Fluorometric techniques

The recognition of their structure permits the determination of vitamins by the tools of analytical chemistry, but while such methods are widely used in industrial production, the minute quantities in body fluids and tissues limit the purely chemical approach to a few members of this group present in relatively high concentration, e.g., vitamin C (K5). Microchemical methods are in use for the determination of thiamine, riboflavin, and some of the fat-soluble vitamins, based on the most sensitive colorimetric and, in particular, fluorometric techniques. Vitamin D, on the other hand, is determined by animal assay. 

Wehry E. L. and Mamantov G. (1981) Low-Temperature Fluorometric Techniques and Their Application to Analytical Chemistry, in Wehry E. L. (Ed.), Modem Fluorescence Spectroscopy, Vol. 4, Plenum Press, New York, pp. 193-250. 

Principles of steady-state and time-resolved fluorometric techniques... 

A three-step method can be applied for analysis of mixtures of hydroperoxides and peroxides, consisting of RP-HPLC separation, photolytic conversion of the emerging analyte to H2O2, by irradiation with a 254 nm UV lamp and application of the fluorometric technique depicted in equation 34 (w = 1) to determine this analyte, f-Butyl perbenzoate has LOD 0.3 mgL , linearity range 0.9-80 mgL and RSD 6.1% . See a similar method in Section VI.B.l. 

FIGURE 11.8 Ambient H202 concentrations measured by TDLS ( ) and the continuous scrubbing enzyme fluorometric technique ( ) during the period June 24-26 at Research Triangle Park, North Carolina (adapted from Schiff et al., 1994a, 1994b). 

Evidence of Two Enzymatic Activities. We found no formation of NADH spectrophotometrically during decomposition of malic acid by cell-free extracts of L. oenos ML 34. However, using a more sensitive fluorometric technique to search for NADH (88), we found a small, continual formation of NADH (about 5 nmoles NADH/min/mg extract protein) as an end product (89,90). Thus, in addition to the decarboxylation of malic acid to lactic acid, there is another reaction that gives... 

Although radio-immunoassays are rapid and convenient to analyze large numbers of samples, they lack absolute sensitivity and specificity, since cannabinoids cross-react within a given assay. Gas chromatography is no more satisfactory. However, when combined with mass spectrometry it is far more specific and sensitive, although extremely costly. Finally, fluorometric techniques have proved suitable for many applications such as the one based on gallium chelate formation which can only be used for urine (9), and more especially for applications based on cannabinoid dansylation (16-18). However, these last techniques only allow qualitative determination of A9-THC. 

This new fluorometric technique makes the determination of kynurenic acid simpler and that of xanthurenic acid more specific if the ferrous or ferric iron technique is used for the latter, higher results are obtained, probably due to the presence in urine of chelating substances which combine with iron (P8). 3-Hydroxykynurenine is determined by an analogous method (B23). 

Phase fluorometry is another useful fluorometric technique for the determination of substances with overlapping fluorescence spectra. In phase fluorometry the phase angle between the lamp pulse and the emission of fluorescent light allows discrimination between fluorescences of different origins. Phase-sensitive optics and electronics are rather complicated and, as in the case of time-resolved fluorometry, will be mentioned here only in passing. For further information on these areas, the reader is referred to the bibliography. 

The realiability of this fluorometric technique for determination of (H.,o) is demonstrated in Figures 10 and 11. Figure 10 relates the activity of water with the molar concentration of NaCl, as determined by the rate... 

Elucidation of the structure of the B vitamins resxilted in the development of analytical chemical methods. Such methods are useful in industries manufacturing vitamins in which measurements of microgram to milligram quantities are routine, but these same methods are often not applicable to biological fiuids that contain as little as picogram quantities. Colorimetric and fluorometric techniques have been developed for niacin and folate, but these techniques require complicated extraction procedures and blank determinations. They also suflFer from lack of specificity, and the results are often altered by interference from biologically inactive materials that occur naturally or are produced during extraction (16,20). 

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