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Dear Dr. Bajad
I have been working last months on some assays of oxidative stress
biomarkers. One of the candidates I would like to measure in blood is
ascorbic acid in its oxidized as well as in its reduced form. For this
purpose, I have an HPLC equipped with a Coul-Array detector
(manufacturer: ESA). This detector consists of 8 electrochemical cells,
one after another, functioning independently.
I wonder whether you can share with me some of your experience regarding
the following issues:
1. A source of a reference material of dehydroascorbic acid.
2. Pre-analytical handling of blood samples and stabilization of
ascorbic acid and dehydroascorbic acid in blood/plasma samples upon
storage at -70.
3. The method you use to assay both compounds. Do you do it
4. Critical points in the handling, processing and analyzing blood
samples for Vit. C in both forms.
5. Quality control program of Vit C in both forms.
Thank you for your attention and help.
Dr. Shlomo Almog
Head, The Clinical Pharmacology and toxicology Lab
Sheba Medical Center
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Dear Dr. Shlomo,
Here are some of the points about Ascorbic acid
1. Ascorbic acid(AA) and dehydroascorbic acid(DHA) are
stable in metaphosphoric acid (MPA, 5% w/v).
2. DHA is highly unstable in plasma (t1/2 ~2 min)
3. So we immediately add 50% w/v cold MPA to plasma(90
ul plasma + 10 ul 50% MPA).
4. Both AA and DHA can be analyzed simultaneously.
5. Since the samples are expected to sit in the
autosampler for some time (batch analysis), the
processed samples should contain 5% MPA to avoid
degradation in autosampler.
6.We kep autosampler temp at 5 oC.
7. DHA is available from Sigma.
Hope it helps.
Dr. Sunil bajad
Department of Medical Sciences
School of Vet. Medicine
University of Wisconsin-Madison,
2015 Linden Drive west,
Madison 53706, WI,USA
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