Page 72 - Modern Derivatization Methods for Separation Sciences

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The method was applied for the analysis of canned corn, potatoes, green beans, potato chips, cereals
and infant formula and favorable chromatograms with the absence of interfering peaks were observed.
In the pre-column derivatization with OPDA, problems were encountered with the stability of the
derivative in aqueous solutions.

The use of the post-column method is reported; after separation of AA and DHAA by ionexclusion
HPLC, DHAA was reduced with sodium borohydride under alkaline conditions and detected at UV300
nm [124]; after separation by anion-exchange HPLC, AA was oxidized to DHAA with the addition of
HgCl or CuSO reagent followed by DFQ derivatization with OPDA [125,126]. This method was used
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for the analysis of fruit juice and meat-based food.
Vitamin C is the most unstable among vitamins and destroyed by air oxidation and heat. Caution is
required for extraction and clean-up procedure. Extracting solvents of vitamin C are pyrogallol added
citric acid [125], metaphosporic acid [127], EDTA containing sulfuric acid [119], trichloroacetic acid
[128] and acetic acid containing metaphosporic acid [123,126], suppressing oxidase and copper ion.

Folacin (Pteroylglutamic acid)

Folacin is a glycoside of p-aminobenzoic acid and glutamic acid with pteridine skeleton. The pre-
column method is used to separate folic acid by oxidation with potassium permanganate and
hydrogenperoxide to form fluorescence product (2-amino-4-hydroxypteridine-6-carboxylic acid)
followed by HPLC [112]. Sample pretreatment of oral nutrients for the analysis of folic acid employs
extraction under alkaline conditions and clean-up with SPE (Bond Elut SAX).

Biotin

Biotin is a growth factor of yeast and some bacteria and anti-albumin disorder factor. Taking too much
albumin causes nutritious disorder. Biotin occurs in most foods and yeast, liver, egg yolk and tomatoes
are rich sources of this vitamin.

As for biotin analysis in foods, microbiological assay with Lactobacillus plantarum has been
traditionally used. The use of the HPLC method using fluorescence pre-column technique with 4-
bromomethyl-7-methoxycoumarin (Br-MMC) [129], 9-anthryldiazomethane (ADAM) [130], 1-
pyrenyldiazomethane (PDAM) [131], panacyl bromide [132] was developed and applied to the analysis
of biological samples.

The post-column method includes chlorination of the amide group of biotin, introduction of thiamine to
form thiochrome and detection with fluorescence [133]. The analysis of pharmaceutical preparations
employs this method and the detection limit is 10 ng.

Vitamin K

Most 2-methyl-1-naphtoquinone derivatives show vitamin K activity to some extent. Naturally
occurring vitamin K is vitamin K (phytonadione) found in plants and vitamin K (menaquinones) in
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bacteria. The reduction fluorescence method is developed for the analysis of vitamin K. The method
includes post-column reduction of vitamin K with zinc or platina column to form fluorescence product
(corresponding hydroquinones) and fluorescent detection. Application was reported for the analysis of
cereals, fats and vegetables using the post-column method with sodium tetrahydroboric acid [134], for
infant formula with zinc column [135], and for breast milk with platina column [136]. To measure 2,3-
epoxide of vitamin K an electrode of ECD detector was used for the reduction to form a fluorescence
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