Chromatography Forum

Hello,

I want to quantify dehydroascorbic acid by HPLC.In our product we want to quantify how much Ascorbic Acid (AA) is coverting into DHA. Are there any reverse phase methods for DHA ?? Or Reduction of DHA into AA is the only option??

Thanks in advance

Ascorbic acid is very water soluble and will not be retained on a typical reverse phase column.

Other options:
Ion pairing method using an ammonium salt;
An "aqueous-stable" reverse phase column; I think Uwe has recommended a Waters version in the past, and there are others on the market;
A mixed mode column, such as those from SIELC;
A HILIC column (silica with acn/water).

Search the web sites for similar applications.

Now, whether or not these columns will separate both your analytes is another question, but one or more of these should at least get you some retention.

Here are few methods for ascorbic acid (no DHA here) I know that at least two companies who are using Primesep D column to measure DHA/AA. I can't give more details (It is covered by CDA) but they are using ACN water with AcOH for quantifying this pair in biofluids:
http://www.sielc.com/application_165.html

Components of plasma will repel from stationary phase, but you might want to use guard column:
http://www.sielc.com/Technology_DirectP ... lysis.html

Regards,

Vlad

P.S. I represent SILEC Technologies, manufacturer of mixed-mode columns

HILIC is a viable alternative for this purpose.
Below, I have added two references, the first is and in-house prepared application note, and the other is a recently published study on Ascorbic Acid stability.


HILIC Separation of Dehydroascorbate and Ascorbic Acid
http://www.sequant.com/sn/ufiles/SeQuan ... 00-12A.pdf


Hydrophilic Interaction Liquid Chromatography Method for the Determination of Ascorbic Acid
L. Nováková, D. Solichová, S. Pavloviová, P. Solich
J. Sep. Sci., 31 (2008) 1634-1644

This was mentioned many times before: I had no trouble with HPLC of ascorbic in tomatoes on C-18 columns (RP!). I finally gave up doing Ascorbic acid in plasma.
Patrik, this is the first time I hear about a direct HPLC analysis of DHA. Since I couldn´t make the link work I wonder how the chromatographer managed to get DHA through the column since it rapidly decomposes irreversibly to gulonic acid, etc., and is in very rapid eqilibrium with ascorbic acid via the radical intermediate in aqu. media (DHA does a disproportionation with ascorbic).

Dear Hans,

One of the advantages with HILIC, is as you know, the proportionally high concentrations of organic solvent in the mobile phase. This, I believe, could sometime be very beneficial for unstable compounds.

For the simultaneous separation of AA and DHA, I can only relate to my own in-house results, which are summarized in the application note found via the previously posted hyperlink. Initially, I did observe some initial trouble with DHA stability, but after introduction of brown sample vials, this was solved and it became a straightforward approach.

Another possibility could be the use of stabilizing agents in the samples.

I quote the last section from the paper by Nováková et al in J. Sep Sci.

"The key problem of AA analysis is its instability in solutions. The influence of individual factors decreasing AA stability (the influence of temperature, pH, degassing the mobile phase, commonly used stabilizing agents, and concentration of AA in solution) taking into account HILIC approach specifics was deeply studied and described. The optimum stability under HILIC conditions could be ensured by decreasing the autosampler temperature to 4 degrees C, measuring more concentrated solutions, if possible, and by the addition of either 10 mM oxalic acid or 5% o-phosphoric acid as stabilizing agents to samples/standards. Both stabilizing reagents gave good results as was verified by SST measurements."

As always, alterations to given experimental conditions might be needed to fit the users specific applications, thus some additional method development could be required.

I wondered about DHA not ascorbic acid. In nonaqu. media DHA is more or less undefined, though I would think that any HILIC technique works with enough water to hydrolyze DHA.

Dehydroascorbic acid and related compounds on Unison UK-Amino:
http://www.silvertonesciences.com/files/TI341E.pdf

Bryan, is there more info available on this? Is it known what species actually elutes as the #1 peak? (I have never seen anybody seriously suggest that the triketone actually exists). Is the same standard used by both methods? Are there real compounds at the unknown peak?

Hans - thank you for your question.

Imtakt has data (under similar conditions) with and without
dehydroascorbic acid:

Dehydroascorbic acid / isoascorbic acid / L-ascorbic acid (LC-ELSD)
http://www.silvertonesciences.com/files/TI341E.pdf

isoascorbic acid / L-ascorbic acid (LC-UV)
http://www.silvertonesciences.com/files/TI317E.pdf

The unknown peak is observed in data with ELSD (and / or)
dehydroascorbic acid.

If Imtakt has any input - I will post it on here.

My guess that "unknown" peak is sodium. With ELSD the void market on this column will elute around 1.6-1.7 minutes and unknown peak is around this time (or before void, due to repulsion).

Vlad

Using Citric acid in the diluent and mobile phase, provides both some degree of solution stability and also retention in reversed phase.

here is fast mixed-mode approach to analysis of ascorbic and dehydroascorbic acids using LC.MS and ELSD compatible conditions:
http://www.sielc.com/application_145.html

Sielc_Tech, is there an MS of dehydroascorbic acid available?