Dear colleagues,
Hi
Anyone could provide a detailed methods for simultaneous HPLC determination of GSH and GSSG in biological sample (e.g liver tissue). please indicate flow rate, sample preparation, mobile phase, column type, etc. Any HPLC diagram for your method?
Thanks
Mehdi
We used HPLC with electrochemical detector. In liver, GSH may be quite high so you might want to take normal liver and conduct dilution curve to ensure within quantitation range. I have attached a paper we published some time ago. It is a methods paper so pretty well described. I used flow rate of 1.0 mL/min with HPLC. Modern methods using mass spectrometry may be better, but the ECD works fine with HPLC grade water filtered through carbon filters to remove LPS, chlorine, etc. Please feel free to contact me should you need more information.
Thank you very much for your response. Our detector is UV. We used iodoacetic acid and dinitroflourobenzene. A NH2 column, flow rate 1 ml/min. the mobile phase is acetate buffer:methanol and methanol:water with a gradient process. However, surperisingly we could just detect mM councentrations of GSH and GSSG.
If you have enough sample, you could measure GSH, then with second sample, reduce GSSG to GSH to get GSSG value? More steps so that may be limiting factor. The nice thing about ECD was it did both simultanously. Liver is nice as it has lots of GSH. If you use smaller column or change sensitivity of detector? What size flow cell does your detector use? The ECD is reportedly more sensitive but required periodic cleaning.
Dear Sir,
we will try this way again. Thank you so much.
mehdi
Dear Prof Jeffrey Lakritz ,
I appreciate your help in this case, we had a very beautiful standard curve under your advise. I will send you this figure in these days to discus about it.
Thank you in advance for your attention to this matter.
Mehdi
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