Sensitivity analysis metabolite quantitation studies

Mass spectrometric approaches are also very useful for the measurement of stable isotopes in drug metabolism studies. The application of MS to the quantitative measurement of stable isotope has been limited due to the high cost and sophistication of the instruments necessary for stable isotope enrichment studies. Nonetheless, recent improvements in instrument design and performance, as well as computer software for instrument control, data acquisition, and analysis, have increased the sensitivity and reliability of stable isotopic enrichment studies. These new MS instruments, including continuous-flow isotope ratio mass spectrometry (CF-IRMS) and HPLC-chemical reaction interface mass spectrometry (HPLC-CRIMS) are increasingly less expensive, easier to operate, and accessible for mass balance/ metabolite identification studies with stable isotopes. 

The combination of LC with MS has created a powerful tool for the analysis and quantitation of a variety of dmgs and metabolites in complex matrices. LC-MS has turned out to be a particularly sensitive and selective technique for the analysis and quantitation of pharmaceuticals. It is a fundamental analytical tool for the studies of drug metabolism of new dmg candidates and the identification and characterization of impurities and degradents in pharmaceuticals. Further transfer of the routine activity of immunosuppressants from immunoassays to LC-MS, which allows the automation of sample preparation, shortens analytical run times and is probably cost-effective despite heavy investment costs. 

Currently, LC-MS is widely used for the analysis of polar compounds, such as medicinal metabolites and bioactive peptides, since the interface has been improved and several new ionization methods have been developed. The sensitivity and reproducibility are sufficient for a daily quantitative analysis. The usefulness of the LC-MS has been demonstrated for studies on Type II pheromones using a time-of-flight MS with electrospray ionization (ESI) [180]. Each epoxydiene derived from the (Z3,Z6,Z9)-triene shows three ion series of [M+NHJ+, [M+H]+, and [M-OH]+ with high resolution and good sensitivity, indicating its molecular formula. In addition to these, characteristic fragment... 

In many instances, such as in studies of metabolism or degradation where the sensitivity is not so important, TLC and radioscanning provide a quick and simple technique for the identification and quantitation of radiolabeled metabolic products. An example of a radiochromatogram scan is shown in Fig. 2.13 for the analysis of some metabolites of the insecticide ethyl parathion in a microsomal enzyme preparation from rat liver [SI]. 

High-sensitivity techniques for the quantitation and characterization of circulating metabolites following administration of radiolabeled compounds are of critical importance to understand the safety and efficacy profiles of novel drug candidates. AMS is one of the most sensitive techniques for the detection of radiolabeled components. However, the high cost and slow throughput of AMS analysis preclude the routine use of the techniques for metabolism studies. 

The use of SRM methods for quantitative bioanalysis represents increased dimensions of mass spectrometry analysis. SRM methods that use APCI-LC/MS/MS for the quantitative analysis of an antipsychotic agent, clozapine, in human plasma were described by Dear and co-workers (Dear et al., 1998). Preclinical development studies of clozapine in rats and dogs used HPLC with fluorescence detection (FLD). With this method, a better limit of quantitation (LOQ) of 1 ng/mL was obtained. As the compound moved into the clinical stages of development, a more sensitive method of analysis was required to obtain rapid metabolic information in support of drug safety evaluation studies. A standard LC/MS/MS method is used for the quantitative analysis of clozapine (I) and four metabolites (II-V) in human plasma (Figure 6.34). 

Cellular Metabolites. - A review of methods for the measurement of ml has been produced with 95 references. It examines the quantitative measurement of ml by mass spectrometry and in vivo NMR. The NMR chemical shifts and /-coupling values of 35 metabolites which can be detected by in vivo or in vitro investigations of the mammalian brain have been published. The principles and recent applications of dynamic nuclear polarisation, which combines the sensitivity to oxygen of EPR and the tractability of NMR imaging, have been reviewed with 244 references. A review of studies of intermediary metabolism, including the use of NMR in the analysis of substrate selection under in vivo conditions, has been produced. A review has been produced, with 74 references, on the study of metabolic flux and subcellular transport of metabolites using NMR. " ... 

---