Kniga na apstrakti book of abstrcts

Prakti~ni aspekti pri transfer na analiti~ki metod
Aneta Dimitrovska, Suzana Trajkovi}-Jolevska
Farmacevtski fakultet, Centar za ispituvawe i kontrola na lekovi, 1000 Skopje, R. Makedonija
Vo reguliranite podra~ja na analitikata na lekovite, razvienite i validirani metodi vo edna
laboratorija (originator ili laboratorija-ispra}a~) naj~esto se prenesuvaat vo druga laboratorija (lab-
oratorija-primatel). Tansfer na analiti~kiot metod ozna~uva potvrda deka metodot se sproveduva vo lab-
oratorijata-primatel na ist na~in kako i vo laboratorijata-ispra}a~ i deka gi ispolnuva site kriteri-
umi za prifatlivost na metodot.
Postojat nekolku na~ini za transfer na anliti~kiot metod, kako {to se: sporedbeni ispituvawa,
celosna ili delumna validacija na metodot ili revalidacija, vzaemna validacija pome|u dve laboratorii
i izostavuvawe na formalniot transfer, poznato kako transfer "odrekuvawe“. Koj od ovie na~ini na
transfer na metodot }e se izbere zavisi od stepenot na razvoj na metodot (rana ili pokasna faza), tipot
na metodot (ednostaven ili slo`en, farmakopejski ili ne-farmakopejski), kako i iskustvoto i sposob-
nosta na personalot vo laboratorijata. So cel da se izvr{i uspe{en transfer na analiti~kiot metod,
potrebno e da se zadovolat odredeni barawa, po~nuvaj}i od prethodno odobreniot protokol za rabota,
opisot na metodot, opisot na barawata za postapkata na ispituvawe, kriteriumite na prifatlivost, doku-
mentacija na rezultatite, pa se do izgotvuvawe na izve{tajot za transfer na analiti~kiot metod. Kako i
vo slu~ajot na sekoj proces na validacija, najzna~ajno za tehni~kiot transfer e celosnoto dokumentirawe
i toa kako na procesot na izveduvawe taka i na dobienite rezultati. Izve{tajot za transfer na anali-
ti~kiot metod potvrduva deka se ispolneti kriteriumite na prifatlovost i deka laboratorijata-prima-
tel e celosno trenirana i kvalifikuvana za izvedba na metodot. Imaj}i vo predvid deka aparaturata,
iskustvoto i treningot na personalot i postapkata na prika`uvawe na rezultatite se razlikuvaat od labo-
ratorija do laboratorija, mnogu od ovie zamki bi mo`ele lesno da se izbegnat so mala odnapred izvr{ena
proverka.
Brziot napredok na visoko-performansnata te~na hromatografija (HPLC) kako glavna tehnika vo
farmacevtskata industrija iziskuva celosno utvrdena postapka za transfer od edna vo druga laboratori-
ja. Vo ovoj pregled se dadeni izvorite na gre{ki {to onevozmo`uvaat da se reproducira HPLC postapka-
ta, po~nuvaj}i od podgotovkata na primerokot, preku aparaturata (injektor, pumpa, kolona i detektor),
mobilnata faza, treningot na personalot, pa se do obrabotkata i prika`uvaweto na rezultatite. Isto
taka, treba da se zemat vo predvid merkite {to treba da se prevzemat koga se razviva metodot so cel da se
namalat na najnisko nivo problemite do koi mo`e da dojde vo idnina, kako i spravuvaweto so situaciii-
te koga se javuvaat problemi pri opredeluvawata. Imaj}i vo predvid deka HPLC metodite se sè pozastapeni
vo analitikata na lekovite, istite se predmet na se po~est transfer od edna vo druga laboratorija, pora-
di {to pri razvojot na HPLC metodot treba da se vodi gri`a istiot da bide robusten i reproducibilen. 
Macedonian pharmaceutical bulletin 53 (1,2) 175-176 (2007)
SOP - 4
176
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Quality standards and quality control of biopharmaceuticals 
Suzana Trajkovic-Jolevska, Jasmina Tonic-Ribarska
Faculty of Pharmacy, Ss Cyril and Methodius University,
Vodnjanska 17, 1000 Skopje, Macedonia
Biopharmaceuticals are pharmaceuticals derived from biological sources, consisting of (glyco) proteins and/or
nucleic acids... They deserve special attention as their active pharmaceutical ingradients (API)-proteins have unique
chemical and physical properties that make them different from low molecular drugs. The activity of proteins is close-
ly connected with their extremely complicated conformation that cannot be fully characterized with the known ana-
lytical techniques. On the other hand, large and heterogeneous molecules with many reactive groups and complex 3D
structure necessary for biological activity, pose difficult stability problems, caused by physical and chemical instabil-
ities of proteins. Critical steps for protein stability are during production of the proteins and finish product, as well;
during transport, storage and handling (climate conditions, shaking). Stability of proteins may vary depending of the
production process and host cells (eucariotic/procariotic). Because of that, it is very important to use suitable analyt-
ical techniques for investigation of biopharmaceutical at every step and which is even more important - to use a com-
bination of different analytical methods for monitoring protein structure and examination of finish product.
Quality requirements and quality control for biopharmaceuticals are defined with global standards and reg-
ulatory environment, which includes standards for production, pharmacopoeia monographs (if there is any), ICH
guidelines (for example: Q5C, Quality of Biotechnological products: Stability testing of Biotechnological/Biological
Products Q6B, Test Procedures and Acceptance Criteria for Biotechnological/Biological Products), national guid-
ances (if there is any). Due to the complexity of proteins and structural differences among different proteins, even
in the ICH guideline is stated that "…there is no single stability-indicating assay or parameter that profiles the sta-
bility characteristics of the biopharmaceuticals. Although these products may be subject to significant loses of activ-
ity, physicochemical changes or degradation, international and national regulations have provided little guidance
with respect to distinct release and shelf-life specifications". Furthermore, according to the relevant institutions and
experts, there is a lack of well defined regulatory framework for analytical techniques for quality control of biopharma-
ceuticals. For batch release purposes the finish product is characterised by a range of physicochemical and biologi-
cal methods. Sometimes, physicochemical assays are more routinely used for bioactivity measurements and in some
case have replaced biological analysis after lengthy validation. 
Macedonian pharmaceutical bulletin 53 (1,2) 177-178 (2007)
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Standardi za kvalitet i kontrola na kvalitet 
na biofarmacevtski preparati
Suzana Trajkovi}-Jolevska, Jasmina Toni}-Ribarska
Farmacevtski fakultet, Univerzitet "Sv. Kiril i Metodij"
Vodwanska 17, 1000 Skopje, Makedonija
Biofarmacevtskite preparati se farmacevtski proizvodi dobieni od biolo{ki izvori i se
sostaveni od (gliko)proteini i/ili nukleinski kiselini. Tie zaslu`uvaat posebno vnimanie, bidej}i
nivnite aktivni komponenti (API)-proteinite imaat edinstveni hemiski i fizi~ki karakteristiki koi
gi pravat razli~ni od nisko molekularnite  lekovi. Aktivnosta na proteinite e tesno povrzana so niv-
nata isklu~itelno slo`ena konformacija, koja ne mo`e vo potpolnost da bide karakterizirana so poz-
natite analiti~ki tehniki. Od druga strana, golemite i heterogeni molekuli so mnogu reaktivni grupi
i slo`enata 3D struktura neophodna za biolo{kata aktivnost, se pri~ina za problemi  so stabilnosta,
predizvikani do fizi~kata i hemiskata nestabilnost na proteinite. Kriti~ni to~ki za stabilnosta na
proteinite se vo tekot na proizvodstvotokako na proteinite, taka i na gotovio proizvod; vo tekot na
transportot, ~uvaweto i rakuvaweto (razli~ni klimatski uslovi, protresuvawe). Stabilnosta na protei-
nite mo`e da bide razli~na vo zavisnost od proizvodniot proces i kletkite doma}ini od kade se dobiva
(eukarioti/prokarioti). Poradi toa, mnogu e va`no da se upotrebi soodvetna analiti~ka tehnika za ispitu-
vawe na sekoj ~ekor od `ivotot na biofarmacevtskiot preparat i {to e u{te pova`no - da se upotrebi
kombinacija na pove}e analiti~ki metodi za monitorirawe na strukturata na proteinot i ispituvawe na
gotoviot farmacevtski preparat. 
Barawata za kvalitet i kontrolata na kvalitetot za biofarmacevtskite preparati se definirani
so globalni standardi i regulativi, {to vklu~uva standardi za proizvodstvo, farmakopejski monografii
(vokolku gi ima), ICH vodi~i (na primer: Q5C, Kvalitet na proizvodi dobieni so biotehnologija:
Ispituvawe na stabilnost na proizvodi dobieni so biotehnologija/biolo{ki proizvodi; Q6B, Proce-
duri za ispituvawe i kriteriumi za prifatlivost za proizvodi dobieni so biotehnologija/biolo{ki
proizvodi)
, nacionalni vodi~i  (vokolku gi ima). Poradi slo`enata struktura na proteinite i struktur-
nite razliki pome|u razli~nite proteini, duri i vo ICH vodi~ite e nazna~eno deka "…ne postoi edno opre-
deluvawe ili parametar koe gi odrazuva karakteristikite vo odnos na stabilnosta na biofarmacevtskite
preparati. Iako ovie proizvodi mo`at da bidat subjekt na zna~ajno gubewe na aktuvnosta, fizi~ko-hemis-
ki promeni ili degradacija, internacionalnite i nacionalnite regulatorni organi davaat malku vodi~i
vo odnos na razlikata pome|u specifikacija za kvalitet pri pu{tawe vo promet i specifikacija za kva-
litet vo rok na upotreba“. U{te pove}e, spored relevantni isnstitucii i eksperti, postoi nedostatok na
dobro definirana regulatorna ramka za analiti~kite tehniki za kontrola na kvalitet na biofarmacevt-
skite preparati. Za pu{tawe na serija vo promet, gotoviot proizvod se karakterizira so golem broj
fizi~ko-hemiski i biolo{ki metodi. Ponekoga{, fizi~ko-hemiskite opredeluvawa se koristat vo ruti-
nskite analizi za opredeluvawe na biolo{kata aktivnost i vo nekoi slu~ai pretstavuvaat zamena za bio-
lo{kite analizi, po nivna opse`na validacija.
Macedonian pharmaceutical bulletin 53 (1,2) 177-178 (2007)
SOP - 5
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,
1
k
Interaction of Quinapril with cationic surfactant micelles using 
micellar liquid chromatography
O. Cudina
1
, I. Jankovi
c
2
, J. Brbori
c
1
, S. Vladimirov
1
1
Institute of Pharmaceutical Chemistry and Drug Analysis, Faculty of Pharmacy, 
Vojvode Stepe 450,P.O.Box 146, 11000 Belgrade, Serbia
2
Laboratory for Radiation Chemistry and Physics, Vin
~
a Institute of Nuclear Sciences, 
P. O. Box 522, 11001 Belgrade, Serbia
Because of its amphiphilic nature, micelles are known to play a vital role in many processes of interest in
both fundamental and applied sciences. Micellar systems can solubilize poorly soluble drugs and thus increase their
bioavailability, can be used as a model system for biomembrane, as well as drug carriers in numerous drug delivery
and drug targeting systems.
In this study, the interaction of quinapril (QUIN), a nonpeptide, nonsulfhydryl ACE inhibitor with cationic
surfactant cetyltrimethylammonium bromide (CTAB) was investigated. Quinapril is a polyfunctional molecule, and
at physiological conditions (pH 7.4) it is an anion. Micellar liquid chromatography (MLC) was used for the deter-
mination of binding constant QUIN/CTAB which is important for quantification of drug/micelle interaction. The
retention of QUIN anion on the polar stationary phase (alkyl nitrile saturated with CTAB) and methanol-phosphate
buffer (5:95 v/v) with 0.01 to 0.035 M of CTAB added in 0.005 M increments, at pH 7.4 as mobile phase, follows
the behavior of class A compounds according to Jandera and Fischer. The retention increases in submicellar mobile
phases and decreases in micellar mobile phases as the concentration of micelles is raised. According to Arunyanart
and Cline Love, by plotting     (k’- capacity factor of the solute) versus [M
m
] ([M
m
] - concentration of micellized
surfactant), the value of the solute/micelle binding constant  per monomer of surfactant can be calculated. From the
slope/intercept ratio the value of QUIN/CTAB binding constant K
2
= (2.4±0.6)·10
2
mol-
1
l is obtained. Binding con-
stants obtained by using MLC and UV spectrophotometry (K
b
=(2.3±0.4)·10
3
mol-
1
l) show great discrepancy of an
order of magnitude. The main drawback of MLC method is the intrinsic error associated with the determination of
K
2
from the slope/intercept ratio. Error propagation of a quotient affects the error in detemining K
b
, the error of the
intercept being large when the intercept is very small, near to zero.
The elucidation of binding constant drug/micelle is important for the understanding of interactions with bio-
membranes, QSAR studies, micellar HPLC or MEKC used in drug quality control. 
Macedonian pharmaceutical bulletin 53 (1,2) 179 (2007)
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Stress degradation studies on DIIODIDA – ligand in
99m
Tc-radiopharmaceuticals for hepatobiliary scintigraphy
J. S. Brboric
1
, M. S. Jovanovic
2
, O. Cudina
1,
S. Vladimirov
1
1
Institute of Pharmaceutical Chemistry and Drug Analysis, Faculty of Pharmacy, 
Vojvode Stepe 450, P. O.Box 146, 11000 Belgrade, Serbia
2
Medicines and Medical Devices Agency of Serbia, Belgrade
The analogs of iminodiacetic acid (IDA) labeled with technetium-99m are used as diagnostic radiopharma-
ceuticals for hepatobiliary imaging. In order to develop a radiopharmaceutical with better hepatobiliary properties
and larger tolerance on bilirubin, a new ligand for complexation of technetium-99m, DIIODIDA (2,4-diiodo-6-
methylphenylcarbamoylmethyl iminodiacetic acid; N-[2-[(2,4-Diiodo- 6-methylphenyl)amino]-2-oxoethyl]-N-(car-
boxymethyl)-glycine) was synthesized. 
Besides quantification of DIIODIDA, determination of possible degradation products and impurities is of
importance in developing of new radiopharmaceutical. The impurities presented in the ligand during the labeling pro-
cedure with technetium-99m, could cause reducing of labeling yield and appearance of radiochemical impurities.
DIIODIDA was subjected to different ICH prescribed stress conditions. Samples of DIIODIDA (drug pow-
der) were exposed to dry heat (24 h at 100
o
C and 6 h at 160
0
C). All stress decomposition studies were performed at
an initial solution of DIIODIDA in methanol (1mg/ml). Acid hydrolysis has been performed in 1M HCl at ~90
0
C
(reflux) during 6 h. The study in alkaline conditions has been carried out in 1M NaOH at ~ 90
0
C (reflux) during 6
h. Oxidative studies has been carried out at room and higher temperatures, in 3%, 10% and 30% hydrogen peroxide
during 24h. 
RP-HPLC method for investigation of chemical purity and determination of DIIODIDA content was devel-
oped. HPLC investigations were performed on Zorbax Eclipse XDB-C18 (150mm x 4.6 mm; 3.5 
µm) column. The
optimal conditions were achieved using binary gradient system with mobile phase (A) acetonitrile; (B) 15 mM tri-
ethylamine, pH 3,0 (adjusted with orthophosphoric acid), at a flow rate 1 ml/min, at 25
0
C and detection at 205 nm
and 230 nm. 
The gradient method was successful in separation of DIIODIDA from its degradation products, as well as
from synthetic precursors/intermediates formed during the DIIODIDA synthesis. The solid-state studies showed that
DIIODIDA is stable to the effect of temperature. The study shows that DIIODIDA is stable to alkaline hydrolysis.
DIIODIDA is not stable to acid hydrolysis and oxidation (hydrogen peroxide). This paper also reports stability results
of DIIODIDA solutions, conditioned at 25
0
C and in the presence of stannous as a reducing agent for the sodium
pertechnetate. None of peaks that indicate degradation products were present in the chromatograms of DIIODIDA
incubated with stannous chloride.
Macedonian pharmaceutical bulletin 53 (1,2) 180 (2007)
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Monitoring the level of chromium in urine of patients with Diabetes
Mellitus by Atomic Absorption Spectrometry 
Fehim Korac, Meliha Lekic, Nermin Hrncic, Zlatan Rimpapa
Department for Medical chemistry, Faculty of Medicine, University of Sarajevo, Bosnia and Herzegovina
Chromium is present in the environment in several different forms. The most common forms are trivalent
and hexavalent. Hexavalent form is genotoxic, while Chromium (III) is an essential nutrient that is an integral part
of glucose tolerance factor. Level of chromium in human body is 10 to 20 mg. In bloodstream it is bound for tran-
spherine and is distributed to tissues, and is eliminated for 80% in the urine with the half time for three months.
Deficit of chromium in organism leads to a reversible insulin tolerance. Level of chromium in blood arise just after
the administration of glucose or insulin, and is dependent on the age. It seems that there is a connection between the
insulin dependent oxidation of glucose, synthesis of glucagon and fatty acids on one side and the presence of chromi-
um in tissues on the other.
For that reasons deficit of chromium in organism can lead us to impeach on disturbed tolerance to carbohy-
drates or to Diabetes Mellitus type I.
Concentration of chromium in blood and urine increases for two or three times after the stimulation with
insulin and in patients with Diabetes type I. If this does not happen there is probably present the deficit of chromi-
um. These conclusions are formed on the observations that were done on the patients with Diabetes and elder peo-
ple that normally have decreased level of chromium comparing with children and adolescents.  
The main framework of this investigation was to investigate the influence of therapy with insulin and oral
antidiabetics on the level of chromium in urine in patients with Diabetes. 
In the work there were three groups: two with the patients with Diabetes mellitus type I and type II respec-
tively, and control group-people that do not have Diabetes or any chronically disease, such are liver or kidney insuf-
ficiency.
The level of chromium was investigated in 24-hours samples of urine. Samples were first examined macro-
scopic (colour, appearance) and than the volume and pH value were measured. 
Prepared samples were investigated on atomic absorption spectrometer VARIAN-SPECTRA 110 with elec-
trothermal atomizer (graphite furnace) and with Cr-lamp with the range of wavelength from 10µA to 357.9 nm.
The data that were obtained from this investigation shows the increased concentrations of chromium in urine
in both groups of patients comparing with the control group. Level of chromium was bit higher in patients that were
treated with oral antidiabetics, than in the group that was administrated insulin. 
In each of three examined groups was significant difference in chromium concentration in men and women.
Level of chromium in the urine of men in control group and group treated with oral antidiabetics was higher com-
paring the level that was measured in women, while in the group treated with insulin results were quite oposit.
Macedonian pharmaceutical bulletin 53 (1,2) 181 (2007)
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Designing, synthesis and defining of morphological characteristics 
of Pyridine derivates
Mirza Nuhanovic, Seherzada Hadzidedic
Bosnalijek d.d. Development Department, Jukiceva 53, 71000 Sarajevo, Bosnia and Herzegovina
Pyridine derivative ( 3 – ethyl – 5 methyl – 2 – (-aminoethoxymethyl) – 4 – (2-chlorophenyl) – 6 – methyl – 1,4
– dihydro – 3,5 – pyridine – dicarboxilate benzene sulphonate is pharmacologically examined and clinically approved
long-acting calcium channel blockers drug substance.
Taking the pharmacological potential of this class of compounds into account we have synthesized a series
of pyridine derivatives .Structure of the synthesized compounds was confirmed by standard analytical methods (HPLC,
TCL, FTIR, T.t). In addition to morphological assessments of crystals we used Scanning Electron Microscopy (SEM).
Crystal morphology is greatly affected by the design of the synthetic pattern of the active component on while,
in the final synthesis phase, the crystal form is directly affected by the technique and the conditions of crystallization.
The type of the solvent selected, its concentration, solvent mixture, temperature, the crystallization rate, initiation of
the crystallization nucleus, the conditions of precipitation, the type of solvents used in the synthesis process (residual
solvents) are the factors that affect the crystal form. The influence of the selected solvent is very important as it has the
ability of adsorption to the crystal surface in the process of crystal growth. Due to that the crystals belonging to the
same crystal system show different physical properties depending on the conditions of crystallization.
The morphology of the crystal is very important as it affects a number of properties of the compound, the
stability, compressibility, the fluidity in the tablet creation process. The morphology of the crystal also affects the
dissolution rate, that is the bioavailability. The said pyridine derivative was synthesized by a selected procedure and
the particles of the product are not of a uniform size. It is rather a mixture/solution of crystals of different size. Diffe-
rent conditions of crystallization were tested and the one that was selected was the one which enabled identical mor-
phology of the substance as well as experimental reproducibility.
Crystal morphology of the synthesized compounds was investigated by Scanning Electron Microscopy
(SEM). The Differential Scanning Calorimetry (DSC) as a poweful tool was used in early stage of drug synthesis in
order to get information about polymorphism.Size characterization was performed according to Particle Size Analysis
(PSA) method.
It turned out that the compounds, produced by the own synthetic put, had almost entirely identical crystal
morphology as the CRS standard, i.e. the other aforementioned raw material.
Through optimization of the crystallization conditions it is possible to produce the said pyridine derivative
which meets the required conditions under the selected granulation process. Further studies are aimed at determin-
ing crystallization conditions required for creation of uniform morphology of the said pyridune derivattive.
Macedonian pharmaceutical bulletin 53 (1,2) 182 (2007)
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