Automated microanalysis of gabapentin in human serum by high-performance liquid chromatography with fluorometric detection

Abstract

An automated high-performance liquid chromatographic method for the determination of gabapentin, 1-(aminomethyl)cyclohexaneacetic acid, in serum is described. The procedure involves protein precipitation with methanol followed by using a robotized derivatization with o-phthaldialdehyde reagent and automated high-performance liquid chromatography. The analog of gabapentin, 1-(aminomethyl)cycloheptaneacetic acid, was used as the internal standard. Blank serum was fortified with gabapentin (0.1–10.0 μg/ml) and internal standard. Separation was achieved on a Waters 5-μm reversed-phase column (10 cm×4.6 mm) with mobile phase consisting of 0.02 M phosphate buffer (pH 4.5)–acetonitrile (50:50, v/v). Eluents were monitored by fluorescence spectroscopy with excitation and emission wavelengths of 230 and 420 nm, respectively. The calibration curve for gabapentin in serum was linear ( r=0.999) over the concentration range 0.1–10.0 μg/ml. The inter- and intraassay variations for three different gabapentin concentrations were ≤10% throughout. The lower limit of quantitation was found to be 0.1 μg/ml. Chromatography was unaffected by a range of commonly employed antiepileptic drugs or selected amino acids.