34 February / March 2016
Review of Microsampling Techniques in Bioanalysis
by Tony Edge (University of Liverpool), Chris Smith (Bioanalytics Elanco)
The pharmaceutical industry strives to generate new revenue and simultaneously reduce costs associated with research activities. The concept of microsampling tackles both of these issues, by addressing the technical challenges associated with the sampling of neonatals, and also addressing the issue associated with composite Pharmacokinetic (PK) profiles, which reduce the need in animal studies for larger sets of animals to be used. Clearly reducing the number of animals required for testing also addresses an important ethical issue.
The article will cover all aspects of microsampling, looking at the use of dried blood spots, and the use calibrated capillaries for the original sample collection. The use of blood or plasma as the sample will be assessed with particular reference to the haematocrit issue, as will be the issue of sample stability in these different forms.
Introduction
Within the pharmaceutical industry bioanalysis is used to determine the concentrations of the parent compound and any active metabolites within the biological entity. By plotting the concentration of these compounds as a function of the time, and interpreting the resulting curve, it is possible to determine the correct dosage for the patient. Factors such as AUC (area under the curve), Cmax
(the maximum observed concentration within the body) and t1/2 (time
required for the concentration to fall by half) allow for the efficacy of the drug to be determined over a period of time after dosing [1,2].
Reasons for Microsampling
There are many scientific and ethical reasons for the interest in microsampling. The pharmaceutical industry has placed substantial emphasis on the 3R’s, (reduction, replacement, and refinement) and this has helped in reducing the volumes of samples taken from animals, which in turn can lead to a reduction of animals required for testing during pharmacokinetic profiling studies of a drug, as well as improving the scientific data.
Early Discovery pharmacokinetic studies routinely use rodents (typically rats or mice) as test animals, and substantial efforts
Sample volumes for Bioanalysis
Sample volume (% of blood volume in 25 g mouse)
Number of animals in study
have been focussed on implementing microsampling into this area. There is clearly a potential issue with blood volume in both of these rodent species and in particular if multiple samples are to be taken from one animal, removing the need for composite studies. These large volumes could cause anaemia or other secondary effects such as bone marrow and haematological changes, which would affect interpretation of primary drug effects. Wilson [3] stated that for a typical 4 week repeat oral dose rat study an additional three to nine satellite animals per dose group per sex might be required (depending upon the sample volume and number of time points required) in addition to ten main study animals. Investigation of longer studies, as seen in Table 1, will dramatically reduce the number of animals required. The reduction in the number of animals also results in a substantial reduction in the labour associated with these studies, as well as improving the scientific credibility of the data due to a reduction in the number of satellite animals.
The concept of microsampling has been around for some time [4-6] although the exact definition of a microsample varies. It has been generally accepted that microsampling refers to volumes less than 50 µL taken from the patient. The challenges associated with microsampling relate to being able to manipulate small volumes in an accurate manner as well as the detection of the analytes under investigation. The performance improvements associated with the detection devices currently available, in particular mass spectrometry, have resulted in the latter being less of an issue.
Approaches to Microsampling Dried Blood Spots (DBS)
Different approaches have been employed to allow the collection of the blood sample. One of the most popular is the use of dried blood spots, which are routinely used on newborns for the so called Heel prick or Guthrie test, named
Table 1 Comparison between 2 different designs of a 6 month toxicology mouse study – a conventional design and a microsampling design without satellite animals [3].
Conventional design with satellite animals
200-300 µL 10-15% 238
Total number of dosing’s 42,840 Hours of work
(husbandry and dosing) 2,100 hours
Microsampling design without satellite animals
32 µL 1.5% 160 28,800 1400 hours Reduction 6 – 10 times less 6 – 10 times less 78 14,000 700 hours
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