Coeluting metabolites

The benefits of further validation of a bioanalytical method by judicious use of incurred samples have been described (Jemal 2002), with particular emphasis on detection of contributions of formerly undetected coeluting metabolites to signal intensity in the SIM or MRM channels used to monitor the target analyte and/or the SIS Table 9.4 summarizes accumulated experience permitting prediction of metabolite types resulting from specific functional groups in the analyte. Such spurious contributions (direct interferences, not matrix effects) will be absent from the samples prepared from control matrix spiked with analytical standard and used for the validation procedures and as QC samples. The following sequence of checks was recommended (Jemal 2002) ... 

In chromatography techniques, selectivity can be proved by the existence of good separation between the analyte and the other components (such as the matrix, impurities, degradation product(s), and metabolites). A consequence of this requirement is that the resolution of the analyte from the other components should be more than 1.5-2.0. In order to detect the possibility of coelution of other substance(s), the purity of the analyte peak should also be determined. For instance, the UV-Vis spectrum of the analyte peak/spot can be used to determine 4the purity of the analyte peak/spot, in this case the correlation coefficient V (this term is used by the software of DAD System Manager Hitachi, and CATS from Camag). With the same meaning and mathematical equation, other terms are used, such as Match... 

Figure 8.9. Overlay of EC-Array data from urine following intraperitoneal administration of vehicle, 20 mg /kg, or 300 mg /kg APAP to rats (n = 5 each group). Peaks labeled E indicate endogenous metabolites while peaks labeled M indicate drug metabolites. Base peak m/z ratios as determined from corresponding MS data are shown. APAP-M + S indicates coelution of sulfate and mercapturate metabolites of APAP. (Reprinted with pemnission from Gamache et a ., 2004b.)...

III.C). Tissues are then ground and subjected to acid extraction and the metabolites are purified by HPLC. The cADPR fraction, identified by its coelution with radiolabeled tracer, is then collected, concentrated, and added to sea urchin egg homogenates or other cADPR-sensitive microsomes. A fluorimetric assay for Ca + release then forms the basis of the mass assay. The authenticity of cADPR in samples can then be validated by inhibition of Ca + release with the specific cADPR antagonist, 8-amino-cADPR (see Section IV. A), or by desensitization of the microsomes by prior release of microsomal Ca2+ by a maximal dose of cADPR (Dargie et ah, 1990). This assay has been used not only to quantify cADPR (Walseth et ah, 1991) in tissues but also to identify ADP-iibosyl cyclase activities (Howard et ah, 1993 Summerhill et ah, 1993 Lee et ah, 1993b). 

Figure 7 illustrates how an isocratic method was developed for the resolution of the two compounds. The compounds coeluted after 2.4 min using an eluent of 65% v/v acetonitrile/water (Fig. 7A). As the acetonitrile concentration was progressively decreased, resolution was improved and retention was increased. It can be seen that this also results in much broader peaks. Partial resolution was observed when the organic modifier concentration was 55% v/v (Fig. 7B), near-baseline resolution was seen at 50% v/v (Fig. 7C), and complete resolution was achieved when the organic modifier concentration was 45% v/v (Fig. 7D). It can be seen from the structures that these metabolites bear an abundance of ionizable phenolic hydroxyls, and this feature necessitated the use of a buffered eluent for preparative work (see Subheading 2.3.2.).

Until recently (93-96), little was known about the arsenic methylation intermediates, MMA(lll) and DMA(lll), in the human system, a result of the lack of techniques for the determination of these arsenic species. Recent developments of more sensitive and improved arsenic speciation techniques contribute to the discovery of these intermediary metabolites in human urine (93-96). Figure 3 shows typical chromatograms obtained from the analyses of arsenic compounds in deionized water and in urine samples. Coinjection of the urine sample with authentic MMA(lll) standard (Fig. 3c) demonstrates the coelution of the suspected MMA(lll) in the sample with that of the standard MMA(III), confirming the identity of MMA(lll) in the urine sample. Similarly, coinjection of the urine sample with standard DMA(III) (Fig. 3d) and As(V) (Fig. 3e) confirms the presence of DMA(lll) in the sample (96). Two other research groups have recently also found MMA(lll) and DMA(llI) in human urine samples (123,124). 

The incomplete chromatographic separation of the isomers in the rat urine and the similarities of the MS" spectra of the coeluted components resulted in difficulties in the structural elucidation of metaboKtes in rat urine. These coeluted components were distinguished on the basis of the metaboKc pathway and biogenic feature. The metabolite Mil was used as an example to iKustrate the analytical procedure. 

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