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A HPLC method with automated column switching and UV-diode array detection is described for the simultaneous determination of Vitamin D3 and 25-hydroxyvitamin D3 (25-OH-D3) in a sample of human plasma. The system uses a BioTrap precolumn for the on-line sample cleanup. A sample of 1 ml of human plasma was treated with 2 ml of a mixture of ethanol–acetonitrile (2:1 (v/v)). Following centrifugation, the supernatant was evaporated to dryness under a stream of dry and pure nitrogen. The residue was reconstituted in 250 μL of a solution of methanol 5 mmol l-1 phosphate buffer, pH 6.5 (4:1 (v/v)), and a 200 μl aliquot of this solution was injected onto the BioTrap precolumn. After washing during 5 min with a mobile phase constituted by a solution of 6% acetonitrile in 5 mmol l-1 phosphate buffer, pH 6.5 (extraction mobile phase), the retained analytes were then transferred to the analytical column in the backflush mode. The analytical separation was then performed by reverse-phase chromatography in the gradient elution mode with the solvents A and B (Solvent A: acetonitrile–phosphate buffer 5 mmol l-1, pH 6.5; 20:80 (v/v); solvent B: methanol–acetonitrile–tetrahydrofuran, 65:20:15 (v/v)). The compounds of interest were detected at 265 nm. The method was linear in the range 3.0–32.0 ng ml-1 with a limit of quantification of 3.0 ng ml-1. Quantitative recoveries from spiked plasma samples were between 91.0 and 98.0%. In all cases, the coefficient of variation (CV) of the intra-day and inter-day-assay precision was ≤2.80%. The proposed method permitted the simultaneous determination of Vitamin D3 and 25-OH-D3 in 16 min, with an adequate precision and sensitivity. However, the overlap of the sample cleanup step with the analysis increases the sampling frequency to five samples h-1. The method was successfully applied for the determination of Vitamin D3 and 25-OH-D3 in plasma from 46 female volunteers, ranging from 50 to 94 years old. Vitamin D3 and 25-OH-D3 concentrations in plasma were found from 4.30–40.70 ng ml-1 (19.74 ± 9.48 ng ml-1) and 3.1–36.52 ng ml-1 (7.13 ± 7.80 ng ml-1), respectively. These results were in good agreement with data published by other authors. [Copyright &y& Elsevier]

This report describes the determination of paraquat (PQ) in human blood plasma samples by a direct-injection reversed-phase ion-pair chromatographic method. Blood plasma filtrate was injected directly into the LiChrospher® RP-18 alkyl-diol silica (ADS) precolumn integrated in a column switching system using a mixture of 3% 2-propanol and 10 mM sodium octane sulfonate (SOS) in a 0.05 M phosphate buffer (pH 2.8). After washing with this phase, the ADS precolumn was back-flushed with the analytical mobile phase consisting of 40% of methanol and 10 mM SOS in a 0.05 M phosphate buffer (pH 2.8) at a flow rate of 1.0 ml min−1, in order to carry the analyte to a conventional reversed-phase analytical column, where the separation of PQ was achieved and finally detected by UV at 258 nm. The recoveries of PQ from human blood plasma samples ranged between 95.0 and 99.5% at nine different concentrations (from 0.05 to 3.00 μg of PQ ml−1) with coefficients of variation <2.5% (n=3). The precision expressed as relative standard deviation was below 3.5% for between-day and below 4.3% for within-day measurements (n=5). The detection limit (signal-to-noise ratio, S/N>3) was 0.005 μg ml−1 with an injection volume of 200 μl. The proposed method is promising for the identification and quantification of PQ at low concentration levels and is suitable for its analysis in human blood plasma samples from intentional or accidental poisonings cases with a sample throughput of 5 samples per hour. [Copyright &y& Elsevier]

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 C18 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 C18 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. [Copyright &y& Elsevier]