While I have never measured thiamine by the HPLC thiochrome assay it
is the usually quoted method.Difficulties lie in the fact that the
reference range for thiamine is very low and therefore distinquishing
these from deficient states would in theory require an assay that is
extremely sensitive not to mention specific. With the need for
derivitisation the assay sensitivity is unlikely to be adequate for
clinical needs.This is why ETK has long been an easier alternative for
establishing thiamine deficient states.This in turn however is not a
very robust assay and there is really no satisfactory QC material
available to allow for the setting up of a routine service that is
properly quality assured.Both are probably best employed in a research
capacity at present.There is the old chestnut of giving the ? vitamin
deficient patient the vitamin in question and watching the clinical
response.Hope this doesn't sound too negative.I do believe sometimes
that no result is better than an inaccurate/imprecise one.
______________________________ Reply Separator _________________________________
Subject: Thiamine by HPLC
Author: [log in to unmask]
Date: 10/09/1999 12:04
I would like to make contact with anyone measuring blood thiamine levels by
HPLC. I have a Masters student doing his project in the above area and have
only found one other laboratory in Australia which has looked at this and
they have opted for a
microbial bioassay as the HPLC method had problems - we have trialled the
Biorad thiamine assay but are not completely
happy with its performance. The method showing the greatest promise is that
published by JW Brunnekreeft et al. J Chrom 491
(1989) 89-96 - a precolumn derivatisation method using ferricyanide to
produce the thiochrome. We have kindly been given some material from SKZL
along with the EQAP results - we were intrigued that the majority of
laboratories reporting results used cyanogen bromide derivatisation - is
this for some historical reason or can it be safer to use or better
performing than ferricyanide?
Do laboratories measuring thiamine measure total thiamine by individual
quantitation of TMP,TPP,TTP and Thiamine and summation or by the
prechromatographic cleavage of the phosphate moieties to give one thiamine
peak or by just quantitating the TPP peak (80-90% of total and presumably
that fraction measured by the ETK assay)? Which sample (urine, blood,
fasting plasma) shows the best correlation with brain levels?.
The decision by Roche to discontinue supporting the Cobas Bio has led us
scrambling to our HPLC looking for an alternative
to the current ETK assay - by far the most popular method used to assess
the thiamine status in our at risk patient population
here in Australia.
Thanks
Michael Freemantle
Sullivan Nicolaides Pathology
PO Box 344
Indooroopilly Q4068
Brisbane
Australia 4068
ph +61 (0)7 33778638
fax +61 (0)7 38705989
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