Description:FORM 2
The Patent Act 1970
(39 of 1970)
&
The Patents Rules, 2003
(See section 10 and rule 13)

Complete Specification

Title: A taste masked and rapidly disintegrating ultra-thin orodispersible film of vitamins and a process thereof

Name of the Applicants:
1) AAVISHKAR ORAL STRIPS PRIVATE LIMITED, 109/3, IDA, Phase 2, Sector 2, Lane 6, Cherlapally, Hyderabad-500051, RR District, Telangana, India.
2) NUTRIZOE NUTRIIFOODS PRIVATE LIMITED, Unit No 611, Reliables Pride, Anand Nagar, Opp Heera Panna, Jogeshwari W, Mumbai – 400 102, Maharashtra, India.

Nationality: Indian(s).

The following specification particularly describes the invention and the manner in which it is performed.
Technical field
The present disclosure is in relation to oral thin film formulations. Particularly, the disclosure provides Orodispersible Film (ODF) formulation of vitamins and a method to prepare the same for preconception and pregnancy in women.
Background
Generally speaking, gestation or pregnancy is nothing but a time during which one or more offspring(s) develops inside a woman’s womb. Gestation lasts approximately for forty weeks and particularly it is divided into about three trimesters with each one lasting about three months long. Starting from preconception till breastfeeding the mother is both mentally and physically stressed out thereby requiring quality nutrition. Optimal nutrient intake is critical both during preconception and during pregnancy to support foetal growth and development. Vitamins such as vitamin B12 (Cyanocobalamin), Vitamin D3 (Cholecalciferol), Folic acid (Vitamin B9), Pyridoxine (Vitamin B6) and Vitamin B4 (Choline) are most essential and play vital roles in epigenetic regulation, skeletal development and central nervous system development.
At present, these vitamins are administered in the form of different oral dosage forms to subjects who are in need thereof. Nonetheless, dysphagia (difficulty in swallowing) is seen in some rare case of pregnancy, termed as esophageal achalasia. In this rare condition, the esophagus becomes paralyzed and dilated over time and eventually loses the ability to squeeze food down into the stomach. Besides this rare pharmacological condition, pregnancy nausea poses a significant challenge to consume multiple medications. Lack in medication compliance would eventually lead to rise in percentage of miscarriage (pregnancy loss) and/or infertility or alternately could even lead to neural tube defects if conceived. Therefore, administering/ consuming traditional oral dosage forms (tablets or capsules or liquids like syrups or elixirs) in such conditions is quite a daunting task to the pregnant women. Administration of vitamins is utmost essential for various reasons and administering multiple vitamins in an easily dissolvable formulation is the need of the hour, which is quite a challenging task to the formulator to develop such an acceptable formulation. All in all, there is a strong need for a novel drug delivery system that do not require water to swallow the oral dosage forms and also that do not require mastication by the subjects. The present disclosure is focused in this direction to offer a novel formulation, that rapidly disintegrates, for administration to pregnant women. Additionally, the information disclosed in this background section is only for enhancement of understanding of the general background of the disclosure and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Objectives
First and foremost objective of the present disclosure is to develop a taste masked and rapidly disintegrating Orodispersible Film (ODF) formulation of vitamins in general and particularly vitamin combination comprising of vitamin B12 (Cyanocobalamin), Vitamin D3 (Cholecalciferol), Folic acid (Vitamin B9), Pyridoxine (Vitamin B6) and Vitamin B4 (Choline).
Secondly, the disclosure is aimed at developing a process for preparation of taste masked and rapidly disintegrating ODF formulation of vitamins, particularly vitamin combination comprising of vitamin B12 (Cyanocobalamin), Vitamin D3 (Cholecalciferol), Folic acid (Vitamin B9), Pyridoxine (Vitamin B6) and Vitamin B4 (Choline).
Summary
One or more shortcomings of conventional drug delivery system (tablets/ capsules) for offering vitamin supplemental formulation to women are overcome and additional advantages are provided through the ODF formulation of vitamins or combination of vitamins as claimed in the present disclosure. Additional features and advantages are realized through the techniques of the present disclosure. Other embodiments and aspects of the disclosure are described in detail herein and are considered a part of the claimed disclosure.
The present disclosure provides a taste masked and rapidly disintegrating ultra-thin orodispersible film formulation that is a combination of vitamins. Most importantly, the combination comprising of vitamin D3 concentration of 0.44 % w/w, vitamin B12 at a concentration of 0.004% w/w, glucosamine salt of (6S)-5-methyltetrahydrofolate at a concentration of 0.30% w/w, vitamin B6 (pyridoxine hydrochloride) at a concentration of 1.37% w/w, vitamin B4 (choline as choline bitartrate) at a concentration of 51.90% w/w along with vitamin overages and pharmaceutically acceptable excipients; and the present disclosure also provides a process for preparing a taste masked and rapidly disintegrating ultra thin orodispersible film formulation of vitamins or vitamin combination as explained in the above embodiment. The first step of the process begins with microencapsulating choline bitartrate using poloxamer to obtain granules of microencapsulated choline bitartrate. Thereafter, different vitamins namely vitamin D3, vitamin B12, microencapsulated choline bitartrate or vitamin B4, vitamin B6 and glucosamine salt of (6S)-5-methyltetrahydrofolate are dissolved in water along with colorant sunset yellow, sodium bicarbonate under continuous stirring for a time period of thirty minutes to obtain a fist solution. At this time, magnesium aluminometasilicate, maize starch, mannitol, neohespiridine, malic acid and steviol glycoside are added to the first solution under continuous stirring for 15 to 20 minutes to obtain second solution. Following this, sorbitol, glycerol, tween 80 and orange flavor are added to the second solution under continuous stirring for about 10 to 20 minutes to obtain third solution. Further, lecithin and carrageenan gum are added to third solution under continuous stirring for about 5 to 10 minutes followed by addition of pullulan and hydroxypropyl methyl cellulose under continuous stirring for 60 minutes to obtain a casting solution. It is pertinent to state a polymer combination comprising of pullulan and hydroxypropyl methylcellulose are employed to overcome any limitation associated with pullulan (hygroscopic). Above all, the casting solution obtained was subjected to deaeration under vacuum for a time period of 2 to 3 hours to remove air bubbles from the casting solution followed by layering in an automatic film-forming to obtain orodispersible film of suitable thickness. Lastly, drying the wet orodispersible film followed by slitting using a slitting machine, cutting and packaging the orange colored square shaped orodispersible films.
The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.
Brief description of the accompanying drawings:
The features of the present disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments in accordance with the disclosure and are therefore not to be considered limiting of its scope the disclosure will be described with additional specificity and detail through use of the accompanying drawings.
Figure 1: shows HPLC chromatograms pertinent to vitamin B12 standard and sample ODF formulation
Figure 2: shows HPLC chromatograms of pertinent to vitamin D3 – standard and sample ODF formulation
Figure 3: shows HPLC chromatograms pertinent to vitamin B6 and folic acid – standard and sample ODF formulation
Detailed Description
Before explaining any one embodiment of the present disclosure by way of drawings, experimentation, results, and pertinent procedures, it is to be understood that the disclosure is not limited in its application to the details as explained in below embodiments set forth in the following description or illustrated in the drawings, experimentation and/or results. The disclosure is further capable of other embodiments which can be practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary and not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
Definitions:
The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
‘Nutrients’ or ‘Micronutrients’ shall mean organic molecules that are essential in small quantities for normal functioning of metabolism in human body. Vitamin B complex is nothing but water soluble vitamins and are a supplement that delivers eight of the B vitamins namely B1 (Thiamine), B2 (Riboflavin), B3 (Niacin), B5 (Pantothenic acid), B6 (Pyridoxine), B7 (Biotin), B9 (Folate) and B12 (Cyanocobalamin). Vitamin D is a group of fat-soluble vitamins – most important in this group is Vitamin D3 (Cholecalciferol).
‘Orodispersible film(s)’ or ODF or ODFs shall mean thin sheets that disintegrate rapidly when placed/ administered to the tongue as they easily get hydrated due to the saliva in oral cavity.
“Film-forming substance” shall mean a substance capable of forming a cohesive, solid or gelatinous film or layer. The film or layer in particular can be formed by casting or otherwise applying a formulation containing the film-forming substance solved or dispersed in a solvent, and optionally further ingredients onto a surface. Preferably, the film-forming substance is a water-soluble polymer such as hydroxypropyl cellulose, pullulan and others that are obvious to a person skilled in the art.
“Plasticizer” shall mean a substance/ agent used to improve the flexibility of the orodispersible film. The plasticizer may be one selected from the group consisting of sorbitol, malitol, xylitol, glycerol, polyethylene glycol, propyleneglycol, hydrogenated starch syrup, starch syrup, triacetin, glycerol oleate, glycerol, sucrose fatty acid ester and double chain fatty acid. Preferably, the plasticizer used in the present invention is glycerol and sorbitol.
“Organoleptic agents” shall mean sweetening agents, coloring agents, and flavoring agents. No coloring agents were employed in the present disclosure.
“Sweetening agents” shall mean agents that impart more pleasant taste to the orodispersible film formulation. Generally, the sweetener may be at least one selected from the group consisting of sucrose, glucose, maltose, sucralose, oligosaccharides dextrin, alpha cyclodextrin, beta cyclodextrin, gamma cyclodextrin, methyl beta cyclodextrin, aspartame, cluster dextrin, invert sugar, fructose, lactose, galactose, starch syrup, sorbitol, malitol, xylitol, erythritol, hydrogenated starch syrup, mannitol, trehalose. Preferably, the orodispersible film of the present invention comprises sucralose, steviose and neohespiridine.
“Flavoring agents” shall mean any natural or artificial flavor or a combination thereof. The natural flavor may include aromatic plants, especially extracts and/oils obtained from leaves, flowers or fruits of such plants and can include spearmint oil, cinnamon oil, peppermint oil, lemon, oil, clove oil, bay oil, thyme oil, nutmeg oil, sage oil, almond oil etc. The artificial flavoring may include synthetic fruit flavors such as lemon, orange, grape, lime, strawberry, etc and other synthetic flavors such as vanilla, chocolate, coffee, cocoa, ginseng, citrus etc. Preferred flavoring agent employed in the present disclosure is peppermint oil.
“Acidic agent” shall mean a substance that serves to control taste together with the sweetener. Besides, it stimulates secretion of saliva in order to dissolve the orodispersible film. The acidic agent may be at least one selected from the group consisting of citric acid, malic acid, fumaric acid, tartaric acid, ascorbic acid, succinic acid, adipic acid, lactic acid. Preferable acidic agent is citric acid.
The present disclosure is in relation to a taste masked and rapidly disintegrating ultra thin ultra-thin orodispersible film composition comprising: vitamin D3 at a concentration of 0.44 % w/w, vitamin B12 at a concentration of 0.004% w/w, glucosamine salt of (6S)-5-methyltetrahydrofolate at a concentration of 0.30% w/w, vitamin B6 at a concentration of 1.37% w/w, vitamin B4 at a concentration of 51.90% w/w along with vitamin overages and pharmaceutically acceptable excipients. In addition, vitamin overages as vitamin D3 at a concentration of 30 %, vitamin B12 at a concentration of 25%, glucosamine salt of (6S)-5-methyltetrahydrofolate at a concentration of 25% are employed.
In another embodiment of the present disclosure, the vitamin B4 or choline bitartrate is microencapsulated choline comprising of choline and poloxamer are at a ratio of 1:0.137.
In yet another embodiment of the present disclosure, the pharmaceutically acceptable excipients are poloxamer at a concentration of 7.14% w/w, sodium bicarbonate at a concentration of 3.33% w/w, magnesium aluminometasilicate at a concentration of 4.76% w/w, pullulan and hydroxypropyl methyl cellulose each at a concentration of 9.52% w/w, maize starch and mannitol each at a concentration of 1.19% w/w, sorbitol and erythritol each at a concentration of 0.48% w/w, carrageenan at a concentration of 0.38% w/w, glycerol at a concentration of 0.71% w/w, orange flavor at a concentration of 2.38% w/w, neohespiridine at a concentration of 0.24% w/w, lecithin at a concentration of 1.90% w/w, malic acid at a concentration of 0.53% w/w, steviol glycoside at a concentration of 1.43% w/w, sucralose at a concentration of 0.55% w/w, tween 80 at a concentration of 0.24% w/w, sunset yellow 0.005% w/w and purified water at a concentration of 95.24% w/w.
The present disclosure is in relation to a process for preparing a taste masked and rapidly disintegrating ultra thin orodispersible film formulation as explained in above embodiments, comprising steps of microencapsulating choline bitartrate using poloxamer to obtain granules of microencapsulated choline bitartrate; dissolving vitamin D3, vitamin B12, microencapsulated choline bitartrate or vitamin B4, vitamin B6 and glucosamine salt of (6S)-5-methyltetrahydrofolate in water along with colorant sunset yellow, sodium bicarbonate under continuous stirring for a time period of 30 minutes to obtain a fist solution; adding magnesium aluminometasilicate, maize starch, mannitol, neohespiridine, malic acid and steviol glycoside to the first solution obtained previous embodiment under continuous stirring for 15 to 20 minutes to obtain second solution; adding sorbitol, glycerol, tween 80 and orange flavor to the second solution obtained under preceding embodiment under continuous stirring for about 10 to 20 minutes to obtain third solution; adding lecithin and carrageenan gum to third solution obtained under the above embodiment under continuous stirring for about 5 to 10 minutes followed by addition of pullulan and hydroxypropyl methyl cellulose under continuous stirring for 60 minutes to obtain a casting solution; deaerating the casting solution obtained under the aforementioned embodiment under vacuum for a time period of 2 to 3 hours to remove air bubbles from the casting solution followed by layering in an automatic film-forming to obtain orodispersible film of thickness 300µ; and drying the orodispersible film at a temperature of 1200 ± 5℃ for a time period of 10 to 15 minutes followed by slitting using a slitting machine, cutting and packaging the orange colored square shaped orodispersible films.
In yet another embodiment of the present disclosure it provides microencapsulation process that involves dissolving poloxamer in a stainless steel vessel followed by mixing with choline bitartrate, cooling and sifting using 100 number mesh to obtain granules of microencapsulated choline bitartrate.
In yet another embodiment of the present disclosure it provides the processing temperature of the mixing vessel which is maintained at a temperature ranging from 25℃ to 30℃ and the bottom stirrer speed is maintained at 1000 rpm at all stage and is increased to 2000 while addition of the polymer. In addition, the anchor speed of the vessel is maintained at a range starting from 20 to 60 rpm.
Additionally, the disclosure is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope of the present invention. On the contrary, it is to be clearly understood that various other embodiments, modifications, and equivalents thereof, after reading the description herein in conjunction with the drawings and appended claims, may suggest themselves to those skilled in the art without departing from the spirit and scope of the presently disclosed and claimed invention.
Example 1: Method to prepare orodispersible films (ODFs) of vitamins
Table 1 provides the list of ingredients employed in preparing ODF formulation of vitamins that are used by women during preconception and during pregnancy. Generally speaking, folic acid (vitamin B9) plays a pivotal role in cell division and DNA synthesis to promote growth and development. Conventional folates undergo metabolism leading to formation of Unmetabolized Folic Acid (UMFA) that enters systemic circulation causing toxic effects to the subjects. To circumvent this potential toxicity associated with usage of conventional folates and as one of the unique features of the film formulation of present disclosure it employs an active version of folic acid as L-methyl folate (5-methyl tetrahydro folate) which prevents accumulation of UMFA in the systemic circulation. Yet another unique feature of the present disclosure is masking the bitter taste of choline bitartrate using polymers as detailed below to gain subject compliance.
The method employed to fabricate the ODFs is called as ‘solvent casting method’ and the steps involved in the process include first and foremost step of encapsulating choline bitartrate using poloxamer. Poloxamer is dissolved in stainless steel vessel by applying heat followed by combining the same with choline bitartrate and the mixed mass is subjected to sieving using 100 mesh sieves to obtain granules of microencapsulated choline bitartrate.
Secondly, all the vitamins namely vitamin D3, vitamin B12, microencapsulated choline bitartrate or vitamin B4, vitamin B6 and glucosamine salt of (6S)-5-methyltetrahydrofolate are dissolved in water along with colorant sunset yellow, and sodium bicarbonate under continuous stirring for a time period of 30 minutes to obtain a first solution. Thirdly, magnesium aluminometasilicate, maize starch, mannitol, neohespiridine, malic acid and steviol glycoside are added to the first solution under continuous stirring for 15 to 20 minutes to obtain second solution. Thereafter, sorbitol, glycerol, tween 80 and orange flavor to the second solution under continuous stirring for about 10 to 20 minutes to obtain third solution. Following this, add lecithin and carrageenan gum to third solution obtained in the previous step under continuous stirring for about 5 to 10 minutes followed by addition of pullulan and hydroxypropyl methyl cellulose (grade E5) under continuous stirring for 60 minutes to obtain a casting solution. The casting solution is subjected to deaeration under vacuum strictly at 600 to 700 mm Hg to prevent any physical damage to the finished product. In addition, deaeration is carried out for a time period of 2 to 3 hours to remove any air bubbles. The deaerated casting solution s subjected to casting by using an automatic layering machine to obtain ODFs of thickness ranging from 110 to 125 microns. The wet layered films are dried followed by slitting using a slitting machine and cutting to obtain a predetermined size and packed until further use. It is pertinent to state that the wet layered film is dried at a temperature of 120℃ ± 5℃ for a time period ranging between 10 to 15 minutes.
 
Table 1: List of ingredients employed in the ODF
Sl. No Name of the Ingredient % W/W Use/ Role
1 Vitamin D3 (Cholecalciferol) 0.44 Active agent
2 Vitamin B12 as Cyanocobalamin 0.0004 Active agent
3 Glucosamine salt of (6S)-5-methyltetrahydrofolate (Vitamin B9) 0.30 Active agent
4 Vitamin B6 (Pyridoxine HCl) 1.37 Active agent
5 Choline as choline bitartrate (Vitamin B4) 51.90 Active agent
6 Poloxamer 7.14 Taste masking agent
7 Sodium bicarbonate 3.33 pH adjusting agent
8 Magnesium aluminometasilicate 4.76 Adsorbent
9 Pullulan 9.52 Film-forming agent
10 Hydroxypropyl methylcellulose 9.52 Encapsulating agent
11 Maize starch 1.19 Absorbent
12 Mannitol 1.19 Anti-caking agent
13 Sorbitol 0.48 Humectant
14 Carrageenan 0.38 Thickener
15 Glycerol 0.71 Plasticizer
16 Natural Orange Flavor 2.38 Flavorant
17 Erythritol 0.48 Sweetener
18 Neohespiridine 0.24 Sweetener
19 Lecithin 1.90 Emulsifier
20 Malic Acid 0.53 Acidifying agent
21 Steviol glycoside 1.43 Sweetener
22 Sucralose 0.55 Sweetener
23 Tween 80 0.24 Surfactant
24 Sunset yellow 0.005 Colorant
25 Purified water 95.24 Solvent

It is pertinent to state that the concentration of the ingredients shown under Table 1 are the optimized concentrations arrived after a systematically designing the formulation experiments by Box-Behnken design. Different batches were tested and the batch that showed highly promising results are shown in example 1 and the same batch characterization studies are provided under the following examples. It is pertinent to state that negative data pertinent to different batches of the formulation is not shown here. Any variation in the concentration of the ingredients would not help in obtaining ODFs of vitamins which are acceptable to the subjects who are in need thereof. Some of the process parameters are tabulated below under Table 2.
Table 2: Critical process parameters to fabricate ODFs of vitamins
Parameter Item Range
Deaeration Vacuum 600 to 700 mmHg
Time 2 to 3Hours
Layering and drying machine parameters
Thickness 110 to 125 microns
RPM 7.0 to 9.0
Temperature 120±5ºC
Strip weight 235 mg ±7.5%
Slitting Tension 8 to 12Kg

Example 2: Physical methods for characterization of ODFs of vitamins
The orodispersible films of vitamins prepared under example 1 is subjected to the following physical methods of characterization.
(a) Visual inspection: Visual inspection was carried out from sampled ODFs. The colour of the films was orange color and free from air bubbles.
(b) Shape: The ODFs could be cut into desired shape. For instance, square shaped ODFs were cut using the cutting machine of size ranging from 3 cm2 to 3.4 cm2. These sizes of ODFs are highly comfortable for self-administration by subjects across all the age groups.
(c) Thickness: Thickness of the ODF is measured using a micrometer (digital) which was found to be ranging from 0.110 to 0.125 Mm or 110 to 125 microns.
(d) Average weight: ODFs having an area of 700 mm2 were weighed using an electronic balance. The average weight obtained is a mean weight variation of the film. This gives a general confirmation of the fact that both the drug and excipients are uniformly distributed in the ODF and one has obtained an ODF weighing about 210 mg ±7.5 mg.
(e) Folding endurance (FE): This test is performed manually. The ODF of the uniform cross-sectional area is folded repeatedly until it breaks. FE value is the number of times the sample ODF is folded repeatedly without cracking. High FE value is a direct indication to establish the fact that ODF is indeed associated with higher mechanical strength. The FE value for the ODFs was ranging from 15 to 20. Various other in-vitro methods are explained in the subsequent examples.
Example 3: In-vitro methods for characterization ODFs of vitamins
(a) Disintegration Test: Disintegration of ODF is critical quality attribute that helps in gaining patient compliance. The ODFs are expected to rapidly disintegrate when administered to tongue. There exist several methods for determining the disintegration time of an ODF. Most popular methods are petri dish method, slide frame method, drop method, hollow glass cylinder method, slide frame and ball method and others. The most popular LDR-LED sensing method can also be utilized for predicting both the start time and end disintegration time of an ODF. In the present disclosure, PharmaTest® - ODF disintegration tester was employed to study the disintegration time of the vitamin ODF. Standard procedure was followed in testing the disintegration time using disintegration medium - ‘phosphate buffer’ having pH 6.8. It was observed that the disintegration time of all the films was not more than 60 seconds.
In addition, petri dish method was also used in studying the disintegration time of microencapsulated ODFs. The Petri dish method is much simpler compared to the other methods as it just involves placing a film of size 2X2 cm in a petri dish with 10 mL of water followed by recording the time required for the complete disintegration of the film. In order to simulate the movement of tongue, ‘orbital bath shaker’ was used by maintaining the speed of about 50 rpm at a temperature of 370C. The disintegration time of all the ODFs was found to be not more than 60 seconds.
(b) Estimation of moisture content by Karl Fischer (KF) titration method:
This method helps in determining even the lowest amount of water content in any ODF sample. It employs methanol or anhydrous dimethyl sulfoxide as a solvent. The selected solvent determines the solubility of an ODF for the analysis. In the present method, suitable amount of ODF sample, say 500 mg of ODF sample is transferred into titration vessel and the titration was continued till the electrometric end point. Every time, before adding the sample, titrate the vessels content to electrometric end point to neutralize the moisture interference during the process. The water content in ODF samples was found to be ranging between 3.0 to 8.0% w/w.
(c) Identification of vitamins by HPLC: The retention time of the peak of the test solution correspond to the chromatogram of the standard solution. The chromatographic conditions, mobile phase, blank, sample and standard solution preparations are explained in the below section.
(d) Assay by Titration Method: Choline as Choline Bitartrate by Titration: Transfer the sample equivalent to 200 mg of Choline bitartrate to a conical flask, and dissolve it in 50 ml of glacial acetic acid. Titrate with 0.1N perchloric acid volumetric solution. Determine the end point potentiometrically. Perform a blank determination and make any necessary correction. Each mL of 0.1N perchloric acid is equivalent to 25.32 mg of C9H19NO7.
(e) Vitamin B6 & B9 by HPLC method:
Principle- Vitamin-B6 and B9 are separated by liquid chromatography and measured by HPLC.
Apparatus: Analytical balance, Spatulas (SS), Pipettes, Measuring cylinder (1000ml), Volumetric flasks (100ml,500ml), Sonicator and HPLC with UV detector. HPLC Column: Spursil C18 with length 150mm, ID: 4.6mm and Particle size: 5µm equivalent.
Chemicals and reagents: Water and methanol
Preparation of mobile phase: Mobile phase A: (20 mM ammonium formate in water): 1.2612g of ammonium formate in 1 lit of water. Mobile phase B: 100% methanol, Preparation of methanol: water (1:1): Fill 500 ml of methanol in 1000 ml measuring cylinder, fill up to mark with water.
Preparation of standard solutions: Weigh approximately 25 mg of Vitamin B6 and 5 mg of Vitamin B9 standards in a 100 ml of volumetric flask. Dissolve and dilute to 100 ml with methanol. Transfer 1 ml of the above solution into a 10 ml volumetric flask and dilute to volume with methanol.
Sample Preparation: Weigh and transfer five strips or sample ODFs of the formulation of the present disclosure into a 500 ml volumetric flask, add about 300 ml of diluent (water and methanol 1:1 ratio mixture). Shake well and dilute to 500 ml with methanol: water (1:1). Filter the solution through 0.22µ filter.
Chromatographic conditions:
HPLC Method - Parameters
Detector UV
Column Spursil-C18 5.0µm, 150mm X 4.6mm
Column oven temperature 40 ºC
Auto sampler temperature 10 ºC
Mobile phase Eluent A – water +20mM ammonium format,
Eluent B Methanol.
Flow rate (0.500 mL/min)
Volume of injection 10 µL
Wavelength 285nm

Inject five standard injections and two sample injections, record the chromatograms
and calculate the assay percentage.
Vitamin D3 by HPLC:
Principle: Vitamin-D3is separated by liquid chromatography and measured by HPLC-UV detector.
Apparatus: Analytical balance, spatulas (SS), pipette, measuring cylinder (1000ml), volumetric flasks (100ml, 500ml), sonicator, HPLC with UV Detector. HPLC Column: Spursil C18 with length 150 mm, ID: 4.6mm and Particle size: 5 µm equivalent.
Chemicals and reagents: Water and methanol.
Preparation of solutions
Preparation of mobile phase: 3.9.3.4.2 Mobile phase A: (20 mM ammonium format in water): 1.2612g of ammonium format in 1 lit of water. Mobile phase B: 100% methanol. Preparation of methanol: water (1:1): Fill 500 ml of methanol in 1000 ml measuring cylinder, fill up to mark with water.
Preparation of standard solutions: Weigh approximately 8 mg of Vitamin D3 standard in a 100 ml of volumetric flask. Dissolve and dilute to 100 ml with methanol. Transfer 1 ml of the above solution into a 10 ml volumetric flask and dilute to volume with methanol.
Sample Preparation: Weigh and transfer five strips into a 500 ml volumetric flask, add about 300 ml of diluent (Water and methanol 1:1 ratio mixture). Shake well and dilute to 500 ml with methanol: water (1:1). Filter the solution through 0.22µ filter.

Chromatographic conditions:
HPLC Method Parameters
Detector UV
Column Spursil-C18 5.0µm, 150mm X 4.6mm
Column oven temperature 40 ºC
Auto sampler temperature 10 ºC
Mobile phase Eluent A – water +20mM ammonium format,
Eluent B Methanol.
Flow rate (0.500 mL/min)
Volume of injection 10 µL
Wavelength 226nm

Inject five standard injections and two sample injections, record the chromatograms and calculate the assay%.
Vitamin B12 by HPLC:
Principle: Vitamin-B12 are separated by liquid chromatography and measured by HPLC-UV.
Apparatus: Analytical balance, spatulas (SS), pipette, measuring cylinder (1000ml), volumetric flasks (100ml, 500ml), sonicator, HPLC with UV detector. HPLC Column: Spursil C18 with length 150mm, ID: 4.6mm and Particle size: 5µm equivalent. Chemicals and reagents: Water and methanol.
Preparation of mobile phase: Mobile phase A: (20 mM ammonium format in water): 1.2612g of ammonium format in 1 lit of water. Mobile phase B: 100% methanol. Preparation of methanol : water (1:1) (diluent for sample preparation): Fill 500 ml of methanol in 1000 ml measuring cylinder, fill up to mark with water. Preparation of standard solutions: Weigh approximately 25 mg of Vitamin B12 standard in a 100 ml of volumetric flask. Dissolve and dilute to 100 ml with methanol. Transfer 1 ml of the above stock solution into a 100 ml volumetric flask, make up the volume with methanol. Transfer 1 ml of the above diluted standard solution into a 100 ml volumetric flask, make up the volume with methanol.
Sample Preparation: Weigh and transfer five strips/ ODFs of vitamins of the present disclosure into a 500 ml volumetric flask, add about 300 ml of diluent (Water and methanol 1:1 ratio mixture). Shake well and dilute to volume with diluent (Water and methanol 1:1 ratio mixture). Filter the solution through 0.22µ filter
Chromatographic conditions:
HPLC Method Parameters
Detector UV
Column Spursil-C18 5.0µm, 150mm X 4.6mm
Column oven temperature
40 ℃
Auto sampler temperature
10 ℃
Mobile phase Eluent A – water +20mM ammonium format,
Eluent B Methanol.
Flow rate (0.500 mL/min)
Volume of injection 10 µL
Wavelength 361nm

Inject five standard injections and two sample injections, record the chromatograms and calculate the assay percentage. The assay results are provided in below table 2. The chromatograms of the HPLC analysis are provided in Figure 1 to Figure 3.
Table 2: Assay values
Assay: Each strip/ ODF contains
Vitamin B4 (Choline) Ranging between 90.0 % to 110.0 %
Pyridoxine (Vitamin B6) Not less than 90.0 %
Vitamin D3
(Cholecalciferol) Not less than 90.0 %

Folic acid (Vitamin B9) Not less than 90.0 %

vitamin B12
(Cyanocobalamin) Not less than 90.0 %

Example 4: Dissolution studies for ODFs – method to study release of Vitamin B6 and B9:
Dissolution Parameters: Dissolution Apparatus: USP II (Paddle) (Samples – ODFs of vitamins are inserted into mesh sinkers); RPM: 100; Volume: 500 mL; Dissolution medium: Simulated saliva at pH 6.7 buffer. Time interval: 5, 10, 15, 20, 30 minutes.
HPLC instrument Parameters, Chromatographic conditions & Preparation of Mobile phase: Refer Assay by HPLC method for Vitamin B6 and B9 under Example 3.
Standard Preparation: Transfer about 4.5 mg of Vitamin B6 working Standard and 1.0 mg of vitamin B9 working standard into a 100 ml volumetric flask. Dissolve and dilute to volume with methanol. Transfer about 1 ml of the above solution into a 10 ml volumetric flask. Dilute to volume with dissolution medium. Filter the standard solution through 0.45µm filter and inject into the HPLC system. (Five replicate injections).
Sample Preparation: Transfer 500 mL of dissolution medium into each dissolution vessel and start the instrument. After reaching the required temperature (37°C ± 0.5°C), transfer one strip (ODF of vitamins) into each dissolution vessel with sinkers and start the dissolution instrument. Collect 10 ml of the dissolution sample after each specified time interval and replace the same volume in the each vessel to maintain sink conditions. Filter the samples solutions through 0.45µm filter and inject into the HPLC system. The percentage release is calculated using the below formula.
Calculation:
Spl. area Std. Wt 1 500 Std. Purity 100
------------ x --------- x ---------- x ---------- x ---------- x ----------- =
Std. area 100 10 1 100 L.C

Dissolution studies for Vitamin D3:
Dissolution Parameters: Dissolution Apparatus: USP II (Paddle) (Samples -ODFs of vitamins are inserted into mesh sinkers are kept into mesh sinkers) RPM: 100, Volume: 500 mL, Dissolution medium: Simulated saliva at pH 6.7 buffer, Time interval: 5, 10, 15, 20, 30 minutes.
HPLC instrument Parameters, Chromatographic conditions & Preparation of Mobile phase: Refer Assay by HPLC method for Vitamin D3 – under Example 3.
Standard Preparation: Transfer about 1.5 mg of Vitamin D3 Working Standard into a 100 ml volumetric flask. Dissolve and dilute to volume with methanol. Transfer about 1 ml of the above solution into a 10 ml volumetric flask. Dilute to volume with dissolution medium. Filter the standard solution through 0.45µm filter and inject into the HPLC system. (Five replicate injections)
Sample Preparation: Transfer 500 mL of dissolution medium into each dissolution vessel and start the instrument. After reaching the required temperature (37°C ± 0.5°C), transfer one strip (ODF of vitamins) or into each dissolution vessel with sinkers and start the dissolution instrument. Collect 10 ml of the dissolution sample after each specified time interval and replace the same volume in the each vessel. Filter the samples solutions through 0.45µm filter and inject into the HPLC system. The percentage release is calculated using the below formula.
Calculation:
Spl. area Std. Wt 1 500 Std. Purity 100
------------ x --------- x ---------- x ---------- x ---------- x ----------- =
Std. area 100 10 1 100 L.C

Dissolution study for Vitamin B12:
Dissolution Parameters: Dissolution Apparatus: USP II (Paddle) (Samples – ODFS of vitamins are kept into mesh sinkers), RPM: 100, Volume: 500 mL, Dissolution medium: Simulated saliva at pH 6.7 buffer, Time interval: 5, 10, 15, 20, 30 minutes.
HPLC instrument Parameters, Chromatographic conditions & Preparation of Mobile phase: Refer Assay by HPLC method for Vitamin B12 –Example 3.
Standard Preparation: Transfer about 4.5 mg of Vitamin B12 Working Standard into a 100 ml volumetric flask. Dissolve and dilute to volume with methanol. Transfer about 1 ml of the above solution into a 100 ml volumetric flask. Dilute to volume with dissolution medium. Again transfer about 1 ml of the above solution into a 100 ml volumetric flask. Dilute to volume with dissolution medium. Filter the standard solution through 0.45µm filter and inject into the HPLC system. (Five replicate injections)
Sample Preparation: Transfer 500 mL of dissolution medium into each dissolution vessel and start the instrument. After reaching the required temperature (37°C ± 0.5°C), transfer one strip into each dissolution vessel with sinkers and start the dissolution instrument. Collect 10 ml of the dissolution sample after each specified time interval and replace the same volume in the each vessel to maintain sink conditions. Filter the samples solutions through 0.45µm filter and inject into the HPLC system. The percentage release is calculated using the below formula.
Calculation:
Spl. area Std. Wt 1 1 500 Std. Purity 100
------------ x --------- x ---------- x ---------- x ---------- x ----------- x ----------- =
Std. area 100 100 100 1 100 L.C
Dissolution studies for Choline as Choline bitartrate:
Dissolution Parameters: Dissolution Apparatus: USP II (Paddle) (Samples – ODFs of vitamins are kept into mesh sinkers), RPM: 100, Volume: 500 mL. Dissolution medium: Simulated saliva at pH 6.7 buffer. Time interval: 5, 10, 15, 20, 30 minutes.
Sample Preparation: Transfer 500 mL of dissolution medium into each dissolution vessel and start the instrument. After reaching the required temperature (37°C ± 0.5°C), transfer one strip into each dissolution vessel with sinkers and start the dissolution instrument. Collect 20 ml of the dissolution sample into a conical flask and add 0.5 ml of acetic acid into the same conical flask. Titrate with 0.1 N perchloric acid volumetric solution. Determine the end point potentiometrically. Perform a blank determination and make any necessary corrections. Each ml of 0.1N perchloric acid is equivalent to 25.32 mg of C9H19NO7. The dissolution release profile data is tabulated in Table 3.
Table 3: Dissolution release profile of ODFs of vitamins

Active agent Dissolution release profile (%)
5 min 10 min 15 min 20 min 30 min
Vitamin B4 20.4 58.3 87.5 96.7 97.2
Vitamin B6 53.5 71.8 102.8 105.3 106.2
Vitamin D3 8.3 15.7 107.3 109.5 111.5
Vitamin B9 51.7 98.4 102.9 104.0 109.7
Vitamin B12 52.2 64.7 103.5 108.1 106.3

From the above table, it is evident that more than 95% release profile is achieved for all the vitamins within 30 minutes of time.
Example 5: Other characterization tests
(a) Pesticide residue testing for Characterization of ODFs of vitamins: The ODF samples were tested for presence of pesticides by in-house standard testing procedure. The test procedure involves testing the samples by gas chromatography- mass spectroscopy (GC-MS) method. Almost 144 pesticides were found to be well below the quantification limits (0.01 mg/Kg).
(b) Test for heavy metals: The ODF samples were tested for presence of heavy metals by in-house standard testing procedures. The test procedure involves testing the samples by ‘Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for presence of arsenic, cadmium, mercury and lead. The tested heavy metals include but not limiting to lead (not more than 3.0 ppm), cadmium (not more than 1 ppm) and arsenic (not more than 1 ppm) and mercury (not more than 0.1 ppm).
(c) Microorganism: The ODF samples were tested for presence of microorganisms by in-house standard testing procedures. The test procedure involves testing the samples for presence of microorganisms namely yeast and moulds, E.coli (bacteria), Salmonella, Staphylococcus aureus and pseudomonas aeruginosa. Except yeast and moulds (with <10 CFU/g), THE remaining microorganism were absent.
Advantages:
 The orodispersible film formulation of vitamins of the present disclosure is boon to pregnant women with dysphagia or esophageal achalasia.
 One of the unique and inventive aspects of the ODFs of the present disclosure is that they are delivery systems that don’t need water for consumption by the subjects / patients. Thus, they are definitely of great advantage for third world countries that don’t always have clean drinking water readily available to consume medication.
 The formulation of the present disclosure are very portable and easy to carry and can be consumed on the go.
 The microencapsulated vitamins orodispersible films of the present disclosure are the most powerful and transformational alternative to the extensively used solid and parenteral dosage forms (tablets, capsules, intravenous and intramuscular injections).
 The film formulation of the present disclosure rapidly release and can be self-administrable without the assistance of any nurse or paramedic.
 The vitamin ODFs of the present disclosure can be used in situations where vitamins are required as a supplement to pregnant women.
 ODFs of the present disclosure are excellent in gaining patient compliance in general and particularly in patients with dysphagia (difficulty in swallowing), Parkinson’s disease, mucositis and vomiting tendency.

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.
, Claims:We claim:
1) A taste masked and rapidly disintegrating ultra-thin orodispersible film composition comprising: vitamin D3 at a concentration of 0.44 % w/w, vitamin B12 at a concentration of 0.004% w/w, glucosamine salt of (6S)-5-methyltetrahydrofolate at a concentration of 0.30% w/w, vitamin B6 at a concentration of 1.37% w/w, vitamin B4 at a concentration of 51.90% w/w along with vitamin overages and pharmaceutically acceptable excipients.
2) The composition as claimed in claim 1, wherein said vitamin B4 or choline bitartrate is microencapsulated choline comprising of choline and poloxamer are at a ratio of 1:0.137.
3) The composition as claimed in claim 1, wherein said ultra-thin orodispersible film is a pharmaceutical formulation comprising of vitamin overages as vitamin D3 at a concentration of 30 %, vitamin B12 at a concentration of 25%, glucosamine salt of (6S)-5-methyltetrahydrofolate at a concentration of 25%.
4) The composition as claimed in claim 1, wherein said pharmaceutically acceptable excipients are poloxamer at a concentration of 7.14% w/w, sodium bicarbonate at a concentration of 3.33% w/w, magnesium aluminometasilicate at a concentration of 4.76% w/w, pullulan and hydroxypropyl methyl cellulose each at a concentration of 9.52% w/w, maize starch and mannitol each at a concentration of 1.19% w/w, sorbitol and erythritol each at a concentration of 0.48% w/w, carrageenan at a concentration of 0.38% w/w, glycerol at a concentration of 0.71% w/w, orange flavor at a concentration of 2.38% w/w, neohespiridine at a concentration of 0.24% w/w, lecithin at a concentration of 1.90% w/w, malic acid at a concentration of 0.53% w/w, steviol glycoside at a concentration of 1.43% w/w, sucralose at a concentration of 0.55% w/w, tween 80 at a concentration of 0.24% w/w, sunset yellow 0.005% w/w and purified water at a concentration of 95.24% w/w.
5) A process for preparing a taste masked and rapidly disintegrating ultra thin orodispersible film formulation as claimed in claim 1 to 4, comprising steps of:
a) microencapsulating choline bitartrate using poloxamer to obtain granules of microencapsulated choline bitartrate;
b) dissolving vitamin D3, vitamin B12, microencapsulated choline bitartrate or vitamin B4, vitamin B6 and glucosamine salt of (6S)-5-methyltetrahydrofolate in water along with colorant sunset yellow, sodium bicarbonate under continuous stirring for a time period of 30 minutes to obtain a fist solution;
c) adding magnesium aluminometasilicate, maize starch, mannitol, neohespiridine, malic acid and steviol glycoside to the first solution obtained under step (a) under continuous stirring for 5 to 15 minutes to obtain second solution;
d) adding sorbitol, glycerol, tween 80 and orange flavor to the second solution obtained under step (b) under continuous stirring for about 10 to 20 minutes to obtain third solution;
e) adding lecithin and carrageenan gum to third solution obtained under step (c) under continuous stirring for about 5 to 10 minutes followed by addition of pullulan and hydroxypropyl methyl cellulose under continuous stirring for 60 minutes to obtain a casting solution;
f) deaerating the casting solution obtained under step (d) under vacuum for a time period of 2 to 3 hours to remove air bubbles from the casting solution followed by layering in an automatic film-forming to obtain orodispersible film of thickness 300µ; and
g) drying the orodispersible film followed by slitting using a slitting machine, cutting and packaging the orange colored square shaped orodispersible films.
6) The process as claimed in claim 5, wherein said step of microencapsulation includes dissolving poloxamer in a stainless steel vessel followed by mixing with choline bitartrate, cooling and sifting using 100 number mesh to obtain granules of microencapsulated choline bitartrate.
7) The process as claimed in claim 5, wherein said orodispersible films are dried at a temperature of 1200 ± 5℃ for a time period of 10 to 15 minutes.
8) The process as claimed in claim 5, wherein said slitting is carried industrially under a tension of 8 to 12 kg.
9) The process as claimed in claim 5, wherein said vitamins include its overages as vitamin D3 at a concentration of 30 %, vitamin B12 at a concentration of 25%, glucosamine salt of (6S)-5-methyltetrahydrofolate at a concentration of 25%.
10) The process as claimed in claim 5, wherein the processing temperature was maintained at a temperature ranging from 25℃ to 30℃.