The paper deals with the concept of simple automated creation of gradient profile of the mobile phase for gradient-elution sequential injection chromatography (GE-SIC). The feasibility and merits of this concept are demonstrated on the separation and simultaneous assay of indomethacin as active principle and of its two degradation products (5-methoxy-2-methylindoleacetic acid and 4-chloro-benzoic acid) in a topical pharmaceutical formulation. The GE-SIC separation was performed with a FIAlab(®) 3000 SIC set-up (USA) equipped with an Onyx™ Monolithic C18 (25 mm × 4.6mm, Phenomenex(®)) column, a six-port selection valve, a 5-mL syringe pump and a fiber-optics UV CCD detector. Ketoprofen was used as an internal standard (IS). The gradient elution was achieved by automated reproducible mixing of acetonitrile and aqueous 0.2% phosphoric acid in the holding coil of the SIC system. Different profiles of the gradient elution were tested. The optimal gradient using two mobile phases 30:70 and 50:50 of acetonitrile/0.2% phosphoric acid (v/v) was achieved under the optimum flow rate 1.2 mL min(-1). The chromatographic resolution R between the peaks of all solutes (including the IS) was >2.00. The repeatability of retention times was characterized by the RSD values 0.18-0.30% (n=6). Net separation time was 3.5 min and the mobile phase consumption was 4.5 mL for a single GE-SIC assay. The figures of merit of the novel GE-SIC method compared well with those of conventional HPLC.

Department of Analytical Chemistry Faculty of Pharmacy Charles University Heyrovského 1203 50005 Hradec Králové Czech Republic

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