I am trying to adapt an HPLC method to measure cortisol in fish plasma. My main problem is that my standard and sample retention times don't match. I have a 20 mg/L hydrocortisone standard (Sigma Aldrich) diluted in 60% acetonitrile. To precipitate the proteins in the sample, the fish plasma was centrifuged in acetonitrile (aceto:plasma 60:40) at 13,000 g for 10 min prior to injection into the HPLC. I suspect that the cortisol in the plasma is still bound to a protein (probably transcortin), which could explain the different RT. Some methods use SPE cartridges to extract cortisol, but I would prefer not to use them if possible to reduce the analysis cost.
Hi Sebastien,
I'm following your question out of general interest. In Gothenburg, we run a classic cortisol RIA (with antibodies validated for little cross-reactivity with corticosterone, of course), but my understanding is that e.g. many clinical laboratories where steroid levels are being measured in humans are moving over to HPLC methods instead of immunological methods for various reasons.
I have no specific advise to give you about your technical problem. There are of course a lot of papers on the use of HPLC for analyzing cortisol, but I'm sure you're aware of these.
You mention the possibility/fact that a part of the cortisol is bound. While this may well be a root of your technical problem, it also opens up the question - which fraction do you want to measure - the free, the bound or the total?
It is intriguing that while for some hormones such as IGF-I, the binding proteins have received a lot of attention, but for other hormones such as steroids and thyroid hormones, which we know are to large extent bound to binding proteins, most of us still measure only total plasma levels. Perhaps we should only be measuring the free levels?
Maybe Sébastien I can help you out. We will talk about it very soon. Your Colleague Etienne Dako
I don't know the answer to your question, but I know HPLC has been used to measure cortisol in fish plasma in Gary Anderson's lab at U. Manitoba, although I can't find the related publication at the moment.
Hello sebastien, i read your question, i think that first of all HPLC is not a right method to quantify Cortisol, if still you want to go for HPLC go for LC-MS as quantity of cortisol is very low in mammals so you need to have a instrument like LC-MS as it can identify cortisol up to pg level. Also for cortisol instead of ACN go for Methanol:water combination. Thanks
Dear Plante,
It is hard to accept the retention time of a certain compound differs in different medium (acetonitril and plasma). specially when fluorescent detection method is applied. However, I faced same problem in my assay of amino acids (gradient mobile phase). By changing the percentage of mobile phases, my problem was solved.
Hope it helps.
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