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November 15, 2009 (Vol. 29, No. 20)

Dried Blood Spots for Toxicokinetic Studies

Reduction of Sample Volumes Required Is One of the Key Benefits of DBS Assays

  • Toxicokinetic (TK) studies play an important role in the preclinical phase of the drug discovery process, determining the relationship between the systemic exposure of a compound in an animal model and its toxicity. Parameters such as bioavailability and dose proportionality of the compound are analyzed, from which the toxicity may be predicted and on which the doses to be used in clinical trials can be decided.

    Plasma or serum samples have traditionally been used for generation of TK data due to the difficulties associated with handling whole blood. However, this approach requires relatively large volumes (100–500 µL) of blood in order to produce the required volume of matrix for bioanalysis, which makes it difficult to generate serial profiles in rodent test models. Composite profiles are therefore necessary, which can result in lower quality data and requires the use of a greater number of animals.

    In recent years, dried blood spot (DBS) assays have received increasing attention as an alternative to venous blood sampling. Although the technique has been around for over 40 years and is widely used in the screening of newborn babies for metabolic abnormalities and clinical trials in remote areas, it is only now starting to be adopted in preclinical safety assessments.

    Sample collection for a preclinical DBS assay involves spotting a small volume of blood (typically between 10-20 µL per sample) onto the DBS collection card (Whatman FTA DMPK from GE Healthcare). These cards consist of a specialized matrix that lyses cells on contact, denatures proteins, and inactivates bacteria and viruses. The resultant samples can then be air dried and stored or shipped at room temperature.

    When required, a disc of typically 3 mm in diameter is punched from the card and the analytes isolated by liquid extraction. Analysis can then carried out by LC-MS/MS, which offers great sensitivity, analysis speed, and selectivity.

  • Advantages of DBS Assays

    Click Image To Enlarge +
    Figure 1. TK profile of dexamethasone in male and female rats over 24 hours by using DBS

    DBS assays offer many advantages in TK studies, from ease of sample collection, storage, and post-collection processing, to quality of data generated. While plasma samples must be stored in a freezer, DBS cards can routinely be stored at room temperature. 

    Stability may be analyte-specific and should be checked, but experience has generally been good. Successful analysis after 56 days on DBS at room temperature has been shown. In addition, the use of DBS presents a low biohazard risk, due to minimal handling of the whole blood and the inactivation of viral and bacterial contamination when the blood sample comes into contact with the card. This pathogen inactivation results from the chemical coatings of FTA DMPK-A and FTA DMPK-B cards and is not available with uncoated cellulose cards.

    The number of animals required for rodent toxicity/TK studies can be drastically reduced by using this approach, as less blood is required per time point and a full time point profile can be collected from each animal. This reduction in animal use is both ethically and economically desirable. Serial sampling also enables better correlations of exposure with toxicity to be drawn, which leads to higher quality rodent TK data than would be possible with plasma or serum sampling. Figure 1 shows an example of a TK profile of dexamethaone in rats over a time period of 24 hours.

    Researchers have also compared DBS sampling with the traditional whole blood sampling method and demonstrated that quantitative analysis of a drug extracted from DBS provides higher quality PK data while reducing the volume of blood required by an order of magnitude lower than the current practice used in the pharmaceutical industry. 

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