Kniga na apstrakti book of abstrcts

Determination of phosphates and phosphites using reverse phase
HPLC and indirect UV detection
Katerina Brezovska, Zoran Kitanovski, Suzana Trajkovic Jolevska, Aneta Dimitrovska
Center for drug quality control, Faculty of Pharmacy, 
University Ss. Cyril and Methodius,Vodnjanska 17, Skopje, Macedonia
A method based on reverse phase chromatography with indirect UV detection was developed for determi-
nation of phosphates and phosphites as impurities in sodium risedronate. The partitioning characteristics of the ions
are improved by adding the ionic, hydrophobic reagents in the mobile phase, resulting in better retention, efficacy,
selectivity and better resolution. Reverse phase separation of the phosphates and phosphites was achieved by adding
tetrabytilammonium hydroxide in the mobile phase. A substance showing a high UV absorption (potassium hydrogen
phthalate), was added to the mobile phase in order to obtain high background absorption of the mobile phase. Detection
was accomplished using indirect UV detection, wherein the decrease in absorbance observed when an analyte ion
displaces the chromophoric ion in the mobile phase is monitored. The composition of the mobile phase and the pH
were adjusted in order to obtain optimal resolution and optimal peak area of the phosphates and the phosphites for
minimal analysis time.
The sample was injected in an Agilent 1100 HPLC system. Phosphates and phosphites were separated on
Purospher Star RP 18e, 150 x 4,6 mm, 5 
µm, column, using a buffer (pH 8.2) containing 1 mM phthalate and 0,5 mM
tetrabytilammonium hydroxide and 5 % acetonitrile as a mobile phase. UV detection was performed on 248 nm, with
indirect mode of detector. The validation of the method included determination of specificity-selectivity, linearity (in
the concentration range from 5 
µg/ml to 18 µg/ml), limit of detection and quantification, accuracy and precision. 
The results have shown a good separation of phosphates (Rt =3,3) and phosphites (Rt =3,9) and there was
no interference from sodium risedronat. The limit of detection for phosphates was 0,86 
µg/ml and for phosphites
0,76 
µg/ml. The limit of quantification for phosphates was 2,60 µg/ml and for phosphites 2,29 µg/ml. 
Validation results have shown that the method is selective, linear, accurate and precise, and it’s suitable for
determination of phosphates and phosphites as impurities in sodium risedronat.
Macedonian pharmaceutical bulletin 53 (1,2) 205-206 (2007)
PP - 96
205
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Opredeluvawe na fosfati i fosfiti so primena na reverzno
fazen HPLC metod i indirektna UV detekcija
Katerina Brezovska, Zoran Kitanovski, Suzana Trajkovi} Jolevska, Aneta Dimitrovska
Centar za ispituvawe i kontrola na lekovi, Farmacevtski fakultet, 
Univerzitet Sv. Kiril i Metodij, Vodwanska 17, Skopje, Makedonija
Razvien e metod za opredeluvawe na koncentracija na fosfati i fosfiti kako one~istuvawa vo
natrium risedronat, koj se bazira na reverzno-fazna hromatografija i indirektna UV detekcija.
So dodavawe na jonski, hidrofobni reagensi vo mobilnata faza, mo`e da se podobrat particionite
karakteristiki na jonite, {to rezultira so podobra retencija, efikasnost i selektivnost i podobra
rezolucija. Razdeluvaweto na fosfatite i fosfitite so reverzno fazna hromatografija e postignato so
dodavawe na tetrabutil amonium hidroksid vo mobilnata faza. Na mobilnata faza i se dodava i supstan-
cija (kalium hidrogen ftalat) koja poka`uva visoka absorptivnost vo UV podra~je, so cel da se dobie viso-
ka apsorpcija na mobilnata faza. Detekcijata e postignata so primena na indirektna UV absorpcija, preku
merewe na namaluvaweto na absorpcijata {to se zabele`uva koga analitot go zamenuva hromoforniot jon
od mobilnata faza. Sostavot na mobilnata faza i pH bea prilagodeni so cel da se da se dobie optimalna
rezolucija i optimalna povr{ina na pikovite na fosfati i fosfiti za minimalno vreme na analiza.
Primerocite za analiza bea injektirani vo Agilent 1100 HPLC sistem. Fosfatite i fosfitite bea
razdeleni so primena na Purospher Star RP 18e 150 x 4,6 mm, 5 mm, kolona, i smesa od pufer pH 8,2 (1 mM kali-
um hidrogen ftalat i 0,5 mM tetrabutilamonium hidroksid) i acetonitril vo odnos 95:5, kako mobilna
faza. Detekcijata be{e izvr{ena preku indirektna UV apsorpcija na 248 nm. Metodot be{e validiran
preku opredeluvawe na selektivnost-specifi~nost, linearnost (vo opseg na koncentracija od 5 
µg/ml do
18 
µg/ml), limit na detekcija i kvantifikacija, to~nost i preciznost.
Rezultatite dobieni so primena na predlo`enite hromatografski uslovi poka`aa dobro razdelu-
vawe na fosfatite (Rt =3,3) i fosfitite (Rt = 3,9), bez interferencija od natrium risedronatot. Limitot
na detekcija na fosfati iznesuva 0,86 
µg/ml, a na fosfiti iznesuva 0,76 µg/ml. Limitot na kvantifikaci-
ja na fosfati iznesuva 2,60 mg/ml, a na fosfiti iznesuva 2,29 
µg/ml.
Rezultatite od validacijata na metodot poka`aa deka metodot e selektiven, linearen, to~en i
precizen i mo`e da se primenuva za opredeluvawe na fosfati i fosfiti kako one~istuvawa na natrium
risedronat.
.
Macedonian pharmaceutical bulletin 53 (1,2) 205-206 (2007)
PP - 96
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Control of separation in Ion-Pair Chromatography 
L. Ugrinova, J. Petrusevska, Z. Kitanovski, K. Brezovska, A. Dimitrovska
Center for drug quality control, Faculty of Pharmacy, 
The “St. Cyril and Methodius” University, Vodnjanska 17, 1000 Skopje, Macedonia
Ion-pair chromatography is special type of reverse phase chromatography which is used for separation and
determination of ionic or ionogenic species. Use of ion-pair agents can enhance peak shape and retention time when
common approaches, such as modifying eluent ratios or changing stationary phase, fail to improve resolution of polar
and ionized analytes, especially when they are in large numbers, e.g. impurities of active pharmaceutical ingredient.
Changing one ion-pairing agent with another (which is the same type as the previous one) also changes the chro-
matographic separation. This change may lead to different resolution, inversion of retention and fail in achieving the
system suitability. When using ion-pairing agent that slightly differs in structure with the prescribed ion-pairing agent
in the method, questions arise whether the changed method should or should not be revalidated.
The main advantage of ion-pair chromatography is the high degree of flexibility in adjustment of the chro-
matographic conditions in order to tailor them to a specific separation. Cations and bases are separated with anionic
counter-ions such as pentane or heptane-sulfonic acid or with perchlorate. Anions and acids are usually separated with
a tetra-alkyl-ammonium counter-ion, such as tetra-butyl-ammonium ion. There are few general principles for control-
ling separation that should be followed when developing and transferring an ion pair chromatography method. In order
to achieve the best results, the most important principle is to select an appropriate ion-pair agent and for this matter,
alkyl chain lengths must be taken into consideration. The chain lengths enable selective separation of the analytes.
The longer is the chain, the more hydrophobic is the counter-ion, and therefore, the greater is the retention. Retention
may increase by a factor of almost 20 when going from pentyl to dodecyl alkyl chain. In either case, increase in the
alkyl chain length of the counter-ion increases retention in reverse-phase ion pair chromatography by up to 2.5 times
per added - CH
2
- group in the counter-ion. This is the most common pitfall in transfer of ion pair chromatography
methods, if the different laboratories haven’t got the prescribed ion pair reagent at disposal.
The other principles that must be considered while developing the method with ion pair are not that tricky
and are easy to control in method transfer. These includes: pH value of the mobile phase, the ion pair reagent con-
centration in the mobile phase, buffer concentrations, addition of different organics, such as acetonitrile and methanol
to the mobile phase and the temperature of the LC system. The validation should include robustness of the method
on the influence of several ion pair reagents with different chain lengths. The system suitability criteria must predict
the critical resolutions, k’ and symmetry factor. And very important, during the application of the method, the sys-
tem should be physically stable, i.e. the stationary phase must be preequilibrated with mobile phase. 
If all the above considerations are taken into account during the method development, there should be few
problems in method transfer. Partial revalidation, i.e. selectivity and the system suitability criteria are the sufficient
proof of successful method transfer. 
Macedonian pharmaceutical bulletin 53 (1,2) 207-208 (2007)
PP - 97
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Kontrola na razdeluvawe kaj jon-par hromatografski metodi
L. Ugrinova, J. Petru{evska, Z. Kitanovski, K. Brezovska, A. Dimitrovska
Centar za ispituvawe i kontrola na lekovi, Farmacevtski Fakultet, 
Univerzitet Sv. Kiril i Metodi, Vodwanska 17, 1000 Skopje, Makedonija
Jon-par hromatografija e poseben tip na reverzno fazna hromatografija koja se upotrebuva za
razdeluvawe i odreduvawe na jonski i jonogeni vidovi. Upotrebata na jon-par reagensi mo`e da gi podobri
izgledot na pikot i retencionoto vreme, toga{ koga so voobi~aenite pristapi, kako na primer promena
na odnosot na eluentite ili promena na stacionarnata faza, ne e vozmo`no da se podobri rezolucijata
na polarni i jonizirani analiti, osobeno koga se prisutni vo pogolem broj, kako na primer one~istu-
vawa na aktivna komponenta. Promena na jon-par reagensi (od ist tip) vodi do promena na hromatograf-
skoto razdeluvawe. Ovaa promena mo`e da dovede do namaluvawe na rezolucijata, inverzija na retenci-
ite i nepostignuvawe na soodvetnost na hromatografskiot sistem. Pri prenos na metodi, dokolku se
upotrebuva jon-par reagens koj strukturno se razlikuva od propi{aniot, se postavuvuva pra{aweto dali
promenetiot metod treba da se revalidira ili ne.
Glavnata prednost na jon-par hromatografijata e golemiot stepen na fleksibilnost pri prilago-
duvawe na hromatografskite uslovi so cel najdobra separacija. Katjonite i baznite komponenti se
razdeluvaat so anjonski jon kako {to e pentan ili heptan sulfonska kiselina, ili pak so perhlorat.
Anjoni i kiseli komponenti voobi~aeno se razdvojuvaat so tetraalkil amoniumovi joni, kako {to e tetra-
butilamoniumov jon. Postojat nekolku principi za kontrola na separacijata koi mora da se po~ituvaat
pri razvoj i prenos na jon-par hromatografski metod. Za da se dobijat najdobri rezultati, najva`no e da
se izbere soodveten jon-par reagens i pritoa mora da se zeme predvid dol`inata na alkilniot lanec.
Dol`inata na lanecot e klu~nata varijabla koja go odreduva razdeluvaweto na analitite vo dvata tipa
na jon-par hromatografija. Kolku e podolg lanecot, tolku e pohidrofoben jonot so sprotiven polne` od
analitot, pa so toa i retencijata e pogolema. Retencijata mo`e da se zgolemi i za faktor od skoro 20 pri
promena od pentil na dodecil alkil lanec. Vo sekoj slu~aj, so zgolemuvawe na dol`inata na alkilniot
lanec na jon-par reagensot za edna -CH
2
- grupa, retencionoto vreme se zgolemuva duri i do 2,5 pati. Ova
voedno e naj~estata zamka pri prenos na metodite, dokolku ne e dostapen identi~niot jon-par reagens.
Drugite principi na koi mora da se vnimava pri razvoj na metod so jon-par ne se mnogu prob-
lemati~ni i lesno se kontroliraat pri prenos na metodite. Tuka se vklu~eni: rN vrednost na mobilna-
ta faza, koncentracija na jon-par reagensot vo mobilnata faza, koncentracija na puferskiot sistem,
dodavawe na razli~ni organski rastvoruva~i, kako acetonitril ili metanol vo mobilnata faza i tem-
peraturata na hromatografskiot sistem. Vo validacijata treba da bide vklu~ena robustnosta na metodot,
odredena preku vlijanie na nekolku jon-par reagensi so razli~na dol`ina na alkilnite lanci.
Kriteriumite za soodvetnost na sistemot mora da gi predvidat kriti~nite rezolucii, k’ vrednostite na
soodvetnite pikovi, kako i faktorite na simetrija. Za vreme na aplikaciite, mnogu e va`no sistemot da
bide fizi~ki stabilen, odnosno stacionarnata faza da e pre-ekvilibrirana so mobilnata faza.
Dokolku site prethodni napomeni se zemeni predvid pri razvivaweto na metodot, ne treba da se
o~ekuvaat problemi pri prenos na metodot. Delumnata revalidacija, odnosno selektivnosta i ispol-
netite kriteriumi za soodvetnost na sistemot se doovolen dokaz za uspe{en prenos na metodot. 
Macedonian pharmaceutical bulletin 53 (1,2) 207-208 (2007)
PP - 97
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HPLC method for determination of lamotrigine impuriities 
in Lamal
®
tablets
I. Bozinovska, D. Kafediska, C. Dolikoska, M.Simjanovska, 
B. Samarova-Stoev, B. Sapkareva, H. Babunovska
ALKALOID AD- Skopje, Pharmaceutical, Chemical and Cosmetics Company,
Aleksandar Makedonski br.12;1000 Skopje, Republic of Macedonia
Lamotrigine is a drug of the phenyltriazine class, chemically unrelated to existing antiepileptic drugs. It is rec-
ognized as an anticonvulsant drug used in the treatment of epilepsy (Lennox-Gastaut syndrome) and bipolar disorder.
It is the first medication since lithium granted FDA approval for the maintenance treatment of bipolar type I. It’s avail-
able in four dosage forms, as Lamal tablets from 25mg, 50mg, 100mg and 200mg, approved for adults and children.
Our work was to develop and validate reliable and sensitive HPLC method using UV detection to determine
Lamotrigine related compounds. The optimal chromatographic separation has been achieved using Inertsil ODS-3 250
x 4,6mm; 5
µm column, with a mobile phase of 0.05M NH
4
(CH
3
COO) (mobile fase A) and a mixture of methanol and
acetonitril (97.5 : 2.5) (mobile fase B), using gradient mode. Detection was at 225 nm and total run time of 35 minutes.
This conditions allowed the complete separation of:
1)Impurities due to the presence of un-reacted starting materials: Schiff base (RRT 2.04);
2)Impurities formed during synthesis: Impurity A (RRT 1.15); Impurity B (RRT 2.34);
3)Impurities due to the degradation of the main product: Acid impurity (RRT 0.74);
According ICH guideline Q2(R1), the HPLC method has demonstrated to be specific for determination of
impurities, linear within reasonably wide range of concentration, accurate, precise and robust.
Validation of the HPLC method was performed with concentration of 0.002mg/ml of the Lamotrigine stan-
dard solution, as a target concentration, corresponding to 0.1% of the Lamotrigine test solution used in the determi-
nation of chromatographic purity.
To demonstrate specificity,a mixed standard of Lamotrigine and Impurity A was prepared and the achieved
resolution was 3.34. Furthermore, specificity was established by demonstrating that there is no interference between
peak of interest and peaks from diluent and placebo.
Linear correlations were obtained between the response of Lamotrigine peak related to the concentrations
of standards over the range of 0.0008-0.0032mg/ml (20
µl injected) and correlation coefficient of 0.99918 was
obtained.The accuracy expressed as a percent of recovery was good in all cases, ranging in the predetermined accept-
ance criteria (90.0% - 110.0%), as well as RSD values less than 5%.
The precision expressed as RSD was 1.84%.Quantitation limit is 0.26 g/ml. Detection limit is 0.09 g/ml.
The validated method was applied for impurities determination in commercially available tablets of Lamo-
trigine in routine quality control as well as for stability studies.
Macedonian pharmaceutical bulletin 53 (1,2) 209-210 (2007)
PP - 98
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Odreduvawe na srodni i degradacioni produkti na Lamotrigin 
vo Lamal
®
tableti so HPCL metoda
I. Bo`inovska, D. Kafexiska, C. Doli}oska, M. Simjanovska, B. Samarova- Stoev, 
B. [apkareva, H. Babunovska
ALKALOID AD-Skopje, Farmacevtska, Hemiska i Kozmeti~ka Industrija,
Aleksandar Makedonski br. 12, 1000 Skopje, Republika Makedonija
Lamotrigine e lek od Feniltriazinskata grupa na lekovi i po svojata hemiska struktura se razliku-
va od ostanatite antiepilepti~ni lekovi. Se upotrebuva kako antikonvulziven lek vo tretman na epilep-
sija (Lennnox-Gastaut-ov sindrom) i vo lekuvawe na bipolarni naru{uvawa. Pretstavuva prv lek posle
Litiumot, zdobien so FDA odobruvawe za odr`uvawe i tretman na bipolarni naru{uvawa od tip I. Dosta-
pen e vo ~etiri doza`ni formi kako Lamal tableti od 25 mg, 50 mg, 100 mg i 200 mg, odobreni za upotreba
i kaj vozrasni i kaj deca.
Na{a zada~a be{e da razvieme i validirame soodvetna verodostojna i senzitivna HPLC metoda
koristej}i UV detekcija za odreduvawe na srodnite i degradacioni produkti na Lamotriginot. Optimalno
hromatografsko razdeluvawe se postignuva so koristewe na Inertsil ODS-3 kolona 250 x 4,6mm; 5mm; kako
mobilna faza: 0.05M NH
4
(CH
3
COO)(mobilna faza A) i me{avina od metanol i acetonitril (97.5:2.5 )(mobil-
na faza V) koristej}i gradient. Detekcijata e na 225 nm a vremetraeweto na ranot e 35 minuti. 
Vakvite hromatografski uslovi ovozmo`uvaat celosno razdvojuvawe na :
1) One~istuvawa koi se dol`at na prisustvo na neizreagirani po~etni supstancii: [ifova baza
(RRT 2.04)
2) One~istuvawa sozdadeni pri tekot na sintezata : One~istuvawe A (RRT 1.15); One~istuvawe B
(RRT 2.34);
3) One~istuvawa nastanati kako rezultat na degradacija na glavniot proiavod: Kiselo one~istu-
vawe (RRT okolu 0.74).
Soglasno ICH vodi~ot Q2(R1), HPLC metodot se doka`a deka e specifi~en za odreduvawe na
one~istuvawata, linearen vo ramkite na {irok opseg na koncentracii, to~en, precizen i robusten.
Validacijata na ovoj HPLC metod e izvedena so standarden rastvor na Lamotrigin so koncentraci-
ja od 0.002 mg/ml kako celna koncentracija, na koja odgovara 0.1 % Lamotrigin test rastvorot, koj se
koristi za determinacija na hromatografskata ~istota. 
Za da se prika`e specifi~nosta, podgotven e me{an standarden rastvor od Lamotrigin i One~istu-
vawe A i pritoa e dobiena rezolucija od 3.34. Ponatamu, doka`ano e deka ne postoi interferencija pome|u
pikot od interes i pikovite od placeboto i rastvoruva~ot.
Linearen soodnos e postignat pome|u odgovorot od pikot na Lamotrigin vo odnos na koncentracii
na standardot vo koncentracionen opseg od 0.0008-0.0032 mg/ml (injektirawe 20 
µl), i postignat e korela-
cionen koeficient od 0.99918. To~nosta, izrazena kako percent recovery dade dobri rezultati vo site
slu~ai, vo granicite na prethodno utvrdeniot prifatliv kriterium (90.0-110.0%), kako i RSD vrednosti
pomali od 5%. Preciznosta e doka`ana so toa {to e dobiena RSD vrednost od 1.84 %. Limitot na kvan-
tifikacija e 0.26 g/
µl. Limitot na detekcija e 0.09 g/µl.
Validiranata metoda e primeneta za odreduvawe na one~istuvawa vo komercijalno dostapnite
tableti Lamal, kako vo redovnata kontrola na kvalitet, taka i vo ispituvawata pri sledewe na stabil-
nosta na lekot.
Macedonian pharmaceutical bulletin 53 (1,2) 209-210 (2007)
PP - 98
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Forced degradation study performed on Lisinopril dihydrate active
pharmaceutical ingredient
K. Brzilova, H. Babunovska, V. Dubrova-Koceva, S. Janeva, D. Damcevska, B. Debarlieva, 
F. Butikoski, T. Kovacevik-Novakova
A.D. Alkaloid, Department of Quality Control, Bul. Aleksandar Makedonski br .12,1000 Skopje, Macedonia
Lisinopril dihydrate is active pharmaceutical ingredient which belongs in the group of angiotensin convert-
ing enzyme inhibitors.It is active pharmaceutical ingredient in Skopryl tablets, product of Alkaloid-Skopje, which
are primarily used in treatment of hypertension,congestive heart failure and heart attacks.
The goal of this testing was determination of Lisinopril dihydrate degradation products formed under the
influence of several stress conditions:heat degradation(105
°C);VIS degradation(450-650nm); UV degradation (254nm)
;acid hydrolysis(1N HCl);base hydrolysis(1N NaOH) and oxidation(2% H
2
O
2
).
In determination of degradation products HPLC method was used. This HPLC method includes: LiChrospher
100 RP-8 250mmx4mm,5µm column;wavelength of 210nm;temperature of 50
0
C;flow rate of 1.0ml/min;injection
volume of 20µl and 100µl;mobile phase A:mixture of 30volumes acetonitrile R and 970volumes of 3.12g/l sodium
dihydrogen phosphate R solution adjusted to pH=5.0 with 50g/L solution of sodium hydroxide R; mobile phase
B:mixture of 200ml acetonitrile R and 800ml of 3.12g/l sodium dihydrogen phosphate R solution adjusted to pH=5.0
with 50g/L solution of sodium hydroxide R; percentage of mobile phase changes with linear gradient.
With this study it was determined that:
• Lisinopril dihydrate active substance undergoes degradation influenced by temperature of 105
0
C. Known
impurity A and unknown impurity are formed. Major detected peak is unknown impurity with RRT 4.08;
• Lisinopril dihydrate active substance is not influenced by VIS light at 450-650nm (degradation products
are not formed);
• Lisinopril dihydrate active substance is influenced by the UV light at 254nm. Known impurity A and impu-
rity D, and unknown impurities are formed. Major detected peak is known impurity A;
• Solution of Lisinopril dihydrate in 1N HCl is stable (degradation products are not formed);
• Solution of Lisinopril dihydrate in 1N NaOH is stable (degradation products are not formed);
• Solution of Lisinopril dihydrate in 2% H
2
O
2
is not stable. Known impurity A and impurity D,and unknown
impurities are formed.Major detected peak is known impurity A.
It was concluded that described HPLC method has shown good selectivity and that is why it could be used
in determination of potential known and unknown impurities which might be formed during the degradation process
of active pharmaceutical ingredient Lisinopril dihydrate.
Macedonian pharmaceutical bulletin 53 (1,2) 211-212 (2007)
PP - 99
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