Kniga na apstrakti book of abstrcts

Razvoj i optimizacija na RP RR-HPLC metod za razdvojuvawe 
na haloperidol i srodni supstancii so primena na hemometriski
pristap pri eksperimentalno dizajnirawe
R. Petkovska, J. Petru{evska, A. Dimitrovska
Institut za hemija, Farmacevtski fakultet, 
Univerzitet ”Sv. Kiril i Metodij”, Vodwanska 17, 1000 Skopje, R. Makedonija
Metodot za analiza so primena na reverzno fazna visoko efektivna te~na hromatografija so brza
rezolucija (RP RR-HPLC) be{e primenet za razdvojuvawe na haloperidol i negovi srodni supstancii koi
mo`e da se javat kako one~istuvawa vo terapevtski aktivnata supstanca ili vo gotov farmacevtski proizvod.
Hemometriski pristap pri eksperimentalnoto dizajnirawe be{e primenet vo tek na razvoj (pot-
poln faktorski dizajn) i optimizacija na metodot (dizajn na povr{ina na odgovor). Vo tek na razvoj na
metodot potpoln faktorski 2
3
dizajn be{e primenet za utvrduvawe na eksperimentalnite faktori koi
imaat najgolemo vlijanie na procesot na razdvojuvawe na ispituvanite supstancii. Be{e utvrdeno vlijani-
eto na: promena na sostav na mobilna faza vo tek na gradientno eluirawe, promena na vreme na gradient-
no eluirawe i protok na mobilna faza. Za presmetuvawe na koeficientite na linearniot model i procen-
ka na zna~ajnosta na vlijanieto od ispituvanite eksperimentalni faktori be{e koristena vrednosta na
rezolucijata (R
s
) me|u dobienite pikovi. Optimizacija na vrednostite na zna~ajnite eksperimentalni fak-
tori be{e izar{ena so primena na dizajn na povr{ina na odgovorot (RSM). Optimalnite vrednosti na
zna~ajnite eksperimentalni faktori pri koi se postigna razdvojuvawe so najgolema mo`na rezolucija za
najkratko mo`no vreme bea proceneti preku presmetuvawe na vrednosta na funkcija na hromatografski
odgovor. Funkcijata na hromatografski odgovor (CRF) dava mo`nost za kvantitativna procenka na razd-
vojuvaweto so predhodno definirawe na kriteriumi za rezolucija i vreme za razdvojuvawe. Poedine~na
vrednost za R
s
od najmalku 2.5 be{e primenet kako kriterium za selekcija. Médium za ispituvawe be{e
rastvor na haloperidol i pet negovi srodni supstancii vo koncentraciski odnos 100:1 soodvetno.
Optimalno razdvojuvawe be{e postignata so linearno gradientno eluirawe so promena na udel
acetonitril vo mobilna faza od 20% do 80% za vreme od 7 minuti na Eclipse XDB C18 Rapid Resolution HT
4.6 mm x50 mm,1.8 
µm kolona, na temperatura od 25 °C, protok 1.5 ml min
-1
. UV apsorpcijata be{e merena na
230 nm. Metodot be{e validiran preku opredeluvawe na linearnost, to~nost, preciznost, limit na detekci-
ja i limit na kvantifikacija.
Primenata na hemometriski pristap pri eksperimentalno dizajnirawe ovozmo`i definirawe
na hromatografskoto odnesuvawe na ispituvanite supstancii kako i planirano i sistematsko defini-
rawe na eksperimentalnite uslovi za nivno razdvojuvawe. 
Macedonian pharmaceutical bulletin 53 (1,2) 197-198 (2007)
PP - 92
198
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SEC-HPLC used for detection of aggregate formation in recombinant
human granulocyte - colony stimulating factor (rHuG-CSF),
Lenograstim
Jasmina Tonic-Ribarska, Suzana Trajkovic - Jolevska, Aneta Dimitrovska
Faculty of Pharmacy, Ss Cyril and Methodius University, Vodnjanska 17, 1000 Skopje, Macedonia
Human granulocyte - colony stimulating factor (G-CSF) is a hematopoietic growth factor that plays a major
role in the stimulation of the proliferation and maturation of granulocyte neutrophil cells. Lenograstim is glycosy-
lated form of the protein, derived from Chinese hamster ovary (CHO) cells and it is indistinguishable from natural
human (endogenous) G-CSF. It is a 19,6 kDa glycoprotein consisting of 174 amino acids and possesses an O-linked
carbohydrate chain attached to threonine-133 of the molecule.
A major disadvantage of recombinant protein drugs is their highly susceptibility to chemical and physical
degradation resulting in a decrease or complete loss of biological activities. Protein aggregation is a dominant degra-
dation pathway for therapeutic proteins, potentially occurring during all phases of production, purification, shipping,
storage and administration. The presence of aggregates in therapeutic protein pharmaceuticals can cause adverse
effects within patients, ranging from immune response to anaphylactic shock. Many analytical techniques are avail-
able to monitor protein instability. Among these techniques, size-exclusion high – performance liquid chromatogra-
phy (SEC-HPLC) was found to be an appropriate method for detecting and quantifying protein aggregation. No SEC-
HPLC methods for assessing aggregation in lenograstim and detecting aggregates and intact protein have been
published to date.
In this study a simple and sensitive SEC-HPLC method for detection and separation of aggregates from mo-
nomer, lenograstim was developed. The aggregation of lenograstim was occurred under conditions that do not dif-
fer greatly from physiological, in the absence of chemical denaturants (the sample was incubated during 5 days at
37
0
C in pH 6,9 phosphate buffered saline). The experiments were carried out by size exclusion chromatography on
a Fractogel
®
EMD BioSEC, Merck, column (superformance 600 – 16 mm). The HPLC system was operated isocra-
tically at ambient temperature using phosphoric acid (pH 2,5; 0,1M) as a mobile phase, run at a flow rate of 2.0 ml/min
and UV detection at 215 nm. Under the proposed chromatographic conditions successful separation of aggregates
from intact monomer was obtained. 
SEC provides information about the total aggregate content. To get a deeper insight into the nature of the
aggregation mechanism, SDS-PAGE was performed, allowing a differentiation between covalently and non-cova-
lently linked aggregates (under reducing and non-reducing conditions, respectively). The SDS-PAGE results have
demonstrated that if any aggregates were attached covalently, the bonds must be disulfide.
In conclusion, the proposed SEC-HPLC method is able to detect and separate the aggregates and the intact
protein. This method can be successfully used for assessing protein aggregation and allows detection of aggregate
formation at the very beginning, signaling the stability changes important for pharmaceutical quality and biological
activity of lenograstim.
Macedonian pharmaceutical bulletin 53 (1,2) 199-200 (2007)
PP - 93
199
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SEC-HPLC metod za detekcija na produktite na agregacija na
rekombinaten human granulocit kolon stimulira~ki faktor
(rHuG-CSF), Lenograstim
Jasmina Toni}-Ribarska, Suzana Trajkovi} - Jolevska, Aneta Dimitrovska
Farmacevtski fakultet, Univerzitet Sv. Kiril i Metodij, 
Vodwanska 17, 1000 Skopje, Makedonija
Granulocit kolon stimulira~kiot faktor (G-CSF) e hematopoetski faktor na rast i e odgovoren
za stimulacija na proliferacijata i diferencijacijata na granulocitnite neutrofilni kletki.
Glikoziliranata forma na rHuG-CSF, lenograstim, e izolirana od kleto~en sistem na doma}in cica~,
Chinese Hamster Ovaries. Lenograstimot e glikoprotein so molekulska masa od 19,6 kDa. Vo sostav na lenogras-
timot vleguvaat 174 amino kiselini i eden {e}eren lanec koj e O- povrzan za treoninot na polo`ba 133.
Strukturata na lenograstimot ne se razlikuva od strukturata na prirodniot human (endogen) G-CSF.
Golem problem koj se javuva kaj rekombinantnite proteinski farmacevtski preparati e nivnata
nestabilnost, odnosno podlo`nosta na proteinite na fizi~ka i hemiska degradacija, {to rezultira so
namaluvawe ili celosno gubewe na nivnata biolo{ka aktivnost. Procesot na agregacija pretstavuva
nesomneno glaven i naj~est problem koj mo`e da se javi vo site fazi od proizvodstvoto na proteinskite
farmacevtski preparati, vo tek na transportot i ~uvaweto. Prisustvoto na agregati vo proteinskite
farmacevtski preparati mo`e da dovede do pojava na niza nesakani efekti kaj pacientite, od imunolo{ki
odgovor do anafilakti~en {ok. Za sledewe na nestabilnosta na proteinite se primenuvaat pove}e anal-
iti~ki tehniki, me|u koi najsoodvetna tehnika za detekcija i kvantifikacija na produktite na agregaci-
ja e SEC-HPLC . Nema literaturni podatoci za razvoj na SEC-HPLC metod za sledewe na procesot na agre-
gacija kaj lenograstim i za detekcija i separacija na formiranite agregati i intaktniot protein. 
Celta na ovoj trud be{e da se vospostavi ednostaven i osetliv SEC-HPLC  metod za detekcija i
separacija na formiranite agregati od monomerot-lenograstim. Agregacijata be{e predizvikana vo uslo-
vi koi ne se razlikuvaat mnogu od fiziolo{kite uslovi (primerocite bea rastvoreni vo fosften pufer
rN 6,9 i ~uvani na temperatura od 37
0
C, 5 dena). Ispituvwata bea izvedeni na Fractogel
®
EMD BioSEC kolona,
so upotreba na fosoforna kiselina (pH 2,5; 0,1M) kako mobilna faza, protok od 2 ml/min i UV detekcija na
215 nm. Pod utvrdenite hromatografski uslovi, postignata e zadovolitelna separacija na agregatite od
intaktniot monomer. So primena na SEC se detektiraat site rastvorlivi agregati. So cel da ja utvrdime
prirodata na formiranite agregati, odnosno dali se kovalentno ili nekovalentno povrzani, be{e prime-
neta SDS-PAGE pod reducira~ki i nereducira~ki uslovi. Dobienite rezultati uka`uvaat na prisustvo na
kovalentno povrzani agregati so disulfidni vrski. 
Predlo`eniot SEC-HPLC metod mo`e da se koristi za detekcija i separacija na formiranite
agregati od intaktniot protein. Ovoj metod ovozmo`uva sledewe na procesot na agregacija i detekcija
na agregatite vo rana faza od nivnoto formirawe, {to }e signalizira za promeni vo stabilnosta na
molekulot, zna~ajni za farmacevtskiot kvalitet i biolo{kata aktivnost na lenograstim. 
Macedonian pharmaceutical bulletin 53 (1,2) 199-200 (2007)
PP - 93
200
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Influence of dwell volume of HPCL systems on determination 
of tegaserod and its imputities
J. Petrusevska, Z. Kitanovski, R. Petkovska, L. Ugrinova, A. Dimitrovska
Center for drug quality control, Faculty of Pharmacy, 
„St. Cyril and Methodius“ University, Vodnjanska 17, 1000 Skopje, Macedonia
Simple and rapid reverse-phase HPLC method was developed for determination of tegaserod and its impu-
rities: 515-91 (5-methoxy-1H-indole-3-carbaldehyde); 203-91 (1,3-bis-(5-methoxy-1H-indole-3-ylmethyleneamino)-
guanidine) and 001-94 (1-(5-methoxy-1H-indole-3-ylmethylene-5-dodecyl isosemi-carbazide). The aim of this work
was to avoid the problems of method transfer including availability of buffers for mobile phase, system suitability
criteria for gradient elution and dwell volumes of different HPLC instruments.
Separation of the four components was performed on HPLC Rapid Resolution instrument with a binary pump
and a constant-flow solvent-delivery system, a reverse phase C 8, 150 x 4.6 mm, 5µm column, maintained at 35
0
C
with a column heater. The detection was performed with a diode array detector set at 315 nm. The mobile phases
consisted of acetonitrile (mobile phase A) and 0.05 M ammonium acetate buffer with 0.5 ml/l triethylamine, adjust-
ed to pH 7.5 with concentrated acetic acid (mobile phase B), delivered at a flow rate of 2.0 ml/min. Best separation
was achieved with a step gradient elution after an initial isocratic phase: time zero to time 3 minutes, A = 25%, B =
75%; time 3.01 to time 9 minutes, A = 70%, B = 30%; time 9.01 minutes, back to initial conditions. Validation of
the method included determination of selectivity, linearity, accuracy, precision, limit of detection and quantification.
In order to minimise the effect of differences in dwell volumes, instead of initial rapid gradient described in
currently available studies, the less strongly retained component 515-91 is eluted with an initial isocratic phase fol-
lowed by a gradient for elution of the more strongly retained analytes: 203-91, tegaserod and 001-94. The change of
the detection on 315 nm, (instead of 220 nm as found in the published methods) allows stable baseline during the
gradient changing. Results have shown good separation of the four components: 515-91 (RT 3.7; k' 2.7), 203-91 (RT
6.0; k' 5.0), tegaserod (RT 6.9; k' 5.9) and 001-94 (RT 8.7; k' 7.7). Resolutions between peaks are 12.7; 4.4 and 8.0
respectively. The removal of the gradient mixing chamber result in decreasing the retention of all the peaks for about
0.3 minutes and even better k', especially for the most retained analyt. 
Since is not possible to employ isocratic method for determination of tegaserod and its impurities, the gra-
dient method was unavoidable. The modifications of the currently published gradient methods serves to avoid the
potential pitfalls of significant differences in dwell volumes of the different gradient pumping systems in the con-
text of method transfer. This method allows more easy adjustment of chromatographic parameters on different HPLC
instruments for meeting the system suitability criteria. The proposed method is simple, rapid and is suitable for deter-
mination of tegaserod and its impurities. 
Macedonian pharmaceutical bulletin 53 (1,2) 201-202 (2007)
PP - 94
201
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Vlijanie na zadocnetiot volumen na HPLC sistemite 
vrz odreduvaweto na tegaserod i one~istuvawa
J. Petru{evska, Z Kitanovski, R. Petkovska, L. Ugrinova, A. Dimitrovska
Centar za ispituvawe i kontrola na lekovi, Farmacevtski fakultet, 
Univerzitet Sv. Kiril i Metodij, Vodwanska 17, 1000 Skopje, Makedonija
Razvien e ednostaven i brz metod za odreduvawe na tegaserod i negovite onegovite one~istuvawa:
515-91 (5 - methoxy-1H-indole-3-carbaldehyde); 203-91 (1,3-bis-(5methoxy-1H-indole-3-ylmethyleneamino)-guanidine) i
001-94 (1-(5-methoxy-1H-indole-3-ylmethylene-5-dodecyl-isosemi-carbazide). Celta na trudot e da se izbegnat proble-
mite koi nastanuvaat pri transfer na metodi, vklu~itelno dostapnosta na puferi za mobilna faza, ispol-
nuvaweto na kriteriumite za prifatlivost na sistemot pri gradientno eluirawe i zadocnetite volumeni
na razli~ni HPLC sistemi.  
Razdeluvaweto na ~etirite komponenti be{e postignato so primena na visoko efektivna te~na hro-
matografija so brza rezolucija, na reverzno fazna kolona S 8 150 h 4,6 mm, 5
µm, termostatirana na 35
0
C.
Analitite bea detektirani na branova dol`ina od 315 nm, so pomo{ na detektor so podvi`na dioda.
Mobilnata faza be{e sostavena od acetonitril (mobilna faza A) i 0,05 M ammonium acetaten pufer so 0,5 ml/l
trietilamin, doteran do rN 7,5 so ocetna kiselina (mobilna faza B), so protok od 2,0 ml/min. Eluiraweto  e
izvr{eno so gradienten ~ekor: od po~etok do treta minuta: A = 25 %, B = 75 %; od treta do devetta minuta,
A = 70 %, B = 30 %; posle devetta minuta povtorno po~etnite uslovi. Validacijata na metodot be{e izvr{ena
vo odnos na selektivnost, linearnost, to~nost, preciznost, limit na detekcija i na kvantifikacija.
Za da se namali efektot na razlikata vo zadocnetite volumeni pome|u razli~ni HPLC sistemi,
komponentata so najmala retencija, 515-91, se eluira so po~etna izokratska faza, namesto so direkten brz
gradient opi{an vo publikuvani trudovi. Posle po~etnata izokratska faza sledi gradient za eluirawe
na komponentite so pogolema retencija: 203-91, tegaserod i 001-94. Promenata na branovata dol`ina od
220 nm na 315 nm, kade apsorbiraat site komponenti, ovozmo`uva stabilna bazna linija za vreme na gradi-
entnite promeni. Rezultatite poka`uvaat dobro razdeluvawe na ~etirite komponenti: 515-91 (Rt 3,7; k´
2,7), 203-91 (Rt 6,0; k´ 5,0), tegaserod (Rt 6,9; k´ 5,9) i 001-94 (Rt 8,7; k´ 7,7). Rezoluciite pome|u pikovite bea
12,7; 4,4 i 8,0, soodvetno. Otstranuvaweto na komorata za me{awe na rastvoruva~ite rezultira{e so namalu-
vawe na retencijata na site pikovi za okolu 0,3 minuti i podobri kapacitetni faktori, osobeno na anali-
tot so najgolema retencija. 
Bidej}i e nevozmo`no da se upotrebi izokratski metod za razdeluvawe na tegaserod i negovite
one~istuvawa, gradientnoto eluirawe ne mo`e da se izbegne. Modifikaciite na gradientnite metodi od
literatura ovozmo`uvaat izbegnuvawe na potencijalnite zamki predizvikani od zna~ajni razliki vo zadoc-
net volumen pome|u razli~ni gradientni pumpi vo kontekst na transfer na metodolo{ki postapki. Ovoj
metod ovozmo`uva polesno prilagoduvawe na hromatografskite parametri na razli~ni HPLC instrumen-
ti so cel zadovoluvawe na kriteriumite za soodvetnost na sistemot. 
Macedonian pharmaceutical bulletin 53 (1,2) 201-202 (2007)
PP - 94
202
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Optimizing gradient elution by contolling the dwell volume
Z. Kitanovski, J. Petrusevska, N. Cukic, S. Gjoseva, L. Ugrinova, A. Dimitrovska
Center for drug quality control, Faculty of Pharmacy, 
The "St. Cyril and Methodius" University, Vodnjanska 17, 1000 Skopje, Macedonia
Dwell volume is the system volume from the point at which the mobile phase solvents are mixed until they
reach the head of the column. It is simply the time delay for a gradient front to reach the top of the LC column. Each
LC system has its own dwell volume and will affect the separation results. The gradient dwell volume of the system
is the main instrumental factor complicating the method transfer and limiting rapid high resolution in gradient LC.
To avoid these problems and to make possible precise prediction of the gradient elution data by calculation, the gra-
dient dwell volume should be taken into account during method development and appropriate calculations should
be adopted for the instrumental gradient delay. 
The simplest way to cope with dwell volume differences between LC systems is to develop the method with
sufficient resolution that it will tolerate the changes encountered on systems of different dwell volume. Another approach
that is used for compensation of differences in dwell volume, such that the chromatogram is unchanged when a dif-
ferent system is used, is to develop methods with maximum dwell volume. In this case, just an increase in the dwell
volume of the development system would match the maximum dwell volume and then development of the method
will be by usual manner. And the third approach to addressing dwell volume differences is to set the dwell volume to
zero for all methods. Of course this cannot be done from a plumbing standpoint, but many, if not most, new LC sys-
tems have a feature that allows sample injecting after gradient start. If this approach is taken, sufficient time for equi-
libration of the column has to be allowed before the next injection occurs, because the timing will be a bit different
than normal. In this work, different options of solving the dwell volume problems during the method development
and transfer between different systems are presented through experimental work and calculation examples.
The practical impact of the system dwell volume on retention and resolution is not something to take light-
ly. By measuring the dwell volume for each LC system and planning the use of gradient methods on different sys-
tems future problems in method transferring would be minimized. 
Macedonian pharmaceutical bulletin 53 (1,2) 203-204 (2007)
PP - 95
203
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Optimizirawe na gradientnoto eluirawe preku kontrola 
na zadocnetiot volumen
Z. Kitanovski, J. Petru{evska, N. ^uki}, S. \o{eva, L. Ugrinova, A. Dimitrovska
Centar za ispituvawe i kontrola na lekovi, Farmacevtski fakultet, 
Univerzitet Sv. Kiril i Metodij,Vodwanska 17, 1000 Skopje, Makedonija
Zadocnet volumen e volumenot vo sistemot za te~na hromatografija (LC sistem) od mestoto na
me{awe na rastvoruva~ite na mobilnata faza do po~etokot na kolonata. Poednostaveno, izrazeno preku
vreme, toa e vremeto za koe e potrebno frontot na gradientot da stigne do kolonata. Sekoj sistem za te~na
hromatografija ima sopstven zadocnet volumen {to mo`e da vlijae vrz razdeluvaweto. Zadocnetiot vol-
umen na eden LC sistem e glavniot instrumentalen faktor {to go ote`nuva prenesuvaweto na metodite
me|u sistemite i go ograni~uva brzoto efikasno razdeluvawe vo gradientnata hromatografija. Za da se
izbegnat problemite i da se ovozmo`i to~no presmetkovno predviduvawe na vrednosta na parametrite na
gradientnoto eluirawe, potrebno e zadocnetiot volumen da se zeme v predvid pri razvojot na metodite i
da se usvojat na~ini na presmetuvawe na zadocnetiot volumen kaj sistemite za te~na hromatografija.
Najednostaven na~in na spravuvawe so razlikite vo zadocnetite volumeni me|u razli~ni LC sis-
temi e razvoj na metod so dovolno golema rezolucija. Na toj na~in se kompenziraat razlikite me|u sis-
temite so razli~ni zadocneti volumeni. Drug pristap za dobivawe hromatogram {to nema da se izmeni
pri prenos na metod na razli~ni sistemi e razvoj na metodi so maksimalen zadocnet volumen. Na ovoj na~in,
maksimalniot zadocnet volumen }e se postigne so zgolemuvawe na zadocnetiot volumen na sistemot na koj
se razviva metodot, a potoa metodot }e se razvie na voobi~aen na~in. I tretiot pristap koj se odnesuva
na razlikite vo zadocnetite volumeni, e doteruvawe na nivnata vrednost na nula za site metodi. Ova mo`e
da se postigne imaj}i go predvid faktot deka mnogu, ako ne i najgolem broj od novite LC sistemi nudat
mo`nost za injektirawe na primerokot po zapo~nuvawe na gradientot. Ako se primeni ovoj pristap na
spravuvawe so razlikite vo zadocnetite volumeni, potrebno e da se ovozmo`i podolgo vreme za ekvili-
bracija na kolonata pred po~etokot na sekoe hromatografsko razdeluvawe. Vo ovoj trud, preku eksperi-
mentalno presmetkovni primeri, prika`ani se mo`ni   na~ini na re{avawe na problemite so zadocne-
tiot volumen pri razvoj i prenesuvawe na metodite na razli~ni sistemi.
Vlijanieto na zadocnetiot volumen na retencijata i rezolucijata vo praksa ne e za zanemaruvawe.
Preku mereweto na zadocnetiot volumen za sekoj LC sistem i planiranata upotreba na gradientnite meto-
di na razli~ni sistemi bi se namalile problemite pri prenesuvaweto na metodite na razli~ni sistemi.
Macedonian pharmaceutical bulletin 53 (1,2) 203-204 (2007)
PP - 95
204
^ETVRTI KONGRES NA FARMACIJATA NA MAKEDONIJA SO ME\UNARODNO U^ESTVO
FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION