Biomedical Engineering Reference
In-Depth Information
The internal standard chosen for the experiments plays a role in
determining the accuracy of the extraction process. As a matter of
standard practice a compound that is structurally similar to the
analyte is chosen, such that their retention times on the HPLC
column are similar. Similar solubility characteristics ensure concur-
rent extraction with the drug under analysis. The role of the inter-
nal standards is to verify the degree of extraction, as well as for
normalization.
Dounce homogenizer or Potter-Elvehjem homogenizer.
Methanol and/or acetonitrile.
Phosphate-buffered saline (PBS) pH 7.4.
Micro-centrifuge tubes.
Table top centrifuge.
3.1 Extraction of
Drug from Tissue
3.1.1 Materials
1. Place tissue into a Dounce homogenizer or in a Potter-
Elvehjem homogenizer.
2. Add 1 volume of the tissue PBS, and add the appropriate
amount of internal standard and homogenize the tissue
completely and no more tissue chunks are visible.
3. Add 2 volumes of acetonitrile or methanol and mix well.
Transfer mixture to a centrifuge tube.
4. Centrifuge 10,000
3.1.2 Methods
g
for 10 min at room temp or at 4
Cif
drug is thermolabile.
5. Transfer the organic phase to a clean tube.
6. Inject an appropriate amount into HPLC or LC-MS/MS or
use the electrospray method.
4
Transporters
Several transporters exist in the vasculature of the BRB. These
transporters play a central role in the influx and efflux of several
substrates including drugs, toxins, metabolites, and nutrients into
the retinal spaces [
7
,
12
,
13
]. Table
1
shows a summary of the
transporters commonly found in the BRB and their main metabo-
lites. For drug discovery purposes, several methods are available to
measure substrate affinity for a specific transporter. The simplest
form is the use of radiolabeled substrates known to be high affinity
substrates for specific transporters. In such cases, competition with
the novel drug will show any potential interaction. These types of
studies can be done in vitro using a cell line such as TR-iBRB2 cells
[
14
] or using the carotid artery single injection method [
14
].
Additionally, these types of experiments can be done using an LC-
MS/MS instrument if available.
