A single spectrometer provided excitation at 365 nm, and a double spectrometer was positioned to monitor fluorescence at 444 nm. The slits on each spectrometer were adjusted to give a bandpass of 5 nm. For each calibration standard, the computer acquired a data file representing the fluorescence intensity during the reaction, measured at intervals of 100 ms over a total time of 50 s. The fastest integration time for data acquisition is 1 ms.
Results and Discussion
Figure 2 shows four time-base fluorescence scans obtained with the FLUOROLOG spectrofluorometer during the oxidation of thiamine to thiochrome. The four scans trace the reaction for standard concentrations of 2.5, 1.25, 0.625, and 0.313 ppm. The reaction rates, determined from the slope of the intensity-versus-time plots in Figure 2, are given in Table 1.
Using the data-processing capability of the DM3000F, reaction rates were plotted against concentration, and a linear-regression program was applied to determine the best fit. The resulting calibration curve (Figure 3) can be used selectively to quantify trace amounts of thiamine in samples containing other vitamins and minerals.
Ryan and Ingle demonstrated that their reaction-rate method for determination of thiamine gave reasonable results for commercial and synthetic vitamin-mineral preparations. Kinetic analysis of a synthetic preparation compared favorably with results obtained by the USP standard method of analysis. Moreover, the kinetic method was shown to be faster and less expensive.