Posterske teme Poster topics - Biochemia Medica

P15-1 (UP10-1) Toksikologija i TDM
P15 – Toksikologija i TDM, P15-1 (UP10-1)
Određivanje lamotrigina u plazmi metodom
tekućinske kromatografi je visokog
razlučivanja uz primjenu ultraljubičastog
detektora
Petek-Tarnik I1 , Coce I1 , Petek M1 , Cvitanović-Šojat LJ2 , Kusić Z3 1Endokrinološki laboratorij, Zavod za nuklearnu medicinu i onkologiju,
KB Sestre milosrdnice, Zagreb, Hrvatska
2Klinika za pedijatriju, KB Sestre milosrdnice, Zagreb, Hrvatska
3Zavod za nuklearnu medicinu i onkologiju, KB Sestre milosrdnice,
Zagreb, Hrvatska
Lamotrigin (LTG) je antiepileptik novije generacije djelovanje
kojega se očituje u blokiranju potencijala ulaznih
vrata natrijskih kanala. Lamotrigin suzbija patološko otpuštanje
glutamata i glutamatom izazvano izbijanje akcijskog
potencijala. Terapeutsko praćenje LTG je važno kako
bi se izbjeglo njegovo toksično djelovanje, kao i utjecaj na
ostale antiepileptike. Cilj rada bio je uvesti metodu HPLC
za određivanje LTG te istražiti kakav je odnos između primijenjene
doze LTG i koncentracije u plazmi. Uzorku plazme
(100 µL) dodaje se unutarnji standard (kloramfenikol)
te se LTG ekstrahira dodatkom smjese kloroform:izopropanol
u omjeru 95:5. Nakon odvajanja organski sloj se odpari
do suhog, a LTG otopi u metanolu. Dobiveni ekstrakt
(5 µL) injicira se na sustav HPLC. Odvajanje se provodi na
koloni C18 uz mobilnu fazu sastava: fosfatni pufer, pH 6,0,
acetonitril i metanol u omjeru 70:20:10, pri protoku od 1,0
mL/min. LTG se određuje UV detekcijom pri 214 nm. Vrijeme
zadržavanja LTG na koloni je 4,7 minuta, a unutarnjeg
standarda 7,8 minuta. Preciznost metode ispitana je određivanjem
kontrolnog uzorka plazme kojem su dodavane
poznate koncentracije LTG. Koefi cijenti varijacije kretali su
se između 3,1% i 4,5% za uzastopne analize unutar dana, te
između 2,3% i 6,7% među opetovanim analizama tijekom
nekoliko dana. Iskorištenje ekstrahiranog LTG kao i unutarnjeg
standarda bilo je između 88,1% i 91,7%. Metoda je
primijenjena određivanjem koncentracije LTG u 39 djece
oboljele od epilepsije, od čega ih e 17 bilo na monoterapiji
lamotriginom (4,66 mg/kg), a ostali na kombiniranoj terapiji
LTG (3,92-5,66 mg/kg) i ostalim antiepilepticima (VPA,
CLB, CZP). Prekoračena dozvoljena koncentracija LTG u
plazmi (5 mg/L) pronađena je u 5 bolesnika na monoterapiji
i 11 bolesnika na kombiniranim terapijama. Najviše
vrijednosti LTG nađene su u bolesnika na kombiniranoj
terapiji LTG i VPA. Također je zaključeno kako nema međusobnog
odnosa između primijenjene doze LTG i koncentracije
u plazmi. Metoda HPLC uz UV detektor može se
preporučiti kao metoda izbora za određivanje LTG u plazmi
zbog osjetljivosti, preciznosti i jednostavnosti.
E-mail: petektarnik@net.hr
Biochemia Medica 2006;16(Suppl 1):S1–S268
S162
P15 – Toxicology and TDM, P15-1 (UP10-1)
Toxicology and TDM
Determination of lamotrigine in plasma by
high performance liquid chromatography
with UV detection
Petek-Tarnik I 1 , Coce I 1 , Petek M 1 , Cvitanović-Šojat LJ 2 , Kusić Z 3
1 Laboratory of Endocrinology, Department of Nuclear Medicine and
Oncology, Sestre milosrdnice University Hospital, Zagreb, Croatia
2 University Department of Pediatrics, Sestre milosrdnice University
Hospital, Zagreb, Croatia
3 Department of Nuclear Medicine and Oncology, Sestre milosrdnice
University Hospital, Zagreb, Croatia
Lamotrigine (LTG) is a novel antiepileptic, which has a
phenytoin-like membrane stabilizing mechanism via
blockade of voltage-dependent sodium channels and
inhibition of glutamate release. Therapeutic monitoring
of lamotrigine is useful for patient management and
avoidance of toxicity. The aim of the study was to develop
HPLC method for determination of plasma LTG and
to investigate the relationship between oral dose and
plasma concentration of LTG. To 100 µL of plasma sample
the internal standard (chloramphenicol) was added. The
extraction was performed by a mixture of chloroform
and isopropanol (95:5% v/v) in the presence of phosphate
buff er. After evaporation the residue was reconstituted
with methanol and injected to HPLC system. The separation
was performed on a C18 stainless steel column.
Mobile phase consisted of phosphate buff er pH 6.0, acetonitrile
and methanol (70:20:10 v/v/v), with a fl ow rate of
1 mL/min. The detection was obtained by UV detector at
214 nm.The retention times for LTG and IS were 4,7 and
7,8 minutes, respectively. Within- and between-day precision
was examined by analysis of control plasma sample
with coeffi cients of variation of 3.1%-4.5% and 2.3%-
6.7%, respectively. The absolute recovery of LTG and of
IS ranged from 88.1% to 91.7%. The method was used to
analyze plasma LTG concentrations from 39 children with
epilepsy; 17 samples were obtained from patients receiving
LTG monotherapy (4.66 mg/kg), and the others received
combined therapy with LTG (3.92-5.66 mg/kg) and
other antiepileptics (VPA, CLB, CZP). In 16 plasma samples
LTG level exceeded the reference range of 5 mg/L (5 on
monotherapy and 11 on combined therapy). The highest
plasma LTG levels were found in 4 patients receiving VPA.
There was no relationship between oral dose and plasma
concentration. The HPLC method with UV detection is the
method of choice for the measurement of lamotrigine in
plasma because of its sensitivity, selectivity and simplicity.
E-mail: petektarnik@net.hr