Determination of thiamine in wheat flours using a validated isocratic HPLC-fluorescence method previously optimized by Box-Behnken design

NICOLÁS MICHLIG; FRANCO VAN DE VELDE; MARCELINO FREYRE; CELILA MARÍA DEL HUERTO BERNARDI

FOOD ANALYTICAL METHODS

SPRINGER

Lugar: Berlin; Año: 2014 vol. 7 p. 828 - 835

1936-9751

The main purpose of this work was to optimize and validate a new isocratic HPLCmethod for thiamin analysis in wheat flours. Thiamine was previously derivatized intothiochrome and determined by fluorescence detection (excitation wavelength: 366 nm,emission wavelength: 435 nm). The mobile phase consisted of buffer phosphate (0.02M) and organic modifier (methanol 88%: acetonitrile 12%). Optimization was doneusing a Box-Behnken design (3 factors at 3 levels) where organic modifier proportion(A), varying from 20 to 90%, mobile phase pH (B), varying from 5.8 to 7.8, and flowrate (C), varying from 0.5 to 1 mL min-1 were the studied variables. Peak width was thestudied response. Optimal chromatographic conditions that minimized response wereA = 90%, B = 6.00 and C = 1 mL min-1. Using optimal conditions, thiocromeextractions with isobutanol and solid phase extraction (SPE) employing styrenedivinylbenzeneadsorbent cartridges were compared. Method validation, usingthiamine standard solutions and both types of thiocrome isolation, included linearitystudies, limits of detection and quantification and calibration and analytical sensitivityquantifications. Standard addition method was employed to quantify thiamine in wheatflours. Good precision results were obtained by both extraction methods. Recoveriesranged from 63 to 65 % for isobutanol and 84 to 101% for SPE, including sampleextraction, thiamine derivatization, thiochrome purification and chromatographicseparation. Taking into account better obtained SPE recoveries, acceptable precisionand isobutanol irritant action on eyes and skin operator, SPE using styrenedivinylbenzeneadsorbent could be chosen for routine thiamine analysis in wheat floursamples.