University of Szeged ANALYSIS OF PHARMACEUTICAL INGREDIENTS, EXCIPIENTS AND DOSAGE FORMS (PRACTICAL WORKBOOK) Edited by: Dr. Zoltán Aigner Dr. Piroska Szabó-Révész Authors: Dr. Mária Budai-Sz"cs Dr. Erzsébet Csányi Dr. Katalin Kristó Dr. Péter Láng Szeged, 2015 This work is supported by the European Union, co-financed by the European Social Fund, within the framework of "Coordinated, practice-oriented, student-friendly modernization of biomedical education in three Hungarian universities (Pécs, Debrecen, Szeged), with focus on the strengthening of international competitiveness" TÁMOP-4.1.1.C-13/1/KONV-2014-0001 project. The curriculum can not be sold in any form!
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University of Szeged
ANALYSIS OF PHARMACEUTICAL INGREDIENTS,
EXCIPIENTS AND DOSAGE FORMS
(PRACTICAL WORKBOOK)
Edited by:
Dr. Zoltán Aigner
Dr. Piroska Szabó-Révész
Authors:
Dr. Mária Budai-Szűcs
Dr. Erzsébet Csányi
Dr. Katalin Kristó
Dr. Péter Láng
Szeged, 2015
This work is supported by the European Union, co-financed by the European Social Fund, within the framework of "Coordinated, practice-oriented, student-friendly modernization of
biomedical education in three Hungarian universities (Pécs, Debrecen, Szeged), with focus on the strengthening of international competitiveness" TÁMOP-4.1.1.C-13/1/KONV-2014-0001
project.
The curriculum can not be sold in any form!
Analysis of pharmaceutical ingredients, excipients and dosage forms 2_______________________________________________________________________________
I. Preformulation ............................................................................................................6
1. Determination of the dissolution rate of the active substance ....................................7 2. Determination of the spectral transmission of glass containers................................13 3. Investigation of the gel formation of Carbomera......................................................19 4. Investigation of the consistency of suppository bases – Determination of the resistance to rupture and flow point of suppositories ...............................................22
II. Investigation of dosage forms...................................................................................28
5. Determination of the drop-weight of dosage forms used dropwise ..........................29 6. Determination of the viscosity of dextran infusion and the average molecular weight of dextran .....................................................................................38 7. Investigation of emulsions ........................................................................................43 8. Investigation of suspensions .....................................................................................49 9. Investigation of ointments I. – Investigation of drop point, solidifying point, oil
number and water absorbing capacity.......................................................................56 10. Investigation of ointments II. – Investigation of the washability and rheological
characteristics of ointments.......................................................................................61 11. Investigation of ointments III. – Characterisation of consistency by viscosity,
spreadability and adhesion investigations.................................................................66 12. Investigation of the water uptake of tablets ..............................................................74 13. Investigation of tablets ..............................................................................................80 14. Investigation of the disintegration of rectal and vaginal suppositories.....................85 III. Investigation of stability ...........................................................................................88
15. Determination of the decomposition (caramelization) of glucose solution ..............89
16. Investigation of the viscosity changes of hydrophilic sols I. ...................................94 17. Investigation of the viscosity changes of hydrophilic sols II....................................99 18. Influence of humidity on the geometric parameters of tablets ...............................103 19. Investigation of the stability of tablets containing acetylsalicylic acid I. – Investigation of decomposition kinetics, calculation of shelf life using (real time) long-term stability test...........................................................................108 20. Investigation of the stability of tablets containing acetylsalicylic acid II. – Investigation of decomposition kinetics, calculation of shelf life using stress test .......................................................................................................113 IV. Biopharmaceutical investigations ...........................................................................117
21. Investigation of the drug release of emulsions by static diffusion method.............118
22. Investigation of drug release by agar plate diffusion method.................................124 23. Investigation of drug release from suppositories by dynamic diffusion method....131 24. Investigation of the mucoadhesivity of hydrogels ..................................................136
Analysis of pharmaceutical ingredients, excipients and dosage forms 3_______________________________________________________________________________
Helios Alpha UV-Vis spectrophotometer ...............................................................143 Anton Paar RHEOLAB MC 1 rheometer ...............................................................145
Analysis of pharmaceutical ingredients, excipients and dosage forms 4_______________________________________________________________________________
Introduction
Medicine is a special commodity, which means that besides the active pharmaceutical
ingredients (API) (equivalent terms: active substance, active ingredient, drug substance,
medicinal substance) and the excipients, the product also involves the sum of technological
processes and additional information which leads to effective therapy.
For the formulation of a medicine with the desired effect and stability, comprehensive
physical, physical-chemical and biopharmaceutical knowledge is needed, which must be
applied already during the API research and development, and later through the process of
development, licensing and production as well. It is very important for the professionals
participating in development, production and control to adopt the approach which leads to the
solution of the above problem.
As the first step of development, preformulation studies are made. During the
preformulation studies the physical-chemical properties of the API and excipients are
investigated, they are qualified from the aspect of pharmaceutical technology and the
compatibility test of the ingredients is performed. The investigation of polymorphism, the
selection of API salts, early method developments and the analysis of the effects of
technological procedures are carried out in this stage of development. Examples for
preformulation, such as determination of solubility and dissolution rate as physical-chemical
tasks; studying of the effect of salts and pH on hydrophilic sols and gels as compatibility tests,
and investigation of the consistency of suppositories are all included in this book.
After – or possibly parallel to – the preformulation studies, stability tests are started in the
formulation development. With regard to stability, we can speak of chemical, physical-
chemical, microbiological, therapeutic and toxicological stability. The book discusses the
degradation kinetics of the API in the dosage form and its interpretation; and then in view of
kinetics, the calculation of shelf life is presented by using long term and stress tests.
Parameters indicating stability need to be determined in order to establish the physical-
chemical stability of dosage forms, because international guidelines (ICH, EMA) regulating
stability tests do not specify special parameters for the description of systems, they must be
ascertained during the development. The book provides some examples for the
characterization of dosage form stability, such as phase separation rate (emulsions), half-time
(suspensions), oil number (ointments), rheological parameters (yield point, viscosity,
thixotropy), disintegration time (tablets, suppositories).
Analysis of pharmaceutical ingredients, excipients and dosage forms 5_______________________________________________________________________________
The investigation of dosage forms can be conducted on the basis of the methods of the
Pharmacopoeia (disintegration, dissolution, viscosity measurements), but in many cases there
are no specific investigations for the dosage form, and then we can opt for investigations not
listed in the Pharmacopoeia. Such investigations are also presented in this book, for example,
redispersibility of suspensions, determination of the spreadability, adhesion and washability
of ointments.
In the book, great emphasis is laid on biopharmaceutical investigations. Dissolution and
mucoadhesivity tests belong to this type of investigations. In case of dissolution and diffusion
tests, pharmacopoeial and non-pharmacopoeial investigations of emulsions, ointments and
suppositories are presented. As regards mucosal drug delivery, the adhesion of the form on
the mucosa is of key importance, because in the absence of this drug absorption is
questionable. The book presents in vitro rheological methods for the investigation of
mucoadhesion, which can indicate the in vivo adhesivity of the system.
The investigation of containers is discussed in the Pharmacopeia in great detail. This type
of measurements is represented by the light permeability measurements of glass containers.
The book summarizes the main pharmacopoeial and non-pharmacopoeial investigations.
Because of the complexity and manual needs of the tasks, the students perform them in pairs.
The execution of the tasks, the preparation of the report and the (statistical) evaluation of the
results help the students to adopt the proper approach and to acquire manual skills.
Szeged, 1st June, 2015
Dr. Mária Budai-Szűcs Dr. Piroska Szabó-Révész Dr. Zoltán Aigner
Analysis of pharmaceutical ingredients, excipients and dosage forms 6_______________________________________________________________________________
I. Preformulation
Analysis of pharmaceutical ingredients, excipients and dosage forms 7_______________________________________________________________________________
1. Determination of the dissolution rate of the active substance
Introduction
The development of the effect of an active substance depends on several factors. One of
the most important of these is dissolution rate, which determines what quantity of the
pharmaceutical product is dissolved during its residence time in the gastrointestinal tract. The
bioavailability of the active substance may be impaired greatly by its possibly slow
dissolution rate. During the practice, we are going to examine the time course of the
dissolution of active substances, which can provide important data for formulation.
The rate of dissolution is the amount of the dissolved material (dw) that diffuses on surface
(A), perpendicularly to the surface, through layer thickness (d(x)), at constant temperature, as
a result of concentration change (d(c)) in time dt:
d(x)d(c)AD-=
dtdw
⋅⋅ (1)
where D is the diffusion constant (Fick’s law).
The dimension of the dissolution rate constant is: mg · cm-2 ·hour-1.
According to the Noyes-Whitney equation, the dissolution rate constant can be determined
with the following equation in the case of first order kinetics:
][][
][log303.2CC
Ct
ks
s
−= (2)
where t is the time, C is the current concentration of the solute, Cs is the saturation
concentration of the solute (e.g. at room temperature 0.50 g of theobromine dissolves in 1000
mL of distilled water).
The rate of dissolution depends on the following factors:
1. During the practice, you are going to examine the dissolution rate of a specific quantity
of theobromine with the rotating paddle apparatus described in the Hungarian
Pharmacopoeia. Determine the active substance concentration of the samples taken at
the given times spectrophotometrically. Include the obtained results in a table. (c* is the
amount of dissolved theobromine expressed as the percentage of the measured amount.)
2. Calculate the dissolution rate constant of theobromine (k).
Measurement of theobromine Test 1 Test 2 Test 3
_________ with material (g)
_________ empty (g)
Measurement (g)
Results of the dissolution test of theobromine Measurement Time (minute) 5 10 20 30 60 90
A
dilution
c (µg/mL)
dissolved theobromine (mg)
1.
c*
A
dilution
c (µg/mL)
dissolved theobromine (mg)
2.
c*
A
dilution
c (µg/mL)
dissolved theobromine (mg)
3.
c*
c* average
k
Analysis of pharmaceutical ingredients, excipients and dosage forms 12 _______________________________________________________________________________
3. Plot the average of c* against sampling time.
4. Write a short evaluation of the results of the test.
Analysis of pharmaceutical ingredients, excipients and dosage forms 13 _______________________________________________________________________________
2. Determination of the spectral transmission of glass containers
Introduction
Glass containers are most commonly used for storing and dispensing liquid dosage forms.
A great number of APIs, excipients and dosage forms are light-sensitive, that is why they
must be dispensed and stored in glass containers protecting against light, which are tested
according to the Pharmacopeia.
According to Ph. Hg. VIII., glass containers for pharmaceutical use are glass articles
intended to come into direct contact with pharmaceutical preparations. Coloured and
colourless glass can be distinguished. Colourless glass is highly transparent in the visible
spectrum. Coloured glass is obtained by the addition of small amounts of metal oxide(s),
chosen according to the desired spectral absorbance.
The Hungarian Pharmacopeia (Ph. Hg. VIII., 3.2.1.) makes the following
recommendations about glass containers: Preparations for parenteral administration are
normally presented in colourless glass, but coloured glass may be used for substances known
to be light-sensitive. Colourless or coloured glass is used for the other pharmaceutical
preparations. It is recommended that all glass containers for liquid preparations and for
powders for parenteral administration permit the visual inspection of the contents.
Concerning glass containers, the Hungarian Pharmacopeia prescribes physical and
chemical investigations.
Physical investigations:
– determination of filling volume – spectral transmission for coloured glass containers
Chemical investigations:
– investigation of hydrolytic resistance A. hydrolytic resistance of the inner surface of glass containers (surface test) B. hydrolytic resistance of glass grains (glass grains test) C. to determine whether the containers have been surface-treated (etching test) D. surface hydrolytic resistance - determination by flame atomic absorption
spectrometry (faas) – investigation for arsenic compounds
Analysis of pharmaceutical ingredients, excipients and dosage forms 14 _______________________________________________________________________________
Task
Break the glass container or cut it. Select sections representative of the wall thickness and
trim them as suitable for mounting in a spectrophotometer. Make sure the length of the
specimen is greater than that of the slit. Before placing in the holder, wash, dry and wipe the
specimen with lens tissue. Take care to avoid leaving fingerprints or other marks.
Apparatus: UV-VIS spectrophotometer, in transmittance mode
Place the specimen in the spectrophotometer in such a way that the light beam is
perpendicular to the surface of the section (Figure 2.1). Measure the transmission
(transmittance, T) of the specimen with reference to air in the spectral region of 290-450 nm,
at intervals of 20 nm.
Figure 2.1: Placing of the specimen into the cuvette holder of the spectrophotometer
Analysis of pharmaceutical ingredients, excipients and dosage forms 15 _______________________________________________________________________________
Limits (Ph. Hg. VIII.; 3.2.1.)
The observed spectral transmission for coloured glass containers for preparations that are
not for parenteral use does not exceed 10 per cent at any wavelength in the range of 290 nm to
450 nm, irrespective of the type and the capacity of the glass container. The observed spectral
transmission in coloured glass containers for parenteral preparations does not exceed the
limits given in the Table below.
Limits of spectral transmission for coloured glass containers for parenteral preparations
(Ph. Hg. VIII, Table 3.2.1.-5.)
Maximum percentage of spectral transmission at any wavelength between
290 nm and 450 nm Filling volume (mL) Flame-sealed
containers Containers with
closures Up to 1 50 25 Above 1 and up to 2 45 20 Above 2 and up to 5 40 15 Above 5 and up to 10 35 13 Above 10 and up to 20 30 12 Above 20 25 10
1. Measure each specimen in 4 different positions, in this way you will get 4 values for
each sample. Calculate the average of 4 measurements, and plot the data against the
wavelength. Evaluate the results according to the pharmacopoeial limits.
2. Correlate the different glass samples in such a way that their transmission refers to a
wall thickness of 1 mm, by using the following equation:
''%
1
''%
'%
% lglglglg Tll
TTT +⋅−= (1)
where T% is the light permeability of the minimum thickness of the glass container in %
transmittance, T’% is the maximum % light permeability measured in the investigated
wavelength range, T”% is the theoretical light permeability (92%) of the glass container
of a wall thickness of 0 mm (lg T’’% = 1.9638), l1 is the average thickness of the glass
sample in mm, l = 1 mm.
Measure the thickness of the glass samples at 10 points of the specimen with a
micrometer screw. Calculate the average values of wall thickness.
Analysis of pharmaceutical ingredients, excipients and dosage forms 16 _______________________________________________________________________________
Report 2. Determination of the spectral transmission of glass containers
1. Prepare 50 mL of 0.9 % sodium chloride solution. How much NaCl should be used for
the preparation? .................................... g
2. Determine the density of the dextran infusion by using a pycnometer according to the
Hungarian Pharmacopoeia, with three parallel measurements, and fill in the table.
Number of pycnometer Weight of empty pycnometer (g) Weight of pycnometer filled with water (g) Weight of pycnometer filled with dextran infusion (g) Weight of water (g) Weight of dextran infusion (g) Density (g/cm3)Average of density (rounded to three decimal places)
3. Determine the dynamic viscosity of the diluted dextran infusion with the help of a
modified Ostwald-Fenske capillary viscometer according to the Hungarian
Pharmacopoeia, with 5 parallel measurements. Include the results in the table below:
Number of measurement Flow time (s) Average of flow time (s) Viscosity (mPa·s)
1. 2. 3. 4. 5.
4. Calculate the average molecular weight of the dextran infusion according to the formula
given. The average molecular weight of dextran: ............................................
Evaluation:
Analysis of pharmaceutical ingredients, excipients and dosage forms 43 _______________________________________________________________________________
7. Investigation of emulsions
Introduction
Emulsions are thermodynamically unstable systems. The reason for this is the large
specific surface area of the particles. The emulsified state lasts until the layers separating the
liquid drops retain their structure. Thus the constancy of the degree of dispersion is one of the
most important aspects concerning the stability of emulsions, because depending on the drop
size distribution – depending on time – they may float. The drop size is increasing with the
confluence of the floated or flocculated drops (coalescence); this leads to the breaking of the
emulsion, then the dispersed part begins to form a continuous phase, therefore a
thermodynamically favourable condition results. From the point of view of pharmaceutical
production, an emulsion is regarded stable if its degree of dispersion remains unchanged or
changes only within the prescribed limits until use.
During the investigation of the stability of emulsions, the rheological properties, which are
determined by the following factors, are also of great significance: the dispersed phase, the
viscosity of the dispersion medium, the amount and the material quality of the emulsifier
used. If the volumetric concentration of the dispersed phase is small enough, the system
behaves as a Newtonian or ideal viscous fluid. The yield curve of ideal viscous fluids is
characterized by a baseline starting from the origin, and they give a straight line. The flow
becomes pseudoplastic if the volumetric concentration is increased, as a result of which the
resistance of the system to flow increases. The yield curves of such materials also start from
the origin, but they are not linear. Viscosity changes depending on shear stress. The reason for
this is the instantaneous and reversible disaggregation or deformity of the emulsified drops
caused by shear force. The aim of this practice is the investigation of the characteristic
properties of emulsions in case of changing the factors influencing stability.
Task
1. The determination of the yield properties and viscosity of emulsions
The Hungarian Pharmacopoeia (Ph. Hg. VIII., 2.2.10.) lists the following types of
instruments for the determination of viscosity by using a rotating viscometer: spindle
Analysis of pharmaceutical ingredients, excipients and dosage forms 44 _______________________________________________________________________________
viscometers, cone-plate viscometers and concentric cylinder viscometers. During the practice,
an Anton Paar Rheolab MC 1 Rheometer is used, which is a concentric cylinder type
viscometer. The fluid to be examined has to be located in the gap between the inner and outer
cylinders. Viscosity can be measured by rotating the inner cylinder ("Searle" type viscometer)
or by rotating the outer cylinder ("Couette" type viscometer).
Apparatus: Anton Paar Rheolab MC 1 Rheometer
Investigation method
Sample holder "Z3" (Figure 7.1) is filled hermetically with the emulsion to be examined up
to the inner mark. The cylindrical probe is affixed with the help of the retaining ring. The
device is installed according to the instructions of the manual and the required program is
started.
Figure 7.1: Sample holder "Z3" of the Anton Paar Rheolab MC 1 Rheometer
2. Investigation of spontaneous separation (not an official investigation method in Ph. Hg.
VIII.)
Apparatus: 25.0-mL measuring cylinder with a ground stopper
Analysis of pharmaceutical ingredients, excipients and dosage forms 45 _______________________________________________________________________________
Investigation method
25.0 mL of emulsion is filled into a measuring cylinder with a ground stopper. The sample
is shaken a few times. The volume of the water separated and collected in the bottom of the
measuring cylinder is determined after a certain time (one and a half hours).
Calculation of the speed of separation:
V-VV+V
t1=v
t
t
0
0log (1)
where v is the speed of separation (mL/hour), t is the time of observation (hour), Vt is the
volume of water separated during t time (mL), V0 is the original volume of the emulsion (25
mL).
Compositions to be examined:
Materials I. II. III. IV. V.
Paraffinum liquidum 10.0 g 30.0 g 60.0 g 30.0 g 30.0 g
Aqua purificata 68.0 g 48.0 g 8.0 g 47.0 g 49.5 g
Polysorbatum 20 2.0 g 2.0 g 2.0 g 3.0 g 0.5 g
Mucilago methylcellulosi 20.0 g 20.0 g 20.0 g 20.0 g 20.0 g
Apparatus: Mixer (stage 1, 1 min of mixing time)
Analysis of pharmaceutical ingredients, excipients and dosage forms 46 _______________________________________________________________________________
1. The effect of the weight ratio of the dispersed phase on the properties of the emulsion:
Composition Speed of separation Viscosity (D = 656 1/s)
II. (10 %)
I. (30 %)
III. (60 %)
Evaluation:
2. The effect of the concentration of emulsifiers on the properties of the emulsion:
Composition Speed of separation Viscosity (D = 656 1/s)
V. (0.5 %)
I. (2 %)
IV. (3 %)
Evaluation:
Analysis of pharmaceutical ingredients, excipients and dosage forms 47 _______________________________________________________________________________
3. Rheological characterization of the compositions tested (determination of flow type):
I. II. III. IV. V.
D
(1/s)
α τ
(N/m2)
D
(1/s)
α τ
(N/m2)
D
(1/s)
α τ
(N/m2)
D
(1/s)
α τ
(N/m2)
D
(1/s)
α τ
(N/m2)
Analysis of pharmaceutical ingredients, excipients and dosage forms 48 _______________________________________________________________________________
4. Plot and evaluate the yield curves of the compositions.
Evaluation:
Analysis of pharmaceutical ingredients, excipients and dosage forms 49 _______________________________________________________________________________
8. Investigation of suspensions
Introduction
The two most important requirements to be met by suspensions are: the homogeneous
distribution of the active ingredient at least for the period between the mixing of the
suspension and the pouring of given dose, and the easy redispersibility of the sediment
formed during storage. Sedimentation cannot be prevented, but it can be delayed with various
technological methods. One possibility for this is to reduce the size of the non-dissolving
particles, or to enhance the viscosity of the dispersion medium with various macromolecular
materials (e.g. mucilages). With respect to the sediment formed, suspensions can be
flocculated or deflocculated. Flocculation occurs if the macromolecules adsorbed on the solid
particles are cross-linked, or if the surface parts of the particles not covered with
macromolecules stick together due to the high adhesion. The sediment of flocculated
suspensions is loose and bulky. Its advantage is that the floccules (grain aggregates) form a
sediment which is easily redispersible, but sedimentation is fast in this case. In contrast, the
sediment of deflocculated suspensions consists of particles in a close packed arrangement (so-
called "cemented sediment"), therefore it is difficult to shake, but its sedimentation is slower.
The sedimentation of suspensions can be free or hindered (see Figures 8.1 and 8.2).
Sedimentation is free if the suspension is sufficiently washy, in which the particles do not
hinder each other during sedimentation and a sediment with a gradually increasing volume is
formed, where the supernatant is cloudy because of the floating particles. Free sedimentation
is characterized by a high half-life and a low sedimentation rate. However, hindered
sedimentation is more typical of pharmaceutical suspensions. In this case, the particles hinder
each other during sedimentation. The reason for this is the higher concentration of the
particles in the given volume and the interactions between them, and also the presence of the
viscosity increasing excipient. Hindered sedimentation is characterized by a clear supernatant
and a gradually decreasing phase boundary between the sedimenting particles. The aim of this
practice is to determine the half-life (t1/2) of flocculated and deflocculated suspensions, and to
investigate the resuspensibility of the sediment.
Analysis of pharmaceutical ingredients, excipients and dosage forms 50 _______________________________________________________________________________
Figure 8.1: Hindered settling suspension
Figure 8.2: Free settling suspension
Task
1. Investigation of the sedimentation of suspensions (not an official investigation method
in Ph. Hg. VIII.)
Apparatus: 25.0-mL measuring cylinder with a ground stopper
Analysis of pharmaceutical ingredients, excipients and dosage forms 51 _______________________________________________________________________________
Investigation method
Pour the homogenous suspension into a measuring cylinder with a ground stopper and
shake it well. Observe the separation of the dispersed part and the dispersion medium. Note
the height of the sediment column in every 5 minutes during 30 minutes, then in every 15
minutes during 75 minutes. If the suspension becomes foamy after being poured into the
cylinder, the total distance should be measured from the bottom of the foam layer.
Determination of half-life: plot the height of the sediment column against time. In case of
hindered sedimentation:
2
l-l-l=l E1/2
00 (1)
In case of free sedimentation:
2l=l E
1/2 (2)
where l1/2 is the height of „half-distance”, l0 is the initial height, lE is the equilibrium height.
Plot the sediment volume against time. Mark the l1/2 values on the curve, and project them
onto the time axis. The value thus obtained is the half-life. During the determination of half-
life it is also acceptable if the volume of the sediment column is measured instead of its
height. The last measured value is regarded as the equilibrium height (lE) and the equilibrium
volume (VE) .
2. Redispersibility of the sediment (not an official investigation method in Ph. Hg. VIII.)
Apparatus: resuspension device
Investigation method
Carefully fix the cylinder containing the sedimented suspension into the stand of the
sample holder. Rotate the measuring cylinder until the sediment is uniformly distributed in the
medium. The number of rotations is the value of resuspension. The sediment in the bottom of
the measuring cylinder can be observed better if the device is stopped during the investigation
when the measuring cylinder is with its mouth downwards (make sure that the glass stopper
closes the measuring cylinder).
Analysis of pharmaceutical ingredients, excipients and dosage forms 52 _______________________________________________________________________________
Compositions to be examined
Instrument: Mixer (stage 1, 1 min of mixing time)
Series „A” A/1 A/2 A/3 A/4
Bismuthi subgallas 5.0 g 5.0 g 5.0 g 5.0 g Natrii dihydrogenophosphas dihydricus
0.0 g 0.5 g 10.0 g 20.0 g
Aqua purificata ad 50.0 g ad 50.0 g ad 50.0 g ad 50.0 g Series „B” B/1 B/2 B/3 B/4
Bismuthi subgallas 5.0 g 5.0 g 5.0 g 5.0 g Mucilago hydroxyaethylcellulosi 0.0 g 0.5 g 10.0 g 20.0 g Aqua purificata ad 50.0 g ad 50.0 g ad 50.0 g ad 50.0 g
Attention! Start the preparation of the suspensions in series „A” with the dissolution of
NaH2PO4.
Analysis of pharmaceutical ingredients, excipients and dosage forms 53 _______________________________________________________________________________
sodium) is also official, which may contain short fibrils. The calcium salt of partially O-
carboxymethylated cellulose is also official. It swells in water and becomes opalescent, it
forms a viscous colloidal solution. Its application is diverse: for internal use it can be used
only in the form of a colloidal aqueous solution. It is beneficial as a coating agent in the case
of gastro-intestinal disorders. As it can adsorb several substances, it can also be used as an
antidiarrheal drug. It also has an antacid effect, and it is used as a food additive as well (E
466).
In pharmaceutical technology, it is used as a binder agent during the production of
granules. In tablets, Carmellosum natricum conexum (croscarmellose sodium) containing
cross-links can be applied as a disintegrant and it has good swelling properties. In different
concentrations it can also be used as a hydrogel, as the stabilizer of emulsions and
suspensions, furthermore, as a protective colloid, too. It is incompatible with acidic medium,
heavy metal salts, cationic surfactants, alkaloid salts and phenols.
The purpose of the task is to present an incompatibility: to study the viscosity change of 3
% of carmellose sodium mucilage due to the effect of hydrochloric acid.
Task
Apparatus: Höppler-type viscometer
Investigation method
Determine the apparent viscosity of the different available – stored for 24 hours –
carmellose sodium mucilages containing hydrochloric acid of 2 M (1, 2, 3 %) with the
Höppler-type viscometer. The hydrochloric acid-free mucilage serves as the basis for
Analysis of pharmaceutical ingredients, excipients and dosage forms 100 _______________________________________________________________________________
comparison. Plot the calculated viscosity values against the load. Make 3 parallel
measurements and always use a new sample for each measurement.
Process of viscosity measurement
Fill the measuring cup with the colloidal solution to be tested carefully, in a bubble-free
way. Choose the ball rod and mount it onto the measuring arm. Bring the device state ready to
measure. (levelling, offsetting the buoyancy with sliding weight). Put the smallest weight on
the weight holder plate, release the locking screw, measure the time needed to cover the
distance corresponding to the 0-30 scale. After that, remove the weight, pull back the ball rod
carefully and fix the measuring arm with the eccentric. Wait for a short time and repeat the
measurement with greater load. You can increase the load as long as the descent time is
higher than 4 seconds.
The equation of the calculation of viscosity:
tpk= ⋅⋅η (1)
where η is the apparent viscosity (mPa·s), (Pa = 0.01 g/cm2), k is the instrument constant
(cuvette labelled 01, k = 0.1053), p is the load, shear stress (p = 10, 20, 30, 40 and 50 g/cm2), t
is the descent time (s).
Analysis of pharmaceutical ingredients, excipients and dosage forms 101 _______________________________________________________________________________
Report 17. Investigation of the viscosity changes of hydrophilic sols II.
Analysis of pharmaceutical ingredients, excipients and dosage forms 102 _______________________________________________________________________________
2. Plot the viscosity against the load.
3. Evaluation:
Write down what you experienced and find a correlation between the concentration of
hydrochloric acid and the viscosity of the mucilages based on the results. What kind of
colloidal physical phenomenon is it? Give reasons for your answer.
Analysis of pharmaceutical ingredients, excipients and dosage forms 103 _______________________________________________________________________________
18. Influence of humidity on the geometric parameters of tablets
Introduction
Humidity in the air may affect the stability, the physical parameters of tablets, such as their
mass, geometric parameters, mechanical strength, disintegration and also active substance
release. The geometric parameters of tablets (diameter, height) must be within the threshold
limits prescribed in the detailed specifications of the given preparation. The uniformity of size
and shape has great significance when packaging with automatic packaging machines.
The reason for the effect of humidity lies in the hygroscopicity and water uptake capacity
(Enslin number) of the excipients and active substances of the tablets. For example, excipients
used as disintegrants or superdisintegrants, such as starch and sodium starch glycolate, have a
high Enslin number. Because of this, these tablets must be stored carefully away from air, or
with the use of a moisture absorber.
The aim of this investigation is to examine how 100% relative humidity influences the
mass and the geometric parameters of tablets containing various excipients.
Task
1. Measure the mass, diameter and height of the disintegrant- and superdisintegrant-
containing tablets given by your practice leader. Measure the mass with an analytical
balance and the geometric parameters with a micrometer screw.
2. Measure the above parameters also for tablets stored at room temperature, under normal
circumstances and for tablets stored for different periods of time at room temperature at
measurements for each. Include your results in a table. Calculate the averages, the mass
and volume changes.
3. Plot the volume change against time.
Analysis of pharmaceutical ingredients, excipients and dosage forms 104 _______________________________________________________________________________
Report 18. Influence of humidity on the geometric parameters of tablets
Analysis of pharmaceutical ingredients, excipients and dosage forms 105 _______________________________________________________________________________
Analysis of pharmaceutical ingredients, excipients and dosage forms 106 _______________________________________________________________________________
Average values of the tested parameters of Tablet 2:
Storage time (hours)
Average mass (mg)
Average diameter
(mm)
Average height (mm)
Average volume (mm3)
Mass change (mg)
Volume change (mm3)
024 48 72 96
Analysis of pharmaceutical ingredients, excipients and dosage forms 107 _______________________________________________________________________________
3. Plot the volume change against time.
Evaluation:
Analysis of pharmaceutical ingredients, excipients and dosage forms 108 _______________________________________________________________________________
19. Investigation of the stability of tablets containing
acetylsalicylic acid I.
– Investigation of decomposition kinetics, calculation of shelf life
using (real time) long-term stability test
Introduction
The suitable stability of the dosage form provides the efficacy of the medicine up to the
expiration date. A basic requirement is that neither the effect, nor the side effect of the
medicine is allowed to change. The drug content may change between the values prescribed
by law, the maximum decrease permitted is usually 10%.
The kinetic investigation of drug stability gives information about the time course of drug
decomposition. The main decomposition ways of drugs follow first or zero order kinetics.
In case of zero order kinetics, the reaction rate is independent of the concentration of the
reaction partners (Figure 19.1):
[ ] [ ] tkCCt ⋅−= 0 (1)
where Ct is the concentration of the drug in time t, C0 is the initial concentration of the drug, k
is the rate constant.
Figure 19.1: Decomposition of drug following zero order kinetics
Analysis of pharmaceutical ingredients, excipients and dosage forms 109 _______________________________________________________________________________
According to the above, the reaction rate is constant during the reaction.
[ ]0k
dtCdv =−= (2)
On the basis of equation (1), in case of zero order kinetics, the values of t1/2 (half-time) and
t10% (shelf life) can be calculated as follows:
[ ]0
02/1 2k
Ct = (3)
5
2/1%10
tt = (4)
In case of first order kinetics, the reaction rate depends on the concentration of the
reaction partners (Figure 19.2):
[ ] [ ] tkt eCC 1
0−= (5)
[ ] [ ]Ckdt
Cdv 1=−= (6)
On the basis of equation (5), in case of first order kinetics, the values of t1/2 and t10% can be
calculated as follows:
11
2/1693.02lnkk
t == (7)
90
100lg303.2
1%10 k
t = (8)
Figure 19.2: Decomposition of drug following first order kinetics
Analysis of pharmaceutical ingredients, excipients and dosage forms 110 _______________________________________________________________________________
Task
The aim of the investigation is to determine the half-time and the shelf life (t1/2, t10 %) of
tablets containing acetylsalicylic acid (ASA).
Measure the ASA content of tablets stored in well-sealed containers for different periods of
time by means of spectrophotometry. Make reaction kinetics calculations on the basis of 3
parallel measurements. Determine the order of the reaction (zero or first order). Knowing the
rate constant, calculate the values of t1/2 and t10 %.
Investigation method
Pulverize 5 tablets from each batch of tablets stored for a given time. Weigh the amount
corresponding to 1 tablet from the pulverized sample by using an analytical balance and wash
it into a 100-mL volumetric flask with 15 mL of 96% alcohol. Dilute it with purified water,
shake it for 5 minutes and complete it to 100.00 mL with purified water. Filter the solution
immediately and prepare a dilution in the given way.
Make 3 parallel measurements, preparing 3 dilutions from each filtered solution. Measure
the absorbance of the solutions (A) at λ = 276 nm. The reference solution is purified water.
On the basis of the calibration curve, 0.100 A corresponds 25.66 µg/mL of ASA.
Analysis of pharmaceutical ingredients, excipients and dosage forms 111 _______________________________________________________________________________
Report 19. Investigation of the stability of tablets containing acetylsalicylic acid I.
– Investigation of decomposition kinetics, calculation of shelf life using (real time) long-
Nominal weight of 1 tablet: ................................... g
ASA content of fresh tablet: ................................... g
Tablet number
Storage time (month)
2
10
30
60
Pulverize 5 tablets from each batch of tablets stored for a given time. Weigh the amount
corresponding to 1 tablet from the pulverized sample by using an analytical balance and wash
it into a 100-mL volumetric flask with 15 mL of 96% alcohol. Dilute it with purified water,
shake it for 5 minutes and complete it to 100.00 mL with purified water. Filter the solution
immediately and dilute 5 mL of the filtered solution to 100 mL with purified water. Prepare 3
dilutions from each filtered solution.
1. Results of the spectrophotometric measurements:
Measurement 1 Measurement 2 Measurement 3
Sample A
conc. (µg/mL) A
conc. (µg/mL) A
conc. (µg/mL)
Average conc.
(µg/mL)
ASA content
in 1 tablet (mg)
Analysis of pharmaceutical ingredients, excipients and dosage forms 112 _______________________________________________________________________________
2. Calculation of reaction kinetics parameters:
sample C0 (%) Ct (%) k1 k2 t1/2 t10%
AVERAGE:
Evaluation:
Analysis of pharmaceutical ingredients, excipients and dosage forms 113 _______________________________________________________________________________
20. Investigation of the stability of tablets containing
acetylsalicylic acid II.
– Investigation of decomposition kinetics, calculation of shelf life
using stress test
Introduction
Tablets are usually stored at room temperature. Investigations lasting for months or years
are often needed to determine how long they remain stable and contain the effective drug
concentration (>90%) when stored at room temperature. Stress tests can be performed in order
to shorten these investigations.
There is proportionality between the decomposition rate of the drug and the storage
temperature, which is known as the Arrhenius equation:
RT
EAk a
303.2lglg −= (1)
where k is the rate constant of decomposition, A is the pre-exponential factor/frequency factor,
Ea is the activation energy (kJ/mol), R is the universal gas constant (8.314 J/mol·K), T is the
temperature (K).
If log k is plotted against 1/T, it will result in a linear correlation, which is called Arrhenius
plot (Figure 20.1). The slope of this line is -Ea/2.303·R, from which the activation energy can
be calculated.
Figure 20.1: Arrhenius plot
Analysis of pharmaceutical ingredients, excipients and dosage forms 114 _______________________________________________________________________________
Knowing the value of the activation energy (Ea) and the rate constant (k1) at a given
temperature (T1), the rate constant (k2) at a desired temperature (T2) can be calculated and the
shelf life (t10%) can also be determined at this temperature.
−−=
121
2 11303.2
logTTR
Ekk a (2)
2
%,10105.0
2 kt T = (3)
In case of first order kinetics, if the drug concentrations are known, the rate constant can be
calculated as follows:
tC
Ct
k 0log303.2= (4)
where C0 is the initial drug concentration, Ct is the drug concentration in time t, t is the time
(days).
Task
The aim of the investigation is to determine the half-time and the shelf life (t1/2,) of tablets
containing ASA by using stress test. The tablets were stored at different temperatures (40, 50,
60, 70, 80 °C) and the drug content was measured after 1, 2 and 3 days.
1. Calculate the average drug content of the tablets from the data at the given time and
temperature. On the basis of equation (4), calculate the rate constant of the reaction as a
first order reaction at each measuring point, by using the substitution method. Calculate
the rate constant for each temperature.
2. Draw the Arrhenius plot from the mean value of the rate constants belonging to the
given temperature. Calculate the activation energy from the slope of the plot.
3. Calculate the rate constant for 25 °C on the basis of equation (2). Knowing the rate
constant, calculate the shelf life of the tablet referring to 25 °C.
Analysis of pharmaceutical ingredients, excipients and dosage forms 115 _______________________________________________________________________________
Report 20. Investigation of the stability of tablets containing acetylsalicylic acid II.
– Investigation of decomposition kinetics, calculation of shelf life using stress test
1. The ASA concentrations in tablets with different storage times and temperatures are
listed below. Calculate the rate constant for each measuring point and for each
temperature.
ASA concentration (%) Temp.
(T)(°C)
1/T
(K-1)
Storage time (t)(days)
1 2 3 mean k kmean log kmean
1 99.4 99.9 99.8
2 99.1 98.4 98.9
40
3 97.8 98.1 98.7
1 98.9 99.2 98.9
2 97.0 96.9 96.5
50
3 95.4 95.4 95.7
1 96.4 96.3 95.9
2 92.8 92.9 92.7
60
3 89.9 89.9 92.0
1 88.7 89.1 89.2
2 82.0 82.2 81.8
70
3 78.9 78.7 78.2
1 76.2 75.9 75.9
2 60.5 59.9 59.6
80
3 47.2 46.8 47.0
Analysis of pharmaceutical ingredients, excipients and dosage forms 116 _______________________________________________________________________________
2. Arrhenius plot, determination of activation energy:
Ea = ...................................................................
3. Rate constant and shelf life of tablet referring to 25 °C:
Analysis of pharmaceutical ingredients, excipients and dosage forms 117 _______________________________________________________________________________
IV. Biopharmaceutical investigations
Analysis of pharmaceutical ingredients, excipients and dosage forms 118 _______________________________________________________________________________
21. Investigation of the drug release of emulsions by static
diffusion method
Introduction
Although the excipients used in formulation do not have an effect on their own, they can
influence the release of the API, and thereby the efficacy of the drug significantly. The type of
the emulsion is chosen on the basis of the physical-chemical properties of the API. Based on
these factors, the proper choice of the type of the emulsions and the emulsifiers applied is
very important. In the case of emulsions for external use, both the o/w and the w/o types are
used, depending on the extent of penetration into the deeper layers of the skin and on the
duration of drug release.
Polysorbate 20 is a nonionic, water-soluble surfactant, which can be used in o/w emulsions
and emulsion ointments. It increases the solubility of poorly water-soluble drugs. In water it
forms micelles belonging to the colloidal size range and it encloses the non-soluble solid and
liquid components (e.g. essential oils). It is used as a wetting agent in suspensions and
suppositories. It is applied in a concentration of 1-10 % as an emulsifier and 0.1-3 % as a
wetting agent.
Span 80 is a nonionic, practically water-insoluble surfactant, which can be used to produce
w/o type emulsions by itself. It can increase the water absorption of creams. It is also applied
as a solubilizer and a wetting agent in lipophilic medium.
The static diffusion method is a model by which the release of the drug can be determined.
It consists of three main parts: a donor and an acceptor phase, and a semipermeable dialysis
membrane separating the two phases. The donor phase contains the emulsion to be tested,
from which only the API can get through the dialysis membrane into the acceptor phase.
Concerning the latter one, the physical-chemical properties of the API should be considered
and the appropriate temperature of the phase is also an important aspect (it is not an official
method in the Hungarian Pharmacopoeia).
Because the HLB-value significantly influences drug release, during the task the rate of the
release of the API incorporated in different types of emulsions is determined by the static
diffusion method under in vitro conditions, using an emulsifier with a lower (Span 80) and a
higher (Polysorate 20) HLB-value.
Analysis of pharmaceutical ingredients, excipients and dosage forms 119 _______________________________________________________________________________
Figure 21.1: Investigation of the drug release of emulsions by static diffusion
Task
The release and the membrane diffusion of lidocaine hydrochloride, as a model API, is
determined using different types of emulsions (o/w and w/o). The API is dissolved in the
aqueous phase in every case. During the investigations, the drug release of the emulsion is
measured, which means the diffusion of the API through the dialysis membrane into the
acceptor phase in such a way that the amount of lidocaine hydrochloride is determined
spectrophotometrically in the acceptor phase (Figure 21.1).
For the investigations, the students have to make the emulsions and arrange the membranes
themselves.
1. Preparation of the diffusion tests
Cut a 10-cm piece from the membrane used, soak it in water for 15 minutes and affix the
plastic closures to the membrane. The two emulsions to be prepared are investigated
Analysis of pharmaceutical ingredients, excipients and dosage forms 120 _______________________________________________________________________________
simultaneously, so altogether 6 membranes should be arranged for the 3 parallel
measurements each. Put the membranes into 6 beakers of 250 mL, previously filled with 100
mL of distilled water of room temperature (~ 25 °C).
2. Preparation of the emulsions to be examined
Prepare the emulsions with the given ingredients properly by using 2 medicine bottles of
100 g.
Sample "A" weight (g)
Lidocaini hydrochloridum 5.00
Polysorbatum 20 1.00
Mucilago hydroxyaethylcellulosi 6.00
Paraffinum liquidum 20.00
Aqua purificata 18.00
Preparation: dissolve the lidocaine hydrochloride in the aqueous phase, mix the emulsifier,
then emulsify the oily phase into this in portions. Shake vigorously.
Sample "B" weight (g)
Lidocaini hydrochloridum 5.00
Span 80 1.50
Mucilago hydroxyaethylcellulosi 5.00
Paraffinum liquidum 20.00
Aqua purificata 18.50
Preparation: dissolve the lidocaine hydrochloride in the aqueous phase. Mix the emulsifier
into the oily phase, then emulsify the previously prepared aqueous phase in portions. Shake
vigorously.
Analysis of pharmaceutical ingredients, excipients and dosage forms 121 _______________________________________________________________________________
3. The carrying out of dialysis
Carefully measure out 5 mL of the emulsions prepared into each dialysis membrane with a
digital pipette. Make sure that the dialysis membranes are closed bubble-free.
When you have finished the insertion of the sample, the investigation is started. Start the
first measurements after 15 minutes by taking out 5 mL of each sample. Filter it on a filter
paper, then determine spectrophotometrically the amount of lidocaine hydrochloride dissolved
in the acceptor phase and replace the amount of the sample taken out (5 mL) with distilled
water.
The spectrophotometric evaluation is made possible by the light absorption maximum of
the model API at 263 nm. At this wavelength, the values of the calibration curve were
recorded previously, and by using them the measured extinction values can be recalculated
into the concentration of the API (0.100 A = 154.262 µg/mL). During the measurements,
make dilutions if necessary and consider them when making the final calculations. Repeat the
sampling at the times given (15, 30 and 60 minutes).
Analysis of pharmaceutical ingredients, excipients and dosage forms 122 _______________________________________________________________________________
Report 21. Investigation of the drug release of emulsions by static diffusion method
1. Fill in the table below with the results of the investigation and calculate the appropriate
values. Give the total amount (in mg!) of the API dissolved after 60 minutes.
Sample Time
(min) A dilution
c
(mg/mL)
API in the sample
(mg/5 mL)
API in the dissolution
medium (mg/95
mL)
Total API dissolved
(mg)
A
B
Analysis of pharmaceutical ingredients, excipients and dosage forms 123 _______________________________________________________________________________
2. Show your results graphically by plotting the total amount of the API diffused against
diffusion time.
Evaluation:
Analysis of pharmaceutical ingredients, excipients and dosage forms 124 _______________________________________________________________________________
22. Investigation of drug release by agar plate diffusion method
Introduction
The ointment bases and excipients used in formulation can modify drug release, and
thereby the efficacy of the drug significantly. Their proper choice is very important with
regard to effect.
During the experiment the rate of the release of the incorporated API depending on the
properties of the ointment bases and drug concentration is determined under in vitro
conditions.
Task
Investigate the drug release from ointments containing salicylic acid. The iron (III)
chloride dissolved previously in the agar medium will form a coloured complex with salicylic
acid after its diffusion from the ointment samples. The size and the intensity of this violet-
coloured ring around the samples are proportional to the amount of salicylic acid released
(Figure 22.1).
Figure 22.1: Salicylic acid diffusion in iron (III) chloride containing agar gel
Analysis of pharmaceutical ingredients, excipients and dosage forms 125 _______________________________________________________________________________
1. Preparation for the diffusion investigation
For the investigation, the students get the ready agar gels in Petri-dishes and the different
ointment samples in marked tubes. In each Petri dish six cylindrical holes should be punched
with a special puncher, spaced at equal distances from each other and not too close to the wall
of the Petri dish. Carry out 2 parallel measurements with the same ointment with different
salicylic acid contents. Mark the holes on the bottom of the Petri dish to avoid mixing up the
samples. Use the short markings on the tubes.
2. Measurement of drug (salicylic acid) release from different ointments
Carry out the experiments with 4 different ointments containing 3 different salicylic acid
concentrations (1 w/w %, 5 w/w %, 10 w/w %). Fill each hole completely with the samples.
Use a small spatula to help the filling. Take care to avoid the ointment getting onto the surface
of the agar gel. The samples must be in contact with the agar gel on the side of the holes.
Start measuring the release time as one Petri dish is filled. The first reading time is after 30
minutes. Measure 2 perpendicular diameters of the coloured ring by means of a graph paper.
Be careful to read the Petri dishes in the same order as they were filled. Measure the size of
the ring by putting a graph paper under the Petri dish and reading the diameter of the coloured
ring through the transparent agar gel.
The reading times of the coloured rings are: 30, 60, 90 and 120 minutes.
Analysis of pharmaceutical ingredients, excipients and dosage forms 126 _______________________________________________________________________________
Report 22. Investigation of drug release by agar plate diffusion method
1. Fill in the table below and calculate the average of the two parallel measurements.
30 min.
average diameter
(mm)
60 min.
average diameter
(mm)
90 min.
average diameter
(mm)
120 min.
average diameter
(mm) Ointment API
%1. 2. 3. average 1. 2. 3. average 1. 2. 3. average 1. 2. 3. average
Evaluation:
Analysis of pharmaceutical ingredients, excipients and dosage forms 127 _______________________________________________________________________________
Analysis of pharmaceutical ingredients, excipients and dosage forms 128 _______________________________________________________________________________
Analysis of pharmaceutical ingredients, excipients and dosage forms 129 _______________________________________________________________________________
Analysis of pharmaceutical ingredients, excipients and dosage forms 130 _______________________________________________________________________________
Analysis of pharmaceutical ingredients, excipients and dosage forms 131 _______________________________________________________________________________
23. Investigation of drug release from suppositories by dynamic
diffusion method
Introduction
Suppository masses do not have an effect on their own, but they can modify drug release –
and thereby therapeutic effect – significantly. In this practice the rate of the release of the API
incorporated in different suppository masses and in different concentrations is determined by
dynamic diffusion.
Task
The drug release of readily water-soluble chloroquine phosphate and its diffusion through a
membrane from different suppository masses are determined. During the investigation the
drug release from the suppositories through a membrane is measured by determining the
chloroquine phosphate content in the acceptor phase spectrophotometrically.
Investigation method:
1. Measure the 3 suppositories received from your practice leader with mg accuracy and
mark them as Sample A, B, and C. You have 3 Petri dishes for the storage and the
identification of the suppositories. The base and the API content are the same in all 3
samples.
2. Cut off a piece from the membrane tube equalling the suppository length + 2 cm. Place
the suppositories into the tubes and close them with the closing device.
3. Place the samples prepared in this way into the apparatus.
4. Start the apparatus. The rotational speed is 1 turn / 30 s.
5. Take samples after 15, 30 and 60 minutes in a volume of 20 mL from the acceptor
phase with the rubber tube connected to the equipment. You will need 3 x 3 pieces of
Erlenmeyer flasks for the sampling.
6. Use an aliquot of the 20-mL samples and make a dilution to measure the value of
quenching. The spectrophotometric evaluation is made possible by the light absorption
maximum of chloroquine phosphate at 220 nm. At this wavelength, the values of the
Analysis of pharmaceutical ingredients, excipients and dosage forms 132 _______________________________________________________________________________
calibration curve were recorded previously, and by using them the measured quenching
values can be recalculated into the concentration of the API. Consider the dilutions
when making the calculations.
7. The volume of the acceptor phase taken during sampling was supplied; do not forget
about it when you make the final concentration calculation.
Apparatus: Apparatus for the disintegration of suppositories
Use of apparatus
1. Switch on the thermostat, set the knob on the right side to VAR position, and the one on
left side to the desired temperature.
2. Measure 3.00 kg of distilled water into 3 glass beakers (3,000 mL) and place them into
the apparatus. Wait until the desired temperature is reached in the acceptor phase.
3. Place the wrapped suppositories into the metal baskets, immerse them into the acceptor
phase and fix the rotating device.
4. Switch on the timer.
5. Switch the knob to ALAP position.
6. Press the START knob (to set the rotating device to the right position).
7. Press the ‘STOP’ knob.
8. Switch the knob to ‘STOP’ position.
9. Set the timer: 00, 30. (In ALAP position the two numbers mean hours and minutes, in
TIMER position minutes and seconds.)
10. Press the LOAD knob.
11. Switch the knob to ‘TIMER’ position.
12. As the switch is turned to ‘TIMER’ position, the investigation is started. Half a
rotation is made, repeated every 30 seconds.
The apparatus should work continuously during the 1 hour of investigation time. Take the
samples after 15, 30 and 60 minutes. When the experiment is finished, switch off the
apparatus and the thermostat.
Analysis of pharmaceutical ingredients, excipients and dosage forms 133 _______________________________________________________________________________
Start the work by switching on the thermostat and measuring the acceptor phase. You have
time to prepare the suppositories for investigation till the acceptor phase reaches the desired
temperature.
Analysis of pharmaceutical ingredients, excipients and dosage forms 134 _______________________________________________________________________________
Report 23. Investigation of drug release from suppositories by dynamic diffusion method
Number of composition: .............................................
API concentration: ...................................................... %
Weight of suppository A: ............................................ mg
Weight of suppository B: ............................................ mg
Weight of suppository C: ............................................ mg
2. Fill in the table with the results of the three parallel measurements (aliquot, dilution,
quenching values).
Sampling time (min)
15 30 60 Supp.
Aliquot Dilution Quenching Aliquot Dilution Quenching Aliquot Dilution Quenching
A
B
C
3. Calculate the released amount of API in µg/mL. Use the values of quenching, and the
slope and the intersection of the calibration curve. Give the released amount of API in
mg and w/w%, too. During the calculation, take care to consider the dilution, the
starting amount of the acceptor phase and the supplied amount of the sampling volume.
Equation of the calibration curve:
bxay +⋅= (1)
where y is the quenching, a is the slope (0.0618), x is the concentration (µg / mL), b is the
intersection (- 0.0776).
The absorption maximum of chloroquine phosphate is at 220 nm.
Analysis of pharmaceutical ingredients, excipients and dosage forms 135 _______________________________________________________________________________
Released API (mg) Sampling time
(min) Supp. A Supp. B Supp. C Average
15
30
60
Released API (%) Sampling time
(min) Supp. A Supp. B Supp. C Average
15
30
60
3. Plot the released API % against sampling time.
Evaluation:
Analysis of pharmaceutical ingredients, excipients and dosage forms 136 _______________________________________________________________________________
24. Investigation of the mucoadhesivity of hydrogels (Calculation task)
Introduction
The mucoadhesivity of a pharmaceutical preparation means the adhesion between the
preparation and the mucosa, in the course of which physical and chemical interactions can
form. Physical interactions mean the diffusion of the polymer chains into the mucin chains
and their interpenetration. Chemical interactions mean the formation of primary (e.g.
covalent) and secondary (e.g. hydrogen, van der Waals) bonds between the entangled chains
(Figure 24.1).
Figure 24.1: Mechanism of mucoadhesion
A: contact stage; B: consolidation stage;
C: formation of primary and secondary chemical bonds between the mucin and polymer chains
As mucoadhesion is a complex mechanism, numerous theories exist to explain it, such as:
– electric;
– adsorption;
– wetting;
– diffusion and
– fracture theory.
Analysis of pharmaceutical ingredients, excipients and dosage forms 137 _______________________________________________________________________________
Because of these complexities, there is no standardized method of measurement; numerous
methods are known and developed to investigate mucoadhesive behaviour. One of the most
accepted methods is rheology. The rheological synergism between the polymer and the mucin
chains can be regarded as an in vitro parameter of mucoadhesion. The base of the
methodology is that the rheological parameters change after mixing the polymer gels with
mucin. This change is greater than it could be expected from the sum of the rheological
parameters.
Hassan and Gallo were the first to describe the relationship which uses a viscosity
parameter (ηb) to characterize the strength of mucoadhesivity:
pmtb ηηηη −−= (1)
where, ηt is the viscosity of gels containing polymers and mucin, ηp is the viscosity of the
polymer gels without mucin, and ηm is the viscosity of mucin dispersion.
Besides the equation above, the relative synergism parameter can be also used, where the
changes in viscosity refer to the original viscosity:
pm
brelb ηη
ηη+
=, (2)
Task
The mucoadhesivity of polymer gels containing different amounts of hydroxyethyl
cellulose polymer (HEC) is investigated by means of rotational viscometry. From the
measured data listed below, the synergism and relative synergism parameters can be
calculated, and the changes of mucoadhesivity can be characterized by increasing the polymer
concentration.
The flow and viscosity curves of the mucin (porcine stomach type II) dispersion, and gels
containing HEC alone and HEC and mucin together were recorded in the range of 0.1-100 1/s
shear rate.
Analysis of pharmaceutical ingredients, excipients and dosage forms 138 _______________________________________________________________________________
The composition of the samples is listed in the table below:
Analysis of pharmaceutical ingredients, excipients and dosage forms 139 _______________________________________________________________________________
Report 24. Investigation of the mucoadhesivity of hydrogels
1. Shear stress (τ) and viscosity data (η) (at 100 1/s) derived from the flow and viscosity
curves can be seen in the table below. Three parallel measurements were made.
Calculate the mean values.
Measurement 1 Measurement 2 Measurement 3 Mean Sample τ
(Pa) η
(mPa·s) τ
(Pa) η
(mPa·s) τ
(Pa) η
(mPa·s) τ
(Pa) η
(mPa·s) 1 1.5 15.1 1.6 15.5 1.3 13.2
2.1 2.1 21.1 2.1 21.3 2.1 20.8
2.2 14.3 143 14.4 144 14.3 143
2.3 43.5 435 45.5 455 50.7 507
2.4 163 1630 182 1820 148 1480
3.1 7.32 73.2 7.6 75.7 8.9 88.8
3.2 35.6 356 35.1 351 33 330
3.3 89.6 896 88.1 881 90.8 908
3.4 244 2440 306 3060 296 2970
Analysis of pharmaceutical ingredients, excipients and dosage forms 140 _______________________________________________________________________________
2. Plot the values of the shear stress against the HEC concentration both for the sample
containing HEC alone and for the sample containing HEC and mucin together.
3. Evaluation:
Analysis of pharmaceutical ingredients, excipients and dosage forms 141 _______________________________________________________________________________
4. Calculate the value of ηb and ηb rel.
HEC conc. (%) ηb(mPa*s)
ηb rel (-)
1
2
3
5
5. Plot ηb and ηb rel against the HEC concentration. Use a different y-axis for the ηb (left
side y-axis) and the ηb rel (right side y-axis) values.
Evaluation:
Analysis of pharmaceutical ingredients, excipients and dosage forms 142 _______________________________________________________________________________
Appendix
Analysis of pharmaceutical ingredients, excipients and dosage forms 143 _______________________________________________________________________________
A.1. Helios Alpha UV-Vis spectrophotometer
The spectrophotometer is an optical instrument, which measures the intensity of
monochromatic light and its changes.
Spectrophotometers can be classified on the basis of:
– applied wavelength:
ultraviolet
visible
infrared
– operation way:
single beam
double beam
The Helios Alpha UV-Vis spectrophotometer (Figure A.1.1) is a double beam
spectrophotometer, which means the optical system divides the light from the light source into
two paths, one of them goes through the reference, and the other one goes through the sample.
This operation way allows the simultaneous measurements of light intensity passing through
Analysis of pharmaceutical ingredients, excipients and dosage forms 144 _______________________________________________________________________________
concentration (knowing the correlation between the concentration and the absorbance).
– plotting of the calibration curve
Analysis of pharmaceutical ingredients, excipients and dosage forms 145 _______________________________________________________________________________
A.2. Anton Paar RHEOLAB MC 1 rheometer
The Anton Paar Physica RHEOLAB MC 1 rheometer is a rotational rheometer, which can be
used to measure the flow curve, yield point, structure breakdown and recovery (CREEP test),
viscosity of Newtonian and non-Newtonian systems. The instrument can be used in CSR
(controlled shear rate) and CSS (controlled shear stress) mode, too (Figure A.2.1).
Figure A.2.1: Anton Paar Physica RHEOLAB MC 1 rheometer
The measuring device is a concentric cylinder. The sample is placed in a gap between a fixed
cup and a rotating cylinder (Searle system). The measuring controller developed for the
device is based on a dynamic system which consists of a drive unit and an optical encoder
without gear wheel and mechanical power converter, therefore it measures the rotary torque
without deformation, so without loss. The measuring controller is such that the revolving
body rotates at the desired speed and it measures the rotary torque – which comes from the
flow resistance of the sample – acting on the measuring body. The investigation of shear
stress or the CREEP test is also possible by setting the desired shear stress, and the shear
deformation of the sample is measured through the angle rotation.
Controlled shear investigations (CSS) for the determination of the flow properties of
plastic materials can also be performed by the Anton Paar Physica RHEOLAB MC 1 device,
Analysis of pharmaceutical ingredients, excipients and dosage forms 146 _______________________________________________________________________________
which allows the accurate measurement of the flow point without deformation, so without the
shearing of the internal structure of the sample (rotation).
Handling of the device, measurement:
1. In case of the investigation of emulsions, sample holder "Z3" is filled hermetically with
the sample up to the inner mark (Figure A.2.2).
1. In case of the investigation of the gel-forming of polymers and the investigation of
ointments, sample holder "Z4" is filled hermetically with the sample up to the inner
mark (Figure A.2.3).
2. The cylinder measuring head is fitted and fixed with the help of the retaining ring.
3. The device is switched on at the back.
4. The "PROG" function is chosen and the "OK" button is pressed.
5. In case of the investigation of emulsions, the "PROG 02" function is chosen and the
"OK" button is pressed.
5. In case of the investigation of the gel-forming of polymers, the "PROG 01" function is
chosen and the "OK" button is pressed.
6. The measurement is started by pressing the "ST" (START) button.
7. A piping sound will indicate the end of the measurement.
Figures A.2.2 and A.2.3: Measuring heads "Z3" and "Z4"