Metabolic products, mass spectra

When working with non-radiolabeled drugs the major challenge is to find metabolites in the biological matrices. Because the enzymes responsible for metabolism are quite well characterized metabolic changes can partially be predicted. For example hydroxylation of the parent drug is in many cases the principal metabolic pathway. From a mass spectrometric point of view it results in an increase of 16 units in the mass spectrum. In the full-scan mode an extracted ion current profile can be used to screen for potential metabolites. In a second step a product ion spectrum is recorded for structural interpretation. Ideally, one would like to obtain relative molecular mass information and the corresponding product ion spectrum in the same LC-MS run. This information can be obtained by data dependant acquisition (DDA), as illustrated in Fig. 1.39. 

These model studies permitted demonstration of metabolic a-hydroxylation of NPy by isolating 2-hydroxytetrahydrofuran as a metabolite of NPy. This was accoitplished by trapping 4-hydroxy-butyraldehyde as its 2,4-dinitrophenylhydrazone (DNP) derivative. For in vitvo studies, NPy-2,5-l C was incubated with rat liver microsomes, 02i and an NADPH generating system. After the incubation was complete, DNP reagent was added to the mixture and the products were extracted and examined by preparative TLC. A radioactive band corresponding to the DNP of 4-hydroxybutyralde-hyde was observed. This band was not present in controls in which NADPH was omitted or in which boiled enzyme was used. The mass spectrum was identical to that of a reference sairple. In addition, a minor metabolite with mass spectrum identical to that of the DNP of 2-butenal was also isolated. These results showed conclusively that NPy underwent metabolic a-hydroxylation in this in vitro system. 

The metabolism of ticlopidine, 45, has been investigated by mass spectral analysis. The principal metabolic pathways can be proposed based on supporting mass spectrometry (MS) fragmentation patterns. Chlorotropylium ion 46 (mlz= 125) is the base peak in the product ion mass spectrum of ticlopidine <2004MI49>. 

In practical application scanning can be manipulated on-the-fly within a chromatographic separation to obtain maximum information. In metabolism studies or as a chemical dosimeter, the structural feature of the parent compound and its unique neutral loss occurring on collisional activation, marks the metabolite. The mass of the metabolite is then obtained from the TSP mass spectrum at Q1 and the product ion spectrum of the metabolite molecule ion is obtained by product ion scanning. Recent publications have discussed additional applications of both tandem mass spectrometry (26. 271 and thermospray tandem mass spectrometry (28) in metabolite structure elucidation. 

The chemical ionization mass spectrum of the permethylated pure sample revealed that the base peak corresponds to the molecular ion at m/e=452. A cell incubation experiment was carried out, a crude extract was prepared, and a total ion chromatogram obtained (Fig. 6). On the basis of initial experiments with the pure compound, the metabolic product was expected to appear, if at all, around 200 C, which would correspond to about scan 200. A Mass chromatogram of m/e=452 was next obtained (Fig. 7) which showed that phosphorylation did, indeed, take place inside the cells. Incidentally, an important option in the cathode-ray-tube- output of this kind of information is the possibility to amplify any part of the display so that both observation and subsequent quantification by computerized area measurement can be made more conveniently and accurately. A series of pictures by Watson t l. (8) in a paper on display-oriented data systems, nicely illustrates the power of this feature. 

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