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Topic: HPLC analysis on compounds prone to oxidation  (Read 562 times)

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Offline moorufous

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HPLC analysis on compounds prone to oxidation
« on: December 06, 2023, 10:33:47 AM »
What are your thoughts on the accurate quantification of compounds in their reduced form prone to oxidation during HPLC analysis?

I've been on the search for r-dihydrolipoic acid on the market and have found a company that claims they cannot perform a certificate of analysis due to rapid oxidation of R-DHLA back into RLA during HPLC analysis. Is that a valid claim?

I would like to independently analyze this R-DHLA since I've already purchased some, any suggestions on how I can proceed for the most accurate analysis?

Offline Babcock_Hall

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Re: HPLC analysis on compounds prone to oxidation
« Reply #1 on: December 06, 2023, 04:12:05 PM »
I cannot say whether or not the claim is valid.  If I were trying to develop an assay using HPLC, I would consider working under slightly acidic conditions, and I would try to remove metal ions, with the goal minimizing the rate of oxidation.  If you just need to know whether the compound is oxidized or reduced, I suggest using Ellman's reagent or an equivalent compound in a spectrophotometric assay.  There is an article about Ellman's reagent by Riddles in Methods in Enzymology, circa 1980.  The molar absorptivity of the chromophore produced in the assay is a little greater than 14,000.

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