Kniga na apstrakti book of abstrcts

Opredeluvawe na Alantoin vo farmacevtski i kozmeti~ki
preparati so spektrofotometriski metod vo vidlivo podra~je
Maja Veli~kovska
1
, Vasil Kar~ev
2
,  Liljana Ugrinova
2
, Aneta Dimitrovska
2
1
Galafarm dooel, ul. 51 br. .23, Skopje, Makedonija
2
Centar za ispituvawe i kontrola na lekovi , Farmacevtski fakultet, 
Vodwanska 17, Skopje, Makedonija  
Vo ovoj trud e razraboten spektrofotometriski metod za kvantitativno opredeluvawe na alantoin
(baziran na metodot opi{an od Young i Conway za kvantitativno opredeluvawe na alantoin vo biolo{ki
materijali) vo farmacevtski preparat (mast) i vo kozmeti~ki preparat (krema). Metodot na Young i Conway
se zasnova na promena na ispituvanata supstanca so hemiski reakcii so cel dobivawe visoko apsorbira~ko
soedinenie. Alantoinot e hidroliziran do glioksalna kiselina, koja reagira specifi~no so fenilhidrazin.
Nastanatiot fenilhidrazon vo silno kisela sredina se oksidira so kalium heksacijanoferat i dava pur-
purno oboen kompleks, ne mnogu stabilen, so apsorpcionen maksimum vo vidlivo podra~je na 522 nm.
Reakciite se odvivaat stehiometriski, {to dava mo`nost za spektrofotometrisko opredeluvawe na alan-
toinot. Zna~ajni momenti za kvantitativno opredeluvawe na alantoinot vo metodot se: ekstrakcijata na
alantoin od matriksot (krema, mast) nerastvorliv vo voda, uslovite koi gi kontroliraat hemiskite reakcii,,
kako i vremeto na inkubacija pred ~itaweto na apsorbancijata (ili vremeto na inkubacija po dodavaweto
na kalium heksacijanoferat). Primeneta e ekstraktivna separativna tehnika i toa prvo te~no-cvrsta
ekstrakcija so dihlormetan kako organski rastvoruva~ za otstranuvawe na podlogata od ispituvanite
preparati vo oblik na mast i krema, a potoa te~no-te~na ekstrakcija so topla destilirana voda (okolu 45°C)
za ekstrakcija na  alantoinot. So primena na celokupniot metod za opredeluvawe na alantoin (vklu~uvaj}i
ja i ekstrakcijata) na placebo ve{ta~ki smesi na masta i kremata se potvrdi  deka ne postoi zna~itelna
interferenca koja proizleguva od sostavot na preparatite. Kvantitativna hidroliza na alantoinot vo
alantoinska kiselina be{e postignata so zagrevawe to~no 7 minuti vo vodena bawa na 90°C vo bazna sredi-
na (so dodavawe 0,5 M NaOH). So zagrevawe to~no 7 minuti vo vodena bawa na 90°S, vo kisela sredina (postig-
nata so dodavawe na 0,5 M HCl) alantoinskata kiselina celosno hidrolizira do glioksalna kiselina. So
naglo ladewe nekolku minuti vo ledena alkoholna bawa (-10°C) se inhibiraat sporednite reakcii i se
postignuva kvantitativno odvivawe na reakcijata me|u glioksalnata kiselina i fenilhidrazinot. Eksperi-
mentalno be{e ispitano vlijanieto na vremeto na inkubacija na dobienite vrednosti za apsorpcijata i
potvrdeno deka so merewe na apsorbancijata na primerocite posle to~no 20 minuti se obezbeduva to~nost
na metodata. Metodot e validiran preku ispituvawe na povtorlivost, linearnost, opseg na metodot, limit
na detekcija, limit na kvantifikacija , to~nost i preciznost.
To~nosta i preciznosta na metodot se potvrdeni i so dobienite vrednosti za analiti~kiot prinos
pri opredeluvawe na alantoin vo ve{ta~ki prigotveni probi od kremata i masta vo koncentracisko podra~je
od 50 - 150 % od rabotnata koncentracija i toa za kremata iznesuvaat od 96,1 - 102,8 % (RSD: 1,3 - 1,8 %), a
za masta 95,8 - 102,4 % (RSD: 1,5 - 2,2 %). Metodot se bazira na specifi~na reakcija i se karakterizira so
zadovolitelna osetlivost. Mo`e da se primeni za rutinski analizi pri opredeluvaweto na alantoin vo
farmacevtski i kozmeti~ki preparati, kade e zastapen vo niski koncentracii.
Macedonian pharmaceutical bulletin 53 (1,2) 228-229 (2007)
PP - 108
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FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION

Determination of UV protection substances 
in cosmetic products by RP - HPCL with UV - DAD detection
Gorica Vukovic
1
, MarinelaTadic
1
, Marija Saric
1
1
Institute of Public Health of Belgrade Bulevar Despota Stefana, 54 a, 11000 Belgrade, Serbia
UV protector substances, are complex organic compounds (benzophenons, biphenyl cianoacrilates, cinna-
mates, p-aminobenzoic acid derivates, dybenzoil methanes, etc.). They are ingredients of many cosmetic products
for skin and hair protection against harmful UV radiation, but they are also used for conservation of active com-
pounds in cosmetic products. The aim of this work was to develop and validate a HPLC method for determination
of UV filters in cosmetic products. 
A HPLC method has been developed for the simultaneous determination UV protection substances  (3-ben-
zophenone, 4-benzophenone, benzyl-o-hydroksibenzoate, ethylhexyl methoxycinnamate, butyl methoxydibenzoyl
methane, phenylbenzimidazole-5-sulphonic acid, 4-methylbenzylidene camphor, 2-ethylhexyl-4 dimethylaminoben-
zoate) in cosmetic products.
Sample is dissolved in methanol-2M sulphuric acid mixture (40/60, v/v). After homogenization, centrifuga-
tion and dilution, sample was filtered and injected to the HPLC system. UV filters have been seperated using Zorbax
Rapid Resolution SB-C18 column, 4,6x75mm 3,5
µm, and gradient elution with mobile phase consisting of 10mM
phosphate baffer, (with ion par reagent 1mM tetrabutylammoniumdihydrogen phosphate), and acetonitrile, HPLC
grade. Detection was performed using DAD detector set at several detection wavelengths (305, 254, 272, 280, 340nm).
Flow rate was 0,7ml/min.
Analytical method was validated considering linearity, precision, accuracy, limit of detection and limit of quan-
tification. The external standard calibration curves were linear within the range from 0,4-4%, except for benzil-o-
hidroksibenzoat, that was linear from 2 to 20% expressed as final concentration . Linearity coefficient and RSD (%)
were: 0,9999 (3,1%) for phenylbenzimidazole-5-sulphonic acid, 0,9965 (9,8%) for 4-benzophenone, 0,9998 (9,9%) for
3-benzophenone, 0,9999 (4,9%) for benzyl-o-hydroksibenzoate, 0,9999 (5,7%), for 4-methylbenzylidene camphor,
0,9999 (5,7%) for butyl methoxydibenzoyl methane, 0,9999 (4,0%) for ethylhexyl methoxycinnamate, and 0,9999
(4,2%) for 2-ethylhexyl-4 dimethylaminobenzoate.Recoveries ranged from 97-107%, with RSD 2,7% maximum, per-
formed at three different concentration levels at three probes. Limit of detection and quantification, depending of sub-
stance and chosen wavelength, varied from 0,01% for 3-benzophenone, to 0,1% for benzyl-o-hidroksibenzoate.
Macedonian pharmaceutical bulletin 53 (1,2) 230 (2007)
PP - 109
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Validation and quantitative detection of bacterial endotoxins with
kinetic turbidimetric method on Heparin inj. 5000 IU/ml
E. Popovska, S. Ilioska Zlatanovic, H. Babunovska, V. Jovanovska, M. Ilievska
Alkaloid  A. D., Bul. A. Makedonski 12, 1000 Skopje, Macedonia
Endotoxins are constituents of the membranes of Gram-negative bacteria ( LPS = lipopolysaccharide ) which
retain their activity even after death of the bacteria (negative sterility). If contaminated solution/API is/are used for
the manufacture of parenteral products, the patient may experience a pyrogenic reaction which may range from low
fewer via shock to death. A reliable method for quantitative detection of bacterial endotoxins is therefore of funda-
mental importance for pharmaceutical quality control.
For quantitative determination of bacterial endotoxins in Heparin inj. 5000 IU/ml, kinetic turbidrimetric
method PYROGENT- 5000 has been used, along with Microplate reader Elx808. The results are calculated and
stored with WIN QCL 3.0.1 software. The kinetic turbidrimetric method is based on measuring the turbidity (opti-
cal density) of an LAL/sample mixture at regular interval troughout the test. The time required before the appear-
ance of turbidity (Reaction time) is inversely proportional to the amount of endotoxin present. The concentration of
endotoxin in unknown samples can be calculated from a standard curve.
Heparin sodium is an anticoagulant chelator of calcium ions. This property makes it a powerful inhibitor of
the LAL assay. In order to overcome this inhibition due to calcium depletion, a cation buffer (Tris buffer and MgSO
4
) is utilized to make the first dilution; all subsequent dilutions are made using LAL regent water. Validation has been
made firstly with screening (Inhibition / Enhancement) test. For that purpose LONZA reagents have been used. Lysate
sensitivity 
λ = 0,01 EU/ml ; MVD = ELC/λ = 50 / 0,01 = 500 . Dilutions used from a Preliminary Inhibition Test :
1/2 in Tris buffer and MgSO
4
, then 1/4; 1/8; 1/16 … up to 1/4192 in the LAL reagent water. The product is inhibito-
ry up to 1/8 dilution inclusive. The inhibition was overcome at 1/16 dilution which gave PPC recovery 70%. Upon
received results the validation further was done on three series of Heparin inj. 5000 IU/ml with dilution 1/100. The
endotoxin concentration reported is less than 2 x 10
-4
IU/IU. Upon validation results the routine test further has been
conducted on the 1/100 dilution, a cation buffer is utilized to make the first dilution.
Macedonian pharmaceutical bulletin 53 (1,2) 231-232 (2007)
PP - 110
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Validacija i kvantitativno odreduvawe na bakteriski
endotoksini so kineti~ko turbidimetriska metoda 
na preparatot Heparin inj. 5000 IE/ml
E. Popovska, S. Ilioska Zlatanovi}, H. Babunovska, V. Jovanovska, M. Ilievska
1
Alkaloid A. D.
, Bul. A. Makedonski 12, 1000 Skopje, Makedonija
Endotoksinite se lipopolisahariden kompleks (LPS) koj e glaven sostaven del na nadvore{nata
membrana od pove}eto Gram-negativni bakterii ~ija aktivnost prodol`uva i po smrtta na bakteriite (ster-
ilen proizvod). Dokolku se upotrebi kontaminiran rastvor/aktivna supstanca za proizvodstvo na parenter-
alni produkti, kaj pacientot se pojavuva pirogena reakcija vo opseg od slaba treska preku {ok do smrt.
Zatoa e neophodna sigurna metoda za kvantitativno odreduvawe na bakteriskite endotoksini vo farma-
cevtskata kontrola na kvalitetot.
Za odreduvawe na bakteriski endotoksini vo Heparin inj. 5000 IU/ml e upotrebena Kineti~ko tur-
bidimetriska metoda, PYROGENT- 5000, koja se izveduva so Microplate reader Elx808 i koristi WIN QCL 3.0.1
softver za presmetuvawe i memorirawe na rezultatite. Principot na ovaa metoda e merewe na turbidite-
tot (opti~kata gustina) na smesata LAL/mostra vo odredeni vremenski intervali za vreme na testot. Vremeto
neophodno za pojava na turbiditet (Reakciono vreme) e obratno proporcionalno so koncentracijata na bak-
teriski endotoksini. Koncentracijata na endotoksini od nepznati aproizvodi se presmetuva so pomo{ na
standardna kriva. 
Heparin sodium e antikoagulans, helator na Ca ++ joni. Ova svojstvo go pravi mo}en inhibitor na LAL
analizata. So cel da se nadmine inhibicijata koja se dol`i na vrzuvawe na Ca ++ joni, se upotrebuva katjon-
ski pufer (Tris buffer i MgSO
4
) za prvo razreduvawe na produktot, site posledovatelni razreduvawa se vr{at
so LAL reagent water. Validacijata na metodata e napravena taka {to prvo e izraboten skrining (Inhibicija/
Zabrzuvawe). Pri toa e upotrebeni reagensi od LONZA. Lizat so senzitivnost l = 0,01 EU/ml; MVD = ELC/l
= 50 / 0,01 = 5000. Za Testot na Inhibicija/Zabrzuvawe napraveni se slednite razreduvawa: 1/2 vo Tris buffer
i MgSO
4
, potoa 1/4; 1/8; 1/16 ... se do 1/4192 vo LAL reagent water. Produktot poka`uva inhibicija vklu~uvaj}i
go razreduvaweto 1/8. Inhibicijata se nadminuva pri razreduvawe 1/16 koe dade PPC recovery 70%. Vrz osno-
va na dobienite rezultati od skriningot izvr{ena e validacija na tri serii Heparin inj. 5000 IU/ml so dilu-
cija 1/100. Dobienata endotoksin koncentracija kaj trite serii e < 2 x 10
-4
IU/IU. Vrz osnova na rezultatite
od validacijata preparatot ponatamu rutinski se testira so razdeduvawe od 1/100, a pri toa prvata dilu-
cija se vr{i so katjonski pufer.
Macedonian pharmaceutical bulletin 53 (1,2) 231-232 (2007)
PP - 110
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^ETVRTI KONGRES NA FARMACIJATA NA MAKEDONIJA SO ME\UNARODNO U^ESTVO
FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION

Efficacy of antimicrobial preservation in 
Pholcodin 15 mg/15 ml oral solution
Ljiljana Krsteska
1
, J. Dimitrovska
2
, S. M. Sejfulah , Sonja Ugarkovic
1
, 
2
Research & Development
1
,Quality Control, Alkaloid AD,
bul. Aleksandar Makedonski 12, Skopje, Macedonia
Antimicrobial Preservatives are used to prevent or inhibit the growth of microorganisms wich could present
a risk of infection or degradation of the Drug Product. These microorgnisms may proliferate during normal condi-
tions of use of the product by the patient particularly in multidose prepartions.
The aim of this work is to obtain the lowest concentracion of preservatives which provide protection againts
microbial contamination of multidose Drug Product.Model Drug Product is Pholcodin 15mg/15 ml oral solution.
The antimicrobial efficacy of the preservatives had been assessed during product develpment using Pharmacopeia
Europian test 5.1.3.
In examined product Pholcodin 15mg/15 ml oral solution a combination of preservatives Methylparaben
and Prolpylparaben were used because of their synergistic effect. 
For performance of antimicrobial efficacy of preservatives, Pilot batches were prepared in accordance EMEA
QWP/CPMP/419 guideline. The concentration of preservatives in the Pilot batches are displayed in the Table 1.
Method:
• Pharmacopoeia European test 5.1.3. for Efficacy of antimicrobial preservation, were criteria of acceptance
for antimicrobial efficacy of used preservatives are in accordance to antimicrobial activity request given in Table
5.1.3.–3.oral preparation, expressed as a log. reduction of number of viable microorganisms in certain terms, against
the value obtained for the inoculum at the start of the test. 
Test was performed with microorganisms of American Type Culture Collection(ATCC):
• Pseudomonas aeruginosa 
ATCC   9027
• Staphylococcus aureus 
ATCC   6538
• Escherichia coli 
ATCC  8739
• Candida albicans 
ATCC 10231
• Aspergillus niger 
ATCC 16404
In accordance with Criteria of acceptance for antimicrobial efficacy in multidosed Drug Product performed
on Pilot Batches No: 01122, 02122, 03122, 04122, 05122 it is obvious that the Pilot Batch  04122 possessed suffi-
cient antimicrobial activity.
Due to obtain results for antimicrobial efficacy during pharmaceutical development a formulation with con-
centration in Pilot batch 04122 was chosen.
Macedonian pharmaceutical bulletin 53 (1,2) 233-234 (2007)
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Pilot batches 
Methylparaben 
Propylparaben 
01122 
- 
- 
02122 
20mg/100 ml 
             5 mg/100 ml 
03122 
40 mg/100 ml 
10 mg/100 ml 
04122 
80 mg/100 ml 
20mg/100 ml 
05122 
120mg/100 ml 
30mg/100 ml 

Antimikrobna efikasnost na konzervansi vo 
Pholcodin rastvor 15mg/15 ml rastvor za oralna upotreba
Qiqana Krsteska
1
, J. Dimitrovska
2
, S. M. Sejfulah
1
, S. Ugarkovi}
1
,
1
Istra`uvawe i razvoj,  
2
Kontrola na kvalitet, 
Alkaloid AD, bul. Aleksandar Makedonski 12, Skopje, Makedonija
Konzervansite se upotrebuvaat za da go preveniraat ili go inhibiraat rastot na mikroorganizmite
kako rizik faktor na zagaduvawe ili degradacija na lekot. Ovie mikroorganizmi mo`at da proliferiraat i
vo normalni uslovi na upotreba na proizvodot od strana na pacientot osobeno kaj multidoznite proizvodi.
Cel e odreduvawe na najniska koncentracija na konzervansi koja }e go za{titi prizvodot od kon-
taminacija vo multidozen proizvod. Model preparat e Pholcodin 15mg/15 ml rastvor za oralna upotreba
Antimikrobnata efikasnost na konzervansi e izvedena vo faza na farmacevtsko-tehnolo{kiot
razvoj na preparatot koristej}i go Pharmacopoeia European test - ot 5.1.3. 
Vo ispituvaniot proizvod Pholcodin 15mg/15ml rastvor za oralna upotreba za prezervativna za{tita
upotrebena e kombinacija na konzervansi i toa Methylparaben i Propylparaben zaradi sinergisti~kiot efekt.
Za ispituvawe na antimikrobna efikasnost izraboteni se Pilot serii soglasno vodi~ot EMEA
QWP/CPMP/419. Koncentraciite na konzervansi na Pilot seriite se prilo`eni vo Tabela 1.
Metoda za ispituvawe na antimikrobna efikasnost:
• Test for Efficacy of antimicrobial preservation, Ph.Eur. 5.1.3. kade kriterium za procenka na antimikrob-
nata efikasnost na primenetite konzervansi se vr{i spored barawata dadeni vo Tabela 5.1.3.-3 oralni
preparati, koj predstavuva logaritamska redukcija na brojot na `ivotosposobni mikroorganizmi vo opre-
deleni termini, nasproti vrednosta na inokulumot dobiena na po~etokot na testot. 
Testot e izveden na mikroorganizmi od American Type Culture Collection(ATCC):
• Pseudomonas aeruginosa 
ATCC   9027
• Staphylococcus aureus 
ATCC   6538
• Escherichia coli 
ATCC  8739
• Candida albicans 
ATCC 10231
• Aspergillus niger 
ATCC 16404
Spored kriteriumot na prifatlivost za procenka na antimikrobnata efikasnost za multidozni
oralni preparati izveden na Pilot seriite: 01122, 02122, 03122, 04122, 05122 konstatirano e deka koncen-
tracijata na konzervansi vo pilot probata 04122 poseduva zadovolitelno antimikrobno dejstvo.
Vrz baza na dobenite rezultati za antimikrobna efikasnost na konzervansi pri definirawe na
formulacija, odbrana e koncentracijata na konzervansi od Pilot serija 04122
Macedonian pharmaceutical bulletin 53 (1,2) 233-234 (2007)
PP - 111
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^ETVRTI KONGRES NA FARMACIJATA NA MAKEDONIJA SO ME\UNARODNO U^ESTVO
FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION
 
Pilot Serija 
Methylparaben 
Propylparaben 
01122 
- 
- 
02122 
20mg/100 ml 
             5 mg/100 ml 
03122 
40 mg/100 ml 
10 mg/100 ml 
04122 
80 mg/100 ml 
20mg/100 ml 
05122 
120mg/100 ml 
30mg/100 ml 

Evaluation of the microbiological quality of medicines prepared 
in pharmacies
Dzenita Softic, Diana Jerg-Simanovic, Tamara Bosnic, Vesna Simic
Institute for Quality Control of Medicines F BiH, Titova 9, Sarajevo, Bosnia and Herzegovina
Medicines prepared in pharmacies in certain cases are irreplaceable. Those medicines are being prepared on
the basis of the prescription or standard prescriptions listed in expert manuals or Pharmacopoeia Nowadays in the
pharmacies, located at Sarajevo area, 30 various preparations are being prepared in this way. Most frequently pre-
pared preparations are: nasal drops, solutions, liquid powders, creams, ointments and oral powders. All these are
being prepared in small series or are being prepared just for a patient so they cannot be kept for a long period of time. 
This group of medicines is terminologically included in legislation. However, due to its purpose as well as due
to the small series of production it is almost impossible to conduct testing of its quality, a part form inspection control.
Having in mind the fact that process of medicine preparation in pharmacies is being conducted on the basis
of the GPP and GLP principles, goal of the conducted activities was to evaluate critical points in the preparation
process in relation to the microbiological quality of prepared medicine. 
Research showed that all components that are being used in the process of medicine preparation in pharma-
cies are of good quality, proved by appropriate certificates. However, it was found out that water solutions, which
preparation does not include thermal processing, often do not have appropriate microbiological quality. Additional
examination of filtered water, used in the preparation process, showed that there is only small number of microbio-
logically proper samples (around 20%, by TSA, that is 0% by R2A). No cases of contamination with pathogen microor-
ganism are recorded. However, due to the purpose of those preparations special attention should be paid to those results.
Macedonian pharmaceutical bulletin 53 (1,2) 235 (2007)
PP - 112
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^ETVRTI KONGRES NA FARMACIJATA NA MAKEDONIJA SO ME\UNARODNO U^ESTVO
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Storage time influence on purified water Bioburden 
A. Isovic, M. Mihajljica, S. Caklovica 
Bosnalijek d.d., Jukiceva 53, 71000 Sarajevo, Bosnia and Herzegovina
Water is one of the key utilities in most pharmaceutical facilities. It is used as solvent, product ingredient,
cleaning agent, and for many other applications. Some of those applications require water of higher purity than tipi-
caly found in municipal water supplies.
It must meet the requirements for ionic and organic chemical purity and microbiological quality. 
Purified water in bulk is prepared by distillation, by ion exchange, by reverse osmosis or by any other suit-
able method from water that complies with the regulations on water intended for human consumption laid down by
the competent authority. During production and storage appropriate measures are taken to ensure that the total viable
aerobic count is adequately controlled and monitored. Appropriate alert and action limits are set so as to detect adverse
trend. Under normal conditions an appropriate action limit is a total viable aerobic count of 100 microorganisms per
millilitre.
Consideration should be given to the timelines of microbiological enumeration testing after sample collec-
tion. Also it is very important to use clean sample container without of some microbial nutrient that could promote
microbial growth within the sample container. Because the number of recoverable bacteria in a sample can change
positively or negatively over time after sample collection it is recommended to test the samples as soon as possible
after being collecting.
The aim of this study was to investigate microbiological quality of water tested two and six hours after sam-
pling. It was analysed seventy two samples of purified water during twenty four days. 
Purified water was collected in sterile reagens botlles (volume of botlle is 1000 ml) and it was filtered 200
ml of sample through membrane filter (Cellulose nitrate filter 0,45 micron Lot. No 090613806 060 1913). It was
used a Sartorius vacuum pump. The choosen medium was medium B (Caso agar). Samples of purified water were
incubated at 35 ºC during three days (72 hours). After incubation has determined number of colony forming units.
The results showed that there is no significant influence of storing time at 4?C between two and six hours
on microbiological quality of water.
Macedonian pharmaceutical bulletin 53 (1,2) 236 (2007)
PP - 113
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^ETVRTI KONGRES NA FARMACIJATA NA MAKEDONIJA SO ME\UNARODNO U^ESTVO
FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION