Typical Analytes Small Molecules

Given the foregoing discussion of some of the unique characteristics of macromolecules that lead to clear differences in their pharmacokinetics compared to those typical of small-molecule drugs, there is a subset of the entire group of bioanalytical assay validation parameters that are of key importance in support of pharmacokinetics of candidate macromolecular therapeutics. Assuming demonstration of accuracy and precision of sufficient quality for the intended application of the assay (e.g., non-GLP discovery support or GLP toxicokinetic support, as discussed above), the most important characteristics of a given assay in support of pharmacokinetic studies are likely to be selectivity, specificity, and reproducibility for analysis of incurred samples. These are all related to the ability of the LBA to detect and quantitate solely, or as closely as possible to solely, the analyte of interest. 

The two examples of sample preparation for the analysis of trace material in liquid matrixes are typical of those met in the analytical laboratory. They are dealt with in two quite different ways one uses the now well established cartridge extraction technique which is the most common the other uses a unique type of stationary phase which separates simultaneously on two different principles. Firstly, due to its design it can exclude large molecules from the interacting surface secondly, small molecules that can penetrate to the retentive surface can be separated by dispersive interactions. The two examples given will be the determination of trimethoprim in blood serum and the determination of herbicides in pond water. 

In practice, then, it is fairly straightforward to convert the potential energy determined from an electronic structure calculation into a wealth of thennodynamic data - all that is required is an optimized structure with its associated vibrational frequencies. Given the many levels of electronic structure theory for which analytic second derivatives are available, it is usually worth the effort required to compute the frequencies and then the thermodynamic variables, especially since experimental data are typically measured in this form. For one such quantity, the absolute entropy 5°, which is computed as the sum of Eqs. (10.13), (10.18), (10.24) (for non-linear molecules), and (10.30), theory and experiment are directly comparable. Hout, Levi, and Hehre (1982) computed absolute entropies at 300 K for a large number of small molecules at the MP2/6-31G(d) level and obtained agreement with experiment within 0.1 e.u. for many cases. Absolute heat capacities at constant volume can also be computed using the thermodynamic definition... 

Unfortunately, for the majority of small molecule LC-MS/MS analyses, stable isotope labelled internal standards are not available so far. In such cases, compounds with a very similar molecular structure typically serve as internal standard ( homologues or analogues ). Since the ionization properties are substantially determined by functional groups of a molecule, ionization behaviour may differ significantly—even between compounds with very similar over-all molecular structure. Differential clustering, e.g. with sodium, ammonium or formate ions often present in mobile phases may as well impact the parity of ionization yield between analyte and internal standard. Hence the availability of an appropriate homologue is crucial and critical for the development of reliable LC-MS/MS methods in TDM [51]. 

Only up to 58% of natural rubber can be practically depolymerized to isoprene during pyrolysis. A random chain scission may also take place along the polymeric chain. The result is the formation of molecules of lower molecular weight. However, in order to be volatile enough to be analyzed by typical analytical techniques associated with analytical pyrolysis, these fragments have to be relatively small. The formation of monomers as a final step in the random chain scission is not uncommon, and sometimes it is difficult to decide if a depolymerization or a random chain scission was the first step in pyrolysis. 

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