vitamin K; water soluble Vitamin B1, Vitamin B2, Vitamin B3, Vitamin B5, Vitamin B6, Vitamin B12, folic acid and vitamin C. Minerals include zinc, copper, magnesium, potassium, calcium as calcium phosphate or calcium carbonate, iron and b-carotene etc. e. buffers comprise one or more selected from the group consisting of sodium citrate and sodium carbonate, f. flavoring agent comprise one or more flavors selected from the group consisting of orange, strawberry, raspberry, mango, peach, vanilla, lime flavors, g. sweetening agents comprise one or more selected from the group consisting Sorbitol, xylitol, mannitol, Sucralose, Stevia, Aspartame, Neotame, Acesulfame K and mixtures thereof, h. preservative comprises one or more selected from the group consisting of rosemary extract, sodium benzoate, sodium azide, Methyl and propyl Paraben,

26. The oral Omega-3-fatty acid-rich oil emulsion according to claim 9, wherein: a. the natural emulsifiers comprise one or more selected from the group consisting of i. Vitamin E TPGS (d-α-Tocopheryi polyethylene glycol 1000 succinate); ii. phospholipidstone or more selected from the group consisting of soya- and egg phosphatidylcholine, distearyl phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine; iii. gums consisting of gum acacia, guar gum, xanthan gum, and gum targacanth, and iv. polymers consisting of pectin, gelatin and alginate, b. emulsion stabilizers comprise one or more selected from the group consisting of Xanthan gum, guar gum, gum acacia, Bentonite, glycerol and mixture thereof, c. antioxidants comprise one or more selected from the group consisting of butylated hydroxytoluene, rosemary oil, sodium ascorbate, Vitablend (consisting of Vitamin E and ascorbyl palmitate), sodium metabisulphite, ascorbyl palmitate and Vitamin E, d. vitamins comprise one or more selected from the group consisting of oil soluble vitamin A, vitamin D, vitamin E, vitamin K; water soluble Vitamin B1, Vitamin B2, Vitamin B3, Vitamin B5, Vitamin B6, Vitamin B12, folic acid and vitamin C. Minerals include zinc, copper, magnesium, potassium, calcium as calcium phosphate or calcium carbonate, iron and b-carotene etc. e. buffers comprise one or more selected from the group consisting of sodium citrate and sodium carbonate, f. flavoring agent comprise one or more flavors selected from the group consisting of orange, strawberry, raspberry, mango, peach, vanilla, lime flavors, g. sweetening agents comprise one or more selected from the group consisting Sorbitol, xylitol, mannitol, Sucralose, Stevia, Aspartame, Neotame, Acesulfame K and mixtures thereof, h. preservative comprises one or more selected from the group consisting of rosemary extract, sodium benzoate, sodium azide, Methyl and propyl Paraben,

Description

BRIEF DESCRIPTION OF FIGURES AND LEGENDS

[0057] FIG. 1: Illustrates the Transmission electron micrograph (TEM) of Emulsion formulation.

[0058] FIG. 2: Globule size analysis of emulsion formulation using Dynamic light scattering method.

[0059] FIG. 3: Illustrates the rheological analysis of O3FA-rich oil formulation (A) at constant shear rate at 40 s.sup.−1 (B) at variable shear rate 0-60 s.sup.−1 (C) thixotropic analysis of O3FA-rich oil emulsion.

[0060] FIG. 4: Illustrates the peroxide value of O3FA-rich oil emulsion A) at real time condition B) at refrigerated conditions.

[0061] FIG. 5: Illustrates the induction time at which secondary oxidation products are formed in Rancimat analysis in emulsion (induction time 2.57 h) in comparison to pure oil (induction time 0.45 h), indicating the higher oxidative stability of emulsion.

[0062] FIG. 6: Illustrates the biosafety of emulsion formulation in MTT assay against A) Caco-2 cell line B) Fr2 cell line.

[0063] FIG. 7: Illustrates A) Paracellular permeability B) TEER measurement of emulsion formulation. (Both parameter are the measures of changes in the tight junctional and paracellular spacing of gastrointestinal cells).

[0064] FIG. 8: Permeability of DHA from DHA emulsion formulation in comparison to DHA oil.

[0065] FIG. 9: Pharmacokinetic of optimized DHA emulsion formulation in comparison to DHA oil by measuring DHA content in phospholipids.

[0066] FIG. 10: HPLC chromatogram for method developed and validated for estimation of DHA.

[0067] FIG. 11: Process flow diagram for commercial production of DHA emulsion.

EXAMPLE 1

[0068] Preparation of a O3FA-Rich Oil Emulsion Formulations

[0069] Emulsions were prepared using O3FA-rich microalgae oil making 5-25% of the total content of emulsion. In first step, oil phase was prepared by mixing 0.4% w/v of soya phosphatidyl choline, 0.05% of Vitablend, 0.1% of butylated hydroxytoluene with 12.5-25% w/v of DHA-rich microalgae oil in stainless steel Jacketed Manufacturing tank with stirrer at 40° C. with stirring speed of 100 RPM. In second step, aqueous phase was prepared in stainless steel Jacketed Manufacturing tank with stirrer. Xanthan gum (0.4% w/v) was soaked in purified water at 40° C. for 2-3 h period. In another vessel, Vitamin E TPGS (2% w/v) was dissolved under mechanical stirring at 1000-1200 RPM at 40° C. and other water soluble ingredients sodium benzoate (0.05% w/v) and Sucralose (0.1% w/v) were mixed with this solution. Then both gum and Vitamin E TPGS solutions were mixed under mechanical stirring at 1000-1200 RPM at 40° C. for 30 minutes to form uniform mixture. Finally, oil phase was added to the aqueous phase under mechanical stirring at 1000-1200 rpm, maintaining both phases at 40° C. Stirring was continued for 0.5-1.0 hr after addition of orange oil (0.5% w/v) as flavoring agent. Process flow diagram for commercial production of DHA emulsion in strength of 50-100 mg/ml is shown in FIG. 9.

TABLE-US-00001 TABLE 1 Composition of O3FA-rich oil emulsion formulation in strength 50 mg/ml (prototype 1) Ingredients Quantity for 100 ml % w/v OIL PHASE DHA oil (40%) 12.500 g  12.500%  Soy Phosphatidylcholine 0.400 g 0.400% Ascorbyl Palmitate plus Vitamin E 0.050 g 0.050% Butylated Hydroxytoluene 0.100 g 0.100% Orange 5 Fold Royal Oil Flavour 0.500 g 0.500% WATER PHASE Xanthan gum 0.400 g 0.540% D-α-Tocopheryl polyethylene 2.000 g 2.000% glycol 1000 succinate (Vitamin E TPGS) Sodium Benzoate 0.050 g 0.050% Sucralose 0.100 g 0.100% Purified water QS for 100 ml  71.4%

TABLE-US-00002 TABLE 2 Composition of O3FA-rich oil emulsion formulation 75 mg/ml (prototype 2) Ingredients Quantity for 100 ml % w/v OIL PHASE DHA oil (40%) 18.750 g  18.750%  Soy Phosphatidylcholine 0.400 g 0.400% Ascorbyl Palmitate plus Vitamin E 0.050 g 0.050% Butylated Hydroxytoluene 0.100 g 0.100% Orange 5 Fold Royal Oil Flavour 0.500 g 0.500% WATER PHASE Xanthan gum 0.400 g 0.400% D-α-Tocopheryl polyethylene 2.000 g 2.000% glycol 1000 succinate (Vitamin E TPGS) Sodium Benzoate 0.050 g 0.050% Sucralose 0.100 g 0.100% Purified water QS for 100 ml 77.650% 

TABLE-US-00003 TABLE 3 Composition of O3FA-rich oil emulsion formulation 100 mg/ml (prototype 4) Ingredients Quantity for 100 ml % w/v OIL PHASE DHA oil (40%) 18.750 g  25.000%  Soy Phosphatidylcholine 0.400 g 0.400% Ascorbyl Palmitate plus Vitamin E 0.050 g 0.050% Butylated Hydroxytoluene 0.100 g 0.100% Orange 5 Fold Royal Oil Flavour 0.500 g 0.500% WATER PHASE Xanthan gum 0.400 g 0.400% D-α-Tocopheryl polyethylene 2.000 g 2.000% glycol 1000 succinate (Vitamin E TPGS) Sodium Benzoate 0.050 g 0.050% Sucralose 0.100 g 0.100% Purified water QS for 100 ml 71.400% 

TABLE-US-00004 TABLE 4 Composition of O3FA-rich oil emulsion formulation with Vitamins using combination of natural emulsifiers (Prototype 4) Ingredients Quantity for 100 ml % w/v OIL PHASE DHA oil (40%) 12.750 g  25.000%  Soy Phosphatidylcholine 0.400 g 0.400% Ascorbyl Palmitate plus Vitamin E 0.050 g 0.050% Butylated Hydroxytoluene 0.100 g 0.100% Orange 5 Fold Royal Oil Flavour 0.500 g 0.500% WATER PHASE Xanthan gum 0.500 g 0.400% Pectin 0.100 g 0.100% Bentonite 0.500 g 0.500% D-α-Tocopheryl polyethylene 2.000 g 2.000% glycol 1000 succinate (Vitamin E TPGS) Sodium Benzoate 0.050 g 0.050% Sucralose 0.100 g 0.100% Purified water QS for 100 ml 69.800% 

EXAMPLE 2

[0070] Preparation of O3FA-Rich Oil Emulsion Fortified with Vitamins and Minerals

[0071] Method of making the basic emulsion was as provided in example 1, to which Vitamins and minerals and folic acid were added. The composition of this product has been given in Table 5 and 6.

TABLE-US-00005 TABLE 5 Composition of O3FA-rich oil emulsion formulation fortified with Vitamins and Minerals Ingredients Quantity for 100 ml % w/v OIL PHASE DHA oil (40%) 12.500 g  12.500%  Soy Phosphatidylcholine 0.400 g 0.400% Ascorbyl Palmitate plus Vitamin E 0.050 g 0.050% Butylated Hydroxytoluene 0.100 g 0.100% Orange 5 Fold Royal Oil Flavour 0.500 g 0.500% Retinal (as vitamin A palmitate) 0.040 g 0.040% Ergocalciferol solution 0.004 g 0.004% WATER PHASE Xanthan gum 0.400 g 0.400% Thiamine hydrochloride 0.040 g 0.040% Nicotinamide 0.800 g 0.800% Ascorbic Acid 4.000 g 4.000% Zinc (as zinc gluconate) 0.697 g 0.697% Mangnese (as manganese gluconate) 0.810 g 0.810 g D-α-Tocopheryl polyethylene 2.000 g 2.000% glycol 1000 succinate (Vitamin E TPGS) Sodium Benzoate 0.050 g 0.050% Sucralose 0.100 g 0.100% Purified water QS for 100 ml 77.509% 

TABLE-US-00006 TABLE 6 Composition of O3FA-rich oil emulsion formulation fortified with Vitamins and Minerals Ingredients Quantity for 100 ml % w/v OIL PHASE DHA oil (40%) 12.500 g  12.500%  Soy Phosphatidylcholine 0.400 g 0.400% Ascorbyl Palmitate plus Vitamin E 0.050 g 0.050% Butylated Hydroxytoluene 0.100 g 0.100% Orange 5 Fold Royal Oil Flavour 0.500 g 0.500% Retinal (as vitamin A palmitate) 0.040 g 0.040% Ergocalciferol solution 0.004 g 0.004% WATER PHASE Xanthan gum 0.400 g 0.400% Pectin 0.100 g 0.100% Bentonite 0.500 g 0.500% Thiamine hydrochloride 0.040 g 0.040% Nicotinamide 0.800 g 0.800% Ascorbic Acid 4.000 g 4.000% Zinc (as zinc gluconate) 0.697 g 0.697% Mangnese (as manganese gluconate) 0.810 g 0.810% Folic acid 0.010 g 0.010% D-α-Tocopheryl polyethylene 2.000 g 2.000% glycol 1000 succinate (Vitamin E TPGS) Sodium Benzoate 0.050 g 0.050% Sucralose 0.100 g 0.100% Purified water QS for 100 ml  76.9%

EXAMPLE 3

[0072] In Vitro Characterization for Quality Control Tests

[0073] Following are the properties/characteristics studied/determined and the results are provided in Table 7 below.

[0074] Globule Size and Zeta Potential Analysis

[0075] Globule size, size distribution and zeta potential of emulsion was measured on O3FA-rich oil emulsion prepared in Examples using particle size analyzer (Malvern Zetasizer Nano ZS90, UK) by 100 times diluting the sample with triple distilled water. In addition to average particle size the intensity distribution of the particles and polydispersity index (PDI) which is a measure of uniformity in size distribution were also measured.

[0076] pH of Emulsion Formulations

[0077] O3FA-rich oil emulsions were developed for the oral administration; therefore the pH of the emulsion should be within the acceptable range required for the oral administration i.e. in between pH 5-8. The pH of the prepared emulsion formulations was determined using pH meter (Toshcon CL-54).

[0078] Transmission Electron Microscopy (TEM) Analysis

[0079] The size and shape of dispersed phase in the emulsion system was observed by Transmission electron microscopy (TEM), (HRTEM, JEM 2100, JEOL, Japan) operated at an accelerating voltage of 200 kV with beam current of 100 μA. The sample was diluted in ratio of 1:100 with triple distilled water and then a drop of diluted emulsion was placed directly onto a carbon-coated copper grid. The grid was air dried and was observed under transmission electron microscope. Results are provided in FIG. 1.

[0080] Thermodynamic Stability Study

[0081] The thermodynamic stability of the O3FA-rich oil emulsion formulations was determined by storing the emulsion samples at different temperature conditions 4±1° C. and 45±1° C. for 24 h storage at each temperature. Finally, after cooling and heating cycle sample was subjected to centrifugation stress at 3000 rpm for 10 min and the extent of any oil separation was monitored.

[0082] Viscosity

[0083] The viscosity of the prepared emulsion was determined by Brookfield viscometer using spindle no. 5 at constant shear rate of 40 rpm/min.

[0084] Dispersibility Test and Robustness to Dilution

[0085] Five ml of the prepared formulations were added to 500 ml of distilled water in USP Type II Dissolution apparatus (Lab India DS 8000) at 37±0.5° C. and 50 rpm. The formulations were visually examined for water dispersibility immediately after addition to the vessel using following grading system: Grade A—Rapidly dispersing milky emulsion (<1 minutes); Grade B—Moderate dispersing milky emulsion (>2 minutes); Grade C: Slow dispersion milky emulsion (>5 minutes) with appearance of oil. Robustness to dilution is an important parameter for emulsion to ensure that the prepared emulsion has similar properties at different dilutions after oral administration. 1 ml of each emulsion was diluted to 100 and 1000 times with distilled water and 0.1 N HCl. The diluted emulsions were observed for 24 h in order to determine the separation of oily phase at higher dilutions and globule size. Robustness to dilution is an important parameter to understand the behaviour of emulsion globules under in vivo conditions. Separation of oily phase leads to poor intestinal absorption. The results obtained are provided in Table 7.

TABLE-US-00007 TABLE 7 In vitro characterization of O3FA-rich oil emulsion formulations O3FA emulsion O3FA emulsion formulation fortified with Characteristic formulation Vitamins and Minerals Particle size (nm)  54.6 nm 146 nm after 10 fold dilution with water Particle size (nm) 566.1 nm 612 nm after 100 fold dilution with water Zeta potential −17.0 mV −17.2 mV pH 6.52 5.74 Thermodynamic Stable and Stable and stability No creaming and No creaming and no cracking. no cracking Heat cooling cycle Pass Pass Shape Globular Globular Viscosity (Brookfield 410 cp 990 cp Viscometer spindle no. 5, 40 RPM at RT) Dilution integrity 0.1 N HCl Good (No precipitation) Good (No precipitation) PBS 6.8 Good (No precipitation) Good (No precipitation) PBS 7.4 Good (No precipitation) Good (No precipitation) Water Good (No precipitation) Good (No precipitation) Dispersibility grade Grade A (Rapidly Grade A (Rapidly dispersing milky dispersing milky emulsion (<1 minutes) emulsion (<1 minutes)

EXAMPLE 4

[0086] Rheological Analysis

[0087] Rheological properties are important parameters for accessing the physical stability and thixotropic behavior of emulsion when subjected to different shear rates and stress. Viscosity and thixotropic profiles of O3FA emulsion was determined by using rheometer (Rheolab QC, Anton paar, Germany). Viscosities were determined at a constant shear rate (40 s.sup.−1) and varying shear rate (0-60 s.sup.−1) at 25° C. temperature conditions. The thixotropic behavior of O3FA emulsion was determined at shear rate of 80 s.sup.−1 in order to evaluate the strength recovery ratio of the prepared emulsion after application and removal of shear stress. Results obtained are given in Table 8 and FIG. 3.

TABLE-US-00008 TABLE 8 Rheological behavior of emulsion formulation. S Apparent Viscosity at Thixotrophy no. constant shear rate (40 s.sup.−1) (% Recovery) Breakdown 1 150- 850cp 97.0-100.25 No

EXAMPLE 5

[0088] Peroxide Value Estimation

[0089] Peroxides are the primary products of oxidation formed during oxidation of oils and lipids. They were measured according to the official method followed by Association of Analytical Communities (AOAC) at time interval of 0, 30, 60 and 90 days. During this study the samples were stored under accelerated storage conditions and refrigerated condition. Acetic acid-chloroform mixture (30 mL) in the ratio 3:2 was added to the 5 ml of emulsion in conical flask and the content was properly shaken. 0.5 mL saturated solution of potassium iodide (PI) was added and conical flask was placed in dark with occasional shaking for 1 min. 0.5 mL of 1% w/v freshly prepared starch solution was added and titration was done with 0.01 N sodium thiosulphate with vigorous shaking until blue color was disappeared. The peroxide value was calculated by using the following equation. Results obtained are given in FIG. 4.

[00001]$\mathrm{Peroxide}\mathrm{value}\left(\mathrm{meq}\right)=\frac{\left(S-B\right)\times N\left({\mathrm{Na}}_2{S}_2{O}_3\right)}{\mathrm{Weight}\mathrm{of}\mathrm{sample}}\times 1000S=\mathrm{titration}\mathrm{volume}\mathrm{of}\mathrm{sample}B=\mathrm{titration}\mathrm{volume}\mathrm{of}\mathrm{blank}$

EXAMPLE 6

[0090] Rancimat Analysis

[0091] Deterioration of taste and odor (rancidity) due to oxidation is slow process at ambient temperature; rancimat method accelerates the natural autooxidation process and determine the oxidative stability of the products with time. It was determined using 892 Professional Rancimat apparatus from Metrohm, Herisau, Switzerland at temperature condition of 90° C. with air flow rate of 20 mL air/h. 3 ml of emulsion formulation was kept in reaction vessels which were attached with measuring vessel and air flow pipes. Secondary metabolites formed were transfer to measuring vessel with the air flow and change in the conductivity of measuring solution as inflection point was measured. Results are provided in FIG. 4.

EXAMPLE 7

[0092] Stability Study

[0093] Accelerated stability testing is going on as per ICH (International Conference for Harmonization) guidelines by storing the O3FA-rich emulsion in sealed amber colored bottles at refrigerated condition (2 to 8° C.), and at accelerated conditions (40° C./75% RH), real time (25° C./75% RH) by using stability chamber and results are provided in Table 9.

TABLE-US-00009 TABLE 9 Results of stability studies under real time and refrigeration storage conditions. Parameters Initial 1month 2months 3months 6 months Storage Conditions: 25° C. ± 2° C./60% ± 5% RH (Room temperature) Appearance Light yellow NC NC NC NC coloured emulsion having sweet taste and pleasant odour. Physical Stable Stable Stable Stable Stable Stability Viscosity 410 410 430 460 520 (cP) pH 5.46 5.60 5.61 5.61 5.69 Z-Average 566.1 560.8 539.1 565.8 585.1 (nm) after 100× dil Z-Average 54.6 50.5 51.7 52.2 55.7 (nm) after 10× dil Specific 0.9934 0.9920 0.9932 0.9961 0.9978 Gravity g/ml Thixotropy 99.41 99.23 97.63 96.1 94.5 (%) Peroxide 1.13 1.88 1.98 2.77 2.92 value (meq/Kg) Assay (% 109.1 108.4 105.3 103.4 102.8 DHA Content) Refrigerated Storage Condition: 5° C. ± 3° C. Appearance Light yellow NC NC NC NC coloured emulsion having sweet taste and pleasant odour. Physical Stable Stable Stable Stable Stable Stability Viscosity 410 410 425 445 490 (cP) pH 5.46 5.65 5.65 5.69 5.72 Z-Average 562.5 556.4 565.8 539.1 546.9 (nm) after 100× dil Z-Average 49.5 42.7 52.2 55.7 55.5 (nm) after 10× dil Specific 0.9934 0.9967 0.9901 0.9886 0.9812 gravity g/ml Thixotropy 99.41 99.23 97.63 96.1 96.22 (%) Peroxide 1.13 2.17 2.12 2.05 2.27 value (meq/Kg) Assay (% 109.1 105.9 104.6 104 103.8 DHA Content) All value is expressed as mean ± SD (n = 3). “*” represents the significant change in parameter where p < 0.05.

EXAMPLE 8

[0094] Cell Cytotoxicity Assay

[0095] Cell viability assay was performed in Human colorectal (Caco2) and Normal Breast Epithelial (Fr2) cell lines with standard MTT (3-(4,5-Dimethylthiazol-2-yl)-2,S-diphenyltetrazolium bromide) method by using Human colorectal cell line (Caco2). Both the cell line were seeded in 96-well plate and Caco2 cell line was treated with 10, 20, 40, 80, 100 and 200 μM O3FA concentrations whereas Fr2 was treated with 25, 12.5, 6.25, 3.12, 1.56 and 0.78 mg/ml O3FA concentration present in emulsion formulations for 24 hour. After O3FA-rich oil treatment, media of each well was supplemented with 20 μL of MTT [3-(4,5-dimethylthaiazole-2-yl)-2,5-diphenyltetrazolium bromide] reagent and were further allowed to incubate for 4 h. Formed formazan crystals were solubilized by adding 200 μL of DMSO (Dimethyl Sulphoxide) into each well. In proportion of viable cells violet crystals were visible and absorbance was taken at 570 nm in microplate reader (Model: Omega fluostar, BMG Labtech Ltd., Germany). The results of % cell viability are summarized in FIG. 6. and it clearly indicated that the developed DHA emulsion formulation have more than 90% cell viability at all treated concentrations

EXAMPLE 9

[0096] Transepithelial Electrical Resistance

[0097] Human colorectal cell line, (Caco-2) were cultured in Dulbecco's modified Eagle's medium (DMEM) high Glucose supplemented with 10% FBS and 10% antibiotic-antimycotic in a humidified incubator with 5% CO.sub.2 supply at 37° C. Cells were grown up to 60-70% confluency and harvested with trypsin-EDTA (0.25%) and seeded at a density of 2×10.sup.5 cells/mL onto polycarbonate membrane Transwell inserts with pore size of 0.4 μm. Cells were cultured for 14 days to reach differentiation, and growth media was refreshed every 2-3 days. Differentiated Caco-2 cells were treated with two concentrations of optimized formulation (200 μg/mL) and lipopolysaccharides (1 μg/mL), taken as a positive control. After incubation period of 48 h, TEER was measured using epithelial volt-ohm meter with a chopstick electrode (Millicell ERS-2, EMD Millipore, Billerica, Mass.) against control. Unit area resistance was calculated by dividing the resistance values with effective membrane area (4.52 cm.sup.2). Observed findings are shown in FIG. 7. Treatment with developed emulsion formulations and microalgae DHA oil did not show any significant change in TEER value in comparison to negative control, whereas as significant (p<0.001) decrease in TEER value in positive control (lipopolysaccharides) was observed (FIG. 7). Thus, confirming the developed formulations did not alter the integrity of the cellular barrier of Caco-2 cells

EXAMPLE 10

[0098] Determination of Paracellular Permeability

[0099] After measurement of TEER value, treated Caco-2 cells were further analyzed for paracellular permeability by using fluorescein isothiocyanatedextran (FD, 4 kDa). FD was dissolved in phosphate buffer saline (PBS) to make concentration of 1 mg/mL. 0.2 mL of dye was added to the apical compartment of each insert, while 1.0 mL of PBS was added to the basolateral well.

[0100] The plate was covered with foil to prevent light inactivation of the fluorescent markers and placed in a shaker incubator at 37° C. at 150 rpm. After 12 h, 0.3 mL aliquots were taken from the basolateral chamber and fluorescence intensity was measured using a black 96 well plate in multiplate reader (Fluostar Omega, BMG Lab tech, Germany). Observed findings are shown in FIG. 7

[0101] Intestinal Permeation Measurement

[0102] The study was performed goat ileum for developed emulsion formulation and DHA oil. With the Tyroid solution of pH 7.4, tissue was separated and cleaned. The thread was tied to one end of the ileum and placed in the student organ bath containing the Tyrode solution of pH 7.4. To prevent peristaltic muscle contractions, the liquid solution was packed into a bag and the weight of 1 g was attached to the unlined part of the lever. Aeration and bath temperatures around the ileum bag were established at 37±0.5° C. Samples were taken from the organ tube at various intervals of 0.25, 0.5, 1, 2, 4, and 5 h, and the DHA content was measured. Results are shown in FIG. 8.

[0103] Pharmacokinetic Study

[0104] Pharmacokinetic study was done for the period of 24 h with DHA emulsion formulation in comparison to DHA oil and 2.2 fold increases in the absorption of DHA was observed form the developed DHA formulation (FIG. 9). This increase of DHA in plasma was depicted from DHA content analysis by Gas Chromatography. The improved plasma levels of DHA observed during pharmacokinetic study resulted from the increased permeability of DHA from intestine, which was clearly demonstrated from the results of ex vivo intestinal permeability study.

EXAMPLE 11

[0105] Development and Validation of HPLC Assay for Omega 3 Fatty Acids

[0106] New HPLC assay method has been developed with following chromatographic conditions have been optimized as specified in Table 10.

TABLE-US-00010 TABLE 10 Advantages of HPLC over GC for Analysis of DHA content in DHA-rich oil Emulsion and Oil S.No. Parameters HPLC Method GC Method 1 Type of Its non-destructive It is a destructive Method method, If the method, If the sequence run not sequence run not completed due to completed due to errors like Instrument errors like Instrument error, power failure error, power failure etc. the same sample etc. the same solutions can be sample solutions cannot re-injected. be re-injected. 2 Accuracy More precise Less precise and accurate and and accurate. comparing to HPLC. Precision RSD limit: NMT 2.0%. RSD limit: NMT 5.0%. 3 Analysis 20 Min (RT 11.89 Min) More comparing Time to HPLC 4 Solutions Stable Less stable stability 5 Mobile Water:Methanol (950:50) GC Gases (Nitrogen, Phase (General and Hydrogen and Zero Regular solvents Air). Handling of used in Lab and less cost) Gases is difficult. 6 For HPLC Systems Generally GC Instruments routine readily available in generally not analysis Analytical laboratories readily available hence can be preferred. in Analytical laboratories hence cannot prefer over HPLC.  7. Column C18 (250 × 4.6 mm), Dedicated column: 5μ Thermo Synchronis Fused silica or other equivalent capillary column column, readily (Cyanopropyl available in 90%, phenyl Analytical Laboratory. siloxane 10%). Column cost is less More cost comparing comparing to to HPLC columns. GC columns.  8. Handling Handling is easy Handling of gases is comparing to GC. difficult in GC analysis.

[0107] Chromatographic Conditions:

TABLE-US-00011 Column : Thermo Syncronis C18 (250 × 4.6 mm), 5 μm or Equivalent Pump mode : Isocratic Flow rate : 1.0 ml/min Detection : UV, 210 nm Injection volume : 20 μl Column oven temperature : 45° C. Run time : 20 minutes

[0108] Optimized method was found to accurately measure the DHA content in experimental protocols with high accuracy and validated for following parameters with RSD value of less than 2%. 1: Specificity and Identification, 1.1: Forced Degradation study; 2: Solution stability, 3: Linearity, 4: Accuracy, 5: Precision—5.1: System precision, 5.2: Method precision/Repeatability, 5.3: Intermediate precision/Ruggedness, 6: Range, 7: Robustness—7.1: Effect of Variation in flow rate, 7.2: Effect of Variation in Wavelength of detection, 7.3: Effect of Variation in Column oven temperature, 8: System suitability.

[0109] Preparation of Standard Solution:

[0110] Weigh accurately 250 mg of DHA working standard (625 mg of 40% DHA-rich microalgae oil) into a round bottom flask, add 10 mL n-Heptane by using pipette and sonicate for 15 minutes with intermittent shaking. Add 20 mL of 0.5N Methanolic Sodium hydroxide solution, connect to the condenser through Claisen adapter. Reflux the contents for 10 minutes with Magnetic Stirrer and cool for 5 minutes in ice bath without removing the round bottom flask. Slowly with cautiously add 20 mL of Boron Triflouride Methanol Complex Solution (13-15%) through the Claisen adapter and reflux for further 30 minutes with magnetic stirrer. Cool for 5 minutes in ice bath without removing the round bottom flask. Slowly with cautiously add 10 ml of n-heptane through Claisen adapter and reflux for further 5 minutes. Cool the mixture and remove the round bottom flask. Add 5 mL of saturated Sodium chloride solution, shake well and transfer the contents to a centrifuge tube. Centrifuge with low speed (500 RPM) for 5 minutes. Dilute 2 mL of upper Heptane layer to 50 mL with Isopropyl Alcohol and mix. Further dilute 5 mL of this solution to 50 mL with Methanol and mix.

[0111] Preparation of Sample Solution: (DHA-Rich Oil Emulsion)

[0112] Weigh the sample accurately equivalent to 250 mg of DHA-rich oil Emulsion into a round bottom flask, add 10 mL n-Heptane by using pipette and sonicate for 15 minutes with intermittent shaking. In illustrative work, DHA-rich microalgae oil was used; however, any other DHA-rich oil can be used. Add 20 mL of 0.5N Methanolic Sodium hydroxide solution, connect to the condenser through Claisen adapter. Reflux the contents for 10 minutes with Magnetic Stirrer and cool for 5 minutes in ice bath without removing the round bottom flask. Slowly with cautiously add 20 mL of Boron Triflouride Methanol Complex Solution (13-15%) through the Claisen adapter and reflux for further 30 minutes with magnetic stirrer. Cool for 5 minutes in ice bath without removing the round bottom flask. Slowly with cautiously add 10 ml of n-heptane through Claisen adapter and reflux for further 5 minutes. Cool the mixture and remove the round bottom flask. Add 5 mL of saturated Sodium chloride solution, shake well and transfer the contents to a centrifuge tube. Centrifuge with low speed (500 RPM) for 5 minutes. Dilute 2 mL of upper Heptane layer to 50 mL with Isopropyl Alcohol and mix. Further dilute 5 mL of this solution to 50 mL with Methanol and mix.

[0113] Sample Solution: (DHA-Rich Oil)

[0114] Weigh the sample accurately equivalent to 250 mg of DHA-rich algal oil into a round bottom flask, add 10 mL n-Heptane by using pipette and sonicate for 15 minutes with intermittent shaking. Add 20 mL of 0.5N Methanolic Sodium hydroxide solution, connect to the condenser through Claisen adapter. Reflux the contents for 10 minutes with Magnetic Stirrer and cool for 5 minutes in ice bath without removing the round bottom flask. Slowly with cautiously add 20 mL of Boron Triflouride Methanol Complex Solution (13-15%) through the Claisen adapter and reflux for further 30 minutes with magnetic stirrer. Cool for 5 minutes in ice bath without removing the round bottom flask. Slowly with cautiously add 10 ml of n-heptane through Claisen adapter and reflux for further 5 minutes. Cool the mixture and remove the round bottom flask. Add 5 mL of saturated Sodium chloride solution shake well and transfer the contents to a centrifuge tube. Centrifuge with low speed (500 RPM) for 5 minutes. Dilute 2 mL of upper Heptane layer to 50 mL with Isopropyl Alcohol and mix. Further dilute 5 mL of this solution to 50 mL with Methanol and mix.

[0115] Procedure:

[0116] Separately inject 20 μl of solvent blank (solvent system without analyte), standard solutions-1 (containing DHA in known amount) five replicates), standard solutions-2 (containing DHA sample for analysis (three replicates) and sample solution (single) into the chromatograph, record the chromatograms and measure the peak responses for DHA.

[0117] Retention Time of DHA peak is about 11.5 minutes.

[0118] System Suitability:

[0119] 1) The column efficiency should be not less than 2000 theoretical plates for principal peaks.

[0120] 2) The Theoretical plates should be not more than 2.0 for principal peaks.

[0121] 3) The relative standard deviation for area response of principal peaks in five replicate injections of standard solution should be not more than 2.0%.

[0122] 4) Asymmetry (Tailing factor) in each condition is not more than 2.0.

TABLE-US-00012 TABLE 11 Validation of developed HPLC method for analyzing DHA Acceptance S. No. Parameter Experiment criteria Results 1. Specificity Blank, There should There is no interference due to diluent and placebo placebo, not be any at the retention time of DHA peak in standard run. standard interference The identified impurities peaks are well separated solution and due to blank, from DHA peak. test solution. placebo and Sample name Retention time (minutes) palmetic acid Standard solution 11.87 at the Control sample 11.87 retention time of DHA peak Peak purity DHA Peak factor should Sample Purity Factor Result be within the Standard solution The purity factor Pass calculated is within the threshold limit calculated for DHA peak threshold limit in standard Control test The purity factor Pass and control solution is within the test solution calculated threshold limit Assay of Control Sample ID % Assay control sample sample result Control sample 101.4 should be report 2. Forced No peaks DHA Peak degradation should be Sample Purity Factor Result Stressed detected at Control sample The purity factor Pass sample the retention is within the time of DHA calculated peak in the threshold limit chromatogram Acid Degradation The purity factor Pass of the diluent is within the and placebo. calculated Peak purity threshold limit factor should Base The purity factor Pass be within the Degradation is within the calculated calculated threshold limit threshold limit for DHA peak Oxidation The purity factor Pass in final Degradation is within the stressed test calculated solutions. threshold limit 3. Solution Standard For standard: Standard Solution stability solution and Recovery for Interval Area % Recovery Test solution solution Initial 437770 99.7 stability 3.sup.rd day 5° C.-8° C. 23° C.-27° C. standard 99.8 99.7 against freshly prepared standard solution should be in the range of 98.0% to 102.0%. For sample Absolute solution: Interval Area % Assay % Diff. Absolute Initial 7331860 100.6 Not difference Applicable between % 3.sup.rd day 5° C.-8° C. 23° C.-27° C. assay of n.sup.th Absolute Absolute time point and % Assay % Diff. % Assay % Diff. initial time 100.9 0.3 101.2 0.6 point should be NMT 2.0%. 4. LOD and LOD & LOQ Recorded the Component Concentration Percentage LOQ Prediction results Name (μg/ml) w/w LOD LOQ LOD LOQ 0.742 2.250 1.484 4.500 5. Linearity Correlation r should be 0.99973 coefficient (r) NLT 0.999 Slope Record the 148112.06011 results Intercept Record the −24516.53392 results % deviation of Should be not −0.2 the Y- more intercept than ±2.0% 6. Accuracy Percentage Each recovery: individual % Level % Recovery Mean % RSD Level 50% recovery and  50% 100.4 100.6 0.3 the mean 100.9 recovery at 100.5 Level 100% each level 100% 101.6 101.6 0.1 should be 101.7 between 101.6 Level 200% 98.0%- 200% 99.3 99.2 0.2 102.0%. 99.0 Relative 99.4 standard deviation for % recovery of triplicate preparation (each level) should be not more than 2.0% 7. Range Method is linear of 20% to 100%, precise and accurate in the range of 50% to 200% of test concentration. 8. Precision System % RSD of DHA peak from six replicate 0.1 precision injections of standard Standard Solution-1 is NMT 2.0%. solution Method % Assay should comply as per Complies as per Precision specification. specification % Relative standard deviation (% RSD) % RSD (Method of 6 samples should be NMT 2.0% precision) Assay 0.6 Inter mediate % Assay should comply as per Complies as per Precision specification. specification % Relative standard deviation (% RSD) % RSD (Intermediate of 6 samples should be NMT 2.0% precision) Assay 0.8 Overall relative standard deviation (RSD) % RSD (Method of 12 samples from method precision precision and and intermediate precision should be not intermediate precision) more than 2.0%. 0.6 9. Robustness Standard System suitability should System suitability parameters were solution pass. met as per the set acceptance criteria Flow rate variation Robustness parameters were met as Wavelength variation per the set acceptance criteria Column temperature Variation 10. System Standard 1. % RSD of area ratio of DHA System suitability suitability solution (Docosahexaenoic acid) peak from five parameters were met replicate injections of standard solution-1 is as per the set not more than 2.0. acceptance criteria 2. Calculate the % similarity factor of for all the validation standard-2 against standard-1 as per the parameters. following formula. The % Similarity factor should be between 98.0%-102.0%. 3. Asymmetry (Tailing factor) for DHA (Docosahexaenoic acid) peak is not more than 2.0. 4. Asymmetry (Tailing factor) in each condition is not more than 2.0.