Analysis of drugs and other toxic substances in biological samples for pharmacokinetic studies

Abstract

The importance of the role of analysis of drugs and other toxic substances in biological samples (bioanalysis) in medicine, toxicology, pharmacology, forensic science, environmental research and other biomedical disciplines is self-evident. Among these disciplines, bioanalysis plays a special pivotal role in pharmacokinetics. The pharmacokinetic parameters, such as half-life, volume of distribution, clearance and bioavailability, of drugs and other compounds are derived from the concentrations of these analytes assayed in the biological samples collected at specified time points. The capability of analysts to develop sensitive and specific analytical methods for the assay of low concentrations of drugs and other toxic compounds in small amounts of biological samples has contributed significantly to the theoretical advances in pharmacokinetics and its applications in clinical pharmacology and the management of drug therapy in patients. The increased demands for pharmacokinetic applications in turn have stimulated the innovation and improvement in bioanalytical technologies. The reliability of the pharmacokinetic conclusions depends on the accuracy and precision of the analytical methods employed to assay the biological samples. Factors that affect the integrity of the bioanalytical data should therefore be controlled in analysis of biological samples for pharmacokinetics studies. The biological samples for drug concentration determination should be collected as specified in the study protocol with respect to the time and site of sampling. These samples should be processed to avoid extraneous interactions between the analytes and sampling devices or additives resulting in the redistribution of the analytes between components of the biological samples, such as displacement of drug binding and changes in the distribution of the analytes between plasma and red blood cells. The stability of the drugs and other analytes in the samples should also be evaluated to establish the conditions suitable for the transportation and storage of the samples to avoid chemical, photochemical and enzymatic degradation of the analytes. Various technologies have been utilized to assay biological samples for pharmacokinetic studies. The most frequently used are chromatography (high-performance liquid chromatography, gas chromatography and thin-layer chromatography), immunoassays and mass spectrometry. Except for some immunoassays, the biological samples are usually prepared by liquid—liquid or solid-phase extraction taking advantage of the pH ionization and partition characteristics of the analytes for preliminary separation of the analytes from other components in the sample matrices. Such extraction procedures should be optimized to obtain high and, most importantly, reproducible recovery. Approaches to simplify the sample preparation procedures have been actively pursued and some advances such as immunoaffinity purification prior to chromatographic separation or other means of quantification and direct introduction of the biological samples for chromatography using column-switching techniques and special stationary phases have been made. Validation of bioanalytical methods is an important task to assure the reliability of the assay data. In addition, validation of analytical methods has also been required by various regulatory agencies. Such validation generally requires the demonstration of the specificity, sensitivity, calibration linearity, extraction recovery, precision and accuracy of the assay methods. However, despite the importance of assay method validation, many of these terms have been defined differently and the criteria for validation of bioanalytical methods are often dependent on the intended application of the assay. For pharmacokinetic evaluations, it is most important that the analytical methods should be specific with no interferences from endogenous and exogenous substances; that the methods should have a low limit of quantification capable of accurate measurement of the compounds over a sufficient period of time following drug dosing or exposure necessary to characterize the pharmacokinetic parameters; and that the accuracy and precision of the assay are demonstrated by appropriate calibration standards and seeded control samples. Analysis of drugs and other toxic substances in biological samples is a challenging task because the concentration of the analytes in the complex matrices are often very low. The interest in free drug concentrations in plasma further requires that the assay method is sensitive to measure even lower concentrations of the free drugs and metabolites. The renewed interest in stereoselectivity of drug disposition has also added the demands for measurement of drug enantiomers. New bioanalytical technologies in chromatography, immunoassays, radioreceptor assays, mass spectrometry and biological sensors are emerging. These, together with the development in automation in bioanalytical laboratories in sample preparation, analysis and data management, provide analysts with an ample armamentarium to meet the present analytical requirements for pharmacokinetic studies. These changes also present an opportunity for innovation and improvement of the bioanalytical technologies to meet the ever demanding requirements of pharmacokinetics and other biomedical sciences that rely on accurate measurement of lower and lower levels of drugs and toxic substances in biological samples.