Metabolites fluorescence chromatogram

Figure 2. Fluorescence chromatogram of BaP metabolites formed by microsomes from livers of rats fed the diets described in "Methods". Key A, various tetrols B, 9,10 dihydrodiol ...

Fluorescence chromatograms from the blank, test compound control, and sample incubations are superimposed. Additional peaks present only in the sample chromatogram are eonsidered adducts. Mass spectra (full scan and MS") of the peak provide additional confirmation. Quantitation is based on comparison of fluorescence peak area of adduct versus that of dGSH standard (external calibration). Quantitative analysis should not be performed if the test compound or its metabolites have fluorescence interference at the wavelength... 

While Funk et al. did not use temperatures above 30 °C during the irradiation times discussed above, Sistovasis combined UV irradiation with simultaneous heating (70 °C, 2 h) of the TLC layers [24]. After this treatment nomifensine and its metabolites appeared as intense yellow fluorescent chromatogram zones on a dark background. 

Frdns has demonstrated this possibility with reserpine and rescinnamine hy irradiating at the start zone for two hours and obtaining a characteristic zone pattern (fingerprint) after TLC separation of the photochemically produced derivatives [20J. Huck and Dworcak exposed developed chromatograms with vanilmandelic acid and homovanillic acid zones to diffuse daylight and observed the formation of fluorescent metabolites that were suitable for direct quantitative analysis [21]. 

Figure 9.147 HPLC chromatogram of 7-hydroxycoutnarin generated from incubation with HepG2 liver cells. A 10,000g supernatant was obtained from approximately 1 X 10 cells and incubated (IS min) with 4.2 /xM 7-ethoxycoumarin. Following extraction with CHC13, 7-hydroxycoumarin metabolite formed was quantified by the HPLC-fluorescent method. (From Rosenberg et al., 1990.)...

Figure 10.13 HPLC of FoA and its metabolites produced by activities present in an S-30 fraction prepared from rat liver FoB is a formycin analog of inosine. Operating conditions mobile phase, 0.1 M KH2P04 adjusted to pH 5.5 with NaOH with 10% methanol flow rate, 2 mL/min room temperature Qg /xBondapak column packing fluorescence, excitation at 300 nm, emission about 320 nm. (A) Chromatogram obtained with standards as indicated at approximately 10 /xg of each. Sensitivity of fluorescence detection, 0.5 AUFS units full scale. (B) Chromatogram obtained immediately after addition of S-30 to a reaction mixture containing FoA and ATP. (C and D) Chromatograms obtained after 8 and 16 minutes of incubation, respectively. [From Dye and Rossomando, 1982. Reprinted by permission from Bioscience Reports, 2 229-234 (1982).]...

Figure 6.5 Chromatogram of tamoxifen and its major metabolites using fluorescence detection with the post-column photochemical reactor switched on (above) and off (below, with attenuation reduced by 10 times). Insert structure of tamoxifen. Peaks are 4-hydroxy-tamoxifen (32 min), tamoxifen (40 min), desmethyltamoxifen (44 min), didesmethyltamoxifen (58 min), tamoxifenol (62 min), others unknown.

Amount of Information Delivered. There are two barriers here, one in the single-analyte focus of IA which predudes its use as a multi-residue screen or even to cover all metabolites of a single toxicant, and the other is in the limited information contained in the final IA color or fluorescence intensity. For multi-residue screens, IAs have a limited role through such techniques as test batteries (several IAs in an integrated format) or antibody miring. These are reasonable fields for future research and development. As far as information delivery is concerned, this limitation of IA will disappear when IA is coupled with conventional resolution techniques such as HPLC. The use of affinity columns and immobilized antibody-based detectors will produce a chromatogram with the peripheral vision analysts have come to expect from GC and HPLC. 

Column iiBondapak Phenyl (300x4 mm 101, mobile phase 0.0015 M aqueous phosphoric acid - acetonitrile (9 1), flow rate 2 ml/min, fluorescence detection (excitation 320 nm, emission 418 nm) Chromatogram A blank plasma chromatogram B plasma standard ( 10 umole/1 quinidine, 5 umole/ 1 3-hydroxyquinidine) chromatogram C plasma from patient on chronic oral quinidine therapy. Peaks 1, polar metabolites 2, 3-hydroxyquinidine 3, internal standard (cinchonidine) 4, unidentified metabolite 5, quinidine 6, dihydroquinidine. 

The nonanionic fluorescent [i C]metabolites derived from N-acetylcysteinyl -[ C]riboflavin migrated on paper chromatograms in a manner expected for decarboxylated rather than deacylated catabolites. This was verified by absence of reactivities with both acid indicator and ninhydrin sprays. 

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