Journal of Chromatography B, 830 (2006) 368–371 Short communication Development and validation of a sensitive assay of valproic acid in human plasma by high-performance liquid chromatography without prior derivatization Hossein Amini a,∗ , Mohammad Javan b , Abolhassan Ahmadiani a a Department of Pharmacology, Neuroscience Research Center, Shaheed Beheshti University of Medical Sciences, P.O. Box 19835-355, Tehran, Iran b Department of Physiology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran Received 11 September 2005; accepted 11 November 2005 Available online 1 December 2005 Abstract Sensitive and selective determination of valproic acid in plasma by high-performance liquid chromatography (HPLC) is usually achieved with pre-column derivatization. In the present work, the derivatization is omitted due to using a simple but highly selective plasma extraction procedure and an optimized chromatographic condition. Valproic acid and the internal standard octanoic acid were extracted from plasma samples with n-hexane under acidic condition followed by back-extraction into diluted triethylamine. Chromatography was performed on a CN column (250 × 4.6 mm, 5 m) under isocratic elution with acetonitrile–40 mM aqueous sodium dihydrogen phosphate (30:70, v/v), pH 3.5. Detection was made at 210 nm and analyses were run at a flow-rate of 1 ml/min. The method was specific and sensitive with a quantification limit of 1.25 g/ml and a detection limit of 0.1 g/ml in plasma. The mean absolute recovery for valproic acid using the present plasma extraction procedure was 75.8%. The intra- and inter-day coefficient of variation and percent error values of the assay method were all in acceptable range. Calibration curves were linear (r > 0.999) from 1.25 to 320 g/ml in plasma. © 2005 Elsevier B.V. All rights reserved. Keyword: Valproic acid 1. Introduction Valproic acid (2-propylpentanoic acid) is a simple eight- carbon branched-chain fatty acid with unique anticonvulsant properties against several types of epileptic seizures [1]. Due to the widespread usage of valproic acid as an antiepileptic drug, there is a constant need for simple and specific meth- ods for the determination of this substance in patients’ plasma. Since valproic acid is volatile, it is mostly determined by gas chromatography (GC) without derivatization or following alky- lation [2]. High-performance liquid chromatography (HPLC) is an attractive alternative to GC for the analysis of most drug, however, its application for the analysis of valproic acid in plasma is limited by the fact that the drug has no suitable chro- mophore. The very non-specific absorption, where substantial ∗ Corresponding author. Fax: +98 21 22403154. E-mail address: hamini@sbmu.ac.ir (H. Amini). UV absorption occurs, makes it difficult to develop a specific, selective and sensitive HPLC–UV method, particularly when using complex matrices such as biological fluids. Only a few HPLC methods with UV detection at 210 nm are available for valproic acid [3,4] based on deproteination with acetonitrile, which in turn leads to decreased sensitivity and selectivity. It could be suggested that a more specific extraction of valproic acid from plasma would improve HPLC determination of the drug. There are existing HPLC methods that offer sufficient sen- sitivity and selectivity but require prior derivatization of valproic acid to add either a chromophore or a fluorophore [5–10]. Chro- matography of valproic acid without prior derivatization would significantly simplify the method and thus shorten the analysis time. This paper describes a simple, rapid and efficient (in term of recovery and removal of interferences) liquid–liquid extraction procedure for valproic acid from plasma. The method allows determination of valproic acid at low concentrations without the need for prior derivatization. 1570-0232/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2005.11.028