Bioanalytical assay

Online SPE LC/MS/MS is commonly used for bioanalytical applications in the pharmaceutical industry. Column switching systems and TFC systems are easy to build and control. Sophisticated commercial systems and SPE cartridges are readily available. Compared to offline sample preparation, the online approach can save time and labor. However, the development of online SPE bioanalytical assays remains analyte-dependent. Generic methods can be applied to many analytes. For extremely hydrophobic, hydrophilic, and ionic analytes at normal pH range and analytes with a variety of hydrophobicity and pKa values, analyte-specific methods must be developed. An understanding of the chemistry of the analytes and SPE is critical. 

With decreasing LC run times, sample pretreatment, and the associated method development, sample analysis may be the rate-limiting step in bioanalytical assays. Both semi-automated and automated... 

It is therefore essential that before pivotal (repeat dose) preclinical studies are initiated, bioanalytical assay development must be completed. This has to cover potential test species, normal and diseased humans. The assays must be validated in the sampling matrix of the toxicity test species, and one should also develop suitable assays for antibodies to the test article. 

Soukka T, Rantanen T, Kuningas K (2008) Photon upconversion in homogeneous fluorescence-based bioanalytical assays. Ann N Y Acad Sci 1130 188-200, Fluorescence Methods and Applications Spectroscopy, Imaging, and Probes... 

R. A. Evangelista, A. Poliak, and E. F. G. Templeton, Enzyme-amplified lanthanide luminescence for enzyme detection in bioanalytical assays, Anal. Biochem. 197, 213-224 (1991). 

Hemmlia, 1., Mukkala, V.-M. Time-resolution in fluorometry technologies, labels, and applications in bioanalytical assays. Crit. Rev. Clin. Lab. Sci. 2001, 38, 441-519. 

Korfmacher, W. Bioanalytical assays in a drug discovery environment, in Using Mass Spectrometry for Drug Metabolism Studies, CRC Press, Boca Raton, 2005, pp 1-34. 

Gruber, H. J. Kada, G. Pragl, B. Riener, C. Hahn, C. D. Harms, G. S. Ahrer, W. Dax, T. G. Hohenthanner, K. Knaus, H. G. Preparation of thiol-reactive Cy5 derivatives from commercial Cy5 succinimidyl ester. Bioconjug. Chem. 11(2), 161-166. Gudgin Dickinson, E. F. Poliak, A. Diamandis, E. P. Time-resolved detection of lanthanide luminescence for ultrasensitive bioanalytical assays. J. Photochem. Photobiol. B Biol. 1995,27, 3-19. 

Stereoisomer Assays. There are many drugs that are administered as racemic mixtures. They may undergo stereoselective metabolism and/or elimination, and one isomer may be more active than the other. Therefore, there is the need to develop and validate bioanalytical assays for stereoselective determination in bioavailability/bioequivalence studies. All methods used for measurement of stereoisomer should be validated (with emphasis on stereospecificity). For bioequivalence studies of an existing racemic product, a stereospecific assay is not required if the rate and extent of profiles are superimposable (within the usual statistical boundaries) [3,23]. 

H. Pang, Impact of Biotransformation in Bioanalytical Assays CVG LC-MS Discussion Group, Mar. 21, 2002 (available from www.cvg.ca). 

As shown in Fig. 1.7, the method for evaluating ion suppression/enhancement encountered during a bioanalytical assay involves injection of a processed blank matrix sample on the column with continuous postcolumn infusion of a mixture of an analyte and an internal standard into the LC stream. The analyte and the internal standard are monitored (MRM or SRM scan) throughout the entire LC ran time while the matrix components are eluting from the column. Data from a matrix effect experiment obtained using the postcolumn addition method are given in Fig. 1.8. 

TABLE 2.1. Recommendations to Engineer Quality into Fit-for-Purpose Bioanalytical Assay Characterization in Drug Discovery... 

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