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Simple determination of riluzole in rat brain by high-performance liquid chromatography and spectrophotometric detection

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Abstract

A simple method was developed for separation and quantification of riluzole in rat brain. The analyses were performed by high-performance liquid chromatography using a C18 reversed-phase column (Hypersil ODS) with UV detection at 264 nm. The mobile phase consisted of methanol–water containing 1% triethylamine adjusted with orthophosphoric acid to pH 3.2. The retention time was 8.6 min. A simple liquid–liquid extraction with ethyl acetate was used to obtain riluzole from brain samples. The limit of quantification was 10 ng/g. The recovery was about 80%. The relationship between peak areas and concentrations was linear over the range between 0.01 and 0.8 μg/g, with r2 value over 0.99. The assay provided good reproducibility and accuracy and proved to be suitable for pharmacokinetic studies of riluzole.

Introduction

Riluzole (Fig. 1), 2-amino-6-trifluoromethoxybenzothiazole, is an anti-glutamatergic agent, which was found to be protective in several models of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) [1], multiple sclerosis (MS) [2], Parkinson's disease (PD) [3], and ischemia [4]. Riluzole was demonstrated to modulate the anti-glutamatergic activity through glutamate and sodium receptors. Several studies showed that riluzole inhibits the release of glutamate and l-aspartate from nerve terminals, modulates the N-methyl-d-aspartate (NMDA) ionotropic receptors and stabilizes the voltage-dependent sodium channels in myelinated fibers [5]. Due to its interesting pharmacology properties and potential therapeutic applications a new and easy HPLC method for the determination of riluzole in rat brain could be useful to support pharmacokinetic studies. Previous studies described methods for determination of riluzole in human plasma and urine by high-performance liquid chromatography with ultraviolet detection [6] or coupled with tandem mass spectrometry [7], which were unsuitable to determine riluzole in rat brain. Pharmacokinetics studies of riluzole done in rat and monkeys used a radiolabeled assay [8]. However, this method of quantification is not very convenient for routine analysis. A recent method [9] based on high-performance liquid chromatography with ultraviolet detection has been described for riluzole. The method used a sample preparation procedure based on solid-phase extraction and a relatively long analysis time by HPLC. Therefore, the aim of this study was to develop and validate a rapid and sensitive method for the determination of riluzole in rat brain, with a simple liquid–liquid extraction, which could be easily applicable routinely.

Section snippets

Chemicals

Riluzole was obtained from Sigma (Milan, Italy). All solvents and chemicals were of HPLC or analytical grade. Methanol and ethyl acetate were obtained from Merck (Milan, Italy). Triethylamine, 85% orthophosphoric acid, and perchloric acid were purchased from Aldrich (Milan, Italy).

Instrumentation

The pH was determined using a Metrohm 691 pH meter (Metrohm Ltd., Herisau, Switzerland). The sample preparation procedure utilized a Heidolph REAX 2000 vortex mixer, a Nüve NF 800 R centrifuge with RA 200 Swing Out

In vivo rat study

In order to demonstrated the suitability of our newly developed riluzole HPLC assay in brain for pharmacokinetic studies, we performed a pilot study in the rat. A solution of riluzole (5 mg/kg) was injected intraperitoneally in the rat. Brain samples were collected 8, 16, and 30 h post-dose and stored at −20 °C until analysis by HPLC.

Results and discussion

This study was performed to determine the optimal conditions for extraction of riluzole from rat brain and for chromatographic separation.

Under the chromatographic conditions described in our study, retention time of 8.6 min for riluzole was observed. No interfering peaks were observed in the blank brain chromatogram, even at LOQ value of riluzole as shown in Fig. 2, indicating the efficient clean up method used. Representative chromatograms for riluzole in rat brain are shown in Fig. 3. Good

Conclusions

A specific HPLC method using spectrophotometric detection was developed and fully validated for the determination of riluzole in rat brain. The assay is rapid, selective and accurate. Furthermore, the extraction procedure is simple, allowing sufficient sample throughput to be applied to pharmacokinetic studies.

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