Ascorbic acid by hplc

[Jimmy Boily]

Hi everybody

Can someone give me some ideas for trace analysis of ascorbic acid. I have to quantify tablets containg about 0.5 mg ascorbic acid (otal tablet weight = 50 mg). I Use the usp method (titration) but the end point is not easy and bring me some variations depending who's doing the analysis...

I just want to this is feasable, if yes...I would need some basin information about the mobile phase, wavelengh and colomn...I have a UV detector.

Thank You !

Jimmy, Québec

[Vlad Orlovsky]

Here are few applications for ascorbic acid:
http://www.sielc.com/compound_150.html

Let me know if you have questions.

[grzesiek]

if you search the web, you'll find some methods

[Bryan Evans]

You can do normal phase on Unison UK-Amino:
http://www.imtaktusa.com/site_media/fil ... TI317E.pdf

[krickos]

Hi

Here is breifly the EP monograph related substances test for sodium ascorbate + chromathogram from the knowledge database. Perhaps it can be used as a start unless you find something better.

http://extranet.pheur.org/4DLink1/pdfs/ ... s/1791.pdf

Related substances. ►Liquid chromatography (2.2.29). Prepare the solutions immediately before use.
Phosphate buffer solution. Dissolve 6.8 g of potassium dihydrogen phosphate R in water R and dilute to about 175 ml with the same solvent. Filter (porosity 0.45 µm) and dilute to 1000 ml with water R.
Test solution. Dissolve 0.500 g of the substance to be examined in the phosphate buffer solution and dilute to 10.0 ml with the phosphate buffer solution.
Reference solution (a). Dissolve 10.0 mg of ascorbic acid impurity C CRS in the mobile phase and dilute to 5.0 ml with the mobile phase.
Reference solution (b). Dilute 2.5 ml of reference solution (a) to 100.0 ml with the mobile phase.
Reference solution (c). Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase. Mix 1.0 ml of this solution with 1.0 ml of reference solution (a).
Column:
— size: l = 0.25 m, Ø = 4.6 mm;
— stationary phase: aminopropylsilyl silica gel for chromatography R (5 µm);
— temperature: 45 °C.
Mobile phase: phosphate buffer solution, acetonitrile R1 (30:70 V/V).
Flow rate: 1.0 ml/min.
Detection: spectrophotometer at 210 nm.
Injection: 20 µl of the test solution and reference solutions (b) and (c).
Run time: twice the retention time of sodium ascorbate.
Relative retention with reference to sodium ascorbate (retention time = about 8 min); impurity C = about 1.4.

[rhaefe]

If memory serves me well there were some discussions about ascorbic acid in the past (you can do a search).
Someone cited a paper dealing with the degradation of ascorbic acid in glass autosampler vials etc. Probably interesting information especially at low levels.

Robert Haefele

[HW Mueller]

It has been discussed to death here and the literature is overwhelming, yet I am shocked at the myths still circulating. For instance, I am still convinced that you need a special gift to have ascorbic react with glass. Or a retention time of Na-ascorbate, there is no such a thing. Why 210nm? There is a good max at around 240nm of ascorbic acid (which is being chromatographed). If your matrix reacts with ascorbic one can stabilize it via derivatization (I don´t recommend dinitrophenylhydrazine).
I don´t consider 0.5mg/pill a trace amount. Unless your other stuff in the pill is highly hydrophilic there should be no trouble.

[krickos]

Or a retention time of Na-ascorbate, there is no such a thing. Why 210nm? There is a good max at around 240nm of ascorbic acid (which is being chromatographed).

Agreed the monograph writer should be smacked (although chromatogram names the acid)

As for the wavelength in the monograph, I think (not 100% sure) it was chosen due to a specific impurity.

[Uwe Neue]

Looking at the procedure, I nearly guarantee that it will give you random retention times, even if you buy two columns that are made the same way from the same batch of packing material. I do not understand why one finds the best collection of the worst methods in the world in the pharmacopoeial compendia.

[mbicking]

I do not understand why one finds the best collection of the worst methods in the world in the pharmacopoeial compendia.

Science by committe has never been a good way to get a good result.

Anecdotal story follows: I once was discussing USP with a pharma scientist. He said that in a previous job, he had been told to develop the worst and most difficult method possible, that would still pass validation, and then submit it to USP. And then go ahead and develop a useful one for internal use. It seems that no one wants to let any of their secrets out.

Regarding the specific ascorbic acid method, I recently tested this on C18, silica, and PFP, and got little or no retention under either acidic or neutral conditions. An amide phase might work, but the amino looks like a better choice.

[danko]

Merlin, I don’t know your acquaintance (the pharma scientist) so I can’t judge him – I’m sure he’s a smart guy. But to the most of the people (pharma scientists) that submit to pharmacopoeias, developing a bad method comes rather natural. Maybe that’s all they can do. Regarding the validation of these methods, the procedure is to headhunt a scrupulous statistician, who will manipulate the generated data (valid or not and without a trace of understanding anything of it) to end up with the desired conclusion.
I’ve seen it more often than I care about!

Best Regards

[mbicking]

Sad.

[rhaefe]

@Hans:

I have never done any work with ascorbic acid but here is one reference that floats around this board:

Clinical Chemistry. 2001;47:1463-1464
Technical Briefs
Stability of Ascorbic Acid in Solutions Stored in Autosampler Vials
Sam A. Margolis and Edward Park
1 NIST, Analytical Chemistry Division, Gaithersburg, MD 20899-8392

[Antje_Traub]

Hallo Jimmy,
you can stabilize your solution with m-phosphoric acid. This solution is stable up to 24 ours.
We use a HILIC-column for the analysis of ascorbic acid in aqueous solutions. (www.sequant.com). It works very well.
Bye!

[HW Mueller]

Robert, this must be the ref. which I saw before, it´s possible that there are wizzards among people who publish also?
More seriously: It is no surprise to me to see recurring reports on the instability of ascorbic acid when there is this very bad stability of ascorbate and when government agencies don´t make a clear distinction between Ascorbate and ascorbic acid.