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LUČKA BIBIČ
FORMULATION OF ALBENDAZOLE MICROCAPSULES FOR THE ORAL
ADMINISTRATION IN A SEMI-SOLID CARRIER
MASTER’S THESIS
Ljubljana, 2014
LUČKA BIBIČ
FORMULATION OF ALBENDAZOLE MICROCAPSULES FOR THE ORAL
ADMINISTRATION IN A SEMI-SOLID CARRIER
RAZVOJ MIKROKAPSUL Z ALBENDAZOLOM V POLTRDNEM VEHIKLU ZA
PERORALNO JEMANJE
Academic supervisor: Prof. Dr. Mirjana Gašperlin
Academic co-supervisor: Assoc. Prof. Dr. Daniel Bar-Shalom
Ljubljana, 2014
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This Master’s thesis was done at the Faculty of Pharmaceutical Sciences (Rational Oral Drug Delivery
Group), University of Copenhagen under the academic supervision of Prof. Dr. Mirjana Gašperlin
from the Faculty of pharmacy, University of Ljubljana, Slovenia and co-supervision of Assoc. Prof. Dr.
Daniel Bar-Shalom, from the Faculty of Pharmaceutical Sciences, University of Copenhagen,
Denmark.
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Statement
I hereby declare that this Master’s thesis was done by me, Lučka Bibič, under the academic
supervision of Prof. Dr. Mirjana Gašperlin from the Faculty of pharmacy, University of Ljubljana,
Slovenia and co-supervision of Assoc. Prof. Dr. Daniel Bar-Shalom from the Faculty of Pharmaceutical
Sciences, University of Copenhagen, Denmark.
Lučka Bibič
Head of Committee: Prof. Dr. Janko Kos
Member of Committee: Doc. Dr. Jurij Trontelj
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Just keep swimming.
[Nemo]
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ACKNOWLEDGEMENTS
I’m not sure if anyone reads the acknowledgments, but I hope so because without the following
people, it would have been nearly impossible to create this piece of work.
First of all, I would like to acknowledge my co-supervisor, Assoc. Prof. Daniel Bar-Shalom. Your
brilliant ideas and pearls of wisdom, accompanied with the enormous experience and knowledge
inspired me through my Master thesis’s path and helped me realize the power of critical and creative
thinking. I would like to thank you for general guidance on writing scientific papers and putting up
patiently with my long barrages of e-mails while providing amazingly timely feedback. I really
appreciate your willingness to treat me as a future colleague during my stay at the Faculty of
Pharmaceutical Sciences in Copenhagen and let me experience this research project beyond the
scope of the textbooks.
My next greeting goes to Prof. Mirjana Gašperlin, my supervisor at the Faculty of Pharmacy in
Ljubljana. It was my privilege to have you as my supervisor and I would like to say one big “Thank
you!” - for your patient, care and editorial guidance. Your kindness and reassuring words mean a
world to me.
I am indebted to all the great people working at the Department of Pharmaceutics and Analytical
Chemistry at the Faculty of Pharmaceutical Sciences in Copenhagen. Special thanks go to the RODD
group on the 3rd floor for providing a great working atmosphere with plenty of support, genuine
interest and good company. It would be a lonely lab without all of you.
I also want to express my sincere gratitude to Kaisa, Jette, Charlotte, Tonie, Dorte, Alan, Martin and
Yun. I really appreciate that you always manage to be available when discussions and guidance were
needed and that some of you always found the time for a coffee break, when I was buffeted by the
winds of uncertainty.
I am also very grateful to all the children and their caring parents at the Hvidovre Hospital in
Denmark, Pediatric Department, for letting us close. Thank you kids for the invaluable insights into
this thesis’s topic.
Pharmaceutical study turned out to be a long 5-years battle against the small molecules. Therefore, I
wish to thank to all of my great friends for keeping me sane. I am superlatively thankful that you
understood my mental pauses from time to time. Nina, Rok, Vid, Katarina, Špela, Barbara, Maruša,
Mariša and Maša, thanks for reminding me, what really matters in life.
It is hard to overstate my gratitude to my father Vojko and both my brothers, Klemen and Jošt, who
are probably saying they do not need thanking. I am also grateful to both my grandmothers and
grandfathers – I bet you are proud of me.
Finally, a loving thanks to my mother, Renata, who helped and lead me through my first
pharmaceutical breakthrough and who still managed to save some nerves. Thanks mom for putting
up patiently with my frantic Danish calls at 2am, when I needed to know why my HPLC method is not
working properly.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ........................................................................................................................... 6
TABLE OF CONTENTS ............................................................................................................................... 7
LIST OF TABLES ........................................................................................................................................ 9
LIST OF FIGURES .................................................................................................................................... 10
LIST OF GRAPHS ..................................................................................................................................... 10
ABSTRACT .............................................................................................................................................. 11
POVZETEK .............................................................................................................................................. 12
LIST OF ABBREVIATIONS ........................................................................................................................ 14
1. BACKGROUND ............................................................................................................................... 15
1.1. Parasitic Helminths in the Third World Countries ................................................................. 15
1.2. Pediatric drug formulation .................................................................................................... 15
1.2.1. UNICEF gel ..................................................................................................................... 16
1.3. Active Pharmaceutical Ingredient (API) ................................................................................. 17
1.3.1. Albendazole (ALB).......................................................................................................... 18
1.3.2. Caffeine (CAF) ................................................................................................................ 18
1.4. Microcapsules as a Drug Delivery System ............................................................................. 19
1.4.1. Definition ....................................................................................................................... 19
1.4.2. Microencapsulation (MEC) operation ........................................................................... 20
1.4.3. Microencapsulation (MEC) materials ............................................................................ 22
1.4.4. Pediatric patients and taste concealing ........................................................................ 23
1.5. Pharmacokinetic barriers in anthelmintic action and the role of dissolution ....................... 25
1.5.1. BCS Class II Drugs and IVIVC .......................................................................................... 26
1.5.2. Solubility ........................................................................................................................ 26
1.5.3. Taste-Concealing drives Dissolution .............................................................................. 27
1.5.4. Dissolution rate ............................................................................................................. 27
1.5.5. In vitro methods ............................................................................................................ 28
1.6. HPLC Analysis ......................................................................................................................... 28
1.6.1. Method Development for RP-HPLC ............................................................................... 29
1.6.2. Method Validation ......................................................................................................... 30
2. AIM ................................................................................................................................................ 30
3. MATERIALS and METHODS ............................................................................................................ 32
3.1. Equipment ............................................................................................................................. 32
3.2. Materials ................................................................................................................................ 33
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Methyl Cellulose (MC) Taste-Concealing .............................................................................................. 33
3.3. Methods ................................................................................................................................ 35
3.3.1. Characterization of ALB ................................................................................................. 35
3.3.2. Microencapsulation (MEC) ............................................................................................ 37
3.3.3. Dissolution ..................................................................................................................... 41
3.3.4. HPLC Analysis ................................................................................................................. 43
3.3.5. UV Spectroscopy ............................................................................................................ 47
3.3.6. Student t-test................................................................................................................. 47
4. RESULTS and DISCUSSION ............................................................................................................. 48
4.1. Characterization of ALB ......................................................................................................... 48
4.1.1. Identification of ALB by LC/MS ...................................................................................... 48
4.1.2. Particle size distribution (PSD) ...................................................................................... 48
4.1.3. Determination of bulk, tapped densities and flow properties ...................................... 50
4.1.4. Morphology determination of powders, granules and MIC ......................................... 51
4.2. Microencapsulation ............................................................................................................... 53
4.2.1. Determination of wall thickness of the MICs ................................................................ 53
4.3. Dissolution ............................................................................................................................. 54
4.3.1. Granules ........................................................................................................................ 54
4.3.2. Microcapsules for taste-concealing............................................................................... 58
4.3.3. ALB dissolution studies ................................................................................................. 61
4.3.4. Microcapsules and granules/ MICs in 0,1M HCl and in MilliQ Water ........................... 62
4.4. UV Spectroscopic measurements and t-test ......................................................................... 65
4.5. HPLC Analysis ......................................................................................................................... 65
4.5.1. Analysis for validation ................................................................................................... 65
4.5.2. Analysis for drug content estimation ............................................................................ 70
5. CONCLUSION ................................................................................................................................. 71
6. FUTURE RESEARCH SUGGESTIONS ................................................................................................ 73
7. REFERENCES .................................................................................................................................. 74
8. APPENDICES ................................................................................................................................... 84
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LIST OF TABLES
Table 1: Physicochemical properties of ALB and CAF -------------------------------------------------------------- 17
Table 2: The main variables that affect the granule's quality ---------------------------------------------------- 21
Table 3: Apparatuses for manufacturing and analysis of MEC -------------------------------------------------- 32
Table 4: Substances for manufacturing the granules -------------------------------------------------------------- 33
Table 5: Substances for manufacturing the coated granules ---------------------------------------------------- 33
Table 6: Chemicals for the UNICEF gel --------------------------------------------------------------------------------- 34
Table 7: Chemicals for HPLC analysis ---------------------------------------------------------------------------------- 34
Table 8: Chemicals used in the dissolution test --------------------------------------------------------------------- 34
Table 9: Twelve different formulations were prepared in the granulation experiments ----------------- 38
Table 10: the composition of the MC coating suspension -------------------------------------------------------- 39
Table 11: Process conditions for coating in fluid bed -------------------------------------------------------------- 39
Table 12: Excipients used for preparation of UNICEF gel ------------------------------------------------------ 43
Table 13: Calculated Bulk, Tap Densities and flow properties (CI and HR) of ALB, Dextrin powder,
granules and MIC ----------------------------------------------------------------------------------------------------------- 50
Table 14: Measured and calculated variables for the determination of the microcapsules’s wall
thickness ---------------------------------------------------------------------------------------------------------------------- 53
Table 15: Precision results from the nine replicate injections of ALB with their mean tr and Area with
the calculated RSD value -------------------------------------------------------------------------------------------------- 67
Table 16: Precision results from the nine replicate injections of CAF with their mean tr and Area with
the calculated RSD value -------------------------------------------------------------------------------------------------- 68
Table 17: Ruggedness results from the nine replicate injections of ALB with their mean tr and Area
with the calculated RSD value ------------------------------------------------------------------------------------------- 68
Table 18: Rugedness results from the nine replicate injections of CAF with their mean tr and Area
with the calculated RSD value ------------------------------------------------------------------------------------------- 69
Table 19: Results from the six replicate injections of ALB and CAF with their mean Area, LOD and LOQ
---------------------------------------------------------------------------------------------------------------------------------- 69
Table 20: Albendazole encapsulation efficiency for granules and microcapsules -------------------------- 70
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LIST OF FIGURES
Figure 1: Granulation process ------------------------------------------------------------------------------------------- 20
Figure 2: The process of coating in the fluid bed with a Wurster partition. ---------------------------------- 22
Figure 3: Modified Kenwood Food Processor -------------------------------------------------------------------- 32
Figure 4: Fluid bed MiniGlatt -------------------------------------------------------------------- 32
Figure 5: The pouch with the UNICEF gel ------------------------------------------------------ 43
Figure 6: Granules FOR6 size distribution with the granules diameter range from 75 μm to 177 μm- 49
Figure 7: MICs size distribution with the diameter range from 134 μm to 340 μm ------------------------ 49
Figure 8: SEM images magnification series (x75and x1000 respectively) of the granules ---------------- 52
Figure 9: SEM images magnification series (x35 and x750 respectively) of the MIC ----------------------- 52
LIST OF GRAPHS
Graph 1: Drug release profiles of ALB formulations FOR1-FOR5 in water within 30 min time range - 54
Graph 2: Drug release profiles of ALB formulations FOR6-FOR12 in water within 30 min time range 54
Graph 3: ALB release from uncoated, coated FOR6 granules and coated FOR6 in UNICEF gel with ALB
pure drug release ----------------------------------------------------------------------------------------------------------- 58
Graph 4: ALB release without and with the incorporation into the UNICEF gel ----------------------------- 61
Graph 5: ALB release from MICs and granules in 0,1M HCL and water --------------------------------------- 62
Graph 6: Standard curve for ALB ---------------------------------------------------------------------------------------- 66
Graph 7: Standard curve for CAF ---------------------------------------------------------------------------------------- 66
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ABSTRACT
Albendazole (ALB) is an anthelmintic drug, classified as BCS type II compound, mainly used in the
pediatric population in the third world countries. As the main setback of ALB is its low aqueous
solubility (1 μg/ml), the enhancement of ALB release rate is essential. Another main disadvantage of
oral drug delivery for pediatrics is the children’s inability to swallow. That is why a semi-solid
formulation might be a better oral dosage form. Because the bitter taste of ALB might deter children
from consuming the medicine, a microencapsulation technique would be a valuable achievement in
order to avoid ALB unpleasant taste. Furthermore, it is predicted that semi-solid formulation,
consisting of “UNICEF gel”, an HPMC based gel, with the incorporated taste-concealedmicroparticles,
should overcome this major problems.
In the first part of this thesis, a quantitative particle-size analysis, determination of flow properties
and LC/MS identification of ALB were carried out. Following that step, the granulation process, with
the various compositions of the numerous excipients, was conducted. After the granulation, the
appropriate polymer, dextrin, was chosen, according to the dissolution studies. In order to
quantitatively determine the ALB content and release from granules and microcapsules (MICs), a
rapid RP-HPLC method was developed, optimized and validated. In the second stage, a fluid bed
technique with a Wurster partition was selected for the coating with the Methyl Cellulose (MC) as a
potential taste-concealing polymer. Once the ALB microcapsules were MC-coated, they were
incorporated into UNICEF-gel and further dissolution studies were conducted at the neutral pH and
pH 1.2. Finally, spectroscopy was used in order to evaluate the ALB release in the oral cavity and
stomach from the MICs and the final semi-solid formulation.
The results obtained from our studies, suggest that it is possible to achieve a micro sized product of
poorly soluble drug ALB with the defined and narrow particle size distribution. Furthermore, dextrin
not only gives a good coverage of the core material, resulting in the smooth and uniform surface and
provides high entrapment efficiency, but also greatly improves the ALB release. The ALB dissolution
of the pure drug was 7,80% ± 0,39% in 30 min, whereas 93,15% ± 0,34% and 67,36% ± 1,19% of the
ALB present in uncoated (dextrin granules) and coated MICs, respectively, dissolved within the same
time range. Thus, a remarkably surprising ALB release from the MICs was achieved. It has also been
found that MICs incorporated into UNICEF gel might sufficiently conceal the bitterness of ALB for the
first 2 min.
Keywords:albendazole, microencapsulation, oral drug delivery, fluid bed, coating, granulation,
dissolution, taste-concealing, methyl-cellulose, dextrin, HPLC, semi-solid, pediatric
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POVZETEK
Albendazol (ALB) je antihelmintična zdravilna učinkovina, uvrščena v skupino II po biofarmacevtskem
klasifikacijskem sistemu (BCS), ki se pretežno uporablja v nerazvitih državah sveta pri pediatrični
populaciji. Ker je ena izmed glavnih pomanjkljivosti ALB je njegova nizka topnost v vodnem mediju
(manj kot 1 μg/ml), je ključnega pomena povečati sproščanje učinkovine, katere absorpcija je
omejena s hitrostjo raztapljanja. Druga pomanjkljivost peroralne dostave učinkovine pri pediatrični
populaciji pa je nezmožnost požiranja trdnih oblik pri mlajših otrocih. Eno od ustreznih rešitev tega
problema predstavlja poltrden vehikel. Z metodo mikrokapsuliranja sem želela izboljšati grenak okus
ALB, saj ta odločilno vpliva na otrokovo percepcijo jemanja zdravila. Zaradi tega poltrdna formulacija,
sestavljena iz UNICEF gela (HPMC gela), skupaj z vgrajenimi mikrokapsulami ALB, predstavlja možno
rešitev pri premagovanju problema požiranja pri otrocih.
V prvem delu naloge je bila izvedena kvantitativna analiza velikosti delcev, določitev pretočnih
lastnosti in test identifikacije molekule ALB z LC/MS. Sledili so številni procesi granuliranja z različnimi
količinami in vrstami pomožnih snovi. Po granuliranju je bil, glede na rezultate študij raztapljanja,
izbran najprimernejši polimer – dekstrin. Z namenom kvantitativne določitve količine ALB v granulah
in mikrokapsulah ter odstotka sproščene učinkovine iz obeh farmacevtskih oblik smo razviliRP-HPLC
metodo in potrdili njena ustreznost. V drugem delu študije smo izvedli oblaganje zrnc z
vrtinčnoslojno tehnologijo(Wurster). Kot polimer za oblaganje je bila uporabljena metilceluloza (MC)
s potencialno sposobnostjo prekrivanja neželenega okusa. Po oblaganju z MC smo mikrokapsule z
ALB vgradili v UNICEF gel in opravili številne študije raztapljanja pri nevtralnem in kislem pH. Na
koncu smo uporabili UV spektroskopijo za ovrednotenje sproščanja količine ALB iz ustne votline in
želodčnega soka, tako iz samih mikrokapsul kot mikrokapsul, predhodno vgrajenih v poltrdni vehikel.
Rezultati magistrske naloge potrjujejo, da je možno narediti mikrokapsule s težko topno zdravilno
učinkovino ALB z definirano velikostjo in ozko porazdelitvijo delcev. Hkrati je bilo ugotovljeno, da
dekstrin ne samoda omogoča ustrezno pokrivnost jedra mikrokapsul, kar se kaže v gladki in
enakomerni površini, temveč tudi zagotavlja dobro ujetost učinkovine v zrncih ter močno izboljša
količino sproščenega ALB, kar potrjujejo rezultati testa raztapljanja (po 30 min. je bilo količina
sproščenega ALB v le 7,80% ± 0,39%, medtem ko se je zrnc z dekstrinom in obloženih mikrokapsul z
MC sprostilo 93,15% ± 0,34% in 67,36% ± 1,19% v ALB enakem času).
Doseženo je bilo presenetljivo sproščanje ALB iz mikrokapsul in ugotovljeno, da mikrokapsule,
predhodno vgrajene v UNICEF-gel, zadovoljivo prekrijejo grenkobo ALB v prvih dveh minutah
sproščanja učinkovine.
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Ključne besede: albendazol, mikrokapsuliranje , peroralna dostava, vrtinčnoslojna tehnologija,
oblaganje, granuliranje, raztapljanje, prekrivanje okusa, metilceluloza, dekstrin, HPLC,poltrdni
vehikel, pediatrija
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LIST OF ABBREVIATIONS
AIDS = Acquired Immune Deficiency Syndrome
ALB = Albendazole
API = Active Pharmaceutical Ingredient
ATP = Adenosine Triphosphate
BCS= Biopharmaceutical Classification System
CAF = Caffeine
CCS = Croscarmellose Sodium
CI = Carr’s Compressibility Index
CIKDEX = Cyclodextrin Beta
DEX = Dextrin
EPA = Environmental Protection Agency
ESI = Electro-Spray Ionization
FDA = Food and Drug Administration
FOR = Formulation
GR = Granules
GR FOR6 = Granules of Formulation 6
H= Wall thickness
HAc = Acetic Acid
HIV = Human Immunodeficiency virus
HPMC = Hydroxypropylmethycellulose
HPLC = High-Performance Liquid
Chromatography
HR = Hausner Ratio
ID = Internal Diameter
IR = Immediate Release
ISO = International Standards Organization
IVIVC = In Vitro In Vivo Correlation
LC = Liquid Chromatography
LOD = Limit of Detection
Log P = Partition Coefficient
LOQ = Limit of Quantification
KCl = Potassium Chloride
MEC = Microencapsulation
MC =Methyl Cellulose
MIC = Microcapsule
MICs = Microcapsules
MP = Mobile Phrase
MS = Mass Spectroscopy
NO. = Number
OTC = Over the Counter
PH EUR = European Pharmacopoeia
PIP = Pediatric investigation Plan
PSD = Particle Size Distribution
PVP = Polyvinylpyrrolidone
RP = Reverse Phrase
SA = Surface Area
SD = Standard Deviation
SEM = Scanning Electron Microscope
SP = Stationary Phrase
STH = Soil-Transmitted Helminth
UNICEF = United Nations Children’s Fund
USP = United States Pharmacopoeia
VH20 = Volume of Water
WAR = Water Addition Rate
WHO= World Health Organization
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1. BACKGROUND
1.1. Parasitic Helminths in the Third World Countries
Globally, soil-transmitted helminth (STH) infections constitute one of the most significant neglected
infection clusters of our time. (1,2) It is estimated that there are more than two billion cases
worldwide, of which 450 million have attributed a major morbidity due to their infection; the
majority of whom are children between 5-14 years. (3) In addition, the infectious disease is the most
common reason for death of children, which are younger than 5 years, predominantly in the low-
income countries. (4)Black et al.estimated that among the 8.80 million children’s deaths, all under
the age of 5, two-third were due to infectious diseases. (5)
The World Health Organization (WHO) and the United Nations Children's Fund (UNICEF), among
others, suggested deworming programs targeting school-age children to reduce STH burden of
disease. (6)(7)The principal intervention available for controlling helminthes infections is the periodic
administration of one of the four anthelmintic drugs recommended by WHO, of which
benzimidazoles – mebendazole and albendazole are the most regularly used anthelmintics for the
treatment of STH infections. (1)
Furthermore, Bentwichet al. (3) outlined that intestinal helminth infections might have an effect on
the vaccination efficacy for HIV and tuberculosis due to the increased susceptibility of the host’s
immune system and spread of the diseases. Consequently, eradication of helminth infections may
have a major impact on AIDS and tuberculosis in the developing countries and also carry possible
benefits in the disease pathogenesis.
1.2. Pediatric drug formulation
Pediatric drug delivery is a major challenge in the drug development. Focusing on the factors such as
solubility and taste of the formulation, development might be technically demanding and time
consuming. Yet, some drugs compulsory for pediatric patients are not commercially accessible in
dosage forms that should be suitable for use in this population.(4,8)
There is a safety concern when unsuitable medicines are administrated to children.
It is highly improbable that a “one-size-fits-all” solution will provide the best outcome, but with the
current increase in interest and research, an upward trend for better pediatric dosage forms is likely
to follow. (4,9)
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A new pediatric regulation came into operation in the European Union on the January 2007 and the
regulatory environment for pediatrics in Europe was dramatically changed. Its objective is to improve
the children’s health by promoting the development of medicines as well as ensuring the high quality
and appropriate authorization. These pediatric trials must be conducted according to a detailed
pediatric investigation plan (PIP), which ought to demonstrate the quality, safety and efficacy of the
therapeutic product for the pediatric population. It is truly optimistic to know that new campaigns
have been launched to accelerate pediatric drug development. For example, “Make medicines child
size” is a worldwide initiative under the leadership of WHO, which is promoting the need for the age-
appropriate formulation developments. To sum up, this initiatives aim at that pediatric drug
development will become an integral part of the development of medicinal products. (4,10,11)
So far, there is a lack of suitable and safe drug formulations for children. (4) Pediatric oral dosage
forms include mostly tablets, emulsions and suspensions. (12) However, with the liquid formulations,
a number of drawbacks have been shown: difficulties in transport, storage and handling, stability
issues and poor dose uniformity when using spoons and dosing cups. (13) In addition, finding non-
toxic excipients and masking the taste is harder to achieve than for solid formulations. (4)
The main disadvantage of oral drug delivery for the young children is difficulty or inability to swallow.
(9) Hence, there is a need for a semi-solid composition that behaves like a liquid when consumed and
yet, acts like a solid in many other ways. In such formulation, chewing and biting would not be
necessary, which explains their ideal nature for being use in the pediatric field. Moreover, semi-solid
formulations enable better taste, mouth feel and storage stability. (12,14)In Krause&Breitkreutz(4)
review it had been noted that small-sized multiparticles can be swallowed together with the liquid
food by very young children, aged from 6 months, who are unable to receive solid oral dosage form.
In this thesis, a semi-solid drug formulation with incorporated microparticles will be developed. The
dosage form would primary be meant for toddlers and children, between two and five years,
regarding that the main disadvantage of oral drug delivery them is difficulty or inability to swallow,
especially in the case of the bitter drugs.
1.2.1. UNICEF gel
It was reasoned before, that children are not fond of syrups as a liquid formulation. (15,16)
In addition, Bar-Shalom (17) proposed to formulate a dosage form that should resemble something
children are used to take, for instance a dry pudding, where the microencapsulated drug would swell
into the pudding-like vehicle - UNICEF gel. (17,18)
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Small-sized multiparticles are thus a valuable alternative to the liquid formulations. Since the drug
will be suspended in the pudding, the particles should be microparticles (75 – 200 µm). If smaller, the
particles can be trapped between the taste buds on the tongue, whereas if bigger, a child could find
the grittiness objectionable. (17)
To produce a gel, a fast hydrating quality of Hydroxypropyl methylcellulose (HPMC), as a gelling
agent, was used in the vehicles. The vehicle should be attractive for the target group in terms of
taste, viscosity, texture and appearance, and possess properties like being fast hydrating and
easy to prepare.(18)
1.3. Active Pharmaceutical Ingredient (API)
In this thesis, albendazole (ALB) has been investigated as a main compound and caffeine (CAF) as a
model drug. In Table 1, the physicochemical properties of ALB and CAF are seen.
Table 1: Physicochemical properties of ALB and CAF
Physicochemical property / Drug ALBENDAZOLE(19,20) CAFFEINE (19,21,22)
BCS Class II I
MW 265,3 194,2
Structural formula
Chemical Formula C12H15N3O2S C8H10N4O2
Melting Point 209 °C 238 °C
pKa 2,68 (pKa1)and 11,83 (pKa2) 14,0
Aqueous solubility1in buffer pH 6,0 1 μg/ml 20 mg/ml
Log P 3,5 (predicted) and 2,7
(experimental)
-0,07 (predicted)and
-0,55 (experimental)
Absorption Poorly absorbed from the
gastrointestinal tract
Readily absorbed after
oral administration
1According to Wu, Zimei, et al, water solubility is 0,61 μg/L. (23)Nevertheless, the study from Bassani, V.L., et.al., shows that
water solubility at pH 7,2 is even lower; 0,2 μg/L. (24)
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1.3.1. Albendazole (ALB)
ALB is a broad-spectrum anthelmintic which is used to treat infections caused by worms. ALB is a
benzimidazole drug whose selectivity is known to be 250 – 400 times more potent in helminth than
in mammalian tissue.(2)
ALB mechanism of action is to keep the worm from absorbing glucose, which leads to the decreased
production of adenosine triphosphate (ATP). ATP then causes the depletion of vital energy for the
worm’s endurance. Due to diminished energy production, the worm is immobilized and ultimately
dies.(20)
Furthermore, ALB is a potential anticancer agent that is currently under development for the
treatment of cancer. (25)
The majority of the orally administrated drugs taste very bitter and ALB is no exception.
(24,25,26,27) Thus, one of the main drawbacks of ALB is its bitter taste and oral administration of
ALB is a key issue for the formulation scientist since the drug acts on the parasites in the
gastrointestinal lumen.
An additional challenge, when ALB is concerned, includes ALB’s low aqueous solubility. (26)Regarding
low ALB solubility and its high partition, the drug is classified as BCS type II compound. Moreover,
ALB is known to be practically insoluble in both gastric and intestinal fluids. Slow release from the
stomach increases the time for ALB to dissolve before entering the intestine, thus favoring increased
bioavailability. (20,24)Thereby, achieving better dissolution in the stomach would decrease the time
for ALB to dissolve before entering the small intestinal lumen, where the helminth parasites are
presented.
As with other poorly soluble compounds, the dissolution rate is likely to be dependent on the
formulation. (24) One of the aims in this work is to improve the dissolved percentage of ALB in the
stomach, which could consequently lead to better exposure to the parasites in the intestine and
enhanced antiparasitic activity of ALB.
1.3.2. Caffeine (CAF)
In this study, CAF has been chosen as a model drug. This high-water soluble and bitter xantine
derivate was mainly selected to investigate the patency of the taste concealing.
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That is to ensure if the taste concealing coat could remain unobstructed and, consequently, if the
drugs release rate from a microencapsulated dosage form, containing ALB and CAF, can be limited for
that reason.
1.4. Microcapsules as a Drug Delivery System
1.4.1. Definition
“Microencapsulation is both an art and a science.” (28)
By the Allen et al. (29), the microencapsulation (MEC) is a drug delivery technique, defined as the
entrapment of solids, liquids or gases in the microscopic particles. This encapsulation is achieved by
the deposition of a thin layer of coat material around the core. Others, Banker et al. (30) define MEC
as a process where the particles of core material, either solid or liquid in nature, are enclosed within
a thin wall of the polymer.
There are numerous reasons for MEC: concealing the bitter taste of drugs; reducing GIT irritation;
easier handling and storage; reducing the hygroscopic properties of core material; reducing the drugs
odor and volatility; protecting the core material against atmospheric effects and separating the
incompatible substances. In addition, MEC can be used as a controlled release delivery system. (28)
In this study, the microcapsules (MICs) have been prepared by two separated production processes:
wet granulation, with a modified Kenwood Food Processor, followed by film coating using fluid bed
equipment.
1.4.1.1. Wet granulation
Benali et al. (31) define the granulation as the process where particles agglomerate into bigger
aggregates (granules) where the initial particles could, yet, be identified. (31,32) With the
granulation, we may: improve flow properties of the powders and hence the uniformity of the dose;
increase the bulk density of a product; decrease dust generation and, last but not least, improve the
product appearance.(29) That is why this process is one of the powerful unit operations in the
production of pharmaceutical oral dosage forms. (33)
Granulation methods are divided into two separate types: dry granulation and wet granulation. (34)
In the wet granulation process, the granulation is typically performed in a tumbling drum, a fluidized
bed, a high shear mixer or similar apparatus.
P a g e | 20
Wetting and nucleation consolidation and growth attrition and breakage
After the particles are placed in the equipment, the agitation starts and the addition of a liquid,
commonly water, is added in a pre-defined range of time. (31)
The major advantage of wet granulation is, among others, its feature to make hydrophobic surfaces
hydrophilic with increased sphericity and uniformity. (32,34,35)
However, the preferred product’s attributes are not controlled just by choosing the right powder
mass and liquid, since the granulator type and operating parameters, as seen in Table 2, are also of a
great importance.(35)It is fairly common to see granulation as three-stage process, starting with the
wetting and nucleation, followed by the consolidation and growth, and at the end there comes the
attrition and breakage:
Figure 1: Granulation process
1.4.1.1. Coating
Coating is defined as the process of application when a liquid material, which is applied on the
surface, results in either a continuous or discontinuous film after drying. (36,37) This shell-like barrier
structure provides a shelter of a core material that may be enclosed in the particle, its surface or
entrapped within the coating layer. (29,38) Coating of the solid particles is considered to have
countless advantages, among them also the taste concealing that was the main reason for MEC in
this thesis.
1.4.2. Microencapsulation (MEC) operation
1.4.2.1. Granulation in a modified food processor Kenwood
The use of the smaller scale mixer (food processor) was choose according to the time and material
efficiency issues as more experiments can be conducted using smaller quantity of material and less
time.
The modified food processor is, when compared with the high-shear mixers, the equipment without
the chopper and with the different operating impeller.
P a g e | 21
In recent years, many experiments, for instance from Michaels et al. (39), established a belief that
chopper has no effect on the granule size distribution. On the other hand, Schaefer et al. (40)well –
documented study suggested that chopper’s effect vaguely decreases the mean particle size and
narrows the PSD.
Fu, J. S., et al.(41)has established a belief that when different features of granules, made with high-
shear mixer and food processor, are questioned, a good correlation between the results can be
established.
Therefore it can be suggested that granulation process in the food processor resembles the one use
in the high shear mixer. In high shear granulation, as well as in the food processor, the powder is
constantly agitated by the impeller and a binder solution is sprayed from the top. (42,43,44)The first
nuclei is rapidly made as the liquid droplets are dispersed into the powder, while the growth of the
inseparable, and thus larger, granules (agglomerates) is prevented by the agitation force. When
mixing and spraying persist, the agglomerates develop into harder particles with more adhesive
surface and hence enlarge. (42,45) The principal difficulty, concerning the wet granulation in the
both of the mixers, is the control of the end point. (46) Whilst an excess of liquid can form a slurry or
unmanageable growth of the particles, the granulation process is supposed to be stopped. (47)
The main variables affecting the quality of the granules are seen in Table 2. (31,45,47,48,49,50)
Table 2: The main variables that affect the granule's quality
1.4.2.2. Coating in a fluid bed with a Wurster partition
The requirement for the coated particles is constantly emerging in the pharmaceutical area. (51)
There are few apparatus which can be applied for the coating of pellets, granules and powders, with
the fluidized bed as the most common one. (52)
Material parameters Granulation condition in the
mixer Granule properties
Powder particle size distribution (PSD) Impeller and Chopper Speed PSD
Wettability of the solid by the liquid Massing time Bulk density and
porosity
Binder concentration and viscosity Load of mixer (generally 2/3) Flow properties
Solid solubility and degree of swelling and
binder liquid
Liquid spray rate Flow properties
Quantity of the solvent Drug content
uniformity
Temperature Binder distribution
P a g e | 22
When spraying of the coating material is concerned, three basic designs are commonly known: top-
spray, side spray and bottom-spray (Wurster). (52) The last configuration (Wurster on Figure 2) has
been extensively studied to be a powerful and most acceptable tool for the coating of small
particles. (33,53) A nozzle and a bottom-spray tube are located right in the middle of a perforated
distributor plate, which allows the granules to flow in a predictable circulating trajectory. (51,54)
During the coating process there is a short distance between the coating materials and particles,
which greatly contributes to the high coating uniformity and coating efficiency. (55)
Numerous investigations, regarding the particle circulation in the Wurster, have been published.
Experiments from Teunouet al. (52), Becheret al. (56), Ström et al.(57), Tzika et al.(58), Arimoto et al.
(59) and Christansenet al. (60) have evaluated the Wurster’s impact of the coating thickness and
quality of the layer.
They all hold a common belief that the
bottom spray design (Figure 2) is the most
proficient fluidized bed configuration for
coating when the high-quality of the coated
layer is essential. Furthermore, Hampel et al.
(51) and Watano et al. (61) have confirmed
that when uniform layer is concerned, the
Wurster equipment surely provides the most
excellent outcome.
All this features determines the highest coating quality, which is mandatory if one requires producing
the defined and reproducible drug delivery profiles. (29)
1.4.3. Microencapsulation (MEC) materials
1.4.3.1. Core materials
The core material, which can be either liquid or solid, is identified as the particular material that
requires to be coated. (28) The solid core should consist either of the active constituents, stabilizers,
diluents or other excipients and release-rate retardants. In order to achieve a certain design of MICs
with the efficient properties, changing the core material’s composition is vital. (36)
Figure 2: The process of coating in the fluid bed with a
Wurster partition.
P a g e | 23
1.4.3.2. Coating materials (polymers)
Selecting the most suitable coating material is of a key importance as it indicates the physical and
chemical characteristics of the MICs. (54)
The classic feature of the appropriate coating polymer is to be inert and chemically compatible with
the core material as forming a cohesive film with the core material is fundamental. In addition, the
coating material should provide the optimal layer properties such as strength, flexibility,
impermeability, optical properties and stability. (54)
For MEC, hydrophilic or hydrophobic polymers can be used. (33) When MEC process is used, as either
a taste masking or a taste concealing technique, the selection of the appropriate taste concealing
polymer is crucial. Vummaneni et al. (62) state that hydrophobic polymers are more popularly used
for coating of bitter drugs than hydrophilic polymers. Ideally, the taste concealing polymer should be
such that is prevents the release of API in the oral cavity, following per oral intake, but allows its
release in stomach.
Polymers, which are not soluble at salivary pH 6,8 but rapidly dissolve at gastric fluid (pH 1,2), play a
major part in taste concealing’ techniques. (63)
Coating agents used in microencapsulation are gelatin, povidone, HPMC, ethylcellulose, carnauba
wax, acrylics (different grades of Eudragit) and shellac. (63,64) From Ayenew Z. et al. (65),Sharma et
al (63) and Vummaneni et al. (62), it is evident that there have been a few successful studies how to
either mask or conceal a bitter taste of a drug by MEC using a Wurster fluid bed coating. However,
using hydrophilic methyl cellulose (MC), as a potential taste-concealing polymer, is fairly an
unexploded area.
1.4.4. Pediatric patients and taste concealing
Taste is one of the traditional five senses and is the ability to detect the flavor of substances, like
food, drugs etc. (62)Moreover, taste can surely refer to a subjective perception which arises from the
taste buds stimulus on the tongue’s surface. (65)
Human beings are able to differentiate among five types of taste: sourness, saltiness, sweetness,
umami (savory) and bitterness,(66) which is stimulated at the back of the tongue. (65)
In case of pediatrics and geriatrics, taste – masking and taste-concealing of a bitter drug might
represent a key role in the formulation development when unwillingness to swallow solid dosage
forms, is concerned. (62,64)
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Bar-Shalom (17) postulated that children have to be taught how to prevail their instinctive reactions,
and their organ development might prevent the swallowing of solid oral dosage formulations.
Moreover, Iwai N. (67) confirmed that more than half of the subjects in his study had certain
complications in taking the medicines.
In addition, the majority of troubles regarded taste, primarily bitter taste, followed by roughness on
the tongue, too large quantity and odor. Bitter taste was a major criticism within all age groups.
(67)It was reasoned that children are more sensitive to the bitter and unfamiliar taste of drugs than
are adults.(17)That is why it is prerequisite for the pharmaceutical industry to find new ways to
soften the bitterness of the drugs as the commercial success of the product depends upon the
patient compliance.
Taste masking is defined as the apparent reduction of an unpleasant taste by using suitable
agents.(68)It is, however, important to note that Bar-Shalom (17)proposed a distinction between
taste masking and taste concealing. According to his definition (17) taste masking represents the
addition of chemicals (sweeteners and flavors) to modify the taste of the medication, whereas taste
concealing approach implicates the coating of the drug to make it unavailable to the taste buds.
The major taste mask/conceal technologies yearn for a reduction of solubility of the drug in the saliva
hence the drug concentration in saliva will remain below taste threshold value. (63) These
technologies include two aspects: selection of suitable taste masking/concealing substance such
polymers, sweeteners, flavors, amino acids etc., as well as selection of suitable taste concealing
techniques, which can powerfully impact the quality of taste concealing and process effectiveness.
(68)
There are many techniques developed for, either taste masking or taste concealing of bitter drug, for
example the addition of flavoring and sweetening agents, ion-exchange complexation, inclusion
complexes, gel formation, emulsion techniques, granulation and microencapsulation or incorporation
of the bitterness inhibitor. (62)
For now, MEC seems to be the one method, among others, to conceal the taste of drug. (69) Fast-
hydrating dry granules which swell into pudding-like carriers have been developed for the
microencapsulated drugs to limit their exposure to the taste receptors. (17,69)That is why MEC has
been chosen as a taste-concealing technique in the present study.
P a g e | 25
When the small drug particles are encapsulated by using a suitable polymer, which can reduce the
drug solubility in saliva, the core material (API) cannot contract to the taste buds in the mouth and
hence the unpleasant taste of bitter drug is concealed. (63,68) When the distasteful taste is avoided,
this results in the increase of the patient compliance. (64,68)
1.5. Pharmacokinetic barriers in anthelmintic action and the role of
dissolution
It is generally accepted that API has to be initially dissolved in the GIT’s aqueous medium in order to
be effortlessly absorbed. (70)
Within drug delivery, drug formulation and quality control, dissolution testing is an essential device,
primarily chosen for immediate release dosage forms. In addition, when the evaluation of
performance in vivo is questioned, the dissolution testing’s goal is to offer the rapid dissolution in the
test medium. (71)
In this thesis, ALB, an anthelmintic drug, was chosen in order to improve its release and,
consequently, attain elevated ALB concentrations where the intestinal parasites are located. This
should further permit the delivery of effective ALB concentrations in the worm’s cell and adequate
time to provoke its anthelmintic effect.
It is widely accepted that dissolution of the drug in aqueous content of GIT is a limited step which can
further provide absorption through the GIT and thus limit the amount of ALB for efficiently reaching
the bloodstream. (72) After the reversible exchange of ALB between the systemic circulation and
tissues, the entrance of ALB molecules should be able to penetrate into the parasite’s cells.(2)
That is why the drug delivery can only be efficient if the ALB molecule is capable to reach the
parasite’s structure in the anthelmintically effective concentrations. (73)
Therefore, the dissolution as rate-limiting step of ALB’s absorption, greatly influence the
concentration of poor soluble ALB, which enters the parasites.
In the last decade, the improvement of the solubility of poor water soluble drugs has been one of the
main targets of drug development. (74) Because the taste-concealing is a main target of the dosage
form, it is crucial to note that the taste-concealing aspect also greatly influences dissolution method
development, specifications and testing.(75)
The two most widespread dissolution tests (basket and paddle method) are based on the principle of
having a solid dosage form in a vessel filled with the dissolution medium that is agitated by a paddle
or by rotation of the basket. (76)
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1.5.1. BCS Class II Drugs and IVIVC
There remains a need of how to correlate in vitro drug release of numerous drug formulations to the
in vivo drug profiles. (70) The dissolution rate for in vivo prediction can only be improved when the in
vitro in vivo correlation (IVIVC) is fully settled. (71)
When it comes to the BCS Class 2 Compounds, as ALB, the drug dissolution could be the rate limiting
step for the drug absorption and IVIVC might be predictable. In this case, the dissolution critically
affects the bioavailability of BCS class 2 drugs which have very low water solubility and high human
intestinal permeability.(33) Consequently, even a diminutive increase in dissolution rate might result
in a great increase in bioavailability.(72,77) For that reason, the enhancement of the dissolution rate
of the drugs is a fundamental aspect when improving the bioavailability of BCS class II drugs is
preferred.
In this thesis, the improvement of ALB release rate on the basis of its encapsulation into polymeric
MIC would be a useful achievement.
1.5.2. Solubility
Solubility is a thermodynamic feature of a chemical substance, existing in a solid state, which takes
contact with a liquid solvent. (78)
As the result of BCS Class 2 drug’s poor water solubility, as in the case of the API - albendazole (ALB),
its oral delivery is a common challenge for pharmaceutical scientists. (79)
One of the aims in this thesis was to improve the drug release rate of ALB by its encapsulation into
polymeric MIC in order to see if it is possible to enhance the initial ALB release.
Several strategies have been considered to be beneficial for the enhancement of ALB aqueous
solubility including complexation with povidone and cyclodextrins (27,80,81) as well as using the
dendrimers as solubility enhancers for ALB. (23)
The use of microencapsulation as a technique to enhance the solubility of ALB is considerably
unexplored area and only a few studies have focused on this aspect; for instance, the preparation of
enteric microparticles of poorly soluble and bitter drugs (79) and hyaluronic microspheres of
cyclosporin A (82), both for the improvement of the ALB oral bioavailability. It is important to note,
however, that micronization in terms of the particle size’s reduction increases the surface area and
dissolution rate, but does not affect the solubility.(20)
P a g e | 27
1.5.3. Taste-Concealing drives Dissolution
As previously described, taste-concealing of bitter drug substance is important for any orally-
administrated dosage form. However, it is important to note that the dissolution performance of
these dosage forms is influenced by their wettability, particle size determination and surface area.
(71)
Because granulation of the bitter drug, followed by a polymer coating, can achieve taste-concealing,
it is vital to understand that several polymer coatings are known for their pH-dependent solubility,
which is capable to influence the dissolution profiles.(75) In addition, the polymers can swell, dissolve
or become permeable throughout the dissolution; it is important to acknowledge that, however, the
dissolution medium is of imperative importance here. (75)
Because the taste-concealing is an essential element of the microencapsulated dosage form, a multi-
point profile in a neutral pH medium with early points of analysis (e.g. ≤5min) is recommended. Such
a dissolution criterion (typical ≤10% dissolved in 5min) primarily depends on the taste intensity of the
drug, but might, however, allow the in vitro estimation of the taste concealing characteristics. (71)
1.5.4. Dissolution rate
“Dissolution rate may be defined as an amount of drug substance that goes in the solution per unit
time under standard conditions of liquid/solid interface, temperature and solvent composition.”(83)
According to the Noyes-Whitney equation (20,83), the rate of dissolution may be modified primarily
by altering the surface area of the solid with the micronization. (84)
When designing a rapidly-dissolving dosage form, the formulation scientists want to omit even the
slightest possibility of delaying or prolonging the drug’s release value after the application.
It has been generally accepted that for immediate-release (IR) products, more than 85% of the API
must dissolve in half an hour (30 min), using either USP Apparatus I (basket) or USP Apparatus II
(paddle) in 900 ml of aqueous medium. (33,71)
For poorly soluble compounds, solubility in the conventionally used dissolution medium may be
insufficient. It has been suggested that this might be normal; however, if that’s the case, dissolution
tests should be carried out in more robust medium with various testing settings. If that what the
conditions are, the surfactants could be used in order to improve the drug’s solubility. (70)
P a g e | 28
1.5.5. In vitro methods
According to the European (76) and US (83) Pharmacopoeia it is crucial to determine and measure
the solubility of both pure pharmaceutical ingredient and solid formulation, where the compounds
must be dissolved in a special vessel. The apparatuses that are foremost appropriate for performing
the dissolution test of solid oral dosage forms are Apparatus I (basket) and Apparatus II (paddle).
Both of these apparatuses basic equipments are positioned perpendicularly to the vessel. Dissolution
medium is placed into the vessel and its temperature could be further maintained at the required
level. (76,83)
When Apparatus I is concerned, the dosage form is positioned in a basket and the agitation is
achieved by rotating the basket, whether in Apparatus II, the dosage form is dropped straight into
the vessel. If this is the case, a paddle is used to stimulate the material’s movement with the usual
rotation speed, agitated commonly around 50-100 rpm. If the dosage form may float, Apparatus I is
more suitable. (76)
The pH of the dissolution medium is also an importing factor, considering which in vivo environment
is representing.
Dissolution testing is generally performed within the specific pH range. Normally that is from pH 1.2
(gastric pH) to pH 7.5 (intestinal pH). Even though purified water is commonly acceptable, it has no
buffering capacity that is why it is not an ideal medium. On the other hand, water is believed to be
the optimal medium if pH-sensitivity, either of a drug or excipients, is excluded from the dissolution
testing.(76,83)
1.6. HPLC Analysis
HPL Chromatographic separation is a technique, based on the distribution of a sample between two
phases. One phase is stationary, where the small surface-active solid particles are captured in a
chromatographic column, whilst the second phase – mobile phase, consists of a liquid that passes
through the first phase, as an eluent. (85)
Within the pharmaceuticals, High Performance Liquid Chromatography (HPLC) is one of the most
important analytical tools, frequently used for the quantitative and qualitative analysis of drug
substances and drug products. (85) Since the analytical methods are expected to be accurate and
reliable, it is fundamental to provide the high degree of assurance through the documented
evidence, known as the method validation. (85,86,87)
P a g e | 29
In practice, scientists are keen on using RP-HPLC since its versatility, reliability and ease to use have
been thoroughly verified.(86) The term “reversed phase” (RP) has been applied as the first HPLC
separation, consisting of a polar stationary phase (SP) and a non-polar mobile phase (MP). In a
common RP-HPLC, the stationary and the mobile phrases are reversed. Normally, SP consists of
several silica particles, previously bonded with alkyl chains (for example C8, C18 columns)or phenyl
rings (known as phenyl columns),representing a non-polar entity. Typical RP-HPLC mobile phases are
water, methanol, or acetonitrile, making a mobile phase a polar constituent in RP-HPLC separation.
(85)
Retention of small and poorly-water soluble molecules by RP-HPLC strictly occurs when the
hydrophobic portion of the molecule reversibly absorbs to the bonded phase of the column and after
an increase in the concentration of organic modifier, the poorly water-soluble molecule is desorbed
and eluted from the column. (86)
1.6.1. Method Development for RP-HPLC
When developing a method, it is one of the main priorities to choose the most suitable stationary
and mobile phase in order to sufficiently retain and separate the molecules.(88) While the majority
of the RP-HPLC stationary phases (C8, C18) can form weak van der Waals interactions with the
molecules in the sample, the other RP-HPLC phases, for instance phenyl columns, are capable of
forming the different ones.
Nevertheless, while in the process of a RP-HPLC method development, it might be useful to try as
many different particle chemistries as possible. By doing so, the RP-HPLC separation can be
effectively optimized. (85,88)The suitable mobile phase also needs to be appropriately chosen. RP-
HPLC mobile phases usually consist of an organic modifier (methanol or acetonitrile) and water,
which usually contains a very small portion of an ionic additive as the acetic acid.
After the suitable stationary phase (column) and a mobile phrase system (organic and ionic modifier)
are reasonably chosen, the elution mode should be determined. This can be either isocratic, where
the composition of the MP is unchanged during the entire elution process or gradient elution with
the increasing strength of MP during the elution. (89,90) This can provide an optimal separation
within an adequate analysis time. In addition, isocratic elution is preferable as the development of
gradient elution can be found difficult. (86,89,90)
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1.6.2. Method Validation
In the pharmaceutical field, the analytical methods might be in widely in use; hence they must be
precise, reliable, accurate, specific and robust. To establish that kind of an analytical tool, there is an
important regulatory requirement in pharmaceutical analysis named “method validation” in which
the documented validation ensures that the material produced meets the required specifications.
(85,87,91)
Typically method validation parameters are dependent on the type and applications of the method.
In case of the identification tests, where the aim is to confirm the identity of the API in the samples,
the typically required parameters, according to the USP (83), are specificity, accuracy, precision,
detection limit, quantification limit, linearity and range. Other analytical tests often include other
parameters as system suitability and solution stability. (83)
2. AIM
A considerable number of experiments have proved that producing a pediatric drug formulation is a
unique challenge. The ideal medicine must be effective, stable, affordable, have a good palatability,
acceptable taste and smell.(8) However, some drugs crucial for pediatric patients are not
commercially obtainable in dosage forms suitable for use in this population. (9)
Hence, there are substantial benefits to be gained from developing an age-appropriate dosage form
since there is a growing concern that children are at risk when they are administered unsuitable
medicines.
For patient acceptability, oral drug delivery is the preferred route of administration. Nevertheless,
the main obstacle of oral drug delivery for babies, toddlers and children is difficulty or inability to
swallow.(9) Children do not like novelty in their food as well as its bitterness and, contrary to the
belief of the industry; they are not so fond of syrups. (15,67) It has been reported that the most
accepted consistency for children from 6 months to 5 years, is the semi-solid formulation (16),where
mastication is not necessary. That explains the ideal nature of such formulations for in the pediatric
field. For that vital reason, the potential development of age-adapted dosage form with its beneficial
taste-concealing properties for the oral administration is a redoubtable challenge for the formulation
researchers.(92)
Over the past years, masking unpleasant taste with sweeteners and flavors has been extensively
studied.(62,63,65,93)However, if this is not feasible, more sophisticated formulation approaches
such as encapsulation of drug particles are desirable. (94)
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What is more, the microencapsulation of drugs has been widely used in pharmaceuticals, but only a
few studies have used this technique in order to increase the drug release and bioavailability of poor
soluble drugs. (95) Nevertheless, the performance of formulation of poorly soluble drugs depends
not only on the feature of the manufacturing procedure used.
A close attention should be paid to the fact that uniform microparticles with control size distribution
play an essential role in the drug release of poorly soluble drugs. (96)Moreover, the types of
excipients, applied in a manufacturing process, are also of great importance. (24)
In this thesis, a poorly soluble drug, albendazole (ALB), which is very frequently used in the third
world countries as an anthelmintic (24), was used in order to microencapsulate. Therefore, the focus
of this thesis is to microencapsulate ALB the purpose to conceal its unpleasant taste, initially
decrease drug release in the mouth and on the other hand, enhance the drug release in the stomach,
all on the basis of ALB encapsulation into the polymeric microparticles.
The main aims in this formulation study are:
Achieve PSD of the granules in the range of 70-180 μm
Improving the drug release from the granules of poorly soluble drug ALB
Microencapsulating to obtain efficient taste concealing of the ALB
Allowing easy swallowing by integrating microparticles into an UNICEF gel, a HPMC based gel,
resulting in a production of a semi-solid formulation
Developing simple production process for the dosage form (primarily) appropriate for
children in the third world countries
P a g e | 32
3. MATERIALS and METHODS
3.1. Equipment
Table 3: Apparatuses for manufacturing and analysis of MEC
These apparatus were used in manufacturing and analysis of MEC (Table 3, Figure 3 and Figure 4):
Figure 3: Modified Kenwood Food Processor Figure 4: Fluid bed MiniGlatt
Apparatus Process Manufacturer
Kenwood FP250 food processor Manufacturing the granules KENWOOD
Microscope Characterization Carl Zeiss
Malvern Mastersizer 2000 Particle size distribution Malvern Ins.
HPLC Analysis, separation Dionex
Paddle apparatus USP In vitro dissolution test ERWEKA DT70
Scanning Electron Microscope Characterization LEICA, S340
Ultrasonic Bath (GS13) Dissolving, degassing Qteck GmbH
Weight (AG 204 Delta Range) Weighting Mettler-Toledo
Oven Drying Lytzen
Meterlab pH meter pH measurements Radiometer analytical
Magnetic Stirrer Agitation Ika
Sieve No.75, No.125, No.180 USP Sieving Retsch
Microcentrifuge Centrifuging LabnetInternation
UV Imager Dissolution Test Actipix SD130, Paryatec Ltd.
Mass spectrometer (MS)
SingleQuad Characterization
Agilent Technology LC/MS 6150
MSD module connected to the
1200 series
Fluidized bed (Wurster) Coating Glatt GmbH, Germany
P a g e | 33
3.2. Materials
These substances were used in manufacturing and analysis of MEC (Table 4 – Table 8):
Chemicals for granulation
Table 4: Substances for manufacturing the granules
Substances Batch number Manufacturer
API
Albendazole 620130311 Hangzhou Dayangchem Com, Ltd.
Caffeine powder 22234.187 VWR Prolabo
Excipiens
Avicel pH 101 61515033 Fagron
Povidone BCBJ4899V Sigma-Aldrich
MilIiQ Water / FARMA
Dextrin from corn SLBD3699V Sigma-Aldrich
Sucrose 22545001 Fagron
Ethanol 70% 1680766 Kemetyl
Maize starch 61515051 Fagron
Tween 80 (Polysorbate 80) BCBG4438V Sigma-Aldrich
Ciklodextrin Beta / Fagron
Lactosum 03455011 Fagron
PEG400 0072500 Fluka Analytical
KCl K42278904121 Merck
Croscarmellose sodium 432801 BASF
Chemicals for coating
Table 5: Substances for manufacturing the coated granules
Substances Batch number Manufacturer
API
Caffeine granules 2377500 Fagron
Excipiens
Methyl Cellulose (MC) Taste-Concealing RAD-27-01-SL-131 Dow Company
Glycerol 00344LH Sigma-Aldrich
Talcum 20365001 Fagron
MilliQ Water / FARMA
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Chemicals for the preparation of UNICEF gel
Table 6: Chemicals for the UNICEF gel
Substances Batch number Manufacturer
HPMC K250 RAD – 57-02-LL-555 Dow Company
Sucrose 22545001 Fagron
Vanillin 22529627 Fagron
MilliQ Water / FARMA
Chemicals used for HPLC analysis
Table 7: Chemicals for HPLC analysis
Substances Batch number Manufacturer Quality
Methanol / VWR prolab HPLC isocratic grade
MilliQ Water / FARMA demineralized
Acetic acid (glacial) 100% K42116163112 MERCK Ph. Eur.
Chemicals used for dissolution test
Table 8: Chemicals used in the dissolution test
Substances Batch number Manufacturer Quality
Hydrochloric Acid 37% 12E020010 VWR prolab Ph. Eur.
Na2HPO4 BCBD8825V Sigma-Aldrich Ph. Eur.
NaH2PO4 BCBG8983V Sigma-Aldrich Ph. Eur.
MilliQ Water / FARMA demineralized
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3.3. Methods
3.3.1. Characterization of ALB
3.3.1.1. Identification of ALB by LC/MS
In order to confirm that the main API is undoubtedly ALB, the specific identification test for the active
substance is essential.
The LC-MS analysis of ALB was performed as a part of the characterization of API. A mass
spectrometer with electro-spray ionization (ESI) interface was used for MS analysis. The analyte
infusion experiments were performed using a built-in syringe pump with the mass spectrometer.
The ESI parameters were as follows: ionization mode, positive; scan 100-300 m/z; fragmentor 70;
capillary voltage, 4.1 kV; drying gas temperature, 300°C; vaporizer temperature, 200 °C; dissolvation
gas flow rate, 400 L/h and cone gas flow rate, 20 L/h.
A stock solution containing 0,5mg/ml of ALB in methanol was prepared just before the analysis and
was put on the magnetic stirrer for 45min. Standard solution containing ALB was prepared from one
dilution from the stock solution, yielding final concentration of 1,20 µg/ml.
3.3.1.2. Particle size distribution
A qualitative analysis of drug particle size was carried out using optical microscopy, where a small
amount of each sample was placed on a slide.
A more detailed particle size analysis of the samples was performed using a Malvern Mastersizer,
where different pressures (from 0,6 to 1,6 bar) were used in order to classify the potential presence
of primary agglomerates and then break them. A small, but sufficient mass of samples were added
on the measuring cell and then the Mastersizer measured the volume percent of the particles by
laser ensemble light scattering.
The results are given as a volume median diameter D(0,5) which is the diameter where 50% of the
distribution is above; in case D (0,1), 10% of the volume distribution is below and in case of D(0,9),
90% of the volume distribution is below a given value.
In all cases, the obscuration (light intensity absorbed by the sample) was between 0,1-0,3, which is
known as an optimal and reliable interval for the measurements. (97)
P a g e | 36
3.3.1.3. Determination of bulk, tapped densities and flow properties
Bulk and tapped densities of powders and granules have to be measured as a part of flowability
indicators as the powder and granule flow is one of the most important considerations in the
pharmaceutical industry manufacturing solid dosage forms. (69)(70)(71)(72)
According to ICH Guideline (72) determination of Bulk and Tapped Densities is a method to
determine the bulked densities of the powdered drugs under loose and tapped packing conditions
respectively. The bulk and tapped densities measurements were used to calculate the compressibility
index and Hausner ratio, from which the powder flow was further characterized. (72)(73) In the
equation below, V0 stands for the apparent volume and Vf for the final tapped volume after 1250
taps.
(
)
Both densities were calculated in accordance with the method described in Section 2.9.15. of
European Pharmacopoeia (74) and ICH Guidelines. (72)
100,0g of each powder and finished batch of the granules (50,0g in case of ALB and MC powder,
because 100 g of ALB has not fit into the 250ml cylinder as the powder density of ALB is too low)
were weighted and gently introduced into a graduated cylinder of 250ml. As the bulk density is
determined by measuring the volume of a known powder in a graduated cylinder, the unsettled
apparent volume V0was read and the bulk density was calculated by the formula m/V0.
The cylinder was put into the holder and 250, 500 and 1250 taps were carried out.
The volume after 500 and 1250 taps was read as V500 and V1250. Because difference between these
two measurements was less than 2 ml, more taps were not necessary.
Therefore, the tapped density was obtained after mechanically tapping a cylinder with the powder
sample until the apparent volume of the sample becomes almost constant. The tapped density was
calculated using the formula m/Vf where Vf is the final tapped volume after 1250 taps. The
determinations of bulk and tapped volume were done in triplicate.
The Carr’s compressibility index (CI, %) and Hausner ratio (HR) were calculated based on the
equations above and the ratios were be used to estimate the flow characteristics of the powders and
granules.
P a g e | 37
3.3.1.4. Morphology determination of powders, granules and MICs
Morphology of all samples, used for produce the MICs, was observed by scanning electron
microscope (SEM) after the samples were mounted directly on to the SEM sample stub, using double
sided sticking tape, and then sputter-coated with gold to create electric conductivity on the surface
on the samples.
3.3.2. Microencapsulation (MEC)
Twelve different formulations (FOR1-FOR12) were prepared by granulation technique. One
formulation was chosen and further coating of MICs was then followed.
3.3.2.1. Granulation
The powders of drugs (ALB and CAF) were mixed together in the modified Kenwood Food Processor
FP250 with Avicel /Maize starch/Lactose/Sucrose/Dextrin/Ciklodextrin, and Croscarmellose sodium
(CCS) for 2 minutes at the lower speed. Then, a granulating liquid composed of PEG400/ Polysorbate
80 (Tween 80)/Potassium Chloride (KCl) or Povidone (PVP) in MilliQ Water (in FOR1-FOR5) or in
Ethanol (FOR6-FOR12) was slowly (with the rate of 1.1 – 3.5 mL/min, depending from formulation to
formulation) added at the same speed until granules were formed.
After all the granulation liquid was added, 7-9 min, powders were forming into granules for
approximately 6-7 minutes on lower speed, which was, for the first minute, manually increased to
the highest speed.
The formulations used in the granulation experiments are listed in Table 9. Each formulation was
granulated using the 1L bowl of the Kenwood at a batch size of 50 g. The polymeric binder was added
dry and its concentration was selected based on previous investigations. Granulation was stopped
several times to mix granules which had been trapped to the Kenwood wall and to collect samples
for the microscope. The samples were collected every 1-3 min to observe them under the
microscope whether the end point of granulation had been reached. Granulation was terminated
and end point was reached when a satisfactory granulation was observed – this was mainly after 1-2
minutes on granulation on the highest speed. The granules were then spread on a tray and left to dry
overnight (25°C for 12h). The granules were automatically sieved with SIEVES NO.180, NO.125 and
NO.75 and these sieve shakers were used to break the agglomerates, obtained after drying, into
smaller particles. The percentage of particles between 75-180 µm was then calculated and it was
almost in all cases above 70%. This fraction was used for the following studies.
P a g e | 38
Table 9: Twelve different formulations were prepared in the granulation experiments
3.3.2.2. Coating
In the preliminary experiments, trial settings had been investigated and, as a model compound, CAF
granules (instead of ALB granules, previously produced in the granulating process) were used and the
experimental conditions for coating were optimized.
Preparation of coating suspension:
Methyl Cellulose (MC) was dispersed and dissolved in 1/3 of the whole amount of cold water, which
had been pre-heated to 40°C. The dispersion was continuously mixed on the magnetic stirrer till all
the particles were thoroughly wetted. No lumping was observed, so there was no incomplete wetting
of the individual powder particles. The slurry was formed and it was on the magnetic stirrer for 3
hours at 750rpm.
2Dextrin
3Ciklodextrin Beta
Name ALB CAF AVICEL STARCH SUCROSE DEX2 CIKDEX3 PVP TWEEN80 PEG400 KCl CCS V H20
( w / w % ) (ml)
FOR1 35 2 20 20 - - - 3 10 7 - 3 19
FOR2 38 2 25 22 - - - 3 8 - - 3 22
FOR3 38 2 20 20 - - - 7 10 - - 3 24
FOR4 35 2 24 16 - - - 5 10 - 5 3 15
FOR5 35 2 33 - - - - 5 10 7 5 3 10
FOR6 38 2 - - - 40 - - 15 - - 5 19
FOR7 38 2 - - - 35 - - 10 10 - 5 22
FOR8 38 2 15 20 - - - - 10 10 - 5 21
FOR9 33 2 - - 50 - - - 10 - - 5 20
FOR10 38 2 10 30 - - - - 15 - 5 21
FOR11 33 2 - - - - 40 - 10 - - 15 20
FOR12 33 2 - 10 30 - - - 10 10 - 5 21
P a g e | 39
The rest of the water, previously cooled to 4°C was then added as cold water to lower the
temperature of the dispersion for complete solubilization, improved clarity and reproducible control
of viscosity. Once the dispersion had reached the temperature at which MC became water soluble,
the powder began to hydrate and viscosity increased.
Then Talcum and Glycerol were added and the spray suspension was stirred gently for 1 hour at
750rpm. After preparation, the solution needed to deaerate, so it was put on in the refrigerator
(T=4°C) overnight and it was used the next day. During the coating process, the coating solution was
continuously and gently mixed on the magnetic stirrer.
The composition of the coating suspension can be seen in the Table 10 below:
Table 10: the composition of the MC coating suspension
Ingredient Quantity (w/w %) Function
MC Taste Concealing 5 Polymer
Glycerol 1 Plasticizer
Talcum 2,5 Anti-tacking
Water 91,5 Diluent
The coating procedure:
For coating, 50g quantities of drug-loaded granules were used. Fluid Bed Coating was performed by
bottom spray technique in a Mini-Glatt fluid bed with a 2L chamber fitted with a standard Wurster
partition, using process conditions as seen in Table 11.
Table 11: Process conditions for coating in fluid bed
Before coating, the fluid bed was preheated to a
temperature of 40 °C. Atomization air flow was set to
0,29bar and then liquid feed flow with 0,5 ml/min
started until 10% w/w (solids) coating level was
reached. Bag filters were shaken for 10s every
minute.
There were some minor variations of the inlet air
temperature, but a steady temperature of 34 °C was
maintained after the initial requirement of excess
energy for heating of the substrate.
Process variable Value
Inlet air T (°C) 40
Liquid flow (ml/min) 0,5
Atomization air flow (bar) 0,29
Wurster height (mm) 1,5
Inlet air pressure (bar) 0,26
Batch size (g) 50
Start product T (°C) 22
End product T (°C) 28
Inlet air pressure (bar) 0,26
P a g e | 40
The granules were coated to a 10% weight gain which is acceptable according to the taste-masking
guidelines in Evonik.(102) According to Alkire et al.(64), the coating should preferably constitute at
least about 10 percent by weight of the microparticle.
After the end point was reached, the MICs were allowed to dry for an additional ten minutes to drive
off any residual solvents. Initial weight of the granules was noted and during different intervals in
between coating, the weight of the coated granules was, yet, acknowledged and the coating
percentage was calculated as:
3.3.2.3. Determination of wall thickness of the MICs
As seen from the SEM Images, the microcapsules are uniform and spherical. Consequently, the wall
thickness of the microcapsules was determined by the method described by Si‐Nang, Luu, et al. (103)
using the equation:
( )
[( ) ( ) ]
Where, h is the wall thickness, is the arithmetic mean radius of the microcapsule, d1is the density of
core material, d2 is the density of the coat material and ‘p’ is the proportion of the medicament in the
microcapsules, which had been determined by Particle Size Mastersizer.
The density (δ in g/m3) of the core (d1) and coat (d2) material was also estimated from
measurements by the Particle Size Mastersizer, which determined the mean diameter (D) and
specific surface area (SA) of the uncoated granules and MC as the coat material. The density was
then calculated using the equitation:
For the mean diameter and mean radius, the values from Volume weight means [D 4.3] was used
according to Loveland et al. (97), when the most common mean value noted when using laser
diffraction is the volume mean or D [4.3]. The surface weighted mean D [3.2] can also be used, but it
is more typically used if the product is an aerosol or spray.
P a g e | 41
3.3.3. Dissolution
Preparation of sodium phosphate buffer solutions pH 6,80 :
First, stock solutions of disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate
(NaH2PO4) were prepared, both at 0, 1 mol/L. 7L of buffer solutions at 0,1 mol/L were then prepared
at the pH 6,80 by mixing together 324,5ml Na2HPO4 and 375,9 ml NaH2PO4 and then diluting to a
final volume of 7L using Milli-Q Water. Buffer solutions were placed on the magnetic stirrer for 15min
and then in an ultrasonic water-bath to remove gases from the solution, because dissolved gases
could form bubbles which may change the results of the test. The pH of dissolution medium solution
was checked with a pH-meter and it was between 6, 75 – 6, 85 for each buffer solution.
Measurement of ALB release from MICs:
Because HPLC method was not suitable for estimation of ALB release from MICs, UV Spectroscopy
was used. In addition, the comparison between ALB release from granules by HPLC and Spectroscopy
was made, using a Student t-test.
3.3.3.1. ALB solubility estimations in the dissolution vessels
ALB powder (m=80mg) was introduced and incorporated into the 2,5 ± 0,1 g UNICEF gel just before
the test. The preparation of UNICEF gel is described below (Section 3.3.3.4.).
The dissolution was performed in 900ml medium consisted of phosphate buffer pH 6.8, using USP
dissolution apparatus I equipped with a basket which was operated at the speed of 75 rpm and
temperature of 37 ± 0,5°C.The amount of ALB dissolved was measured at 5min; 10min; 20min;
30min; 40min, 50min and 60min and was then determined with HPLC.
In further experiments, Tween80 was added into the dissolution medium of phosphate buffer pH 6,8
and ALB was incorporated into the gel. The amount of ALB dissolved was measured at 5min; 10min;
20min; 30min; 40min, 50min, 60min, 120min, 150min and 180min and was then determined with
HPLC. Each dissolution study was performed in triplicate and the following sample preparation
procedure was then the same as the one described in the Section “3.3.3.2.”
3.3.3.2. ALB release from granules and microcapsules
Accurately weighed granules of 200mg (equivalent to a specific amount of ALB and 4mg of CAF in
FOR1-FOR12) were used for dissolution studies.
P a g e | 42
In case of MICs, accurately weighed MICs of 200mg (equivalent to 80mg of ALB in FOR6) were used
for dissolution studies. The dissolution was performed in 900ml dissolution medium Mili-Q® Water
(with the pre-checked pH of 6,9 ± 0,1) or 0,1M HCl with pH 1,2) using USP dissolution apparatus II
equipped with a paddle which was operated at the speed of 75 rpm and temperature of 37 ± 0,5°C.
The peak vessels were used as they reduce the hydrodynamic problems associated with the
traditional vessels by eliminating the unstirred region underneath the paddle. (104)
The amount of the drug dissolved was measured at the suitable time interval for every 5minutes in
30min time range for granules and at 5min; 10min; 20min; 30min; 40min and 60min for MICs and
was then determined with HPLC. After each sampling, the aliquots were replaced with the equal
volume of fresh dissolution medium to correct the volume change for calculation. The vessels were
covered for the duration of the test and the temperature was checked at suitable times. The volume
of each sample was 5ml and it was diluted with methanol up to 10ml. Before measuring on the HPLC,
the samples were centrifuged for 5 minutes on 13000 rpm (17900 x g) speed in order to eliminate
the undissolved particles present during the sampling.
Each dissolution study was performed in triplicate for granules, whether the ALB release from MICs
was studied in the six peak vessels.
3.3.3.3. ALB release from the granules and MICs in the semi-solid formulation
Accurately weighed granules and MICs of 200mg (equivalent to 80mg of ALB in FOR6) were used for
dissolution studies and incorporated into the 2,5±0,1 g UNICEF gel, just before the test. The
preparation of UNICEF gel is described below (Section 3.3.3.4.)
The dissolution was performed in 900ml of medium consisting of phosphate buffer pH 6.8, using USP
dissolution apparatus I equipped with a basket which was operated at the speed of 75 rpm and
temperature of 37 ± 0,5°C.The amount of the drug dissolved was measured at 5min; 10min; 20min;
30min; 40min, 50min and 60min and was then determined with HPLC. Each dissolution study was
performed in triplicate for granules+gel, whether the ALB release from MICs+gel was studied in the
six peak vessels. The following sample preparation procedure was then the same as the one
described in the Section “3.3.3.2.”
P a g e | 43
3.3.3.4. Preparation of UNICEF gel
The mixture of 13g HPMC K250, 200g Sucrose and 0.2g of Vanillin was added into a transparent
pouch as seen in Table 12. Dry blending with sweetening agents such as sucrose was used as
dispersion method for the vehicles. Then 1L of Milli-Q Water was slowly added into a pouch, as seen
on Figure 5, which was continuously shaken and after 3 minutes a homogeneous viscous material
was obtained. The air bubbles were removed by a vacuum pump and the gel was prepared for the
further use. The single-dosing vehicle was further optimized by addition of effervescent agents in
order to achieve a fast gelation without shaking.(18)
Table 12: Excipients used for preparation of UNICEF gel Figure 5: The pouch with the UNICEF gel
3.3.4. HPLC Analysis
3.3.4.1. Analysis for method development
Apparatus:
The method development was performed with a Dionex auto sampler HPLC system using 20µL
sample loop. The detector was set at 273nm for CAF and 295nm for ALB and peak areas were
integrated by computer. The maximum absorbance values for CAF and ALB were determined by UV
Spectrum Measurement (Appendix 10).
Separation was carried out at ambient temperature using a Phenylhexyl column. A guard column
was also used to safeguard the analytical column. All the calculations concerning the quantitative
analysis were performed with external standardization by the measurements of peak areas.
Excipient Amount
[g] Purpose
HPMC K250 13,0 Fast hydrating polymer
Sucrose 200,0 Sweetener for taste masking;
separation and hydration of
polymer
Vanillin 0,2 Increased taste perception of
the Sucrose
Water 1000,0 Diluent
P a g e | 44
Method development:
The mobile phase was chosen after several trials with methanol (+0,1% Acetic Acid [HAc]), methanol,
water, water (+0,1% HAc) and acetonitril in various proportions. A mobile phase consisting of
methanol (+0,1%HAc) and water (+0,1% HAc) (80:20) was selected to achieve maximum separation
and sensitivity of CAF and ALB.
A flow rate of 1.0 ml/min gave an optimal signal to noise ratio with a reasonable separation time.
Various columns are available for RP-HPLC and therefore different columns were studied. Among
reversed-phase C8 and C18 column, the Phenylhexyl column (150 x 4,6 mm) with 5 μm particle size
was chosen. It showed a good resolution, lower back pressure, excellent peak symmetry, sharp
separation peaks of ALB and CAF and the good degree of retention. In addition, retention times were
observed to be less than 4 min for ALB and CAF respectively.
Under these defined conditions, all peaks were well defined, without any tailing observed.
λ(Amaxalbendazol) = 295nm
λ(Amaxcaffeine) = 275nm
Preparation of mobile phases:
Water + 0,1% HAc:1ml of 100% acetic acid are added to 1000ml MilliQ water and then degassed in
ultrasonic bath for 15min
Methanol + 0,1% HAc: 1ml of 100% acetic acid are added to 1000ml Methanol and then degassed in
ultrasonic bath for 15min
3.3.4.2. Analysis for validation
Chromatographic conditions
HPLC analysis was performed by isocratic elution with a flow rate of 1.0 ml/min. The mobile phase
composition was methanol with 0,1% HAc and water with 0,1% HAc (80:20).
Volumes of 20 µL prepared solutions and samples were injected into the column. Quantification was
made by measuring at 273nm for CAF and at 295nm for ALB. The chromatographic run time was 5
min.
P a g e | 45
Preparation of stock solutions:
A stock solution containing 0,5mg/ml of ALB in methanol was prepared just before the analysis.
100mg of ALB was dissolved in methanol in a 200ml volumetric flask and then the stock solution was
put on the magnetic stirrer for 45min.
A stock solution containing 0,05mg/ml of CAF in methanol was prepared just before the analysis.
5mg of CAF in methanol was dissolved in a 100ml volumetric flask and then the stock solution was
put on the magnetic stiller for 45min.
Linearity
Standard solutions containing ALB and CAF in methanol, yielding final concentrations of 5, 20, 40, 80,
120, 150 and 200 µg/ml for ALB and 1, 2, 4, 6, 8, 10 and 12 µg/ml for CAF were prepared from one
dilution from the stock solution. The samples were injected in triplicate and standard curve was
made.
Accuracy
Standard solutions containing ALB in methanol with concentrations 10, 30, 50, 100,140 and 180
µg/ml and for CAF with concentrations 3, 5, 7, 8, 9 and 11 µg/ml in methanol were prepared from
one dilution from the stock solution. The prepared solutions were injected 3 times. From the
respective area counts, the concentrations of ALB and CAF were then calculated using the equitation
obtained from the standard curve.
Precision
The precision of this method was checked by nine injections of ALB at concentration 25, 50 and 100
µg/ml and nine injection of caffeine at concentration 2, 6 and 10 µg/ml.
Ruggedness
The ruggedness of the HPLC method was evaluated by carrying out the analysis using a stock
solution, standard solutions (same as solutions described in “Precision”), the same chromatographic
system and the same column on two different days. The prepared solutions were injected 9 times at
three different concentrations.
P a g e | 46
LOD and LOQ estimation
For estimating LOD and LOQ the “from the noise” approach was chosen. (105)
The noise magnitude was taken as an estimate of the blank standard deviation and then the noise
and signal were measured manually on the chromatogram printout.
The seven concentrations of ALB and CAF were prepared in concentration range from 0,5μg/ml –
0,04μg/ml. Based on 6 injection (V = 20 μL) of the same concentration, the average area was then
calculated. The concentration of the prepared sample has corresponded to LOD and the ratio
between the mean area and SD was 3,3.
Then the concentration of c (ALB) = 0,1μg/ml and c(CAF) = 0,5 μg/ml were chosen.
LOD and LOQ were calculated based on standard deviation of the response (SD) and the slope of the
calibration curve (slope) at levels approximating the LOD or LOQ, according to the below formulas:
3.3.4.3. Analysis for drug content estimation
100mg granules were weighted (which is similar to 35mg ALB in FOR1 and FOR4; to 38mg of ALB in
FOR2, FOR3, FOR6 – FOR8 and FOR10; and to 33mg ALB in FOR9, FOR11 and FOR12), crushed with a
mortar and pestle until a fine powder was obtained, then transferred into 20ml volumetric flask,
filled up with methanol to 20ml and put on the magnetic stirrer for 15min. The samples were put on
ultrasonic bath for 10 minutes to achieve breakdown and homogenization. 1ml of this sample
solution was diluted with methanol in a 10ml volumetric flask and the solution was stirred for 15min.
The samples were prepared in five replicates and then averaged. Before measuring on the HPLC, the
samples were centrifuged for 5 minutes on 13000 rpm (17900 x g) speed.
The same procedure was followed when MICs (coated granules) were estimated for ALB content. The
only difference was in number of replicates, which were 5 in case of granules; whereas for MICs they
were 10.
P a g e | 47
This variation in the number of samples was done intentionally since smaller standard deviations
were needed when MICs were evaluated. Therefore, it was demonstrated that with 10 MICs
replicates, the results were reasonably representative.
3.3.5. UV Spectroscopy
Measurement of ALB absorbance spectrum
ALB and CAF absorbance spectra was measured after the samples were prepared by the same
method procedure as for the standard solution preparation of ALB and CAF, described in HPLC
Methodology Section.
Standard solution preparation and standard curve for ALB
A stock solution containing 0,25mg/ml of ALB in methanol was prepared by dissolving 12,5mg of ALB
in methanol in a 50ml volumetric flask and placed on a magnetic stirrer for 30min. Eight standard
solutions were prepared in concentration range from 45 µg/ml to 10 µg/ml from one dilution of the
stock solution in methanol. Solutions were further diluted because the Beer-Lambert Law is most
accurate between absorbance of 0,05 to 1,00. For example, the diluted (1:2) ALB solution (c=45
μg/ml) had absorbance 1,06, meaning that undiluted solution had an absorbance of 1,06 x 2= 2,12.
The standard curve for the spectrophotometric measurements was linear (R= 0,9985) under work
concentration interval.
The absorbance spectrum and standard curve for UV Spectroscopy measurements can be seen in
Appendix 10.
3.3.6. Student t-test
In order to compare the analytical results obtained with two different methods (HPLC and UV
Spectroscopy), from which HPLC is validated and confirm that UV Spectroscopy provide similar
analytical results, a statistical tool was used, known as a Student t-test. The data were obtained by
the HPLC and Spectroscopic determination of ALB release from uncoated FOR6 granules. The paired
t-test was made with a predefined confidence interval (CI) of 99%, using Microsoft Excel 2007 as an
analytical tool and the null hypothesis being that both methods provide the same analytical results
and that differences (if any) are purely due to random errors.
P a g e | 48
4. RESULTS and DISCUSSION
4.1. Characterization of ALB
4.1.1. Identification of ALB by LC/MS
Liquid chromatography with mass spectrometer was used to characterize and thus identify the drug
Albendazole (ALB) that has been used throughout this thesis. The identification spectrums of ALB,
obtained by MS can be seen in Appendix 1. The mass spectrum showed a main peak at m/z 266.1
which is in good agreement with the ALB standard spectrum obtained by MassBank’s database. (106)
One can conclude that ALB was therefore determined and it can be further applied in this study.
4.1.2. Particle size distribution (PSD)4
Data obtained in previous studies suggested that reducing the particle size have a positive impact on
the dissolution rate (72,81,84,107), stability in drug formulation (108), efficacy of delivery (109),
children’s projection of texture and feel of the drug (67,110,111), appearance (67), flowability and
handling (70). One of the specific research problems was getting the PSD of the granules between
70-180 μm.
In this study, the evaluation of granule size distribution was accurately measured using Malvern laser
diffraction and results were obtained as the volume percentage of the material.
Both Figure 6 and Figure 7 represent symmetrical and narrow normalized distribution (volume
based) which show that the diameter of the granules and MICs ranged from 75 to 177 μm and from
134 to 340 μm, respectively. The term D(0,9) represents the particle diameter at which 90% of the
distribution is below 177 μm for granules and 340μm for MICs. As noted, there is a significant
difference between their particle sizes, resulting in the size enlargement due to the coat forming. As
the ALB particle size remains unchanged, reduction of the granules and MICs size increases the
specific surface area (and might enhance the drug’s dissolution). However, that does not affect the
solubility. In addition, the narrow distribution of granules is one of the crucial material parameters
for the coating process in the fluidized bed. (54)
However, in Appendix 2 can be seen that ALB particles of pure drug are noticeably small (>90%,
<38μm) and present non-symmetrical bimodal distribution with a wider PSD.
4 The whole analysis report from Malvern Mastersizer of granules, MICs and ALB powder can be seen in
Appendix 2.
P a g e | 49
Additionally, the specific surface area of ALB particles is forty and seventy-five times larger than the
area of granules and MICs, respectively.
Mosharraf and Nyström(84) have proved that increase in particle size (which resulted in decreased
surface area) enhances the dissolution rate in case of poor-water soluble drug Griseofulvin.
Moreover, from a manufacturing perspective it is preferable to have taste-concealed MICs that are
larger in size (0,25-1 mm) due to the lower cost in the manufacturing process.(112)
In the Figure 6and 7it can be seen that is possible to achieve a micro sized product of poorly soluble
drug ALB with defined and narrow distribution range.
Figure 6: Granules FOR6 size distribution with the granules diameter range from 75 μm to 177 μm
Figure 7: MICs size distribution with the diameter range from 134 μm to 340 μm
P a g e | 50
4.1.3. Determination of bulk, tapped densities and flow properties
As generally accepted, many powders do not flow well. When the material has poor flowing
characteristics, a greater interparticulate interactions and differences between the bulk and tapped
densities can be inspected. (100)That can be due to their small size, irregular shape or surface
characteristic. For that reason the microencapsulation, consisting of granulation and coating, should
improve the flow properties of powders and granules respectively. For the coating process, a good
product flow is an important characteristic, because it influences on the MIC quality. (51)
Before the flow properties were calculated (based on the equations explained in the Method section
and Appendix 3, where the average results for V0 as unsettled apparent volume (bulk volume) and Vf
as the final tapped volume of ALB, excipients and granules can be seen), the bulk and tapped volume
were measured as shown in Table 13.
Table 13: Calculated Bulk, Tap Densities and flow properties (CI and HR) of ALB, Dextrin powder, granules and MIC
Parameter Albendazole Dextrin MC GR FOR65
Bulk density (g/ml) 0,23 0,59 0,27 0,49
Tapped density (g/ml) 0,30 0,68 0,42 0,55
Carr’s index (%) (CI) 25 13,53 35,68 9,9
Hausner ratio (HR) 1,33 1,16 1,56 1,10
Flow character Passable Good Very poor Excellent
From the same Table 13 it can be demonstrated that the order of decreasing flowability, based on HR
and CI, is:
G r a n u l e s F O R 6 > D e x t r i n > A L B > M C .
While MC powder has very high HR and CI and thus poor flow character, dextrin has a good
flowability. One can claim that FOR6 granules have an excellent flowability as having the lowest CI
and HR. Its free flowing behavior is probably due to spherical particle shape, larger particle size and
narrow PSD.
According to the bulk and tapped values in the Handbook of Ph.Excipiens, (113), MC has HR of 1,68
and Dextrin of 1,14, which is slightly different than in this experiment.
5GR FOR6 = granules of formulation 6
P a g e | 51
Nevertheless, dextrin flow properties, according to the previous reference, can still be described as
“Good”, where MC properties have superior flow in our experimental.
In literature (113) MC flow properties are described as “very, very poor” and as “very poor” in our
study. This might be due to the different particle size and particle shape of MC used in this report as
the flow is highly dependent on these values. Poor flow properties are caused by smaller particle
size, different particle shapes and its wide PSD. (41)
Despite this, a good approximation of flow properties of excipients was made in this experimental
study. Therefore it can be concluded that granulation greatly improved the flow properties of
powder mix, which should result in better quality of the final product after the fluidized bed coating.
4.1.4. Morphology determination of powders, granules and MIC
The surface of ALB, MC, DEXTRIN, FOR6 granules and coated granules were observed by using
scanning electron microscope (SEM) on two different magnifications.
Granules before and after coating were spherical as shown in Figure 8 and Figure 9, respectively, and
a smooth surface was found for both products. That is strongly related to the fact that children and
infants like more spherical and uniform particles than rough and uneven.(67) Moreover, the
roundness also resulted in the excellent granules flowability, as discussed previously. According to
expectations, the surface texture of the uncoated granules is less spherical than the coated surface
which confirms that the Wurster apparatus is valuable in achieving small spherical particles. (51) The
incomplete spherical shape of uncoated granules can be due to the granulation process or the
surface nature of dextrin powder and its insoluble behavior when adding ethanol as the granulation
liquid. Regarding this, from SEM Images in Appendix 14 can be concluded that dextrin balloon-like
particles have smooth surface which also resulted in their good flowability behavior, indicating that
dextrin is an appropriate material for the granulation process. However, in previous investigations
when using AVICEL pH 101 as a binder, more spherical and smoother granules were observed, which
Law et al. (114)also demonstrated in their study.
SEM Images also confirmed the enlargement of the particles after the coating process and
revealed potential holes on the granule’s surface, which may resulted from the manufacturing
process, either the granulation or coating. The holes on the surface can also indicate that the pores
had been formed throughout the granules. Those crevices could favor ALB release with enhancing
the inward water penetration in the drug release study. In addition, on the surface of the both
products, there are very few irregular particles, which have adhered to the polymer coat.
P a g e | 52
It is possible, that ALB is not completely trapped into the dextrin/MC polymer which should be
further investigated as this may affect, not just the dissolution rate of ALB, but also the taste-
concealing properties of the formulation. This process of adhesion can also affect the liquid uptake
and therefore the hydration rate, swelling of the MC and finally, the ALB release from the eroded
surface to the dissolution medium.
Nevertheless, it can be concluded that dextrin gives good coverage of the core materials. This
resulted in the smooth and uniform surface morphology which is preferred for a further fluid-bed
coating. In addition, in Julian’s and Radebaugh’s patent (115), the granules with irregular shape and
broad PSD have led to poor taste concealing efficiency and varying dissolution of coated particles.
The SEM Images of powders (ALB, DEXTRIN and MC) are shown In Appendix 14 just for comparison. It
can be concluded that their particle size are smaller and thus their PSD is more heterogeneous as can
be seen in Appendix 2 and discussed before.
Figure 8: SEM images magnification series (x75and x1000 respectively) of the granules
Figure 9: SEM images magnification series (x35 and x750 respectively) of the MIC
P a g e | 53
4.2. Microencapsulation
4.2.1. Determination of wall thickness of the MICs
Wall thickness (h) of the microcapsules was determined by the equation explained in the Method
Section and then, on the basis of the variables seen in Table 14, calculated as:
( )
[(
) ( )
]
In the coating process, the surface of solid particles is covered by a solid layer, where the coating
thickness is, according to Guignon et al. (38), generally between 0,01 and 0,05 mm with a size of
particles from 0,1 to 10 mm. The study of Pearnchob et al. (116) has demonstrated that the taste-
concealing efficiency is enhanced with increasing coating thickness, but on the other hand this can
significantly alter the ALB release rate.
Therefore, there is a need, in the art of taste-concealing formulation, to develop a coat which is not
too thick and, yet, not too thin. As the more round particles were obtained after MC taste-concealed
coating, this gives a fairly good thin layer around the granules and thus the better coverage of MICs.
Furthermore, a thin layer of MC, 29,5 is confirmed. This should achieve, according to the Guignon
et al. (38), sufficient taste-concealing properties of the microcapsules. Further dissolution studies
would confirm if the MC coat had altered the diffusion rate of ALB and consequently decreased its
release rate.
Material Core material
(uncoated granules)
Coat material (polymer - MC) MICs
Variables SA D d1 SA D d2 p SA
Values 0,055
m2/g
121.562
µm
0,897
g/cm3
0,108
m2/g
128,200
µm
0,433 g/cm3 0,37 113,48
µm
0,03
m2/g
Table 14: Measured and calculated variables for the determination of the microcapsules’s wall thickness
Legend: h = wall thickness, 𝒓 = arithmetic mean radius of the microcapsule, d1 = density of core material, d2 = the density of the coat material, ‘p’ = proportion of the medicament in the microcapsules. D = mean diameter, SA= specific surface area
P a g e | 54
4.3. Dissolution
In Appendix 4 the numeric values of ALB and CAF release from the different granules’ formulations in
water are fully provided. Furthermore, in Appendix 12, the example of calculation for ALB release can
be seen.
4.3.1. Granules6
Graph 1: Drug release profiles of ALB formulations FOR1-FOR5 in water within 30 min time range
Graph 2: Drug release profiles of ALB formulations FOR6-FOR12 in water within 30 min time range
6In Appendix 4 and Appendix 5 the individual graphs of each formulation can be seen and thus, the ALB and also the CAF release can be
observed in more details.
0%
10%
20%
30%
40%
50%
60%
70%
80%
0 5 10 15 20 25 30 35
% d
rug
rele
ase
time (min)
% ALB release from FOR1-FOR5 in water
ALB in FOR1 ALB in FOR2 ALB in FOR3 ALB in FOR4 ALB in FOR5
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 5 10 15 20 25 30 35
%d
rug
rele
ase
time (min)
% ALB release from FOR6 - FOR12 in water
ALB in FOR6 ALB in FOR7 ALB in FOR8 ALB in FOR9 ALB in FOR10 ALB in FOR11 ALB in FOR12
P a g e | 55
The development of microparticulate formulation is hampered by the fact that ALB is very sparingly
soluble in water (app. 1μg/ml). It has been suggested that the enhancement of ALB release in the
water can be due to, for instance, the improvement of wetting and solubilization by a hydrophilic
carrier. Gilis et al. (117) showed that the incorporation of a poorly soluble substance into a
hydrophilic polymer results in excellent dissolution properties. In the preliminary investigations, a lot
of different hydrophilic materials have been used in the granulation procedure. Examples of such are
sucrose, dextrin, maize starch and microcrystalline cellulose (Avicel pH101).
Avicel pH101 was used as a polymer in seven different formulations out of twelve: FOR1 – FOR5,
FOR8 and FOR10, where the ALB release was the lowest for FOR5 (37,38% ± 0,80% in 30min) while
the highest release was achieved with FOR10 (88,47% ± 1,27% in 30min). In this study, the
improvement of ALB release rate was found to be greater from granules where Avicel and Maize
starch were used together with superdisintegrant Cross-Carmellose Sodium (CCS) and non-ionic
surfactant Tween80.
It was observed that lower concentration of Avicel and thus higher concentration of Maize starch
resulted in greater ALB release. This was confirmed with formulation FOR5 (having Avicel/Maize
Starch mass ratio of 2,5) from which ALB release was twice less than for FOR10 with the same ratio of
0,33. The reason for this occurrence can be found in different swelling mechanism of Avicel and
Maize starch with starch absorbing water via a capillary system, between and into the particles.(118)
Because Avicel can only absorb water into the capillary system, wetting of ALB is not easy and thus
lower ALB release rate has been noticed. Since the model drug Caffeine (CAF) was almost fully
released (above 91% in all formulations in 30 minutes as seen in Appendix 5) and ALB is not, it can be
suggested that ALB had been entrapped into the microcrystalline cellulose’s water insoluble chain,
which delay the wetting and leads to the poor dissolution. Although Avicel has been found as the
excellent binder, diluent and disintegrant in the wet granulation process (113), researches have also
proved its lack of disintegration. (118,119) They found that Avicel granules do not disintegrate and
drug release occurs by diffusion through an insoluble matrix. In addition, it is generally accepted that
unlike starch, Avicel cannot be wet granulated without losing some of its disintegration properties.
(113) In this dissolution study, this was confirmed with the high soluble drug caffeine (CAF) as its
lowest release of 91% ± 0,76% in 30 min was noted for FOR5. Therefore the lack of disintegration of
Avicel is an important issue for immediate drug release formulation.
The differences in release can be further explained by the diversity in ALB and CAF solubility since the
drug release mechanism depends primarily on the solubility of the drug.
P a g e | 56
When the uncoated particles were exposed to the water, the liquid penetrated into the granules,
dissolved the both drugs and thus form a saturated solution (as long as undissolved drugs were
present). Therefore it is reasonable to expect a faster CAF release since it is far more soluble.
The other reason might be the difference in the mechanism of diffusion. Because CAF is 20 000x
more soluble in water than ALB (19,22), migration of CAF into the dextrin polymer during granulation
process is possible. Thus, faster dissolution of that drug is probable as the CAF particles might be
caught on the granule surface.
It has to be noted that a majority of studies and patents have confirmed that pellets prepared by
microcrystalline cellulose (MCC) show tendency to have a prolong drug release. (120,121,122,123)
On the other hand, researches have also found that combining MMC, CCS and Tween80 with poor
water soluble drug Curcumin, the disintegration of pellets was noted to be within 4 min.(124)
Moreover, Newton et al. (125) have proved that Tween80 is able to solubilize water-insoluble drugs.
On that basis, the Tween80 was chosen properly as this surfactant can absorbs onto the surface of a
hydrophobic ALB particle with its hydrophobic chain and thus promote ALB release.
Although it was found that release can be increased up to 80% when using Avicel and Maize Starch
with Tween80 and CCS, the ALB release was even higher when using dextrin as a carrier. In addition,
dextrin has the same general formula as starch, although it’s smaller and less complex. (113) On the
Graph 1 (look in Appendix 4 for the more detailed graph values) one can compare the ALB release
from the dextrin granules in FOR6 and FOR7. As it is presented, nearly 88% ALB was released in the
first 10 min, 93,15% ± 1,59% at 30 min and 95,04% ± 0,34% after one hour from FOR6. This
corresponds to the USP Guidelines for Immediate release formulation.(126)On the other hand, ALB
release from FOR7 was 60,00% ± 0,26% at 30 min. In the case of CAF, its release was at 30 min
98,31% ± 1,81% and 99,45% ± 1,57% for FOR6 and FOR7, respectively. Dextrin, together with
Tween80 and CSS, is obviously much more appropriate polymer for improving the ALB release rate
which can be due to the rapid solubilization of the polymer in the water.
Generally, the dissolution is explained by the rate of solid-solvent interaction leading to the
solubilization of the molecule and the diffusion rate of the solvated molecule into the water. Because
of dextrin’s polar O-H groups, water easily forms hydrogen bonds with polar O-H groups in the
polymer. What is more, the strong electrophilic carbamat group in ALB might spontaneously react
with the nucleophilic groups. After the contact, the water molecules solvate the Tween-solubilized
dextrin vehicle (with entrapped ALB) and then break the hydrogen bonds between the ALB-dextrin
complexes. As a result, a layer of dissolved dextrin and ALB is formed, promoting the ALB release.
P a g e | 57
However, this may not be the case with Avicel, probably due to its poor disintegration properties.
(127)
When Cyclodextrin Beta was used in FOR11, 88,47% ± 1,27% ALB was released at 30 min and 93,06%
± 0,19% in case of CAF. This result can further confirm that dextrin can be a suitable polymer for
improving the apparent solubility of poor water soluble drugs. This occurrence might be interpreted
due to a change in the cyclodextrin confirmation, which may be unfolded and acted as a linear
dextrin. (127) Gharibi et al. (128)proposed that the nonionic surfactant’s head absorbs on the PVP
chain, which leads to an expansion of the polymer and results in its unfolding. However, as Tween80
acts as a nonionic surfactant, the unfolding from the cyclic to dextrin’s linear form should be further
investigated.
In case of FOR9 and FOR12, the sucrose was used as a binder. Nonetheless, sucrose was not found to
be as good excipient as the dextrin. From the Graph 2 (look in the Appendix 4 for more in-depth
view) it can be seen that ALB release at 30 min was 77,90% ± 1,45% and 66,04% ± 0,03% for FOR9
and FOR12, respectively. The CAF release was noted to be above 98% at 30 min in both formulations.
Comparing the ALB release from formulation with and without PEG400 (FOR1 in comparison with
FOR2; FOR8 with FOR3 and FOR10; FOR7 with FOR6 and FOR12 with FOR9), it can be concluded that
with PEG-formulations, ALB release is significantly lower. On both graphs, Graph 1 and Graph 2, it
appears that only 60,83% ± 1,57%; 60,00% ± 0,26%; 70,00% ± 1,25% and 66,04% ± 0,03% ALB release
was noted at 30 min with FOR1, FOR8, FOR7 and FOR12, respectively. In contrast with their parallel
formulations, without PEG, it can be found that ALB release is notably greater: 72,75% ± 2,51%;
71,35% ± 0,82% / 88,47% ± 1,27%; 93,15% ± 1,59% and 77,90% ± 1,45% in FOR2; FOR3/FOR10; FOR6
and FOR9, respectively. Thus, the ALB release rate has been found to be greater when only Tween80
was incorporated into the formulation FOR2, FOR3, FOR6, FOR9 and FOR10, respectively. This may
be due to the findings reported by Newton et al.(119,125) and Ghembremeskel et al. (129), when
incorporation of Tween80 as a non-ionic surfactant has lead to the more porous pellets with
increased drug release rate.
Apart from that, in the Jachowicz’s study (130), PEG400 was found as the carrier for increasing the
dissolution rate of several poorly water-soluble drugs. Moreover, Nicklasson and Alderborn(131)
have found the evidence that the PEG resulting pellets are less rigid than MMC pellets.
Nevertheless, the use of PEG400 in this study was found to decrease the ALB release from granules
and thus was not suitable for immediate release formulations. It may be reasonable to conclude that
the greatest difference has appeared between FOR6 and FOR7, 93,15% ± 1,59% and 60,00% ± 0,26%
at 30 min, where the enhanced ALB release is linked with FOR6 (formulation without PEG).
P a g e | 58
It can be summed up that ALB granules containing PEG400 diminished the apparent solubility from
the particles. This effect was further investigated as the interaction between ALB and PEG can be
likely a reason since it was found by Cory et al., (132) Carvalho et al.,(133) Vecchio et al.,(134) and
Mura et al. (135) that PEG show interactions with some major APIs on the market.
In conclusion, 12 formulations with different excipients have been developed by the wet granulation
process. The ALB release was greatly improved when using dextrin polymer; nonetheless, the
incorporation of Tween80 and CCS allowed granules to disintegrate within a very short time, while
PEG400 was found to decrease the ALB release in the water. The results suggest that the dextrin
granules containing ALB can be a promising carrier for enhancing drug dissolution of poorly water
soluble drugs.
4.3.2. Microcapsules for taste-concealing7
In Appendix 6, the ALB and CAF release can be observed in more details.
Graph 3: ALB release from uncoated, coated FOR6 granules and coated FOR6 in UNICEF gel with ALB pure drug release
It was reasoned before that with the great improvement in the social standards of living, it is no
longer acceptable for drugs to have bitter taste.
Among various technologies, the easiest method is to add sweeteners, but this may not be effective
enough to conceal the drug’s bitterness. (63,64,65)
7Because the HPLC method was not suitable for ALB release estimation from coated microparticles, UV Spectroscopy was used.
Furthermore, the statistical t-test has been made between ALB release from granules by HPLC and Spectroscopy in order to confirm the
comparability of both methods.
0%
20%
40%
60%
80%
100%
0 10 20 30 40 50 60 70 80
%d
rug
rele
ase
time (min)
% ALB release from uncoated and coated FOR6 granules in comparison with coated FOR6 MICs incorporated in UNICEF gel in water
FOR6 uncoated granules Coated FOR6 Coated FOR6 in UNICEF gel Albendazole powder
P a g e | 59
Therefore, microencapsulation with the methyl cellulose coating was made in order to avoid
albendazole’s unpleasant taste. In this regard, severe considerations must be made to prevent any
remarkable change in the immediate release kinetics after the formulation dissolves in the saliva and
goes to the stomach.
It is important to reiterate that if taste concealing (using MC as polymer coating in this study) is a key
aspect of the dosage form, a multi-point profile in a neutral pH medium with early point of analysis
(e.g. ≤5min) may be recommended. Dissolution criteria of ≤10% dissolved drug in 5 min may enable
the in vitro evaluation of the taste concealing properties. (71)
In order to evaluate whether the goal of improving the ALB release rate from uncoated and coated
granules was reached, in vitro dissolution profiles were compared with that of pure ALB powder. The
ALB dissolution in Milli-Q® Water (with the pre-checked pH of 6,9 ± 0,1) was very low. As can be seen
in Graph 3, only 7,80% ± 0,39% was dissolved in the first 30 minutes, whereas 93,15% ± 0,34% and
67,36% ± 1,19% of the ALB present in uncoated and coated microparticles, respectively, dissolved
within the same time range. Thus, a major ALB release from the microparticles was achieved.
However, the coating polymer methyl cellulose (MC) had retarded the drug release, resulting in
67,36% ± 1,19% ALB release at 30 min from the coated granules. In addition, ALB release was noted
to be 5,88% ± 0,42% and 15,31% ± 1,99% after initially 2 min and 5 min, respectively. Although this
not follows the taste-masked guidelines (71), the microparticles may sufficiently mask the bitterness
of ALB for the first 2 min.
The aforementioned theory explains that for the therapeutic effect it is essential to deliver a
sufficient amount of drug to the site of action, which can be hampered in the pediatric population
especially if the drug is bitter. (9,92,110,136) After my own observations and reports by the nurses at
the Pediatric Department in the Hvidovre Hospital in Denmark, the majority of parents solve this
problem by mixing the bitter drug with the food. Therefore, there is a need for efficient drug delivery
system as there is no proof whether the medicine interacts with food constituents.
Therefore, the coated MICs were incorporated into the UNICEF gel, a semi-solid carrier, having the
consistency of porridge. In the patent, granted by Cuff (136), it was reported that a drug delivery
system of porridge/pudding consistency can sufficiently help to mask the unpleasant taste. In
addition, the pudding formulation is also more acceptable to the children (136). After the
incorporation, the ALB release was, against our expectations, initially vaguely higher; it has reached
9,01% ± 1,44% and 19,45% ± 1,69% in 2 min and 5 min, respectively.
P a g e | 60
It can be proposed that, even though this is not in accordance with the taste-masked guidelines (71),
the semi-solid formulation, with the incorporated MICs, may sufficiently mask the bitterness of ALB
for the first 2 min.
Hence, it could be clearly seen that the ALB release from the semi-solid formulation was faster than
from the MICs. The reason for this might be in the linkages between hydrophobic portions of MC
chains between the MICs and UNICEF-gel or in faster solubilization of MICs, initially promoted by the
gel.
However, the ability of the polymer coating to conceal the bitterness depends on various factors,
such as its permeability for ALB and water, mechanical stability, water-solubility and coating
thickness. (116) It was confirmed by Pearnchub et al. (116) that taste-concealing is efficiently
improved with increasing the coating thickness. In this thesis, the coating thickness was confirmed to
be 29, 47μm. For that reason, the future work should include the coat dimensions up to 50 μm in
order to provide better taste concealing.
It might be reasonable to propose that with greater coating levels of MC, the diffusion pathways for
water should also be increased. Once the microparticles come into the contact with the saliva in the
mouth, water leads to the swelling and dissolution of the coating polymer. While CAF, a water
soluble drug, is released by diffusion through a swollen polymer, ALB as a water insoluble drug is
released by erosion of the polymer. (116,137) Finally, ALB releases from MICs and causes the bitter
taste. Therefore, higher coating levels than 10% should be required when coating with MC as this
can result in more sufficient outcome in the taste concealing.
Although hydrophobic polymers have been popularly used for coating bitter drugs to achieve taste-
concealing (65), hydrophilic polymers may also provide good taste concealing. According to Sohi et
al. (93), Ayenew et al. (65), Sharma et al. (63) and Vummaneni et al. (62), there were few polymers
used for MEC in order to conceal the taste. The polymers are mainly used in the combination of
water soluble, like gelatin and water insoluble polymers like Ethyl Cellulose/Hydroxyl Propil Methyl
Cellulose. However, there were no microencapsulation studies which could affirm the sufficient
coating with MC so far.
To sum up, as the ALB release of the pure drug was only 7,80% ± 0,39% at 30 min, ALB presented in
the uncoated and coated MICs was 93,15% ± 0,34% and 69,36% ± 1,19% within the same time range.
Thus, an exceptional increase in ALB release was achieved in the both formulations. It was, however,
found that the coating polymer MC had somehow retarded the ALB release when compared to the
granules.
P a g e | 61
When MICs were incorporated into the UNICEF gel, it was indicated that MICs would sufficiently
mask the bitterness of ALB in the first 2 minutes. This does not follow the guidelines, but knowing the
fact that poor taste masking is not due irregular shape, nor either broader PSD, the unsuitable factor
may be the MC. In addition, the coating levels should be increased for higher taste-concealing
properties as this might be in correlation with increased coat thickness. In future, care should be
taken when considering the appropriate coating polymer.
4.3.3. ALB dissolution studies 8
In Appendix 7, the ALB and CAF release can be observed in more details.
Graph 4: ALB release without and with the incorporation into the UNICEF gel
The analysis in Rogers et al. reviews (138,139) obviously showed that only 5% of all MICs references
till 2012 reported the use of methylcellulose (MC) to achieve MEC. No references were identified
where MC microcapsules were used for taste concealing, although the numerous preparation
technologies for MEC of core material have been reported. (138) However, there were a few studies
where MC was used as a polymer film for coating, but none of these include fluidized bed
coating.(138)
8In Appendix 8 more detailed information about ALB release in the gel at pH 6,8 can be seen. Moreover, another graph was
made in order to pinpoint the difference between ALB release with and without the UNICEF gel.
0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
0 20 40 60 80 100 120 140 160 180 200
dru
g re
leas
e
time (min)
ALB and ALB incorporated in the gel at pH 6,8 with the addition of Tween80
Albendazole Albendazole in gel Albendazole + Tween80 Albendazole + Tween80 + GEL
P a g e | 62
Beyond the scope of this study, dissolution of ALB and ALB incorporated into the UNICEF gel can be
seen from the Graph 4. As noted before, the ALB release was higher when MICs were incorporated
into the gel. However, when ALB dissolution of a pure drug was plotted as a reference to the
dissolution of ALB in the UNICEF gel, it was clearly confirmed that UNICEF gel had altered the drug
release. As can be seen that 13,93% ± 1,87% ALB was dissolved in 120 min, whereas only 7,56% ±
1,27% at the same time when ALB was incorporated in the gel. In the later investigations, when ALB
was mixed with Tween80 and then incorporated into the UNICEF gel, it was found that ALB had
dissolved rapidly when Tween80 was added.
The data shows that when ALB+TWEEN80 was incorporated in the gel, just 34,50% ± 1,50% ALB had
dissolved, whereas 41,39% ± 1,46% ALB+TWEEN80 had dissolved without the gel incorporation. As
mentioned previously, Newton et al. (125) have proved that Tween80 is able to solubilize water-
insoluble drugs. This confirms that whilst the specific semi-solid carrier (UNICEF gel) slightly alters the
ALB release, Tween80 is the efficient solubilizer for promoting the drug’s release.
4.3.4. Microcapsules and granules/ MICs in 0,1M HCl and in MilliQ Water
In Appendix 8, the ALB and CAF release can be observed in more details.
Graph 5: ALB release from MICs and granules in 0,1M HCL and water
0%
20%
40%
60%
80%
100%
120%
0 10 20 30 40 50 60 70
% d
rug
rele
ase
time (min)
ALB release from microencapsules and granules in 0,1M HCl and in water
Microencapsules in 0,1M HCl Granules in 0,1M HCl Granules in Milli-Q Water Microencapsules in Milli-Q Water
P a g e | 63
The objective of the present investigation was to conceal the bitterness of ALB by encapsulation in
microparticles and lower ALB release in neutral medium (in order to emulate the oral cavity) within
the first 5 min. The complete release of ALB should be then achieved in the stomach at much more
lower pH.
As outlined before in the Background section, once the coated microparticles reach the stomach, the
taste-concealed coat, consisting of MC polymer, should be rapidly dissolved or ruptured to assure
immediate release kinetics. However, it is generally accepted that when MC, being a cellulose ether,
is exposed to an aqueous medium, it undergoes fast hydration and chains relaxation to form a gel
layer, which control water uptake and therefore modify the drug release.(140,141) When polymer
begins to swell, the drug starts to dissolve and then diffuse out of the system due to the different
concentration gradients. (141)
Nevertheless, failure to form the gel layer may cause immediate drug release.(142) From the Graph 5
it is visible, that ALB release from MICs after 30 min, under acidic conditions, was only 71,26% ±
0,51%; whether from the granules it was 92,92% ± 1,54% at the same time, respectively. It appears
that when ALB from the coated granules was dissolved under acidic pH in stomach, the release was
slower in comparison with ALB release from granules under the same conditions. Consequently, the
gel layer was formed as the immediate release was not achieved. This might be due poor solubility
of ALB, as poor soluble drugs are primary released thought erosion of the gel layer. (141) In the case
of ALB, the release mechanism is dictated by the low solubility of the drug and thus dissolve and non-
dissolved drug coexist within the polymer matrix, whilst non-dissolve drug is not available for
diffusion/erosion. On the other hand, it could also be due to the inability of ALB to release from
microcapsules in the restricted fluid space. As the coated granules were exposed to the medium, the
water penetrated through MC’s coat and dextrin’s barrier into the microcapsules and dissolved ALB
to form a saturated solution. Therefore the non-dissolve ALB becomes entrapped inside the MC coat
which lowers its release.
Furthermore, the interactions between ALB and methyl cellulose polymer can also alter the barrier
properties of the coat. Nevertheless, this is less probable because the dextrin subcoating around ALB
was sealed before coating with MC.
The release of ALB from FOR6 granules was abundantly achieved within 30 min at acidic (92,92% ±
1,54%) and neutral pH (93,15% ± 0,34%), followed by a plateau as a result of the high porosity of the
granules, the hydrophilic nature of polymer, improved wettability, excellent PSD and spherical shape,
as noted in the previous sections.
P a g e | 64
Graph 5 shows ALB release from uncoated and coated granules in 0,1M HCl, whilst their release in
water is plotted as a reference. From the results it is evident that after changing a dissolution
medium from water to 0,1M HCl, there were approximately no variations between percent of ALB
release from the uncoated granules. However, a slight difference in the slopes of these two curves
when comparing ALB release from MICs in water and in 0,1M HCl, can be observed in Graph 5.
It is interesting that ALB release from the coated granules in the acidic pH was 54,76% ± 1,71% and
61,02% ± 0,91%; whilst at the neutral pH was noted to be 15,31% ± 1,99% and 27,54% ± 1,36% after
5 and 10 min, respectively. Hence, the initial release after 5 and 10 min was 2-3 folds lower in the
water with the pre-checked pH 6,9 ± 0,1. However, ALB release in the acidic environment at 30 min
was 71,26% ± 0,51%, which is not radically higher than in salivary pH with 67,36% ± 1,19%.
Therefore, ALB release from the coated granules was initially influenced by pH of the release media.
As METHOCEL Dow Handbook suggests, MC is stable between the pH range of 2.0 to 13.0 (143). It
can be speculated that at pH less than 2, acid-catalyzed hydrolysis of the glucose-glucose linkages in
MC occurs, which significantly reduce the viscosity of the polymer. (113) Because the erosion plays
the key role in promoting drug release from poor soluble drugs, MC viscosity is one of the essential
factors in the poor water soluble drug’s release mechanism. (142,144,145) Thus, polymer chain
relaxation occurs rapidly at lower pH, leading to faster ALB release in the acidic environment than in
the neutral dissolution media.
As reported by Dashevskyet al. (146) many drugs, being weak bases (as ALB) demonstrate pH-
dependent solubility from the coated pellets. On the contrary, the release rate of ALB for the
uncoated granules was relatively unaffected by pH of the release media, suggesting that this must be
due to the MC coat which had prolonged ALB release in the water in the first 10 min. However, after
10 min, the coat starts swelling and releases ALB. This occurrence might be directly related to the
lower ALB solubility in water than in 0,1M HCl and, hence it could be explained by the pH-dependant
solubility of MC.
To sum up, it is evident that there were insignificant variations between the percent of ALB release
from the uncoated granules in different medium. However, when coated MICs were exposed to the
neutral medium, the ALB release was decreased. As the MC is expected to behave as insoluble at
salivary pH, it can be suggested that MC coat swells and erodes more slowly in neutral, than in the
acidic, environment. For this reason, the ALB release from the coated MICs was found to be MC
dependent.
P a g e | 65
4.4. UV Spectroscopic measurements and t-test
Because the HPLC method was not suitable for ALB release estimation from MICs, UV Spectroscopy
was used. When comparing the measurements of the validated HPLC method with the Spectroscopic
method, the issue was if there had been a significant difference between the results of the same
samples. Since Bland and Altman(147) showed that plotting a difference between measurements
obtained by two methods against a validated method is misleading, the more accurate Student t-test
was made.
T-tests are commonly used in health studies to determine differences between two paired groups.
The significance level (or p value) is used to quantify how probable it is that the null hypothesis is
true. (148) In this comparison, a p value of 0,01 was used, indicating that there is 1% chance that the
null hypothesis could be mistakenly rejected.
In this study, the null hypothesis is that both methods provide the same analytical results. As seen
from Appendix 11, the P two sided experimental value is 0,0126, which is more than the tabulated
value of P= 0,0100, indicating that the null hypothesis can be accepted at the 99% confidence
interval. As the null hypothesis is accepted, we can conclude that the both methods provide the
same analytical results with 99% probability that our conclusion is correct.
4.5. HPLC Analysis
4.5.1. Analysis for validation
Linearity
Tables in Appendix 9.1 presents triplicate concentrations of ALB (λ=295 nm) and CAF (λ=273nm) and
their measured areas. Good linearity was obtained for ALB with equitation Y= 0,8248x + 0,785 (r2
=0,9997) with standard deviation (SD) of 0,2% , relative standard deviation (RSD) for slope 0,24% and
for intercept 4,33% and for CAF with equation Y= 1,0648x-0,2223 (r2 =0,9993) with standard
deviation (SD) of 0,72%, relative standard deviation (RSD) for slope 0,68% and for intercept 6,72%.
Therefore, the method was found linear over the concentration range 5 – 200 μg/ml for ALB and 1 –
12 μg/ml for CAF and the regression coefficients are for both drugs ≥0,999, which follows the ICH
guidelines. (149,150)In Appendix 9.2 and Appendix 9.3, the chromatogram peaks for ALB and CAF can
be seen.
P a g e | 66
y = 0,8248x + 0,785 R² = 0,9997
0
20
40
60
80
100
120
140
160
180
0 50 100 150 200 250
Are
a (m
Au
*min
)
conc. (µg/ml)
Standard curve for albendazole
y = 1,0648x - 0,2223 R² = 0,9993
0
2
4
6
8
10
12
14
0 2 4 6 8 10 12 14
Are
a (m
Au
*min
)
conc. (µg/ml)
Standard curve for caffeine
Accuracy:
Accuracy is given in terms of % deviation of the calculated concentrations, an average-measured
concentration ± SD and RSD, which according to the FDA guidelines is inside the recommended limit
(<2%). However, following ICH Guidelines, the accuracy criterion is the mean recovery of 100 ± 2%
over the range of 80 – 120% of target concentration over three concentration levels at three
replicates. (150) In this study, data were collected over five concentration levels with three replicates
at each level. The % of deviation for ALB and CAF are below 2%, as well as RSD and from this is
evident that the test results are close to the true value. Therefore, the method is accurate within the
desired range.
Graph 7: Standard curve for CAF
Graph 6: Standard curve for ALB
P a g e | 67
ALBENDAZOLE:
Spiked conc.
(µg/ml) Area 1 Area 2 Area 3
Average
Area
Measured conc.
(Mean ± SD)
RSD
%
Deviation
%
30 26,075 26,166 25,731 25,99 30,56 ± 0,23 0,88% 1,87%
50 43,065 42,837 42,549 42,82 50,96 ± 0,26 0,60% 1,92%
100 83,529 84,237 82,651 83,47 100,25 ± 0,79 0,95% 0,25%
140 118,332 117,883 119,363 118,53 142,75 ± 0,76 0,64% 1,96%
180 149,741 146,986 144,857 147,19 177,51 ± 2,45 1,66% 1,38%
% Deviation= (Spiked Concentration – Mean Measured Concentration) x 100 / Spiked Concentration
CAFFEINE:
Spiked conc.
(µg/ml)
Area
1
Area
2
Area
3
Average
Area
Measured conc.
(Mean ± SD) RSD %
Deviation
%
5 5,203 5,137 5,276 5,21 5,10 ± 0,07 1,34% 1,95%
7 7,149 7,043 7,121 7,10 6,88 ± 0,05 0,77% 1,70%
8 8,105 8,109 8,164 8,13 7,84 ± 0,03 0,41% 2,00%
9 9,299 9,285 9,387 9,32 8,97 ± 0,06 0,59% 0,39%
11 11,325 11,536 11,13 11,33 10,85 ± 0,20 1,80% 1,37%
% Deviation= (Spiked Concentration – Mean Measured Concentration) x 100 / Spiked Concentration
Precision:
The precision is expressed as the RSD % and the results can be seen in tables below. ICH (150)
recommends that the instruments’ precision (RSD) is ≤1%. In this study, precision of the technique
was evaluated through the repeatability by assaying nine replicate injections of ALB and CAF at three
different concentration levels. The RSD value for the retention times and peak areas are found to be
≤0,40% for ALB and CAF, respectively.
9.5.2013 for ALBENDAZOLE
Table 15: Precision results from the nine replicate injections of ALB with their mean tr and Area with the calculated RSD value
conc.ALB[μg/ml] tr 1 tr 2 tr 3 tr 4 tr 5 tr 6 tr 7 tr 8 tr 9 Mean tr RSD
%
25 2,667 2,663 2,655 2,657 2,65 2,645 2,645 2,647 2,643 2,65 0,32%
50 2,685 2,675 2,672 2,673 2,667 2,67 2,663 2,67 2,665 2,67 0,24%
100 2,693 2,695 2,703 2,678 2,697 2,675 2,69 2,678 2,688 2,69 0,36%
conc.
ALB[μg/ml] Area 1 Area 2 Area 3 Area 4 Area 5 Area 6 Area 7 Area 8 Area 9
Mean
Area
RSD
%
25 17,197 17,129 17,082 17,116 17,154 17,134 17,111 17,151 17,196 17,14 0,22%
50 37,461 37,462 37,441 37,464 37,48 37,466 37,518 37,513 37,514 37,48 0,08%
100 78,672 78,677 78,645 78,621 78,54 78,689 78,568 78,658 78,7 78,64 0,07%
P a g e | 68
9.5.2013 for CAFFEINE
Table 16: Precision results from the nine replicate injections of CAF with their mean tr and Area with the calculated RSD value
conc. CAF
[μg/ml] tr 1 tr 2 tr 3 tr 4 tr 5 tr 6 tr 7 tr 8 tr 9 Mean tr
RSD
%
2 2,003 2 2,012 2,01 2,003 2,015 2,007 2,002 2,003 2,01 0,26%
6 2,007 2 2,017 2,017 2,008 1,998 1,993 2,002 2,005 2,01 0,40%
10 2,012 2,01 2,003 2 2,01 2,007 2,015 2,013 2,01 2,01 0,24%
conc. CAF
[μg/ml] Area 1 Area 2 Area 3
Area
4 Area 5 Area 6 Area 7 Area 8 Area 9
Mean
Area
RSD
%
2 1,985 1,984 2 1,992 1,999 1,999 2,005 1,989 2 1,99 0,37%
6 6,391 6,398 6,399 6,415 6,405 6,405 6,407 6,419 6,414 6,41 0,14%
10 11,139 11,132 11,208 11,21 11,163 11,154 11,159 11,151 11,143 11,16 0,25%
Ruggedness:
Ruggedness, expressed as the intermediate precision (inter-day variation) is the result from within
lab variations, due to different analyzing days. (91) Ruggedness criterion is expressed in RSD and it
should be, according to the ICH, less than 2%. (150)
As seen in the Table 17 and Table 18, an RSD of less than 1,52% for ALB retention times and less than
2,03% for ALB areas were obtained. For CAF, RSD for areas was less than 0,95% and for retention
time less than 0,38%. Nevertheless, the experimental values are not radically higher than
recommending values, which indicates that the method is capable of producing results with high
precision on different days.
10.5.2013 for ALBENDAZOLE
Table 17: Ruggedness results from the nine replicate injections of ALB with their mean tr and Area with the calculated RSD value
conc. ALB[μg/ml]
tr 1 tr 2 tr 3 tr 4 tr 5 tr 6 tr 7 tr 8 tr 9 Mean tr RSD
%
25 2,795 2,715 2,695 2,693 2,677 2,68 2,667 2,663 2,668 2,69 1,52%
50 2,78 2,703 2,702 2,705 2,69 2,673 2,682 2,678 2,673 2,70 1,23%
100 2,773 2,733 2,733 2,738 2,722 2,71 2,717 2,717 2,705 2,73 0,75%
conc. ALB[μg/ml]
Area 1 Area 2 Area 3 Area 4 Area 5 Area 6 Area 7 Area 8 Area 9 Mean Area
RSD %
25 13,568 13,578 13,593 13,197 13,217 13,89 13,854 13,532 13,147 13,51 2,02%
50 30,664 29,796 29,538 29,941 29,18 29,66 29,03 29,329 30,742 29,76 2,03%
100 78,375 77,335 76,318 77,99 75,527 77,634 75,568 75,673 77,862 76,92 1,49%
P a g e | 69
10.5.2013 for CAFFEINE
LOD and LOQ determination:
The LOD and LOQ of the method were determined by calculating the signal to noise (S/N) ratio of 3:1
and 10:1 respectively. LOD and LOQ values for ALB were estimated to be 0,085 μg/ml and 0,26μg/ml,
respectively and similarly LOD and LOQ values for CAF were estimated to be 0,38 μg/ml and 1,16
μg/ml, respectively.
In Appendix 9.4 – 9.7., the chromatograms with LOD and LOQ for ALB and CAF can be observed.
A simple and rapid isocratic RP-HPLC method was developed for the determination of ALB and CAF.
The method was validated for various statistical parameters, including linearity, accuracy, precision,
and ruggedness and LOD/LOQ estimation. It is found to be linear, precise, accurate, and therefore
reliable for the estimation of ALB and CAF. It has runtime of 5 min which allows analysis of large
number of samples in a short period of time. This method can be used for further determinations.
Table 18: Rugedness results from the nine replicate injections of CAF with their mean tr and Area with the calculated RSD value
conc. CAF [μg/ml]
tr 1 tr 2 tr 3 tr 4 tr 5 tr 6 tr 7 tr 8 tr 9 Mean
tr RSD
%
2 2,02 2,023 2,022 2,015 2,018 2,022 2,008 2,017 2 2,02 0,38%
6 2,022 2,012 2,02 2,017 2,027 2,018 2,015 2,013 2 2,02 0,38%
10 2,017 2,017 2,025 2,013 2,017 2,007 2,017 2,017 2,008 2,02 0,27%
conc. CAF [μg/ml]
Area 1 Area 2 Area 3 Area 4 Area 5 Area 6 Area 7 Area 8 Area 9 Mean Area
RSD %
2 22,757 22,919 22,919 23,393 22,976 22,965 22,931 23,234 23,34 23,05 0,95%
6 70,552 70,738 70,738 71,282 70,811 70,68 70,898 71,159 71,838 70,97 0,56%
10 122,396 122,644 122,644 123,403 122,086 122,188 122,654 122,687 122,9 122,62 0,32%
Table 19: Results from the six replicate injections of ALB and CAF with their mean Area, LOD and LOQ
Area1 Area2 Area3 Area4 Area5 Area6
Average Area
SD LOD
[μg/ml] LOQ
[μg/ml]
ALB 0,863 0,467 0,769 0,861 0,405 0,861 0,704 0,212 0,0848 0,257
CAF 0,567 0,655 0,606 0,298 0,604 0,332 0,510 0,154 0,382 1,16
P a g e | 70
4.5.2. Analysis for drug content estimation9
Drug content estimation is an important factor when determining the incorporation efficiency and
thus, the MICs quality. From Appendix 13, it can be seen that ALB encapsulation efficiency was in
FOR1 – FOR3, FOR6 and FOR8 – FOR10 above 95%, while in all other was noted to be above 80% with
the exception of FOR12, when the efficiency was above 100%. In addition, the CAF granulation
efficiency was also in most cases above 90% as seen in Appendix 13.
From the results in Table 20, it can be observed that ALB content was 96,92 ± 0,35% and 92,43 ±
0,74% for uncoated and coated granules respectively. It is essential to note that the main purpose of
this estimation study was not investigating the reasons for either low or high encapsulating
efficiency. Therefore, the data obtained from this study was strictly used for the correct estimation
of ALB and CAF release in dissolution studies.
Nevertheless, due to immensely entrapment efficiency, especially in FOR6, it can be suggested that
the granulation and the coating method, done by food processor and fluid bed, respectively, are
suitable for the preparation and estimation of ALB microcapsules. In addition, the high efficiency can
also be due interactions between the core and the coat material, as reported by Boury et al. (151) On
the other hand, Crotts et al. (152)indicated that these interactions can, thus, limit the drug release
from the microparticles. Johansen et al. (153) proposed that a co-encapsulated excipient can
mediate the interactions between the core and coat material. Therefore, this might explain the
slighter lower ALB efficiency value in coated granules when compared to the uncoated ones.
To sum up, the entrapment efficiencies were, in both formulations, unprecedentedly high. Since it is
evident from the dissolution studies that MC delays the ALB release, the interaction between MC
polymer and ALB can be reasonably suggested. However, the variation between the granulation and
coating efficiency is not significant, which can be due to the different excipients used in the both
production processes.
9In Appendix 11, the example of the calculation for the estimation of ALB drug content was made and the efficacy of the
granulation process can be predicted. These calculations were used for the calculations of drug release in dissolution
studies.
Table 20: Albendazole encapsulation efficiency for granules and microcapsules
ALB content EFFICACY SD
FOR6 granules 96,92% 0,35%
Microcapsules (coated granules) 92,43% 0,74%
P a g e | 71
5. CONCLUSION
The use of microencapsulation as a technique to conceal the albendazole’s bitter taste and enhance
the solubility is a considerably unexploded area. Hence, this study is among the first of its kind. It was
the initial attempt to provide the insights into the improvement of ALB release rate on the basis of its
granulation in dextrin microparticles, further fluid bed coating with methyl cellulose and, finally, their
incorporation into the semi-solid formulation, known as UNICEF gel.
The surprising outcome of the research lends support to the belief that it is achievable to attain the
micro size product of poorly soluble drug ALB with defined and narrow distribution range, where the
granules and MICs size range from 75-174 μm and 134-340 μm, respectively. According to the
measured bulk and tapped density values, granulation in Kenwood Food Processor greatly improved
the flow properties of the powder mix and 12 formulations, all with various excipients, had been
designed.
For the dissolution studies, as well as for the entrapment efficiency studies, it was fundamental to
have a linear, precise, accurate and reliable determination method for the quantification of ALB.
Therefore, a simple and rapid isocratic RP-HPLC method for the estimation of ALB (and a model drug
CAF) was developed and validated.
After the dissolution studies, using USP Apparatus II, the ALB release has been greatly improved
when using a dextrin as a granulation polymer. From the SEM Images, it can be further concluded
that dextrin gives a good coverage of the ALB, resulting in the smooth and uniform surface
morphology which has been preferred for fluid bed coating. What's more, the incorporation of
Tween80 and CCS allow granules to disintegrate within a very short time, while PEG400 has been
found to decrease the ALB release in the water. After the taste-masked MC layer of 29,5μm had been
formed in the fluid bed onto the granules, even more round MICs were obtained. As the ALB release
of the pure drug was only 7,80% ± 0,39% in water at 30 min, ALB presented in uncoated and coated
MICs was 93,15% ± 0,34% and 67,36% ± 1,19% within the same time range. Thus, an exceptional
increase has been achieved in the both formulations. Nonetheless, the coating polymer MC had
slightly retarded the ALB release from MICs when compared to the granules.
When MICs were incorporated into UNICEF gel, it was found that MICs could sufficiently conceal the
bitterness of ALB in the first 2 minutes. This, however, does not follow the taste-masked guidelines,
which suggest a broader time range of 5min. Furthermore, it was confirmed that while UNICEF gel
slightly alters the ALB release, Tween80 is the efficient solubilizer for promoting its release.
P a g e | 72
It was also demonstrated that with the dissolution media change from the neutral media to 0,1M HCl
(pH 1,2) the ALB release from the MC-coated MICs was initially influenced by the pH environment.
Conversely, this occurrence does not happen with the uncoated granules.
Therefore ALB release was found to be MC dependent. As the immensely high entrapment
efficiencies were confirmed in granules and MICs, 96,92% ± 0,35% and 92,43% ± 0,74%, this might
also offer an understanding why MC delays the ALB release. As reported by Boury et al. (151),
Crotts&Gwan-Park(152) and Johansen et al.(153), the interactions between the core and coat
material could be the reason for the high entrapment efficiency.
To sum up, the novel results provide striking evidence that it is feasible to attain the round and
uniform granules of poorly soluble drug ALB for the immediate release formulation. In addition, the
dextrin granules, containing ALB, with the incorporation of Tween80 and CCS, can be a fruitful and
promising carrier for enhancing the drug dissolution of poorly water soluble drugs. In case of MC,
there is an overwhelming need to optimize the level of coating in order to completely conceal the
taste of a bitter ALB while not adversely affect the immediate drug release profile.
P a g e | 73
6. FUTURE RESEARCH SUGGESTIONS
A number of open problems need to be solved in order to allow the microencapsulation
development. Hence, some suggestions and a few possible research directions would be made in
order to provide the future steps along the microencapsulation of poor soluble drugs, particularly
albendazole.
One such direction would be to investigate the rupturability of a microparticle, as the SEM Images
clearly suggested there are a few visible cervices on the MIC’s surface. In order to get the
quantitative effect of the above mentioned limitation, the rupturability/mechanical robustness can
be measured on a practical basis, by subjecting the microcapsules to, either a chewing test, or a
texture analyzer.
The idea of albendazole not being completely trapped into the dextrin/MC polymer was addressed in
the Section 4.3. This might affect, not just the dissolution rate of ALB, but also the taste-concealing
properties of the formulation. Therefore, this suggestion is certainly the one that would need further
investigation.
It would also be preferable to optimize the level of coating by coordinating the right type of the
coating material in order to completely conceal the taste of a bitter drug while at the same time, not
adversely affect the immediate drug release profile.
Another possibility would be to form a multiparticulate system, using different anthelmintic drugs as
the resistance to single active antihelmintic is nowadays an arising problem. However, several
stability studies should be done beforehand.
P a g e | 74
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8. APPENDICES
