Column-switching high-performance liquid chromatographic analysis of carbamazepine and its principal metabolite in human plasma with direct sample injection using an alkyl-diol silica (ADS) precolumn

• Published in: Talanta (Oxford) 2002-09, Vol.58 (3), p.535-542
• Main Authors: Brunetto, M.R., Obando, M.A., Fernández, A., Gallignani, M., Burguera, J.L., Burguera, M.
• Format: Article
• Language: English
• Subjects: Analysis; Biological and medical sciences; Carbamazepine; Carbamazepine-10,11-epoxide; General pharmacology; Medical sciences; Pharmacology. Drug treatments; Plasma samples; Restricted access packings; Sample preparation
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/S0039-9140(02)00326-0

In this paper, the on-line coupling of solid-phase extraction, based on a restricted-access support with high-performance reverse phase chromatography for the analysis of carbamazepine (CBZ) and carbamazepine-10,11-epoxide (CBZ-E) in human plasma samples is described. A precolumn packed with 25 μm C 18 alkyl-diol support is used for direct plasma injection. Using column-switching techniques, the analytes were enriched on the precolumn by a 5 mM phosphate buffer (pH 7) with 2% of methanol solution at a flow-rate of 0.8 ml min −1, while proteins and endogenous hydrophilic substances in plasma were washed off to waste. The enriched analytes were then back-flushed onto the analytical C 18 column, separated by a mixture of 10 mM phosphate buffer (pH 7) acetonitrile (70:30 v/v) solution at a flow-rate of 1.0 ml min −1 and detected by the ultraviolet absorbance set at 212 and 285 nm and without transfer loss. Linear calibration graphs were obtained for sample injection volumes of 50 (0.2–4.0 of μg of CBZ ml −1 and 0.1–5.0 μg of CBZ-E ml −1, respectively), and 20 μl (5.0–20.0 μg of CBZ ml −1); in either case the r-value was >0.9963. Recoveries from spiked plasma samples were quantitative for both analytes and the coefficients of variation were below 3.83%. The lowest samples concentrations that can be quantified with acceptable accuracy and precision was 0.2 μg CBZ ml −1 and 0.1 μg CBZ-E ml −1 when a sample volume of 50 μl was injected. Concentrations of 0.08 and 0.05 μg ml −1 of CBZ and CBZ-E were considered the limit of detection for a signal-to-noise ratio of 3. Furthermore, the developed column-switching method was successfully applied to the determination of CBZ and CBZ-E in plasma samples of patients submitted to CBZ therapy.