Kniga na apstrakti book of abstrcts

Opredeluvawe na Klindamicin vo farmacevtski formulacii 
so kapilarna zonska elektroforeza
Z. Kavrakovski, Z. Kitanovski, K. Mladenovska
Farmacevtski fakultet, Univerzitet ”Sv. Kiril i Metodij”, 
Vodwanska 17, 1000 Skopje, Republika Makedonija
Kapilarna zonska elektroforeza e komplementarna i alternativna tehnika na naj~esto koristeni-
te hromatografski tehniki za detekcija, identifikacija i kvantifikacija na aktivni materii vo dozi-
rani farmacevtski preparati. Tehnikata se karakterizira so ednostavnost, visoka efikasnost i selek-
tivnost, realativno golema brzina i avtomatizam na izveduvawe na analizata, visoka rezolucija, malo
koli~estvo na injektiran volumen, £uvstvitelnost, reproduktivnost i niska cena na £inewe na analiza-
ta. Mo`e da se primenuva za ispituvawe na razli~ni grupi soedinenija, me|u koi i antibiotici. 
Klindamicinot (kako fosfat ili hidrohloridna sol) e antibiotik koj se primenuva za tretman na
Gram-pozitivni bakteriski infekcii vo forma na kapsuli, rastvori za nadore{na primena, losioni, gelovi,
kremi. Objaveni se pove}e (spektrofotometriski, hromatografski, elektrohemiski, mikrobiolo{ki)
metodi za kvalitativno i kvantitativno opredeluvawe na klindamicinot vo surovini i farmacevtski
preparati. Odredeni metodi se karakteriziraat so slaba specifi£nost, preciznost i to£nost, dodeka za
drugi e potrebna slo`ena postapka na ekstrakcija i pro£istuvawe.
Cel na istra`uvaweto e da se vospostavi i validira analiti~ka postapka za identifikacija i
opredeluvawe na klindamicinot so kapilarna zonska elektroforeza i UV detekcija posle rastvoruvawe
na klindamicinot vo voden rastvor so rN vo interval od 2 do 5 vo koja klindamicinot poka`uva maksimal-
na stabilnost. Ispituvawata se vr{eni so primena na BioRad model (BioFocus 3000, USA), opremen so kapi-
larna kolona (35 cm x 100 
µm i.d x 375µm o.d) i BioRad softver Ver.5.2 (za Windows NT). UV detekcijata e vr-
{ena na 190 nm pri napon 11 kV i temperatura 30
o
C. Kako elektroliten rastvor, koristen e 0.07 M fosfaten
pufer so rN mnogu poniska od rKa na klindamicinot (rKa 7.6), odnosno pH 3.28 doterana so 0.1 M fosfor-
na kiselina. Primerocite za analiza se injektirani vo sistemot so hidrodinamski pritisok od 5 s na anod-
niot kraj od kapilarata. Kapilarata pred sekoja analiza se ispira 1 min so rastvor na 0.05 M fosforna
kiselina, a potoa 1 min so elektrolitniot rastvor. 
Pod ovie uslovi separacija na klindamicinot se postignuva za pomalku od 10 min so RSD na migra-
ciskoto vreme od 0.18 - 0.30% (n = 6). Linearnosta e ispituvana vo podra~je od 0.007 - 1.01 mg/ml (R = 0.9896).
Limitot na kvantifikacija e 0.14% (m/m). Preciznosta, limitot na kvantifikacija i detekcija, speci-
fi~nosta i linearnosta na metodot e potvrdena so kvantitativna analiza na klindamicinot (vo oblik na
hidrohloridna sol) vo kapsuli (R = 96.5 - 98.9%).
Predlo`eniot CE metod so UV detekcija e mo{ne ednostaven i brz metod za opredeluvawe na klin-
damicin vo doziran farmacevtski preparat i mo`e da se primeni kako alternativen metod na ve}e propi-
{anite metodi vo Evropskata i Amerikanskata Farmakopeja.
Macedonian pharmaceutical bulletin 53 (1,2) 190-191 (2007)
PP - 88
191
^ETVRTI KONGRES NA FARMACIJATA NA MAKEDONIJA SO ME\UNARODNO U^ESTVO
FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION

Determination of Aminoglycoside antibiotics 
with capillary zone electrophoresis
Z. Kavrakovski
Faculty of Pharmacy, University „Ss. Cyril and Methodius“, Vodnjanska 17, Skopje, Macedonia
Aminoglycosides are important class of antibiotics active against gram-positive and gram-negative bacterial
infections, which are extensively used in clinical and veterinary medicine and in agriculture as well. Chemically, it is
a large and diverse class of antibiotics with two or more aminosugars linked by glycoside bonds to an aminocyclitol
component. For qualitative analysis, X-ray crystallography, NMR and MS techniques were used, while for quantita-
tive determination, microbiological, radiochemical and radioimmunoassay were used as well as UV/VIS and other
separative methods, such as GC, TLC, HPLC and CE. One of the major problems when analyzing aminoglycoside
antibiotics is lack of strong UV chromophore or fluorophore. For this reason, direct absorbance or fluorescence is not
possible, so derivatization is commonly used for quantification. The aim of this work was to explore the potential of
CE with direct UV detection for quantification of aminoglycoside antibiotics in pharmaceutical dosage forms.
A simple and fast capillary zone electrophoretic method was developed for simultaneous analysis of three
non-UV active compounds directly without derivatization. The usefulness of the method was demonstrated by detect-
ing neomycin, gentamicin and streptomycin in aminoglycoside antibiotic mixtures. All experiments were performed
on Bio Rad system model (Bio Focus 3000, USA) equipped with UV absorbance detector at 191 nm. Data acquisi-
tion and control were preformed using BioRad software (Ver.5.2) for Windows NT. Untreated fused silica capillar-
ies (BioRad, USA) with an inner diameter of 100 ?m id x 50 cm were used. The influence of pH (5.0-9.8), concen-
tration of disodium tetraborate in the running buffer (0.02-0.1 M) and the temperature (25-40°C) on separation efficacy
was analyzed.
The optimal conditions for quantitative analysis included 0.06 M disodium tetraborate buffer, pH 9.4, tem-
perature of 30° C and applied voltage of 15.0 kV. Under these conditions, baseline separation of the selected com-
pounds neomycin, gentamicin and streptomycin was achieved in less than 14 min with RSDs of migration times
from 0.21 to 0.44% (n=6) for each component. Linear correlation in the concentration range from 0.01 to 0.5 mg/ml
has been observed. Detection of non-UV-absorbing aminoglycosides through in-situ complexation with borate ions
has been achieved. Migration behavior was significantly affected by their chemical structure (number and position
of hydroxyl groups). The method showed good validation data in terms of precision, limits of quantification and
detection, specificity and linearity and it was found to be suitable for analysis of bulk pharmaceutical samples. 
Considering the results obtained, one can conclude that CE with direct UV detection shows potential for
identification and quantification of aminoglycoside antibiotics as an alternative method to the assays given by the
Ph Eur and USP.
Macedonian pharmaceutical bulletin 53 (1,2) 192-193 (2007)
PP - 89
192
^ETVRTI KONGRES NA FARMACIJATA NA MAKEDONIJA SO ME\UNARODNO U^ESTVO
FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION

Opredeluvawe na Aminoglikozidi 
so kapilarna zonska elektroforeza
Z. Kavrakovski
Farmacevtski fakultet, Univerzitet ”Sv. Kiril i Metodij”, 
Vodwanska 17, 1000 Skopje, Republika Makedonija
Aminoglikozidite se va`na grupa na antibiotici koi se koristat vo tretman na Gram-pozitivni
i Gram-negativni bakteriski infekcii vo klini~kata i veterinarnata medicina i vo zemjodelstvoto.
Hemiski, stanuva zbor za golema i heterogena grupa na lekovi so dva ili pove}e amino{e}eri povrzani
so glikozidni vrski za aminociklitolna komponenta. Za kvalitativna analiza na aminoglikozidnite
antibiotici se koristat nekolku tehniki, me|u koi kristalografija so H-zraci, NMR i MS, dodeka za
kvantitativno opredeluvawe, na raspolagawe se mikrobiolo{ki, radiohemiski i radioimunolo{ki meto-
di, kako i UV/VIS i drugi separativni metodi (GC, TLC, HPLC i CE). Koga se analiziraat aminoglikozid-
nite antibiotici, posebna pote{kotija e otsustvoto na potenten UV hromofor ili fluorofor. Od tie
pri~ini, direktnata apsorpcija ili florescenicija ne mo`e da se odredat t.{. za kvantitaivno oprede-
luvawe se koristi derivatizacija. Cel na trudot e da se ispita potencijalot na tehnikata na kapilarnat
zonska elektroforeza so direktna UV detekcija za kvalitativno i kvantitativno opredeluvawe na aminog-
likozidnite antibiotici vo farmacevtski dozirani formi.
Razvien e ednostaven i brz kapilarno zonski elektroforetski metod za istovremeno odreduvawe na
tri UV neaktivni komponenti direktno bez derivatizacija. Primenata na ovoj metod e ispituvana na prime-
roci koi sodr`at neomicin, gentamicin i streptomicin vo me{avina. Site eksperimenti se izvedeni na
BioRad sistem (Bio Focus 3000, USA) opremen se UV detektor (191 nm). Obrabotkata na podatocite se vr{i so
koristewe na BioRad softver (Ver.5.2) za Windows NT. Koristeni se neoblo`eni kapilari (BioRad, USA) so
vnatre{en pre~nik 100 
µm id x 50 cm. Ispituvano e vlijanieto na rN (5.0-9.8), koncentracijata na dinatri-
um tetraborat vo vode~kiot pufer (0.02-0.1 M) i temperaturata (25-40
0
C) vrz separaciskata efikasnost.
Optimalnite uslovi za kvantitativna analiza vklu~uvaat 0.06 M dinatrium tetraboraten pufer,
pH 9.4, temperatura od 30
0
C i napon od 15.0 kV. Vo ovie uslovi, postignata e separacija na antibioticite
neomicin, gentamicin i streptomicin za pomalku od 14 min so RSD vrednosti za migraciskite vremiwa od
0.21 to 0.44% (n = 6) za sekoja komponenta. Dobiena e linearna korelacija vo koncentraciski interval
od 0.01 do 0.5 mg/ml. Obezbedena e detekcija na UV neapsorbira~kite aminoglikozidi preku in-situ kom-
pleksacija so boratni joni. Migraciskoto vreme e pod zna~ajno vlijanie na hemiskata struktura na antibio-
ticite t.e. brojot i pozicijata na hidroksilnite grupi. Metodot e precizen i poka`uva dobar limit na
kvantifikacija i detekcija, specifi~nost i linearnost i e soodveten za analiza na aminoglikozidnite
antibiotici vo farmacevtski surovini. 
Kako zaklu~ok, kapilarnata zonska elektroforeza so direktna UV detekcija poka`uva potenci-
jal za identifikacija i kvantitativno opredeluvawe na aminoglikozidnite antibiotici i pretstavuva
alternativa na metodite navedeni vo Evropskata i Amerikanskata Farmakopea.
Macedonian pharmaceutical bulletin 53 (1,2) 192-193 (2007)
PP - 89
193
^ETVRTI KONGRES NA FARMACIJATA NA MAKEDONIJA SO ME\UNARODNO U^ESTVO
FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION

Capillary zone electrophoresis of monosaccharides 
by direct UV detection
Z. Kavrakovski 
Faculty of Pharmacy, University „Ss. Cyril and Methodius“, Vodnjanska 17, Skopje, Macedonia
Carbohydrates play an important role in many diverse research and industrial domains (e.g. biochemistry,
clinical chemistry, pharmacy, biotechnology and food science). The considerable number of stereoisomers, the
immense combination possibilities of carbohydrate monomers and the lack of chromophores make sugar analysis a
challenge. The most commonly used techniques for the determination of carbohydrates are TLC, GC of volatile
derivatives, HPLC in various models such as reverse phase, ion exchange, and affinity. 
Capillary zone electrophoresis (CZE) represents an alternative to the commonly used chromatographic tech-
niques for the determination of carbohydrates, with detection of the nanoliter sample volume bringing speed, quan-
tification, reproducibility and automation to the intrinsically high-resolution techniques of electrophoresis. 
The aim of the research was to establish direct CE method for simultaneous detection of monosaccharides
glucose, fructose and mannose in orange juice and honey.
Analyses were performed on BioRad system model (Bio Focus 3000 capillary electrophoresis, USA), which
was equipped with a 25 cm x 100 
µm i.d. coated silica capillary. Detection was carried out by on-column measure-
ment of UV absorption at 191 nm. Analyses were carried out at a constant temperature of 35
0
C. Running electrolyte
solution was disodium tetraborate on voltage 10.0 kV. The influence of pH (7.0 - 10.0) adjusted by NH
4
OH, con-
centration of disodium tetraborate in running buffer (0.02 - 0.10 M) and temperature (25 - 40
0
C) on separation effi-
cacy was analyzed.
The enhanced absorption of sugars in the presence of borate allows their UV detection without derivatiza-
tion and consequently, electrophoretic separation in a capillary electrophoresis system can be achieved. The results
obtained when above-mentioned conditions used showed efficacious separation with different migration times when
mixtures of compounds analyzed. For CE determination of glucose, fructose and mannose in the concentration range
of 1.0 - 10 mg/ml, linear regression was observed (0.9889 
<
R 
<
0.9957). Precision expressed by relative standard
deviation ranged from 1.44 to 4.37% of glucose, 1.44 to 4.37% of fructose and 1.00 to 4.20% of mannose. Recoveries
were in the region of 95.3 - 103.0%. 
As a conclusion, the low detection limit and RSD values and the high percentage of recovery confirm that
the CZE technique is a sensitive and selective method. Therefore, CZE by direct UV detection of monosaccharides
under established conditions permits quantification at lower level without complicated extraction and further deriv-
ative reaction.
Macedonian pharmaceutical bulletin 53 (1,2) 194-195 (2007)
PP - 90
194
^ETVRTI KONGRES NA FARMACIJATA NA MAKEDONIJA SO ME\UNARODNO U^ESTVO
FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION

Opredeluvawe na monosahsaridi so kapilarna zonska 
elekrtoforeza i direktna UV detekcija
Z. Kavrakovski 
Farmacevtski fakultet, Univerzitet ”Sv. Kiril i Metodij”, 
Vodwanska 17, 1000 Skopje, Republika Makedonija
Jaglehidratite imaat zna~ajna uloga vo mnogu istra`uva~ki i tehnolo{ki domeni vo medicina-
ta, farmacijata, biotehnologijata, hemiskata industrija i naukata za hrana. Za jaglehidratite postoi
mislewe deka se „diva grupa“ na soedinenija za ispituvawe i karakterizacija. Strukturno, tie pretstavu-
vaat zbir na hidroksilni grupi vo relativno mala molekula so mo{ne sli~ni fizi~ko-hemiski osobi-
ni. Razlikite pome|u niv, ~estopati se nao|aat vo nivnata trodimenzionalna molekulska struktura, a
golemiot broj na stereoizomerni formi i mo`ni kombinacii na jaglehidratni monomeri, kako i nedosta-
tokot na hromoforni i fluoroforni grupi gi pravat nivnite ispituvawa isklu~itelno te{ki, no vo
isto vreme i vistinski predizvik za analiti~arite. Za analiza na jaglehidratite se primenuvaat brojni
hromatografski (TLC, GC, HPLC), spektrometriski (UV/VIS, FABS, NMR), elektroforetski, enzimski,
mikro- i PCR analizi. Kapilarnata zonska elektroforeza (CZE) e alternativna tehnika na naj~esto
koristenite hromatografski tehniki za detekcija, identifikacija i kvantifikacija na jaglehidratite.
Se karakterizira so relativno golema brzina i avtomatizam na izveduvawe, visoka rezolucija, £uvstvitel-
nost, reproduktivnost i malo koli~estvo na injektiran volumen. 
Cel na istra`uvaweto e da se vospostavi analiti~ka postapka za istovremena identifikacija i
opredeluvawe na monosaharidite glukoza, fruktoza i manoza vo sok od portokal i med, so primena na CZE.
Ispituvawata se vr{eni na BioRad sistem (Bio Focus 3000, USA), opremen so oblo`ena kapilarna kolona
(25 cm x 100 
µm i.d.). UV detekcijata e vr{ena na 190 nm pri napon od 10 kV i temperatura 35 
0
C. Kako nose~ki
elektrolit, koristen e natrium tetraboraten pufer. Ispituvano e vlijanieto na pH vrednosta, koja se
doteruva so NH4OH (7.0 - 10.0), koncentracijata na nose~kiot elektrolit (0.02 - 0.10 M), kako i temperatu-
rata (25 - 40
0
C) vrz efikasnosta na separacijata. Ispituvawata se vr{eni vo koncentracisko podra~je od
1.0 - 10.0 mg/ml, so koeficient na korelacija (0.9889 < R < 0.9957).
Jaglehidratite kako slabi kiselini joniziraat i se separiraat vo sistemot samo vo alkalen medi-
um. So dodavawe na boraten pufer, neutralnite monosaharidni molekuli se transformiraat vo negativno
naelektriziran boraten kompleks. Kompleksiraweto ja pomestuva ramnote`ata kon karbonilnata forma,
{to e edna od pri~inite za porastot na apsorptivnosta na ispituvanite komponenti vo rastvorot.
Reakcijata opfa}a sozdavawe na ester na borna kiselina so tri hidroksilni grupi i koordinacija na
~etvrta hidroksilna grupa so atomot na borot. Na toj na~in se ovozmo`uva fotometriska detekcija bez
prethodna derivatizacija. Jaglehidratite koi sodr`at pogolem broj hidroksilni grupi i karboksilni
funkcii imaat povisoka mobilnost vo sistemot poradi visokata mo`nost na formirawe na boraten kom-
pleks. Formiraweto na kompleksite e brzo, a detekcijata e vo nisko UV podra~je. RSD (%) se dvi`i od
1.44-4.37 za glukoza, 1.40-1.47 za fruktoza i 1.00-4.20 za manoza. Analiti~kiot prinos e vo granici od 95.3-
103.0%. Metodot ovozmo`uva opredeluvawe na monosaharidite direktno bez primena na slo`ena postap-
ka na ekstrakcija ili derivatizacija.
Macedonian pharmaceutical bulletin 53 (1,2) 194-195 (2007)
PP - 90
195
^ETVRTI KONGRES NA FARMACIJATA NA MAKEDONIJA SO ME\UNARODNO U^ESTVO
FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION

HPLC analysis of acetylsalicylic acid, paracetamol, caffeine and their
degradation products in Malophenum
®
tablets
V. Ilijev
2
, M.Crevar
1
, Z.Vuji
c
1
, B.Ivkovi
c
1
Faculty of Pharmacy, Belgrade, Serbia,  Apotekarska ustanova Nis, Nis, Serbia
Acetylsalicylic acid, paracetamol and caffeine are very often used together in many combinated formula-
tions. Paracetamol and acetylsalicylic acid are valuable analgoantipiretics and anti inflammatory drugs. Their com-
bination with central analeptic caffein has less side effects than every component separately but sinergistic therapeu-
tic effects.
Para-aminophenol is process-related impurity that may be present in paracetamol drug substance. Salicylic
acid is product of hydrolysis of acetylsalicylic acid. Both can cause important side effects and has to be discovered
in formulations on time.
Aim of this work was to define optimal conditions for separation, identification and quantitative analysis of
active components and their degradation products in combined formulations by HPLC method.
It was impossible to develop one method for all mentioned components. Conditions suitable for acetylsalicylic
acid are not suitable for paracetamol and caffeine. Two different HPLC methods are developed. Good separation of
paracetamol, caffeine and p-aminophenol was obtained on Zorbax-Extend-C
18
column (150 mm x 4.6 mm, 5 
µm).
Column temperature was 25
0
C, flow rate 1 ml/min, UV detection performed on 215 nm with mobile phase consist-
ed of methanol / 0.01M KH
2
PO
4
(20:80 v/v). Phenason was used as internal standard. Separation of acetylsalicylic
acid and its degradation product salicylic acid was carried out on the same column, temperature, UV detection wave-
length and flow rate. Mixture of methanol and 1.5% CH
3
COOH (40:60 v/v) was used as mobile phase. Internal stan-
dard was benzoic acid.
There are known methods for separation of active components paracetamol, caffeine and acetylsalicylic acid
by HPLC. This is the first method for identification and determination of all, active components and their degrada-
tion products. Both given methods are simple, economic (small percentage of organic phase) and rapid. Validation
results show that these methods are accurate, selective, sensitive and reproductive so they can be used for the quan-
titative analysis of caffeine, paracetamol, acetylsalicylic acid and its degradation products not just in Malophenum
tablets but also in various combinations of these components in their routine control.
Macedonian pharmaceutical bulletin 53 (1,2) 196 (2007)
PP - 91
196
^ETVRTI KONGRES NA FARMACIJATA NA MAKEDONIJA SO ME\UNARODNO U^ESTVO
FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION

Development and optimization of RP RR-HPLC method 
for determination of haloperidol and related compounds 
using Chemometric approach
R. Petkovska, J. Petrusevska, A. Dimitrovska
Department of Chemistry, Faculty of Pharmacy, University “Ss.Cyril and Methodius”, 
Vodnjanska 17, 1000 Skopje, R.Macedonia
A Reversed Phase Rapid Resolution High Performance Liquid Chromatography (RP RR-HPLC) method for
simultaneous determination of haloperidol and related compounds which are specified as impurities has been devel-
oped and validated. 
Experimental design has been used during development (full factorial design) and method optimization
(Response surface methodology). For experimental screening a full factorial design 2
3
was applied. Three factors:
organic phase variation during gradient elution, gradient flow rate and gradient rise time during gradient elution were
independent variables or factors to be investigated. R
s
values for all consecutive peak pairs were applied to estimate
coefficients of the linear model. After the most important factors were identified, the separation was optimized using
Response surface methodology (RSM). The separation quality of haloperidol and its impurities for achieving the
maximum resolution with the minimum assay time was assessed by calculating the value of Chromatographic response
function (CRF). The CRF is coefficient which characterizes the quality of the separation in quantitative manner and
allows desirable time and resolution criteria to be specified. Minimum obtained value of individual R
s
-values of 2,5
as a selection criterion was used. Investigation matrix was laboratory mixture of therapeutic active supstance haloperi-
dol and its five related compounds in concentration ratio 100:1 respectively. Chromatography was performed with a
mobile phase containing phosphate buffer pH 8.5 and acetonitrile as organic modifier. Separation was achieved using
a gradient elution (organic phase fraction changed linearly during gradient elution from 20 % to 80 % over 7 min).
A Zorbax Eclipse XDB C18 Rapid Resolution HT 4.6 mm × 50 mm, 1,8
µm particle size column was used at 25
0
C
with a flow rate of 1.5 ml min-
1
. UV detection was performed at 230 nm. The total time for the separation was 5.5 min.
The method was validated statistically for its selectivity, linearity, precision, accuracy and robustness. 
Applying the chemometrical approach enables a relatively limited number of experiments to define factors which
affect the chromatographic behavior of investigated substances and obtain optimum conditions for their analysis.
Macedonian pharmaceutical bulletin 53 (1,2) 197-198 (2007)
PP - 92
197
^ETVRTI KONGRES NA FARMACIJATA NA MAKEDONIJA SO ME\UNARODNO U^ESTVO
FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION