Toxicokinetic assays

An appropriate tool to enhance the selectivity and the feasibility of the work-up procedure using liq-uid/liquid extraction is the subsequent back extraction into aqueous phase. For example, Los et al. (1996) described a toxicokinetic assay for diltiazem and its two metabolites. To 0.2 mL rat plasma, internal standard solution and a pH 7.3 phosphate buffer and methyl-t-butyl ether were added. The analytes were... 

For sample preparation, protein precipitation or liq-uid/liquid extraction can also be applied instead of solid phase extraction. Gluth et al. (1988) described for a toxicokinetic assay for Sotalol a threefold combination of these principles. A protein precipitation using 5 M perchloric acid was followed by a liquid/liquid extraction into a mixture of n-pentanol-chloroform 1/3 at pH 9. Thereafter, the organic phase was transferred to another glass tube and the analyte back extracted into 0.05 M sulfuric acid. 

For HPLC MS/MS assays the use of stable isotope labeled internal standards is by far the best method to overcome any potential matrix effects and random variation in the MS/MS detector. If for any reason this stable isotope internal standard is not available, an analog compound with a mass different from the analyte can also be used. The chromatographic retention time of the internal standard, however, should be as close as possible to the retention time of the analyte. This ensures, that time dependent random variation in the ionization chamber, or whereever else in the MS/MS detector, are compensated by the internal standard. In a toxicokinetic assay described by Chi et al. (2003), for example, an internal standard was used which showed the same retention time as the analyte. 

Instead of the sandwich ELISA-system, which was applied by Damle et al. (1998), Hildebrand et al. (1996) and Audebert et al. (1994), the competitive RIA-system was applied by Wring et al. (1996) and Basu et al. (1996) for toxicokinetic assays. The competitive RIA-system needs only one antibody but this may lead to a loss of selectivity. 

GLP regulations require confirmation of the potency of all formulations used in nonclinical studies. Furthermore, current ICH guidelines also require toxicokinetic data (i.e. animal pharmacokinetics determined at one or more time points during a nonclinical toxicology study). Both the potency and the toxicokinetic assays require an analytical method to determine the parent drug... 

The problem of potentiation was discussed earlier (Chapter 2, Section 2.5). Potentiation is often the consequence of interactions at the toxicokinetic level, especially inhibition of detoxication or increased activation. The consequences of such potentiation may be evident not only at the whole animal level but also in enhanced responses of biomarker assays that measure toxicity (Figure 13.3). By contrast, biomarkers of exposure alone are unlikely to give any indication of potentiation at the toxicokinetic level. 

Operational and metabolic considerations generally make urine sampling and assay of limited value for toxicokinetic purposes. 

An enzyme-linked immunosorbent assay (ELISA) was developed by Van der Schans and colleagues for detection of the mustard adduct within DNA, using monoclonal antibodies raised against N7-HETE-guanosine-5 -phosphate coupled to keyhole limpet hemocyanin (n). The ELISA was successfully applied in toxicokinetic studies in which levels of adducted DNA were followed in conjunction with measurement of intact sulfur mustard (12). 

In Note 8 of the ICH Guidance Toxicokinetics 1994 it is stated It is often considered unnecessary to assay samples from control groups. Samples may be collected and then assayed if it is deemed that this may help in the interpretation of the toxicity findings, or in the valida-... 

The described method is a typical HPLC fluorescence assay for drug level determination for toxicokinetic purposes. However, the conditions of sample extraction, the choice of the internal standard substance, the choice of the HPLC stationary and mobile phase and the combination of excitation and emission wavelength has to be adjusted specifically to the properties of the analytes. Particularly, the lipophilicity, the pKa value and the pH stability of the analytes have to be considered. 

Levels of drug and/or its metabolite have to be determined in plasma samples collected during the course of a toxicity study in order to evaluate the toxicokinetics of that compound. One of the most selective and sensitive assay method is the LC-MS/MS technique. Due to the high intrinsic selectivity of the detection method there is no necessity for a selective or compound specific work-up procedure. However, proteins and matrix... 

The described methods are typical immunoassays for drug level determination for toxicokinetic purposes. However, for each analyte specific monoclonal or polyclonal antibodies have to be generated in animals, which takes a relatively long time period. Having these antibodies available, the work up and the assay conditions have to be adjusted for the combination of antibody and analyte (antigen). The use of immuno-... 

An example from our own laboratory is the methylphenidate bioanalyti-cal chiral LC-MS/MS assay in support of toxicokinetics (TK) and PK studies (vide infra). Attention-deflcit hyperactivity disorder (ADHD) is a recognized medical problem characterized by symptoms of inattention, hyperactivity, and impulsivity. Methylphenidate (MPH Ritalin methyl-alpha-phenyl-2-piperid-inacetate hydrochloride) is prescribed for the treatment of ADHD. MPH has two chiral centers yielding enantiomers with distinct pharmacokinetic and pharmacodynamic properties in humans. It is known that the t/-threo [2R,2 R]-MPH (i.e., (-i-)-threo) exhibits greater pharmacological activity than the... 

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