Bioanalytical Techniques

In vitro techniques for studying DDI potential are based on the metabolism of known marker substrates. Two assay types are typically used to study DDIs the turnover of drug-like probes monitored by LC-MS/MS methods or the use of spectrophotometer (plate reader) based methods. As each technique has unique advantages and shortcomings, assay use has not been standardized across the industry. Although techniques based on the turnover of radiolabeled substrates have also been developed [94—97], these methods are used infrequently and will not be discussed further. 

Gentest (now BD Biosciences) was the first to develop spectrophotometric assays to study CYP inhibition [98]. These assays are based on the turnover of mildly fluorescent substrate probes to moderately fluorescent metabolites, where metabolite formation is monitored by an increase in fluorescence using a plate reader [99,100]. Problems with these methods include background interference due to low signal-to-noise ratio, chemotype-specific interference and fluorescence quenching. Aurora Biosciences (now Vertex) has designed probes that exhibit larger fluorescence 

Other techniques to improve throughput are instrumentation based and may involve multiple HPLC systems. The simplest method involves the automated use of solid phase extraction cartridges for sample cleanup followed by direct injection into the mass spectrometer [114], Coupling of multiple HPLC systems to one mass spectrometer allows one column to equilibrate and separate while another column to flow into the mass spectrometer. Multiple HPLC systems may be configured such that the mass spectrometer is only exposed to each serial HPLC eluent as the analyte of interest is eluted [115,116]. Although multiple H P LC-based methods may increase throughput, they also typically decrease sensitivity and may confound data workup and interpretation. 

Both plate reader and mass spectrometry based methods are commonly used for screening. The selection of a technique depends on instrument availability, throughput needs and the stage of compound advancement. For the characterization of compounds in drug development and clinical candidates, assays carried out using drug-like probes and analyzed by LC-MS/MS methods are considered the gold standard [117]. 

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The realization of sensitive bioanalytical methods for measuring dmg and metaboUte concentrations in plasma and other biological fluids (see Automatic INSTRUMENTATION BlosENSORs) and the development of biocompatible polymers that can be tailor made with a wide range of predictable physical properties (see Prosthetic and biomedical devices) have revolutionized the development of pharmaceuticals (qv). Such bioanalytical techniques permit the characterization of pharmacokinetics, ie, the fate of a dmg in the plasma and body as a function of time. The pharmacokinetics of a dmg encompass absorption from the physiological site, distribution to the various compartments of the body, metaboHsm (if any), and excretion from the body (ADME). Clearance is the rate of removal of a dmg from the body and is the sum of all rates of clearance including metaboHsm, elimination, and excretion. 

The development of biological tools to support DDI studies has paralleled the development of bioanalytical techniques. To better understand in vitro-in vivo (IVIV) correlations, the effects of differences in enzyme preparations and incubation conditions must be understood. Differences between enzyme preparations include nonspecific binding, the ratio of accessory proteins (cytochrome b5 and reductase) to CYPs and genetic variability differences in incubation conditions include buffer strength, the presence of inorganic cations and solvent effects. Understanding how biology influences enzymatic activity is crucial to accurate and consistent prediction of the inhibition potential. 

Methods employing low sample volumes (< pL) have been explored, which are aimed at the high-throughput and cost-effective exploration of the design space for chromatographic separations, especially related to bioanalytical techniques focused on large biomolecules analyses (proteins and peptides). 

In bioanalytical techniques based on triggerable dioxetanes, the chemiluminescence emission is generated on command by treating the dioxetane solution with an analyte. 

Van Emon JM, Gerlach CL, Bowman K. Bioseperation and bioanalytical techniques in environmental monitoring. Journal of Chromatography B 1998 715 211-228. 

Pedraglio, S., Rozio, M. G., Misiano, P., Reali, V., Dondio, G., and Bigogno, C. (2007). New perspectives in bioanalytical techniques for preclinical characterization of a drug candidate UPLC-MS/MS in in vitro metabolism and pharmacokinelic studies. J. Pharm. Biomed. Anal. 44 665-673. 

Marjam Behar, National Institutes of Health I joined the faculty of the University of Pennsylvania School of Medicine, Department of Anesthesiology, in December 1962 to work with a group of physicians who were doing studies of cerebral blood flow, and they needed a chemist to do their metabolic studies. I didn t have a tenure-track position. As a matter of fact I was not in the faculty track, but as we advanced in the studies (I was there for 17 years), they made me director of the Core Facility for Analytical Chemistry. I had 12 technicians that I supervised and taught. I also taught residents, faculty members, and medical students who needed to learn bioanalytical techniques to pursue their research. 

However, as already mentioned earlier, high pressure liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) has evolved in the last few years as the major bioanalytical technique for the bioanalysis of analytes in biological matrices. This is reflected also in a number of LC-MS/MS assays for the determination of dihydropyridine calcium antagonists in biological fluids (Carvalho et al. 2001, Schug et al. 2002 Kang et al. 2004). 

TABLE 2 Approximate Protein Impurity Detection Limits for Common Bioanalytical Techniques... 

A diverse range of bioanalytical techniques have been used to study the basic composition and characteristics of biomolecules. Typically these techniques focus on measures of distinct physical and/or chemical attributes, to identify and determine... 

In the arena of large molecule drug development, ligand-binding assays are the bioanalytical techniques of choice. Assays supporting preclinical and clinical studies... 

Chemiluminescence is also found in fireflies. The male firefly uses the reaction of a luciferin substrate and the enzyme luciferase with oxygen, with adenosine triphosphate (ATP) as an energy source, to create the illumination it uses to attract a mate. Because the detection of very minute amounts of light is possible, chemiluminescence and bioluminescence have become the basis of many sensitive analytical and bioanalytical techniques or assays used to quantify particular compounds in samples. Indeed, the use of these techniques is broad enough to justify the existence of a journal devoted to them, the Journal of Bioluminescence and Chemiluminescence. 

The availability of an accurate, precise, and specific bioanalytical technique for the quantification of active drug moieties in plasma, hlood, or other hiological fluids is an essential prerequisite for the evaluation of the relationship between dose, concentration, and effect of hiotech drugs. In analogy to small molecules, these analytical techniques have to he validated and have to meet prespecified criteria regarding accuracy, precision, selectivity, sensitivity, reproducihihty, and stahihty, for example, those recommended hy the US Food and Drug Administration [10-12]. Additional requirements for bioanalytical method validation for macromolecules have recently been published [11]. 

Table 17.3 Some common bioanalytical techniques for stability testing of biologies...

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