Page 66 - Modern Derivatization Methods for Separation Sciences

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Fig. 1.2.4.
HPLC chromatogram of fatty acids in beer. (1) Lauric acid (C12:0); (2)
linolenic acid(C18:3); (3) myristic acid (C14:0); (4) palmitoleic
acid (C16:1); (5) linoleic acid (C18:2); (6) palmitic
acid (C16:0); (7) oleic acid (C18:1); (8) n-heptadecanoic acid
(C17:0); (9) stearic acid (C18:0). (Reproduced
from ref. 103: J. Agric. Food Chem., (1990) 38, p. 1365, Fig. 5.).

1.2.2.4—
Organic Acids

Organic acids are compounds which have a carboxylic group in the molecule, including fatty acids and
amino acids. Organic acids in foods are often called fruit acids, mainly contained in fruit and vegetable
and tasting sour. Organic acids are also intermediate or end products in biological metabolism. As for
foods, such agricultural products as fruit, vegetable and grains, such fermented products as Japanese
sake and bean paste(miso), meat and dairy products of milk, and such sea foods as fish, shellfish and
seaweeds are good sources of organic acids. These organic acids are not only associated with food taste
but are used for additives for some purposes of pH adjustment, nutrition fortification, sour agents and
preservatives.

Recently the use of HPLC or CE for the analysis of organic acid, has been applied, but derivatization
methods have not been well reported. The pre-column method includes esterification with phenacyl
bromide [104,105] and O-(4-nitrobenzyl)N,N'-diisopropylisourea (PNBDI) [106] followed by
separation with reversed-phase HPLC and detection at UV (254 or 265 nm) has been traditionally
applied to the analysis of organic acids in fruit juice (Fig. 1.2.5). However, PNB derivatization could
not produce characterizable derivatives of oxalic acid and pyruvic acid.

Fluorescent derivatization with pre-column method uses the reaction of lactic acid with ceric sulfate to
form acetaldehyde followed by condensation of 1,3-cyclohexadione to produce fluorescent 9-methyl-
1,8-dioxaoctahydroacridine(λex366 nm, λem455 nm). This method was applied to the analysis of lactic
acid in wine [107]. Pretreatment includes 10-fold dilution of the sample with water, boiling down to
half the volume of the diluted sample to eliminate native acetaldehyde and ethyl acetate in wine,
followed by clean-up with Bio-Rex 5 anion-exchange column. Fluorescent derivatives were separated

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