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Fig. 7.10 Representative binding curve obtained by nonlinear regression from a competitive MS-binding assay for dopamine D2 receptors, in which (+)-butaclamol competes with spiperone as marker. The points describe nonbound spiperone quantified by LC-ESI-MS/MS. Data reflect means (Gs) from binding samples each performed in triplicate.

Fig. 7.10 Representative binding curve obtained by nonlinear regression from a competitive MS-binding assay for dopamine D2 receptors, in which (+)-butaclamol competes with spiperone as marker. The points describe nonbound spiperone quantified by LC-ESI-MS/MS. Data reflect means (Gs) from binding samples each performed in triplicate.

high amount of nonspecific binding [defined as the remaining binding in the presence of 10 mM (+)-butadamol].

Nevertheless, they easily allowed the determination of IC50 values of the test compounds and the calculation of the respective Ki values [according to Eq. (7), Section 7.3.1, Table 7.2].

The reliability of the SPE-LC-ESI-MS/MS quantitation method of the non-bound marker spiperone was verified in identical binding assays employing [3H]spiperone as marker and (S)-sulpiride as test compound, again quantifying the nonbound marker but this time by scintillation counting. Both the run of the competition curve and the Ki value determined for (S)-sulpiride were in good accordance with the results from the MS binding assays (Table 7.2).

The entire binding experiment was further validated by characterizing the test compounds in a conventional [3 H]spiperone radioligand binding assay. This control experiment was conducted in analogy to the MS binding experiment except

Table 7.2 Affinities (mean G SEM, n = 3) for dopamine antagonists at D2 receptors obtained by MS-binding assays and by radioligand binding assays, respectively [62]. n.d. Not determined.

Spiperone [3H]Spiperone [3H]Spiperone

(nonbound) (nonbound) (bound)

IC50 (nM) Ki (nM) IC50 (nM) Ki (nM) IC50 (nM) Ki (nM)

(+)-Butaclamol 140 G 50 43 G 10 Chlorpromazine 560 G 90 220 G 20 (S)-Sulpiride 210 G 30 65 G 8

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