ORIGINAL PAPER Modulation of mobile phase composition in flow-injection/sequential-injection chromatography exploiting multisyringe flow analysis Mailen Fernández & Manuel Miró & Hilda M. González & Víctor Cerdà Received: 20 September 2007 / Revised: 10 December 2007 / Accepted: 8 January 2008 / Published online: 1 February 2008 # Springer-Verlag 2008 Abstract In this paper, a time-based multicommutated flow system is proposed for appropriate selection and modulation of mobile phase composition in flow-injection (FI)/sequential-injection (SI) chromatography. The novel flow assembly involves the on-line coupling of a short monolithic reversed-phase chromatographic column with a multisyringe flow injection set-up furnished with a set of solenoid valves. The proposed hyphenated technique was applied to the simultaneous spectrophotometric determina- tion of thiamine (B 1 ), pyridoxine (B 6 ) and cyanocobalamin (B 12 ) which were taken as model analytes. The separation method capitalizes on a dual isocratic elution protocol involving the use of a single forward stroke of the multisyringe pump for initial delivery of 50 mmol L -1 ammonium acetate (pH 7.0) for 2.4 min followed by 50 mmol L -1 ammonium acetate–methanol (80:20, v/v) for 6.4 min at 0.5 mL min -1 and room temperature. Detection was performed at the maximum wavelength for each target vitamin—280 nm for B 1 , 325 nm for B 6 , and 360 nm for B 12 . A first-order, two-level full-factorial design was utilized to ascertain the significant variables influenc- ing the chromatographic separation and the magnitude of the interaction effects. The experimental design method revealed that resolution of the target vitamins is highly dependent on the pH, percentage of organic modifier, and their second-order interaction. The multisyringe flow- injection-based monolithic column separation method, which should be viewed as an expeditious and cost- effective alternative to the high-performance liquid chro- matography counterpart, was applied to the separation and determination of B 1 ,B 6 , and B 12 in different pharmaceu- tical dosage forms in less than 9 min. Statistical comparison of the results from the proposed procedure with those from the HPLC method endorsed by the US Pharmacopeia revealed there were no significant differences at the 95 % confidence level. Keywords Monolithic column . Multisyringe liquid chromatography . Pyridoxine . Thiamine . Cyanocobalamin . Pharmaceutical preparations Introduction Vitamins are a group of organic compounds essential for the normal growth and development of human and animal bodies. Vitamins can be classified into two main groups— water-soluble and fat-soluble. Most of the water-soluble vitamins generate the B-complex, each member of this series has a unique structure and specific activity within the human body [1]. As a result of their chemical instability, determination of water-soluble vitamins in biological matrices and pharma- ceutical formulations can be problematic [2]. Several analytical methods have been proposed for individual Anal Bioanal Chem (2008) 391:817–825 DOI 10.1007/s00216-008-1848-8 M. Fernández : M. Miró : V. Cerdà (*) Department of Chemistry, Faculty of Sciences, University of the Balearic Islands, Carretera de Valldemossa km. 7.5, 07122 Palma de Mallorca, Illes Balears, Spain e-mail: victor.cerda@uib.es M. Fernández Cuban Sugar Research Institute, Carretera al Central Martínez Prieto km 2.5, Boyeros, La Habana, Cuba H. M. González Department of Quality Assurance and Pharmaceutical Technology, Food and Pharmaceutical Sciences Institute, University of Havana, Havana, Cuba