62

Journal of Applied Pharmaceutical Research

Volume 8, Issue 4, Year of Publication 2020, Page 62 – 69
DOI: 10.18231/j.joapr.2020.v.8.i.4.62.69

Research Article
JOURNAL OF APPLIED PHARMACEUTICAL RESEARCH | JOAPR
www.japtronline.com ISSN: 2348 – 0335

FORMULATION BY DESIGN APPROACH FOR EFFERVESCENT

GRANULES OF VITAMIN C USING STATISTICAL OPTIMIZATION
METHODOLOGIES
Abdu Faisal 1, 2*, Maher Al-Absi1, 2, Safwan Alagbarri1, 2, Mofeed Al- Nowihi 3

Article Information ABSTRACT

Received: 4th January 2020 Introduction: Ascorbic acid (vitamin C), is a potent antioxidant, and the human body cannot synthesis
Revised: 26th August 2020 it, so ascorbic acid can be obtained from external sources as food and pharmaceutical products.
Accepted: 19th September 2020 Objective: This study was using formulation by design (FbD) concept to prepare effervescent granules
of vitamin C with high quality and more patient compliance in less effort and cost. Methods: A 23
factorial design was used to study the physical and chemical characteristics of the granules
Keywords
Formulation, Effervescent (disintegration time (DT), taste of product (TP), carbon dioxide amount (CO2), and pH of the solution).

granules, FbD, Factorial design, Also the effects of and interaction between selected independent variables (citric acid, tartaric acid, and

Vitamin C. sodium bicarbonate) evaluated, while, their concentrations were optimized by application of
optimization model methodology for factorial design. Formulations were prepared by non-aqueous wet
granulation method. Results: From the results of designed batches, the disintegration time ranged from
33±2.7 to 153±3.3sec while the amount of carbon dioxide ranged from 0.360±0.004gm to
1,512±0.002gm also the pH range was from 2.9±0.09 to 6.42±0.07 but the taste of product was coded
numerically in proportional with acceptability from 1 to 5. According to the experimental design, both
of studied independent variables have significant effect in determining the physicochemical
characteristics of effervescent granules. Conclusion: FbD concept effectively was utilized for the
development of vitamin C effervescent granules as per modern quality practices used in pharmaceutical
industries.

_______________________________________________________________________________________________
1
Faculty of Pharmacy, Sana’a University, Yemen
2
Research & Development Center of Modern and Global Pharma Companies
3
Biology Department, Faculty of Science, Sana’a University, Yemen.

*For Correspondence: [email protected]

©2020 The authors
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 Journal of Applied Pharmaceutical Research 8 (4); 2020: 62 – 69 Faisal et. al

INTRODUCTION gain a high-quality product in shorter time with using minimum

Ascorbic acid (vitamin C) is a water soluble vitamin and trails [5,6]. The above shows the importance of vitamin C for
possesses a potent antioxidant activity. Vitamin C presents health and due to the most of naturally occurring species that
naturally in Citrus and most of fruits and vegetables. It has many consist vitamin C are seasonal and difficult to stay for long time.
functions like protects against respiratory tract infections and Therefore, vitamin C has been formulated as pharmaceutical
reduces risk for cardiovascular diseases and even Cancer. dosage form such as effervescent granules and tablets that
Moreover, the processes of collagen synthesis and iron already has been available in the market many years ago.
absorption are enhanced in presence of vitamin C. on the other However, as mentioned before the optimization process in
hand, when no intake of vitamin C, deficiency vitamin C formulation development was OVAT in past time but the aim of
diseases will occur that named scurvy, the scurvy manifestations this study use the FbD concept for development and
are bleeding gums and increased hemorrhage potential due to optimization of vitamin C effervescent granules that makes
blood capillaries fragility, in addition to tired feeling and understanding and treating the formula statistically with high
psychological issues like depression, hysteria, and social accuracy and precision to obtain the desirable properties of the
introversion [1,2]. Today, Oral administrated drugs occupied the product in less time and effort and the dose of vitamin C intended
majority of manufactured medicines in the world especially solid to develop was 1000mg.
dosage forms, and oral delivery of drug is the most used route of
administration among all the routes that were utilized for the MATERIALS AND METHOD
systemic delivery of drugs via diverse pharmaceutical products Materials
of different dosage forms. The ease of administration and greater The active ingredient vitamin C (CSPC Weisheng
stability are the most reasons that made orally taken drugs more Pharmaceutical Shijiazhuang Co. Ltd) and the excipients
popular than liquid and semi-solid forms [3,4]. anhydrous citric acid (Weifang ensign industry Co. Ltd), tartaric
Effervescent granules are one of the solid dosage forms that is acid (Yantai Taroke Bioengineering Co. Ltd), sodium
taken orally, and noticeably the effervescent preparations are bicarbonate (TOSOH Manufacturing Co. Ltd), sodium saccharin
becoming increasingly due to many advantages such as good (Tianjin Changjie Chemical Co. Ltd), aspartame(Ajinomoto
stability, quickly dissolve, masking of unpleasant taste and ease Aspartame), sunset yellow (Senset Colors Inc.), orange flavor
administration with highly compliance in patients with difficulty (Aromsa), and absolute ethanol were received as a gift sample
in swallowing of pills and tablets, moreover the bioavailability from Modern and global Pharma companies for manufacturing
of low absorbed drugs can be increased. However, the of medicines. Other used reagents in this study were of analytical
disadvantages are needing controlled preparation and packaging grade.
area to protect from moisture and light that makes the medicine
more expensive. The major of effervescent products are Pre-formulation studies
analgesics, antacids, supplements and flu-cough formulations Determination of melting point
[4,5]. The melting point for both of Ascorbic acid and excipients were
Experimental design is statistical experiments aim to improve determined according to British pharmacopeia 2017 method 1.
the quality of product so is named Quality by Design (QbD) and
one of the modern approach that is involved in the formulation Fourier transform infrared spectroscopy FTIR
of a pharmaceutical dosage forms (FbD). Design of experiment FTIR spectroscopy was utilized on each samples to determine
(DOE) is an optimization technique meant for products and/or the structure of Ascorbic acid as alone and with other used
processes, and developed to evaluate all the potential influential excipients and to identify the presence of specific functional
factors simultaneously, systemically and quickly. On the other groups in a sample. The ascorbic acid-excipients interaction was
hand, the traditional approach of optimizing a formulation or examined by using the results of spectra (figure 1) [7]
process basically consists studying the influence of one variable
at a time (OVAT), whereas keeping all others at constant. Preparation of Vitamin C granules
Therefore, Design of experiment (DOE) has being applied The effervescent granules have been prepared by wet non-
widely in product and process optimization and validation to aqueous granulation method in witch absolute ethanol was used.

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 Journal of Applied Pharmaceutical Research 8 (4); 2020: 62 – 69 Faisal et. al

Vitamin C tartaric acid, calcium carbonate, sodium bicarbonate, Amount of Carbon Dioxide
aspartame, sodium saccharin and sunset yellow were weighted This present study determined the amount of carbon dioxide by
and triturated geometrically and sufficiently blended with using method developed by G. Rajalakshmi et al. In which
absolute ethanol to obtain a wet mass, then this dough mass was solution of 10% sulfuric acid was prepared in purified water.
passed through sieve No. 18. The resulted granules were dried Then to the previously weighted beaker 250 about 100ml of
in an oven at 35°± 5°C until the moisture content was below prepared sulfuric acid solution was taken. One dose of vitamin
0.3%. Precisely, the amount of ethanol was taken and the C granules were added in a beaker and the granules were
granulation step was timed. The eight products were prepared observed for complete release of carbon dioxide from the
3 granules. Again weight of the beaker was taken and the
according to full 2 factorial design that shown in table 1.
difference in weight before and after release of carbon dioxide
FbD based statistical optimization of effervescent shows the amount of carbon dioxide generated. [9-10]
formulation
For the optimization of effervescent formulation, Minitab pH of the solution
(version 18) software was used to design experimental recipe for About one dose of each vitamin C effervescent granules batch
statistical optimization. Statistical analysis of experimental data was placed in 200ml of water at 25Co. The effervescent granules
was performed using this software as well. Excipients under were kept in the beaker which allowed it completely to dissolve.
study were added to the formulation according to the The pH was measured using digital pH apparatus (Model:
experimental recipes developed by Minitab software while Metrohm, Make: Herisau). [10]
concentrations of rest of the components were similar.
Taste of product
Factorial experimental design The taste for each experimental batch was evaluated by giving
Effervescent granules of vitamin C were made according to a about one dose of product for every 40 volunteers [11]. The
3
full 2 factorial design. An amount of anhydrous citric acid-(X1), expressions of volunteers about the tasting were recorded, then
tartaric acid-(X2), and sodium bicarbonate-(X3) were selected each expression was coded proportionally based on acceptability
as independent variables. The formulation compositions and the feelings by numerical code from 1 – 5 as it illustrated in table 5.
effervescent mixture composition s are listed in table 1. The
responses evaluated were the disintegration time (DT), pH value, RESULTS AND DISCUSSION
Physical- chemical characterization
amount of carbon dioxide (CO2), and taste of product (TP).
Routine experimental studies include the utilize OVAT, whereas
Levels of independent variables and dependent variables
other variables are keeping constant. This type of strategy shows
(response) were listed in table 2. The best fitting mathematical
limited and sometime inaccurate information about the influence
model was selected for each response. The model predictor
of the variables included in the study. Factorial design permits a
equations were estimated by stepwise multiple regression
well-planned experiment that produces more information for
analysis. Validation of the model was performed by analysis of
less effort and cost. This strategy enables the identification of the
variance with a significance level of 0.05 and by the coefficient
individual contribution of each variable and the potential
of determination (R2). All statistical calculations and graphic
interactions among them. [12-13]. The 23 factorial design was
plots were performed using the Minitab version 18 software.
used in this current study to evaluate the feasibility of obtaining
vitamin C effervescent granules and to characterize the most
Physical and chemical characterization
influential factors included in the physical and chemical
Disintegration time
properties of the formulations. All of the possible combinations
About one dose of each vitamin C effervescent granules batch
of three factors and two levels were studied, leading to rise eight
was placed in 200ml of water at 25Co. the granules were
different formulations (table 1). Due to the properties of the
considered disintegrated when completely dispersed fragments
formulations, binding agents were not necessary to utilize. The
were gained and the liberation of gas stopped. This test was
independent factors and their ranges were selected from previous
performed according to the British pharmacopeia 2017. [8]
tests. This present study conducted to study the proportional

Journal of Applied Pharmaceutical Research (JOAPR)| October – December 2020 | Volume 8 Issue 4 | 64
 Journal of Applied Pharmaceutical Research 8 (4); 2020: 62 – 69 Faisal et. al

variation of effervescent mixture citric acid anhydrous (X1), interactions. The regression equation for dependent variables
tartaric acid (X2) and sodium bicarbonate (X3). Factorial design was made based on the observed responses for independent
allows the evaluation of the studied factors and their combine variables.
Table 1: Composition of vitamin C effervescent granules
Ingredients F1 F2 F3 F4 F5 F6 F7 F8
Vitamin C 1000 1000 1000 1000 1000 1000 1000 1000
Citric acid 1000 100 1000 100 100 1000 100 1000
Tartaric acid 100 1000 1000 100 100 1000 1000 100
sodium bicarbonate 1500 1500 100 1500 100 1500 100 100
Cal. Carbonate 203 203 203 203 203 203 203 203
Sod. Saccharin 15 15 15 15 15 15 15 15
Aspartam 30 30 30 30 30 30 30 30
Orange flavor 30 30 30 30 30 30 30 30
Sunset yellow 2 2 2 2 2 2 2 2
3880 3880 3380 2980 1580 4780 2480 2480
3
Table 2: variable and their levels in 2 factorial design
Levels
Independent variables
Low High
X1 = Citric acid (anhydrous) (mg) 100 1000
X2 = Tartaric acid (mg) 100 1000
X3 = Sodium bicarbonate (mg) 100 1500
Dependent Variables (response)
Y1 = Disintegration time (DT) Y2 = Taste of product (TP) Y3 = pH Y4 = CO2
3
Table 3: 2 factorial design observed responses
Code X1 X2 X3 pH DT(sec.) CO2 TP
F1 1000 100 1500 4.8±0.01 97.5±3.5 1.46±0.004 4
F2 100 1000 1500 4.5±0.16 153.5±3.3 1.52+0.002 3
F3 1000 1000 100 2.9±0.09 33±2.8 0.58±0.001 2
F4 100 100 1500 6.4±0.07 33±2.7 1.45±0.001 1
F5 100 100 100 4.3±0.04 40±3.1 0.52±0014 3
F6 1000 1000 1500 4.1±0.06 152.5±3.5 1.13±0.001 4
F7 100 1000 100 3.4±0.04 28±4.2 0.76±002 5
F8 1000 100 100 3.3±0.04 32±3.9 0.36±0.004 4
DT= Disintegration time , TP= taste of product, X1= citric acid, X2= tartaric acid, X3= sodium bicarbonate
26
46
vitamin C alone vitamin C with excipients
24 44

42
22
40
20
38

18 36

34
16
%Transmittance

%Transmittance

32
14 30

28
12
26
10
24

8 22

20
6
18

4 16

14
2
12

3800 3600 3400 3200 3000 2800 2600 2400 2200

Wavenumber
2000 1800 1600 1400 1200 1000 800 600
(A) 3800 3600 3400 3200 3000 2800 2600 2400 2200
Wavenumber
2000 1800 1600 1400 1200 1000 800 600
(B)
Figure 1: Fourier transform infrared spectrum of vitamin C alone (A) and vitamin C with excipients (B).

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Effect of Disintegration time Carbon dioxide amount

The effervescent granules of vitamin C resulted from all The amount of carbon dioxide ranged from 0.360 gm to 1,52 gm
formulations were found to conform the pharmacopoeial for all the formulations F1 to F8 table 3. From the analysis of
requirements related disintegration time (table 3). Based on the variance for factorial regression revealed both citric acid (X1),
analysis of variance for factorial regression displayed this tartaric acid (X2), and sodium bicarbonate (X3) variable had a
response was affected by all the independent variables studied in significant effect (P < 0.05, table 4) on the amount of carbon
this research particularly the sodium bicarbonate gave the dioxide. As shown on Pareto chart the largest effect was for
strongest influence, due to the P < 0.05 (table 4). Conversely, sodium bicarbonate. While the effect for citric acid by sodium
interaction of citric acid by tartaric acid (AB) showed the bicarbonate (AC) was the smallest. In contrast, the main effect
smallest effect. While, the main effect for each variable on the for each variable was the high concentration of sodium
mean of disintegration time illustrated in the figure 2, by bicarbonate and tartaric acid produced more carbon dioxide than
comparing the slopes of the lines indicated individually both low concentration but the magnitude of effect for sodium
variables produced longer disintegration time whenever bicarbonate was greater than tartaric acid. Similar results
concentration of citric acid, tartaric acid, and sodium bicarbonate obtained by Amit A. Patel et. Al. Reversely, the citric acid at low
transfer from low level to high level but the effect for citric acid concentration produced more carbon dioxide than high
was less than effects of tartaric acid and sodium bicarbonate concentration figure 4. As well as, the interaction between these
[14]. Additionally, the interaction between these factors was three factors was statistically significant as revealed in Pareto
statistically significant as shown in Pareto chart by reference line chart figure 5.
figure 3.
pH measurement
The pH value for all experiment ranged from 2.83 to 6.42 as
displayed on (table 3) and the statistical analysis of variance for
DOE showed significant effect and interaction (P < 0.05) for the
three studied variables X1, X2, and X3 on the pH value, and the
strongest effect was for sodium bicarbonate followed by tartaric
acid then citric acid figure 6. Whereas, the mean value of pH
decreased as the concentration of citric acid and tartaric acid
transferred from 100mg to 1000mg due to an acidity of solution
increased, and the pH increased as concentration of sodium
bicarbonate transfer from 100mg to 1500mg because the
alkalinity increased as shown on figure 7.
Figure 2. Main effects plot for effervescent time
Taste evaluation
The obtaining data from volunteers was subjected for analysis of
variance and the resulted information exhibited significant effect
and interaction (p < 0.05) between the both of three variables.
As it displayed on Pareto chart figure 8, the effect citric acid by
tartaric acid interaction (AB) and citric acid by sodium
bicarbonate interaction (AC) were the strongest on the taste,
while the effect of citric acid alone was the smallest. On the other
hand, the main effect for each variable individually was
desirable taste for volunteers at high concentration of citric acid
and tartaric acid than low concentration; oppositely, the taste
acceptability was better at low sodium bicarbonate concentration
Figure 3. Effects Pareto for effervescent time than high concentration figure 9.

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 Journal of Applied Pharmaceutical Research 8 (4); 2020: 62 – 69 Faisal et. al

Table 4: results of p value and regression coefficient

p values of coefficients
Responses
p value R2
Effervescence time 0.000 0.9971
pH 0.000 0.9955
CO2 0.000 1
Taste 0.000 0.9977

Figure 7. Main effects plot for pH

Figure 4. Main effects plot for Carbon dioxide amount

Figure 8: Effects Pareto of Taste

Figure 5: Effects Pareto of carbon dioxide amount

Figure 9. Main effects plot for Taste

Optimization of formula for vitamin C effervescent granules
The optimized formulation has chosen after considering the
results of dependent variables of the experimental design
batches. The batch with short disintegration time and moderate
amount of carbon dioxide with acceptable taste and pH will be
considered as optimized batch. The selected dependent variables
are correlated with each other because the high amount of
liberated carbon dioxide produces violent effervescence for
Figure 6: Effects Pareto of pH

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 Journal of Applied Pharmaceutical Research 8 (4); 2020: 62 – 69 Faisal et. al

granules that lead to loss some of the vitamin C dose. As well, Table 5: numerical codes for taste evaluation
as the pH and disintegration time also highly affected by the ratio Taste code Taste code
of effervescent mixture X1, X2, to X3. Therefore, using the Very 1 Sweet/acid 4
optimization model has been applied to predict the optimized sweet
formula of vitamin C effervescent granules, and the predicted Acid 2 Balanced/neutral 5
amounts for optimized formula with its predicted and observed Sweet 3
responses were showed in following table 6.

Table 6: composition of optimized formulation, expected observed values for response variable of ET, pH and TP

Composition Model predicted values Observed values

Citric acid Tartaric acid Sodium bicarbonate DT

DT (Sec) pH TP pH TP
(anhydrous) (mg) (mg) (mg) (Sec)
100 1000 113٫5 35 3٫5 5 30 3٫37 5

CONCLUSION CONFLICT OF INTEREST

Present study may be considered as a model study to identify and The authors declare no conflict of interest.
understand the effect of critical formulation parameters on
desired product quality attributes for selected formulation. The AUTHOR CONTRIBUTION
FbD concept was successfully applied for development and Safwan Alagbarri contributed in the conception of this work. He
optimization of the effervescent granules. Citric acid also contributed in arranging materials and instruments used in
(anhydrous), tartaric acid, and sodium bicarbonate were selected the work. Abdu Faisal and Maher Al-Absi have designed and
to have a most critical effect on the quality attributes, i.e., DT, performed the studies in the laboratories. They also recorded
pH, TP, and CO2 amount of effervescent granules. Factorial observations, analyzed and interpreted the data of experiments.
design was used to generate a highly significant statistical In addition, they contributed in drafting the manuscript. Mofeed
model, which can sufficiently describe or predict the formulation Al- Nowihi has revised the manuscript. All authors read and
optimization of effervescent granules for described quality. approved the final manuscript.
Generally, the concept of FbD was effectively applied to develop
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