Bioanalytical methods

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 a single enantiomer as a new active substance should be described in the same manner as for any other new chemical entity. Studies should be carried out with the single enantiomer, but if development began with the race-mate then these studies may also be taken into account. Chiral conversion should be considered early on so that enantiospecific bioanalytical methods may be developed. These methods should be described in chemistry and pharmacy part of the dossier. If the opposite enantiomer is formed in vivo, then it should be evaluated in the same way as other metabolites. For endogenous human chiral compounds, enantiospecific analysis may not be necessary. The enantiomeric purity of the active ingredient used in preclinical and clinical studies should be stated. 

Generic applications for chiral medicinal products should be supported by bioequivalence studies using enantiospecific bioanalytical methods unless both products contain the same, stable, single enantiomer or both products contain a racemate where both enantiomers show linear pharmacokinetics. 

There will be a continued need for enantiospecific methods of preparation and analysis, not only to ensure the quality of the final drug substance and reference materials, but also to control starting materials used for their manufacture, and key intermediates during synthesis. Likewise, specific and sensitive bioanalytical methods will be required to follow the fate of individual enantiomers after their administration. 

US FDA, Bioanalytical Method Vahdation, US Department of Health and Human Services, Public Health Service, Food and Drug Administration, Washington, DC (2001). Also available on the World Wide Web http //www.fda.gov/cder/guidance/index.html. 

In a bioanalytical method, analyses of blank samples (plasma, urine, or other matrix) should be obtained from at least six sources. Each blank sample should be tested for the possible interference of endogenous substances, metabolites, or degradation products. The response of the peaks interfering at the retention time of the analyte should be less than 20% of the response of a lower quantitation limit standard, and should be less than 5% of the response of the internal standard that was used [18, 19]. For dissolution studies, the dissolution media or excipients should not give a peak or spot that has an identical Rt or Rf value with the analyte [20]. 

F. Garofolo, Bioanalytical method validation, in Analytical Method Validation and Instrument Performance Verification (eds. C. C. Chan, H. Lam, Y. C. Lee and X.-U. Zhang), Wiley-Interscience, Hoboken, NJ, 2004, pp. 105-138. 

Guidance for Industry, Bioanalytical method validation, US Department of Health Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Veterinary Medicine. 

Pharmacokinetic/ ADME (absorption, distribution, metabolism, elimination) studies including bioanalytical method development... 

Bioanalytical resources (including bioanalytical methods to support PK studies), biomarker studies... 

For high-throughput analysis, it is important to increase the specihcity of each bioanalytical method. The enhancement of chromatographic resolution presents various limitations. Better selectivity can be obtained with TOF mass analyzers that routinely provide more than 5000 resolution (full width at half-mass or FWHM). The enhanced selectivity of a TOF MS is very attractive for problems such as matrix suppression and metabolite interference. In one report of quantitative analysis using SRM, TOF appeared less sensitive than triple quadrupole methods but exhibited comparable dynamic range with acceptable precision and accuracy.102... 

It is important to keep in mind that safety assessment is only one of many components involved in the discovery and development of new pharmaceuticals. The entire process has become enormously expensive, and completing the transit of a new drug from discovery to market has to be as efficient and expeditious a process as possible. Even the narrow part of this process (safety assessment) is dependent on many separate efforts. Compounds must be made, analytical and bioanalytical methods developed, and dosage formulations developed, to name a few. One needs only to refer to Beyer (1978), Hamner (1982), Matoren (1984), Sneader (1986) (a good short overview), Zbinden, (1992) or Spilker (1994) for more details on this entire process and all of its components. 

Boulanger, B., Chiap, P., Dewe, W., Crommen, J., Hubert, P. An analysis of the SFSTP guide on validation of chromatographic bioanalytical methods progresses and limitations. J. Pharm. Biomed. Anal., 32, 2003, 753-765. 

Because bioanalytical methods constantly improve, development scientists ability to find impurities increases. The cycle could go on forever, and a drug may never be considered truly pure. Developers must strike a balance between creating a process that is far too complex and expensive (both for the manufacturer and ultimately for the patients) and one that will produce a safe drug. 

Matuszewski, B. K. Constanzer, M. L. Chavez-Eng, C. M. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal. Chem. 2003, 75, 3019-3030. 

Olah, T. V The development and implementation of bioanalytical methods using LC-MS to support ADME studies in early drug discovery and candidate selection. Ernst Schering Res Found Workshop 2002, 155-183. 

Close cooperation for a year or more before the first administration to humans is likely to lead to a smooth transfer of the compound and the rapid movement of a compoimd out of preclinical into man. This lead time can be used to devise the ED plan, design the first studies and, when appropriate, to select and develop methodologies that will contribute to the drug s evaluation in man. This may include validation of pharmacod)mamic measures to be used in the clinical pharmacology unit, assessment of various imaging techniques, development of bioanalytical methods. Not infrequently, the assays that were perfectly adequate to support preclinical work are insufficiently sensitive, specific or accurate to quantify the comparatively low concentrations of parent... 

Selectivity in a detector is most often required for sensitive bioanalytical methods where trace amounts of compounds are being analysed in the presence of interferants which are also present in the sample matrix. The properties of some commonly used detectors are summarised in Table 11.3. 

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