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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]

Abstract: A method for the determination of cocaine (COC) and benzoylecgonine (BZE) in human urine using a column-switching liquid chromatography system is reported. A homemade precolumn (20mm×4.6mm i.d.) dry-packed with Alltech ODS-C18 (35–750μm) was employed as an extraction precolumn in order to extract and concentrate the COC and BZE from the human urine sample. The analytes were continuously transferred to the analytical column (Spherisorb-C8, 250mm×4.6mm i.d.; dp=5μm) by means of the switching arrangement in the backflush mode. Detection was carried out at 235nm in a UV-diode array detector. The validation of the method revealed analytes quantitative recoveries (96–102%) at three concentrations in the range from 0.25 to 4.00 and from 0.5 to 12.0μg/mL for COC and BZE, respectively. These values demonstrate the excellent extraction efficiency of the precolumn. The detection limits for COC and BZE at a signal-to-noise ratio of 3 were 0.08 and 0.15μg/mL when a sample volume of 50μL was injected. The overlap of sample preparation, analysis and recondition of the precolumn increases the sample throughput to four samples per hour. The proposed method has been applied to the determination of COC and BZE in human urine samples from 73 suspecting drug addicts. Urine concentrations of 1.0–118.10μg of BZE/mL and 0.1–41.0μg of COC/mL were found. [Copyright &y& Elsevier]