Short communicationDetermination of lamotrigine in whole blood with on line solid phase extraction
Introduction
Lamotrigine is an anticonvulsant drug used in the treatment of partial and generalized epilepsy.
Although some analytical methods to estimate lamotrigine content in biological fluids have been reported in the peer-reviewed literature an HPLC method for the determination of lamotrigine in whole blood is still lacking [1], [2], [3], [4]. Determination of drugs in whole blood is often necessary in forensic analysis because of the difficulty in obtaining serum or plasma. HPLC analysis of drugs in complex matrices such as whole blood usually involves time consuming liquid–liquid extraction. Such conventional procedure may involve tedious, time consuming, expensive, and complex steps, and finally even sample loss and contamination problems are not unusual. In the HPLC analysis, the preceding on-line solid phase extraction may solve these problems [5], [6]. The sample is diluted with water or mobile phase to avoid clogging of the column filters and then directly injected onto a primary column which results in a preliminary sample clean-up by SPE. The sample dilution does not reduce the sensitivity, because the sample loop volume does not affect the peak width in a on-line solid phase extraction. After the SPE step, a small fraction of the effluent from the extraction column is selectively transferred to the analytical column for the final separation. Because of minimal sample manipulation no internal standard is necessary. In this paper we describe a rapid, accurate, precise, and inexpensive method to determine lamotrigine in whole blood using an on-line solid phase extraction procedure followed by analysis by HPLC.
Section snippets
Chemicals and reagents
Lamotrigine (P.N. L3791-10MG), Acetonitrile HPLC grade (CHROMASOLV® Plus, P.N. 34998-2.5L), Sodium phosphate monobasic dihydrate (BioChemika Ultra, P.N. 71502-1KG) and phosphoric acid (BioChemika Ultra, P.N. 438081-500ML) were of analytical grade and purchased from Sigma Aldrich (Sigma-Aldrich, St. Louis, MO, USA). The water was reagent grade (18.2 MΩ cm at 25 °C of resistivity) obtained from a Milli-Q system (Millipore, Billerica, Massachusetts, USA).
HPLC instrument
The HPLC system consisted of a Varian Vista
Results and discussion
The method to determine the lamotrigine has been reveled to be rapid, accurate and precise; infact the standard curves obtained for the validation of the method were linear within the range 0.2–20.0 μg/ml with a correlation coefficient of R2 = 0.998 and repeatability ranged between 1.2 and 4.5%, while accuracy was found in the range 94.4–103.4% (Table 1). Finally, between-day repeatability (%RSD n = 10) for a nominal concentration of 5.0 μg/ml of lamotrigine was 6.7%. The optimum connection time was
Conclusion
Several HPLC methods used for the determination of lamotrigine have previously been reported, but none for lamotrigine present in complex matrices like whole blood. The proposed assay is sufficiently easy, specific, accurate, and free from interferences of endogenous components. One of the main advantages of this method is the easy and controlled procedure of sample pretreatment. By using the herein presented solid phase extraction, other time consuming extraction procedures can be avoided.
Acknowledgements
This work was partially supported by Università Politecnica delle Marche. Authors are indebted to M. Glebocki for extensive editing of the manuscript.
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