Vitamin C measurement
I know this is some way from the original enquiry for a lab to measure vitamin C, but there are some points arising from recent messages that will be of interest to mailbase readers.
1) Ascorbic acid is not unstable - it is highly reactive, which is quite different. If you don’t give ascorbic acid anything to react with it is suprisingly resistant to oxidation.
2) Hence a pure ascorbate standard (and I don’t mean that brown-ish stuff you’ve had in a cupboard for the past 5 years) made up in ultra-pure water, in a new plastic tissue culture container, will be stable in the fridge for more than 24 hours.
3) Metaphosphoric acid was introduced to de-proteinise samples and as a reagent at a time when ultra-pure trichloracetic acid was not available; the TCA that you can buy now is much more pure than the MPA with regard to metal content and other impurities and would be the acid of choice.
4) Vitamin C is more stable in whole blood on the bench than in separated plasma for 12 hours – possibly longer; this is my experience, but you’ll also find it in the literature.
5) Vitamin C is not infrequently destroyed by procedures designed to preserve it in biological samples.
6) Many of the problems people encounter with vitamin C HPLC methods are caused by impurities in reagents, followed by running the sample at high pressure over metal ferrules etc. – conditions that are excellent for promoting oxidation.
7) One of the best and simplest methods for plasma is the fluorimetric method given in Curtius and Roth (excellent Swiss textbook that was even too expensive for my department to buy when it came out in 1974) - Brubacher G and Vuilleumier JP. Procedure for determination of vitamin C in plasma. In: Curtius H Ch and Roth M, eds. Clinical Biochemistry, Principles and Methods. Berlin: Walter de Gruyter, 1974: vol. 2:992-997.
8) If you want to measure total vitamin C and have a fluorimeter, you will have the manual assay finished before the HPLC is even switched on ! Of course you can measure ascorbic and dehydroascorbic acid by HPLC, but that’s another story.
Nick Miller
London
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