Pharmacokinetic (PK) animal studies provide crucial insight into how drug candidates may be absorbed and distributed in the human body and are, therefore, a critical step in drug development. This type of analysis traditionally requires large volumes of blood from one or more test animals in order to provide sufficient plasma volume over several time points for quantitative bioanalysis. Additionally, the plasma needs to be isolated from whole blood, separated, and prepared for bioanalysis using solid phase extraction, liquid-liquid extraction, or protein precipitation.


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Figure 1a: Sample collection 

This time-consuming process limits throughput and consequently how many samples can be tested. Another limitation is the sampling method. Because only a limited number of serial samples can be taken from each animal, composite sampling is often used, resulting in lower quality PK data and an increase in the number of animals required. Finally, there are practical challenges with shipping and storing blood samples, which require controlled handling and frozen transportation and storage.

The dried blood spot (DBS) method is an alternative technique that overcomes these drawbacks. Widely used to screen for metabolic problems in newborn babies, it is now being successfully employed in PK studies.1-4

Collect, punch, measure


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Figure 1b: Punching out a disc of the sample for analysis (Images: Whatman, GE Healthcare)  
Using DBS is straightforward: the blood from the test subject is collected and applied directly onto the FTA DMPK card from Whatman and dried. (Figure 1a) For analysis, a sample disc is punched and the analytes are immediately extracted using aqueous/organic solvent mixtures. (Figure 1b) This extract can then be analyzed by standard LC-MS/MS. The FTA DMPK card lyses cells and denatures proteins on contact. It also inactivates viruses, making handling and shipping of samples safer. Samples can be shipped and stored at ambient temperature and long term stability has been demonstrated for analytes and metabolites sensitive to plasma enzymes.4

Barfield et al. compared DBS sampling with the traditional blood sampling method for the quantification of acetaminophen (paracetamol) in PK studies.2 Their data showed that quantitative analysis with DBS provided high-quality PK data while requiring 10 times less blood from each test animal than current pharmaceutical practices demand. The concentration data for acetaminophen obtained from the analysis of dried blood spots and blood samples was comparable and the scientists noted a number of benefits to using DBS. The lower blood volume required by DBS means that more serial samples can be taken from each animal and fewer animals are needed. The processing of these samples requires less time and once the cards are dried, they can be easily stored at ambient temperature. Furthermore, the DBS method is both precise and accurate for a variety of compounds from different structural classes with acceptable inter- and intra-assay variability.1-4

At a Glance
Whatman, part of GE Healthcare
Product: FTA DMPK card
Date Introduced: July 2009
Product Description: Whatman FTA DMPK cards for the easy collection of blood or any other biological samples. The matrix instantly captures and stabilizes the analytes and inactivates pathogens.

About the Authors
Dr. Robbins studied biochemistry at the University of Michigan and the Salk Institute. He worked for several years in biochemistry at Merck Research Laboratories.  At Whatman, now part of GE Healthcare, he has been involved in R&D, technical support, and technical marketing.

1. Beaudette P, et al. Discovery Stage Pharmacokinetics Using Dried Blood Spots, J Chromatograph B. 2004;809:153-158.
2. Barfield M, et al. Application of Dried Blood Spots combined with HPLC-MS/MS for the quantification of acetaminophen in toxicokinetic studies, J Chromatograph B. 2008;870:32-37.
3. Liang X, et al. Study of Dried Blood Spots Techniques for the Determination of Dextromethorphan and Its Metabolite Dextrorphan in Human Whole Blood by LC-MS/MS, J Chromatograph B. 2009; 877: 799-806.
4. Spooner N. et al. Dried Blood Spots as a Sample Collection Technique for the Determination of Pharmacokinetics in Clinical Studies: Considerations for the Validation of a Quantitative Bioanalytical Method, Anal Chem. 2009;81:1557-1563.