Radioactivity chromatogram

FIGURE 7.12 Metabolite profile of bile sample obtained from a dog dosed with 20 mg/kg of a 14C-labeled test compound (A) total mass chromatogram of unprocessed LC/MS data (B) total mass chromatogram after mass defect filter processing (C) radioactivity chromatogram. (Source Adapted from Zhang, H.D. and Ray, K J. Mass Spectrom., 2003, 38, 1110. With permission of John Wiley Sons.)... 

Figure 6.3. Metabolite profile of razaxaban (C24H2oNg02F4) in dog bile (a) base peak chromatogram of unprocessed LC-MS data (b) base peak chromatogram of processed LC-MS data (c) corresponding radioactivity chromatogram. The arrows annotate the retention time of the peak shown in Fig. 6.2. Bile was collected from dogs orally administered with 14C-labeled razaxaban (20 mg/kg). HPLC solvents were 10mM NH4HC03 (pH 9.0) and acetonitrile. A portion of the HPLC effluent was collected in 15-s fractions for the radioactivity chromatogram. Another portion of the HPLC effluent was directed to a Q-TOF Ultima mass spectrometer.

The utilization of MDF again allows the TIC to be indicative of metabolite peaks, rendering a sense of simplicity similar to that of working with in vitro samples. As shown in Fig. 6.4, the resulting TIC of the MDF-processed data (Fig. 6.4b) is comparable to the radioactivity chromatogram (Fig. 6.4a). In contrast, the unprocessed TIC (Fig. 6.4c) does not show a correlation to the radioactivity chromatogram. 

Figure 2. Radioactivity chromatogram of sulfur compounds derivatized with monobromobimane. The reversed-phase HPLC separation is based on the hydrophobic properties of the bimane-sulfur adducts but peak area is based on "S-radioactivity of the compounds. At time 0 sulfite and thiosulfate impurities are present before addition of the hepatopancrease tissue homogenate. This was a 60 min experiment to determine the sulfide detoxifying functions of the hepatopancrease of the hydrothermal vent crab Bythograea thermydron. During this time the proportion of radioactivity in sulfide rapidly decreases and thiosulfate and sulfate accumulate as end products. Two intermediates, pi and p2 accumulate then decrease during the experiment. The two intermediates are believed to be polysulfides based on similar elution times of polysulfide standards. (Figure is the unpublished chromatograms from the data in Vetter et al. (24)-) continued on next page.

Preparative purification of radiolabeled DEQUEST 2041 and 2051 was done with the same equipment and by quite similar procedures. Typical radioactivity chromatograms for the two products are given in Figures 5 and 7 respectively. 

In Chapter 7, approaches for visualization of zones in chromatograms are discussed, including use of nondestructive and destructive dyeing reagents, fluorescence quenching on layers with a fluorescent indicator, and densitometry. In Chapter 8, additional detection methods, such as those used for biologically active and radioactive zones, as well as the recovery of separated, detected zones by scraping and elution techniques are covered. 

The analyses involved the exposure of developed chromatograms to x-ray films followed by scraping of radioactive spots and quantifying them. Relative amounts of various compounds in the fractions are indicated on the righthand side against numbered spots. Spots A-6, B-4, and C-5 represent unchanged cis-chlordane A-7 was a mixture of dichloro-chlordene and oxychlordane. A-3, B-3, C-4, and D-4 represent chlordene chlorohydrin. A-2, B-2, C-2, and D-2 were complex spots with the heptachlor diol as the major compound. A-l, B-l, C-l, and D-l were polyhydroxy derivatives or conjugates. Identities of other spots are not known. Recoveries in fractions A, B, C, and D were, respectively, 60.1%, 0.6%, 2.2%, and 2.9% of the applied radiocarbon. 

Introduction of the technique of labeling the phosphate with radioactive P32 finally facilitated the quantitative determination of separate components, the quantities of which were previously determined colorimetri-cally after eluting the chromatogram spots with ammonia (171,192). 

In the case of radioactive isotopes, however, the newcomer in the field is faced with a bewildering array of instruments and manufacturers. Developments in this field have been rapid and papers a few years old probably describe assay methods which are now out of date. Currently there is a vast choice amongst ion chambers, proportional counters, Geiger-Miiller counters, crystal scintillation counters, liquid scintillation counters and semi-conductor counters — all with admirable characteristics for particular purposes. If a worker s interests lie solely in gas chromatographic examination of products then he can obtain a counter specially designed to monitor column effluents. Similarly there are various scanners especially for paper chromatograms or thin layer plates. 

Figure 1. Chromatogram (upper) and corresponding radioactive scan of C 4-...

De Maio, W., Hoffmann, M., Carbonara, M., Moore, R., Mutlib, A., and Talaat, R. E. (2008). Identification of drug metabolites by UPLC-MS with isotope pattern directed mass chromatograms and UPLC with radioactivity flow detection. In Proceedings of the 56th ASMS Conference on Mass Spectrometry and Allied Topics. ASMS, Denver, CO. 

If the retention times of the analytes are known, or there is an efficient method for their detection on-line, such as UV, MS or radioactivity, stop-flow HPLC-NMR becomes a viable option. In the stop-flow technique, all the usual techniques available for high-resolution NMR spectroscopy can be used. In particular, these include valuable techniques for structure determination such as 2-dimensional NMR experiments which provide correlation between NMR resonances based on mutual spin-spin coupling such as the well-known COSY or TOCSY techniques. In practice, it is possible to acquire NMR data on a number of peaks in a chromatogram by using a series of stops during elution without on-column diffusion causing an unacceptable loss of chromatographic resolution. 

Quantitative determinations were reported by densitometry [63,72] or, in the case of Relabelled compounds, by scraping or cutting the chromatograms into strips and measuring the radioactivity in a liquid scintillation spectrometer [44,70]. 

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