4.7. Separation and quantification of biogenic amines by HPLC
For each standard and sample:
1. Load 10 μl of the collected supernatant into the HPLC system.
2. Identify the amines in an HPLC trace by determining the retention time and relative peak sizes for each of the detector channels.
The exact retention times for the amines will vary according to analysis conditions, but they do occur in the specific order given in the example below.
3. For each amine, determine the area of the peak for the one channel on which it was largest.
An example of amine characteristics follows. Although the order in which these peaks appear will remain constant, the specific times and channel responses will vary depending on the HPLC system, flow rate, and applied currents:
Octopamine (OA): 5.2 min, big Channel 1 peak, smaller Channel 2 peak
DHBA: 5.5 min, big Channel 2 peak, smaller Channels 1 and 3 peaks
Synephrine: 6.0 min, big Channel 1 peak, smaller Channel 2 peak
Dopamine (DA): 6.6 min, big Channel 2 peak, smaller Channel 1 and 3 peaks
Tyramine (TA): 9.6 min, big Channel 1 peak, smaller Channel 2 peak
Serotonin (5-HT): 10.2 min, big Channel 2 peak, smaller Channels 1 and 3 peaks
4. If the standards maintained comparable values through multiple batches of samples, a single standard curve can be calculated from their combined data. If the standard values changed over time, each batch of samples will need to be compared to a curve calculated from the standards run just prior to and immediately following each batch.
In order to be sure that any differences in amine content are due to true physiological differences and not just differences in the quantity of tissue from which the amines were extracted, it is necessary to standardize the amine content against tissue content. Several approaches are available to determine protein content, such as the Bradford or Lowry assays (see section 1.2), but the accuracy of many of these assays is compromised by the minute volume of tissue involved in an individual brain. An alternative and simple approach is to determine the dry mass of the brain tissue.
To quantify brain mass:
1. Remove the residual tissue pellet from the sample tube.
2. Place it on a pre-weighed and labelled 1cm square section of foil.
3. Fold these squares over to secure the sample.
4. Place them in an oven set to 100 °C.
5. Bake the samples for 24 h.
6. Weigh the squares on a scale that can measure microgram differences.