Simultaneous photometric microplate assay for free and total thiamine using gold nanoparticles and alkaline phosphatase

A photometric method is described for the determination of free and total thiamine (TH; vitamin B1) in food samples. It is based on the use of gold nanoparticles (AuNPs) which aggregate in the presence of TH owing to the interaction of its sulfur atom with the AuNPs. This is accompanied by a color change from wine red to purple-blue and an increase in absorbance at 590 nm. The effect is not observed for TH phosphates which, however, can be determined, as demonstrated for the example of TH pyrophosphate as a model ester, by treating them with alkaline phosphatase (ALP) which hydrolyses such esters. The use of two sample aliquots, one with and one without ALP, allows the determination of free and total TH, respectively. The dynamic range extends from 0.15 to 3.5 mu M, and the detection limit is 54 nM of TH. The method has been applied to the analysis of spiked food samples and gave recoveries that ranged between 88.8 and 100.7 %.