Analytica Chimica Acta 497 (2003) 165–174 Using on-line solid phase extraction for simultaneous determination of ascorbic acid and rutin trihydrate by sequential injection analysis Z. Legnerová, D. Šat´ ınský ∗ , P. Solich Department of Analytical Chemistry, Faculty of Pharmacy, Laboratory of Flow Analysis, Charles University, Heyrovského 1203, Hradec Králové 50005, Czech Republic Received 5 December 2002; accepted 2 July 2003 Abstract This presented paper deals with a new methodology for the separation and simultaneous determination of two active sub- stances in combined pharmaceutical formulation, composed of ascorbic acid (AA) and rutin trihydrate (RT). The methodology described is based on the sequential injection analysis (SIA) technique analysis coupled with a solid phase extraction (SPE) microcolumn. The SPE microcolumn was used for retention of RT, while the AA was eluted with the solvent front phase. Due to the different acidobasic and polarity properties of both compounds and the principles of reversed phase chromatography, it was possible to separate AA and RT with one eluting solution methanol/water (28:72), pH 2.5, flow rate 10 ls -1 . The spectrophotometric detection of both substances was performed at 262 nm. Under the optimal conditions, the calibration was linear over the range 10–100 g ml -1 for AA with R.S.D. of 0.70% (n = 10) and 2–20 g ml -1 for RT with R.S.D. 0.40% (n = 10), and was described by the following equations: A = 0.0098c + (-0.0005); r = 0.9995 and A = 0.0033c + 0.0080; r = 0.9987, for AA and RT, respectively. The sample throughput was 26h -1 . The proposed SIA–SPE method has been applied for the simultaneous assay and dissolution test of AA and RT in pharmaceutical formulation. © 2003 Elsevier B.V. All rights reserved. Keywords: Sequential injection analysis; Solid phase extraction; Spectrophotometry; Ascorbic acid; Rutin trihydrate 1. Introduction Sequential injection analysis (SIA) has been in- troduced by Ruzicka and Marshall in 1990 [1] as a following generation in the development of the flow injection technique. The principles upon which SIA is based are similar to those of flow injection analysis (FIA), namely controlled partial dispersion and repro- ducible sample handling. In contrast to FIA, SIA em- ∗ Corresponding author. Tel.: +420-495067294; fax: +420-495518718. E-mail address: satinsky@faf.cuni.cz (D. Šat´ ınsk´ y). ploys a computer-controlled multiposition valve and syringe pump operated synchronously. The ports of the multiposition valve are connected to sample and reagent reservoirs. Aliquots of sample and reagents are sequentially aspirated into a holding coil connected to the common port of the multiposition valve by op- erating the pump in the reverse mode. The resultant stack of sample and reagent zones is then propelled towards the detector, via another port on the valve, by the pump operating in the forward mode. The flow reversal leads to mixing of the sample and reagent zones to create a zone of product whose properties are measured at the detector. Characteristic advantages of 0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2003.07.007