Been a bit quiet on this site last few weeks as I have been marking exams.

However …..

my lab has been working on developing and validating a liquid chromatographic assay to detect the drug tramadol in koala plasma.

I’ll divide this work into two parts. This first part will describe what tramadol is and how we have developed and assayed the tramadol this post.  Next post will be about what we do to look at the rate of tramadol metabolism with animal enzymes.

Tramadol is a medicine registered for use in people. It is used for pain relief. It is very closely related to the opioid analgesics such as morphine and methadone. One of the advantages of tramadol is that it can be given by mouth to the patient.  Tramadol is used in veterinary practice. It is also being administered to koalas to provide pain relief to those animals that may have undergone trauma, such as being hit by cars or attacked by feral animals. The Koala Hospital at Port Macquarie, NSW, Australia was interested in how effective tramadol is in koalas and generously provided some funds to develop the assay to detect the drug in the koala plasma.

So we undertook the following steps:

Step 1: we identified the liquid chromatography conditions to identify tramadol and O-desmethyl tramadol (metabolite with known analgesic activity) in koala plasma

Identifying the ‘liquid chromatography conditions’ means that we find the best column and solvent to go through the column with the drug to give narrow drug peaks (as shown by the chromatograms below).

We identified the best internal standard as fluconazole. The internal standard is added to every sample at the same concentration. We measure the concentration of the internal standard as well. If the concentration of the internal standard changes when we detect it, it is a signal that there is a problem with the assay’s conditions.

 Step 2: we then identified the best method to extract the tramadol out of the plasma

We found a ‘liquid-liquid’ extraction to be the best method. This means that we add an organic solvent to the plasma, spin the organic solvent with the plasma, and the drug ‘partitions’ up into the organic solvent, drug in the organic solvent rise to the top of the mixture and we remove this fraction to process further.

Step 3: We then put the drug and internal standard into the mobile phase and this takes it through the column.

The chemical packing in the column allows the target components (drug, metabolite, internal standard to accumulate and progress through the column each at a different speed. Each target component then goes through the machine detector at the same time and the detector output is represented as a consistent peak at that appears at the same time. E.g. tramadol peak occurs at around 7 minutes, the metabolite peak is just after 2.5 mins and the internal standard is at 7.6 mins.


So what the assay looks like at present on the assay readout or ‘chromatogram’:

 40 ng/mL tramadol and its metabolite 0-desmethyl tramadol injected into koala plasma

400_ng_mL tramadol

Note that all the peaks of interest in the chromatograms are separate from each other. That represents good assay ‘selectivity’.

Step 4: Once the tramadol, 0-desmethyl tramadol, and internal standard can be identified as narrow peaks in the chromatogram at consistent times, the assay is validated.  

Validation means that we need to demonstrate the accuracy and the precision of the assay. So we need to show that if we think we have detected 10 ug/mL of drug that it is actually 10 ug/mL.  So to do this we have to make up several different concentrations of the drug and put them through the assay. From the results we plot a standard curve. We then put another concentration through the system and read the predicted concentration from the curve and report how accurate the predicted value is to the actual value.

The closer the predicted value is to the original concentration means the greater the accuracy of the assay. The difference in these two concentrations should not be greater than 20%. The difference between the predicted concentration and the known concentration for our assay is less than 14%.

We also need to report on the assay precision. Precision is a measure of assay reliability. Every time we run the same sample we want the same result (again within 20%).  The variation in the precision of this assay is < 17.38%.

I’ll discuss what we are doing to understand what is happening with tramadol’s metabolite in koalas, next blog.


Thank you to The Koala Hospital at Port Macquarie, NSW, Australia for funds to develop this assay.  


Research projects available in my lab for 2018

Looking for people to undertake a research degree such as a research masters or Doctor of Philosophy (PhD) to take this research and many other projects, further. You will need the equivalent of a good undergraduate degree in biological sciences. For more information see my University of Sydney web page

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