Metabolite identification chemical structures

This chapter has two aims. First, techniques that use mass spectrometry for the identification and structural elucidation of neuroactive chemicals are reviewed. Second, and with greater emphasis, this chapter attempts to describe the routine use and pitfalls encountered when using mass spectrometry— especially GC/MS and LC/MS—for rapid, specific, quantitative analyses of neuroactive compounds and metabolites in a variety of biological matrices. 

Usage of correlation analysis is especially suited for combination with nontargeted analysis. Different combinations offer possibilities for identification of gene function or unknown metabolites. Possibly unknown metabolites correlate with genes of known function, which allows elucidation of functional groups or structural scaffolds of the unknown. These can possibly speed up identification of unknown metabolites and their chemical structure. 

The availability of the chemical structure and NMR assignment s for the parent compound, as illustrated in Section 12.7 for Buspirone, significantly simplifies the identification of related metabolites. A significant amount of information has already been accumulated and presented in the literature that describes numerous metabolic reactions associated with a variety of metabolic pathways. Examples of the most common metabolic reactions are tabulated in Table 12.11. These metabolic reactions identify a range of possible chemical modifications that may be applied to the parent compound and generate a variety of related metabolites. 

NMR spectra were measured with a Varian Unity 300 FT-NMR spectrometer. Liquid chromatography-atmospheric chemical ionization-mass spectrometry (LC-APCl-MS) in positive and negative ion modes was performed using a Hitachi M-1000 spectrometer. Unknown metabolites were isolated and purified from the grape fruits extracts with solid phase extraction method (Porapak Q) and HPLC. Isolated metabolites were analyzed by free form or derivatized (methylated, acetylated) form for identification. All of the non-radiolabelled reference standards 1-6 are synthesized in our laboratory and their chemical structures are shown in Figure 2. 

For metabolite identification, 8 male Cij CD(SD) rats (7 weeks old) were treated orally with [ " C-phenylJ-l at 300 mg/1cg b.w./day for 2 days. Urine and feces were collected for 3 days after the first dose. Feces were not used following isolation. Urine was lyophilized and fractionated by solvent extractions (hexane, ethyl acetate, etc.). Eight metabolites were isolated from extracts by preparative TLC and preparative HPLC. Their chemical structures were determined by NMR ( H, H-H COSY, HSQC, HMBC, etc.) and MS (ESI, El) spectroanalyses. Their chemical structures are shown in Figure 3 (8, 9, 10, 11,13,14,15 and 16). 

Studies on the comparative abilities (13) of B[a]P metabolites to bind to DNA in microsomal systems showed that the 7,8-dihydrodiol was the most efficient. This led to the proposal (69) that dihydrodiol epoxides were the ultimate carcinogenic metabolites. Chemical synthesis of all possible isomers (70.71) has allowed complete structural identification of the adducts (72-74). 

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