NO SUCH THING AS THE BEST TECHNIQUE
“ In our lab, we have opted for the Vitrex technique. ”
Depending on the collected blood volume this can be multiple capillaries, with volumes as low
as 1 µL each. This capillary is transferred to a suitable container and stored then shipped to the bioanalytical facility for further processing. This capillary can be processed in its entirety, or the plasma can be washed-out with a suitable solvent to allow taking multiple aliquots for further processing.
A second technique, the Drummond technique, uses a capillary that contains a gas-open plug on one end, and a thixotropic gel [3]
. After collecting the blood (usually 70 µL), the capillary is
centrifuged. The gel helps separating the blood cells from the plasma, while the gas-open plug prevents leakage of blood from the capillary. However, compounds may migrate into the gel, resulting in a lower recovery of the compound from the plasma. With a piston, through the plug, the entire blood and plasma layer are pushed upwards, until the plasma can be collected into a suitable storage container. From this plasma, accurate aliquots can be taken for further processing. This requires accurate pipetting of volumes of a few microliters from an already small volume.
As aforementioned , after freeze-thawing this may be challenging due to clot formation, which can be an issue with rat and even more so, mouse plasma. Also, this technique requires more than two times the volume of blood compared to the technique with hematocrit capillaries (70 vs 32 µL) to enable a large enough plasma volume from which a series of accurate aliquots (5 µL or more, for smaller volumes the accuracy is reduced considerably) can be taken.
In our lab, we have opted for the Vitrex technique. From 32 µL of blood from rats we easily obtainan accurate 10 µL plasma sample in a capillary. In case of insufficient blood volume, which has happened very rarely, we use a 4 µL capillary to collect the resulting plasma sample. With mice it is also possible to take 10 µL plasma from 32 µL blood, but it may be requiredto switch to 4 µL plasma more often due to the higher hematocrit.
For early phase non-GLP studies, the sample is entirely processed, mostly by the addition of acetonitrile followed by vortex mixing. Clogging of the end-to-end capillary has not been observed and the procedure is quite straightforward. For GLP studies, the sample is not entirely processed as a sufficient sample should be available for eventual re-analysis, incurred sample reproducibility testing or metabolite identification and profiling.
For that reason the sample is washed out with a 2%BSA solution (10 volumes) followed by horizontal shaking for 10 minutes. From the resulting sample, multiple aliquots can then be taken for analysis. With this process, it is important to notice that, unlike the use of a ‘traditional’ plasma sample, there is quite some contact between the cap of the tube and the plasma/buffer mixture during the horizontal shaking while the protein content of the diluted sample is lowered by a factor of ten.
recovery due to adsorption. Using the same set of compounds that we tested for our non- capillary procedure [1]
Depending on the analyte and the type of cap, this might introduce the risk of lowered , we evaluated the recovery with this CMS procedure, using various caps
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