NO SUCH THING AS THE BEST TECHNIQUE
techniques has provided us with the opportunity to adapt procedures to the needs of the project...
“ Having gained experience with multiple ”
If the instability is not so critical that some minutes at room temperature would not influence the concentrations dramatically, there are other options to keep working with CMS:
• Add a stabilizer in the diluent, immediately after plasma sampling, e.g., formic acid, sodium fluoride, or others. In this case the wash-out procedure is done at the in-vivo facility instead of in the bioanalytical lab;
• Immediately process the plasma sample with precipitant, e.g., acetonitrile, after sampling. This is similar to the first situation except that in principle the entire sample preparation is already done at the in-vivo facility. In theory, no further sample processing would then be required and the sample as such is amenable to LC–MS/MS analysis;
• Split samples: instead of collecting plasma into one 10 µL capillary, collect two 4 or 5 µL capillaries and process the samples differently depending on the requirements. This may also be an option in case samples need to be shipped and a back-up sample is required as a precaution in the event of a shipment going wrong. This is not our preferred strategy, as we have never experienced samples being lost during shipment, but it might be common practice in other companies;
• Move to blood CMS, and use one of the options above. Two 15 µL blood capillaries provide the same possibilities as with the plasma CMS procedure without increasing the total blood collection volume.
The above mentioned options are of course also possible with non-capillary microsampling.
SENSITIVITY While with current LC–MS/MS instruments sensitivity is most of the times not an issue for bioanalysis in toxicology studies, there might be situations when for example the compound is administered topically or at low doses and large sample volumes are required to attain the required lower limit of quantification. The above described second option would be possible to avoid extra dilution. Also, one could collect a larger sample, e.g., 64 uL, and modify procedures to end up with a less diluted sample, or take a larger sub aliquot for sample analysis. For non-capillary microsampling, tube volumes of 80 or 100 µL are available, which can also help to resolve the challenges with sensitivity. A volume of 100 µL blood may not sound as a microsample, but often still is a much smaller volume than would be collected via traditional sampling (typically 300 µL of blood from a rat).
METABOLITES
Samples may need to be used for metabolite identification and profiling. This may put higher demands on the sample volumes. However, by selective pooling of samples, the required volume may still be sufficient for this purpose. Option three also provides the possibility to treat a subsample differently from the main sample in case metabolite stability requires this. Taking a slightly larger sample with CMS (and collecting multiple sub samples) or with non- capillary microsampling also offers a way to enable metabolite evaluations if required, while still greatly reducing the impact on animals.
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