Journal of Pharmaceutical Investigation

DOI 10.1007/s40005-014-0167-7

RESEARCH ARTICLE

Microencapsulation using aqueous dispersion of lipid matrix

by fluidized bed processing technique for stabilization
of choline salt
Avinash B. Gangurde • Ritesh A. Fule • Sharadchandra D. Javeer •

Rahul K. Patole • Jaywant N. Pawar • Purnima D. Amin

Received: 21 August 2014 / Accepted: 2 December 2014

Ó The Korean Society of Pharmaceutical Sciences and Technology 2014

Abstract Choline bitartrate (CBT) is a water soluble after 6 months stability studies. Choice of proper carrier to
essential nutrient belongs to vitamin-B family. It is mois- drug ratio and selective formulation technique are critical
ture sensitive in nature and marketed formulation has sta- parameters for dispensing CBT microparticle based for-
bility related problems during storage which curtails its mulation which might significantly enhance its
effectiveness. Waxes such as hydrogenated soya bean oil effectiveness.
(HSO) reported to be an excellent coating carrier to reduce
moisture sensitivity or hygroscopic nature of drug candi- Keywords Choline bitartrate 
dates. However, literature dictates HSO applications has Hydrogenated soya bean oil  Fluidized bed coating 
been explored mostly using non-aqueous methods or hot Microparticles  Odour masking
melt techniques of formulation development. In this work,
microparticles of choline bitartrate with aqueous coating
dispersion of HSO as primary carrier was successfully Introduction
developed using fluidized bed coating technique. Aqueous
dispersion of HSO was prepared using selected binder in a Choline is a water soluble essential nutrient in the form of
homogenizer and formed aqueous dispersion was then white crystalline powder. It is involved in various bio-
sprayed through 0.8 mm gun in fluidized bed processor. chemical reactions at various systems of the body,
The microparticles were evaluated for parameters like flow including the brain, muscles and internal organs (Albright
properties, morphological characteristics, drug content, et al. 1997; Paul and Lucien 2004). Choline is required to
encapsulation efficiency and drug release behaviour. The form healthy cell membranes, breakdown of cholesterol,
solid state characterization of optimized microparticle and aid in the function of neurotransmitters in the brain
based formulation was done by differential scanning cal- (Albright et al. 1999; Habich and Hansen 2006). Choline is
orimetry, X-ray diffractometry, infrared spectroscopy and available in salt form as choline bitartrate (CBT), chemi-
scanning electron microscopy. The results showed that cally it is (2-hydroxyethyl) trimethylammonium-L-(?)-
microencapsulation of choline bitartrate were successfully tartrate salt Fig. 1. Its molecular weight is 253.25 and
done by aqueous wax coating dispersion without using any molecular formula is C9H19NO7 (Krishnamurthy et al.
organic solvent or hot melt techniques. Discolouration, 2012).
fishy odour and drug content variation was not observed Multivitamin mineral tablets such as GesticareÒ and
DHA containing encapsulated choline salts are found to be
unstable during storage. The loss of stability of choline
A. B. Gangurde (&)  R. A. Fule  S. D. Javeer  results in decreased choline activity or fishy odour devel-
R. K. Patole  J. N. Pawar  P. D. Amin opment or discoloration of the tablet at 30 °C/45 % RH
Department of Pharmaceutical Sciences and Technology, and 45 °C/75 % RH storage conditions. This reduces the
Institute of Chemical Technology, NAAC Accredited ‘A’ Grade,
shelf life of the product and also it is undesirable for patient
ELITE Status of Excellence, N. P. Marg, Matunga (E),
Mumbai 400019, India compliance (Paul and Lucien 2004). Therefore there is
e-mail: gangurde.avinash8@gmail.com need to encapsulate a choline salt with suitable carriers

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 A. B. Gangurde et al.

were procured from Sd. Fine chemicals, Mumbai, India.

All other chemicals used were of laboratory and reagents
grade.

Formulation and development

Fig. 1 Chemical structure of CBT
Drug excipient compatibility studies
which will provide desired degree of protection. Encap-
sulation of CBT by hydrophobic polymers found to be CBT was uniformly mixed in 1:1 ratio with the HSO and
useful approach to solve the problems related to its the mixture was placed in glass vials. The vials were
stability. covered with aluminium foil and a small hole was made in
Microencapsulation is a technique for encapsulation of the foil. Drug alone was kept in a similar manner to serve
core material which gets entrapped in a protective layer of as control. Vials were kept at 30 °C/65 % RH and 40 °C/
such as polymers or waxes. Various microencapsulation 75 % RH.
techniques are used such as for encapsulation such as Physical mixture of CBT and HSO were characterized
solvent evaporation/extraction, phase separation/co-acer- by Differential scanning calorimetry (DSC) to understand
vation, spray drying and fluidized bed technique (Freitas the solid state transformation. The DSC of CBT and mix-
et al. 2005). These techniques require organic solvent to tures of drug: HSO were taken. The CBT and CBT:HSO
dissolve or disperse the water insoluble material (Wilson (PM) (1:1) were kept in the dried glass vials under normal
and Shah 2007). Due to regulatory and manufacturing conditions at room temperature. These samples were ana-
issues such as safety hazards, toxicity and high costs lysed for DSC using DSC Perkin Elmer pyris-6 (USA)
associated with organic solvents, pharmaceutical compa- equipped with a thermal analysis computerized data sta-
nies are rapidly replacing the organic solvent based coating tion. Samples (3–4 mg) were placed in an aluminium pan
with aqueous film coating procedure (Bodmeier and Wang and heated at a rate of 10 °C/min with the reference pan in
1993). an atmosphere of nitrogen to a temperature of from 40 to
Microencapsulation of active substance using lipid 300 °C.
matrix have been proposed as an alternative to colloidal
and liposomal systems, due to its flexibility on packaging Preparation of 10 % aqueous dispersion of HSO
of materials and size of particles (Muller et al. 2000).
Various lipids such as phospholipids, triacylglycerols, The aqueous dispersion of HSO contains Tween 80
waxes, fatty acids or their mixtures could be used for (1.4–2 %) as emulsifying agent, PEG 400 (0.5–1 %) as
encapsulation (Gamboa et al. 2011; Jannin and Cuppok plasticizer, HPMC 15 cps or PVP K-30D (1.2 %) as binder,
2012). Waxes such as hydrogenated soya bean oil (HSO) BHT as antioxidant, lemon oil as flavour and colloidal
was used for microencapsulation of nutraceuticals such as silicone dioxide as a flow modifier. Composition for oil
tocopherol, iron (Gamboa et al. 2011; Zimmermann et al. layering was showed in Table 1. Aqueous dispersion was
2003). HSO has been widely used as a coating wax to give prepared by heating the HSO with emulsifying agent
better protection and retardation of drug release due to their Tween 80 at a temperature above melting point of the
hydrophobic nature. But its use has been restricted to non- HSO. PEG 400 was added to this dispersion. This oil and
aqueous methods or hot melt formulation techniques such surfactant solution was added to hot water and maintained
as spray chilling/congealing (Paul and Lucien 2004). at temperature of 80–90 °C with continuous stirring using a
Hence, our aim was to prepare an aqueous wax coating high shear homogenizer. HPMC 15 cps/PVP K-30D, BHT,
dispersion of HSO as a coating carrier to stabilize the
choline salt using fluidized bed coating technique.
Table 1 Composition of aqueous dispersion of HSO1 for layering
Ingredients HSO1 (%w/w)
Materials and methods
Soybean oil 7
Tween 80 2
CBT and HSO were obtained as a gift sample from Bajaj
PEG 400 1.4
Healthcare ltd. Mumbai. Hydroxypropyl methyl cellulose
(MethocelÒ E5 & E15) was obtained as gift sample from BHT 0.2
Dow Chemical Co., USA. PVP K-30 D was obtained as a Lemon oil 0.4
gift sample from Anshul life science ltd. Mumbai. Buty- Distilled water q.s.
rated hydroxyl toluene (BHT), Tween 80, and PEG 400 All quantities are in percentage (%w/w)

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Stabilization of choline salt

lemon oil and colloidal silicone dioxide were then added to Optimization of composition of aqueous wax coating
the dispersion with stirring. The dispersion of HSO was dispersion
screened through 100# sieve with particle size less than
150 lm in order to confirm the absence of any insoluble The composition of the wax coating solution was selected
material and to uniformly distribute fine multiparticulate. such that encapsulation efficiency, drug content and flow
Obtained aqueous dispersion of soya bean oil was studied properties obtained was within standard specifications.
for physical stability such as cracking (separation of pha- Microparticles prepared without binder (i.e. only with wax)
ses) or creaming of oil droplets at the surface after 1, 7, 15 and with binder i.e. PVP K-30 D, HPMC 5cps and HPMC
and 30 days storage at room temperature. Particle size 15 cps with wax were observed for their consistency and
distribution of obtained aqueous dispersion was analysed strength. Amongst the binders used, HPMC 15 cps was
using Malvern mastersizer 2000 and viscosity was analysed chosen on the basis of their binding capacity. Microparti-
by rheometer rheoplus (Anton Parr) instrument. cles obtained with HPMC 15 cps showed good compress-
ibility index and flow properties. Different batches were
designed as shown in Table 3.
Encapsulation of CBT using aqueous dispersion of HSO
in a fluidized bed processor (FBP)
Characterisation of CBT microparticles
Aqueous dispersion of HSO which is used as a coating
Morphological characteristics and flow properties of CBT
agent was sprayed over the bead of CBT (mesh no.20) by
using Miniglatt FBP. A laboratory size R & D model
The size distribution in terms of average diameter of the
APCG-175C coating machine was used for coating the
microparticle was determined by an optical microscopic
CBT. The flow rate was maintained constant such that no
method. A compound microscope fitted with a calibrated
agglomeration of the beads occurred during the coating
ocular diameter and stage micrometer slide was used to
process. The air flow was kept at intermediate level to
count at least 100 particles (Olympus, NWF 10x; Educa-
achieve good drying efficiency. During the layering pro-
tional Scientific Stores, India) (Parmar and Gohel 2011).
cess, the beads were intermittently dried for 10 min at
All the batches were studied with regards to the morpho-
50–60 °C. After, completely spraying the aqueous disper-
logical features such as aspect ratio, particle size, and shape
sion of HSO, beads were dried in expansion chamber for a
using photomicrograph. The prepared micro particles were
period of 10–15 min and then cooled to room temperature.
evaluated for bulk density, tapped density, compressibility
Different trials were taken to select the processing vari-
index (Carr’s index), angle of repose and hausner’s ratio.
ables (Mukharya et al. 2012; Ichikawa and Fukumori
1999).
Particle size distribution

Selection of processing variables for aqueous HSO coating Particle size distribution study was carried out using sieve
analysis method. Approximately 50 g of granules were
The various trials were conducted as shown in Table 2. placed into a sieve shaker equipped with 8, 12, 16, 18, 20,
From, these trials the preliminary processing variables for 30 and 40 mesh US standard sieves (Retsch R Model
wax coating was selected. AS200 Digit, Germany) and shaken for 20 min. Each

Table 2 Trials for the selection

Process parameters Conditions
of process parameters for
aqueous dispersion layering on T1 T2 T3 (optimized) T4 T5
CBT
CBT bead (gm) 450 450 450 450 450
Blower speed 15 18 21 24 27
Inlet temperature (°C) 50 55 60 65 70
Bed coating on temp. (°C) 45 45 45 45 45
Bed coating off temp. (°C) 35 35 35 35 35
Bag delay shaking (min.) 3 3 3 3 3
Bold values indicate the
Peristaltic pump speed (R. P. M.) 1 5 10 15 20
optimized processing and
formulation batch parameters Gasket pressure (bars) 2.8 2.8 2.8 2.8 2.8
which gives satisfactory results Atomizing air pressure (bars) 1.3 1.3 1.3 1.3 1.3
as compared to other Woofer height (mm) 25 25 25 25 25
formulation batches

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 A. B. Gangurde et al.

Table 3 Different composition for aqueous wax coating dispersion

Formulation CBT1 CBT2 CBT3 CBT4 CBT5 CBT6 CBT7 CBT8 CBT9

CBT 90 90 90 90 90 90 90 90 90
Soybean oil 8 8 7 7 6 6 5.5 5.5 5.5
Tween 80 – 0.9 1.4 1 1.8 1.6 1.4 1.4 1.4
PEG 400 1.4 0.5 1.0 0.6 0.8 1 0.5 0.5 0.5
BHT 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Lemon oil 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4
Colloidal silicone dioxide – – – 0.8 0.8 0.8 0.8 0.8 0.8
PVP-K30D – – – – – – 1.2 – –
HPMC 5 cps – – – – – – – 1.2 –
HPMC 15 cps – – – – – – – – 1.2
Distilled water q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s.
All quantities are in percentage (%w/w)
Bold values indicate the optimized processing and formulation batch parameters which gives satisfactory results as compared to other formulation batches

weight fraction was recorded. The mean size and size analysis CBT and CBT 9 were placed in crucible at accelerated
distribution were determined (Sinchaipanid et al. 2004). conditions of temperature 40 ± 2 °C and humidity 75 ± 5 %
RH in environmental test chamber for 24 h (Thermo lab,
Assay India). These samples were then analysed for drug content by
Potentiometric titration. The method was useful to determine
Accurately weighed microparticles equivalent to 200 mg the effect of moisture on degradation of drug and encapsulated
of CBT were transferred to a conical flask and dissolved drug systems (Rasenack and Muller 2002).
with 50 ml of glacial acetic acid. This solution was then
titrated with 0.1 N perchloric acid and the endpoint was Release of encapsulated CBT
determined potentiometrically (Pharmacopoeia US 2007).
Each ml of 0:1 N perchloric acid is The 5 h release of encapsulated CBT in water was deter-
ð1Þ mined. 1 gm of CBT was placed in 250 ml of elementary
equivalent to 25:32 mg of C9 H19 NO7 :
flask. 100 ml of distilled water was added to the flask and the
Percentage of drug encapsulation efficiency flask was sealed with a stopper. The flask was then placed on
a shaker for 5 h with moderate shaking intensity. At the end
The encapsulated micro particles equivalent to 500 mg of of 5 h the contents of the flask was filtered through pre-
CBT were accurately weighed and crushed. The powdered moistened glass wool in a powder funnel. The elute was then
microparticles were dissolved in dichloromethane (5 ml) in collected into second flask. The first flask was rinsed with
100 ml volumetric flask and the volume was made with 10 ml of water and this was added to the second flask through
0.1 N HCl. This solution was then filtered through what- powder funnel. The solution in the second flask was titrated
mann filter paper No. 44. After suitable dilution, the with 0.1 N NaOH. The end point of the titration was deter-
absorbance was measured at 208 nm using UV spectro- mined by measuring the first inflexion in the pH values, at
photometer and the percentage drug entrapped was calcu- approximately neutral pH (Paul and Lucien 2004).
lated (Eq. 2). The drug entrapment efficiency study was The percent release was calculated using following
conducted in triplicate. (Parmar and Gohel 2011). equation
ml of 0:1 N NaoH  0:1  253  100
% Drug encapasulation efficiency Percent Release ¼
Actual drug content mg of encapsulate  ð% of CBT =100Þ
¼ ð2Þ
Therotical drug content ð3Þ

Moisture uptake and stability studies Solid state characterization of optimized CBT

Moisture uptake by neat drug and encapsulated drug was Differential scanning calorimetry (DSC) analysis
studied using Moisture balance MB 50C (Citizen, India).
Moisture content of CBT and encapsulated CBT was calcu- Plain CBT and optimized microparticle of CBT9 were
lated in the form of percentage. After moisture content characterized by DSC to understand the solid state

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transformation. DSC (Pyris 6 DSC, Perkin Elmer, and Stability study

USA) with a thermal analyser, equipped with the Pyris
software, was employed to obtain thermal data. Five mil- Stability studies were conducted by placing samples in
ligrams of sample was placed in a DSC aluminium sample closed glass amber colour vials and in aluminium foil
pan and then crimped. Heat flow rate of 10 °C/min was sachet which were kept in a stability chamber. The storage
used to heat the samples from 40 to 300 °C. Nitrogen was condition was 40 °C temperature and relative humidity
used as a purge gas. An empty crimped pan was used as a (RH) of 75 % (Gavin and Osama 2010) and 30 °C tem-
reference pan. perature and 65 % RH. Samples were tested for 0, 1, 2, 3,
4, 5 and 6 months stability study and tested for physical
Fourier transform infrared spectroscopy (FTIR) analysis appearance, discoloration, fishy odor development resem-
bling amines, moisture content and drug content.
FTIR spectrophotometer from PerkinElmer (USA) was
used in an attenuated total reflectance manner to obtain
FTIR spectra. The samples were ground thoroughly with Results and discussion
potassium bromide at 1:100 (sample/potassium bromide)
weight ratio in a mortar and pestle till a uniform mixture Formulation and development
was observed. Scans were performed in triplicate from
4,000 to 400 cm-1, with threshold of 1.303, sensitivity of Drug excipient compatibility study
50 and resolution of 2 cm-1 range were recorded on KBr
disc. Spectra were recorded for CBT powder and micro- No significant physical change was observed for CBT and
particles of optimized CBT9. excipients which were kept for compatibility study for
15 days at 30 °C/65 % RH and 45 °C/75 % RH in glass
Powder X-ray diffractometry (PXRD) analysis vials thus it was concluded that the active and inactive
ingredients are compatible with each other. Also, the pos-
Plain CBT and optimized microparticle of CBT 9 was sible interaction between CBT with HSO and polymers
further characterized by PXRD. X-ray diffraction patterns were studied using DSC. There was no significant change
were recorded on rigaku miniflex instrument using Ni fil- in DSC endothermic values on comparison of plain CBT
tered, Cu Kalpha as source of radiation, a voltage of 40 kV, and CBT loaded HSO with other polymers observed. The
and a 25 mA current. The samples were run over the most peak value obtained at 155.15 °C which is corresponding
informative range from 5 to 50° of 2h values. The step scan to melting of plain drug. There was no major shifting in
mode was performed with a step size of 0.02° at a rate of this peak value observed. The presence of other excipients
2°/min. Crystal size was determined using the Debye– might have suppressed the peak height. Hence, it is con-
scherrer equation. cluded that there was chemical compatibility between drug
and excipients. The DSC thermogram is showed in Fig. 2.
Scanning electron microscopy (SEM)
Preparation of 10 % aqueous dispersion of HSO
The morphology of microparticle was analysed with the
help of XL 30 Model JEOL 5400 scanning electron For preparation of dispersion of HSO in organic solvent
microscope made in Japan during analysis. Double sided free environment, we used non-ionic Polyethylene glycol
carbon tape was affixed on aluminium stubs over which sorbitan mono oleate (Tween 80) as emulsifying agent
sample of CBT and prepared microparticle was placed. The which has hydrophilic and lipophilic structural portions
radiation of platinum plasma beam using JFC-1600 auto within its molecular structure. Tween 80 has slight odour,
fine coater was targeted on aluminium stubs for its coating taste and colour which did not interfere with the stability of
to make layer of 2 nm thickness above the sample for the active content. PEG 400 was used as plasticizer which
25 min. These prepared coated stubs were then placed in improved the process ability, flexibility and elasticity of
the vacuum chamber of a SEM and adjusted to maximum coating agents (Bouchemal et al. 2004). Obtained aqueous
magnification to obtain excellent quality scanning images. dispersion of soybean oil was not showing any cracking
Later these samples were observed for morphological (separation of phases) or creaming of oil droplets at the
characterization using a gaseous secondary electron surface after 1, 7, 15 and 30 days storage at ambient con-
detector (working pressure: 0.8 Torr, acceleration voltage: ditions. The average particle size of the obtained aqueous
20.00 kV). SEM images were obtained at maximum and dispersion was 70 microns measured by Malvern particle
visible magnification to understand the surface interaction mastersizer 2000 as showed in Fig. 3. And viscosity was
between drug and wax. found to be 4.19 Pa.s at a shear rate of 2 s-1 and shear

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Fig. 2 DSC thermogram of a plain CBT and b CBT loaded with formulation excipients

Fig. 3 Particle size distribution

of aqueous dispersion of HSO

stress of 8.39 Pa as showed in Fig. 4. Various trials were But, the compressibility of the obtained microparticles was
taken as mentioned in Table 2 for the selection of different relatively low therefore addition of binder in the compo-
processing variables of fluidized bed processor. In trials T1 sitions CBT7 to CBT9 showed improvement in com-
and T2 the blockage of nozzle gun was observed because pressibility and binding of the microparticles as indicated
of lower blower speed and low inlet temperature. Increased in Table 4.
in the blower speed up to 27 and inlet temperature up to Assay of CBT was determined by using potentiometric
70 °C in trial T4 and T5 lead to agglomeration of the micro titration. Drug content in all the preparation was in the
particles. This was observed because of melting of HSO at range of 89–91 % which implied that microencapsulation
65 °C. The T3 trial processing variables was taken into produced good reproducibility of assay content. The
consideration for determination of wax coating (Ichikawa encapsulation efficiency of microparticle varied between
and Fukumori 1999). 54 and 61 % Table 4. Results demonstrate that an addition
The dispersion prepared in CBT1 caused cracking or of binder increased the encapsulation efficiency of the
creaming of oil droplets at the surface. Addition of the drug.
emulsifying agent Tween 80 to the dispersion composition The photo micrographic study confirmed that obtained
of CBT2 and CBT3 forms the stable dispersion of HSO. microparticles are non-spherical or cylindrical in shape.
After spraying the aqueous s dispersion of soybean oil The aspect ratio of microparticles varied from 1.70 to 1.26
through 0.8 mm nozzle gun the microparticles showed very Table 4 which indicate non-uniform micro particles while
poor flow properties. Addition of colloidal silicon dioxide CBT 9 Fig. 5 has an aspect ratio of 1.11 showing uniform
to the composition CBT4 to CBT6 showed improvement in micro particles compared to other batches. The optimized
the flow properties of microparticles as shown in Table 4. CBT 9 batch contained 85–90 % particles of uniform

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Stabilization of choline salt

Fig. 4 Viscosity of aqueous dispersion of HSO

Table 4 Evaluation of micro particles for their flow properties, assay, aspect ratio and encapsulation efficiency
Batch Angle of Bulk density Tapped Hausner’s Carr’s index Aspect ratio Assay (%) Encapsulation
repose h (g/ml) density (g/ml) ratio efficiency (%)

Plain CBT 39.53 ± 1.24 0.5339 0.7894 1.47 ± 0.21 32.36 ± 1.52 1.86 ± 0.24 99.95 ± 0.98 –
CBT2 35.43 ± 1.18 0.5923 0.8124 1.37 ± 0.19 27.09 ± 1.24 1.70 ± 0.21 90.33 ± 0.74 54.65 ± 1.21
CBT3 34.42 ± 1.16 0.5834 0.8031 1.37 ± 0.18 27.35 ± 1.21 1.64 ± 0.19 90.64 ± 0.53 56.76 ± 1.24
CBT4 29.24 ± 1.02 0.6134 0.8434 1.37 ± 0.19 27.27 ± 1.20 1.71 ± 0.22 90.21 ± 0.49 54.57 ± 1.19
CBT5 28.54 ± 1.01 0.6189 0.8556 1.38 ± 0.16 28.43 ± 1.21 1.78 ± 0.25 89.9 ± 0.45 56.76 ± 1.23
CBT6 29.78 ± 1.09 0.6123 0.8542 1.39 ± 0.19 28.16 ± 1.24 1.64 ± 0.21 91.34 ± 0.86 58.66 ± 1.25
CBT7 24.56 ± 0.96 0.6334 0.8634 1.36 ± 0.15 26.63 ± 1.25 1.32 ± 0.19 90.23 ± 0.45 59.76 ± 1.29
CBT8 23.89 ± 0.91 0.6645 0.8745 1.31 ± 0.12 24.01 ± 1.18 1.21 ± 0.12 90.51 ± 0.56 60.32 ± 1.30
CBT9 22.68 – 0.81 0.6818 0.8823 1.29 – 0.09 23.40 – 1.09 1.11 – 0.10 90.40 – 0.43 61.44 – 1.33
Bold values indicate the optimized processing and formulation batch parameters which gives satisfactory results as compared to other formu-
lation batches

spherical shape and few were cylindrical shape due to Moisture uptake and stability studies
attrition during processing.
Moisture uptake study was conducted to check hygroscopic
Particle size distribution nature of the prepared microparticles (Rasenack and Muller
2002). No significant change in weight was observed after
The size distribution of the CBT microparticles was as subjecting the sample to accelerated conditions of tem-
showed in Fig. 6. Sieve analysis revealed that 98 % of the perature and humidity.
particles were in a size range of 400–1,190 lm, with the Hygroscopic nature of CBT inside the encapsulated for-
highest proportion of 85 % in a range of 400–841 lm. The mulation and pure CBT was useful to understand the deg-
average diameter of the microparticle was approximately radation effect of moisture. The encapsulated CBT
810 lm (Sinchaipanid et al. 2004). microparticles were heated inside the chamber using IR-

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Table 5 Moisture content and drug content of encapsulated CBT

after accelerated temperature conditions
Batch Moisture content (%) Drug content after accelerated
temperature conditions (%)

CBT2 1.2 ± 0.24 91.5 ± 1.2

CBT3 1.12 ± 0.22 91.98 ± 1.5
CBT4 0.94 ± 0.27 90.67 ± 1.3
CBT5 0.91 ± 0.29 90.51 ± 0.91
CBT6 0.95 ± 0.24 90.34 ± 0.78
CBT7 0.94 ± 0.21 90.28 ± 0.83
CBT8 0.85 ± 0.27 90.23 ± 0.89
CBT9 0.78 – 0.23 90.20 – 0.92
Bold values indicate the optimized processing and formulation batch
parameters which gives satisfactory results as compared to other
formulation batches

Fig. 5 Photomicrograph of CBT9

Table 6 Release of Encapsulated CBT in water
Batch Release of encapsulated CBT
in water for 5 h (%)

CBT2 33.54 ± 0.93

CBT3 34.45 ± 0.90
CBT4 35.43 ± 1.20
CBT5 35.65 ± 1.06
CBT6 34.57 ± 0.98
CBT7 31.56 ± 0.82
CBT8 30.65 ± 0.78
CBT 9 28.11 – 0.72
Bold values indicate the optimized processing and formulation batch
parameters which gives satisfactory results as compared to other
Fig. 6 Particle size distribution of microparticles formulation batches

light source for 3 min at 100, 103 and 105 °C to calculate

the moisture content in percentage which was displayed on
screen. It was hypothesized that the extent of moisture
absorption is directly proportional to the amount of hygro-
scopic surface area on the encapsulated particles (Rasenack
and Muller 2002). Thus, moisture absorption would be
indicative of the intimacy of mixing of HSO with polymer
matrix in encapsulated CBT. The Moisture content in
encapsulated CBT was found to be in the range of
0.78 ± 0.23–1.2 ± 0.24 % as showed in Table 5 which is
negligible and has no degradation effect on drug efficacy.
The drug content analysis was carried out after treatment
and observed to be in the range of 90.0 ± 1.6–91.5 ± 1.2 %
analysed by potentiometric titration method. Fig. 7 DSC thermo gram of a plain CBT b CBT9

Release of microencapsulated CBT

less than 35 % drug release in 5 h as showed in Table 6.
Microencapsulated CBT 9 formulation showed less than Therefore nature of coating offered significant protection
28.11 % drug release, while other formulations showed when the encapsulated choline exposed to water for 5 h.

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Solid state characterization with other excipients in the ratio of 1:1 showed no devia-
tion of endothermic peak of CBT. This revealed that there
Differential scanning calorimetry (DSC) analysis was no chemical interaction between drug and the wax.

The DSC studies revealed that CBT has prominent, char- Powder X-ray diffraction (PXRD) analysis
acteristic endothermic peak at 155.15 °C. Similar scans
were conducted for optimized microparticles which The x-ray diffractograms as shown in Fig. 8 showed sharp
showed no new additional endotherm present in the ther- multiple peaks, indicating the crystalline nature of the drug.
mogram of optimized microparticles as shown in Fig. 7. Plain CBT showed sharp peak at diffraction angle (2h)
This endothermic peak signified that CBT is in pure state in values of 11.280, 15.120, 16.520, 17.980, 19.060, 21.620,
microparticles based formulation. Physical mixture of CBT 21.820, 24.00, 26.300, 26.720 and 29.720 indicating

Fig. 8 X-RD diagrams a plain CBT and b CBT 9

Fig. 9 FT-IR spectra of a plain CBT b CBT 9

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crystalline nature of CBT as shown in Fig. 8. These char- Fourier transform infrared (FTIR) analysis
acteristic peaks indicated crystalline form of CBT. PXRD
study of microparticles revealed retention of crystalline FTIR spectroscopy was used to assess the interaction
property of CBT in final product. No significant change in between drug and polymer in a solid state. In order to
percent crystallinity was observed after coating of wax. evaluate any possible chemical interactions between the

Fig. 10 SEM microscopic images of a, b plain CBT. c–e of CBT9

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 Stabilization of choline salt

Table 7 Stability study of optimized CBT9 micro particle (packed in aluminium foil sachet)
Parameters Results
0 day 1 month 2 month 3 month
30 °C/65 % 40 °C/75 % 30 °C/65 % 40 °C/75 % 30 °C/65 % 40 °C/75 %

Description White free-flowing White free-flowing White free-flowing White free-flowing White free-flowing White free-flowing White free-flowing
Moisture content (%) 0.74 ± 0.21 0.79 ± 0.22 0.82 ± 0.25 0.83 ± 0.24 0.87 ± 0.28 0.94 ± 0.29 0.98 ± 0.31
Assay (%) 90.41 ± 0.42 90.50 ± 0.45 90.55 ± 0.46 90.52 ± 0.46 90.60 ± 0.49 90.60 ± 0.51 90.80 ± 0.53
Parameters Results
4 month 5 month 6 month
30 °C/65 % 40 °C/75 % 30 °C/65 % 40 °C/75 % 30 °C/65 % 40 °C/75 %

Description White free- Yellow spots on White free- Yellow spots Yellow spots on Yellow spots on
flowing granules and flowing on granules and granules and granules and
fishy odour fishy odour fishy odour fishy odour
Moisture content (%) 0.97 ± 0.32 1.40 ± 0.36 1.23 ± 0.29 1.47 ± 0.40 1.35 ± 0.31 1.67 ± 0.42
Assay (%) 90.80 ± 0.54 90.96 ± 0.55 90.82 ± 0.56 91.20 ± 0.58 90.82 ± 0.59 91.34 ± 0.60

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 A. B. Gangurde et al.

Table 8 Stability study of optimized CBT9 micro particle (packed in ambered colored glass bottles)
Parameters Results
0 day 1 month 2 month 3 month
30 °C/65 % 40 °C/75 % 30 °C/65 % 40 °C/75 % 30 °C/65 % 40 °C/75 %

Description White free- White free- White free- White free- White free- White free- White free-
flowing flowing flowing flowing flowing flowing flowing
Moisture content (%) 0.74 ± 0.21 0.64 ± 0.18 0.71 ± 0.19 0.69 ± 0.17 0.87 ± 0.22 0.84 ± 0.21 0.92 ± 0.24
Assay (%) 90.41 ± 0.41 90.43 ± 0.43 90.45 ± 0.46 90.48 ± 0.45 90.60 ± 0.47 90.56 ± 0.48 90.71 ± 0.49
Parameters Results
4 month 5 month 6 month
30 °C/65 % 40 °C/75 % 30 °C/65 % 40 °C/75 % 30 °C/65 % 40 °C/75 %

Description White free-flowing White free-flowing White free-flowing White free-flowing White free-flowing White free-flowing
Moisture content (%) 0.87 ± 0.25 1.10 ± 0.29 0.91 ± 0.31 1.17 ± 0.32 0.98 ± 0.35 1.27 ± 0.35
Assay (%) 90.61 ± 0.51 90.78 ± 0.53 90.79 ± 0.54 90.91 ± 0.56 90.92 ± 0.57 91.02 ± 0.59

drug and carriers, spectra of plain CBT and CBT 9 were conditions. But, after 6 months accelerated stability study
examined. FTIR spectra of plain CBT showed a stretching yellow spots on microparticle and fishy odour was
vibration at 3,322.82 and 3,345.43 cm-1 indicating pre- observed as shown in Table 7. While the microparticles
sence of O–H group in tartrate and choline respectively, C– packed in ambered colored glass bottles exhibited no signs
H stretching at 2,918.82 cm-1 indicating presence of –CH of discoloration and odor was developed after 6 months
group as shown in Fig. 9. FTIR spectra of optimized mi- storage at accelerated conditions as shown in Table 8.
croparticles, showed retention of all the major peaks of Also, there was no significant change in moisture content
which indicate absence of chemical interaction between and drug content. Therefore, for stability purpose obtained
wax and drug during processing. micro particles should be packed in amber colored glass
bottles.
Scanning electron microscopy (SEM)

SEM study was performed to understand the physical Conclusion

morphology of drug and coated microparticle. Plain CBT
Fig. 10a, b was found as irregular shape crystalline powder Encapsulation of CBT using HSO in a completely aqueous
and with higher magnification roughness on the surface of environment, with high encapsulation efficiency was suc-
the powder was observed. The microparticles prepared cessfully done in FBP. Microparticles of CBT was suc-
with HSO of CBT9 Fig. 10c, d showed non-spherical shape cessfully prepared by aqueous wax coating dispersion of
due to premature solidification of some molten wax droplet hydrogenated soya bean oil using FBP without the use of
and non-uniform coating pattern of bottom spray coater, jacketed vessel container which is commonly used for lipid
but as more spherical than plain CBT. The higher magni- coating of drugs. Optimization of processing variables and
fication Fig. 10e showed numerous pores on the texture of concentration of wax was successfully done in FBP.
micro particles because of rapid evaporation of water Obtained microparticles showed good physical and mor-
during drying process. phological properties compared to Plain CBT. Optimized
microparticles of batch CBT 9 was stable for 6 months at
Stability studies accelerated stability conditions when packed in amber
colored glass bottles.
Microparticles of optimized batch CBT9 packed in alu-
minium foil sachet exhibited no sign of discoloration and Acknowledgments This article does not contain any studies with
human and animal subjects performed by any of the authors. All
odor development after 3 months storage at accelerated authors (AB Gangurde, RA Fule, SD Javeer, RK Patole, J Pawar and
conditions. Also, there was no significant change in the PD Amin) declare that they have no conflict of interest.The authors
moisture content and drug content. The obtained micro- are thankful to Bajaj healthcare limited, India, for providing the gift
particles were stable for 3 months at accelerated stability sample of choline bitartrate and Hydrogenated soya bean oil and

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Stabilization of choline salt

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providing all facilities and guidance. microencapsulation by film-coating. Int J Pharm 180(2):195–210
Jannin V, Cuppok Y (2012) Hot-melt coating with lipid excipients.
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