Antioxidant to Ascorbic Acid.

[Jamie]

I have developed a method for Ascorbic acid analysis using Synergi Hydro RP column. My mobile phase is Citric Acid pH 2.5. Flow is 1.0 ml/min. The problem is ascorbic acid oxidizes very fast. The matrix is a alkaline aqueous solution. What is a good antioxidant to this antioxidant? How can I tell what is a better oxygen scavenger from a list of oxygen scavengers. My sample solvent is my mobile phase.

I have read an article that vials that are not washed with acid causes this problem because of ferric impurities. So I washed my vials with acid to see if this true but I did not see an improvement. I tried D Ribose in my calibration standards and matrix but I see only 1% improvement.

[Mattias]

Sodium pyrosulphate (or known as metabisulfite) is a quite powerful antioxidant.

Butylated hydroxyanisole and butylated hydroxytoluene might work also, but they do not dissolve in 100% aqueous. If you have problems with metalions you could try to add some EDTA.

[HW Mueller]

Ascorbic does not oxidize fast at acidic pH (fairly safe: pH 4 or below). So what you do depends on what your problem is. Why is there a basic matrix? If the pH is high enough you immediatly get discoloration (yellow) of ascorbic, indicationg a whole cascade of nearly instantaneous reactions.
You can not get rid of catalyzing metal ions unless you add gobs of EDTA, etc. Also, about any mercaptan, even cystein, will stop oxidation for some time near pH = 7.

[Jamie]

My matrix is a photgraphic developer. It is alkaline, ph 10.5, and already contains EDTA type sequestering agents, and plenty of sulfite. Yes, I notice that my standards in my citric acid solvent has slower oxidation rate than plain water but the next day almost all is gone. You can literaly see the peak area decrease with each injection.

Thanks for the tips. I will try them.

[Fabiano]

It is probable that you don´t have oxidation but hydrolysis. Just put the pH to 4 or more acid.

For avoid oxidation you can try Homocysteine, Dithiothreitol or tri(carboxyethyl)phosphine. Dithiothreitol is the standard chemical for reduce dehydroascorbic to ascorbic in clinical assays.

[Mattias]

Jamie!

Do you have the reference for the article you mentioned (ferric impurities in LC vials)?

I have seen unexpected oxidation in some of my samples as well.

many thanks!

[HW Mueller]

What are you trying to do? Determine how much ascorbic someone put in your solutions or how much of an initially known amount of ascorbic has disappeared after a given time? Two persons have told you now that ascorbic may not be stable at basic pH.

[Jamie]

Mattias:

Look at: http://www.clinchem.org/cgi/content/full/47/8/1463

[Jamie]

Fabiano:

Thank you for your post. In the same website I gave Mattias, the references list an article by Margolis S.A. the use of dithiothreitol or metaphosphoric acid to preserve ascorbic acid in human plasma. I will soon try that.

Hw Mueller:

I am trying to measure the oxidation rate of ascorbic acid or acsorbate with time as it is exposed to air and also how to reduce the oxidation rate in a product.I am using HPLC to measure the deterioration rate. I need to preserve my calibration standards and perhaps dithiothreitol is the answer.

Thank you all for your posts.

Jamie

[HW Mueller]

It could be that Fabiano is right. I don´t know, off the head, at what pH one gets that decoloration I mentioned, it seems to me pH = 10.5 is a good candidate. The instantaneous yellowing of ascorbic is not, or mostly not, due to oxidation. Thus it would be surprising if a -SH species would help (especially since sulfite didn´t).
It would be nice to let us know....
Just remembered: The yellow color immediatly disappears if one acidifies the solution (early on), but this action does not reverse most of the reactions that occurred at the basic pH.

[Fabiano]

Hans:

I think that the color of acidified solution disapear cause some supression in dissociation of enol/double bond. in HPLC we se some hypsochromic shift depending on mobile phase pH. with MP pH at 4-5 we can detect easily in 265 nm but with MP in pH 2,5 we can only use 245 nm.

If anyone is interested Dehydroascorbic hydrolysis (half life) occurs at pH of 6 in 100 minutes.
http://www.clinchem.org/cgi/content/abstract/36/10/1807

Ascorbic acid kinetics should be very similar.

[ym3142]

if you keep ph 10.5, can you run the following:
1) prepare a fresh standard, test by the LC(one injection) immediately ; keep the LC in stabilization status;
2) repeat the above exactly five more time to establish the system suit;
3) test another standard prepared freshly like the above at 0, 10, 20, 30, 100, 500 min ... to find the degradation curve;
4) test a sample with the recording of the time period from sample prepartion to injection. do correction based on the curve from 3).

should this work?

[HW Mueller]

Fabiano, I have been "off" ascorbic for some years, but seem to remember that the max of ascorbic acid is 245nm, that of ascorbate 265nm, that of the radical near 340? All of this is well characterised as well as some of the chemistry of dehydroascorbic (DHA). The irreversible decomposition of DHA toward gulonic acid, etc, etc, is rapid under basic conditions. Some people have looked at these transformations, it is so complex that everybody in this field seems to loose interest after some time.
The point of Fabiano and me seems to be: The disappearance of ascorbic at basic pH is not only due to oxidation.

[dcobice]

Hi all,
I had the same problem with the stabilization of ascorbic acid and I found a chromatographic platform that allow me to run 20 hours ( stabillty of the samples and Std) I was using a SAX columns for agilent and the pH was adjusted to 6.8 with amonium formiate.
The sample diluet was acetonitrile 80% and 20% of water + 1% of metaphosphoric acid to avoid the degradation.
The sample cooler was set at 4 degress.
If you are interest in the method I will send you by e-mail.

thanks

Diego

[Jamie]

To All:

Thank you for your help. I will implement the recommendations you have made and will post my results.

Diego, please send me an email of your successful procedure.: jimmybvilla@hotmail.com

I will ask a previous question: Of the many oxygygen scavengers, are there rules to follow to detemine what is the best for preserving a unstable chemical compound? Or are they determined by experiment?

Jamie