Bioanalytical assay methods

The performance of the presented bioanalytical assay method was assessed following the guideline for biological method validation presented by Shah et al. (Shah 2001). 

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... 

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... 

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]. 

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. 

Cetuximab serum concentrations were measured using validated enzyme-linked immunosorbent assay (ELISA) methods or a validated surface plasmon resonance assay. These bioanalytical assays were crossvalidated to allow pooling of PK data across studies. 

A bioanalytical chemistry method is used to support definitive pharmacokinetic and toxicology studies after the assay has been appropriately validated. The validation... 

If not already available, a bioanalytical chemistry method needs to be defined and characterized for the quantification of the lead in physiological fluids. This assay can then support experiments in some of the other scientific disciplines involved in assessing the developability of the lead and, after appropriate validation, the preclinical, nonclinical, and clinical development of a selected drug candidate. For preliminary studies, a bioanalytical chemistry method should be characterized to demonstrate the range of reliable results, the lower and upper limits of quantification, specificity, accuracy, and precision. In addition, evaluations on the matrix to be used (blood, plasma, serum) should be conducted and the stability of the lead in each matrix should be determined. 

Antibodies analyzed (using a validated bioanalytical chemistry method to be transferred to the service provider) for test article concentration to assess extent of exposure Blood specimens collected prior to the first, seventh, and fourteenth dose to be analyzed (using a developed assay to be transferred to the service provider) for antibodies to the test article... 

LC/MS/MS methods for trace analysis (e.g., bioanalytical assays, trace genotoxic impurities)... 

MS/MS) is the standard detector for bioanalytical assays and drug discovery screening, its use for routine assays of drug substances and products is still limited due to its high cost and lower precision. Nevertheless, LC/MS/MS methods are increasingly used for ultra trace analysis or screening of complex samples. Other detection options include conductivity detection for ionic species and electrochemical detection for neuroactive species in biochemical research. 

Validation of bioanalytical assays in general and LBAs in particular has been the subject of intensive debate for the past 18 years or more. Chapter 4 focuses on the key agreements on a phased approach to the validation of LB As, including evaluation of all critical validation parameters prior to implementation of the method to the analysis of any study samples (prestudy validation) as well as in-study validation to assure high performance of the assay during analysis of actual study samples. Also covered in this chapter are the topics of when and how to conduct full validations, partial validations, and cross-validation. 

Table 8.1 provides an overview of various aptamer-based bioanalytical assays for the detection of thrombin, in order to evidence the improvements achieved in the assay detection limit by the use of some of the reported amplification tools. The various methods are explained in detail by the use of several examples. 

S.3 Selection of Metabolite(s) for Monitoring in Clinical Studies and Nonclinical Toxicological Studies The objective of the metabolite measurement using a bioanalytical assay, such as an LC/MS method, is to determine its pharmacokinetic parameters (such as AUC and Cmax values) of a specific metabolite. The MIST document recommends quantifying certain human plasma metabolites, including major metabolites, important active metabolites, and metabolites associated with structural alerts using a... 

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