pdf 2_3_2007 - Technologia Alimentaria

Modifications of spectrophotometric methods ...
Values of analytical parameters for the developed modifications of all methods
which were obtained during the validation studies discussed above, are presented in the
Table 1. The authors of the original methods did not perform similar studies, any comparative
analysis is therefore impossible.
Table 1. Analytical parameters of the authors’ modifications of spectrophotometric methods for
antioxidative vitamins determination
Tabela 1. Parametry analityczne własnych modyfikacji spektrofotometrycznych metod oznaczania
witamin antyoksydacyjnych
Modification of
determination
method of vitamin
Modyfikacja metody
oznaczania witaminy
DISCUSSION
Correlation
coefficient of
analytical curve
Współczynnik
korelacji krzywej
analitycznej
Technologia Alimentaria 6(3) 2007
Detection
limit
Granica
wykrywalności
µM
Recovery
factor
Współczynnik
odzysku
%
Coefficient of variation, %
Współczynnik zmienności, %
intra-serial
wewnątrzseryjny
23
inter-serial
międzyseryjny
C 0.999 0.05 99.0-101.2 0.7-0.9 5.2
E 0.998 0.04 98.8-100.5 0.8-2.7 4.8
A 0.999 0.06 98.7-100.2 0.9-3.2 4.3
To be able to determine vitamins A, E and C in biological material in a precise, fast
and inexpensive way spectrophotometry was chosen. Despite availability of numerous
instrumental methods, spectrophotometry seems to be most frequently used in analytical
laboratories. This is due to a good precision of the method, availability of spectrophotometers
as not very expensive apparatuses and usage of easily accessible, because relatively
cheap reagents. As a result, spectrophotometric methods are widely used, among others in
food analytics for determination of many natural compounds, including vitamins.
Numerous spectrophotometric methods allow quantitative determination of the reduced
form of vitamin C (L-ascorbic acid) or, so called, the total vitamin C – a sum of the
reduced and oxidised form (dehydro-L-ascorbic acid). A popular method using 2,4dinitrophenylhydrazine
[Roe and Kuether 1943] is used for determination of the total
vitamin C. The method, however is not sufficiently accurate, its results being overstated
due to the presence of 2,3-dioxo-L-gulonic acid (biologically inactive metabolite of the
vitamin C) in the tested samples, besides it also requires 3-hour incubation at 37 o C and
addition of concentrated H2SO4 to the samples. Another well known method which uses
aqueous solutions of 2,2’-bipyridyl, FeCl3 and H3PO4 [Sullivan and Clarke 1955], allows
selective determinations of the reduced vitamin C, but is characterized by low sensitivity,
slow course of the reaction (1 hour) and turbidity of the tested samples which requires
their final centrifugation. Various spectrophotometric adaptations of the Tillmans’ titration
method using 2,6-dichlorophenolindophenol (for example [Omaye et al. 1979]) are similarly
applied. They are easy to perform but not entirely selective and require separate
measurements of absorbance for the resulting colour and following its removal.
Vitamin E can be spectrophotometrically determined with two popular methods
concerning the so called, total vitamin E which is a mixture of four tocopherols and four
tocotrienols. According to the first one [Emmerie and Engel 1938] vitamin E is ex-