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32 Part 1: Principles/Food AnalysisVitamin analysis of foods are completed for numerous rea-
sons, including regulatory compliance, nutrient labeling, inves-
tigating the changes in vitamin content attributable to food pro-
cessing, packaging, and storage (Ball 2000). As such, numerous
analytical methods have been developed to determine their levels
during processing and in their final product.
The scientific literature contains numerous analytical methods
for the quantitation of water-soluble vitamins, including several
bioassays, calorimetric, and fluorescent assays, all of which have
been proven to be accurate, specific, and reproducible for both
raw and processed food products (Eitenmiller et al. 1998). By
far, HPLC has become the most popular technique for quantify-
ing water-soluble vitamins, and the scientific literature contains
an abundance of HPLC-based methodologies (Russell 2000). In
general, it is the nonvolatile and hydrophilic natures of these vi-
tamins that make them excellent candidates for reversed-phase
HPLC analysis (Russell 1998). The ability to automate using
autosamplers and robotics makes HPLC an increasingly popu-
lar technique. Since the vast majority of vitamins occur in trace
amounts in foods, detection and sensitivity are critical consider-
ations. Although UV absorbance is the most common detection
method, both fluorescence and electrochemical detection are
also used in specific cases. Refractive index detection is seldom
used for vitamin detection due to its inherent lack of specificity
and sensitivity.
During the 1960s, GC with packed columns was widely used
for determining the presence and concentrations of various fat-
soluble vitamins, especially vitamins D and E. Unfortunately,
additional preliminary techniques such as thin-layer chromatog-
raphy and open-column techniques were still necessary for the
preliminary separation of the vitamins, followed by derivatiza-
tion to increase their thermal stability and volatility (Ball 2000).
More recently, the development of fused-silica open tubular cap-
illary columns has revived the used of GC, leading to a number of
recent applications for the determination of fat-soluble vitamins,
especially vitamin E (Marks 1988, Ulberth 1991, Kmostak and
Kurtz 1993, Mariani and Bellan 1996). Even so, HPLC is also
the method of choice for the fat-soluble vitamins in foods (Ball
1998). The main advantage of this technique is that the vitamins
need not be derivatized, and the method allows for both greater
separation and detection selectivity. Various analytical HPLC
methods have been introduced for the first time in the 1995 edi-
tion of the Official Methods of Analysis of AOAC International,
including vitamin A in milk (AOAC 992.04 1995) and vitamin
A (AOAC 992.26 1995), vitamin E (AOAC 992.03 1995), and
vitamin K (AOAC 992.27 1995) in various milk-based infant
formulas (Ball 1998).
At the present time, there are no universally recognized stan-
dardized methods that can be applied to all food types for de-
termining the presence, amount, or identity of the fat-soluble
vitamins (Ball 1998).PIGMENT ANALYSIS
The color of food is a very important sensory characteristic
as it is often used as an indicator of both food quality and
safety (Schwartz 1998). A vast number of natural and syntheticpigments are obtained from both plant and animal sources as well
as are developed during processes, for example, carmelization
and Maillard browning reactions. The majority of the naturally
occurring pigments in foods are divided into five major classes,
four of which are found in plant tissues while the fifth is found in
animal tissues (Schwartz 1998). Of those found in plants, two are
lipid soluble, that is, the chlorophylls (green) and the carotenoids
(yellow, orange, and red), with the other two are water soluble,
that is, anthocyanins (color usually depending upon pH, red in
acidic conditions and blue in basic) and betalains (yellow or
red indole-derived). Carotenoids are found in both plant (e.g.,
carotenoids (carrots), lycopene (tomatoes)) and animal tissues
(e.g., milk, egg yolk), but in animals they are derived from
dietary sources, for example, the yellow color in milk/egg yolk
is due to the consumption of green plants during pasture feeding
with various grasses, clovers, etc. (Schwartz 1998), not from
biosynthesis in the animal itself.
Several analytical methods exist for the analysis of chloro-
phylls in a wide variety of foods. Early spectrophotometric meth-
ods permitted the quantification of both chlorophyll a and b by
measuring the absorbance maxima of both chlorophyll types.
Unfortunately, only fresh plant material could be assayed as
no pheophytin could be determined. This became the basis for
the AOAC International spectrophotometric procedure (Method
942.04), which provides results for total chlorophyll content as
well as for chlorophyll a and b quantitation.
Schwartz et al. (1981) described a simple reversed-phase
HPLC method for the analysis of chlorophyll and their deriva-
tives in fresh and processed plant tissues. This method simplified
the determination of chemical alterations in chlorophyll during
food processing and allowed for the determination of both pheo-
phytins and pyropheophytins.
Numerous HPLC methods have been developed for
carotenoid analysis, specifically for the separation of the differ-
ent carotenoids found in fruits and vegetables (Bureau and Bush-
way 1986). Both normal and reversed-phase methods have been
used with the reversed-phase methods predominating (Schwartz
1998). Reversed-phase chromatography on C-18 columns us-
ing isocratic elution procedures with mixtures of methanol and
acetonitrile containing ethyl acetate, chloroform or tetrahydro-
furan have been determined to be satisfactory (Schwartz 1998).
Carotenoids are usually detected in wavelengths ranging from
430 nm to 480 nm. Sinceβ-carotene in hexane has an absorp-
tion maximum at 453 nm, many methods have detected a wide
variety of carotenoids in this region (Schwartz 1998).
Measurements of the water-soluble but pH color-dependent
anthocyanins (red, purple, or blue) have been performed by de-
termining absorbance of diluted samples acidified to approxi-
mately pH 1.0 at wavelengths between 510 nm and 540 nm. Un-
fortunately, absorbance measurements of anthocyanins provide
only total quantification and any further information about the
presence and amounts of various other individual anthocyanins
must be performed by other methods. HPLC methods using
reversed-phase methods employing C-18 columns have been
the methods of choice as the anthocyanins are water soluble.
Mixtures of water, acetic, formic, or phosphoric acids usually
are used as part of the mobile phase.