Determination of Thiamine in Wheat Flours Using a Validated Isocratic HPLC-Fluorescence Method Previously Optimized by Box-Behnken Design

Abstract

The main purpose of this work was to optimize and validate a new isocratic high-performance liquid chromatography method for thiamine analysis in wheat flours. Thiamine was previously derivatized into thiochrome and determined by fluorescence detection (excitation wavelength, 366 nm; emission wavelength, 435 nm). The mobile phase consisted of buffer phosphate (0.02 M) and organic modifier (methanol 88 %; acetonitrile 12 %). Optimization was done using a Box-Behnken design (three factors at three levels) where organic modifier proportion (A), varying from 20 to 90 %, mobile phase pH (B), varying from 5.8 to 7.8, and flow rate (C), varying from 0.5 to 1 mL min-1, were the studied variables. Peak width was the studied response. Optimal chromatographic conditions that minimized response were A = 90 %, B = 6.00, and C = 1 mL min-1. Using optimal conditions, thiochrome extractions with isobutanol and solid-phase extraction (SPE) employing styrene-divinylbenzene adsorbent cartridges were compared. Method validation, using thiamine standard solutions and both types of thiochrome isolation, included linearity studies, limits of detection and quantification, and calibration and analytical sensitivity quantifications. Standard addition method was employed to quantify thiamine in wheat flours. Good precision results were obtained by both extraction methods. Recoveries ranged from 63 to 65 % for isobutanol and 84 to 101 % for SPE, including sample extraction, thiamine derivatization, thiochrome purification, and chromatographic separation. Taking into account better obtained SPE recoveries, acceptable precision, and isobutanol irritant action on the eyes and skin of operator, SPE using styrene-divinylbenzene adsorbent could be chosen for routine thiamine analysis in wheat flour samples.