Accepted Manuscript

Dietary supplements containing vitamins and minerals:

Formulation, optimization and evaluation

R. Rajakumari, Oluwatobi Samuel Oluwafemi, Sabu Thomas,

Nandakumar Kalarikkal

PII: S0032-5910(18)30466-2
DOI: doi:10.1016/j.powtec.2018.06.026
Reference: PTEC 13459
To appear in: Powder Technology
Received date: 8 March 2018
Revised date: 9 June 2018
Accepted date: 12 June 2018

Please cite this article as: R. Rajakumari, Oluwatobi Samuel Oluwafemi, Sabu Thomas,
Nandakumar Kalarikkal , Dietary supplements containing vitamins and minerals:
Formulation, optimization and evaluation. Ptec (2017), doi:10.1016/j.powtec.2018.06.026

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Dietary Supplements Containing Vitamins and Minerals: Formulation, Optimization and

Evaluation
R. Rajakumari a, Oluwatobi Samuel Oluwafemi*b c, Sabu Thomasa, d, and Nandakumar
Kalarikkal*a, e
a
International and Inter-University Centre for Nanoscience and Nanotechnology, Mahatma
Gandhi University, Kerala, 686560, India
b
Department of Applied Chemistry, University of Johannesburg, South Africa

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c
Centre for Nanomaterials Sciences Research, University of Johannesburg, Johannesburg, South

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Africa

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E-mail: oluwafemi.oluwatobi@gmail.com
d
School of Chemical Sciences, Mahatma Gandhi University, Kerala, 686560, India.
E-mail: sabuchathukulam@yahoo.co.uk, Fax: +91-481-2731002, Tel: +91-481-2731002

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e
School of Pure and Applied Physics, Mahatma Gandhi University, Kerala, 686560, India.
E-mail: nkkalarikkal@mgu.ac.in, Fax: +91-481-2731669, Tel: +91-481- 2731669
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*Corresponding Authors E mail: oluwafemi.oluwatobi@gmail.com; nkkalarikkal@mgu.ac.in
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Abstract:
The regular diet of common man is often deficient in certain nutrients, so dietary supplements are
commonly prescribed. In this study, a combination of vitamins and minerals in the form of a
bilayer tablet is proposed as a dietary supplement. A film-coated bilayer tablet in which one layer
contains premixed vitamins (A, D, E, C, B1, B2, B3, B5, B6, B7, B9 and B12) and the other layer
contains premixed minerals (magnesium, manganese, molybdenum, iodine, zinc, silicon and

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copper) was formulated by direct compression method. This uniquely formed bilayer tablet was

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assessed for physiochemical properties, microbial load and stability studies. FTIR studies revealed

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that there was no interaction between the vitamins, minerals and excipients used in the study. In
vitro dissolution test revealed that more than 85 % of folic acid and zinc sulphate were released in
60 mins at pH 6.5 -7.5. The release of folic acid was found to follow a first-order and Higuchi

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model, whereas zinc sulphate followed a first-order and Korsemeyer-Peppas model, indicating
diffusion and erosion mechanism. The results of the long term and accelerated stability studies
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showed that, there were no changes in physicochemical properties and release pattern of bilayer
tablets. Furthermore, the optimized formulation showed increase in the stability period for about
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24 months. This nutritional supplement based on oral delivery of bilayer tablets is expected to gain
more acceptance among the commercially available products due to better release properties and
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combination therapy.
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Keywords:
Vitamins, Minerals, Bilayer Tablet, Film-Coated Tablet, Dietary Supplement, In vitro release.
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1. Introduction
Macronutrients are carbohydrates, protein, fats and are required in large amounts for normal
functioning of body systems. Like macronutrients, vitamins and minerals are essential to life but
they are reguired in small amount and are referred to as micronutrients. Though they are required
in meager amounts when compared to the macronutrients however, they are very essential for day
to day functioning of human system [1,2]. The vitamin and mineral deficiencies are rather difficult

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to diagnose and are largely ignored. Marginal micronutrient deficiency is often associated with the

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destruction of various biochemical reactions that occur in our body and some evidence of illness
can also be perceived. Depression, unhealthy food habits and exposure to harmful pollutants can

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lead to loss of essential nutrient which sequentially leads to other chronic illnesses [3,4].

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Nutrients are components of food that every organism requires to grow, reproduce and maintain
their health. These nutrients have specific functions in the body and are required in varying
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amounts. Though these nutrients are easily available from common foods however, no food has
complete composition of these nutrients [5,6]. The amount of nutrient needed by the body is
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influenced by age, sex, size, activity and state of health [7,8]. According to the Dietary Supplement
Health and Education Act (DSHEA) of 1994, an ideal dietary supplement formulation should
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contain the dietary ingredient(s) such as a vitamin, a mineral, an herb or other botanicals (extract),
an amino acid, a concentrate, metabolite, constituent or combination of the above in any of the
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formulation such as pill, capsule, tablet, powder or liquid form. These supplements are generally
designed to; (i) help fill dietary gaps, ii) provide an antioxidant boost to the body, iii) boosts
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immune function, iv) protect against metabolic stress, v) promote growth in children [9,10] vi)
smooth functioning of all vital body functions, vii) prevents infections and viii) expedite recovery
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after illness [11]. Thus, the nutritional supplements combines advanced food science and essential
nutrients to provide holistic nutrition that helps to maintain a healthy life style [12].

Different regulatory authorities like United States Food and Drug Administration (USFDA), Indian
Council of Medical Research (ICMR) and European Union (EU), has formulated recommended
consumption levels of vitamins and minerals called recommended daily allowances (RDAs) such
as USRDA, ICMRRDA and EURDA. The primary goal of RDAs is to prevent diseases caused by
nutrient deficiencies. However, these allowances have been tailored for maintaining borderline
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health rather than complete health. Hence, in the early 1990s, the Food and Nutrition Board (FNB),
the Institute of Medicine, the National Academy of Sciences (USA), with the involvement of
Health Canada, revised the RDAs, and an updated nutrient reference values were introduced as the
Dietary Reference Intakes (DRIs) [13]. The hectic lifestyles, reduced physical activity, sedentary
jobs and an increase in unhealthy food habits have all played a significant role in the widespread
nutrient deficiency of the people around the world. As a result, significant portion of the
population fails to consume the recommended intake levels of nutrients. The primary goal of the

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supplements is not only to prevent nutrient deficiencies, but also to reduce the risk of chronic

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diseases [14] such as osteoporosis [15], cancer and cardiovascular disease [16–18].

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Vitamin and mineral formulations are usually available in the form of sugar-coated and film-
coated tablets. Most conventional dietary supplement formulations have disadvantages such as

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interactions among the dietary components, high processing time, decreased stability of the
components and, sugar-coating process which decreases the disintegration and dissolution of the
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dosage form [19,20][21]. In addition, delayed break up of the sugar coating may leads to impaired
dissolution characteristics, significant variation in the disintegration time and sensitivity to the
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elevated temperature/humidity storage conditions based on the active ingredient and excipients
[22]. To address these issues, we have developed the film-coated bilayer tablets which contain
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vitamins in one layer and minerals in the second layer. Furthermore, the formulation is optimized
based on its physicochemical properties and in vitro release behavior. Nutritional supplement
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strategies based on oral delivery of bilayer tablets is expected to gain more acceptance among the
commercially available products due to better release properties and combination therapy.
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Furthermore, the vitamins and minerals will provide a daily dose of dietary supplements and in
addition to their specific benefits, the synergistic benefits of all the ingredients can also be
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harnessed. As with the therapeutic formulations, the bilayer tablets containing dietary supplements
in two separate layers, release the nutrients concurrently in a desired release profile to improve the
patient health system. To the best of our knowledge, no similar work has been reported so far
involving vitamins and minerals in the form of bilayer tablets.

2. Materials and Methods

2.1 Materials:
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The active ingredients such as vitamin premix for tablet (VPRTB- A, D, E, C, B1, B2, B3, B5, B6,
B7, B9 and B12) and mineral premix for tablet (MPTB- magnesium, manganese, molybdenum,
iodine, zinc, silicon and copper) were kindly supplied by Dietary Supplement Manufacturers
(DSM) Nutritional products, Mumbai. The pharmaceutical excipients selected for the formulation
are pregelatinised starch (PG starch), croscarmellose sodium (CCS), sodium starch glycolate
(SSG) (Type-A) (FMC Biopolymer), micro crystalline cellulose (MCC) (Pharmatrans), aerosil
(Evonik Degussa, Germany), magnesium stearate (Peter Grevens, Germany). The coating

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materials such as instamoistshield and instaglow (Ideal Cures) were received as a gift. Aluminium

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Aluminium (Alu-Alu) Sealable and Forming foil were procured from Unity Flexi Pack Pvt. Ltd.

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All the chemicals used are of analytical grade.

2.2 Formulation of bilayer tablets containing dietary supplements:

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2.2.1 Premix Details:
To increase the stability, two different types of premixes in a coated form were obtained from
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DSM Nutritional Products. The vitamins and minerals present in each premix are VPRTB - A, B1,
B2, B3, B5, B6, B9, B12, C, D, E & MPTB - Zn, Cu, Mg, Mn, Mo, I, Si. The overages in the
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formulation were added as per the limits prescribed in the European Commission [23].
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2.2.2 Preparation of granules for vitamins layer and minerals layer

The label claim of vitamins and minerals present in the bilayer tablet formulation is mentioned in
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Table 1. The direct compression method is employed for the formulation of bilayer tablets because
the active substances are incorporated in the form of premix. The required quantity of VPRTB
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premix was passed through 16 # mesh screen and the excipients such as PG starch, SSG, MCC,
CCS and Aerosil [24,25] [26] were passed through 40 # screen and mixed in a conta blender for
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about 20 mins. In addition, the lubricant magnesium stearate [27,28] was passed through 40 #
screen and added to the above mixture. These was then blended for about 10 mins and kept in a
separate stainless steel vessel (ss. vessel) as the first layer. For the second layer, the whole process
was repeated except that MPTB premix was passed through 16 # screen instead of VPRTB premix.

2.2.3 Evaluation of granules

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The flow property of blended granules was assesed by angle of repose. The granule properties
were determined by bulk density, tap density, Hausner's ratio and compressibility index (Carr’s
index) as per USP30-NF25, 2007, <1174> (Electrolab Tap Denity Tester) requirements [29,30].

2.2.4 Preparation and evaluation of bilayer tablets

The compression of blended granules was carried out in cadmach double rotary machine with ‘D’
tooling using 16/32” flat punches and dies. Firstly, vitamin layer granules were introduced into the

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die cavity and pre-compression was made for the formation of the first layer. Subsequently the

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mineral granules was added to the other feed for a final compression and for the formation of

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bilayer tablets [31,32]. Compressed tablets were evaluated according to the USP methods. Weight
variation (USP30-NF25, 2007, <905>) (Waters-HPLC), friability (USP30-NF25, 2007, <1216>)
(Electrolab Friabilator), hardness (USP30-NF25, 2007, <1217>) (Three Stage Hardness Tester),

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disintegration (USP30-NF25, 2007, <701>) (Electrolab Disintegration Tester) and dissolution
tests (USP30-NF25, 2007, <711>) (Electrolab Dissolution Tester) were carried out after
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compression [33,34]. Tablet hardness and width were calculated using three stage tester
(Electrolab, India) and thickness was tested by vernier caliper (CD-6, CSX, Mitutoyo, Japan).
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Friability of the tablets was assessed by Friability tester (Electrolab, India) and the disintegration
test for compressed tablets was performed using a disintegration apparatus (Electrolab, India) in
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distilled water at 37± 2ºC.

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2.3 Fourier transform Infrared spectroscopy (FT-IR)

FTIR measurements were carried out to understand the interactions and the reaction progress.
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Perkin Elmer FTIR spectrometer was used in attenuated total reflectance (ATR) mode between a
frequency range of 4000-500 cm-1. All the spectra were baseline corrected and normalized using
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Perkin Elmer Spectrum software.

2.4 In vitro dissolution and kinetic data analysis

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The release studies were performed as per USP method (USP30-NF25, 2007, <711>) using
Electrolab USP Dissolution apparatus II fitted with a paddle and the dissolution was tested for the
index vitamin and index mineral.
The kinetic studies were carried out for the bilayer tablets of vitamins and minerals and it was
evaluated by linear regression analysis. The in vitro release profile of vitamins and minerals were
fitted into the kinetic models for estimating the release mechanism
 Zero-Order Kinetics: % release versus time

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 First-Order Kinetics: log % remaining versus time

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 Higuchi Model: % release versus square root of time

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 Korsemeyer-Peppas Model: log % release versus log Time

2.5 Coating of compressed tablets

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The coating parameters were optimized following trial and error method. The optimal procedure
involved mixing of instamoistshield and water followed by stirring for 45 mins with a propeller
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for vortex formation. The solution was then passed through a #80 mesh screen. In a separate
container, the required quantity of instaglow material for polishing was taken in a vessel and
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stirred with propeller stirrer to form vortex for 15-20 mins. The inlet temperature for drying the
coated tablets was set at 50º C, the pan rotations per minute (RPM) was set at 17-19 rpm, spray
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rate at 0.5 g/min and the air pressure at 0.8 mm Hg. The core tablets were coated by spray coating
technique with a multifunction experimental machine (Ideal Cures, India). Aqueous coating
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material was used to avoid degradation of moisture sensitive nutrients present in the formulation.
In order to protect the tablet from moisture, the coating solution was sprayed until the tablets
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attained a 1.5 % weight gain. Moreover, the tablets were also polished by using Instaglow
polishing material for the glossy appearance.
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2.6 Evaluation of coated bilayer tablets

The coated-polished tablets were studied for their weight variation, thickness, disintegration, assay
(USP30-NF25, 2007, <monographs>) (HPLC), dissolution and microbiological limit tests (USP30-
NF25 <2021>). The microbiological limit tests was designed to, estimate the number of viable
aerobic micro-organisms present in the tablet and for detecting the presence of microbial species in
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the tablet. This was performed for each batch according to standard methods as per US
Pharmacopoeia.

2.7 Packing of coated bilayer tablets

After evaluation of coated tablets, the final product was packed in Alu-Alu forming foil and Alu-
Alu sealing foil by ACG-Pam Pac machinery. Alu forming foil consists of Alu film 45 microns
laminated with polyamide film 25 microns on the dull side and PVC film 60 microns on the bright

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side with total gsm of 220 to 275 and total thickness is 0.10 to 0.15 mm. Alu sealable foil consists

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of Alu film with a thickness of 0.027 to 0.033 mm and total gsm is 68 to 78.6.

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2.8 Scanning electron microscopy
The morphology of the samples were analysed by scanning electron microscope (VEGA 3

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TESCAN, Tokyo, Japan). The samples were used without any treatment and fixed on a brass stub
using double-sided adhesive tape. Coating of about 10.0 Å with platinum was carried out in an
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Argon atmosphere using Hitachi ion sputter (E- 1010) for 5 mins (USP30-NF25, 2007, <1181>)
(Scanning Electron Microscopy). SEM image was obtained at an acceleration voltage of 15 to 20
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kV. The morphology of the samples was analysed after dissolution to understand its release
mechanism. The samples were taken after the dissolution of vitamins layer and after the
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dissolution of minerals layer.

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2.9 Stability testing

The packed tablets were placed in the stability study chamber at 25 °C ± 2 °C /60 % ± 5 % RH and
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at 40 °C ± 2°C / 75 % ± 5 % RH to determine the shelf life of the product [35]. During the stability
study, the tablets were evaluated at different time intervals for physico-chemical parameters such
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as description, weight variation, disintegration, assay, dissolution and microbiological limit test.

3 Results and Discussions

3.1 Fourier transform Infrared spectroscopy (FT-IR):
Figure 2 shows FTIR spectra of VPRTB, MPTB, VLG and MLG. VPRTB show significant peaks
at 3253 cm-1, 2922 cm-1, 1749 cm-1 and 1005 cm-1 which are ascribed to hydroxyl, methylene, keto
and ester groups of vitamins respectively. In addition, the VPRTB exhibited a peak at 1339 cm-1
which correpond to sp3 C-H stretching of saturated carbons. After addition of excipients to VPR
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TB, there is no change in the intensity of hydroxyl peak and also the peaks related to keto and ester
groups. However, the VLG shows significant peaks at 3262 cm-1, 2915 cm-1, 1766 cm-1, 1366 cm-1
and 1005 cm-1 with same intensity indicating the existence of VPRTB. This shows that, there is no
interaction of vitamin molecules after the addition of excipients. MPTB shows peaks at 3165 cm-1
indicating the existence of strong bands associated with OH stretching vibrations of water, 1740
cm-1 correponding to multiple bond stretching and metal-hydrogen stretching vibrations, 1355 cm-1
correponding to triatomic linear molecule (OCO), 1085 cm-1 due to the presence of sulphate ions

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and peak at 858 cm-1 cooreponding to metal-oxygen (M=O) stretching. After addition of excipients

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to MPTB, there is slight increase in the intensity of peaks (MLG). The peak at 1751 cm-1 and 1098
cm-1 with high intensity indicate the existence of metal-hydrogen stretching and presence of

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sulphate ions. FT-IR clearly substantiates that, there is no interaction between vitamin and mineral
molecules after the addition of excipients.

3.2 Formulation and optimization of bilayer tablets US

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Bilayer tablets of vitamins and minerals were prepared by direct compression process [36].
Amongst the techniques used to prepare tablets, direct compression method is the most advanced
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technology because it involves only blending and compression. In addition, it requires less
machinery, fewer unit operations, reduced number of personnel, considerably less processing time,
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less process validation and increased product stability [37]. The composition of vitamin layer and
mineral layer used in this study is listed in Table 2 (a) & Table 2 (b). To optimize the physico-
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chemical properties of the tablet, trials were formulated with different disintegrants [38] and the
blend properties of both the granules was found to be within the prescribed limits according to
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USP. The compressibility index and Hausner’s ratio results are in the range 25.466 to 29.647% and
1.342 to 1.422 (Table 3) respectively. The angle of repose measurements were < 30° indicating
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‘excellent’ flow. All these results confirmed that the blend shows good flow property. In addition,
all the physicochemical properties of the granules were found to be within the limits prescribed by
USP requirements.

The formulations with the same compositions as the excipients in both the vitamins and minerals
layers exhibits very high disintegration for about 1 h. Acoording to previous reports [39,40], the
incorporation of more than one disintegrants (CCS and SSG) in the formulation would help to
decrease the disintegration time (DT). To decrease the disintegration of the tablets, a
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superdisintegrant (ECG505) was further added to the formulations. Surprisingly, its incorporation,
irrespective of the amount, significantly decreased the disintegration time from 1 h to 15 mins 40
secs though it disintegrated at low/medium hardness (MVM-002). In another trial (MVM-003),
SSG was added along with CCS and PG starch, As expected, the DT was observed to be
satisfactory but at higher hardness it crosses above 15 mins. The similar trends in DTs observed
after the addition of ECG505 and SSG may be attributed to the same functional moiety
(carboxymethyl group) present in both superdisintegrants. The presence of carboxymethyl group

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slowed down the penetration of the medium which in turn reduces the porosity created by the

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superdisintegrants [41]. To further improve the release rate, MCC was added to the formulation (

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MVM-004). This balanced the disintegrating factors and DT pattern was found to be excellent
compared to all the trials. Because of the inherent property of MCC, the disintegration effect of
MCC was studied along with SSG, instead of adding CCS and PG starch in the formulation

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(MVM-005). This formulation also showed a very high disintegration pattern. Therefore, the
MVM-004 was reproduced in large scale and labelled as MVM-006 formulation, (Table 2
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(a)&(b)). This reproducible batch shows excellent DT pattern.
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The weight variation test plays a key role in the case of layered tablets. A study by Abebe et al.,
reported that, the formulators have no option other than placing the larger weight material in the
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first layer with all its associated challenges [42]. In another report, it was stated that the upper
punch penetration force during the first layer compression and during the second layer
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compression force would impact the potency of the constituents in the second layer [43,44]. In our
work, the weight ratio of the two layers was 1:3 and this gives no significant impact on the
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breaking force of the bilayer tablets. In addition, the thickness of all the tablets ranged between
3.98 and 5.61 mm which is within the acceptable limit of ± 5 % variation. The friability values of
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all the batches exhibited impressive results which is in the range of 0.013 to 0.05 %. This weight
loss of less than 1 %, indicates that, all the tablets in our formulations are mechanically stable and
will be resistant to chipping and cracking during handling and/or subsequent processing. The tablet
hardness has a profound effect on the disintegrating properties and therefore its influence on DT
was assessed. The DT of the optimised formulation was found to be 3:03, 6:04 and 7:42 for the
tablets with a hardness of 6-7, 7.5-8.5 and 9-11 KP respectively. This clearly indicates that, the
DT increases with an increase in hardness and showed that, the hardness or breaking strength of
the tablets directly relates to the disintegration time. The increased hardness results in high density
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of the tablet constituents thereby reducing the porosity of the tablets and slows down water intake.
Therefore, 9-11 KP was selected for further tableting process to obtain optimal performance.

3.3 Scanning Electron Microscopy

The SEM images reveal that, the premix vitamins Fig 3 (a) are spherical shaped particles whereas
the premix minerals Fig 3 (b) are flower shaped particles. After the addition of excipients (Fig. 3 c
and d), there is no change in the morphology confiming that the bilayer tablet posses good flow

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property. Furthermore, Fig 3 (c) and Fig 3 (d) images reveal that, the excipients are adsorbed on

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the surface of the premix vitamins and minerals.

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The SEM images of the bilayer tablets during dissolution exhibited swelling of the vitamin layer

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and mineral layer, and also reveals slight porous matrix structure (Fig 4 (a)). The diffusion of the
vitamins and minerals out of the swollen tablets can be clearly observed in Fig 4 (b) and Fig 4 (c).
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This indicates the formation of a network between the active ingredients and the excipients in the
matrix. From the swollen matrix, the vitamins and minerals diffused to the surrounding medium
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indicating the involvement of diffusion and erosion mechanisms [45].

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3.4 Film Coating Process:

The aqueous based coating system was employed as it is an ecofriendly, safe and cost-effective
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method. The optimized batch MVM-006 trial was selected for coating process because it shows
excellent release profile. The uncoated tablets were film coated by instamoistshield. The film
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coating was performed particularly to coat the hygroscopic and incompatible substances (MVM-
007). In addition, they also acts as moisture barrier [46] and the film coated tablets were then
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polished using instaglow for a glossy finish. This aqueous based coating system was efficient and
water was removed during and after the coating process by controlling the inlet temperature [47].
The coated-polished tablets showed desired release profile and stability at large scale (MVM-008)
Table 3.

4.4 Evaluation of coated tablets:

4.4.1 Disintegration time of coated bilayer Tablets
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Disintegration test with and without disk was performed for all the trials using water as the
immersion medium. The results of distintegration test (MVM-007) show that at higher hardness,
the disintegration effect of the tablet is found to be 08 mins 20 secs (Fig 5). The average
disintegration time of all the tablets at optimized formulations (MVM-008) ranges between 07
mins 59 secs to 08 mins 26 secs. Compare with previous reports, where nearly 50 % of the
commercially available dietary supplement formulations failed to pass the disintegration test and
did not meet the acceptance criteria of the pharmacopoeia [48,49]. This results show that the as-

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produced bilayer tablet meet the acceptance criteria of the pharmacopoeia.

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4.4.2 In vitro dissolution studies
The dissolution test was performed as per USP dissolution guidelines in distilled water at pH of 5-
6. The tablets were also tested in simulated intestinal fluid (pH 6.5-7.5) and simulated gastric fluid

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(pH 1-2). Furthermore, the dissolution testing of the pertinent index vitamin folic acid and mineral
zinc sulphate were also determined. The release of folic acid and zinc sulphate from the bilayer
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tablets was analyzed by plotting the cumulative percentage of drug release Vs time. Compared to
the previous studies [50–53], our results showed improvements in the dissolution of folic acid. The
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formulation containing SSG, PG Starch, MCC was selected as the optimized batch since it showed
the best release profile up to 60 mins as compared to the other formulations. The filler and
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disintegrant amount incorporated in the formulation were found to be the important factors that
affect the tablets dissolution [54]. The most striking finding was that, at all the pH about 75 % or
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more of folic acid and zinc sulphate were released in 60 mins (Fig. 6) with more than 85 % at pH
6.5 -7.5. This high release might be attributed to the fact that, the solubility of the folic acid
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release in the SGF, SIF and distilled water is pH dependent. Usually, the pH of the bulk liquid
reflects the pH of the stagnant diffusion layer upon the tablet surface which also alter the pH of the
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stationary diffusion layer. Thus, increasing the surface pH will increase folic acid solubility and
favour rapid dissolution of folic acid from the bilayer tablet. The tested film coated bilayer tablets
produced almost similar dissolution profiles in all the mediums studied and meet the USP
standards for folic acid and zinc sulphate dissolution in the multivitamin dosage form. The
dissolution range was 50-110 % release (Fig.6). Our data showed huge improvement in the
disintegration and dissolution of bilayer tablets compared to the previous studies.

4.4.3 Kinetic analysis of dissolution data

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Kinetic models play a vital role to determine the release mechanism of the active components. The
in vitro release data obtained (MVM-008) were fitted into zero order, first order, Higuchi’s model
and Korsemeyer-Peppas model and the values of the slope, intercept and R2 were calculated. Both
the first-order and Higuchi square root of time show acceptable fit to the in vitro release data of
folic acid (Fig. 7) with R2 values of 0.9411 and 0.9901 respectively. While first-order and
Korsemeyer-Peppas model shows acceptable fit to the in vitro release data of zinc sulphate (Fig.
8) with R2 values of 0.9533 and 0.9783 respectively. This shows that, the release rate is dependent

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on the concentration of vitamins and minerals as the appropriate plots (first order and higuchi)

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showed high linearity. The release mechanisms of these bilayer systems could be due to the

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diffusion of vitamins through the porous matrix created by the superdisintegrants. After diffusion
of the medium, the layer becomes fully hydrated which eventually led to erosion and resulted in
the release of minerals (disintegrating the active moieties). The obtained results from the kinetic

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data is well corroborated with our SEM analysis.
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4.4.4 Microbial Analysis Report
Microbial analysis was carried out and the results indicated that, the total aerobic microbial count,
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total combined yeast and mould count were found to be within the limits. The individual pathogens
like E. Coli, Salmonella sp, Staphylococcus aureus, and Pseudomonas aeruginosa results were
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found to be within the limits prescribed by USP. (Table 4).

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4.4.5 Stability Studies

This study was carried out to quantify vitamins and minerals in MVM formulation in order to examine
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the degradation. The results obtained from the accelerated and long term degradation tests of the
studied vitamins and minerals demonstrated that there are no changes in the physico-chemical
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parameters, content, microbiological limit and its in vitro release behavior when compared with the
initial results (Table 4). No organoleptic changes (colour, odour, flavor, texture) or other quality
changes were observed during the proposed shelf life of the product. The results confirmed that,
the percentage degradation of actual ingredients meet the acceptable limit of not less than (NLT)
90 % when compared with the initial period. Comparison of the release profiles of initial tablet
and the accelerated and long-term stability study are described in Fig 9 & 10. The moisture barrier
film coated tablets were found to be the most stable among the tested tablets. From the stability
studies it is determined that the expiration period for the prepared bilayer tablets is 24 months.
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Conclusion
The tablet comprising of vitamin A, C, E and all the B vitamins in combination with the minerals
such as Zn, Mg, Mn, Cu, I, Mo was manufactured by direct compression method. An optimized

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formulation using a direct compression method was found to be most suitable for the

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pharmaceutical/dietary supplement industry as it is time saving and cost effective with increased
production. Our results indicate that, the formulation containing MCC-100, ECG 505, SSG, CCS

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and PG Starch composites have good fluidity, compressibility, disintegration and in vitro release
profile. The amount of folic acid and zinc sulphate released from the tablets were observed to be

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more than 85 % in 60 mins. From the kinetics study, the release pattern of the bilayer tablets
showed the diffusion/erosion mechanism which corroborates with our SEM results. The stability
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studies showed no significant changes in physical characteristics, assay content, disintegration and
dissolution of coated tablets. The quality of the formulated product can be maintained over a
M

storage period of 24 months. Moreover, preclinical and clinical studies are required for the
successful supplementation of the prepared bilayer tablets.
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Acknowledgement
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The authors would like to thank Department of Science and Technology (DST-Inspire Fellowship)
for their financial support, Apex laboratories private limited for providing their facility and DSM,
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Colorcon, Ideal Cures for providing the samples at free of cost.

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Table 1: Label claim of vitamins and minerals in Bilayer Tablet

Label claim
S.No. Ingredients
in mg
First Layer
1 Ascorbic Acid (Vitamin C) 75.00
2 Nicotinamide (Vitamin B3) 50.00
3 Tocopherol (Vitamin E) 15.00
4 Thiamine Mononitrate (Vitamin B1) 10.00
5 Riboflavin (Vitamin B2) 10.00

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6 Calcium D Pantothenate (Vitamin B5) 10.00

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7 Pyridoxine HCl (Vitamin B6) 2.00
8 Folic Acid (Vitamin B9) 1.00

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9 Vitamin A (Acetate) (IU) 5000.00
10 Vitamin D3 (IU) (Cholecalciferol) 400.00
11 d-Biotin (mcg) 150.00

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12 Cyanocobalamin (Vitamin B12) (mcg) 7.50
Second layer
13 Zinc (as Zinc Sulphate Monohydrate) 22.00
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14 Magnesium (Magnesium Oxide Heavy) 18.00
15 Silica (Silicon Dioxide) 1.00
16 Manganese (Manganese Sulphate Monohydrate) 0.90
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17 Copper (Copper Sulphate Anhydrous) 0.50

18 Iodine (Potassium Iodide Pure) (mcg) 150.00
19 Molybdenum (Sodium Molybdate Dihydrate) (mcg) 25.00
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Table 2 (a): Composition of vitamins and minerals layer formulated by direct compression
method

Batch No MVM-001 MVM-002 MVM-003 MVM-004

Ingredients (First Layer)
VPRTB 537.0 537.0 537.0 537.0

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1
SSG (Type-A) ― ― 13.0 13.0

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CCS2 ― 13.6 14.0 15.0
3
PG Starch 13.0 16.0 14.8 14.8
Avicel PH 200 LM4 ― ―

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12.0 12.0
5
MCC ― ― ― 17.0
6
ECG 505 18.0 12.0 ― ―
Aerosil 1.0 1.2 1.2 1.3

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Lubrication
Magnesium Stearate 5.0 2.2 2.0 2.0
First Layer (mg) 586.0 582.0 594.0 600.0
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Ingredients (Second Layer)
MPTB 133.00 133.00 133.00 133.00
1
SSG (Type-A) ― ― 14.0 14.0
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CCS2 ― 15.0 15.0 15.0

3
PG Starch 14.00 17.0 17.0 17.0
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Avicel PH 200 LM4 12.00 ― 13.00 ―

5
MCC ― ― ― 18.00
6
ECG 505 17.00 17.00 ― ―
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Aerosil 1.00 1.00 1.00 1.00

Lubrication
Magnesium Stearate 3.00 2.0 2.0 2.0
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Second Layer (mg) 180.00 185.00 195.00 200.00

1
Sodium Starch Glycolate (SSG), 2Cros Carmellose Sodium (CCS), 3Pregelatinised Starch (PG
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Starch), 4Lactose Monohydrate PH 200 (Avicel), 5Microcrystalline Cellulose (MCC), 6Carmellose

Calcium (ECG 505)
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Table 2 (b): Composition of vitamins and minerals layer formulated by direct compression
method
Batch No MVM-005 MVM-006 MVM-007 MVM-008
Ingredients (First Layer)

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VPRTB 537.0 537.0 537.0 537.0
SSG1 (Type-A)

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12.0 13.0 13.0 13.0
2
CCS ― 15.0 15.0 15.0
PG Starch3 ―

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14.8 14.8 14.8
4
Avicel PH 200 LM ― ― ― ―
5
MCC 18.7 17.0 17.0 17.0
ECG 5056 ― ― ― ―

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Aerosil 1.3 1.3 1.3 1.3
Lubrication
Magnesium Stearate 2.0 2.0 2.0 2.0
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First Layer (mg) 571.0 600.0 600.0 600.0
Ingredients (Second Layer)
MPTB 133.00 133.00 133.00 133.00
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SSG1 (Type-A) 14.00 14.0 14.0 14.0

2
CCS ― 15.0 15.0 15.0
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PG Starch3 ― 17.0 17.0 17.0

4
Avicel PH 200 LM ― ― ― ―
MCC5 18.00 18.0 18.0 18.0
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ECG 5056 ― ― ― ―
Aerosil 1.00 1.0 1.0 1.0
Lubrication
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Magnesium Stearate 2.0 2.0 2.0 2.0

Second Layer (mg) 168.00 200.00 200.00 200.00
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1
Sodium Starch Glycolate (SSG), 2Cros Carmellose Sodium (CCS), 3Pregelatinised Starch (PG
Starch), 4Lactose Monohydrate PH 200 (Avicel), 5Microcrystalline Cellulose (MCC), 6Carmellose
Calcium (ECG 505)
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Table 3: Blend Parameters

Bulk Tapped
Compressibility Hausner`s Angle of
Batch No Layer density density
index (%) Ratio Repose
(g/ml) (g/ml)
I 0.600 0.805 25.4658 1.342 28.7º
MVM-001
II 0.580 0.798 27.3183 1.376 25.7º
I 0.582 0.800 27.2500 1.375 27.6º
MVM-002
II 0.592 0.821 27.8928 1.387 24.5º
I 0.612 0.870 25.8000 1.422 29.3º
MVM-003

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II 0.585 0.804 27.2388 1.374 26.4º
I 0.582 0.807 27.8810 1.387 30.3º

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MVM-004
II 0.617 0.877 29.6465 1.421 27.6º
I 0.624 0.843 25.5042 1.351 27.2º

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MVM-005
II 0.593 0.831 28.6402 1.401 25.3 º
I 0.590 0.818 27.2616 1.386 30.4º
MVM-006
II 0.590 0.832 29.0865 1.410 25.3º

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I 0.588 0.802 26.6833 1.364 28.4º
MVM-007
II 0.578 0.817 29.2534 1.413 25.8º
I 0.634 0.854 25.7611 1.347 29.5º
MVM-008
II 0.596 0.828 28.0193 1.389 24.5º
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Table 4: Evaluation of Tablets (Accelerated)

Batch No.: MVM-010

Test Condition: 40°C±2°C/75%±5%RH
Pack: 15’s Alu - Alu Blister
S.NO Ingredients Initial 1st month 2nd month 3rd month 6th month
Orange/White Orange/White Orange/White Orange/White Orange/White
colored circular colored circular colored circular colored circular colored circular
1 Description film-coated film-coated film-coated film-coated film-coated

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bilayer tablets bilayer tablets bilayer tablets bilayer tablets bilayer tablets
2 Average Weight 824.40 825.00 824.00 824.40 825.20

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VPRTB,
3 Niacinamide 52.51 52.00 52.03 50.09 50.09

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4 Pyridoxine 2.05 2.00 2.00 2.08 2.08
5 Riboflavin 10.62 10.00 10.00 10.05 10.05
6 Thiamine 10.35 10.32 10.32 10.38 10.38

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7 Ascorbic Acid 74.97 75.17 75.11 75.14 75.14
8 Vitamin B12 0.0076 0.0077 0.0078 0.0076 0.0076
9 Vitamin E 14.95 14.83 14.86 15.05 15.05
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10 Folic Acid 1.48 1.43 1.42 1.23 1.23
11 Vitamin A (IU) 4989.72 4998.00 4994.00 5032.00 5032.00
MPTB
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12 Zinc Sulphate 22.00 22.14 22.13 22.03 22.03

13 Copper Sulphate 0.51 0.51 0.50 0.52 0.52
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14 Manganese Sulphate 0.92 0.90 0.91 0.92 0.92

15 Magnesium Oxide 18.02 18.00 18.05 18.04 18.04
Microbial Limit Test (Accelerated)
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1st 2nd 3rd 6th

Tests Specification Initial
Month Month Month Month
Microbial contamination
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Not more than

1 a) Total aerobic microbial 10cfu/g 10cfu/g 10cfu/g 10cfu/g 10cfu/g
100 cfu/g
count
b) Total combined yeast and Not more than
2 <10cfu/g <10cfu/g <10cfu/g <10cfu/g <10cfu/g
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mould count 102 cfu/g

c) Pathogens
3 Should be absent Absent Absent Absent Absent Absent
i) Escherichia coli
Should be
4 ii) Salmonella species Absent Absent Absent Absent Absent
absent/10 g
5 iii) Staphylococcus aureus Should be absent Absent Absent Absent Absent Absent
6 iv) Pseudomonas aeruginosa Should be absent Absent Absent Absent Absent Absent
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Fig 1: Graphical representation of Bilayer Tablets (Vitamins and Minerals)

A. Compression (a) 1st layer fill (b) 1st layer tamping (c) Upper punch withdrawal (d) 2nd layer
fill (e) Main compression (f) Ejection (1) Hopper I Vitamins Blend (2) Hopper II Minerals
Blend
B. Schematic process of formulation (a) vitamins granules (b) minerals granules (c)
blending of vitamins and minerals granules separately (d) compression of bilayer tablets (e)
film coating (f) film coated bilayer tablets (g) thickness (h) disintegration (Swelling and

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disintegrated) (i) dissolution (j) vitamins release (k) release pattern (l) pH dependent

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release (GIT).

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Fig 2: FT-IR spectra of VPRTB, MPTB, VLG and MLG
Fig 3: SEM images of (a) Premix Vitamin-VPRTB (b) Premix Mineral-MPR TB
(c) Vitamin Layer Granules (d) Mineral Layer Granules

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Fig 4: SEM images of (a) Bilayer Tablet after dissolution (b) Diffusion of vitamins from the layer
(c) Swelling of mineral substance from the layer.
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Fig 5: (a) Thickness (b) Disintegration Time of Bilayer Tablets
Fig 6: a) Release pattern of all the trials (Folic Acid) b) Release pattern of all the trials (Zinc
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Sulphate) c) Release pattern of MVM-008 (Folic Acid) at different pH d) Release pattern of

MVM-008 (Zinc Sulphate) at different pH
Fig 7: Release Kinetics of Folic Acid (a) Zero-order (b) First-order
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(c) Higuchi (d) Korsemeyer-Peppas

Fig 8: Release Kinetics of Zinc Sulphate (a) Zero-order (b) First-order
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(c) Higuchi (d) Korsemeyer-Peppas

Fig 9: In vitro release of folic acid and zinc sulphate-Accelerated Study
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Fig 10: In vitro release of folic acid and zinc sulphate – Long Term Study
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Graphical abstract

Highlights:
 Dietary supplement was prepared in the form of a bilayer tablet.
 In vitro test shows that more than 85 % of folic acid and zinc sulphate were release.
 The release mechanism of the active components was determined by kinetic analysis.
 The optimized formulation shows increase in stability period for about 24 months.

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Graphics Abstract
Figure 1
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Figure 10