Column-switching chromatographic determination of itraconazole and its metabolite hydroxy-itraconazole in human serum.

A column-switching HPLC assay is described for the determination of the antifungal agent itraconazole and its main metabolite hydroxy-itraconazole in serum samples. Three precolumns packed with alkyl-diol silica sorbents differing in alkyl chain length are compared in the sample clean-up step. Chromatographic separation is achieved with a Symmetry C8 column. The assay presented shows a robust and selective analytical procedure with low requirements for sample quantity, no manual sample treatment and high sample throughput.

Determination of propafenone and its main metabolite 5-hydroxypropafenone in human serum with direct injection into a column-switching chromatographic system.

Column-switching chromatographic systems using conventional reversed-phase Separon SGX C18 and restricted access media LiChrospher ADS RP-18 precolumns were applied for the determination of propafenone and its main metabolite 5-hydroxypropafenone in human serum samples. The LiChrospher ADS RP-18 precolumn has been found to be more suitable for the sample clean-up. Serum samples were directly injected into the chromatographic system. Proteins and other endogenous compounds were removed by washing with 10% 2-propanol in water and the analytes separated on the Gromsil ODS AB analytical column. The chromatograms were detected at 246 nm. The method validation confirms the suitability of the column-switching system for the quantitation of propafenone and its metabolite. The presented assay shows good linearity with high correlation coefficients (0.992-0.999), high recoveries (96.6+/-6.1-103.5+/-5.8) and excellent values of the repeatabilities (1.23-4.5%). The limits of quantitation are 25-40 ng/ml for the injection volume of 50 microl. The complete analysis including the precolumn reconditioning and the sample clean-up requires 26 min, the sample throughput is approximately four samples in an hour.

SPE-HPLC determination of catecholamines using an affinity principle.

Solid-phase extraction (SPE) with the affinity chromatography principle was applied for high performance liquid chromatography (HPLC) determination of catecholamines in patients urine samples. Electrochemical amperometric detection (ED) was used for all HPLC analyses and both analytical and microbore columns were tested for optimal chromatographic resolution of all analyzed catecholamines. Capacity ratio k' and detection limits were evaluated for all columns used. Precolumn affinity chromatographic procedure of catecholamines with diphenylboronic acid (DPBA) in alkaline medium improved their retention on different commercial SPE reversed-phase cartridges. After clean-up and preconcentration step the complex catecholamine-diphenylboronic acid was degraded by acidic pH and eluted from SPE cartridge. After SPE affinity step catecholamines were analyzed by HPLC-ED. The optimal elution solutions was chosen also as suitable SPE cartridges. Extraction recoveries of catecholamines were 89.6-93.3% with relative standard deviation RSD = 3.8-4.3%. Total analysis time was 30 min including 15 min for the preseparation procedure.

Determination of itraconazole and its metabolite in plasma using SPE-HPLC.

A SPE-HPLC procedure for the determination of itraconazole and its metabolite has been developed. The method was shown to be suitable for routine therapeutic monitoring in leukemic patients.

HPLC-ED determination of catecholamines and their metabolites in urine.

High performance liquid chromatography (HPLC) with electrochemical detection (ED) was applied for the monitoring of catecholamines (epinephrine - E, norepinephrine - NE, dopamine - DA) and their main metabolites (homovanillic acid - HVA, vanillylmandelic acid - VMA) in urine samples of patients with the pheochromocytoma diagnosis. Both amperometric and colorimetric detectors were tested for the clinical applications and the results were compared. The optimization of separation conditions was worked out, especially the effects of mobile phase compositions (pH, ionic strength, organic modifier and ion-pair agent concentrations). Dependences of capacity ratio values k' on all optimized components of the mobile phases were evaluated. Chromatographic conditions were optimized for the suitable chromatographic resolution (Rij > 1.25). Extraction recoveries for liquid-liquid and solid-phase extractions have been worked-out. Detection limits for catecholamines in urine were in the range of 0.3-0.6 ng/ml and 10-20 ng/ml for HVA, VMA using coulometric detector.