Page 70 - Modern Derivatization Methods for Separation Sciences

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of vitamins regulate metabolism. Vitamins must be obtained from food because they cannot be
synthesized in the body. Vitamins have widely different functions in the body, and deficiency of
vitamins may upset the balance and function in the body. Vitamins are used as food additives for
nutrient fortification. Although HPLC is the main method in use for vitamin analysis in recent years, a
few have been reported for the derivatized method.

Vitamin B (Thiamine)
1
The Post-column and pre-column method with cyanogen bromide or potassium ferricyanide is used to
form thiochrome fluorescence (λex375 nm, λem440 nm) after the separation of reversed-phase column,
used for the analysis of water soluble vitamin B [111-114]. The drawback of the precolumn method is
1
the formation of by-products. Although thiochrome fluoresces strongly under alkaline conditions, the
fluorescence intensity of thiochrome decreases when the ODS column is used within the recommended
pH region of the mobile phase. Therefore, preference may be give to using an alkaline-tolerant column
or the introduction of an alkaline solution to the ODS column using the post-column technique [115].
This method is applied for the simultaneous determination of thiamine and thiamine phosphoric ester in
a biological sample.

The post-column method can separate each thiamine phosphoric ester and interfering peaks are seldom
observed. The use of oxidizer for the post-column reaction solution requires such maintenance as
washing in flow. The post-column method with potassium ferricyanide was applied for food analysis
and a peak of hydroxyethylamine, an active form of thiamine, was observed, but thiochrome from
hydroxyethylamine was not recognized when cyanogen bromide was used [111,116].

Vitamin B (Pyridoxine)
6

Three organic compounds of pyridoxine (PN), pyridoxal (PL) and pyridoxamine (PM) act as vitamin B 6
and the most common forms of these compounds in animals and plans are phosphoric esters. They are
widely found in animal and plant foods, and in particular cereals contain large quantities of vitamin B
6.
Phosphoric derivatives of PL and PM exist in nature and work as coenzymes. PN was extracted with
perchloric acid and detected with fluorescence HPLC from juice, fruit, vegetable, grains and dairy
products [117]. This method employs derivatization with potassium cyanide to obtain more than 10
times stronger fluorescence intensity because the sensitivity of the PL ester is low. The use of
fluorescence post-column method with sulfurous acid after extraction with perchloric acid and
separation with ion-paired reversed-phase HPLC was applied to the analysis of PN (6 derivatives) in
pork liver and milk [118]. This method was seldom interfered with from unknown peaks, showing high
sensitivity.

Vitamin C (Ascorbic Acid)

Vitamin C has long been recognized as an important nutrient in several food products. The reduced and
oxidized form of the vitamin are referred to as ascorbic acid (AA) and dehydroascorbic acid (DHAA),
respectively. In humans, both forms are biologically active. The total vitamin C activity is the sum of
both forms.

As for analysis for vitamin C, being electrochemically inactive, DHAA cannot be detected with UV
absorbance. Therefore DHAA is reduced to AA with dithiothreitol [119], DL-homocysteine [120] or L-
cysteine [121] and separated with ionexchange or reversed-phase HPLC followed by ECD detection.
This method is applied for the analysis of fruit juice and green tea [121]. As the method measures total

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