Discovery PK Assay Rules

While the rules that need to be followed when developing and using a bioanalyt-ical assay in a GLP setting are well documented [128, 129], there is no standard set of rules to follow when one is developing or using a bioanalytical assay in a drug discovery setting. It is generally agreed that these nonGLP bioanalytical methods do not need to be validated before they can be used for the analysis of discovery (nonGLP) PK samples. This is important because the validation proce-

Fig. 13.8 Time-dependent MS response for pseudoephedrine when 0.1% Tween 80 was used in the formulation that was dosed in the rats via either intravenous (IV) or per os (PO) routes. The HPLC-MS/MS assay was performed in the ESI mode using: (a) Thermo-Finnigan Quantum MS; (b) AB Sciex 3000 MS; (c) Waters-Micromass Quattro Ultima MS. The PK samples were spiked with

Fig. 13.8 Time-dependent MS response for pseudoephedrine when 0.1% Tween 80 was used in the formulation that was dosed in the rats via either intravenous (IV) or per os (PO) routes. The HPLC-MS/MS assay was performed in the ESI mode using: (a) Thermo-Finnigan Quantum MS; (b) AB Sciex 3000 MS; (c) Waters-Micromass Quattro Ultima MS. The PK samples were spiked with the pseudoephedrine after they were collected from the rats. The dip (below 100%) in the profiles shows the time-dependent nature of this type of matrix effect. It can be seen that the effect varied not only with time, but was also dependent on the instrument vendor and the dosing route. Adapted from [119], with permission from John Wiley and Sons.

dure can take 1-2 weeks to complete. Recently, Korfmacher [6] published a set of rules that can be followed for drug discovery PK assays. These rules are based on the concept of increasing the requirements that must be met depending upon the stage in the discovery process where the bioanalytical assay is to be utilized. Korfmacher [6] describes four stages in the drug discovery process (see Fig. 13.1): level I (compound screening), level II (lead optimization), level III (lead qualification) and level IV (development). When the compound is in the level IV development stage, GLP rules need to be followed in most cases (exceptions may be for certain exploratory studies or ''bridging to development studies'' where nonGLP assays can be used).

The rules for level I (screening) assays are shown in Table 13.1. An example of the type of samples where a level I assay could be used is the CARRS samples [85] that can be used for screening NCEs using a rat PK model (vide supra). The concept behind this assay is that it should use a small number of standards and a simple linear extrapolation. For level II assays (see Table 13.2) that might be used for discovery PK studies in preclinical species, a complete standard curve is required. In this case a complete standard curve is defined as 10-15 standards in duplicate assayed with at least five standards used in the final calibration curve. Neither level I nor level II assays require the use of quality control (QC) standards. When a compound is in the lead qualification stage, then a level III assay would be required. As shown in Table 13.3, the main distinction for level III assays is that they are required to include at least six QC standards. As described in Tables 13.1-13.3, these rules show the requirements for how an assay should be set up before the samples are assayed and then these rules describe the acceptance criteria for the assays after they have been performed.

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