Int. J. Pharm. Sci. Rev. Res., 64(2), September - October 2020; Article No.

03, Pages: 17-21 ISSN 0976 – 044X

Research Article

Formulation, Optimization and Evaluation of Multiple Emulsion of Atorvastatin

Sivapriya.S*, Daisy P.A, Praveen Raj R, Betty Carla

Department of Pharmaceutics, St. Joseph’S College of Pharmacy, Cherthala, India.
*Corresponding author’s E-mail: sivapriyasujith92@[Link]

Received: 18-07-2020; Revised: 22-09-2020; Accepted: 04-10-2020; Published on: 20-10-2020.

ABSTRACT
Multiple emulsions are often stabilized using a combination of hydrophilic and hydrophobic surfactants. The ratio of these
surfactants is important in achieving stable multiple emulsions. Atorvastatin was selected as a model drug to study the potential of
multiple emulsions to improve bioavailability with the hypothesis that improvement of drug release profile will reflect the
enhancement of bioavailability of the drug. The objective of this study was to prepare multiple emulsion of Atorvastatin by two step
emulsification using non-ionic surfactants, and evaluate for stability, percentage drug entrapment, In-Vitro & Ex-Vivo drug release.
The different variables like, rpm, concentration of surfactants and ratio of aqueous and oil phase were optimized to get the stable
emulsion with high drug release and less particle size. The study concluded that stable multiple emulsion with high drug release can
be prepared by two step emulsification method using Span80 as primary emulsifier at 30:70 phase volume ratio of internal phase:
external phase with optimized speed of stirring at 3000 r/min for 15 mins for primary emulsification. The optimized formula were
used for the preparation of multiple emulsion with the optimum drug release characteristics.
Keywords: Multiple Emulsion; Non-ionic Surfactant; Atorvastatin, Optimization.

properties of both w/o and o/w emulsions. These have

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been described as heterogeneous systems of one
immiscible liquid dispersed in another in the form of
DOI: droplets, which usually have diameters greater than 1
10.47583/ijpsrr.2020.v64i02.003 μm.
Multiple emulsions were determined to be promising in
DOI link: [Link] many fields, particularly in pharmaceutics and in
separation science. Their potential biopharmaceutical
INTRODUCTION applications include their use as adjuvant vaccines, as

A n ideal dosage regimen in the drug therapy of any prolonged drug delivery systems, as sorbent reservoirs in
disease is the one which immediately attains the drug overdose treatments and in mobilization of
desired therapeutic concentration of drug in enzymes. Multiple emulsions were also investigated for
plasma (or in the site of action) and maintains it constant cosmetics for their potential advantages of prolonged
for the entire duration of treatment. For many decades, release of active agent, incorporation of incompatible
treatment of an acute disease or a chronic illness has materials and protection of active ingredients by
been mostly accomplished by delivery of drugs to patients dispersion in internal phase.1
using various pharmaceutical dosages forms, including Atorvastatin, a synthetic HMG Co-A Reductase inhibitor,
tablets, capsules, pills, suppositories, creams, ointment is widely used in treatment of primary hypercholestremia
,liquids, aerosols, and injectable, as drug carriers. Even and Dyslipidaemia. Atorvastatin is indicated as adjunctive
today these conventional drug delivery systems are the therapy to diet for the treatment of patients with
pharmaceutical products commonly seen in the elevated serum triglyceride levels (Fredrickson Type IV).
prescription and over-the-counter drug market place. This Oral bioavailability of Atorvastatin is very low (Only 14%)
type of drug delivery system is known to provide a due to its presystemic clearance in gastrointestinal
prompt release of drug. Therefore, to achieve as well as mucosa and first pass hepatic metabolism. However, very
to maintain the drug concentration within the few studies have been reported for enhancement of
therapeutically effective range needed for treatment. bioavailability of poorly water soluble drugs by
When administered into the body, this gives high formulating as multiple emulsions.
therapeutic efficacy with minimal toxicity. It gives better
selectivity of pharmacological activity, and improves The present study is based on the hypothesis that
patient compliance by reducing the dosing intervals. improvement of in vitro as well as ex vivo (using rat
intestines) dissolution profile of AT and it will reflect the
Multiple emulsions are defined as emulsions in which enhancement of bioavailability of the drug.
both types of emulsions, i.e. water-in oil (w/o) and oil-in-
water (o/w) exist simultaneously. They combine the
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Int. J. Pharm. Sci. Rev. Res., 64(2), September - October 2020; Article No. 03, Pages: 17-21 ISSN 0976 – 044X

MATERIALS AND METHODS Preparation of calibration curve

Materials 10mg of AT dissolved in sufficient quantity of methanol
and volume was made up to 100ml with methanol. From
Atorvastatin calcium was obtained as the gift sample from
the stock solution, different dilutions from 2-20µg/ml was
Sance laboratories pvt. limited, Kerala, Tween 80 from
prepared with the same diluting medium to obtain a
Merklimite, Mumbai, Span 80 from Chemdyes
calibration curve. Absorbances of solution were
Corporation and light liquid paraffin from Spectrum
spectrophotometrically determined at 247.5nm.
reagents chemicals. All the reagents and chemicals were
of analytical grade. Microscopic Analysis
Methods Microscopic analysis was carried out using an research
microscope (labomed LX 300) combined with a computer
Preparation of Multiple Emulsions
imaging system, and observations were made at 40 X
Multiple emulsions were prepared by two step magnification after diluting in the appropriate amount of
emulsification process. First primary emulsion is external phase of the emulsion. The shape and
formulated in a high stirring speed. Then the so formed homogeneity of the multiple droplets is followed
primary emulsion is again emulsified to get multiple immediately after the preparation of the multiple
emulsions.3 emulsion formulations. A picture of the multiple emulsion
formulation was taken.
Step 1 (Preparation of Primary Emulsion)
Droplet Size Analysis
• 45ml of distilled water containing 50mg of drug was
gradually added to 55ml of oil phase containing primary The mean droplet size of the multiple emulsion
emulsifier span80(4ml) and 25mg of drug with continuous formulations was determined using particle size analyzer
stirring at 5000rpm for 5min. (Malvern Mastersizer) for the freshly prepared
formulation.
• Primary emulsion(w/o) was formulated.
pH
STEP 2 (Secondary Emulsification)
pH of the freshly formulated emulsion was done using
• 20ml viscous primary emulsion was emulsified further
digital pH meter. Here the digital pH meter is calibrated to
with an external aqueous phase containing secondary
neutral pH by dipping the glass electrode end in freshly
emulsifier (Tween 80) and 25mg drug with continous
prepared distilled water for several minutes. Then the
stirring at 1000rpm for 10 minutes.
glass electrode is dipped in the emulsion and pH reading
• Water / oil/ water (w/o/w) multiple emulsion was was noted.
formed.
Entrapment efficiency
Formulation of multiple emulsions with optimized
Entrapment efficiency was determined by taking freshly
primary emulsion
prepared W/O/W multiple emulsions and immediately
Eight batches (F1-F8) of primary emulsions was prepared centrifuged at 4000 r/min for 10 min. Then 1ml of the
by placing the drug concentration as constant (100mg) aqueous phase (the lower layer) was precisely withdrawn
whereas, the rpm, concentration of surfactant & ratio of through 2 ml hypodermic syringe and diluted properly
aqueous and oil phase had taken as variables. The particle with 0.1N HCl. The solution was filtered with a whatman
size & drug release from the emulsion was kept as the filter paper and drug content was analyzed on UV
response factors. By using the design expert (stat ease) spectrophotometer at 240 nm. The Encapsulation
software, the optimization profiles were obtained. The Efficiency was determined by following equation:
optimized formula had used for the preparation of
% EE = [(Total drug incorporated –Free Drug)/ Total drug]
multiple emulsion of Atorvastatin with good particle size
X 100
& drug release profile. Suitable flavouring agents were
added to obtain palatability to multiple emulsion. Invitro drug release study
Variables for optimization are given in table 1 and
The in vitro drug release study was carried out on a
formula for optimum batch of primary emulsion is given
simple dissolution cell using cellophane membrane. Prior
in table 2.
to release studies, the cellophane membrane was soaked
Table 1: Variables for optimization in distilled water for 6 hours, washed frequently 4 times
by changing distilled water, then immersed in 5% v/v
Variables +1 -1
glycerol solution for at least 60 min and washed finally
X1 RPM 5000 3000 with 5 portions of distilled water. Freshly prepared
X2 Surfactant concentration 4ml 12ml multiple emulsion (15ml) was added to donor chamber,
X3 Ratio of aqueous & oil phase 30:70 45:55
made up of a hollow glass tube (2.5 cm in diameter and
10 cm in length) and membrane was tied on bottom end
of the tube with a nylon string. This tube was dipped into
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Int. J. Pharm. Sci. Rev. Res., 64(2), September - October 2020; Article No. 03, Pages: 17-21 ISSN 0976 – 044X

1000 ml vessel containing 900 ml of PBS pH 6.8 and was observed that the Atorvastatin was white amorphous
stirred at 75 rpm on a magnetic stirrer and maintained at powder. The sample was quantitatively tested for its
37 °C which acted as receiving chamber. Aliquots of 1ml solubility in various solvents. Solubility study in different
were collected from receiving chamber at predetermined solvents revealed that it is freely soluble in methanol,
time intervals and the drug contents determined on UV slightly soluble in alcohol and insoluble in distilled water.
spectrophotometer at 240 nm after suitable dilution.5 The melting point of Atorvastatin Calcium was found to
be 159.2-160.7.
Determination of Drug Release Mechanism6,7
Preparation of calibration curve
In order to understand the mechanism of drug release,
Invitro drug release data were treated to kinetic models Absorbances of solution were spectrophotometrically
such as zero order, first order and Higuchi model and determined at 247.5nm. A standard calibration curve for
korsmeyerpeppa”s model. the drug was obtained by measuring absorbance at
247.5nm and by plotting the graph of absorbance Vs
Ex Vivo Release study
concentration.
Drug release study was performed on the rat ileum by
following perfusion method. The special apparatus consist
1 Absorbance
of U shaped glass tube having 1 cm inner diameter with
cannulated cut on the upper half arm of the U tube.8 0.5

Viscosity study Absorbance

Viscosity is a principal parameter when any flow 0

0 5 10 15 20
measurements of fluids, such as liquids, semisolids. Gases
Concentration(μg/ml)
and even solids are made. Brookfield deals with the
liquids and semisolids. Brookfield viscosity usually refers Figure 1: Calibration curve of Atorvastatin
to a viscosity measurement performed with a Brookfield Evaluation of Multiple Emulsions
Viscometer, sometimes referred to as a Brookfield
viscosimeter. There are several models of viscometer Microscopic Analysis
available from Brookfield but the majority operates in the Microscopic analysis was carried out using an research
same manner: the viscometer motor rotates the spindle microscope (labomed LX 300), and observations were
at a defined speed (measured in rpm) or shear rate and made at 40 X magnification after diluting in the
the viscometer measures the resistance to rotation and appropriate amount of external phase of the emulsion
reports a viscosity value.9 the image taken via microscope.
Stability study of optimized formulation
Formulations were subjected to stability studies for a
period of 30 days. The samples were withdrawn after 30
days and were evaluated pH, entrapment efficiency and
release profile.
RESULTS AND DISCUSSION
The Drug (Atorvastatin) powder was examined for its
organoleptic properties like colour, and odour and it is
Figure 2: Microscopic images of multiple emulsions

Table 2: Formula for optimum batch of primary emulsion

Conc. of Ratio of aqueous& Particle size Drug release
Number Rpm
surfactants (ml) oil phase(ml) (µm) (%)
1 3000 4 30:70 5.26 84.13

Table 3: Mean droplet size of emulsion

Sl. No 1 2 3 4 5 6 7 8 9 10
Size(µm) 12.7 14.7 16.4 11.2 10.3 11.3 5.5 5.4 6.4 7.5

Table 4: Cumulative Drug Release of Atorvastatin Multiple emulsion (Exvivo)

Time 0 15 30 45 60 120 180 240 300
%CDR 0 5.3 10.0 15.5 29.1 38.5 50.6 76.5 88.9

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Int. J. Pharm. Sci. Rev. Res., 64(2), September - October 2020; Article No. 03, Pages: 17-21 ISSN 0976 – 044X

Droplet Size Analysis Viscosity

The mean droplet size of the multiple emulsion Viscosity of ME was determined by using the Brookfield
formulation was determined using stage micrometer and viscometer by selecting the spindle number and rpm. The
eyepiece micrometer for the freshly prepared formulation. viscosity of the formulation at different rpm has
The mean droplet size of multiple emulsions was 7.8µm. performed and is given in table 5.
The obtained droplet sizes are given in table3.
Table 5: Viscosity of the formulation
pH rpm Spindle Number Viscosity(Cp) Torque%
pH of the freshly formulated emulsion was done using pH 100 18 16 53.5
paper and it was found to be 8. 50 18 22 36.6
Entrapment efficiency 60 18 7 14
After formulating Atorvastatin multiple emulsions, the Kinetic Modeling
drug content was estimated by UV spectrophotometer at λ
max 247.5nm. The Entrapment efficiency of multiple The results obtained of in vitro release studies were
emulsions was 88.5%. attempted to fit into various mathematical models. From
the results given in table 6, it is clear that the drug release
shows first order kinetics for the formulation.
Table 6: Model fitting for the release profile of
Atorvastatin Multiple Emulsion
Correlation coefficient(r)value
Formulation Zero First Korsmeyer
Higuchi
order order Peppas
ME 0.823 0.967 0.957 0.952

Stability study
Prepared formulation was subjected to stability study for
30days. Sample was withdrawn after 30 days and was
Figure 3: 3D plot of drug release evaluated for parameters like particle size, pH and in vitro
In-vitro drug release studies drug release. Formulation showed slight decrease in
entrapment after 30 days of storage. The in vitro drug
In- vitro drug release studies were carried out in PBS of pH release from the formulation was also decreased after
7.4 as the dissolution medium. Studies were performed as stability study period. This may be due to decrease in the
per the procedure described in methodology. relative drug content.
Comparative study of multiple emulsion of Atorvastatin
100 with marketed Atorvastatin tablets
Cumulative %.drug release

90
80 The Multiple emulsions was evaluated for in vitro
70 dissolution study and compared with marketed tablet
60
50 under same experimental conditions and the data was
40 cumulative… recorded as a chart in figures.
30
20 100
10 90
0
80
0 50 100 150 200 250 300 350
70
Time(min)
60
%CDR of ME
Figure 4: Invitro Cumulative drug release. 50
40 %CDR of tablet
Ex vivo drug release study 30
Exvivo release studies were carried out by using the U 20
shaped apparatus. The rat ileum was tied in this apparatus 10
and multiple emulsion was filled in it. The Multiple 0
emulsions was filled in the ileum and placed inside the 0 2 4 6
dissolution vessel with pH 7.4phosphate buffer. The drug
released in the media was measured. The cumulative drug Figure 5: Comparative study of %CDR of ME & tablet of
release at various time intervals are mentioned in table4. Atorvastatin

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CONCLUSION 4. Jyothi Wadhva, Anroop Nair and Rachna kumaria. Emulsion

forming drug delivery system for lipophilic drugs. Acta
The study was aimed at the Formulation, Optimization and
Poloniae Pharmaceutica n Drug Research, 69(2), 2012, 179-
evaluation of multiple emulsion of Atorvastatin. Two
191.
different non-ionic surfactants were used for the
formulation. Atorvastatin, a synthetic HMG Co-A 5. Sumithapaul, Abhineshkumar, Pramod Yadurkar,
Reductase inhibitor, is widely used in treatment of primary Kruthikasawant. Design and Development of multiple
hypercholesterolemia and Dyslipidaemia. ME was emulsions for the enhancement of oral bioavailability of
prepared by two step emulsification process. The main Acyclovir. Drug development and Industrial pharmacy,
purpose was to develop stable multiple emulsion with 39(11), 2013, 1808-1817.
higher solubility & drug release. The study revealed that
multiple emulsions can be optimized for good stability and 6. Vani Madaan, Arsh Chanana, Mahesh kumar kataria, Ajay
higher drug release by optimizing different formulation Bilandi. Emulsion Technology and recent trends in emulsion
variables like type & proportion of primary & secondary application. International Research Journal of Pharmacy, 5
emulsifier and phase volume ratio of internal phase: (7), 2014, 533-542.
external phase; and process variables like speed & time of 7. Sumana Khosh: Formulation and characterization of
stirring during primary & secondary emulsification. multiple emulsions with various additives. International
This study concludes that Multiple Emulsion formulation Journal of Research in Pharmaceutical and biomedical
can provide consistent and prolonged release of sciences, 2 (2), 2011, 751-759.
Atorvastatin. It will lead to sustained action of the
8. Naveed Akhtar, Muhammad [Link] and
entrapped drug that reduce the side effects associated
Characterization of Multiple emulsion containing 1% Ascorbic
with frequent administration of the drug and potentiate
[Link]. Chem. Soc. Ethiop, 24(1), 2010, 1-10.
the therapeutic effects of the drug.
9. M Sedef ERDAL, Ahmet ARAMAN. Development and
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Source of Support: None declared.

Conflict of Interest: None declared.

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