Validation of a fully automated on-line SPE-LC method with electrochemical detection for the quantitation of the catecholamines norepinephrine, epinephrine and dopamine in human urine.

A Summit x2 dual gradient HPLC system (Dionex, Idstein, Germany) equipped with: a solvent rack model SOR-100, two low-pressure gradient pumps with integrated online degasser P680, an automated sampler injector ASI-100 and a thermostated column oven TCC-100 was used. The electrochemical detector (EC3000), the detector cell (EC4000) and the thermostate (HT3000) were from Recipe Chemicals & Instruments GmbH, Munich, Germany. A three way low pressure switching valve (Recipe) was placed between the outlet of the analytical column and the electrochemical cell and controlled automatically by the LC-system.

The SPE-column (CAT-PBA, 30 x 4 mm ID, Recipe Chemicals & Instruments GmbH, Munich, Germany) contains a restricted access material (RAM) modified with the affinity ligand nitrophenylboronic acid. The size exclusion limit of the RAM SPE-column amounts to approximately 10 kDa. The analytical column (150 x 4.6 mm ID.; particle size: 5 µm) was packed with a reversed phase material and obtained from Recipe Chemicals & Instruments GmbH (Munich, Germany). The SPE-column and the analytical column were coupled via the ten-port switching valve integrated in the column oven of the Summit x2 dual gradient HPLC system. The method was fully validated using accuracy profiles. The acceptance limits were set at ±15 % of the nominal concentration values.

Validation of a LC-UV method for the determination of a drug substance, fenofibrate and its main impurity fenofibric acid in a drug product.

The chromatographic analysis was performed on a Lichrospher 100 RP-8 column (125 x 4 mm i.d., 5 µm particle size) and kept at 35°C. The mobile phase was prepared by mixing methanol and phosphate buffer (pH 4.5) in a ratio 70:30 v/v and degassed before use. The HPLC system was an Agilent technologies HPLC 1100 series (Hewlett-Packard, Palo-Alto, CA, USA) equipped with a solvent delivery quaternary pump G1311A, an on-line degasser G1322A, an autosampler G1313A, a column oven G1316A and a diode-array detector G1315A. It was operated isocratically at a flow rate of 0.8 ml/min and the injection volume was 50 µl. UV detection was performed at 288 nm and peaks were identified with retention times and UV spectra. Acceptance limits were set at ±5% for fenofibrate and ±10% for fenofibric acid.