LC-MS/MS METHOD FOR THE DETERMINATION OF LEVETIRACETAM
IN RAT BRAIN MICRODIALYSATE
D. Dogrukol-Aka, E. Senera, O. Tansel Korkmazb, Sukru Torunc, Lutfi Gencd and Nese Tuncelb
a
Anadolu University, Faculty of Pharmacy, Department of Analytical Chemistry, 26470 Eskişehir, Türkiye
b
Osmangazi University, Faculty of Medicine, Department of Physiology, 26480 Eskişehir, Türkiye
c
Anadolu University, Faculty of Health Science, 26470 Eskişehir, Türkiye
d
Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 26470 Eskişehir, Türkiye
This is the first study to develope and describe
a simple and rapid method of liquid
chromatography-electrosprey tandem mass
spectrometry
(LC-MS/MS)
for
the
determination of levetiracetam in rat brain
microdialysate samples.
INTRODUCTION
Levetiracetam (LVT) is a new antiepileptic drug used in the
treatment of partial onset seizure in patient. For the
determination of LVT in human plasma a few methods
based on high performance liquid chromatography (HPLC)
with UV detection, micro-emulsion electrokinetic
chromatography with UV detection, gas chromatography
(GC) or in combination with mass spectrometry (GC/MS)
have been described in recent years. Less sensitive LCMS/MS methods were also reported for the determination
of LVT in plasma1. The objective of this study is to develop
and validate a high throughput LC-MS/MS method for
accurate and more sensitive measurement of LVT in
biological fluids especially rat brain microdialysate
samples.
RESULTS AND DISCUSSION
LVT was investigated generated the prominent protonated
molecular ion [M+H]+ in positive ion mode. It was
successfully separated at 3.4 min and monitored MRM
transition of m/z 126>69.2. The method was linear over the
range of 0.139-1390 ng/mL with the correlation
coefficients greater than 0.998 (r2) for three separate
batches in Ringer’s solution. Precision and accuracy were
determined by measuring the concentration of LVT in
Ringer’s solution in six replicates of quality control
standards at three different concentrations (1.39, 139 and
1390 ng/mL) for three separate batches. Intra-day precision
was <2%, and calculated recovery was >93% in Ringer’s
solution. For the inter-day studies precision was <4%, and
calculated recovery was >96% in the Ringer’s solution. To
demonstrate the applicability of the method, rat brain
microdialysate samples were directly analyzed after
administration an i.p. dose of LVT and collection of
samples was performed at the different time points over 12
hours (Fig. 1-2).
MATERIALS AND METHODS
Fig. 1. MRM chromatograms of Ringer’s solution (top),
LVT in microdialysate sample at 2h time point (middle)
and QC sample (100 ng/mL LVT) (bottom)
30000
Concentration (ng/mL)
Chemicals and reagents: LVT was purchased from SigmaAldrich Chemical Co. (St. Louis, MO, USA). Water was
purified using Milli-Q water device (Millipore, Bedford,
MA, USA). All other chemicals and reagents were of
analytical grade and solvents were of HPLC grade.
Equipment: Agilent 1290 series LC system consisted of a
solvent delivery system with binary pump, an autosampler,
and column oven (Agilent Technologies, USA). The
separation of LVT was successfully performed on Supelco
Ascentis Express (100 mm x 2.1 mm, 2.7 µm) with
programmed gradient elution consisting of 10 mM
ammonium formate in water (A) and in 10 ammonium
formate in 90 % (v/v) acetonitrile (B) as mobile phase
components and delivered at a flow rate of 0.3 ml/min to a
triple-quadruple mass spectrometer equipped with a jet
stream electrosprey ionization source (6460 Trip-Quad,
Agilent Technologies, USA). Injection volume was 2 µl.
Data was analysed by using Mass Hunter programme.
Microdialysis samples:Wistar rats were used to obtain
brain microdialysate samples in the region of hippocampus
by using a specific prob (PAES 3 mm membrane length
with cut of 20000 D, CMA). LVT was administrated to rats
at a LVT dose of 50 mg/kg by i.p.2.
25000
20000
15000
10000
5000
0
0
200
400
Time (min)
600
800
Fig. 2. The concentration time profiles in rat brain tissue
after administration of 50 mg/kg of LVT to individual rats
(n=5)
CONCLUSIONS
Rapid LC-MS/MS method for the quantitation of LVT in
rat brain microdialysate samples was developed and
validated. It was successfully applied to the analysis of rat
brain microdialysate samples obtained from brain
hippocampal region after i.p. LVT application and it was
showed the concentration time profile of LVT in rat brain.
ACKNOWLEDGMENTS
This work was supported by the Scientific Research
Projects Commission of Anadolu University (Project No
1301S009). The authors acknowledge the instrumental
support of Anadolu University Medicinal Plants and
Medicine Research Center (AUBIBAM). All animal
experiments were performed in accordance with the
principles of animal use and care approved by the ethical
committee of the Medical Faculty of Osmangazi University
(Approval File No. 04/2007)
REFERENCES
1.
Gou, T., Oswald L.M., Mendu D.R., Soldin S.J.,
Determination
of
levetiracetam
in
human
plasma/serum/saliva
by
liquid
chromatographyelectrosprey tandem mass spectrometry. Clinica Chimica
Acta, 2007, 375, 115-118.
2.
Clinckers R., Smolders I., Meurs A., Ebinger G.,
Michotte Y., Quantitative in vivo microdialysis study on
the influence of multidrug transporters on the blood-brain
barrier passage of oxcarbazepine: Concomitant use of
hippocampal monoamines as pharmacodynamic markers
for the anticonvulsant activity. Journal of Pharmacology
and Experimental Therapeutics, 2005, 314, 725-731.