Elsevier

Journal of Chromatography B

Volume 863, Issue 1, 15 February 2008, Pages 177-180
Journal of Chromatography B

Short communication
Determination of lamotrigine in whole blood with on line solid phase extraction

https://doi.org/10.1016/j.jchromb.2007.12.020Get rights and content

Abstract

A simple, sensitive and reproducible method was developed for the determination of lamotrigine in whole blood with on-line solid phase extraction followed by HPLC separation with UV detection. Whole blood samples were diluted 1:1 with water and then injected directly on a clean-up column dry-packed with 40 μm C8 silica and separated on a C18 reversed-phase column (150 × 4.6 mm) at room temperature. The extraction column was activated with methanol and conditioned with phosphate buffer of pH 4.5. Mobile phases consisted of phosphate buffer of pH 4.5 for the extraction column and of phosphate buffer of pH 4.5 – acetonitrile (60:40, v/v) for the analytical column. At a flow rate of 1.0 ml/min and a connection time of 1.0 min, the complete cycle time was 10.0 min. Detection was carried out at 260 nm. No internal standard was necessary. The method was linear over concentration range 0.2–20.0 μg/ml for lamotrigine. Recovery was 98%. Within-day and between-day coefficients of variation ranged from 1.8 to 6.7%.

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  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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